Non-canonical structures, including G-quadruplexes, formed within expanded repeats have been proposed to drive repeat expansion and pathogenesis of ALS and FTD. Oligonucleotide d[(G4C2)3G4], which represents the shortest oligonucleotide model of d(G4C2) repeats with the ability to form a unimolecular G-quadruplex, forms two major G-quadruplex structures in addition to several minor species which coexist in solution with K+ ions. Herein, we used solution-state NMR to determine the high-resolution structure of one of the major G-quadruplex species adopted by d[(G4C2)3G4]. Structural characterization of the G-quadruplex named AQU was facilitated by a single substitution of dG with 8Br-dG at position 21 and revealed an antiparallel fold composed of four G-quartets and three lateral C–C loops. The G-quadruplex exhibits high thermal stability and is favored kinetically and under slightly acidic conditions. An unusual structural element distinct from a C-quartet is observed in the structure. Two C•C base pairs are stacked on the nearby G-quartet and are involved in a dynamic equilibrium between symmetric N3-amino and carbonyl-amino geometries and protonated C+•C state.Most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is a largely increased number of d(G This leads to a lack of completely transcribed C9orf72 mRNA as well as production of abortive RNA transcripts, which contain (G4C2) and (G2C4) repeats. Decreased level of C9ORF72 protein (4G2) and antisense d(G2C4) repeats. These RNA transcripts, which can also adopt non-canonical structures, accumulate as toxic aggregates in the nucleus and can sequester RNA-binding proteins (C9orf72 repeat expansion.An increase in the number of d(Gia (FTD) ,2. Betwe repeats . DNA andructures . Moreovein vitro ,12. Whil protein ,14, whic protein , has bee protein . Furtherproteins . Additioproteins ,21–23. Rproteins . Differeanti conformation, can drive the folding of a G-rich oligonucleotide towards a particular topology . Accompanied by different molecularity, the number of G-quartets, groove dimensions, and types of loops, G-quadruplexes can achieve tremendous structural diversity. Several different G-quadruplex species often coexist in solution which represents a great challenge for structural studies. Incorporation of a C2′-fluorinated guanosine analogue, which preferentially adopts an topology ,28. Simi studies .4C2) repeats in the mechanism triggering devastating neurodegenerative disorders, we explored structural characteristics of oligonucleotides with four d(G4C2) repeats. Four repeats were chosen as the shortest DNA sequence that can adopt a unimolecular G-quadruplex structure. Earlier studies have shown that a four d(G4C2) repeat DNA oligonucleotide forms a mixture of mostly antiparallel G-quadruplexes in the presence of K+ ions  repeats and were extended or modified at the 5′- and/or 3′-ends (4C2)3G4], showed the most favorable 1H NMR spectrum and was chosen for further study. Preliminary analysis of the 2D NMR and CD spectra showed that two distinct antiparallel G-quadruplexes were formed at higher population in addition to  other G-quadruplexes with lower population  repeats uncover structural details which tune polymorphic behavior of G-rich repeats in general, and might facilitate the design of small molecules that can be used to modulate aberrant transcription of C9orf72 gene that has been linked to ALS and FTD in particular.Due to potentially high relevance of non-canonical structures adopted by d or 14 kHz (on 800 MHz), 128 scans and 1.5 s recycle delay. NOESY spectra were acquired with the spectral width of 12 kHz (on 600 MHz) or 14 kHz (on 800 MHz) in both dimensions, 2048 complex points in t2, recycle delay of 1.5 s, 320 t1 increments and 40 scans per t1 increment. The spectra were apodized with a cosine squared function and zero filled twice before the Fourier transform. Double-pulsed field gradient spin echo (DPFGSE) pulse sequence was used to suppress the water signal. The 2D spectra were acquired at 5, 25, 35 and 45°C with samples in 90% H2O, 10% 2H2O and 100% 2H2O. The standard homonuclear 2D NMR experiments recorded at 25°C in 100% 2H2O including 2D DQF-COSY, TOCSY  and NOESY , were used to assign the non-exchangeable protons. Exchangeable proton resonances were assigned using 2D NOESY  recorded in 90% H2O, 10% 2H2O at 25°C. The details on NMR restraints used for structure calculation, assignment of cytosine residues and additional comments on the assignment of sugar puckering are given in the Supplementary Data. NMR spectra were processed and analyzed using VNMRJ (Agilent-Varian) and Sparky (UCSF) software.NMR experiments were performed on the 600 and 800 MHz Agilent-Varian NMR spectrometers equipped with triple resonance HCN cold probe or OneNMR probe (for variable temperature experiments). 1D 4C2)3GGBrGG], created with LEAP module of the AMBER 14 program. Hydrogen bonds, planarity, chirality and chi torsion angle restraints were included over three steps to generate a topology consistent with the NMR data. In the next step, all NOE and torsion angle restraints were included to generate a total of 100 structures calculated in 130 ps of NMR restrained simulated annealing (SA) simulations -repeat oligonucleotide named wt22, d[(G4C2)3G4], forms multiple structures in the presence of K+ ions. Our preliminary NMR study showed that two G-quadruplexes are formed at higher population in addition to  G-quadruplex structures with lower population (4C2)3GGBrGG] named sl21, compared to its natural counterpart wt22  is favored with a population of around 70%. Its high-resolution structure was reported by us earlier (acidic and quenching) is favored with a population of ∼80%, while NAN is present as a minor species (20%). Noteworthy, concentration of KCl does not influence the relative ratio between the populations of AQU and NAN. However, when sl21 is folded with fast annealing at pH 5.8, the intensity of broad and poorly defined signals is increased at 120 mM compared to 30 mM KCl. This suggests that the population of additional unidentified structures or possibly aggregated forms is increased at higher KCl concentration. In comparison, the 1H NMR spectra of sl21 folded with slow annealing at pH 7.2 in the presence of 120 or 30 mM KCl are very similar , TOCSY, DQF-COSY and 13C-HSQC. Guanine imino (H1) and aromatic (H8) resonances were unambiguously assigned by 15N- and 13C-edited HSQC spectra acquired on partially (8%) residue-specifically 15N- and 13C-labeled sl21 (m = 80 ms) indicate that residues G1, G3, G7, G9, G13, G15 and G19, together with BrG21, adopt predominately syn conformations around the glycosidic bonds  spectrum and displayed expected interruptions in H6/H8(n+1)-H2′/2″(n) connectivities at the 5′ anti-3′ syn steps (vide infra). Assignments of H6 resonances of C11 and C12 were obtained through sequential walk in NOESY spectra and were corroborated by the substitution of dC with 5-methyl-2′-deoxycytidine (5Me-dC). Interestingly, only three strong H5-H6 cross-peaks were observed in the NOESY and TOCSY spectra of sl21, although there are six cytosine residues in the sequence  spectrum  undergo protonation in the acidic solution conditions. Notably, protonation of cytosine residues can be expected considering that the pKa of N3 in free cytosine is ∼4.2  repeat has its merits due to potential roles of the non-canonical structures formed within the expanded d(G4C2)•(G2C4) repeats in the disease mechanism of ALS and FTD. First, formation of G-quadruplexes has been proposed as a cause of genetic instability, which is the most common genetic cause of ALS and FTD. Second, G-quadruplexes (and R-loops) which form in the non-coding region of C9orf72 lead to aberrant gene transcription with a myriad of downstream pathological consequences resulting in the development of ALS and FTD. Here, we present a high-resolution structure of one of the major G-quadruplex structures adopted by the oligonucleotide d[(G4C2)3G4], which represents the shortest model of the d(G4C2) repeats that can form a unimolecular G-quadruplex. Oligonucleotide d[(G4C2)3G4] folds into multiple G-quadruplex structures in the presence of K+ ions. Two distinct antiparallel G-quadruplexes together account for ∼80% of the total G-quadruplexes formed by the wild type oligonucleotide, while the remaining 20% consists of unidentified or, possibly, aggregated forms. Introduction of 8Br-dG residue at position 21 in oligonucleotide sl21, d[(G4C2)3GGBrGG], enabled us to determine the 3D structures of the two major G-quadruplexes. Relative populations at which the two G-quadruplex species are formed by sl21 depend on pH during folding and a rate at which the solution is cooled after heating to 90°C. When sl21 is cooled slowly at pH 7.2, G-quadruplex named NAN  forms at ∼70%, while a distinct G-quadruplex denoted AQU forms at 30%. Ratio of AQU and NAN populations is reversed if solution of sl21 is cooled quickly at pH 7.2, resulting in formation of AQU with population of 67% and NAN at 33%. Further increase in the population of AQU with respect to NAN is achieved when solution of sl21 is cooled quickly under slightly acidic solution conditions (pH 5.8). In this case, AQU (for acidic and quenching) is favored at around 80%, while NAN is present as a minor species at 20%. Remarkably, once stationary state is reached, the ratio between the populations of AQU (80%) and NAN (20%) does not change even under alkaline solution conditions or after months of storage at room temperature. In fact, the two G-quadruplexes inter-convert through unfolded state only. Both structures exhibit high thermal stability, which suggests that when AQU and NAN form in vivo, specialized helicases –G22(anti) base pair, the donor of hydrogen bond in AQU is G1, whereas it is G22 in NAN. This reversal affects the width of the grooves defined by a given set of residues, e.g. the G1(syn)→G22(anti) is in a narrow groove in AQU, whereas analogous G22(anti)→G1(syn) pair in NAN is in a wide groove . Consequently, the first and the third lateral loops in AQU span wide grooves, whereas the middle loop spans a narrow groove. In comparison, the first and third lateral loops in NAN span narrow grooves and the middle loop spans a wide groove.NAN is a compact antiparallel G-quadruplex with four G-quartets and three lateral loops, where one of the cytosine residues in each lateral C–C loop is stacked on the nearby G-quartet . The higComparison of AQU and NAN reveals that the lateral loops which span grooves of the same type (wide vs. narrow) adopt similar structure Figure  and D. I+•C states. In a parallel G-quadruplex adopted by d(TG3CG2T), which contains repeat sequence from SV40 virus (2CG2C), which contains two triplet repeats linked to Fragile X syndrome (2ACNVKGCGTG2) in the presence of Ba2+ (4C2) repeats  and  of Ba2+ ) have no repeats . This st repeats .+•C base pairs in AQU, but not in NAN. A more general explanation as to why certain G-quadruplex forming oligonucleotides respond to pH, while others do not, is found by comparing AQU and NAN with two other structures, whose relative populations in solution depend on pH. Truncated human telomeric repeat sequence d[G3T2AG3T2AG3T2AG2] folds into two antiparallel basket-type G-quadruplexes with two lateral and a middle diagonal loop, named TD and KDH+ . In KDH0 to 4.5 .C9orf72. Based on described structural differences we can expect that AQU and NAN could bind and recruit different proteins. It was shown, for example, that replication protein A, a ssDNA binding protein known to unfold G-quadruplexes (4C2)8in vitro with significantly higher affinity at pH 5.0 compared to 7.8, suggesting it recognizes a structure which is preferred at acidic conditions n•(G2C4)n repeat is protective against neurodegeneration and is associated with longer disease duration and later age of disease onset (4C2)-repeat itself is hypermethylated in patients which carry large d(G4C2) expansions (>90 repeats) (4C2) repeats are methylated exhibit decreased production of toxic dipeptide-repeat proteins and fewer RNA foci, which suggests that repeat methylation diminishes disease severity (in vitro interaction between the C-rich d(G2C4)8 oligonucleotide and heterogeneous nuclear ribonucleoprotein complex protein K (hnRNP K) (in vitro could become favored upon methylation of d(G4C2) repeats in the cell, which would effectively lower the concentration of G-quadruplexes and diminish the level of pathological molecular events they trigger (5-Methyl-cytosine (5Me-dC) is an important epigenetic modification of cytosines found in CpG islands, which can alter expression of associated genes. In se onset . Additiorepeats) . Cells iseverity . In the hnRNP K) , a protehnRNP K) . PerhapshnRNP K) . AlternaAtomic coordinates and list of chemical shifts have been deposited with following accession numbers: PDB ID 5OPH, BMRB ID 34168.Supplementary DataClick here for additional data file.


