CYP6P9a and CYP6P9b) combine to impose additive fitness costs in pyrethroid-resistant Anopheles funestus. Genotyping of the CYP6P9b resistance allele in hybrid mosquitoes from a pyrethroid-resistant FUMOZ-R and the susceptible FANG strains revealed that this gene imposes a fitness cost in resistant mosquitoes similar to CYP6P9a. Homozygote susceptible CYP6P9b_S (SS) significantly lay more eggs than the resistant  and with greater hatching rate (p < 0.04). Homozygote resistant larvae CYP6P9b_R (RR) developed significantly slower than homozygote susceptible from L1-L4  with a late pupation observed for RR compared to both heterozygotes and homozygotes susceptible . No difference was observed between genotypes for adult longevity with no change in allele frequency and gene expression across the lifespan. Furthermore, we established that CYP6P9b combines with CYP6P9a to additively exacerbate the fitness cost of pyrethroid resistance with a greater reduction in fecundity/fertility and increased developmental time of double homozygote resistant mosquitoes. Moreover, an increased proportion of double homozygote susceptible individuals was noted over 10 generations in the insecticide-free environment  suggesting a reversal to susceptibility in the absence of selection. Such greater fitness cost imposed by multiple P450 genes shows that resistance management strategy based on rotation could help slow the spread of resistance.Metabolic resistance driven by multiple P450 genes is worsening insecticide resistance in malaria vectors. However, it remains unclear whether such multiple over-expression imposes an additive fitness cost in the vectors. Here, we showed that two highly over-expressed P450 genes (  •Metabolic resistance to insecticide is major challenge in mosquito control.•Fitness cost of metabolic resistance is poorly characterized.•This is the first study evaluating the fitness cost of duplicated P450 using molecular marker.•Anopheles funestus.We showed that CYP6P9b combines with CYP6P9a to exacerbate the fitness cost of pyrethroid resistance in  IncreasAn. funestus  and FUMOZ-R (resistant). Moreover, DNA from mosquitoes maintained in the insectary for 10 generations was used to genotype the CYP6P9b as previously done for CYP6P9a . For this purpose, genotype frequencies were monitored in each stage of development. The dynamic of pupae formation was evaluated by comparing the genotype and allele frequencies from the starting of pupation (pupae D9), in the third day of pupation (pupae D11) and on the fifth day of pupation (pupae D13).CYP6P9b at each time point. The lifespan of homozygous resistant adult mosquitoes was compared to that of susceptible and heterozygotes mosquitoes by assessing the frequency of CYP6P9b genotypes/alleles and the expression level of CYP6P9a/b (qRT-PCR) at different time points.After the emergence of adults, a set of about 150 mosquitoes was removed from the cages at different time points . On average, 100 mosquitoes were used for genotyping whereas 3 pools of 10 mosquitoes each were used to assess the gene expression level of 2.3CYP6P9b-R resistant allele frequency in the absence of insecticide pressure was assessed by cage experiments. After crosses between female FANG and male FUMOZ, the progeny obtained were let in cages for intercrosses for ten generations. In each generation, all mosquitoes irrespective of their genotypes were mixed in cages for intercrossing to generate the next generation. Each generation consisted of about 3 cages of at least 200 mosquitoes/cage of all genotypes. In the first generation, the frequency of the CYP6P9b_R resistant allele was assessed and then monitored in the following generations by genotyping a set of females aged between 2 and 5 days old.The dynamics of 2.4CYP6P9a resistance allele was done using PCR restriction fragment length polymorphism (RFLP) (using a Taq1 restriction enzyme) method as recently described  (cut site 5′-GTSAC-3′). This restriction enzyme cuts the susceptible mosquitoes in two fragments of 400 bp and 150 bp whereas the resistant is uncut at 550 bp.Genomic DNA was extracted from adult mosquitoes and all larval and pupal stages using the LIVAK method . The genescribed . CYP6P9bescribed . The res2.5CYP6P9a and CYP6P9b, were assessed by a quantitative reverse transcription PCR (qRT-PCR), as previously described  from D1, D10, D20, and D30 after adult emergence was extracted using the Picopure RNA Isolation Kit (Arcturus). The transcription patterns of the duplicated cytochrome P450 genes escribed . RNA  was observed in the distribution of genotypes between females which have successfully laid eggs after blood feeding and those which did not lay eggs although a trend of a higher proportion of homozygote susceptible was observed in those that laid (p = 0.04) (p = 0.3)   B. No difp = 0.3) B. This s = 0.09) B suggestCYP6P9b_SS genotype laid more eggs (76.0 ± 4.4) than RR (55.2 ± 11.2) (p = 0.007) but no significant difference was found between RS (70.8 ± 4.8) and RR (55.2 ± 11.2) (p = 0.09) (p = 0.03). Accordingly, the hatch rate also was lower in RR compared to other genotypes (p < 0.04)  C. Concer < 0.04) C.3.2CYP6P9b was shown not to be necessarily linked to CYP6P9a , we next assessed how combinations of genotypes at both genes affect the fecundity of An. funestus. The independent segregation of genotypes at both genes was confirmed when analysing the influence of combined genotypes on the ability of females to lay eggs. Several combinations of genotypes were observed including RR/RR, RR/RS, RS/RS, SS/RS, and SS/SS (P = 0.02) and to RS/RS . The same trend was seen against other combinations although not significant; RR/RS  and RS/SS   E.3.3CYP6P9b to assess the influence of this marker on larval development. This revealed a significant and consistent decrease of the resistant allele CYP6P9b-R from L1 to L4 as noticed previously for CYP6P9a, indicating greater mortality or slower development of the resistant mosquitoes during this immature stage. The proportion of homozygote resistant RR decreases from L1 (11%) to L4 (5%) although this was not significant   together with a significant increase of the homozygote susceptible genotype SS from L1 (40%) to L4 (68%) . This highlights a significant fitness cost of CYP6P9b on the larval development of resistant mosquitoes.On average, 100 randomly collected larvae for each L1, L2, L3, and L4 stages were genotyped for P = 0.2) A probablCYP6P9b genotypes in the pupae obtained in D9 post-hatching, D11, and D13 showed an apparent decrease of the homozygote susceptible SS genotype from D9 (58%) to D11 (34%)  together with a significant increase of the homozygote resistant genotype RR and heterozygote RS from D9 to D13  confirming that homozygote susceptible mosquitoes developed significantly faster than homozygote resistant and heterozygote mosquitoes  whereas there was no difference with CYP6P9b-RS  (Assessment of the rate of pupae formation by comparing the frequency of the squitoes B. Assessp < 0.6) C.CYP6P9a and CYP6P9b on the time of development revealed a bigger impact than with CYP6P9b alone as the SS/SS genotype developed significantly much faster than RS/RS  and RR/RR . RS/RS developed also significantly faster than RR/RR   (p > 0.4) and RS . Evaluation of the expression level of CYP6P9b at the different time-points showed no significant difference in the level of expression of this gene . The relative expression recorded was 12.03 ± 3.50 fold-change (FC) in D1, 10.4 ± 7.2 FC in D10, 9.7 ± 4.6 FC in D20 and 9.2 ± 3.8 FC in D30 .No difference was observed in the longevity of p = 0.8) A. In addC in D30 C. This s3.5CYP6P9b-R allele over 10 generations in the absence of insecticide selection pressure. A frequency of 50% of the resistant allele was confirmed in the F1 generation of the FANG/FUMOZ as well as a 50% for the susceptible allele. A significant and consistent increase in the proportion of homozygote susceptible mosquitoes was observed from F2 (20%) to F10 (58.1%)   suggesting a reversal to susceptibility. Accordingly, an increase in the frequency of the susceptible allele was observed from F2 (50%) to F10 (73%)  . Furthermore, an increased frequency of SS/SS double homozygote susceptible genotype was observed   A&B.Fig. 4CYP6P9b gene, we investigated the fitness cost imposed by this P450-based resistance on various life-traits of An. funestus. This revealed a significant cost imposed by CYP6P9b and that this marker combines with CYP6P9a to additively exacerbate the fitness cost caused by pyrethroid resistance.In this study, using the recently designed DNA-based diagnostic assay for cytochrome P450 4.1CYP6P9b induced a reduction in mosquitoes' fecundity and fertility as previously observed for CYP6P9a . The proportion of females that laid eggs was lower in the resistant and heterozygote genotypes than for susceptible and females with resistant genotype CYP6P9b-RR showed a significantly reduced number of eggs and larvae compared to females with the CYP6P9b-SS susceptible genotype. Also, we observed a greater mortality/slower development of the resistant mosquitoes during larval development compared to the susceptible ones as previously observed for the CYP6P9a. Such greater mortality associated with slower development of mosquitoes with the CYP6P9b-R resistant allele could be linked to the fact that despite all the three genotypes were maintained in the same larval bowl, larvae with CYP6P9b-R resistant allele were probably less competitive for food and space compared to those with the susceptible allele and therefore, developed significantly slower. As observed previously in resistant Culex pipiens for carboxylesterase-mediated metabolic resistance  at both n dengue  and malan dengue  vectors.n Africa . However4.3CYP6P9a-R, in this study, a significant decrease in the frequency of the CYP6P9b-R resistant allele was observed after ten generations in the insecticide-free environment, which corresponds to around 1 year. Also, a significant increase in the frequency of SS/SS susceptible genotype was observed. This reduction in the resistant allele frequency could be attributed either to the accumulation of deleterious effects observed in some life traits of the vector as noticed for fecundity and larval development here  awarded to CSW.The authors declare no conflicts of interest.