INK4, pRB, cyclin D1 and p53. The objective of this study was to assess cell cycle proteins as potential surrogate markers for HR-HPV DNA testing to identify OPSCC with favorable prognosis after resection.Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) has an improved outcome and may allow for treatment de-escalation. High-risk HPV (HR-HPV) infection is associated with deregulated expression of the cell cycle-associated proteins p16INK4a, pRB, cyclin D1 and p53 using immunohistochemistry. Protein expression was scored as high or low based on the proportion of positive carcinoma cells. Tumor samples were analysed for HR-HPV DNA with polymerase chain reaction-based testing. Associations between cell cycle protein expression and HR-HPV DNA status were evaluated by calculating sensitivity, specificity, predictive values, and diagnostic odds ratios (DOR). Kaplan-Meier and Cox regression analysis were applied to evaluate associations between cell cycle protein expression and patient outcome.Tissue microarray cores of 313 surgically treated OPSCC were stained for p16INK4a, cyclin D1, pRB and p53 in tumor cells were observed in 51.8%, 51.4%, 41.9% and 33.5% of OPSCC, respectively. HR-HPV DNA positive were 158/313 (50.5%) tumor samples . P16INK4a showed a higher DOR to predict HR-HPV DNA positivity than pRB, cyclin D1 and p53. Both the p16INK4a/pRB and the p16INK4a/pRB/cyclin D1/p53 signatures had lower DOR than p16INK4a alone. Improved 5-year overall and disease-specific survival were associated with HR-HPV DNA positivity, high p16INK4a, low pRB, low cyclin D1, and low p53 expression. Associations with improved outcome were also observed for the marker combinations high p16INK4a/positive HR-HPV DNA, high p16INK4a/low pRB and high p16INK4a/low pRB/low cyclin D1/low p53. In a multivariate analysis adjusted for age, smoking history, pT and pN category, high p16INK4a expression showed the lowest hazard ratio for death.High expression of p16INK4a expression is a reliable marker for survival prognostication in surgically treated OPSCC patients. Protein signatures including the pRB, cyclin D1 and p53 proteins do not further increase the prognostic performance of p16INK4a as a single marker.High p16 High-risk human papillomavirus (HR-HPV) infection is a major cause of oropharyngeal squamous cell carcinoma (OPSCC) . HPV-relINK4a and down-regulation of cyclin D1. Disruption of both the p53 and pRB pathways promotes S-phase entry. The tumor cell signature of high p16INK4a expression and low/absent expression of cyclin D1, pRB and p53 correlates with HR-HPV infection [INK4a expression of tumor cells was associated with favorable prognosis and high cyclin D1 and p53 expression of tumor cells was linked to poor outcome [HR-HPV infection of tumor cells is associated with the expression of the viral E6 and E7 oncoproteins [ outcome –19. HoweINK4a as a prognostic marker in a large cohort of surgically treated OPSCC, and to directly compare the performance of p16INK4a with pRB, cyclin D1 and p53 to predict patient outcome. The second objective was to evaluate the cell cycle proteins p16INK4a, pRB, cyclin D1 and p53 as surrogate markers for HR-HPV DNA positivity.The objectives of our study were to validate p16n = 104; 33.2%), group 2 with resection and adjuvant radio(chemo)therapy . Pathological staging was performed using the 7th edition of the AJCC/UICC TNM classification system (2010).The cohort of this study represents a subgroup of a previous reported cohort . DemograOne representative formalin-fixed paraffin-embedded tissue block of each OPSCC resection specimen was retrieved. Hematoxylin and eosin (H&E) staining of sections was performed using standard techniques. Stained sections underwent histological review to confirm the presence of carcinoma.Formalin-fixed paraffin-embedded OPSCC tissue samples were used to construct tissue microarrays (TMA). Three cores (diameter 2 mm) with tumor tissue were punched out of each donor block and transferred to the recipient block. Tissue cores from tonsils of non-tumor patients were included as controls. H&E stained TMA sections were microscopically evaluated for the presence of tumor cells.INK4a , pRB , cyclin D1 , and p53 . Marker expression was scored as high or low based on the proportion of positive carcinoma cells  [Three-μm thick tissue microarray sections were deparaffinized followed by antigen retrieval . Stainings were performed on a Leica BOND MAX instrument (Leica) using the Bond Polymer Refine detection kit (Leica) and the following primary antibodies: p16 nuclei) , 21.Tumor cells were located and marked on H&E stained tissue sections obtained from paraffin blocks of primary OPSCC. Selective dissection of tumor cells was performed from three to five 4 μm thick serial tissue sections of the same paraffin block and genomic DNA was prepared. HPV DNA was detected by polymerase chain reaction (PCR)-based amplification using the L1C1/L1C2 consensus primer set as previously described . PCR proP values of < 0.05 were considered statistically significant.All analyses were performed with the SPSS Statistics 22 software . Demographic and clinicopathological patient characteristics were investigated with descriptive statistics. Associations between cell cycle protein expression and HR-HPV DNA status were analyzed using the chi-square test. Specificity, sensitivity, positive predictive values (PPV), negative predictive values (NPV), and diagnostic odds ratios (DOR) were calculated to evaluate cell cycle proteins and expression signatures as potential surrogate markers for HR-HPV DNA positivity. Overall survival (OS) was defined as time from the date of cancer diagnosis to the date of death, disease-specific survival (DSS) as the time from the date of cancer diagnosis to the date of death from OPSCC. Five-year OS and DSS rates were calculated using the Kaplan-Meier method. Univariate Cox proportional hazard models were used to explore associations between patient characteristics, biomarker results, OS and DSS. Hazard ratios (HR) with 95% confidence intervals were calculated. The Cox proportional hazard model was used for multivariate analysis. Three hundred thirteen patients with surgically treated and histologically confirmed OPSCC were included in the analysis. The clinicopathological characteristics of the patient cohort are shown in Table INK4a reactivity. pRB was strongly expressed in the nuclei of basal and parabasal epithelial cells, which also showed weak to moderate nuclear cyclin D1 and p53 staining (data not shown).In non-neoplastic tonsils, scattered epithelial cells showed weak to moderate p16INK4a staining in more than 70% of carcinoma cells was observed in 52.1% of OPSCC. pRB and cyclin D1 reactivity was low (< 25% of tumor cells) or absent in 48.6% and 57.8% of OPSCC, respectively. Nuclear p53 positivity stronger than in tonsillar epithelial cells was observed in 33.5% of tumors. A high p16INK4a/low pRB signature of tumor cells was present in 45.4% of OPSCC. Thirty-eight percent of the OPSCC displayed a high p16INK4a/low pRB/low cyclin D1/ low p53 signature.We next evaluated cell cycle protein expression in tissue cores of 313 OPSCC Fig. . Strong INK4a, pRB, cyclin D1and p53 in 91.8%, 17.1%, 15.8%, and 14.6%, respectively  were calculated. The value of a DOR ranges from 0 to infinity, with higher values indicating better discriminatory test performance. The largest DOR value was observed for high p16INK4a expression alone  tumors were HR-HPV DNA positive . HR-HPV DNA positive OPSCC were positive for p16INK4a, low pRB, low cyclin D1 and low p53 expression of tumor cells . The 5-year OS and DSS rates were 76.1% and 85.2%, respectively. Kaplan-Meier analysis demonstrated that both improved OS and DSS were associated with HR-HPV DNA positivity, and high p16INK4a expression .INK4a protein overexpression and suppress the expression of the cell cycle proteins pRB, cyclin D1 and p53 in infected cells [INK4a protein overexpression identifies transcriptionally active HR-HPV infection with specificity and sensitivity comparable to those of E6/E7 mRNA expression [INK4a immunostaining for the identification of tumors with positive HPV status, based on the conclusion that p16INK4a is a reliable surrogate marker for oncologically relevant HPV infection [INK4a expression is a direct consequence of pRB inactivation by the HPV oncoprotein E7, which induces degradation through the ubiquitin-proteasome pathway [INK4a expression alone as a biomarker for positive HR-HPV status may have limitations due to HR-HPV-independent mechanisms of up-regulation.Among patients with OPSCC, HR-HPV infection of tumor cells is associated with favorable prognosis, and therefore may identify patients who benefit from treatment de-escalation. Various methods are available for HR-HPV testing. HR-HPV E6/E7 messenger RNA (mRNA) expression demonstrated by reverse transcriptase-PCR or RNA in situ hybridization (ISH) is considered the most specific marker for biologically relevant HPV infection because it identifies transcriptionally active HPV infection and indicates the translation of the viral E6 and E7 oncoproteins . In addipression , 23, 25.nfection . In HR-H pathway . HoweverINK4a alone. In accordance with other studies, we observed that HPV-associated OPSCC are characterized by strong p16INK4a expression and low or absent expression of pRB, cyclin D1, und p53 [INK4a staining showed the strongest association with HR-HPV DNA positivity when evaluated as a single marker . The associations between low/absent expression of pRB, cyclin D1, or p53 alone and HR-HPV DNA positivity were significantly lower. In comparison to high p16INK4a expression alone, we observed higher specificity values for the 2-marker signature high p16INK4a/low pRB and the 4-marker signature p16INK4a/pRB/cyclin D1/p53, but reduced sensitivity values. Using the diagnostic odds ratio as a performance parameter, high p16INK4a expression alone was the best immunohistochemical marker for HR-HPV DNA positivity. Our findings confirm p16INK4a as a reliable surrogate marker for HR-HPV infection in OPSCC. They also indicate that signatures including the pRB, cyclin D1 and p53 proteins do not significantly increase the diagnostic performance of p16INK4a as a single marker.In our study, we aimed to evaluate pRB, cyclin D1 and p53 as alternative marker for HR-HPV DNA positivity, and to address the question whether protein signatures including pRB, cyclin D1 and p53 may increase the diagnostic performance of p16 und p53 , 18, 19.INK4a protein, p16INK4aprotein expression alone, or HR-HPV E6/E7mRNA expression identify OPSCC patients with favorable prognosis [INK4a, low pRB, low cyclin D1, and low p53 expression. Furthermore, HR-HPV DNA positivity and the combined parameters high p16INK4a/positive HR-HPV DNA, high p16INK4a/low pRB, and high p16INK4a/low pRB/low cyclin D1/low p53 were also associated with reduced risks of death. Parameters retained prognostic significance in multivariate analyses except high cyclin D1 and p53 expression. Our results confirm the findings of previous studies and validate them specifically for surgically treated OPSCC patients [INK4a expression provides comparable prognostic information as HR-HPV DNA positivity in surgically treated OPSCC patients and may therefore serve as a reliable surrogate marker for improved prognosis, if HR-HPV DNA testing is not available.Treatment options for patients with early OPSCC include surgical resection with or without adjuvant radio(chemo)therapy, or primary radio(chemo)therapy. In addition to its role in tumorigenesis, HR-HPV infection identifies OPSCC patients with improved survival who may benefit from treatment de-escalation , 20, 28.rognosis , 14, 15.rognosis , 16–18. rognosis –31. In tpatients , 23. TheINK4a expression and HR-HPV E6/E7 mRNA detection in surgically treated OPSCC.Limitations of our study include that cell cycle protein expression analyses were performed on TMA cores that may not be representative of the whole tumor. However, various studies have shown excellent concordance between TMA spots and whole tissue sections in immunohistochemical analyses of multiple tumor types. Furthermore, each tumor was represented by three tissue cores in our study to increase concordance. Two individuals blinded for HR-HPV DNA results and clinical outcome information scored all immunohistochemical stainings. However, digital image analysis may have increased standardization of scoring. We used HR-HPV DNA positivity as the gold standard to evaluate cell cycle proteins as potential surrogate markers for HR-HPV infection. However, HR-HPV E6/E7 mRNA detection is considered the best marker for biologically relevant HR-HPV infection. Further studies are necessary to directly compare the performance of p16INK4a expression in tumor cells is a reliable tool for survival prognostication in surgically treated OPSCC patients. Protein signatures including the pRB, cyclin D1 and p53 proteins do not further increase the prognostic and diagnostic performance of p16INK4a as a single marker. Therefore, p16INK4a may be used to select OPSCC patients with improved prognosis for treatment de-escalation, especially in the absence of HR-HPV DNA and HR-HPV E6/E7 mRNA testing.In conclusion, our results show that immunohistochemical demonstration of high p16


Endemic co-circulation of potentially zoonotic avian influenza viruses (AIV) of subtypes H5N1 and H9N2 (G1 lineage) in poultry in Bangladesh accelerated diversifying evolution. Two clinical samples from poultry obtained in 2016 yielded five different subtypes  and eight genotypes of AIV by plaque purification. H5 sequences grouped with clade 2.3.2.1a viruses while N1 was related to an older, preceding clade, 2.2.2. The internal genome segments of the plaque-purified viruses originated from clade 2.2.2 of H5N1 or from G1/H9N2 viruses. H9 and N2 segments clustered with contemporary H9N2 strains. In addition, HP H7 sequences were detected for the first time in samples and linked to Pakistani HP H7N3 viruses of 2003. The unexpected findings of mixtures of reassorted HP H5N1 and G1-like H9N2 viruses, which carry genome segments of older clades in association with the detection of HP H7 HA segments calls for confirmation of these results by targeted surveillance in the area of origin of the investigated samples. Hidden niches and obscured transmission pathways may exist that retain or re-introduce genome segments of older viruses or reassortants thereof which causes additional challenges for diagnosis, risk assessment and disease control. Based on the pathogenic potential in experimentally infected chickens, phenotypes of low and high pathogenicity are distinguished (LP/HPAIV). Several AIV subtypes and lineages exhibiting explicit zoonotic propensity have emerged as a public health threat2. Notably, these are goose/Guangdong (gs/GD) lineage derived HPAI H5 viruses and 2013-origin Chinese LPAI H7N9 viruses, but also further viruses such as the G1 lineage of subtype H9N2 have caused sporadic human cases4. Within the last decade, gs/GD HPAI H5 and LPAI H9N2 viruses have become endemic in poultry in several Asian and African countries causing huge economic losses5. Domestic duck populations play an important role in establishing and maintaining endemicity as they often subclinically shed and spread these viruses7. Long term co-circulation of multiple influenza virus lineages in different species of poultry and inconsequent control attempts including fragmentary vaccination coverage has facilitated diversifying evolution by genetic drift and reassortment10.Avian influenza viruses (AIVs) remain important pathogens for wild birds, domestic poultry and humans. AIVs group into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes each revealing various lineages and host-specific adaptations14. Several reassortants between HPAI H5N1, H9N2 and HPAI H7N3 viruses have been recorded in the country although some reassortments took place before incursion into Bangladesh13. In addition, Bangladesh suffered from successive incursions and replacements of three different gs/GD HPAIV H5N1 clades, 2.2.2, 2.3.2.1, and 2.3.4.214. Since 2012, clade 2.3.2.1a viruses have replaced previously co-circulating gs/GD viruses16. Exposure of humans to these viruses is emphasized by nine and three cases of human infection with HPAIV H5N1 (including one death) and H9N2, respectively, till to date19.Bangladesh, a south Asian country with a high density of poultry and human populations is no exception in this respect. Potentially zoonotic HPAI H5N1 and H9N2 viruses co-circulate endemically in commercial and backyard poultry shuttling between different poultry species and causing sporadic spill-overs into wild bird populations since early 2007Here we illustrate the intricate epidemiological situation and the evolutionary potential of co-circulating AIV subtypes of different sub-, geno- and phenotypes by molecular analyses of clinically overt AI outbreaks in commercial and backyard poultry in Tangail district, one of the most crowded districts for both poultry and human populations at the northern perimeter of Dhaka, the capital of Bangladesh.20. Clinical presentation and epidemiological considerations thus raised the suspicion of AIV H9N2 infections. This suspicion was corroborated by contemporary, laboratory-confirmed findings of H9N2 cases with similar clinical presentation in the same area (unpublished). This prompted submission of samples directly to FVS-BAU while samples from HPAI suspect cases are to be filed to the National Reference Laboratory at Bangladesh Livestock Research Institute. At necropsy, however, at FVS-BAU, severe congestion in lung and trachea along with severely congested blood vessels at intestinal walls, petechial to ecchymotic haemorrhages at the epicardium, air sacs, intestine, proventricular glands and pancreas dominated  reporting respiratory illness. Carcasses of four chickens (VP02-VP05) were sampled at four different farms keeping grower and layer chickens 6 to 84 weeks old, cage-reared and putatively vaccinated against H5 viruses (unknown source). From neighboring backyard poultry holding the carcass of a duck (VP06) was collected as well. The sampled sector 3 chicken layer farms, each of up to 2000 birds reported morbidity of up to 90% dominated by respiratory disease and reduced egg production. Cumulative mortality approaching 35% at these farms was substantial but still low compared to previously recorded HPAI outbreaks in chickens in Bangladeshted Fig. . Histopated Fig.  and evokAdditional samples, came from a commercial chicken farm (C1/2016) and a backyard duck (D1/2016) sampled in November 2016 in Mymensingh district bordering Tangail. Similar clinical signs and lesions as described above were recorded in the respective chickens and ducks (data not shown). However, mortality in the commercial layer chicken farm of C1/2016 was higher (70–75%). In this farm, in contrast to the flocks in Tangail district, an HPAIV H5N1 mono-infection was diagnosed. Mortality in Tangail farms was lower (35%) and, initially, an H9N2 infection was diagnosed that later turned out to be a mixed infection comprising HPAIV H5 and H7.22  and the backyard duck (VP06) from Tangail district. Sequence analysis suggested presence of H9N2 viruses with a dibasic HA cleavage site motif (KSKR/GLF) in all samples. HA H9 and NA N2 phylogenetic analyses revealed that all sequences were similar to circulating Bangladeshi H9N2 viruses of the G1 lineage  suggested presence of reassorted H9N2 viruses. In contrast, full range RT-qPCR-based subtyping by the “Riems influenza A typing assay” (RITA) carried out on RNA extracted from allantoic fluid of embryonated chicken egg (ECE) passage detected further AIV HA and NA subtypes H5, H7, H9, N1 or N2 for samples VP02-06 from Tangail district. No further co-infection was detected for H5N1 positive samples C1/2016 and D1/2016 obtained from Mymensingh district  clustered with HPAIV clade 2.3.2.1a (HA cleavage site PQRERRRKR/GLF) maintaining a close relationship with H5N1 viruses described in Bangladesh during 2012–2013 but distinct from recently circulating H5N1 sample C1/2016 and D1/2016 originating from Mymensingh district in late 2016 , H9N2 (16%), H5N2 and H7N2 (1.0–1.8%). HP H5N1, H5N2, H7N1 and H7N2 produced higher virus yields  and a shortened mean-death time  compared to purified H9N2 .The rescued reassortant subtypes exhibited distinct plaque morphology. The H5N1 subtype produced the largest plaques at approximately 4.5 mm in diameter. Plaque area measurements using ImageJ50 