Plasmodium parasites. Here we use a newly developed transgenic Plasmodium berghei ANKA (Ama1OVAPbA) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates that -/-Ifnar1 mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8+ T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protected -/-Ifnar1 mice and wild type mice suffering from ECM. Importantly, CD8+ T cells were increased in the spleens of ECM-protected -/-Ifnar1 mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8+ T cell infiltration into the brain and increased ECM induction in Ama1OVAPbA-infected -/-Ifnar1 mice. However, eosinophil-depletion did not reduce the CD8+ T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8+ T cell migration and proliferation during Ama1OVAPbA-infection in -/-Ifnar1 mice and thereby are contributing to the protection from ECM.Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses to  Plasmodium falciparum is responsible for over 99% of malaria cases in Africa and can lead to serious complications such as cerebral malaria (CM)  . This coria (CM) , 3. Sevec system , 4–6. Sec system –12 and ac system , 13–17. c system , 18–21. c system , 21.Plasmodium parasites is of major interest , in the immunopathology and regulation of immune responses towards infection with interest . The ind general  and P. b malaria , 24–28. tivation –32. CD4+n to ECM , 33. Hown to ECM .Plasmodium infection  or transgenic P. berghei ANKA parasites  activation in the spleen and in consequence from abrogated ECM.Type I IFNs are induced early upon nfection  and Ifnaarasites , 35, 36.tivation , we firsin vivo, we generated a transgenic Ama1OVAPbA parasite that expresses a truncated C-terminal fragment of OVA under a blood-stage specific promoter of the apical membrane antigen-1 (AMA-1) gene, a merozoite surface antigen of the parasite. Using the resulting Ama1OVAPbA clonal line, we demonstrate here that ECM protection in Ama1OVA-PbAinfected -/-Ifnar1 mice was not due to an altered antigen-specific cytotoxic CD8+ T cell activity in comparison to infected WT mice. However, infected -/-Ifnar1 mice retained more T cells in the spleen, concomitantly with an induction of large numbers of eosinophils. Importantly, eosinophil depletion reduced the protection of -/-Ifnar1 mice from ECM and led to an increased CD8+ T cell number in the brain.To elucidate antigen-specific T cell responses and their activity Ama1OVAPbA-infected -;/-Ifnar1 mice from ECM was not based on impaired CD8+ T cell generation, but was due to an alternative or regulatory milieu in the periphery – substantially mediated by eosinophils - that prevented the migration of CD8+ T cells into the brain in the absence of type I IFN signaling.Thus, we present a so far unrecognized role of type I IFN signaling in combination with eosinophils in ECM pathology. We conclude that the protection of -/-Ifnar1 mice  mice were purchased from Janvier Labs, Saint-Berthevin, France, and kept under specified pathogen-free conditions within the animal facility at Heidelberg University (IBF) or IMMIP, University Hospital Bonn. /- mice  were brehe RMCBS .Ama1OVAPbA) were generated to express ovalbumin-derived CD8 and CD4 T cell epitopes at distinct stages of their life cycle  as described earlier (For transfection of  earlier . Transfe earlier Table S1Ama1OVAPbA) was tested for its life cycle progression in order to rule out potential growth impairment due to the insertion of ovalbumin into the parasite’s genome. To this end transgenic blood-stage OVA-parasites were allowed to grow in NMRI mice for several days until they have reached a high proportion of sexual stages (gametocytes). Gametocytemic mice were then anesthetized and fed to five to seven days-old female Anopheles mosquitoes for 15 to 20 min. Mosquitoes were kept at 80% humidity and 21-24°C. Vital life-cycle progression of the transgenic parasite was then compared to infections with WT, i.e. sporozoites were dissected from mosquito midguts on day 14 and from mosquito salivary glands on day 17 to 21 after blood meal. 10,000 infectious sporozoites isolated from salivary glands were additionally used for intravenous infection of inbred C57BL/6 mice to test the capacity of the transgenic parasite line to normally infect the mammalian host i. e. to complete the clinically silent intrahepatic phase and initiate the pathological blood-stage infection. To this end infected mice were tested for parasitized erythrocytes by daily blood smears and analyzed after Giemsa staining and light microscopy. The parasites behave indistinguishable from WT parasites throughout the malarial life cycle.The transgenic blood-stage OVA parasite (Plasmodium berghei transgenic parasites (Ama1OVAPbA) containing MHC class I restricted epitopes from chicken ovalbumin were generated as described above. Frozen stabilates of the parasites were stored in liquid N2. Stock mice received 200 µL of the stabilates i. p.. After 4 days blood smears were performed to determine the parasitemia of the stock mice (see below). For experimental infections, 5x104 red blood cells infected with Ama1OVAPbA (iRBC) were then injected intravenously (i. v.) in 100 µL PBS into the mice. From d+3 p. i., the mice were analyzed for symptoms related to ECM with the help of the recently described health score called rapid murine coma and behavior scale (RMCBS)  . BrieflyBlood was collected from the tail vein and thin blood smears were prepared. The blood slides were air dried and fixed in 100% methanol. Giemsa staining solution was prepared by diluting Giemsa stock  1:20 in Giemsa buffer pH 7.2. Slides were stained for 20 mins followed by rinsing in tap water for 1 min, after which they were left to airdry. Approximately 1000 red blood cells were counted, the infected cells noted and the percentage of iRBCs calculated [=(iRBCs/counted RBCs)*100].On d+6p.i. mice received 200 μL of 2% Evans Blue  in 0.95% NaCl i. v.. One hour later, mice were sacrificed and the brains were removed, photographed and weighed, thereafter they were placed in 15 mL tubes containing 2 mL formamide  and incubated for 48 h at 37°C. Quantification of Evans Blue extravasation was performed by transferring 100 μL of the incubated formamide/brain solution in 96 well plates in triplicates. The standard curve was prepared by diluting Evans Blue to 200 μg/mL with 0.95% NaCl (starting concentration), and diluted 1:2 in duplicates with formamide. The concentration of Evans Blue was measured spectro-photometrically at 620 nm. The amount of infiltrated dye was calculated as μg Evans Blue/g brain tissue x2.Vena fascialis and the mice were afterwards anaesthetized with 4:1 Ketamine-Rompun mixture (0.01 mL/10 g body weight). After intracardial perfusion (1 X PBS for 5 minutes), spleens and brains were isolated, cut into small pieces, and digested with 0.5 mg/mL collagenase VIII  at 37 °C for 30 min. The tissues were then gently passed through a metal sieve (Ø 70 µm), single cells were washed with 1X PBS containing 1% FCS and 2 mM EDTA. Leukocytes from the brain were enriched as described before with a Percoll gradient  for 20 min on ice. Intracellular staining was performed using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set , according to the manufacturer’s protocol. To detect SIINFEKL-specific CD8+ T cells in spleens of Ama1OVAPbA-infected mice, 1x106 splenocytes were incubated in 30 μL FACS buffer (1X PBS containing 1% FCS) containing 2.5 μL Rat IgG and 5 μL SIINFEKL-H-2Kb pentamers coupled to allophycocyanin  for 20 min at room temperature. Next, 20 μL FACS buffer containing diluted fluorescently labeled anti-CD8a and CD11a antibodies were added and incubated for another 15 min on ice. Then, the cells were washed with FACS buffer and stored on ice until analysis. For intracellular staining of IFN-γ, CCL5 and Granzyme B (GrzB) the cells were treated with Golgi Plug/Stop (protein transport inhibitors) for at least 4 h to prevent secretion of these effector molecules. For that purpose, 1x106 cells per sample were added to a 96-well cell culture plate and centrifuged for 5 min at 4°C and 400 xg. The cells were resuspended in 200 µL complete RPMI medium + 0.8 µL/mL Golgi Plug + 0.8 µL/mL Golgi Stop (1 x concentrated) and cultured for 4 h at 37°C and 5% CO2. For IFN-γ the cells were additionally stimulated with 50ng/mL PMA , 250 ng/mL ionomycin  during this 4 h. Afterwards the cells were fixed, permeabilized and stained according to the eBioscience protocol mentioned above. The following fluorescently-labelled specific antibodies used for flow cytometric analysis were purchased from BioLegend, San Diego, CA, USA if not stated otherwise: anti-mouse CD3 , CD4 , CD8α , CD11a , CD11b , CD45 , CD49a , CD49b , CD90.2=Thy1.2 , CCL5 , CCR5=CD195 , CCR7=CD197 , CXCR3 , F4/80 , GATA-3 , GrzB , Lineage cocktail , Ly6G , NK1.1 , NKp46=CD335  RELM-α purified polyclonal, rabbit,  in combination with goat anti-rabbit Al488 , Siglec-F , T-bet , TCRβ . Data acquisition was performed on a LSR Fortessa®  and CytoFLEX S  and analyzed with FlowJo® Software V10 .Blood was collected from the gradient . Cells wOrgans were fixed with 0.05 M phosphate buffer containing 0.1 M L-lysine , 2 mg/mL NaIO4 (Roth), and 10 mg/mL paraformaldehyde  overnight at 4°C. Afterwards, the organs were equilibrated in 30% sucrose  solution for 24 h and then frozen in OCT (Weckert Labortechnik) and stored at −80°C. Consecutive sections (5 μm) were mounted on Super Frost Plus glass slides (R. Langenbrinck), dried for 10 min at 70°C, rehydrated with PBS with 0.05% Triton X-100  and blocked for 1 h with PBS containing 1% bovine serum albumin  and 0.05% Triton X-100. The staining was performed in blocking buffer (200 μL volume per section).The antibodies ; CD8a  were incubated for 1h, DAPI  was incubated for 5 min. After each staining step, three washing steps of 5 min with 0.05% Triton X-100 in PBS were performed. Sections were imaged with the Zeiss Axio Observer.Z1 and Apotome  at the Imaging Center Essen and processed by the ZEN Software , ImageJ and R. The image processing was done on ImageJ and R by generating cell- and tissue masks. Masks are images where the background is black , and the foreground, the object of interest, is white . The foreground signals were used to analyze the cell populations of interest, characterized by their emplacement, area and the distance to a point of interest. Tissue and cell signals were then coregistered to determine the location of individual cells, and cell population densities.+ T cells in Ama1OVAPbA-infected animals was determined with an in vivo cytotoxicity assay on day 6 p.i. as described before  for 30 min at 37°C and subsequently with 1 μM of 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester  for 15 min . Reference cells were not pulsed with peptide and labeled with 0.1 μM CFSE for 15 min . Cells were washed with 1 X PBS  and the cell number was determined. The cell populations were mixed at a 1:1 ratio (CFSEhigh/CFSElow). Each recipient received 5x106 cells of each population diluted in 0.9% NaCl  into the tail vein on day 5 p.i.. Mice were sacrificed 18 h later on day 6 p.i. and spleens were isolated to prepare single-cell suspensions as described above. Lysis of peptide-loaded cells was quantified by measuring the ratio of CFSEhigh/CFSElow cells via flow cytometry . The percentage of specific lysis, termed S8L-specific lysis, was calculated using the following equation: 100 - [(CFSEhigh/CFSElow) immunized/(CFSEhigh/CFSElow) naïve] x 100.The lytic activity of SIINFEKL-specific CD8d before . BrieflyAma1OVAPbA, -/-Ifnar1 mice received 1.5 µg/g body weight anti-Siglec-F  i. p. in 100 or 150 μL PBS on day 3, 4 and 5 after infection. Mice were analyzed for symptoms related to ECM according to RMCBS. The depletion of eosinophils was confirmed by flow cytometric analysis of the spleen on day 6 after Ama1OVAPbA infection.After infection with 6 splenocytes or 1.25x106 cells from brain homogenates were cultured in triplicates in 200 μL RPMI 1640  incl. 10% fetal calf serum (FCS)  1% penicillin/streptomycin/L-Glutamin  medium overnight. The supernatants were analyzed by sandwich ELISA for IL-10, IL-13, IFN-γ or TNF , CCL5 and GrzB  according to the manufacturers’ protocols.Organs from experimental animals were isolated on day 6 p.i. and single cell suspensions were prepared as described above. 