of 103/0.2 mL. Independent of the sub- and genotype none of the co-inoculated clones was outcompeted during three consecutive blind passages. In particular, seemingly unconstrained growth of H9N2 clones was noted even in the presence of H5 or H7 HPAI viruses. In competitions between HP H5N1 and HP H5N2, the N2 gene became dominant, i.e. Cq-values for N1 and N2 were similar in the inoculum at passage 0 but differed by 7–9 Cq values at passage 3 with N2 taking advantage over N1 , virus isolation and after plaque purification from two selected clinical poultry samples (ck/VP02 and dk/VP06) of AI outbreaks. The same samples were previously diagnosed based on RT-PCR and direct Sanger sequencing of clinical material, to be due to infection with reassorted H9N2 viruses. Despite presence of gene segments of several sub- and genotypes in the same sample, the corresponding sequence chromatograms showed a high quality; this is probably due to the high subtype specificity of primers used for cycle sequencing which grossly favored a single subtype over the rest. H5N1, H5N2 and H9N2 viruses were purified from ck/VP02 whereas H5N1, H5N2, H7N1 and H7N2 subtypes were purified from dk/VP06. Since plaque purification could not be carried out directly from clinical material, the five subtypes and eight different genotypes may not have been present in this constellation in the clinical samples. Some of the sub- and genotypes detected following plaque purification could have been generated during virus isolation in ECE and/or plaque-passaging. Surprisingly, no replicating H9N2 virus was rescued during plaque purification from dk/VP06 although RITA confirmed the presence of this subtype in this sample as well  obtained in the Mymensingh district in late 2016 and treated similarly in our labs proved to contain each a single AIV strain which carried contemporary 2.3.2.1a HA and NA genome segments  undertook all sample manipulations including virus isolation and this laboratory did never have access to replication-competent HP Pak/H7 and Bangladeshi HP H5N1 viruses of clade 2.2.2. Likewise, none of these viruses were available in the laboratory at FLI where subsequent RITA assays, virus amplification and plaque purification experiments were carried out. Unfortunately, original tissues of these cases were no longer retrievable for confirmation. Thus, the true sources of the HP H7 and HP H5 2.2.2 viruses remain elusive.13. These findings at least indicate independent of our findings that there must have been opportunities of reassortment between HP H5N1 and the Pakistani H7 viruses although it remained unclear where and when such events took place. In turn, our findings could provide a very comprehensible explanation for those observations. Also in Cambodia, extensive co-circulation of and reassortment among H5, H7 and H9 AIV in poultry has recently been pointed out26.HA and NA segments of purified H9N2 clones were similar to circulating contemporary G1/H9N2 viruses although their internal genes originated from clade 2.2.2/H5N1. Previously, it was also reported that Bangladeshi H9N2 viruses shared internal genes with HP H5N1  and with the HP Pak/H7N3in vivo. Yet, it is not possible to extrapolate these results to the field where additional transmission bottlenecks may exist. Stable replication in chickens of an LPAI H7N7 virus even in presence of its HP mutant descendant virus has recently been reported27.The potential of sustained replication in ECE of G1/H9N2 viruses even in competition with HPAI H5 and H7 viruses was demonstrated during a limited number of passages in co-infected ECE. Although no prognosis for the next 10 or 50 passages can be made, it seems conceivable that sets of different AIV sub- and genotypes may be co-transmitted across at least several passages also From a diagnostic perspective, the presence of minor viral populations within extended mixtures as described here would only be detected by using highly sensitive and multiple subtype-discerning diagnostic tools such as RITA. In contrast, more global diagnostic approaches including direct Sanger sequencing based on universal “Hoffmann” primers and strain-specific sequencing primers might yield incomplete and even false conclusions depending on the specificity of the primers. This was initially the case here, when different reassorted H9N2 viruses were diagnosed on basis of full-length genome Sanger sequencing. In addition, also high throughput sequencing approaches might give more insights for those samples in the future.28. Potential masking of HPAIV related clinical signs in vaccinated or LPAIV co-infected poultry should be considered when analyzing spread of HPAIV along trading networks.Presence of HPAIV phenotypes in G1/H9N2 mixtures would be expected to influence pathogenesis and clinical picture. This was evident also here with the detection of signs of systemic infection and lesions in multiple organs  viral populations in clinical samples and corresponding viral isolates originating from AI outbreaks. While next-generation sequencing techniques provide ultimate tools for this approach, available multiplexed or arrayed RT-qPCR applications allow for a rapid screening at more sensible costs and of higher sample numbers.29 were carried out upon arrival of poultry carcasses at the Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University (FVS-BAU). In addition, presence of AIV and Newcastle Disease virus (NDV) RNA, respectively, in lung and trachea tissue was investigated by conventional RT-PCR targeting fragments of the matrix (M) gene of both AIV and NDV31. Full-length genome sequences were established using the Hoffmann primers32 as detailed below. Tissue homogenates  were inoculated into 10-day old embryonated chicken eggs (ECE) following standard procedures33. Allantoic fluid of this egg passage was sent to the Friedrich-Loeffler-Institute (FLI) for further investigations. At FLI, passaged samples were HA- and NA-subtyped using the Riems influenza A typing assay (RITA)34.Necropsy, histopathology and virological investigations as previously described2, viral plaque formation was confirmed and individual clones were selected under the light microscope. Virus clones selected were not amplified between three plaque passages. Selected virus clones of the third plaque passage were finally amplified in ECE for production of virus stocks. Plaque clone harvest at different passages and their selection for further analyses are shown in Supplementary Data; Table Based on the RITA results three rounds of plaque purification were conducted for two selected egg-grown virus isolates (ck/VP02 and dk/VP06) to obtain pure virus clones. Briefly, 100 µL of 10-fold serial dilutions of each isolate were incubated on confluent MDCK-II (ATCC® CRL-2936™) cell monolayers in six well plates for 1 h at 37 °C. Cells were carefully washed twice with phosphate buffered saline (PBS) and then overlaid with modified Eagle’s medium containing 1.8% agar, 3.5% bovine serum albumin and 1 μg/mL N-tosyl L-phenylalanine chloromethyl-ketone (TPCK)-treated trypsin (Sigma T1426). After three days of incubation at 37 °C, 5% CO32 for each genome segment were used for full-length amplification of RNA extracted from clinical samples VP02-VP06. Specific primer sets were used for cycle sequencing. At FLI, Sanger sequencing of full-length HA and NA was also performed for all plaque-purified viruses. In addition, whole-genome sequences were established for the H7N1 and H7N2 plaque-purified clones. For the H5 and H9 plaque-purified clones no full-length sequences of internal genes were generated due to the excessive amount of sequencing work. Instead, as shown below, a SYBR-Green-based RT-qPCR technique was used to define their backbone provenance (either H5 or H9). RT-PCR, amplificate purification and cycle sequencing were carried out as described previously35. Sequences were assembled and edited using the Geneious software, version 10.2.336. Alignment and identity matrix analyses were performed using MAFFT37. Sequences generated in this study were deposited in NCBI GenBank and in the Global Initiative on Sharing All Influenza Data (GISAID) database  and Inkscape 0.92.At FVS-BAU, universal influenza primer sets40. Adjusting the assays and validation of specificity using the qScript™One-Step SYBR-Green kit  was carried out with homologous and heterologous gene segments of fully sequenced reference isolates. Melting point analysis of the amplificates was used as an additional specificity control as described41.For rapid genotyping, segment-specific SYBR-Green RT-qPCR assays were established. Based on full-length genome data of clinical isolates and some of the plaque-purified viruses, two sets of SYBR-Green RT-qPCRs were designed; one specific for internal segments of the H5N1 clade 2.2.2 lineage, the other specific for the G1/H9N2 lineage. Primers were selected using Oligo Calculator, online version 3.26  measurements of plaque-purified viruses, five ECE per virus were inoculated each with a fixed concentration of 103 TCID50 and candled twice daily.The infectivity titer of viruses was determined by microtitration in MDCK-II cells as described before3 TCID50 per inoculated virus was used to compare viral fitness of the different sub- and genotypes in 16 combinations each of two viruses obtained during plaque purification. In these combinations all five subtypes and eight genotypes were included. In an additional co-inoculation experiment, three viruses were combined, HP H5N1, HP H7N2 and H9N2. Three consecutive blind passages each of 48 hours in ECE were carried out using 200 µL of allantoic fluid diluted at 10−4 as inoculum for onward passages. RNA was extracted at passages 0 (P0) and 3 (P3) for sub- and genotyping using RT-qPCRs as detailed above selecting PCRs that are specific for those segments that distinguished between the two co-inoculated virus clones. Cq-values were determined as a relative measure of the proportion of the distinct segments in the mixture.Co-inoculation into 14-days old embryonated chicken egg (ECE) at 10We state that none of the work carried out in this study required approval from ethics committees as neither human samples nor animal experiments were employed. All experiments and methods were performed with approval and in accordance with the respective relevant legal guidelines and regulations of Bangladesh Agricultural University, Bangladesh, and the Friedrich-Loeffler-Institut, Germany.Supplementary Data


Histones are constitutive components of nucleosomes and key regulators of chromatin structure. We previously observed that an extrachromosomal histone H2B (ecH2B) localizes at the intercellular bridge (ICB) connecting the two daughter cells during cytokinesis independently of DNA and RNA. Here, we show that ecH2B binds and colocalizes with CHMP4B, a key component of the ESCRT-III machinery responsible for abscission, the final step of cell division. Abscission requires the formation of an abscission site at the ICB where the ESCRT-III complex organizes into narrowing cortical helices that drive the physical separation of sibling cells. ecH2B depletion does not prevent membrane cleavage rather results in abscission delay and accumulation of abnormally long and thin ICBs. In the absence of ecH2B, CHMP4B and other components of the fission machinery, such as IST1 and Spastin, are recruited to the ICB and localize at the midbody. However, in the late stage of abscission, these fission factors fail to re-localize at the periphery of the midbody and the abscission site fails to form. These results show that extrachromosomal activity of histone H2B is required in the formation of the abscission site and the proper localization of the fission machinery. Histones are the constitutive components of nucleosomes, the basic units of DNA packaging in eukaryotes, and their post-translational modifications regulate chromatin structure and activity. ,2. In maWe previously identified an extrachromosomal localization of histone H2B at the intercellular bridge (ICB) during cytokinesis . This H2Cytokinesis is the final stage of the cell cycle through which a mother cell divides into two daughter cells ,19,20. THere, we set out to elucidate the role of ecH2B at the ICB. Visualization of several cytokinesis factors in cells depleted of ecH2B demonstrated the contribution of this histone in the formation of the abscission site.2 environment. Cells were routinely tested for mycoplasma contamination. For live cell imaging, HeLa cells were cultured in DMEM without phenol red, supplemented with 10% FBS. RNA interference was performed with commercially available H2B variant-specific stealth RNAi sequences  and hTERT-immortalized dermal human fibroblasts  were cultured in DMEM/GlutaMAX with 10% heat-inactivated fetal bovine serum  and maintained in a humid incubator at 37 °C in a 5% COv/v), 2 mM PMSF, and 0.02% (w/v) NaN3) and lysed on ice for 10 min. The nuclei were isolated by centrifugation at 6500× g for 10 min at 4 °C and resuspended in 0.2 N HCl over night at 4 °C to extract histones. The supernatant (which contains the histone proteins) was neutralized with 2M NaOH at 1/10 of the volume of the supernatant. NuPAGE® Novex Bis-Tris Gels (Life Technologies) were used for SDS-PAGE and nitrocellulose membranes  for protein transfer and immobilization. The following Abs were employed for WB: anti-α-tubulin moAb , anti-GST moAb (kindly provided by Maurizio Fanciulli), anti-H2B moAb , HRP-conjugated goat anti-mouse, and anti-rabbit secondary Abs (Bio-Rad). Immunoreactivity was determined using the ECL-chemiluminescence reaction  following the manufacturer’s instructions.Total cell lysates were prepared by using the following denaturing lysis buffer (50 mM Tris-HCl (pH 8), 600 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40, and 1 mM EDTA) supplemented with protease-inhibitor mix  and Halt Phosphatase Inhibitor Cocktail (Life Technologies). Histone extraction protocol was performed following the Abcam protocol. Briefly, cells were harvested and washed twice with PBS. The cells were then resuspended in Triton Extraction Buffer , permeabilized for 10 min with 0.25% Triton X-100 and blocked for 60 min with 5% BSA in PBS. Cells were stained with the Abs reported in Cells seeded on poly-2. DIC images were acquired every 3 min over a 24 hr period by using a DS-Qi1Mc camera and the NIS-Elements AR 3.22 software . Image and video processing were performed with NIS-Elements AR 3.22.Cells seeded on 15 μ-Slide 8 well  were observed under an Eclipse Ti inverted microscope using a Plan Apo 40× objective (Nikon). During the whole observation, cells were kept in a microscope stage incubator  at 37 °C and 5% CO® In Situ Detection Reagents Red DUO92008  in four steps: (1) incubation of fixed cells with primary specific Abs; (2) incubation with secondary Abs conjugated with complementary oligonucleotide tails ; (3) ligase addition when, if the two proteins interact or are very close, the ligation step will produce a DNA circle; and (4) rolling circle amplification. Cells were fixed, blocked, and incubated with primary Abs as for IF; we used combination of mouse and rabbit primary Abs for each protein pair (rabbit anti-H2B-Ser14P or -Ser32P and mouse anti-CHMP4B). Anti-mouse MINUS and anti-rabbit PLUS PLA probes were added on coverslips  and incubated in a pre-heated humidity chamber (60 min at 37 °C). Subsequent ligation (30 min at 37 °C) and amplification (70 min at 37 °C) steps were performed following the protocol. To localize PLA signals, cells were fixed in formaldehyde 2% 10 min at RT and then co-stained using HOECHST 33342 and anti-alpha tubulin FITC-conjugated Ab.Cells seeded on round poly-L-lysine coated coverslips were processed for proximity ligation assay (PLA) using the Duolink32P-ATP .For H2B and CHMP4B binding assays, GST-CHMP4B  was incubated overnight at room temperature with 500 ng of recombinant His-H2B  or histone H2B  in buffer phosphate pH 7.5, 150 mM NaCl. GST-pulldown was performed by incubation for 2 h at 4 °C with Glutathione-Sepharose 4 Fast Flow beads  and three washes with buffer phosphate. Bound proteins were resolved by SDS-PAGE and analyzed by WB. For H2B phosphorylation, recombinant His-H2B was incubated with HIPK2 Kinase domain (kind gift of Dr. Linda Montemiglio), as an enzymatic source, in kinase buffer  at 30 °C for 30 min in the presence of cold ATP or, as a control, of γ-p values were derived from unpaired two-tailed t-tests using GraphPad Prism software . p values < 0.05 were considered significant.Each experiment has been repeated from three to five times and the results obtained presented as mean ± standard deviation of the mean (SD). P). This Ab was employed to detect specifically ecH2B and avoid immunostaining of nucleosomal H2B, which is not phosphorylated at S14 unless in apoptotic cells [To investigate the function of ecH2B in cytokinesis, HeLa cells were treated with control siRNA (CTRi) or a mix of nine commercially available H2B siRNAs (H2Bi) that recognize different H2B isoforms a. Transfic cells ,46. To vic cells . CTRi ceic cells , confirmn = 1000), H2Bi HeLa cells (n = 1000) showed a significant accumulation of cytokinetic cells (n = 151 and H2Bi n = 168) and found that, on average, they were twice as long in the H2Bi than in the CTRi cells  and H2Bi  versus 129.56 ± 35.2  min in CTRi (n = 81); p  <  0.0001; Since no significant alteration in cytokinesis factor recruitment were observed upon H2B depletion, we sought to explain the defect leading to the aberrant telophase phenotype measuring the cytokinesis-time of asynchronous CTRi and H2Bi HeLa cells using time-lapse live-cell imaging . No signn = 100; b. In conP shows a stronger binding with CHMP4B than the non-phosphorylated H2B . To avoid Ab-related nonspecific signals, we employed both anti-phospho-H2B-S14 and anti-phospho-H2B-S32 Abs. No PLA signals were detected with both Abs in early cytokinesis   than theP might facilitate CHMP4B polymerization by releasing the auto-inhibitory activity of its C-term. Due to the expanding roles of the ESCRT proteins in membrane remodeling associated with several physiological and pathophysiological processes, such as biogenesis of multi-vesicular bodies, virus budding, and reformation of the nuclear envelope [At this point, we cannot discriminate whether ecH2B simply contributes to the abscission site maturation that requires local cytoskeletal remodeling, prior to the assembly of ESCRT-III, or whether it also aids the formation of ESCRT-III helices ,43,44,45envelope ,55,56,57


Background: Cardiac fibrosis after myocardial infarction mainly causes cardiac diastolic and systolic dysfunction, which results in fatal arrhythmias or even sudden death. Id2, a transcriptional repressor, has been shown to play an important role in the development of fibrosis in various organs, but its effects on cardiac fibrosis remain unclear. This study aimed to explore the effects of Id2 on cardiac fibrosis after myocardial infarction and its possible mechanisms.Methods: This study was performed in four experimental groups: control group, treatment group , treatment+GFP group and treatment+Id2 group. In vitro anoxic and fibrotic models were established by subjecting CFs or NRVMs to a three-gas incubator or TGF-β1, respectively. An animal myocardial infarction model was established by ligating of the left anterior descending coronary artery followed by directly injecting of Id2 adenovirus into the myocardial infarct’s marginal zone.Results: The results showed that Id2 significantly improved cardiac EF and attenuated cardiac hypertrophy. The mRNA and protein levels of α-SMA, Collagen I, Collagen III, MMP2 and TIMP1 were higher in treatment+Id2 group than those in treatment group as well as in treatment+GFP group both in vivo and in vitro. Immunofluorescence revealed that both α-SMA and vimentin were co-expressed in the treatment group and GFP group, but the co-expression were not detected in the control group and Id2 group. Additionally, our findings illustrated that Id2 had protective effects demonstrated by its ability to inhibit the TGF-β1/Smad3/HIF-1α/IL-11 signaling pathways. Besides, over-expression of Id2 reduced cardiomyocytes apoptosis.Conclusion: In conclusion, this study demonstrated that over-expression of Id2 preserved cardiac function and ameliorated adverse cardiac remodeling, which might be a promising treatment target for cardiac fibrosis and apoptosis. Fibrosis is defined as the accumulation of fibrillar extracellular matrix (ECM) components in a tissue or an organ. The development of