1x107 cells were incubated with anti-CD11b+ antibodies coupled to magnetic beads accordingly to the manufacturer’s protocol . Cell separation was performed with the AutoMACS® . Eluted cells were counted and stained to check the purity. For the arginase assay, 1x106 cells MACS-sorted CD11b+ cells were plated into 96-well plates and centrifuged at 1000 x g at 4°C for 10 min. Cells were then lysed with 100 μL of 10 mM Tris-HCl, pH 7.4 , containing 1 μM pepstatin A , 1 μM leupeptin , and 0.4% (w/v) Triton X-100  for 10 min. Lysates were centrifuged at 14,000 x g at 4 °C for 10 min. Supernatant was used for the arginase assay. Analysis for urea quantity was then performed according to manufacturer’s instructions . Arginase activity was calculated as units per liter of sample (U/L).From digested spleens, single cell suspensions were prepared and 4x10+ cells were homogenized in TRIzol®  in BashingBeads tubes . PeqGOLD HP Total RNA Kit was used for purification according to the manufacturer’s instructions. Thereafter, on-membrane DNase I digestion  was performed. For qPCR, TaqMan® RNA-to-Ct™ 1-Step Kit  was used with 2 μg total RNA in a reaction volume of 20 μL. Each sample was normalized to beta-actin. The expression level was calculated as fold-increase against naïve as described  were used: YM1 FWD 5’ gcccttattgagaggagcttta 3’, REV 5’ tacagcagacaagacatcc 3’; Arginase FWD 5’ cctatgtgtcatttgggtgga 3’, REV 5’ caggagaaaggacacaggttg 3’; Beta actin FWD 5’ agagggaaatcgtgcgtgac 3’, REV 5’ caatagtgatgacctggcggt 3’.post hoc multiple comparisons was used to test for significant differences between multiple groups. Survival experiments were analyzed with Log-rank (Mantel-Cox) test. If not stated otherwise, values from each individual mouse and median with interquartile ranges (IQR) are shown. p values <0.05 were considered as significant. Prior to pooling data were analyzed for homogeneity by not passing Spearman’s test for heteroscedasticity. If data could not be pooled, but statistical trends (p<0.1) were confirmed by several experiments, the trends were included in the figure.GraphPad Prism software version 8  was used for statistical analysis. Mann-Whitney-U-test was used to test for significant differences between two unpaired groups. Kruskal-Wallis test, followed by Dunn’s PbA parasites in susceptible C57BL/6 mice driven by CD8+ T cells and IFN-γ  of ovalbumin (OVA) including both CD8+ T and CD4+ T cell epitopes under the control of the AMA-1 promoter in PbA, termed Ama1OVAPbA  WT mice were detected on day 6 p.i., thus confirming the successful generation of endogenous parasite-specific T cells  and less ECM symptoms (PbA clones (-/-Ifnar1 mice as a possible reason for ECM protection was excluded (-/-Ifnar1 mice correlated with an intact blood-brain barrier (BBB), whereas the brains of ECM-positive WT mice showed a permeable BBB, demonstrated by an intensive staining due to strong extravasation of the i. v. injected Evans Blue dye  from the periphery versus brain-resident microglia (gated in P3 = CD11bhi CD45int) , thus exhibiting a cytotoxic phenotype  of infiltrated cells in samples of PbA-infected WT mice compared to WT naïve, but not of PbA-infected and naïve A clones . The tre of CCR5 , 48. In -/-Ifnar1 mice after 24h. Whereas samples of naïve controls and infected -/-Ifnar1 mice contained low levels of TNF and CCL3, both mediators were significantly increased in PbA-infected WT mice in comparison to the infected -/-Ifnar1 mice  , CD4+ T cells (16.8%) and eosinophils (16.7%), together with M2-like macrophages (9.2%) were the main CCL5 producers in spleens of PbA-infected -/-Ifnar1 mice (+ CD8+ T cell count (p=0.002) in -/-Ifnar1 mice upon PbA-infection (+ eosinophil count was significantly higher (p=0.02) in the spleens of PbA-infected -/-Ifnar1 mice compared to PbA-infected WT mice  was reached (-/-Ifnar1 mice (-/-Ifnar1 mice expressed significantly more CCL5 (p=0.01) than those of PbA-infected WT mice  cells were analyzed  of RMCBS when comparing PbA-infected Ifnar1-/- mice to depleted Ifnar1-/-  were increased in the PbA-infected -/-Ifnar1 mice that were depleted of eosinophils as compared to the -/-Ifnar1 mice that had received the isotype control    macrophage-driven immune responses are commonly induced by helminths and have the potential to downregulate type 1-driven immune responses. Thus, a protection from excessive inflammation in mice lacking type I IFN signaling was associated with the induction of type 2 immune responses that are usually associated with helminth infections. Accordingly, experimental studies described that co-infections with filariae result in the protection from ECM in an IL-10 dependent manner, although the role of eosinophils was not investigated so far . JR and JS were supported by a PhD scholarship from the Jürgen Manchot Stiftung, Düsseldorf, Germany. JK, JR, JS, and BS were supported by BONFOR 2012-01-22, 2013-01-29, 2017-5-02, 2018-7-01, 2018-7-02, 2019-7-01, 2019-7-02, and 2020-7-03. A-KM is a recipient of a DZIF funded Maternity Leave stipend. AH and BS were members of the Bonn Cluster of Excellence ImmunoSensation EXC 1023 and AH and MH are funded by the Deutsche Forschungsgemeinschaft  under Germany’s Excellence Strategy – EXC2151 – 390873048. AH and MH are members of the German Center for Infection Research (DZIF).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plasmodium falciparum. We used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 77 PTMs on H3 and H3.3, of which 34 are newly identified in P. falciparum. Coexisting PTMs with unique stage distinctions were identified, indicating that many of these combinatorial PTMs are associated with specific stages of the parasite life cycle. We focused on the code H3R17me2K18acK23ac for its unique presence in mature gametocytes; chromatin proteomics identified a gametocyte-specific SAGA-like effector complex including the transcription factor AP2-G2, which we tied to this specific histone code, as involved in regulating gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with coexisting partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher-order gene regulation for distinct proliferation and differentiation processes.Histone posttranslational modifications (PTMs) frequently co-occur on the same chromatin domains or even in the same molecule. It is now established that these “histone codes” are the result of cross talk between enzymes that catalyze multiple PTMs with univocal readout as compared with these PTMs in isolation. Here, we performed a comprehensive identification and quantification of histone codes of the malaria parasite,   •Plasmodium falciparum.First middle-down chromatin proteomics compendium of the malaria parasite, •Novel histone PTMs (including arginine methylation) in both asexual parasites and transmissible gametocytes.•Histone PTM cross talk is dynamic life cycle stage stratified.•Gametocytes rely on histone PTM connectivity to allow onward transmission.•AP2-G2 is an important effector of H3K18acK23ac in mature gametocytes. The complex combinatorial histone code of the malaria parasite is revealed through advanced, quantitative middle-down proteomics. Cross talk between histone PTMs (including novel PTMs) is dynamic and stage specific and includes arginine methylation. Chromatin proteomics show that the transcription factor AP2-G2 interacts with the combination of H3K18acK23ac to drive mature gametocyte transmissibility. The connectivity of the histone code in these parasites ultimately points toward a higher order of gene regulation involved in essential development processes than previously thought. Histone N-terminal tails are reversibly modified by an array of covalent histone posttranslational modifications (PTMs). These alter chromatin structure to fine-tune gene expression in most eukaryotes, resulting in changes in cell fate. Although the contribution of individual histone PTMs in specific biological processes are well described , 2, there.g., H3K9ac and H3K14ac affect H3R8me2  impairs the binding of the effector protein heterochromatin protein 1 (HP1) to the well-known repressive PTM, H3K9me3 . This bl H3R8me2 , 16.Plasmodium falciparum is the causative agent of severe malaria in humans and undergoes rapid rounds of cell division during its asexual replication to proliferate every ∼48 h. A small percentage of asexual parasites differentiate to gametocytes through five distinct stages of development (stages I–V) over ∼14 days in P. falciparum, after which mature male and female stage V gametocytes can be transmitted to the mosquito vector -3–9, enhancer-of-zeste, trithorax) domain-containing protein 7 (SET7), only in the presence of already acetylated H3K14  analysis. With this workflow, co-occurrences between histone PTMs can be identified only for those that localize nearby in the amino acid sequence. However, this proteomic approach is not suitable for distal co-occurring PTMs. As such, distinctively modified peptides and co-occurring PTMs cannot be accurately identified and quantified. The development of “middle-down” MS has advanced proteomics and allowed the complexity of PTM combinations to be investigated , 33. AltP. falciparum parasites, which was generated using quantitative, middle-down proteomics. We identify a comprehensive histone code that is dynamic and has distinct fingerprints in different life cycle stages, implying refined functions to allow different biological outcomes associated with parasite pathology and survival. Several coexisting PTMs are involved in direct cross talk, indicative of coordinated function, particularly in gametocytes, with the code in immature gametocytes being most connected and mature gametocytes using unique combinations. This is exemplified by the functional association of H3K18acK23ac that interacts with a unique reader complex in mature gametocytes to enable strategy-specific gene expression. With this first, comprehensive report of a combinatorial histone code in a eukaryotic parasite, we show that P. falciparum relies on dynamic interactions between histone PTMs for development and differentiation. These data could serve as a model for the importance of combinatorial histone PTMs in pathogenesis in protista.Here, we present the systems-level identification and characterization of the combinatorial histone code of in vitro experiments involving human blood donors and human malaria parasites hold ethics approval from the University of Pretoria Research Ethics Committee, Health Sciences Faculty (NAS332/2019). This work abides by the Declaration of Helsinki principles. Intraerythrocytic P. falciparum parasites  were cultivated in fresh human erythrocytes (either A⁺ or O+) in RPMI-1640 culture medium supplemented with 25 mM Hepes , 0.2 mM hypoxanthine (Sigma-Aldrich), 0.024 μg/μl gentamycin (Hyclone), 5 μg/μl Albumax II (Invitrogen), 23.81 mM sodium bicarbonate (Sigma-Aldrich) and 0.2% w/v D-glucose. Cultures were maintained with daily media change and fresh erythrocyte supplementation at 5% hematocrit, 2% parasitemia under hypoxic conditions  with moderate shaking at 37 °C. Parasites were synchronized to more than 90% rings stages with D-sorbitol. Gametocytogenesis production was initiated at 0.5% parasitemia and a 6% hematocrit in a glucose-free medium under hypoxic gaseous  conditions at 37 °C without shaking , 3 mM MgCl₂, 0.2% v/v Nonidet P-40, 0.25 M sucrose, and a cocktail of EDTA-free cocktail of protease inhibitors. This hypotonic buffer wash stage was repeated twice with the mixture centrifuged at 500g and 4 °C for 10 min. The chromatin pellet was next homogenized in a hypotonic buffer lacking Nonidet P-40  and incubated on ice for 10 min. Histones were subsequently extracted from chromatin and enriched through incubation with 0.25 M hydrochloric acid and then rotated for 1 h at 4 °C. The histone-containing supernatant was combined with an equal volume of 20% trichloroacetic acid then pelleted after 15 min on ice. The histone-enriched pellet was washed with acetone, air dried, and reconstituted using dddH2O. A SpeedVac concentrator was used to dry all the samples , which were stored at −80 °C.Histones were enriched using a protocol described  and adapet al. (2020) with a few modifications (18 column disk (3M Empore) with porous graphitic carbon suspended in 100% acetonitrile on top and washed with 0.1% trifluoroacetic acid. Peptides were eluted by the addition of 70% acetonitrile and 0.1% trifluoroacetic acid elution buffer. Histone protein samples were dried and suspended to a concentration of ∼2 μg/μl in the sample buffer consisting of 60% acetonitrile, 20 mM propionic acid, and ethylenediamine.Middle-down MS was performed according to Coradin ications , 41, 45.2O. The HPLC gradient was set up for a nonlinear gradient: 0% to 70% of buffer B for 2 min followed with 72% to 85% buffer B for more than 90 min. The gradient was on hold for 5 min at 95% buffer B to wash the column before sequential sample loading. To minimize sample carryover, blank injections were run between different samples for at least 20 min with high (80%–95%) buffer B.Separation of digested histone peptides was performed by using nanoliter flow liquid chromatography using an EASY-nLC nanoHPLC (Thermo Scientific) equipped with an analytical weak cation exchange–hydrophilic interaction liquid chromatographic resin  column . Buffer A was prepared with 70% acetonitrile and 20 mM propionic acid to adjust the