cardiac fibrosis after myocardial infarction (MI) is a continuous process . In the Inhibitor of differentiation (Id), known as DNA binding inhibitor, is a regulatory protein which functions as a negative transcription factor. This protein has four subtypes: Id1, Id2, Id3 and Id4 . SeveralBased on the above discussion, we hypothesize that Id2 may participate in the development of fibrosis after myocardial infarction. Our study aims to discover the role of Id2 in cardiac fibrosis and reveal it possible mechanisms.Newborn male Sprague–Dawley(SD) rats  were purchased from Disease Control and Prevention of Hubei Provincial Center  2015-0018). Adult healthy male SD rats  was acquired from the Experimental Animal Center of Wuhan University People’s Hospital . The SD rats were kept in four per cage with standard laboratory chow and given sterilized water. The room environment was controlled under a constant temperature (22 ± 2°C), constant humidity (55 ± 5%), and a 12:12 h light/dark cycle. The present study was approved by the Experimental Animal Committee of Wuhan University . All experimental procedures were approved by the Ethics Committee of Animal Research, Wuhan University Health Science Center, and the investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .10 PFU/ml and 8 × 1010 PFU/ml respectively, which were preserved at -80°C.The adenoviral vector expressing GV315Ad-MSC-GFP-Id2 was constructed by inserting the human Id2 gene (positive clone sequence: ATGAAAGCCTTCAGTCCCGTGAGGTCCGTTAGGAAAAACAGC CTGTCGGACCACAGCCTGGGCATCTCCCGGAGCAAAACCCCT GTGGACGACCCGATGAGCCTGCTATACAACATGAACGACTGC TACTCCAAGCTCAAGGAGCTGGTGCCCAGCATCCCCCAGAAC AAGAAGGTGAGCAAGATGGAAATCCTGCAGCACGTCATCGAC TACATCTTGGACCTGCAGATCGCCCTGGACTCGCATCCCACT ATTGTCAGCCTGCATCACCAGAGACCCGGGCAGAACCAGGCG TCCAGGACGCCGCTGACCACCCTCAACACGGATATCAGCATC CTGTCCTTGCAGGCTTCTGAATTCCCTTCTGAGTTAATGTCA AATGACAGCAAAGCACTGTGTGGCTGA) into GV315Ad-MSC-GFP vector  using AgeI/NheI (cat. no. CON267) restriction sites, all of which obtained from Shanghai Genechem Co., Ltd. Ad-GFP and Ad-GFP-Id2 were measured as 105/ml. During the first 48 h after seeding, 0.1 mmol/l bromodeoxyuridine  was added to inhibit the mitosis of fibroblasts. CFs were cultured for 48 h and then were passaged to 3–6 generations for further experiments.Primary rat CFs (cardiac fibroblasts) and primary neonatal rat cardiomyocytes (NRVMs) were isolated from ten 1–3-day-old SD pups as described previously with some modifications . Briefly3 cells/well. After culturing for 24 h, cells were treated with serum-free medium for another 24 h and then were placed on three-gas incubators environment . After treatment with hypoxia for 12 h, 20 ul of CCK-8 reagent was added into each well and CFs were incubated at 37°C for another 2 h. The optical density was measured at a wavelength of 450 nm. Serum-free mediums in normal incubators served as the negative control.Cell proliferation was assessed with Cell Counting Kit-8 (CCK-8)  reagent following the manufacturer’s instructions. CFs were passaged in 96-well plates at the density of 8 × 10The apoptosis of NRVMs after exposed to hypoxia was assessed by staining cells with Annexin V-fluorescein isothiocyanate (APC) Apoptosis Detection Kit (BD Biosciences). The cells were collected and then washed with cold PBS twice. Thereafter, they were resuspended in 100 ul of Annexin V binding buffer and incubated with 5 ul of APC-conjugated Annexin V and 5 ul of propidium iodide for 15 min in the dark. Annexin V binding buffer (200 ul) was then added to each tube. Finally, the cells were examined using a BD FACS-Canto II flow cytometer . All experiments were repeated three times, independently.CFs were transfected with Ad-GFP or Ad-GFP-Id2 for 48 h, then anoxic and fibrotic models were established using a three-gas incubator for 12 h or Recombinant Human TGF-β1 for 24 h, respectively. The hypoxia model was divided into 4 groups: control group (control), hypoxia group (hypoxia), hypoxia+Ad-GFP group (hypoxia+GFP), hypoxia+Ad-GFP-Id2 group (hypoxia+Id2). Fibrosis model: control group (control), TGF-β1 group  (TGF-β1), TGF-β1+Ad-GFP group (TGF-β1+GFP), TGF-β1+Ad-GFP-Id2 group (TGF-β1+Id2). Similarly, after adenovirus transfection for 48 h, NRVMs were cultured and treated with three-gas incubators environment  for 6 h. This model was also divided into four groups: control groups (C), hypoxia groups (H), hypoxia+GFP groups (H+G) and hypoxia+Ad-GFP-Id2 (H+I).9 pfu of Ad-GFP-Id2 or Ad-GFP into the left ventricular wall via a 50-gauge needle. A total volume of 100 ul was injected into five separate areas in the viable myocardium bordering the infarct zone. The sham groups or the MI groups were injected with 100 ul PBS. Rats in the sham group underwent similar surgical procedures as those in treatment groups only differing in having unknotted sutures placed under the left anterior descending coronary artery.Male SD rats weighing 180–200 g were randomly divided into six groups: sham operation group , MI group , MI+Ad-GFP group , MI+Ad-GFP-Id2 group , Ad-GFP-Id2 group (n = 12), Ad-GFP group (n = 3). The detailed protocol was described previously . BrieflyTwo weeks after AMI, the body weights (BW) of the rats were recorded. Rats were anesthetized with sodium pentobarbital 1% (50 mg/kg) by intraperitoneal injection and hemodynamic parameters were recorded. Briefly, the right common carotid artery was dissected and separated from the connective tissues. A catheter was inserted into the carotid, then blood pressure and heart rate were recorded by using LabChart 7. After hemodynamics monitoring, the hearts were removed quickly by thoracotomy, washed with saline, and then lung weight (LW) and heart weight (HW) were measured. Cardiac index = HW/BM (mg/g), Cardiopulmonary index = HW/LM (mg/mg) were calculated.g for 10 min (4°C) and the supernatants were collected. Equal amounts of proteins (40 µg) were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies overnight at 4°C and then probed with horseradish-peroxidase-conjugated secondary antibodies for 30 min at room temperature  equipped with a 12-MHz probe(12S).The hearts and cells were fixed with 4% paraformaldehyde and embedded in paraffin. CFs and NRVMs were permeabilized in 0.2% Triton X-100 in PBS. Deparaffinized sections (6-um thickness) were stained with Masson’s trichrome, Hematoxylin and Eosin (HE) or TUNEL. Deparaffinized sections (6-um thickness) or CFs were stained with primary antibodies overnight at 4°C with anti-α-SMA and/or anti-Vimentin or HIF-1α, and NRVMs were stained with anti-caspase-3, respectively, followed by the secondary antibody for 2 h at 37°C. Nuclei were stained with 4’,6-diamidino-2-phenylindole . The negative controls lacking the primary antibody were included. Fluorescence images were captured by the fluorescent microscope . Semiquantitative analysis of the tissue staining images was performed using the Image-Pro Plus 6.0 System .Data were expressed as means ± SEM and were analyzed by GraphPad Prism 5. One-way analysis of variance (ANOVA) was performed for multiple groups, followed by Bonferroni’s multiple comparison test. Unpaired Student’s t-test was performed to compare two groups. All data were subjected to formal tests for normality. Data not following normal distribution were evaluated by non-parametric tests. A P-value of <0.05 was considered statistically significant.Masson’s trichrome staining showed the presence of collagen deposition in the border and infarct zones in MI rats , MI group , MI+Ad-GFP group , and MI+Ad-GFP-Id2 group . There was no difference in body weight (BW) and lung weight (LW) among the four groups after 2 weeks. The heart weight (HW), HW/BW ratio and HW/LW ratio were higher in MI group and GPF group compared to the sham group   values . CFs of passages 3–6 were digested in culture dishes and then inoculated in 24-well plates and 6-well pates. When cell confluence reached 70–80%, Ad-GFP-Id2 and Ad-GFP in DMEM/F12 were added to the cells at different MOI values . After 12 h of incubation period, a fresh complete medium was added. The cells were observed under a light and fluorescent microscope . After transfection for 24 h, the CFs and NRVMs exhibited a green fluorescence. The results showed that as the MOI increased, the fluorescence intensity of Ad-GFP-Id2 increased. The highest MOI that did not reduce the number of cells was chosen as the optimal MOI value for infection. As shown in Although Id2 expression increased after MI, the cellular source of Id2 was not known. Thus, we treated NRVMs and CFs in hypoxia environment for 6 h and 12 h, respectively. Numerous studies have shown that myofibroblasts derived from CFs are markers of cardiac fibrosis. To clarify whether Id2 directly modulates myofibroblast differentiation, we treated CFs with TGF-β1 or hypoxia. The CFs were placed in incubators with hypoxic or normal conditions for 6, 8, 10, 12, and 24 h, after which the absorbance values were measured at 450 nm. It was observed that the proliferation of CFs increased along with the time of hypoxia , Ad-GFP-Id2 group (Id2), Oltipraz group (Oltipraz) (10 mmol/L), hypoxia group (hypoxia), hypoxia+Ad-GFP-Id2 group (hypoxia+Id2), and hypoxia+Oltipraz group (10 mmol/L) (hypoxia+Oltipraz). in vivo  were higher in MI group and GFP group than in the sham group and Id2 group. This implies that MI promoted cardiomyocyte apoptosis and over-expression of Id2 might prevent these adverse effects. The apoptotic cells displayed yellow-brown nuclei whereas normal cells had light blue nuclei. The rate of apoptosis was higher in MI group and GFP group compared to the sham group (MI 31.00% ± 0.9% vs GFP 31.70% ± 1.49% vs sham 7.20% ± 0.25%), and Id2 reversed these effects to some extent (Id2 17.30% ± 0.25%) .LY and MX-L made substantial contributions to the conception and design of the study, performed the experiments and wrote the paper. WL and F-YW assisted in the performance of experiments. Y-HT participated in research design and coordinated the study. C-XH revised the manuscript and gave final approval of the version to be published. All authors read and approved the final manuscript.The present study was supported by the Fundamental Research Funds for the Central Universities of China (grant no. 2042015kf0229).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.