pH to 6.0. Buffer B consisted of 0.1% formic acid in LC-MS grade Hm/z, as this is the range of the most intense charge states (+8) for histone H3 polypeptides. A charge filter was added to include +8 charge states targeted for fragmentation. The ion transfer tube temperature was set to 300 °C and the spray voltage to 2.3 kV. To fragment peptides while retaining PTM information, electron transfer dissociation was performed (MS detection was performed using an Orbitrap Fusion mass spectrometer (Thermo Scientific) operated in a data-dependent acquisition mode and high mass resolution mode for both MS1 events and MS2 scans for each of the three stages investigated in 2 to 3 independent biological experiments. The full mass spectrum scan was set at 665 to 705 erformed , 47 at aP. falciparum  were searched using Mascot . During individual spectra identification, spectra were filtered on multiple layers: (1) deconvolution discarded all MS/MS ions with no identified charge state; (2) spectral identification was performed using the Mascot algorithm without filters; (3) the Mascot output file was filtered using a custom software named ProteoformQuant  for those identifications that pass the ProteoformQuant filter, all mixed MS/MS spectra without unambiguous fragment ions for differential quantification of isobaric peptides are discarded , including Xtract (Thermo) for spectra deconvolution. Three UniProt entries of histone H3 sequences for ormQuant . This soained in ).http://middle-down.github.io/Software/) was used to confidently identify and quantify modified peptides. Only c/z fragment ions were allowed. PTMs were accepted only if there were at least one site determining ions on both sides of the assigned PTM. Estimation of false discovery rate (FDR) is not applicable for middle-down proteomics as peptides are too long to have any decoy match. A list of all peptide sequences identified and sites of modifications are contained in No missed or nonspecific cleavages were considered; only fully digested N-terminal tails were considered. Endoproteinase GluC was used to generate peptides, which is a serine protease that cleaves peptide bonds at the C terminus of glutamic acid residues. Files were searched with the following dynamic modifications: acetylation (K), phosphorylation (ST), mono and dimethylation (RK), and trimethylation (K). No fixed modifications were considered. The mass tolerance was set to 2.1 Da for precursor ions and 0.01 Da for fragment ions. IsoScale (FPTM1PTM2 is the observed relative abundance (F) of the combination divided by the predicted relative abundance of the combination, calculated by multiplication of the relative abundances of the individual histone PTMs. The relative abundance of the individual PTMs was calculated by summing the relative abundances of all combinatorial peptides containing the particular PTM. If the interplay score is positive, the PTMs are likely codependent or positively related; a negative interplay score indicates that the PTMs are mutually exclusive. Ring plots were visualized using Cytoscape (v. 3.8.2). Other plots were created using GraphPad Prism 9 and R (R Studio v. 4.0.3).Interplay scores between bivalent histone PTMs were calculated according to the following equation:9 cells/ml) was cross-linked with 1% formaldehyde and subsequently quenched with 125 mM glycine. Cross-linked parasite nuclei were suspended in cold lysis buffer  and transferred to a prechilled dounce homogenizer. NP40 was added to a final concentration of 0.25%, and parasites were subsequently lysed with dounce B for ∼100 strokes. Sonication shearing buffer , 10 mM EDTA, and 100 mM NaCl) was added to the nuclei pellet and sonicated with the BioRuptor UCD-200 (Diagenode) for 25 cycles at high power and 30-s intervals. Cross-links were reversed by incubating the input sample overnight at 65 °C. The input sample was suspended to a final volume of 200 μl with ChIP dilution buffer . To dilute the SDS in the dilution buffer, Tris-EDTA buffer  was added with RNase to a final concentration of 0.2 μg/μl and incubated for 1 h at 37 °C. The chromatin soup was incubated with 1 μg of either anti-H3K18ac or anti-H3K23ac, rotating at 4 °C overnight. Protein G magnetic Dynabeads (Invitrogen by Thermo Fisher Scientific) was added and incubated for 2 h rotating at 4 °C. A total of 20% of the eluted chromatin was then retained as the input. The remaining eluted material was then used for the Western blot validation of combination histone PTM. The bead–chromatin complex was washed extensively with each of the following buffers with the aspiration of the preceding buffer before the addition of the next buffer: low-salt immune complex wash buffer , high-salt immune complex wash buffer , LiCl immune complex wash buffer , and TE Buffer . The beads were suspended in fresh ChIP elution buffer , and the supernatant was collected.To identify proteins that associate to individual histone PTMs that occur in combination, chromatin immunoprecipitation (ChIP)-MS was performed using an adapted protocol . A minimg for 3 min and complete removal of methanol:chloroform. The resulting precipitated proteins were dried at room temperature. All samples were dissolved in a solution containing 6 M urea, 2 M thiourea, and 50 mM ammonium bicarbonate, pH 7 to 8. Samples were subjected to reduction, alkylation, and digestion in preparation for MS as follows. Samples were incubated at room temperature for 1 h in 5 mM DTT to reduce disulfide bonds, cysteine residues alkylated in 50 mM iodoacetamide, and subjected to Lys-C (1–1.5 μg) and trypsin (10 μg) in-solution digestion at room temperature for 3 h. Samples were then diluted with 50 mM ammonium bicarbonate to lower the urea and DTT concentrations in solution to prevent trypsin inactivation. The samples were sonicated  for three cycles of 10-s continuous sonication (output 2) followed by 10-s resting on ice to dissolve the pellets completely. Cysteine residues were alkylated by incubation with 40 mM iodoacetamide. This was followed by overnight digestion with 10 μg trypsin at room temperature. The pH of the trypsin digested samples was adjusted with ammonium hydroxide to ∼pH 8 to 8.3 followed by centrifugation at 17,000g for 1 min. StageTip (STop And Go Extraction Tip) cleanup in combination with protein fractionation was performed for all the samples using a dual resin approach, where both Empore C18 disks (3 M) and OligoTM R3 reversed-phase resins were used in combination. This was done to remove unwanted contaminants before MS and to fractionate the samples to ensure optimal protein detection. The column was equilibrated with 1 mM ammonium bicarbonate (pH 8.0), after which the supernatant of each sample was added to the dual resin StageTip and pushed through the column using a syringe (dropwise). All samples were dried down completely under vacuum (SpeedVac concentrator). In preparation for the MS run, 0.1% formic acid was added to the samples followed by ultrasonic bath sonication for 10 min at 4 °C. The samples were centrifuged at 17,000g for 10 min, and the supernatant was loaded onto a Dionex LC system (Thermo Fisher Scientific), coupled online with a Q-Exactive HF mass spectrometer (Thermo Scientific). Peptides were loaded into a picofrit 20-cm-long fused silica capillary column (75 μm inner diameter) packed in-house with reversed-phase Repro-Sil Pur C18-AQ 3-μm resin. A gradient of 105 min was set for peptide elution from 2% to 28% buffer B (100% acetonitrile/0.1% formic acid), followed by a gradient from 28% to 80% buffer B in 5 min and an isocratic 80% B for 10 min. The flow rate was set at 300 nl/min. The MS method was set up in a data-dependent acquisition mode. The full MS scan was performed at 70,000 resolutions  in the m/z range 350 to 1200 and an automatic gain control target of 10e6. Tandem MS (MS/MS) was performed at a resolution of 17,500 with a collision-induced dissociation collision energy set to 20; an automatic gain control target of 5 × 10e4; a maximum injection time of 100 ms; a loop count of 12; an intensity threshold for signal selection at 10e4, including charge states 2 to 4; and a dynamic exclusion set to 45 s.Briefly, protein samples were mixed with an equal volume of methanol:chloroform (3:1 ratio), followed by a brief vortex step and centrifugation at 4 °C. Samples were washed twice with methanol followed by centrifugation at 17,000P. falciparum UniProt FASTA database, containing 33,655 entries. Sequencing grade trypsin  was used to generate peptides. Trypsin is a serine protease that specifically cleaves at the carboxylic terminus of lysine and arginine residues. When proline is at the carboxylic side of lysine or arginine, the peptide bond is almost completely resistant to cleavage by trypsin. Two trypsin missed cleavages were allowed. Carbamidomethyl (C) was set as a static modification. Oxidation (M), as well as acetyl (protein N-term), were selected as variable modifications. Mass accuracy was set at 4.5 ppm for precursor and 0.5 Da for the product mass tolerance. Peptides were filtered for high confidence (FDR < 1%) using a fixed value validator. Intensity-based absolute quantification (iBAQ) enabled for label-free quantification, where iBAQ values are calculated by a MaxQuant algorithm . Match between runs was enabled and set to a 1-min window. All samples were run in triplicate for three independent biological replicates. For data analysis, iBAQ values were log₂-transformed and normalized by subtracting to each value the average value of the respective sample. The peptide relative ratio was calculated using the total area under the extracted ion chromatograms of all peptides with the same amino acid sequence  as 100%. For isobaric peptides, the relative ratio of two isobaric forms was estimated by averaging the ratio for each fragment ion with a different mass between the two species. Next, extracted ion chromatography of those m/z ions with a mass tolerance of 10 ppm was performed. Statistical significance was assessed using a two-tail heteroscedastic t test .MS raw files were analyzed by MaxQuant software version 1.5.2.8. MS/MS spectra were searched against the string.org,  v.10 database  parametid study . Taken tP. falciparum parasite. For the middle-down proteomics, we analyzed two independent biological replicates for immature and mature gametocytes and three independent biological replicates for asexual trophozoite stages. The use of multiple biological replicates allowed for the assessment of variance in the data and the identification of outliers. By normalizing and averaging the relative abundances of proteins identified across biological replicates, the data are more accurately representative of actual protein abundance as compared with technical replicates. For the ChIP-MS experiments, we analyzed all samples  in three independent biological replicates. In all statistical comparisons, p-values were corrected for multiple testing according to Benjamini–Hochberg. Statistical analysis was performed using GraphPad Prism v6.00 and R v3.5.3, assuming normal distributions. Unless otherwise noted all error bars represent the standard error of the mean for at least three independent biological replicates.The overall aim of this study was to identify combinations of histone PTMs and their theoretical cross talk patterns on intact histone N termini across three biologically unique stages of the A). All samples yielded >300,000 cells/sample, resulting in sufficient yields of acid-soluble nuclear protein fractions containing the histones: 17 ± 1.1 μg for the trophozoite population and 58 ± 10 μg and 70 ± 16 μg for the immature and mature gametocytes, respectively (A).To investigate the presence of coexisting histone PTMs with middle-down proteomics, we isolated parasites as enriched trophozoites (92 ± 0.9%), immature early-stage gametocytes , and mature gametocytes (94 ± 2.9% stage V), to allow stage-specific differences to be inferred A. All saectively A.Fig. 1MB) (B) were accurately quantified, with a coefficient of variance ≤33% and average Pearson r2 = 0.69 between biological replicates (C), allowing comparison of PTM quantities between samples. To ensure differentiation of isobaric peptides as a result of the middle-down proteomics, data-dependent acquisition data were further processed using isoScale Slim  , 54. UniB) B were acplicates C, allowiale Slim , which eale Slim . From thale Slim , 56, 57.The high-resolution, comprehensive nature of middle-down proteomics allowed successful identification and quantification of 77 PTMs on histone H3 and H3.3 , as onlyP. falciparum parasites and 66 could be accurately quantified. Histone H3 contained 40 PTMs of which 33 were accurately quantified, including 16 novel PTMs. These include acetylation and methylation of H3K37 and methylation of several arginines . Several PTMs displayed high relative abundances (>20%) in more than two stages , with the novel PTM, H3K37me1, highly abundant in immature gametocytes (70 ± 6%)  . These cP. falciparum.Collectively, we demonstrate that middle-down MS identified >70 unique PTMs and quantified 86% thereof. This includes 34 novel modifications, thereby providing an updated, high-resolution compendium of histone modifications across multiple life cycle stages of P. falciparum. On average, three coexisting PTMs were present on any given peptide for both H3 and H3.3 (A), like what is seen for these two histones in humans and mice  in immature gametocytes . Stage-specific diversity was somewhat less evident for H3.3 (B), with a markedly decreased number of coexisting PTMs present in trophozoites for this histone (C). The stage stratification was characterized by changes in the identity of the most prevalent combinations. Trophozoites are characterized by a large number of combinations involving H3K14ac, H3K4me3, H3K9me2, and novel PTMs H3K37me1 and H3R17me1  on H3, with only a minor proportion (9%) of combinations shared between asexual parasites and immature and mature gametocytes B. Stage-for H3.3 B, with a histone C. The stH3R17me1 . These iH3R17me1 , 60, witH3R17me1  and the H3R17me1 , 62. In These data indicate that histones are rarely modified by single, individual PTMs. Therefore, multiple histone PTMs, as opposed to a single PTM on a particular histone tail, would contribute to a distinct chromatin structure that could promote gene expression. As well, these histone codes are rearranged in position and type of modifications in different life cycle stages of the malaria parasite. The most frequent combinations in gametocytes diverge from those trophozoites, with immature gametocytes associated with the highest number of coexisting PTMs ascribed to the presence of novel PTMs. The parasite, therefore, employs a unique and diverse set of PTM combinations likely to guide stage-specific gene expression.The extent and relevance of the influence between pairs of coexisting PTMs were subsequently interrogated by quantifying the coexisting frequency as an interplay score (IS) for bivalent combinations . This prP. falciparum (A and B). Of these, 11 combinations show negative interplay scores throughout development (A), which includes multiple combinations of the repressive PTM H3K9me3 (C). For example, this PTM is found on the same histone molecule with H3K14ac significantly more rarely than stochastic co-occurrence in all three stages, implying mutual exclusion and thus opposing biological functions. The typically euchromatic signal of H3K14ac is therefore negated in the event of H3K9me3, and this contributes to the HP1-bound heterochromatic state as described for certain gene sets , all of which show codependency only in mature gametocytes. The strongest differential interplay score between asexual parasites and gametocytes was observed for H3K4me3K23me1. This novel combination is mutually exclusive in trophozoites, supporting the fact that H3K4me3 participates in cross talk with H3K9ac in asexual parasites  and asexual parasites (strong negative interplay) . Both H3R40me1 and H3R17me1 mostly show pronounced negative interplay scores for most of their interactions in trophozoites. H3R17me1 activates as a derepressor in mammalian cells  to produce the global euchromatic signal associated with asexual parasites . The novel mark H3K14me2 displays the largest number of interactions with positive cross talk, including that with H3K9ac (IS = 6), H3K9me1 (IS = 4.9), and both K18me1&2 (IS ∼ 5). These associations are influenced by the level of methylation  to define chromatin states and active genes versus inactive genes within the same locus  and H3KC), although the frequency of the combinations somewhat differs from those in immature gametocytes. H3R40me1 remains highly connected in mature gametocytes, again showing independence from other coexisting marks. Activating H3R17me2 is the most connected PTM in mature gametocytes, retaining its interactions and codependency with other activating marks, H3K18ac (IS = 3.9) and H3K4me1 (IS = 5.9), and with H3K9me2 (IS = 4.8) and H3R8me2 (IS = 5.2). Mature gametocytes are, however, additionally marked by combinations that uniquely and exclusively are present only in this stage of development. This includes codependency between H3K27me1K36me1 (IS = 4.3). In embryonic stem cells, H3K27me1 is dependent on H3K36me1 to promote transcription . The presence of both marks found in combination was validated by Western blot analysis in mature gametocytes (A). Since an antibody against H3K18acK23ac is not commercially available, we opted to capture proteins that interact with or are recruited to both H3K18ac and H3K23ac, respectively, in two separate ChIP experiments using antibodies specific to H3K18ac and H3K23ac  compared with the IgG control ChIP, at an FDR ≤5%. Selective enrichment of proteins for H3K18ac and H3K23ac was confirmed by (1) the significant (p ≤ 0.01) enrichment of unique peptides for low-abundance proteins in the H3K18ac and H3K23ac ChIP preparations compared with the IgG sample preparations ; and last, (3) the enrichment in the ChIP population for chromatin associated proteins (>20% enrichment) .We defined proteins significantly associated with H3K18ac and H3K23ac as those with a logichment) , the nucichment) , and traichment)  (supplem2 fold change ≥2 over IgG control ChIP)  . The traol ChIP) . The genol ChIP) , and is ol ChIP) . AP2-G2 ol ChIP) , 102.C)  , 103 (ApC) . TranscrC) . The comd AP2-G2 , implicaP. falciparum histone acetyltransferase, GCN5 . Furthermore, TRIM24 has been described as CBP-associated proteins  and an ISWI chromatin-remodeling complex ATPase , which interacts with a sucrose nonfermentable (Snf2)-related CREB (cAMP response element-binding protein)-binding protein (CBP) activator (PF3D7_0820000). As a result, AP2-G2, H3K18ac, H3K23ac, ISWI chromatin-remodeling complex ATPase , and Snf2-related CBP activator (PF3D7_0820000) share a link. AP2-G2 is associated with H3K18ac and H3K23ac and interacts with ISWI, which has a relatively high homology to TRIM24 (42% identity), which is a reader of H3K18ac and H3K23ac , 107. Fuproteins . This suin yeast . Since Sin yeast , 115, thP. falciparum mature gametocytes. Arginine 17 methylation is achieved systematically: CBP first acetylates H3K18, then H3K23, and this allows the arginine methylase coactivator-associated arginine methyltransferase 1 (CARM1) to associate with chromatin to methylate H3R17 , our data for this combination provide evidence that the combinatorial histone code of the P. falciparum. Middle-down proteomics provided high-resolution quantitative data to describe the histone code in this parasite, which could function as a model for other protista. Our data reveal that the combinatorial histone PTM landscape is dynamic, with clear stage-specific differentiation observed, with gametocyte stages more dependent on histone PTM cross talk than asexual parasites.Here we present the first systems-level evidence of a comprehensive combinatorial histone code for various life cycle stages of the human malaria parasite, P. falciparum asexual parasites resembles that of lower eukaryotes, which has a simple genome organization and fewer histone PTMs. Given the primal function it performs, elaborate epigenetic gene regulation mechanisms may not be as important to these stages, as most of the genome is in a euchromatic state and actively transcribed during proliferation. Therefore, asexual parasites use less, and mutually exclusive, histone PTMs to regulate key functions such as host immune evasion. However, gametocytes share a similar mechanism of epigenetic gene regulation with other higher-order, multicellular eukaryotes where chromatin is predominantly condensed and highly regulated to specify the identity and purpose of a cell through multiple histone PTMs. General positive cross talk between histone PTMs is seen in gametocytes and, given the limited number of transcription factors in P. falciparum parasites, gametocytes could rather switch to a more complex epigenetic code to impress very specific regulation of its biological processes. This would suggest that, at least in some stages of the parasite, epigenetic level gene control is superior to transcriptional level control.Collectively, our study shed light on the difference between the asexual replicating parasite and the differentiated nonreplicative gametocyte. The histone code of P. falciparum is characterized by the presence of novel marks, with several new arginine modifications identified. The advances in proteomics technologies such as middle-down proteomics allow such robust description of arginine methylation marks , but activating marks are frequently found together. The majority of activating PTMs coordinate irrespective of life cycle stages, particularly H3K9acK14ac, and explains the euchromatic permissive nature of asexual parasites, enabled by coordinated binding of effector proteins. Novel combinations such as H3K4me3K23me1 and several combinations with H3R17me1 and H3R17me2  is pronounced during gametocytogenesis. In addition, during these stages of unique differentiation of P. falciparum, the parasite relies on specific and differentiated combinations, including the unique H3K27me1K36me1 and H3R17me2K18acK23ac combinations seen in only mature gametocytes. The connectivity and cross talk between histone PTMs are therefore essentially important to establish general euchromatic regions in the parasite’s genome across all stages. However, cross talk of activating marks is more prevalent in gametocytes and requires differentiation in marks used for euchromatin in these stages. This implies the use of specific histone combinations for activation of strategy-specific gene sets to mediate P. falciparum transmission.Connectivity between PTMs in The functional relevance of the connectivity of histone PTMs is underscored by evidence that the associated interacting proteins are also highly connected to include reader and writer proteins, “flavored” to specific marks . In thisP. falciparum is therefore diverse and dynamic to effect different combinations required for proliferation and differentiation. The complex nature of the combinations and changes in the identity of the combinations during stage transition point to this epigenetic level of regulation as a more important level of regulation that can be easily fine-tuned by variation in the combinations, particularly for gametocytes. With a limited set of effector proteins and only a core of ∼5 full effector protein complexes characterized  and are accessible through project number 1721. All raw files from the ChIP-MS data are freely available on https://chorusproject.org at project no. 1730. The data analysis pipeline meets all MIAPE standards.The middle-down proteomics data generated in this study have been deposited in the Chorus database (via the ProteomeXchange Consortium (http://www.proteomexchange.org/) using the PRIDE partner repository (All proteomics data including annotated spectra of the modified peptides identified in this study are available pository , with daThis article contains The authors declare that they have no conflict of interest with the contents of this article.