The genome of some vole rodents exhibit large blocks of heterochromatin coupled to their sex chromosomes. The DNA composition and transcriptional activity of these heterochromatin blocks have been studied, but little is known about their DNA replication dynamics and epigenetic composition.Microtus cabrerae cells. While the X chromosomes are hypoacetylated and cytosine hypomethylated, they are either enriched for macroH2A and H3K27me3 typical for facultative heterochromatin or for H3K9me3 and HP1 beta typical for constitutive heterochromatin. Using pulse-chase replication labeling and time-lapse microscopy, we found that the heterochromatic block enriched for macroH2A/H3K27me3 of the X chromosome is replicated during mid-S-phase, prior to the heterochromatic block enriched for H3K9me3/HP1 beta, which is replicated during late S-phase. To test whether histone acetylation level regulates its replication dynamics, we induced either global hyperacetylation by pharmacological inhibition or by targeting a histone acetyltransferase to the heterochromatic region of the X chromosomes. Our data reveal that histone acetylation level affects DNA replication dynamics of the sex chromosomes’ heterochromatin and leads to a global reduction in replication fork rate genome wide.Here, we show prominent epigenetic marks of the heterochromatic blocks in the giant sex chromosomes of female Microtus cabrerae cells. Furthermore, we highlighted a conserved role of histone acetylation level on replication dynamics across mammalian species.In conclusion, we mapped major epigenetic modifications controlling the structure of the sex chromosome-associated heterochromatin and demonstrated the occurrence of differences in the molecular mechanisms controlling the replication timing of the heterochromatic blocks at the sex chromosomes in female The online version of this article (10.1186/s13072-019-0262-0) contains supplementary material, which is available to authorized users. Arvicolinae) are a remarkable exception. In some vole species, a bulk of constitutive heterochromatin is coupled to both sex chromosomes. These enlarged X and Y chromosomes are referred to as “giant” sex chromosomes [In higher eukaryotes, the nuclear genome is compartmentalized into distinct chromatin territories to facilitate the regulation of complex processes such as DNA repair, transcription and replication. The DNA replication process is highly regulated both spatially and temporally, resulting in the changing pattern of replication structures throughout S-phase. The temporal order of DNA replication reflects this higher-order organization of the nuclear genome –3. Eu- aomosomes , 12 and Euchromatin includes less-condensed transcriptional active regions, determined by a depletion of methylated DNA, an enrichment in specifically methylated histones such as H3K4, H3K36, H3K79 and a high level of histone acetylation . EuchromMicrotus cabrerae, a species presenting giant sex chromosomes with enlarged heterochromatin blocks and, thus, test the validity and reproducibility of the epigenetic control of replication dynamics across mammalian species. We first determine the epigenetic constitution of the sex chromosome-associated heterochromatic blocks and second dissect the timing and regulation of DNA replication of the different chromatin states. Our study revealed that histone hypoacetylation and strikingly also DNA demethylation are major epigenetic determinants of this sex heterochromatin. We distinguished between X chromosome-associated heterochromatin block enriched for H3K27me3 and macroH2A, and X chromosome-associated heterochromatin block exhibiting H3K9me3 and HP1 accumulation. We found that the former replicated before the latter, and each heterochromatin block replicated rather synchronously. To test whether histone acetylation levels might be involved in setting up the synchronous DNA replication process, we manipulated histone acetylation globally by HDAC inhibition or by site-directed HAT targeting. We showed that an increase in histone acetylation levels affects DNA replication dynamics and leads to a prolongation of total and early S-phase, as well as of X chromosome-associated heterochromatin block replication. Finally, we found a global decrease in replication fork speed in hyperacetylated cells going hand in hand with a prolongation of S-phase.In this study, we assess the DNA replication dynamics in female cells from the vole species Microtus species (M. agrestis and M. cabrerae), the heterochromatic blocks from the sex chromosomes are often visible during interphase as bright dense regions of DAPI-stained chromatin [Microtus cabrerae cells. Since four DAPI bright regions were visible per cell, we controlled the karyotype of the cell line by metaphase spreading followed by chromosome painting using a specific probe for the X chromosome  partly labeled by H3K9me3, in female cells we found, in addition, X chromosomes (X*) marked with H3K27me3 marks along their length . Similarly, in cells transiently transfected with GFP-tagged HP1 beta and simultaneously stained for H3K9me3, enrichment for both signals in the same heterochromatic blocks was observed. Furthermore, we also investigated several euchromatic marks such as H3K4 dimethylation (H3K4me2), H4K8 acetylation (H4K8ac), H3K9 acetylation (H3K9ac), as well as the level of DNA methylation , while the other half showed accumulation of H3K9me3/HP1 beta signal (“constitutive” heterochromatic block)  induces global hyperacetylation  at the HP1-enriched heterochromatic blocks Fig. b. HBO1 tFurthermore, we performed HAT targeting also to the H3K27me3/macroH2A enriched X chromosome, using GBP-macroH2A1 and GFP-HBO1. This targeting approach was less effective, possibly due to the required incorporation of ectopic macroH2A1 into endogenous nucleosomes, which should take place with very slow kinetics, whereas HP1 beta simply needs to bind to H3K9 trimethylated nucleosomes and its exchange rate is relatively fast. Nevertheless, we were able to detect a prolongation of DNA replication of the facultative heterochromatic block at the X chromosome upon HAT targeting  in early, mid- and late S-phase as well as in non-S-phase cells  and cloned into pEGFP-C1 (Clontech). DsRed-HP1 beta was transfected to identify the constitutive heterochromatic block of the X chromosome . GFP-macroH2A1 (pc2101) was used as a backbone and was cut with AgeI and XhoI to create the final plasmid GBP-macroH2A1 (pc2883). To detect the constitutive heterochromatic block of the X chromosome, a construct encoding GBP-HP1 beta (pc3357) was created. MacroH2A1 was removed from GBP-macroH2A1 (pc2883) by restriction with EcoRI and BamHI and replaced by HP1 beta from DsRed-HP1 beta . For si.Microtus cabrerae cells [Microtus cabrerae cells [2 atmosphere at 37 °C. As positive control for 5mC staining, the mouse embryonal fibroblast W8 line [Microtus cabrerae cells were performed using nucleofection  with 1 µg per plasmid. For HDAC inhibition treatment, 50 nM panobinostat  in PBS was added 24 h after seeding cells or nucleofection to the culture medium and incubated for at least 24 h prior to fixation or live-cell experiments . Mitotic cells were enriched by shake-off and collected by centrifugation . Cells were hypotonically treated for 20 min at room temperature at a density of 1x105 cells/ml 0.75 mM KCl. Cells were cyto-spun onto glass slides for 10 min at 400 g. Spread chromosomes were immediately fixed in 3.7% formaldehyde/PBS for 15 min, before cells were incubated in KCM buffer  for 20 min. Subsequent immunodetection was performed as described above.Cells were grown on gelatinized glass coverslips, fixed in 4% paraformaldehyde (10 min at room temperature (RT)) and permeabilized for 20 min at RT in 0.5% Triton X-100/PBS. Immunofluorescence staining was performed in 4% BSA/PBS for 1 h at RT (primary antibodies) and in 4% BSA/PBS for 45 min at RT (secondary antibodies). The following primary antibodies were used: rabbit anti-H3K9ac , rabbit anti-H4K8ac  rabbit anti-H3K4me2 , rabbit anti-H3K27me3  and rabbit anti-H3K9me3 , mouse anti-H3K9me3 , mouse anti-5mC . Secondary antibodies were goat anti-rabbit-IgG Alexa Fluor 488 , donkey anti-mouse IgG-Cy3 , donkey anti-rabbit IgG-Cy5  or donkey anti-mouse IgG-Cy5 . Nuclear DNA was visualized by 4,6-diamidino-2-phenylindole . Cells were mounted in Vectashield antifade . For detection of 5mC modifications in nuclear DNA, the protocol described for 5hmC detection in Ludwig et al.  was used earlier . A paintMicrotus cabrerae cells were transiently transfected with CFP-PCNA and were pulse labeled for 20 min with 10 µM 5′-ethynyl-2′-deoxyuridine (EdU)  followed by a 1-h chase prior fixation with 4% paraformaldehyde. EdU was detected with the ClickIT system (Invitrogen) and AlexaFluor 488 followed by an immunostaining with rabbit anti-H3K27me3  and with mouse anti-H3K9me3 . The following secondary antibodies were used: donkey anti-mouse IgG-Cy3  and donkey anti-rabbit IgG-Cy5 . DNA was counterstained with 1 µg/ml DAPI for 10 min at RT, and cells were mounted afterward in Vectashield antifade (Vector Laboratories)  to label nascent RNA . Subsequhttp://rsb.info.nih.gov/ij/). For live-cell microscopy, transfected female Microtus cabrerae cells were plated on a glass bottom p35 dish and were grown and HDACi treated under standard conditions. Time-lapse experiments were carried out in a closed live-cell microscopy chamber  heated to 37 °C, with 5% CO2 and 60% air humidity. Stacks were acquired at 20-min intervals and taken with a CCD camera. Immunofluorescence images of fixed cells were also captured with an Axiovert 200 microscope  with a 63x/1.4 NA Plan-Achromatic oil objective lens . Grayscale images were pseudocolored and merged using ImageJ. For nucleotide incorporation rate measurements and DNA content analysis, cells were imaged using the Operetta High Content imaging system , equipped with a 40×/0.95 NA air objective.Confocal images were obtained using an UltraVIEW VoX spinning disk system  on a Nikon Ti microscope equipped with an oil immersion Plan-Apochromat x60/1.45 NA objective lens . Excitation was done using the following laser lines: 405, 488, 561 and 640 nm. Images were taken with the appropriate filters for the respective dyes: DAPI: emission wavelength (em): 415–475 nm; GFP: em: 505–549 nm; RFP: em: 580–650 nm and Cy5: em: 664–754 nm. RGB stacks and montages were created using ImageJ (http://code.google.com/p/priithon/). Images were processed using a 3D median filter. Filtered images were threshold and then used to calculate the mean intensity of DAPI compaction, acetylation and methylation level. Analysis of total S, S substage and X*/X replication duration was performed by counting live-cell data time points and categorizing cells into early, mid- or late S-phase stage according to their PCNA pattern. An early S-phase pattern is characterized by DNA replication foci distributed homogenously throughout the nucleus with exclusion of the nucle(ol)ar periphery, whereas a mid-S-phase pattern is identifiable by more organized foci at the nucle(ol)ar periphery and the replication of the H3K27me3/macroH2A enriched X chromosome(s). Late S-phase is clearly recognizable due to the appearance of larger DNA replication foci of the H3K9me3/HP1 beta-enriched X chromosome(s) , the mean values of acetylation were measured in the whole nucleus, at the sex chromosomes and in the whole nucleus excluding the X chromosomes . Whereas EdU shows the synthesized DNA (incorporated nucleotide), PCNA represents the replication machinery and, thus, active replisomes. For the calculation of nucleotide incorporation rate, the ratio of EdU (incorporated nucleotides) to PCNA (replication machinery) was estimated, as a proxy for the fork speed  were given to the cells for 10 min prior to fixation. EdU was detected with the ClickIT system (Invitrogen) followed by an immunostaining with mouse anti-PCNA  and detection with donkey anti-mouse IgG-Cy3 . DNA was counterstained with 1 µg/ml DAPI for 10 min at RT and cells were afterward mounted in Vectashield antifade (Vector Laboratories). Detection thereof and fluorescence microscopy allowed the identification of early, mid- and late DNA replication patterns. Microtus cabrerae cells were imaged using the Operetta High Content imaging system . For imaging constant exposure times and appropriate filter sets  were used. Cell segmentation and quantification of nuclear intensities were performed using Harmony . Subsequently, cells were manually staged for early, mid- or late S-phase based on their PCNA pattern. Based on this classification, the integrated DNA intensity (DAPI) per cell nucleus was plotted for all cells from each replicate  Prominent chromatin marks were analyzed by immunostaining: H3K4me3, H3K9ac, H4K8ac (euchromatin), HP1 alpha (constitutive heterochromatin), MBD (DNA methylation) and 5-methyl-cytosine (5mC). Since 5mC staining was barely detectable in Microtus cabrerae cells, 5mC staining is also shown in MEF W8 cells where prominent staining at pericentromeric heterochromatin blocks is visible. DAPI-stained DNA (blue), immunostaining (red) and merge of both channels are depicted. A ROI around one heterochromatic block was selected, and a line intensity plot analysis was performed. Red arrows point to characteristic hypoacetylation of heterochromatic blocks. Scale bar 5 µm. (B) Transcriptional activity of heterochromatic blocks was assessed by the colocalization of nascent transcripts (10 minutes pulse of 1 mM EU) with the H3K9me3 and H3K27me3 enriched heterochromatin blocks. Quantification of EU signal within the two respective volumes after 3D segmentation showed significant higher levels in H3K9me3 blocks .Additional file 2: Movie 1. Time-lapse analysis of female Microtus cabrerae cells throughout S-phase. Cells were triple transfected with CFP-PCNA (red), GFP-macroH2A1 (green) and DsRed-HP1 beta (blue) and imaged every 20 minutes. For details, see Figure 2. Scale bar 5 μm.Additional file 3. Relevant parts of expression constructs used in this study. Schematic representation of relevant features of plasmids used in this study. Plasmid collection number (pc…), structure of the plasmid and, on the right-hand side, the corresponding reference of the plasmids are shown. Drawings are not scaled.Additional file 4. Manipulation of heterochromatic blocks’ constitution by HDAC inhibitor. (A) Schematic representation of the experimental setup to manipulate the heterochromatic blocks’ constitution of Microtus cabrerae cells by HDACi LBH-589. LBH-589 was expected to increase histone acetylation level and potentially lead to decondensation and a potential effect on DNA replication timing. Cells were either seeded or transfected with the corresponding constructs  and incubated for 24 hours. 50 nM LBH-589 was added to the medium, and cells were again incubated for 24 hours. Control cells were treated with DMSO only. Afterward, cells were either fixed and subjected to immunostaining or used for live-cell imaging with a spinning disk confocal microscope. (B) Untreated and HDACi-treated female Microtus cabrerae fibroblasts were analyzed with a user-independent analysis, and the histone acetylation level in the whole cell nucleus was measured. Bar graphs of the mean acetylation level (red bar) in untreated cells and HDACi-treated cells. Sample sizes are indicated in the bars. Gray bars demonstrate the normalized control. Statistical significance was tested using the t-test, comparing untreated and HDACi-treated cells. Error bars demonstrate 95 Cl. ***P < 0.001.Additional file 5. Schematic rationale of single steps for mask generation used for quantification of nuclear PTM levels in untreated and treated/targeted cells. Confocal images were obtained using an UltraVIEW VoX spinning disk system  on a Nikon Ti microscope equipped with an oil immersion Plan-Apochromat x60/1.45 numeric aperture objective lens . For the calculation of mean DAPI and mean PTM intensities  in the whole nucleus, at the heterochromatic block at the X chromosomes or in the whole nucleus excluding the X, mid-nuclear sections of the DAPI and GFP channel were used to generate nuclear, X and exclusion masks, respectively. Images were processed using a median 3D filter and were threshold in four successive steps. For the generation of the binary masks, all pixels below the final threshold were set to 1, for both masks, respectively. Total PTM level values overlapping with the respective mask were calculated and divided by the total number of pixels corresponding to the area of measurement. To automate this analysis procedure, a routine was written in the programming language Python (https://code.google.com/archive/p/priithon/). Mean values were measured and normalized to either untreated or untargeted samples.Additional file 6. Titration analysis of potential HDAC inhibitors. (A) Overview of different HDAC classes and corresponding HDAC inhibitors of each class. MS-275 only affects HDAC1, HDAC2 and HDAC3 of class I (orange), whereas TSA and LBH-589 inhibit HDACs of class I, II and IV (blue/red). (B) Titration analysis of histone hyperacetylation in response to different HDAC inhibitors in female Microtus cabrerae cells. TSA treatment was performed over three days (72 hours). Previous studies have shown that a concentration of 20 nM was sufficient to hyperacetylate very condensed constitutive heterochromatin in C2C12 mouse cells [Microtus cabrerae cells. Over time and at higher concentrations of TSA, cells showed morphological changes and with 800 nM cells died. In contrast, treatment with LBH-589 leads to hyperacetylation at sex chromatin already after one day of treatment. We were able to achieve significant increase in histone acetylation levels at the sex chromatin. To ensure a sufficient and stable hyperacetylation in the absence of morphological changes, we treated the cells with 50 nM for one day. MS-275 data are not shown as cells either showed same levels as controls or died directly after treatment. Statistical significance was tested using the t-test, comparing untreated and HDACi-treated cells. Error bars demonstrate 95Cl. ***P < 0.001. (C) Effect of HDACi treatment on histone acetylation level of female Microtus cabrerae cells. Treatment with LBH-589 leads to significantly increased H3K9ac and H4K8 mean acetylation level at sex chromatin. Mean H4K8ac level at untreated samples is significantly higher at the X* in comparison with the X. Statistical significance was tested using the t-test, comparing HDACi-treated cells and untreated cells, as well as mean H4K8ac levels in untreated cells at X* versus X. Error bars demonstrate 95Cl. ***P < 0.001.Additional file 7. HBO1 targeting to the heterochromatic block of the H3K27me3 decorated X chromosome leads to prolongation of its DNA replication duration. (A) Schematic representation of targeting assay to specifically target HBO1 to the heterochromatic block of the H3K27me3 enriched X chromosome. Cells were transiently transfected with GFP-tagged HBO1 and GBP-macroH2A1. Upon co-expression, both counterparts of the targeting assay strongly interact, leading to the targeting of HBO1. Targeting was validated by immunostaining with H3K27me3 antibody as a hallmark of facultative heterochromatin. DNA (blue), GFP-HBO1 (green), H3K27me3 (red) and merge as an overlay of all three channels. Scale bar 5 µm. (B) Site-directed targeting of HBO1 to the H3K27me3-enriched X chromosome leads to a significant prolongation of DNA replication duration of the X*. Specificity of the targeting approach was validated by the fact that there was no significant difference in H3K9me3 decorated X replication duration upon X* targeting.Additional file 8. Demonstration of ratiometric analysis of nucleotide incorporation rate in Microtus cabrerae control cell. Modified nucleotides were added to proliferating populations of Microtus cabrerae cells and were incorporated for 10 minutes. Cells were fixed, and EdU was detected via ClickIT chemistry. PCNA was stained with an antibody. Exemplary images of an early S-, mid-S- and late S-phase cell are shown: EdU (green), PCNA (red) and merge of both channels. Line profiles depict the ratio of EdU  and PCNA . Scale bar 5 µm.Additional file 9. DNA content analysis independent of S-phase pattern staging. The data shown in Figure 7A were binned using the DNA content of the total cell population (A) to test for user-dependent S-phase pattern classification biases. Then, the frequency of the S-phase patterns assigned was plotted per DNA content bin. The early (red), mid (blue) and late (green) S-phase patterns corresponded well to the progression of genomic DNA content increase during S-phase in untreated cells (B). In contrast, in LBH-treated cells the distribution of the early replication pattern occurred within cells with a higher DNA content (red), while the late replication pattern appeared unaffected.Additional file 10. Plot statistics of main figures. Overview of results and statistics of data implemented in the main figures.Additional file 11. Plot statistics of Supplementary Figures. Overview of results and statistics of data implemented in the additional files.