Broccoli extract was utilized as a natural surfactant with active surface groups to control nucleation and growth of formed crystals with the creation of spatial barriers around the cations, and finally prevent nano-product agglomeration. Changing experimental parameters in synthesis reaction in turn offers a virtuous control over the nano-products size and shape. The shape and size distribution of particles was considered via diverse characterization techniques of microscopic and spectroscopic. The photocatalytic behaviors along with a kinetic study of the nanoparticles were examined by elimination and degradation of different artificial dyes under the UV waves. Effect of particle size, weight ratio of LSO:CN, type of dye, scavenger kind, dye and catalyst loading was designated on altering proficiency of nano-catalyst function. Also, the probable mechanism of removal dye by photocatalytic function was studied.The deficiency of drinking water sources has become a serious crisis for the future of the world that the photocatalytic process is one of the most favorable methods for removal of artificial dyes and poisonous organic impurities. In the present study, rapid ultrasonic treatment was performed to obtain La There are many methods to remove artificial dyes from drinking water such as nanofiltration 2Sn2O7 Pyrochlore-type oxide as a semiconductor nano-oxide has an appropriate performance in diverse applications such as photocatalysis 2Sn2O7 nano samples is significant. La2Sn2O7 nano-sized structures were created via several chemical techniques 2Sn2O7 structures has not yet been considered. This work offers a rapid eco-friendly sonochemical method to fabrication of La2Sn2O7 nanostructures as Uv-light-sensitive photocatalyst with the aid of Broccoli extract, for the first time. Ultrasonic irradiations by increasing reaction rate owning to create of high temperature and pressure in a liquid medium are led to chemical reaction accomplishment in a short time 2Sn2O7 structures, the effect of sonochemical reaction condition using the broccoli extract as a green surfactant on their photocatalytic properties of remain unclear. In the present work, it is aimed at considering nano-sized La2Sn2O7 structures synthesized by ultrasonic irradiation in presence of green broccoli surfactant. Graphitic carbon nitride (g-C3N4) polymeric component was selected to the formation of La2Sn2O7/CN nanocomposite because of suitable energy gap (2.7 eV), high surface area and cheap synthesis route as well as finally, advanced photocatalytic activity 3N4 has a favorable impact on photocatalytic efficiency 2Sn2O7/ g-C3N4 nanocomposites were fabricated via green and rapid ultrasonic technique in presence of broccoli extract natural surfactant. After characterization of obtained nano-products in various experimental circumstances, photo-degradation efficiency of them was evaluated in several experimental tests such as particle size, weight ratio of LSO:CN, type of dye, scavenger kind, dye and catalyst loading. Moreover, the photo-driven degradation mechanism of erythrosine dye by LSO/CN nanocomposite was studied.Numerous efforts have been prepared in the field of produce various forms of photocatalyst such as graphene-based nanocomposite 22.12Sn2O7/ g-C3N4 nanocomposites such as La(NO3)3·6H2O (99.99%), SnCl4·5H2O (98%) and melamine were commercially available and employed without further purification. A multiwave ultrasonic generator , immersed directly in the reaction solution. X-ray diffraction (XRD) patterns were recorded by a Philips-X’pertpro, X-ray diffractometer using Ni-filtered Cu Ka radiation. Fourier transform infrared (FT-IR) spectra were recorded on Nicolet Magna- 550 spectrometer in KBr pellets. The electronic spectrum of the sample was taken on Perkin–Elmer LS-55 luminescence spectrometer. Scanning electron microscopy (SEM) images were obtained on LEO-1455VP equipped with an energy dispersive X-ray spectroscopy. The EDX analysis with 20 kV accelerated voltage was done. Transmission electron microscopy (TEM) image was obtained on a Philips EM208 transmission electron microscope with an accelerating voltage of 200 kV.All the chemical reagents for the synthesis of La2.2via distilled water and air-dried. Then, the clean leaves were crushed in the food processor and filtered via filter paper. The obtained broccoli extract was kept in a refrigerator for further use.An appropriate amounts of fresh broccoli leaves were carefully washed 2.33)3·6H2O (99.99%) and SnCl4·5H2O (98%) salts were purchased from Sigma-Aldrich Company and weighed based on stoichiometric ratios (1:1) and dissolved in deionized water, separately. Then, two solutions were added to each other. Broccoli extract was utilized as a natural surfactant. Meanwhile, NH3 was added to the cationic solution dropwise till set the pH to 11. Afterwards, A multiwave ultrasonic generator , immersed directly in the reaction solution for 15 min. The attained products were centrifuged, dried and calcined at 900 °C for 5 h. In order to reach favorable dimension and morphology of nano-scaled structures for enhanced photocatalytic performance, the effect of calcination conditions, ultrasonic time and type of alkaline agent was evaluated that has been exemplified in 2Sn2O7 nanoparticle creation by sonochemical route has been displayed in La consisting of UVA (λ = 320 to 400 nm) and UVB (λ = 290–320 nm).The photodegradation process was accomplished in a home-produced glass reactor set containing 100 mL aqueous solutions of 10 ppm of erythrosine and methyl violet. For every reaction, 10 mg La33.12Sn2O7 nano-structures. By increasing temperature to 900 °C (LSO2), the peaks are still presented in non-crystallized form (2Sn2O7 structures (JCPDS No = 73–1686) were created at 900 °C for 5 h that have been illustrated in 2Sn2O7 nanocrystals. Cubic La2Sn2O7 structures are including four main peaks at 2 that of 28.88, 33.47, 48.06 and 57.04 related to (2 2 2), (4 0 0), (4 4 0) and (6 2 2) plans. The XRD pattern of La2Sn2O7 crystals prepared in blank condition without ultrasonic irradiation has been demonstrated in 2Sn2O7 powder along with little amount impurity of SnO2 (JCPDS No = 03–0439) were formed. To the investigation of alkaline agent, ethylene diamine (en) was utilized instead of ammonia . In order to formation of structures with smaller dimensions, the presence of surfactant is vital. 2Sn2O7 nano samples in existing of broccoli extract as a natural surfactant (LSO6) that indexed to pure La2Sn2O7 nanostructures (JCPDS No = 73–1686). Moreover, one specimen was synthesized with an increasing ultrasonic irradiation time of 30 min (LSO7) that has been indicated in 2Sn2O7 nanostructures has increased. The crystalline size of La-Sn-O nano-grains was calculated by the Scherer equation X-ray diffractograms of La-Sn-O sample prepared through sonochemical route in calcination condition of 800 °C for 3 h (LSO1) have been presented in zed form b, but pu ammonia b. The en3.22Sn2O7 nanostructures fabricated in various times and temperatures of heat treatment. Interconnected particles were formed in 800 °C for 3 h  than NH3 and finally, helps to control of nucleation and growth of nanoparticles. 2Sn2O7 nanostructures prepared in presence of Broccoli extract. Spherical nanoparticles formed have homogeneous size and shape. Broccoli was utilized as both green surfactant and alkaline agent including active glucosinolate and polyphenol groups that can conjugate to created nuclei, control their growth and finally create favorable nano-sized samples 2Sn2O7 nano-products. By growing reaction time of ultrasonic to 30 min, larger particles were created with a range size of 20–100 nm  have been demonstrated in 2Sn2O7 nanostructures prepared via broccoli extract and 15 min ultrasonic irradiation (LSO6).Particle size distribution diagrams of La2Sn2O7 nanoparticles fabricated via sonochemical route and broccoli natural surfactant (LSO6). Spherical-shaped nanoparticles have a size about 15–50 nm.3.32Sn2O7 nano-products with optimized features are created through the simultaneous outcomes of ultrasonic irradiation and broccoli as a natural surfactant. Appropriate structures in the nano-sized form are produced via the generated cavitation with ultrasound waves. Excessive temperatures and pressures are created with huge energies according to hot-spot theory and make active components of radicals via the sonochemical method stated as below:La3.42Sn2O7 nano (LSO6) and bulk (LSO4) particles were exposed in 2Sn2O7 nano and bulk specimens was gauged by Tauc’s equation UV–vis absorption mode was utilized to evident the optical features and energy structure in semiconductor materials. The UV–Vis outcomes for La2Sn2O7 nano crystals prepared via sonochemical route in optimized circumstances has been illustrated in 2Sn2O7 nanoparticle, the La and Sn cations with inversion symmetry are coordinated to eight and six oxygen, respectively in a geometry only slightly distorted from a regular octahedron The luminescence characteristic of La3.52Sn2O7 nanocrystals. Owing to the small intensity of CN peak versus great intensity of La2Sn2O7 peaks, existing of LSO is not significantly observed. In 3N4 component (JCPDS No. 75-2078).2Sn2O7/g-C3N4 (LSO/CN) nanocomposites obtained in different LSO:CN weight ratios of 30:70, 50:50 and 70:30 have been specified in 3N4 is confirmed. Moreover, more amount CN sheets are observed in The SEM images of La2Sn2O7. Also, in The results of EDX analyses of pure LSO nanoparticles and divers LSO/CN nanocomposites have been represented in 3.63g−1 and 20.28 nm, respectively. Furthermore, the specific surface area evaluates 19.92 m2g−1. By evaluating obtained BET results, LSO/CN nanocomposite with a weight ratio of 30:70 is a favorable applicant for photocatalysis systems.The nitrogen adsorption–desorption isotherms and pore size distribution curves of LSO/CN nanocomposite with a weight ratio of 30:70 have been revealed in 3.7Due to suitable electronic band gap and high surface area properties of LSO nanoparticles and LSO/CN nanocomposites, the photocatalytic activity of nanostructures was evaluated under UV irradiation for degradation of water pollutants. The impact of various parameters of photocatalytic degradation was investigated in order to achieve the highest degradation percentage. The factors of catalyst type, different weight ratios of LSO:CN, nano-catalyst amount, dye type, dye concentration and different additives as the scavengers were considered. 0 and At are absorbance amounts in times of start and t min. According to Eq. Which A0 and Ct are dye concentration in times of start and t min and k is the pseudo-1st-order rate coefficient (min−1) 0/Ct) versus reaction time, the 1st-order rate constant k has been attained. As observed in 2Sn2O7 (LSO6) and g-C3N4 components. Also, by increasing g-C3N4 amount in nanocomposite, the rate constant of k rise (−-h+ separation and carrying Which Cf k rise d. The ca−1) than 0.01 g LSO6 nanocatalyst (k = 0.0157 min−1).2Sn2O7 nanoparticles for degradation of erythrosine as an anionic dye is better than methyl violet as a cationic dye and photocatalytic efficiency was reported 100 and 82 %, respectively. Also, the degradation speed of erythrosine is higher than methyl violet dye.The photocatalytic activity of LSO6 nanocatalyst was evaluated for degradation of anionic erythrosine and cationic methyl violet dyes to acquire developed efficiency. As indicated in 2Sn2O7 nanoparticles have better function in lower dye concentrations.+, •OH and •O2− active specimens 2Sn2O7 nano-catalyst has noticeably reduced in presence of BQ as a •O2− scavenger. It is concluded that •O2− active component has the most contribution in removal dye pollutant of water. However, according to •OH also plays little role in destruction of toxic dye. The mechanism of erythrosine contamination via optimized nanocatalyst by superoxide anion and hydroxyl radicals has been summarized in follows:−CB + h+VB)LSO nano-catalyst + hν → LSO nano-catalyst*  have better efficiency than pristine La2Sn2O7 nanoparticle (ƞ=72%) and g-C3N4 specimen (ƞ=91%). Moreover, the photocatalytic activity is completely performed in presence of more amount of nanocatalyst. Also, the probable mechanism of removal dye by photocatalytic function was studied using three types of scavengers of EDTA, Benzoic acid and benzoquinone to trap h+, •OH and •O2− active specimens, respectively. Finally, it is found that •OH and •O2− radicals promote photo-degradation of dye.In summary, we were productively designed binary LaZeinab Talebzadeh: Methodology, Investigation, Software, Formal analysis. Maryam Masjedi-Arani: Investigation, Writing – original draft, Writing - review & editing, Formal analysis. Omid Amiri: Visualization, Investigation, Software, Data curation. Masoud Salavati-Niasari: Visualization, Writing – original draft, Writing - review & editing, Validation, Investigation, Data curation, Conceptualization, Methodology, Supervision, Project administration.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a  T-DNA mutants have been widely used for Arabidopsis gene function characterization. Here, by characterizing a null mutant created by CRISPR, the authors show that previous reported function of H2A.W is confounded by a T-DNA insertion induced chromosomal rearrangement and reveal its role in regulating heterochromatin accessibility. Arabidopsis thaliana and mouse nuclei. Yet, heterochromatin has to retain a certain degree of accessibility to allow important DNA-related biological processes to occur, including maintenance of DNA methylation, DNA replication, DNA damage repair, and transcription for small RNA production.Eukaryotic genomes are packaged in chromatin. The basic unit of chromatin is the nucleosome, which contains a protein octamer comprising two of each of the core histones H2A, H2B, H3, and H4, wrapped by ~147 bp of DNA. Chromatin is organized into two distinct domains termed constitutive heterochromatin, which is enriched in transposable elements (TEs) and other types of repetitive DNA, and euchromatin, which comprises mostly protein-coding genes. Euchromatin is more accessible and associated with transcriptional activity, whereas heterochromatic domains prevent transcription and are often compacted into higher order structures such as chromocenters in Arabidopsis, heterochromatin is additionally decorated with histone H3 lysine 9 mono and dimethylation (H3K9me1 and H3K9me2), catalyzed by the SU(VAR)3–9 HOMOLOG-class of histone methyltransferases SUVH4/KYP, SUVH5, and SUVH61. The mechanisms that establish and maintain heterochromatin-specific non-CG methylation and H3K9 methylation are tightly and reciprocally interconnected4. The DNA methyltransferases CMT2 and CMT3 are recruited by H3K9me1 and H3K9me22, while the H3K9 methyltransferases SUVH4/5/6 are recruited to chromatin by DNA methylation7, creating a positive feedback loop that reinforces silencing. DNA methylation in plants is also established and maintained via the RNA-directed DNA methylation (RdDM) pathway, which preferentially targets short euchromatic TEs and the edges of long heterochromatic TEs8. Recruitment of RNA polymerase IV during the early steps of RdDM involves SHH1, which binds methylated H3K9, thereby also linking RdDM targeting to H3K9 methylation10. Arabidopsis heterochromatin is also marked by H3K27me1, which depends on the redundant histone methyltransferases ARABIDOPSIS TRITHORAX-RELATED 5 and 6 11. Although heterochromatin structure is visibly altered in atxr5 atxr6 mutants, DNA methylation and H3K9me2 appear largely unaffected, suggesting that H3K27me1 is maintained independently of these marks.In most eukaryotes, euchromatic and heterochromatic regions can be distinguished by their DNA methylation level, the presence of distinct post-translational modifications of histones, and their association with specific histone variants. In plants, DNA methylation occurs in three sequence contexts . DNA methylation in euchromatic regions tends to be low, except at CG sites over gene bodies of protein-coding genes. Heterochromatic sequences, however, are characterized by dense methylation at all three sequence contexts (CG and non-CG methylation). In Arabidopsis16. In Arabidopsis, H1 associates with chromatin independently of DNA methylation, but loss of H1 leads to chromocenter decondensation, and has varying effects on DNA methylation: pericentromeric heterochromatic TEs gain DNA methylation in h1, while TEs on the chromosome arms lose methylation18. H1 is thought to hinder heterochromatic DNA methylation by restricting the access of DNA methyltransferases to these regions19. The joint action of H1 and CG methylation by the DNA methyltransferase MET1 silences a subset of TEs and prevents the production of aberrant gene transcripts, suggesting DNA methylation and H1 help define functional transcriptional units14. The histone variant H3.3 also plays a role in restricting H1 from associating with active genes in Arabidopsis16, Drosophila20, and mouse21. However, the mechanisms that control H1 deposition and shape its relative enrichment in heterochromatin remain unknown.The linker histone H1, which binds nucleosomes and the intervening linker DNA, is also preferentially associated with heterochromatin in Arabidopsis, the histone variant H2A.W is strictly and specifically localized to constitutive heterochromatin23. H2A variants comprise the most diverse histone family and directly impact biochemical properties of the nucleosome25. In Arabidopsis, nucleosomes typically contain a single type of H2A variant—either replicative H2A, H2A.X, H2A.Z, or H2A.W26. In land plants, the majority of H2A.Z nucleosomes associate with repressive H3K27me3 modifications while a small fraction associates with active H3K36me3 marks found at the transcription start site in transcribed genes27. Most of the body of expressed genes is occupied by replicative H2A and H2A.X nucleosomes22. The exclusive localization of H2A.W at constitutive heterochromatin is unique among Arabidopsis H2A variants28. While specialized histone chaperones mediate the incorporation of specific H2A variants, no chaperone dedicated to H2A.W has been identified so far29. Arabidopsis has three H2A.W isoforms, H2A.W.6, H2A.W.7, and H2A.W.12, encoded by HTA6, HTA7, and HTA12 respectively. To characterize the role of H2A.W in Arabidopsis heterochromatin, a previous study generated a triple-knockout hta6 hta7 hta12 line, referred here to as h2a.w-122. The h2a.w-1 mutants showed severely affected plant growth and were not fertile22. In addition, CMT3 expression and CHG methylation levels were significantly increased in h2a.w-122.In hta6 transfer-DNA (T-DNA) insertion mutant allele used to generate the h2a.w-1 triple knockout line. Using CRISPR-Cas9, we obtain a null h2a.w triple mutant without this rearrangement, referred to here as h2a.w-2. Analyzing h2a.w-2 mutants reveals that the hta6 chromosomal rearrangement is responsible for the severe developmental effects and CHG hypermethylation reported in h2a.w-122. Using the mutant h2a.w-2, we now show that loss of H2A.W results in no visible developmental or morphological phenotypes and has only minor effects on gene and TE expression. In h2a.w-2, heterochromatin exhibits a mild decrease in accessibility accompanied by increased deposition of replicative H2A and H2A.X and of the linker histone H1, as well as decreased levels of non-CG methylation. The combined loss of H1 and H2A.W enhances both chromatin accessibility and DNA methylation in pericentromeric heterochromatin. Based on these results, we propose that H2A.W and H1 jointly regulate DNA methylation and heterochromatin accessibility.Here, we identify a large genomic rearrangement in the h2a.w-1 triple mutants and their corresponding wild type (WT)22. We detected significantly increased copy numbers for several TEs , hta7 (GABI_149G05.01), and hta12 (SAIL_667_D09) DNA confirmed the presence of a genomic rearrangement, likely a translocation of part of chromosome 1, in hta6-1  data of w-1 Fig. . Souther6-1 Fig. . Further6-1 Fig. . Using shta6 allele (hta6-2) carrying a single-base frame shift mutation that causes a stop codon early in the protein  and only a handful of transcriptionally activated TEs in h2a.w-2 ted Fig. . Interested Fig. . Unexpec WT Fig. . This obh2a.w-2, we profiled the genome-wide distribution of H3 and other H2A variants by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Profiles of H3 enrichment determined by ChIP-seq were similar in WT and h2a.w-2 plants, suggesting that nucleosome density was not responsible for the change in accessibility in h2a.w-2 , H2A.Z (H2A.Z.9), and replicative H2A (H2A.1 and H2A.13) using ChIP-seq. In WT, H2A.Z, H2A.X, and H2A showed relative depletion over pericentromeric heterochromatin and at H2A.W-associated regions , which lack both H2A.W and H1 . This chromosome rearrangement, and not the loss of H2A.W, is responsible for the developmental defects and CHG hypermethylation previously reported in h2a.w-1, as well as the lethality of h2a.w-1 cmt3 quadruple mutants22. The absence of these defects in triple mutant h2a.w-2 has now enabled us to analyze the direct impact of H2A.W on heterochromatin composition and accessibility.Chromosomal rearrangements are common in T-DNA insertion lines22 and increases nucleosome stability26. Hence, we expected loss of H2A.W to increase heterochromatin accessibility. Surprisingly, we observed the opposite, with accessibility decreasing in h2a.w-2 in association with increased recruitment of H2A, H2A.X and H1 to heterochromatin, indicating that H2A.W presence favors heterochromatin accessibility. H1 is known to stabilize the wrapping of DNA around the nucleosome, promote assembly of higher order chromatin structures36, and influence nucleosome spacing38. In the presence of H2A.W, loss of H1 leads to increased accessibility and increased DNA methylation in heterochromatin. These phenotypes are enhanced in the absence of both H1 and H2A.W, suggesting that H2A.W also contributes to restricting heterochromatic DNA accessibility in the absence of H1. Gain of H1 in h2a.w-2 heterochromatin correlates well with decreased accessibility in the same regions, suggesting that H2A.W also promotes accessibility in heterochromatin by restricting H1 levels. The antagonism between H1 and H2A.W may originate from a competition for linker DNA binding. The extended C-terminal tail of H2A.W interacts with linker DNA, and this interaction prevents micrococcal nuclease accessibility26. The H2A.W C-terminal tail is characterized by the SPKK motif22, which binds A/T-rich DNA in its minor groove and causes condensation40. Among Arabidopsis H2A variants, the SPKK motif is unique to H2A.W22. Two SPKK-like motifs, SPAK and SP(G/A)K, are also present in the C-terminal tails of Arabidopsis H1.1 and H1.2 , which are located primarily outside of pericentromeric heterochromatin, non-CG methylation levels increase in  h1 Fig. 3. This lw-2 Fig. . It was 43. H2A.W is subject to specific modifications44, and its dynamic deposition likely participates in the regulation of chromatin accessibility through its interaction with H1 and other yet unknown factors.Although heterochromatin was long believed to be highly compact and inaccessible to transcriptional machinery, there is increasing evidence that low levels of accessibility within heterochromatin are required for proper heterochromatin formation by permitting access to various factors, including DNA and histone methyltransferases, that help maintain a heterochromatic statehta6 (SALK_024544C), hta7 (GABI_149G05.01), hta12 (SAIL_667_D09), and cmt3-11 (SALK_148381) mutant lines used in this study were all in the Col-0 genetic background. Plants were grown in long-day conditions  at 23 °C with 50% relative humidity.The https://www.genome.arizona.edu/crispr/index.html). The spacer (GTTTCGAAATCGATGAAAGC) was ligated between the two BbsI sites of the pEn-Chimera vector using annealed oligonucleotides  at the gRNA-targeted HTA6 5′ coding region. The +1 G insertion induces an early frame shift 89 bp downstream from the translation initiation site and a stop codon 195 bp downstream from the translation initiation site. Segregation of the mutant allele was analyzed in the T3 generation and we confirmed that both T2 lines were homozygous for the mutation. The knockout nature of this hta6-2 allele was confirmed by immunoblot analysis using a specific antibody  of the PCR product  sequences was performed using an online bioinformatic tool  following manufacturer’s instructions. 750 ng of DNA was digested overnight with 20 units of high fidelity SspI restriction enzyme (New England Biolabs) in Cutsmart® buffer and electrophoresed through a 1% agarose (w/v) gel for 8 h. The gel was depurinated (10 min in 0.25 N HCl), rinsed, denatured , neutralized  and capillary blotted onto a Hybond-N + membrane (Amersham) overnight. The membrane was UV-crosslinked at 150 mJ. The DNA probe was amplified from Col-0 DNA with primers indicated in Supplementary Table 32P-dCTP using the random hexamer priming method  following manufacturer’s instructions and subsequently purified on illustra MicroSpin S-200 HR columns . Hybridization was performed using the PerfectHyb™ Plus hybridization buffer (Sigma) following manufacturer’s instructions, with overnight hybridization at 65 °C followed by one washing step (10 min) in 2X SSC 0.1% SDS and two washing steps (15 min each) in 0.5X SSC 0.1% SDS, all at 65 °C. The membrane was imaged on a Typhoon FLA 7000 .Genomic DNA was extracted from rosette leaves using the Wizardhta6-1 genomic DNA were digested by SspI and then column-purified using the Neo Biotech gel extraction kit. To favor self-recircularization of the SspI-digested fragments, ligation was performed at 15 °C for 16 h using 100 ng of the digested DNAs and 4.5 U of T4 DNA ligase (Promega) in a final volume of 100 µl. Following column purification (Neo Biotech gel extraction kit), 1/50 of the eluted DNA was used as a template for a first round of inverse PCR (iPCR) with primers that closely match the expected extremity of the translocation. A second round of PCR amplification was done using nested primers and a 1/100th dilution of the first amplification as template. A specific product of around 2.1 kb was obtained for the hta6-1 genomic DNA and sequenced (Eurofins) to identify the left border of the translocation. The primers used for iPCR are reported in Supplementary Table A total of 250 ng of Col-0 and ®) from 30 to 40 mg of fresh material following the manufacturer’s instructions. 8 µg of RNA were treated for 1 h at 37 °C with 12 units of RQ1 DNase (Promega®) followed by phenol-chloroform extraction and ethanol precipitation of RNA which was subsequently dissolved in water. One-step reverse-transcription quantitative PCR (RT-qPCR) was performed with the SensiFAST™ SYBR® No-ROX One-Step kit (Bioline®) on an Eco™ Real-Time PCR System (Ilumina®) with the following program: 10 min at 45 °C, 5 min at 95 °C, and 40 cycles of 20 s at 95 °C and 30 s at 60 °C. A melting curve was generated at the end of the program to control for amplification specificity. Data was normalized to a reference gene and analyzed according to the 2−ΔΔCt method. Means and standard errors of the mean were calculated from independent biological samples. Differences in the means for RT-qPCR data were tested using an unpaired Student’s t test with Welch’s correction with the t.test function of R version 3.4.047.Total RNA was extracted with TRI Reagent (Sigma2 leaf tissue was chopped with a razor blade in 125 μL of extraction buffer from the CyStain UV Precise P kit (Partec) in a petri dish. Extracts were passed through a 30 μm filter to isolate nuclei and kept on ice. The procedure was repeated by adding 125 μL of extraction buffer to the petri dish. After 2 min on ice, 20 μL of nucleus extract were supplemented with an equal volume of 60% acetic acid on a slide and stirred continuously with fine forceps on a 45 °C metal plate for 3 min; 60% acetic acid was added again and stirred for 3 min. The slide was cleared with an excess of an ethanol/acetic acid solution (3:1), air-dried and mounted with DAPI in Vectashield mounting medium (Vector Laboratories). Nuclei were visualized on a Zeiss Axio Imager Z1 epifluorescence microscope equipped with a PL Apochromat 100X/1.40 oil objective and images were captured with a Zeiss AxioCam MRm camera using the Zeiss ZEN software. The relative heterochromatin fraction was computed for each nucleus by calculating the ratio of the signal intensity at chromocenters over that of the entire nucleus using the ImageJ software (1.49 v).Rosette leaves (fifth or sixth) from 3/4-week-old plants were fixed in 4% (v/v) formaldehyde/PBS 1X for 3 h at room temperature. Following fixation, leaves were placed between two layers of kitchen paper to remove excess buffer and a piece of approximately 0.5–1 cm23 with few modifications. For each sample 300 mg of 10-day old seedlings or 200 mg of floral buds , mixed with 50 μL of 4x Laemmli loading buffer and boiled for 5 min. Once the samples reached room temperature, 2 μL of Benzonase (Millipore) was added and incubated for 10 min on bench. Samples are again boiled for 3 min to inactivate Benzonase. Samples were spun at maximum speed for 5 min to pellet down insoluble fraction and supernatant is transferred to fresh Eppendorf tubes. For Western blot analyses, 10 μL for histone variants and 5 μL for H3 (used as a loading control) were loaded per lane. Nuclear proteins were resolved using NuPAGE 4–12% Bis-Tris protein gels. Resolved proteins were transferred onto PVDF membrane using Bio-Rad wet transfer unit. blot analysis was performed using 1:1000 diluted antibodies in 5% milk in TBST. H2A.W.6, H2A, H2A.X and H2A.Z antibodies are affinity-purified rabbit polyclonal antibodies made by GenScript USA Inc  against peptides GGRKPPGAPKTKSVC, CPKKAGASKPSADE, CKVGKNKGDIGSASQ, and KPSGSDKDKDKKKPC, respectively22. H2A.W.7, and γH2A.X antibodies are reported in a previous study23. H2A.W and H1 antibodies were generated using peptides CTTKTPKSPSKATKSP and CRTGSSQYAIQKFIEEK, respectively, at Eurogentec.Nuclear protein extracts for Western blot analyses were prepared as described in ref. Total RNA was isolated with RNeasy Mini kit (Qiagen) from 10-day old seedlings in three replicates. DNase treatment was done on 2 µg of total RNA with DNA free DNase Kit (Invitrogen). From 1 µg of total RNA, rRNA was depleted using RiboZero kit (Illumina). NGS-libraries were generated using NEBnext Ultra II directional RNA library prep kit for Illumina and sequenced as PE75 reads on an Illumina NextSeq550.48 and aligned to the TAIR10 genome using STAR49. Reads aligning equally well to more than one position in the reference genome were discarded, and probable PCR duplicates were removed using MarkDuplicates from the Picard Tools suite50. Alignment statistics for each library are available in Supplementary Table 51, with annotations from araport1152. Annotated TEs overlapping strongly (>80%) with an annotated TE gene were considered TE genes, and the TE annotation was discarded. Differential expression analysis was performed using DESeq253, and genes were considered differentially expressed with an adjusted p-value <0.05 and abs[log2(fold change)] > 1.Reads were trimmed and filtered for quality and adapter contamination using Trim Galore54. Briefly, 0.5 g of freshly collected 10-day old seedlings were chopped in 4 mL of pre-chilled lysis buffer . After chopping, the suspension was filtered through a 40 µM filter. Nuclei were further enriched using a sucrose gradient. Enriched nuclei were resuspended in 0.5 mL of pre-cooled lysis buffer with 4,6-Diamidino-2-Phenylindole (DAPI) and incubated for 15 min. DAPI-stained nuclei were sorted on FACS Aria III (BD Biosciences) with FACSDiva software (version 8). Sorted nuclei  were pelleted and washed once . Tagmentation reaction was carried out using Nextera reagents , TD Tagment DNA Buffer ). Tagmented DNA was isolated using Qiagen MinElute PCR purification kit. NGS libraries were amplified using NEBNext high fidelity 2X master mix and Nextera primers. The number of PCR cycles was determined using a method described in ref. 55. NGS-libraries were PE75 sequenced on an Illumina NextSeq550.ATAC-seq was performed as described in ref. 56. Reads aligning to multiple positions and PCR duplicates were removed (see RNA sequencing). Only properly paired reads were retained for the analysis. Alignment statistics for each library are available in Supplementary Table h2a.w-2 were obtained using Genrich57 with parameters -p 0.01, -a 200, -l 100, and -g 100. ChrM, ChrC, and several rRNA regions with very high coverage were omitted from the analysis. Metaplots of ATAC-seq signal over various genomic regions were created using deeptools58. Plots of ATAC-seq signal over entire chromosomes are based on average signal over 1 kb non-overlapping bins tiled genome-wide, calculated using deeptools. Smoothed conditional mean of the signal was computed using the LOESS smoothing method with bin width span 0.1 and plotted using R47. A comparison of the ATAC-seq data generated in this study with selected published ATAC-seq datasets is provided in Supplementary Fig. Reads were trimmed and filtered as indicated above (RNA sequencing), and aligned to TAIR10 using bowtie2® Genomic DNA Purification Kit (Promega) following manufacturer’s instructions. For WT and h2a.w-2 replicates 1 and 2, sodium bisulfite conversion, library preparation, and sequencing on a Hiseq 4000 were performed at the Beijing Genomics Institute (Hong Kong) from 1 µg DNA, producing paired 100-bp (replicate 1) or 150-bp (replicate 2) paired-end reads. For remaining samples, methylated adapters were ligated prior to bisulfite treatment using the ZYMO EZ DNA methylation Gold kit, libraries were prepared using the Nugen ultralow methyl-seq kit and sequenced on NovaSeq 6000. Reads were trimmed, mapped to the TAIR10 genome, and methylation called using methylpy (version 1.4.3). Only uniquely mapping reads were retained. Alignment statistics for each library are available in Supplementary Table 59.Genomic DNA was extracted from the aerial portions of 10-day old seedlings using the Wizard60. Briefly, 3 g (approx. 0.3 mg for each immunoprecipitation (IP)) of 10-day old seedlings were fixed in 1% PFA. Fixed seedlings were ground to fine powder in liquid nitrogen using a mortar and pestle. Nuclei were isolated using M2 buffer  and M3 buffer . Chromatin shearing was done using a Covaris E220 with the following settings: treatment time 15 min, acoustic duty factor % 5.0, PIP 140 W, Cycles per burst 200 and max temperature 8 °C. IP, washes, and DNA isolation were carried out as described in ref. 60. A total of 5 µl of each antibody (1 mg/mL) is used for IP. H2A, H2A.X and H2A.Z antibodies are reported in a previous study22. H3 (ab1791 Abcam), H3K9me1 (ab8896 Abcam), H3K9me2 (ab1220/Abcam), H3K27me1 (17-643/Millipore), and H1 (AS111801/Agrisera) antibodies were obtained from commercial sources. NGS libraries were generated using Ovation Ultralow Library System V2 (NuGEN) for replicate 1 and NEBNext Ultra II DNA preparation kit for replicate 2. NGS-libraries were SR75 sequenced on an Illumina NextSeq550.ChIP was performed as described in ref. 58 bamCoverage . All samples except for H3 were normalized to their matched H3 sample using deeptools bamCompare. Plots over entire chromosomes were obtained from average ChIP-seq signal over 1 kb non-overlapping bins tiled genome-wide and smoothed using the same approach as the ATAC-seq data. H2A.W ChIP-seq data were re-analyzed from ref. 22.Reads were trimmed, filtered, and aligned using bowtie2, and multi-mapping reads and PCR duplicates were removed, all as indicated above (see ‘ATAC-seq’ section). Alignment statistics for each library are available in Supplementary Table Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary