The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention. Histone deacetylases (HDACs) remove acetyl groups from histones and non-histone proteins. Dysregulated acetylation has been attributed to various disease states, including cancer, immunological and neurological diseases. Due to the potential reversibility of epigenetic or post-translational modifications, the pharmacological targeting of HDACs has high potential. However, most HDAC inhibitors used for clinical trials lack specificity which might contribute to side effects. Despite many years of intense research, surprisingly little is known about the substrate specificity of individual HDAC enzymes. To elucidate the consequences of more specific targeting of individual HDAC isoforms, detailed analysis of the catalytic function of each enzyme is required. Albeit HDACs being well studied in general, cross-comparison between the studies is difficult due to the use of different model systems and the frequent use of inhibitors, which lack specificity. Also, commonly used knockout or knockdown models abolish the structural function of the enzymes, potentially not accurately reflecting the situation of inhibitor treatment which only causes enzymatic inactivation. We suggest that the use of catalytic inactive mutants instead of knockouts might be another step towards mimicking isoform specific enzyme inhibition and provide a detailed comparative, side-by-side analysis of class I HDACs. Following incubation for 1 hour  or 5 hours (immunoprecipitation samples) on a thermomixer , 35 μl of Histone Stop Solution  and 800 μl of ethyl acetate were added. After vortexing for 15 sec, the samples were centrifuged with a swing out bucket centrifuge . 600 μl of the organic phase were mixed with 3 ml of Scintillation Solution . Counts per minute (CPM) were determined with a Liquid Scintillation Analyzer (Packard).The deacetylase assay was conducted on 20 μg of total cellular extract or on beads carrying immunoprecipitated proteins in 20 μl total volume as described previously ,66. 4 μlThree biological replicates of each HAP1 cell line and wildtype cells treated with MS-275 (3 μM for 6 or 24 hours) were prepared. Cells were washed twice in 1x PBS, supplemented with 10 mM sodium butyrate  and 3 mM nicotinamide (NAM) (72340 Sigma-Aldrich), and pelleted prior to lysis.7 cells per replicate were prepared in lysis buffer containing 8 M urea, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM PMSF, 5 mM sodium butyrate, 10 ng/ml NAM, 10 ng/ml trichostatin A (TSA) and 1x cOmplete protease inhibitor cocktail (+EDTA). 250 U of benzonase (Merck) were added to each sample. Lysis was achieved using a Bioruptor sonication device (Diagenode) applying 5 alternating cycles of 30 sec sonication and 30 sec cooling. Power level was set to H. Following lysis, cells were centrifuged at 15,000 rcf for 10 min at 4°C to remove insoluble material. The protein fraction was precipitated by addition of 4x volumes of cold (-20°C) 100% acetone and 4°C incubation overnight. The protein pellet was washed with cold (- 20°C) 80% acetone, air dried for 5 min, and resolved in 8 M urea, 50 mM ammonium bicarbonate buffer (ABC). Protein concentration was determined using the Pierce 660 nm Protein Assay (Thermo Scientific). Proteins were reduced with 10 mM DTT for 45 min at RT, and subsequently carbamidomethylated with 20 mM iodoacetamide for 30 min at RT in the dark. The alkylation reaction was quenched by adding 5 mM DTT for 10 min. Prior to digestion, the urea concentration was reduced to 4 M with 50 mM ABC. Samples were predigested for 90 min at 37°C with Lys-C (Wako Laboratories) at an enzyme-to-substrate ratio of 1:100. Samples were digested overnight at 37°C with sequencing grade trypsin (Promega) at an enzyme-to-substrate ratio of 1:50. Following digestion, samples were acidified using 10% TFA (trifluoroacetic acid) (Sigma-Aldrich) to a final concentration of 0.5%, and desalted on a 50 mg tC18 Sep-Pak cartridge (Waters). Peptide concentrations were determined and adjusted according to UV chromatogram peaks obtained with an UltiMate 3000 Dual LC nano-HPLC System , equipped with a monolithic C18 column (Phenomenex). Desalted samples were dried for 30 min in a SpeedVac concentrator and subsequently lyophilized overnight.2x10300 μg of trypsin-digested and desalted peptides were used for each sample for isobaric labeling. Lyophilized peptides were dissolved in 20 μl 100 mM triethylammonium bicarbonate (TEAB) (Sigma). 20 μg of peptides in TEAB from each sample and replicate were additionally mixed  as reference sample (REF). 500 μg of each TMTpro16plex reagent (Thermo Fisher Scientific) were dissolved in 30 μl of 100% anhydrous acetonitrile and added to the peptide/TEAB mixes. The REF sample was mixed with 1.5 mg (90 μl) of TMTpro16plex reagent 134N and subsequently split in 3 aliquots. Labels used: 126C: wildtype; 127C: MS-275 6 hour; 128C: MS-275 24 hour; 129C: WT HDAC1 CI; 130C: WT HDAC2 CI; 131N: HDAC1 KO; 131C: WT HDAC3 CI; 132N: HDAC2 KO; 132C: WT HDAC8 CI; 133N: HDAC3 KO; 133C: REF; 134N: HDAC8 KO. Samples were labeled for 60 min at RT. 0.1 μl of each sample were pooled, mixed with 10 μl 0.1% TFA and analyzed by mass spectrometry (MS) to check labeling efficiency. For quenching, 5 μl of 5% hydroxylamine  were added and the reaction was incubated for 25 min at RT. Samples were pooled and subsequently desalted with Sep-Pak tC-18 (200 mg) cartridges (Waters). Desalted samples were dried for 30 min in a SpeedVac vacuum centrifuge and subsequently lyophilized overnight.−1. 45 fractions were collected and subsequently pooled in a noncontiguous manner into 8 pools. For example, to generate the first pool, original fractions 1, 9, 17, 25, 33 and 41 were combined. Pools were dried for 30 min using a SpeedVac concentrator and subsequently lyophilized overnight.Peptides were dissolved in 80 μl 10 mM ammonium formate, pH 6.8 and separated on a UltiMate 3000 Dual LC nano-HPLC System  equipped with a XBridge Peptide BEH preparative C18  column (Waters). A maximum of 5 mg was loaded. Peptides were separated using a 60 min gradient of 5 to 50% acetonitrile in 10 mM ammonium formate with a flow rate of 1.0 ml minAcetylated peptides were enriched using the PTMScan Acetyl-Lysine Motif Kit . Each lyophilized peptide pool was dissolved in 175 μl of 1x PTMScan IAP buffer. Aliquots of roughly 10 μg of peptide/pool were removed for proteome analysis. Antibody beads  multiplex setup (~4.8 mg of peptide)) were washed four times with 1x PBS and split into eight aliquots. Peptides resuspended in 1x IAP were added and the mix was incubated for 2 hours at 4°C with end-over-end rotation. Following enrichment, PTMScan antibody beads were washed twice in 1x IAP and subsequently three times with high pressure liquid chromatography (HPLC)-grade water . Peptides were eluted twice from antibody beads with 20 μl of 0.15% TFA, respectively. Additional 5 μl of 0.15% TFA were used to wash out residual peptides. Eluates were united and desalted using two C18 StageTips per pool as described in . Enriche6, and maximum injection time (IT) 50 ms. The top 20 precursors were selected for MS2 analysis (HCD) with the following parameters: resolution 45,000, AGC 2×105, maximum IT 96 ms (proteome) or 200 ms (acetylome), isolation window 0.7 m/z, and normalized collision energy (NCE) 35. The minimum AGC target was set at 8×103 (acetylome) or 1×104 (proteome), which corresponds to an intensity threshold of 4×104 or 1×105, respectively. In addition, unassigned, singly and >6+ charged species were excluded from MS2 analysis and dynamic exclusion was set to 20 or 30 sec, respectively.LC-MS/MS analysis was performed on an UltiMate 3000 Dual LC nano-HPLC System , containing both a trapping column for peptide concentration  and an analytical column , coupled to a Q Exactive HF Orbitrap  mass spectrometer (Thermo Fisher) via a Proxeon nanospray flex ion source (Thermo Fisher). For peptide separation on the HPLC, the concentration of organic solvent (acetonitrile) was increased from 1.6% to 32% in 0.1% formic acid at a flow rate of 230 nl/min, using 2 hours gradient time for acetylome analysis and 3 hours gradient time for proteome analysis. The instruments were operated in data-dependent acquisition (DDA) mode with dynamic exclusion enabled. MS1 survey scans were recorded with the following parameters: resolution 120,000, scan range 375–1,500 m/z, automatic gain control (AGC) target 3×10https://www.ebi.ac.uk/pride/archive/projects/PXD030164).Raw MS data were analyzed using MaxQuant  softwareS1 Material). The analysis procedure covered: correction for isotopic impurities of labels, within-plex median normalization, between-plex IRS-normalization [Data were analyzed in R (4.1.0) using custom scripts  were additionally selected. Peptide selection was performed using Skyline . Sampleshttps://panoramaweb.org/edvjs1.url, PXD030272).Data of the targeted approach have been deposited to the PanoramaWeb  (https:/Heatmaps were created using python packages pyplot (Matplotlib), pandas, numPy and seaborn. GraphPad Prism version 5.00 for Windows  was used to generate bar graphs and growth curves and to determine IC50 values of drugs or drug combinations for SCC cell lines as described . In caseS1A Fig). Immunoprecipitated HDAC1, HDAC2 and HDAC3 displayed robust deacetylase activity, while HDAC8 was not active in this assay. The latter result is consistent with a previous report showing that HDAC8 prefers long chain fatty acyl lysines over acetylated lysine as substrate [Fig 1), we wanted to determine if the specific catalytic inactivation of HDAC1, HDAC2 and HDAC3 enzymes causes different effects on cellular functions and reflects the effects of HDACi treatment. In detail, we performed a full comprehensive approach, including global lysine acetylome profiling and gene expression analysis, to compare inactive mutant expressing cells and cells treated with the class I HDAC is MS-275 and JQ12. The class I HDACi entinostat (MS-275) is a synthetic benzamide derivative with preference for HDAC1 and HDAC3 enzymes undergoing clinical trials for several types of cancer [To systematically address the catalytic functions of class I HDACs, we employed the nearly haploid human tumor cell line HAP1 ,74 as moubstrate . Based of cancer –78. JQ12f cancer ,79.AAVS1 safe harbor locus . In our established cell lines, the CI mutants were either expressed in the presence or absence of endogenous wildtype enzyme  . We hypothesized that these two scenarios would mimic partial or full inhibition of the respective HDAC.The inactivation of individual HDAC enzymes based on the mutation of a single conserved histidine residue in the catalytic center to alanine  ,80,81. WS1C Fig). In addition, we compared the total cellular histone deacetylase activities of cells expressing the HDAC1/2/3 CI mutants to wildtype control cells and HDAC1/2/3 KO cells. Cells expressing HDAC1 CI in both wildtype or KO background showed a trend towards lower cellular activity, while expression of HDAC2 CI led to a significant reduction of total cellular histone deacetylase activity . In contrast, HDAC3 inactivation or knockout of HDAC1, HDAC2 or HDAC3 had no effect. It was shown previously that expression of HDAC1 CI or HDAC2 CI causes dominant negative effects, potentially due to their incorporation into HDAC complexes [Using cells expressing the transgenes in the respective KO background, we confirmed that the deacetylase activity was highly reduced in the HDAC CI mutants over HDAC WT transgenic control enzymes . Cell viability was significantly reduced upon MS-275 treatment at 1.5 μM or higher concentration, but not upon JQ12 treatment. Next, we analyzed how cell viability is affected upon expression of HDAC1/2/3 CI, either in the presence (WT) or absence (KO) of the respective endogenous wildtype enzyme . The expression of HDAC1 CI and HDAC2 CI in both settings led to a slight but significant reduction. HDAC3 inactivation in the KO background strongly reduced cell viability, while the observed change for HDAC3 CI in WT cells was not significant. Therefore, we tested also the viability of HDAC3 KO cells, which was significantly reduced, confirming the crucial cellular function of HDAC3  [S2A Fig). Given the significant effects by additional HDAC3 ablation, we included HDAC3 KO cells in all further analyses. Since class I HDACs have a role in the maintenance of genome integrity [S2B and S2C Fig). Inactivation or loss of HDAC3 led to a strong increase of γ-H2AX foci compared to wildtype cells. Taken together, catalytic inactivation of HDAC1 and HDAC2 causes a mild decrease in cell viability, whereas loss of HDAC3 and its activity leads to significant DNA damage, apoptosis and strongly reduced cell viability.Class I HDACs have an important role in the control of cell growth and proliferation ,9. To de ,40,82. Cntegrity ,84, we aS1A and S1C Fig and Fig 2B) [Fig 3A and S1 Material). In total, we identified 8,788 proteins and 33,968 acetylation sites, out of which 6,627 proteins and 13,187 acetylation sites could be reliably quantified in all three replicates. Changes in protein lysine acetylation sites were further normalized to the corresponding protein levels and are provided in S1 Table. Acetylation sites showing a fold change of ≥ 1.5 over wildtype cells and a padj-value of ≤ 0.05 were considered as significantly regulated.Previous studies have revealed changes in protein lysine acetylation upon pharmacological inhibition of HDACs ,85,86. H Fig 2B) ,44,87. T Fig 2B)  , which is the expected effect of deacetylase inactivation. Few non-histone targets, such as co-repressor proteins or other chromatin-associated factors, displayed elevated acetylation sites already after 6 hours of MS-275 treatment (22 hyperacetylated proteins) . However, 24 hours treatment evoked a further, large increase in acetylation on histones and non-histone targets (224 hyperacetylated proteins) . Proteins that were hyperacetylated upon MS-275 treatment show a large overlap with previously published HDAC inhibitor targets [Upon MS-275 treatment, the majority of proteins with affected sites (90%) showed an increase in acetylation . Acetylation sites increased upon HDAC1 or HDAC2 inactivation were highly similar to each other and particularly resembled profiles of 6 hour MS-275 treated cells (82% overlap of 6 hour MS-275 treated cells to HDAC1/2 CI cell lines). In contrast, acetylation sites increased by HDAC3 inactivation differed from those affected by HDAC1/2 inactivation and were less responsive to pharmacological HDAC inhibition (32% overlap of 6 hour MS-275 treated cells to HDAC3 CI cell lines). Among the HDAC cell lines, KO HDAC3 CI cells displayed the largest number of significantly hyperacetylated proteins (175), followed by HDAC3 KO cells (104) and only a minor fraction was found to be hyperacetylated in WT HDAC3 CI cells (38). Importantly, we identified confident HDAC1/2- and HDAC3-specific histone and non-histone substrates, which were independent of the level of HDAC inactivation: 15 proteins with increased acetylation sites were overlapping in both HDAC1 CI clones, while 19 and 25 targets were identified in both HDAC2 CI clones and HDAC3 CI clones, respectively. Of the 25 overlapping targets of HDAC3 CI clones, 20 also displayed an overlap with the HDAC3 KO.Inactivation of individual HDACs reflects to large extent the acetylation profiles of pharmacological inactivation . We determined acetylation sites which were elevated upon expression of HDAC1/2 CI mutants (upper panel), inactivation or deletion of HDAC3 (middle panel), or which were regulated by all three class I deacetylases (lower panel). The selected sites were also found hyperacetylated upon MS-275 treatment. HDAC1/2 inactivation evoked stronger effects on histone acetylation sites compared to HDAC3 CI or KO clones.Next we specifically analyzed effects of HDAC1/2/3 inactivation on histone acetylation . 2 hours of MS-275 treatment already caused a slight, but significant increase of few acetylation sites such as K9ac and K14ac on histone H3, and K5ac, K8ac, K12ac and K16ac on histone H4. Following a gradual increase after 6 hours of treatment, some acetylated peptides were elevated 20-90-fold at the 24 hours time point. We observed again that the majority of detected peptides were multi-acetylated. MS-275 responsive acetylation sites identified by the targeted approach ( and S2 Table) were in accordance with the results from the initial shotgun experiment (summarized in  and listed in S1 Table).To expand our findings on histone acetylation and to additionally get insight into the dynamics, we performed a targeted proteomics approach based on PRM, using cells that were treated for 2–24 hours with MS-275 . We determined the top deacetylation targets of HDAC1/2 (upper panel) and HDAC3 (lower panel) and grouped them according to their biological functions.We further examined changes in acetylation patterns of non-histone proteins .Upon inactivation of HDAC1 or HDAC2, we identified hyperacetylated sites on multiple components of HDAC1/2 associated co-repressor complexes, such as MTA2 and CHD4 of the NURD complex, COREST (RCOR1) and RCOR3 of the COREST complex, ELMSAN1 of the MIDAC complex and RERE, which acts as scaffold for several HDAC complexes , 90. Ace and S1 Table). In addition, acetylated lysine residues within components of HAT (histone acetyltransferase) complexes, such as MEAF6, the bromodomain containing reader proteins BRPF1, BRPF3, and HAT enzymes  [Interestingly, our global acetylation analysis further revealed that one particular acetylation site (K412) within the HDAC1 enzyme itself was elevated upon MS-275 treatment and upon expression of inactive HDAC1, but not HDAC2 or HDAC3 ( CREBBP)  were pre and S1 Table), we identified elevated acetylation sites on components of HDAC3-associated NCOR/SMRT nuclear receptor corepressor complexes, including NCOR1, NCOR2 and TBL1XR1 [Upon inactivation and/or deletion of HDAC3 . To compare changes in gene expression resulting from genetic inactivation to effects caused by pharmacological inactivation, we analyzed cells with partially of fully inactivated HDAC1/2/3 enzymes and cells treated with the HDACis JQ12 or MS-275. Due to the strong effects caused by HDAC3 deletion in previous parts of the study, we again included HDAC3 KO cells. A sample distance heatmap based on the Euclidean distance  and a principal component analysis  show that the three biological replicates for each cell line or treatment were in good agreement with each other, confirming the technical quality of the data set. HDAC3 cell lines, including WT HDAC3 CI, KO HDAC3 CI and HDAC3 KO cells, are highly similar to each other. They differ considerably from cells expressing inactive HDAC1/2 transgenes, indicating distinct functions in gene expression regulation. Cell lines expressing HDAC1/2 CI enzymes in KO as well as in WT background each form neighboring clusters . This confirms the high degree of overlapping functions of HDAC1 and HDAC2 enzymes as orthologous genetic alterations cause similar effects on gene expression. In addition, cells treated with the HDACis MS-275 and JQ12 are more similar to HDAC1/2 cell lines than to HDAC3 cell lines. The high similarity of HDAC1/2 CI expressing cells to each other and their distinction from cells with inactivated and/or ablated HDAC3 enzyme resembles the findings of the acetylome study.Increased acetylation sites on histones and chromatin-modifying enzymes might impact the transcriptional landscapes of the individual HDAC1/2/3 cell lines. Therefore, we performed RNA-seq analysis . Next, we assessed the GO enrichment for upregulated genes upon expression of the inactive HDAC enzymes. Therefore, we focused on the intersection of genes significantly increased upon both partial and full inactivation. In particular, upon expression of inactive HDAC2, and also HDAC1, we found GO terms related to neuronal functions enriched , similar to observations for HDACi-treated cells.To get an overview of altered biological processes upon HDAC1/2/3 inactivation, we performed gene ontology (GO) term enrichment analysis of significantly upregulated genes . Thereby, pharmacological HDAC inactivation by MS-275 and JQ12 particularly increased the expression of genes associated with the nervous system . Interestingly, the COREST complex is known for its repressive function on neuronal-specific genes in non-neuronal cells, mediated via specific binding through the associated transcription factor REST [. Other HDAC1/2 CI cell lines showed a similar effect using the same analysis  = 1.25, FDR = 0.111; WT HDAC1 CI: NES = 1.23, FDR = 0.164; KO HDAC1 CI: NES = 1.10, FDR = 0.267). In line with this, we also observed a significant decrease of COREST-associated deacetylase activity upon expression of inactive HDAC1/2 .Notably, our acetylome analysis has revealed that members of individual HDAC1/2 complexes, such as the COREST complex components COREST (RCOR1) and its less well characterized paralogue RCOR3 , displaytor REST ,105. Gen), we next investigated whether genes regulated by nuclear receptors are enriched upon HDAC3 deletion/inactivation. Expression profiles of HDAC3 KO cells and cells treated with MS-275 displayed a significant enrichment of genes involved in the nuclear receptor metapathway  and expression of inactive HDAC3 caused similar effects .Given that HDAC3 associated complexes function in nuclear receptor-mediated transcriptional repression , and thaIn summary, these findings suggest that changes in gene expression profiles caused by HDAC1/2 and HDAC3 inactivation/deletion are connected with the annotated function of their non-histone deacetylation targets.ex vivo chemosensitivities in tumor biopsies in the context of the clinical trial EXACT  [Fig 6A). The HDACi 4SC-202, which showed no effect in the pilot experiment, was included as control. We included HAP1 wildtype cells, next to cell lines with partial of full inactivation of HDAC1/2/3 and cells pre-treated with MS-275 or JQ12 before addition of the tumor drugs. Following 72 hours of treatment with the different doses, the viability of the cells was determined. Pre-treatment with the HDACis or inactivation of HDAC1 or HDAC2 sensitized HAP1 cells to decitabine and similar trends were observed for alisertib and axitinib . Cells expressing inactive HDAC3 in the KO background, as well as HDAC3 KO cells were slightly more sensitive towards axitinib. None of the transgenic HDACs or treatments sensitized the cells to the negative control 4SC-202.Global acetylome and transcriptome analysis suggest that genetic inactivation of individual HDACs effectively mimics pharmacological inactivation. Based on the fact that HDACis can have synergistic effects with tumor drugs , we testeatment) ,107. BasFig 6B). Class I HDAC inhibition sensitized all cell lines to the three tumor drugs, although the effect on UM-SCC104 was mild. This cell line with episomal HPV 16 expresses high levels of the viral oncoprotein E7 [To extend our analysis to more relevant tumor cell lines, we examined the sensitizing effect of class I HDAC inactivation in HNSCC (head and neck squamous cell carcinoma) cell lines. We used the human papillomavirus (HPV)-positive head and neck cancer cell lines UPCI-SSC152 and UM-SCC104 , in addiotein E7 , which motein E7 –112 and otein E7 ,113,114.Herein, we describe consequences of loss-of-activity mutations of the class I HDACs HDAC1, HDAC2 and HDAC3 in comparison with class I HDAC inhibitors. Our detailed approach suggests that the expression of particular catalytically inactive mutants reflects inhibition of the enzyme, allowing to identify isoform-dependent catalytic functions.HDAC inhibitor treatment affects tumor cell viability by induction of cell death and inhibition of proliferation . InactivFigs 4). The catalytic activity of all three enzymes seems essential for deacetylating histone sites, with HDAC1/2 exerting the strongest effects. This is in agreement with the higher associated deacetylase activity of precipitated HDAC1/2 towards acetylated histones compared to HDAC3 enzyme , negative effects of inactive HDAC2 on total cellular histone deacetylase activity  and significant effects of HDAC1/2 deletion on total cellular activity [Fig 4A). Interestingly, many of the preferred sites of HDAC1/2 correspond to linker histone H1 and variants of histones H2A and H2B. Increased acetylation of these sites was also observed upon pharmacological HDAC inhibition in our study and previous studies [In addition to cell-based assays, we integrated multi-OMICS approaches to determine effects of HDAC1/2/3 inactivation. We examined differences in cellular acetylomes upon treatment with MS-275 or expression of particular inactive HDAC1/2/3 isoforms . HDAC1/2 catalytic inactivation resulted in increased acetylation sites of numerous components of HDAC1/2 associated co-repressor complexes, reader proteins and transcription factors. Notably, many of these chromatin associated factors were already hyperacetylated upon 6 hours of MS-275 treatment, suggesting that acetylation of proteins closely interacting with HDACs is highly dynamic.Importantly, we unraveled multiple non-histone deacetylation substrates of individual HDAC1/2/3 enzymes . This suggests that specific class I HDACs might affect the cellular function of HATs and vice versa.In this context it is remarkable that multiple histone acetyltransferases (HATs) and HDAC1 itself are potential HDAC1/2 targets, suggesting a cross-talk between these antagonistic enzymes. We detected multiple increased acetylation sites within the CREBBP (CBP) protein upon HDAC1/2 inactivation or MS-275 treatment. Interestingly, the corresponding sites are part of an autoregulatory loop of the enzyme ,124. Accg 4B and ), and geg 4B and  and thisFig 4B). For example, ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, as well as the H3K9 demethylase JMJD1C have been identified as interactors of nuclear hormone receptors [Upon inactivation or deletion of HDAC3, we detected hyperacetylated sites on components of NCOR/SMRT complexes ,131, in eceptors –95. Botheceptors ,133. ACTeceptors ,134,135.via several nuclear receptors including the retinoic acid receptor and thyroid hormone receptor [The proto-oncogene SKI, by interactions with HDAC3 containing NCOR/SMRT complexes, is also involved in the transcriptional regulation receptor ,136–138.receptor ,97–100. receptor , which areceptor ,141, whireceptor ,102.Altogether, our acetylome analysis revealed novel HDAC1/2/3 deacetylation substrates that differ in their cellular function. HDAC3 cellular targets were more diverse while HDAC1/2 inactivation primarily induced hyperacetylation on histones and chromatin-associated non-histone substrates. Importantly, many increased acetylation sites detected upon HDAC1/2 inactivation were also hyperacetylated upon MS-275 treatment, suggesting that HDAC1/2 CI mutants effectively mimic pharmacological HDAC inhibition.Figs S4) furthermore revealed that gene sets related to similar biological processes are derepressed by both HDAC inhibitor treatment and by genetic HDAC inactivation. Similar to the acetylome analysis, different processes were affected by HDAC1/2 and HDAC3 inactivation suggesting a division of labor among class I HDACs. Importantly, gene sets that were overrepresented in the individual HDAC cell lines are regulated by factors, which were identified as hyperacetylated upon HDAC1/2 or HDAC3 inactivation. Future experiments will be required to evaluate whether the reversible acetylation of these factors has functional consequences. RCOR1 (COREST) and also its homologue RCOR3, were among the top targets of HDAC1/2 deacetylase activity  and are components of the HDAC1/2 associated COREST complex, a well-known transcriptional regulator of neuronal genes [Figs S4C). The derepression of neuronal genes upon pharmacological HDAC inhibition is consistent with previous work [S5A and S5B Fig), REST target genes were enriched in expression profiles of HDAC1/2 CI expressing cells and MS-275 treated cells . Notably, the strongest effects were observed upon inactivation of HDAC2, which is in accordance with the previously reported importance of HDAC2 for function of the COREST complex [Our transcriptome analysis . Importantly, they function by tethering HDAC3 to transcriptional repressors including unliganded nuclear receptors [Fig 5C).Our acetylome analysis revealed a strong link between HDAC3 and annotated nuclear receptor binding-associated proteins. Upon inactivation/deletion of HDAC3, NCOR1 and NCOR2, components of HDAC3 associated NCOR/SMRT complexes, displayed the highest increase of acetylated sites . Using a panel of clinically approved cancer drugs we tested whether the inactivation or deletion of individual HDAC enzymes can sensitize cells to these drugs . We identified the DNMT inhibitor decitabine, the Aurora A kinase inhibitor alisertib and the tyrosine kinase inhibitor axitinib as synergistic hits for class I HDACs. In all cases, the expression of either inactive HDAC1 or HDAC2 was sufficient to sensitize cells for tumor drug treatment. In contrast, the inactivation of HDAC3 showed no consistent effects. In addition, especially in case of decitabine treatment, HAP1 cells were also sensitized upon treatment with JQ12 or MS-275. These findings were confirmed in several HNSCC tumor cell lines . The DNMT inhibitor decitabine is an FDA-approved effective chemotherapeutic agent for acute myelogenous leukaemia with anti-cancer effects also in head and neck cancer cells [Finally, we showed that our novel HDAC model is suitable for chemical screens (er cells . Aliserter cells . Axitinier cells ). Of noter cells ,156–158)er cells ,113,114.Throughout our comprehensive analyses, results obtained by inactivation of HDAC1 and HDAC2, and to a lesser extent HDAC3, overlap with those obtained by HDAC inhibitor treatment. Of note, the observed effects for the inactivation of each of the analyzed HDACs was very similar in wildtype and KO background and inactivation of HDAC1 and HDAC2 caused overlapping effects. These results are compatible with the idea that these mutants mimic isoform-specific pharmacological inhibition. Consistent with these data, expression of inactive HDAC1 or HDAC2 evoked an isoform-specific effect on mouse development . Due to In line with a unique cellular function, deletion of HDAC3 has a strong effect on cell viability, the acetylome and the transcriptome. Mouse studies have identified an activity-independent function of HDAC3 in transcriptional regulation in the liver  and duriS1 Fig(A) Immunoprecipitation analysis of endogenous HDAC1, HDAC2, HDAC3 and HDAC8 enzymes from cellular extracts of HAP1 wildtype (WT) cells. Cells with a knockout (KO) of the respective HDACs were used as negative controls. Immunoprecipitates were examined by Western blot analysis using specific antibodies against the enzymes (upper panel) and HDAC activity assays (lower panel). (B) Illustration of the targeting strategy to establish transgenic HDAC1/2/3 CI cell lines. HDAC transgenes (red) provided by the pVJV vector, were integrated into the AAVS1 locus within the HAP1 genome (blue) via CRISPR/Cas9 technology and homologous recombination . Transgenes were expressed under control of the EF-1α promoter (brown). (C) Pair-wise immunoprecipitation of comparable amounts of FLAG-tagged HDAC1/2/3 WT and CI enzymes to assess their deacetylase activity. FLAG immunoprecipitates were analyzed on Western blots (upper panel) using the FLAG antibody. Precipitates of wildtype HAP1 cells are shown as negative control. Associated deacetylase activities of HDAC WT and CI enzymes are presented below. (A+C) Bar graphs represent mean values ± standard deviation (SD) of 4 (A) or 3 (C) biological replicates and the significance was determined by Welch‘s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.(TIF)Click here for additional data file.S2 Fig(A) Bar graph representing the percentage of apoptotic cells of cultured cell lines, including wildtype cells, HDAC1/2/3 CI expressing cells (wildtype and knockout background) and HDAC3 KO cells. Apoptotic cells were determined by flow cytometry based on signals for cleaved caspase 3. (B) Representative immunofluorescence images of wildtype control cells and cells with inactivated/deleted HDAC3. DNA damage was analyzed with an antibody specific for γ-H2AX and DNA was stained with Hoechst dye. (C) Quantification of γ-H2AX foci determined by immunofluorescence analysis from WT HDAC1/2/3 CI cells (left panel), KO HDAC1/2/3 CI cells (middle panel) and HDAC3 KO cells (right panel) next to wildtype cells as control. Mean values ±SD of 3 (A) or 4–6 (C) biological replicates are shown. Significance was determined by one-way ANOVA. The pairwise comparison of HDAC3 KO cells to wildtype cells (in (C), right panel) was performed using Welch‘s t-test. **p < 0.01, ***p < 0.001.(TIF)Click here for additional data file.S3 Fig(A) Dynamics of histone acetylation. A selected set of histone acetyl(K) sites, based on findings from the MS-shotgun experiment (left panel), was further investigated by targeted proteomics to monitor changes after 2, 6 and 24 hours of MS-275 treatment . Increased acetylation is shown as log2 fold change over untreated wildtype cells. Individual hits from histone sub-variants are summarized according to their main types (indicated on the right). Positions of acetyl-lysines in the histone protein are indicated on the left. N-terminal methionines are not considered, to fit the commonly used histone site code. *, **: The information for quantification of this site comes from a *dual or ** triple acetylated peptide where only one site was confidentially allocated (for MS shotgun data only). (B) Assessment of COREST (RCOR1) acetylation by acetyl(K)-IP and subsequent Western blot analysis using untreated or 24 hour MS-275 treated cells. The blot was incubated with indicated antibodies (on the left). Vinculin was used as loading control. n = 2(TIF)Click here for additional data file.S4 Fig(A) Sample distance heatmap of RNA seq replicas from HDAC1/2/3 CI expressing cell lines (wildtype and knockout background), MS-275 or JQ12 treated cells (24 hours) and wildtype cells. The color code is based on the Euclidean distance of read counts after variance stabilizing transformation, while the dendrogram represents a hierarchical cluster analysis with the complete linkage method. (B) Two-dimensional principal component analysis (PCA) of RNA-seq transcriptome profiles of wildtype, MS-275 or JQ12 treated cells, HDAC1/2/3 CI expressing cells (wildtype and knockout background) and HDAC3 KO cells. (C) Enriched gene ontology terms of significantly upregulated genes by JQ12 treatment (upper panel) and HDAC1 inactivation (lower panel) , determined with the Enrichr tool. Only genes elevated in both HDAC1 CI clones (wildtype and knockout background) were considered for analysis.(TIF)Click here for additional data file.S5 Fig(A-B) Analysis of COREST (RCOR1) associated HDAC activity upon incorporation of inactive HDAC1 or HDAC2 enzymes. COREST was immunoprecipitated from extracts of (A) wildtype, WT HDAC1 CI and KO HDAC1 CI cells and of (B) wildtype, WT HDAC2 CI and KO HDAC2 CI cells. Inputs (left panels) and immunoprecipitates (middle panels) were analyzed by Western blotting using antibodies indicated on the left. Bar graphs (right panels) represent changes of COREST associated deacetylase activity, measured by incubating the precipitates with acetylated histones. Mean values ± SD of 3 biological replicates are shown and significance was determined by Welch‘s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.(TIF)Click here for additional data file.S1 TableS1 Material) has been processed using MS-Excel. The table covers information on MS-275 treated wildtype cells, HDAC1-3 catalytic inactive mutants expressed in wildtype and corresponding KOs, and HDAC3KO cells. Results obtained from additional experimental conditions covering HDAC1KO and HDAC2KO cells as well as different HDAC8-related strains are hidden columns BQ-CH).The table includes results from the differential expression (DE) testing analysis of protein-normalized acetyl(K)sites (columns C-AL), protein-normalized TMTpro reporter ion intensities of quantified acetyl(K) sites in different conditions , as well as results from DE-testing of proteinGroups (hidden columns CI-DI), and selected columns from the MQ output tables acetyl(K)sites.txt (hidden columns DJ-EB). To generate the table the output from R-script "acetylKSites_analysis_prot_norm" ((XLSB)Click here for additional data file.S2 TableThis table includes Differential Expression (DE) Testing analysis of acetylated peptides of histones  (columns D-F), corresponding statistical analysis (G-J), summed fragment ion intensities (K-Z) and additional information (AA-AB). The table covers information on MS-275 treated wildtype cells .(XLSB)Click here for additional data file.S3 TableThe different worksheets show results obtained by DESeq2 analysis for JQ12 or MS-275 treated wildtype cells, HDAC1-3 catalytic inactive mutants expressed in wildtype and corresponding KOs, and HDAC3 KO cells. Relative expression was calculated relative to wildtype cells. RNA seq experiments were performed using biological triplicates.(XLSB)Click here for additional data file.S1 Material(ZIP)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


E2F4 contains an evolutionarily conserved Thr-motif that, when phosphorylated, modulates its activity, thus constituting a potential target for intervention. In this study, we generated a knock-in mouse strain with neuronal expression of a mouse E2F4 variant lacking this Thr-motif (E2F4DN), which was mated with 5xFAD mice. Here, we show that neuronal expression of E2F4DN in 5xFAD mice potentiates a transcriptional program consistent with the attenuation of the immune response and brain homeostasis. This correlates with reduced microgliosis and astrogliosis, modulation of amyloid-β peptide proteostasis, and blocking of neuronal tetraploidization. Moreover, E2F4DN prevents cognitive impairment and body weight loss, a known somatic alteration associated with AD. We also show that our finding is significant for AD, since E2F4 is expressed in cortical neurons from Alzheimer patients in association with Thr-specific phosphorylation, as evidenced by an anti-E2F4/anti-phosphoThr proximity ligation assay. We propose E2F4DN-based gene therapy as a promising multifactorial approach against AD.Alzheimer’s disease (AD) has a complex etiology, which requires a multifactorial approach for an efficient treatment. We have focused on E2 factor 4 (E2F4), a transcription factor that regulates cell quiescence and tissue homeostasis, controls gene networks affected in AD, and is upregulated in the brains of Alzheimer’s patients and of APPThe online version contains supplementary material available at 10.1007/s12035-022-02764-z. Alzheimer’s disease (AD) is characterized by progressive neurodegeneration that leads to cognitive impairment and eventually to dementia, in association with somatic alterations that include body weight loss . Two maiA potential multifactorial target for AD is E2 factor 4 (E2F4), a transcription factor proposed as a major regulator of most AD-specific gene networks . Other eMAPK [swe/presenilin 1 (PS1)dE9 (APP/PS1) mice, a known AD mouse model, in association with phosphoThr immunoreactivity [E2F4 is a phosphoprotein , and preMAPK , a majorMAPK . In the MAPK . AccordiMAPK . E2F4 exactivity . Remarkaactivity  and in hactivity  as well activity , anotheractivity .MAPK activity, while a dominant negative form of chick E2F4 (E2F4DN) containing Thr261Ala/Thr263Ala substitutions blocks NGF-induced cell cycle re-entry in these cells [MAPK of the Thr conserved motif alters the normal functioning of E2F4 as a quiescent regulator, a process that could participate in the etiology of AD, given that p38MAPK is activated in AD-affected neurons [MAPK expressing cells is expected to conserve E2F4 functionality. The p38MAPK-dependent phosphorylation of E2F4 may also alter other homeostatic processes regulated by this transcription factor, and if this is the case, then, E2F4DN may well be a potential therapeutic tool for AD.We have demonstrated that a phosphomimetic form of chick E2F4 with Thr261Glu/Thr263Glu mutations leads to cell cycle re-entry in differentiating chick neurons that lack p38se cells . This in neurons . TherefoIn this study, we generated a knock-in mouse strain expressing mouse E2F4DN in neurons, which were mated to 5xFAD mice. We show that neuron-specific expression of E2F4DN in 5xFAD mice prevented NT and induced a transcriptional program that includes markers of synapse formation, improved glucose metabolism and vascular integrity, and decreased oxidative stress, glycophagy, and cell starvation. This program is also compatible with the attenuation of the immune response and of Aβ processing, accumulation, and toxicity. Consistently, both microgliosis and astrogliosis were reduced in 5xFAD/E2F4DN mice. Moreover, although the attenuation of the neuroinflammatory response initially correlated with larger Aβ deposits, Aβ deposition was lessened at later stages, and cognition was preserved in 5xFAD/E2F4DN mice, as is the case in asymptomatic AD individuals . FurtherConsejo Superior de Investigaciones Científicas (CSIC).Cryosections of the parietal cortex from AD patients were provided by the Banco de Tejidos Fundación CIEN (BT-CIEN) . These cryosections were obtained from the right half of the brain, which was cut in slices and frozen in −60 °C isopentane immediately after post-mortem brain extraction. A full neuropathological examination of each brain was conducted on the left half of the brain. The severity of the Alzheimer pathology was scored according to the “National Institute on Aging-Alzheimer’s association guidelines for the neuropathologic assessment of Alzheimer’s disease,” following the “ABC” protocol . ConsequMapttm1(EGFP)Klt knock-in mice expressing enhanced green fluorescent protein (EGFP) in neurons (EGFP mice) [Mapt gene, in which the coding sequence of EGFP has been inserted into the first exon, thus disrupting the expression of the tau protein. This results in the neuron-specific expression of cytoplasmic EGFP. Tau is expressed at high levels in neurons [Mapttm(mE2F4DN-myc) knock-in mice (E2F4DN mice) were generated following the procedure described by [n = 25) carried the transgene at the expected Mendelian frequency. The subsequent progeny was analyzed by genomic PCR with primers #1 and #2 (E2F4DN band) and the wild-type (WT) primers described by The Jackson Laboratory for EGFP mice (5′-CTCAGCATCCCACCTGTAAC-3′ and 5′-CCAGTTGTGTATGTCCACCC-3′). The knock-in strain was maintained on a mixed background of C57BL/6 and 129Sv or backcrossed to the C57BL/6 background. Homozygous E2F4DN mice were created by inbreeding mice containing one copy of the E2F4DN transgene. Homozygous E2F4DN mice are viable, fertile, normal in size, and do not display any gross physical or behavioral abnormalities, even though the tau protein has been deleted [Egfp and E2f4dn transgenes to avoid the observed effects of full Mapt null mutation in the phenotype of APP and APP/PS1 transgenic mice [Experimental procedures with mice were approved by the CSIC animal ethics committee and the Autonomous Government of Madrid, in compliance with Spanish and European Union guidelines. Double transgenic mice in C57BL/6J genetic background expressing under the control of the Thy1 promoter both mutant human APP695 with the Swedish , Florida (I716V), and London (V717I) FAD mutations, and human PS1 harboring the M146L and L286V FAD mutations (Tg6799 or 5xFAD mice)  were purFP mice)  were pur neurons , and hom neurons . Homozygribed by . These mribed by . To this deleted . Homozygnic mice . E2F4DN Rabbit anti-Myc tag polyclonal antibody (pAb)  was used at 1:1000 for western blotting and at 1:500 for immunohistochemistry. Mouse anti-α-tubulin monoclonal antibody (mAb) clone DM1A  was used at 1:10,000 for western blotting. The rabbit anti-NeuN pAb  was diluted to 1:800 for flow cytometry and 1:1000 for immunohistochemistry. The mouse anti-NeuN mAb, clone A60  was used at 1:1600 dilution for immunohistochemistry. The rabbit anti-GFAP pAb  was diluted to 1:1000 for immunohistochemistry. The rabbit anti-Iba1 pAb  was used at 1:800 dilution for immunohistochemistry. The rabbit anti-E2F4 polyclonal antibody  was used at 1:400 for proximity ligation assay (PLA) and immunohistochemistry. Mouse anti-E2F4 mAb clone LLF4-2  used at 1:400 for PLA and 1:6,000 for western blot. Mouse anti-phosphoThr mAb clone 20H6.1  was used at 1:750 for PLA. The chicken anti-NeuN pAb  was diluted 1:500 for PLA. The mouse anti-Synaptophysin mAb SY38 (PROGEN) was diluted 1:500 for western blot. The mouse anti-Actin monoclonal antibody (mAb), clone C4  was used at 1:10,000 for western blotting. The rabbit anti-β-Amyloid pAb #2454  was diluted 1:1,000 for western blotting. The APP mAb 22C11 (Invitrogen) was used at a dilution of 1:3,000 for western blotting. The anti-Tau pAb ab64193 (abcam) was diluted 1:1000 for western blotting.The donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (Invitrogen) was used at 1:1000 dilution for immunohistochemistry and 1:400 for flow cytometry. The goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568 (Invitrogen) was diluted 1:1,000 for inmunohistochemistry. The goat anti-chicken IgY (H + L) secondary antibody, Alexa Fluor 647 (Invitrogen) was used at 1:800 dilution for PLA. The IRDye 800CW Goat anti-Rabbit IgG (H + L) and IRDye 680RD Goat anti-Mouse IgG (H + L) antibodies (LI-COR) were diluted 1:15,000 for western blotting.Total RNA was extracted using QIAzol Reagent (Qiagen), and cDNA was synthesized using SuperScript IV Reverse Transcriptase (ThermoFisher Scientific) following the manufacturer’s instructions.Total RNA was extracted from the cerebral cortex of two 5xFAD/EGFP and two 5xFAD/E2F4DN female mice using TriZOL (ThermoFisher Scientific) according to the recommended protocol. Residual genomic DNA was removed with DNase I recombinant, RNase-free (Roche), following the manufacturer’s instructions. The concentration and quality of the total RNAs were measured using an Agilent Bioanalyzer 2100 with RNA 6000 nano Chips (Agilent Technologies). All samples had an RNA integrity value of 7 or greater. 1000 ng of RNA from the combination of two mouse brains of each genotype were used for each RNA-Seq library, which was created using the “NEBNext Ultra Directional RNA Library preparation kit for Illumina” (New England Biolabs) following the manufacturer’s instructions. We followed the indications of “Chapter 1: Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module.” We performed the library amplification included in the manufacturer’s instructions using a 12-cycle PCR. The quality of the RNA-Seq libraries was assessed with an Agilent Bioanalyzer and Agilent DNA7500 DNA chips to confirm that the insert sizes were 200–400 bp (average size: 299-317) for all individual libraries. The individual libraries obtained were also quantified by an Agilent 2100 Bioanalyzer with a DNA7500 LabChip kit and an equimolecular pool of libraries were titrated by quantitative PCR using the “Kapa-SYBR FAST qPCR kit for LightCycler 480” (Kapa BioSystems) and a reference standard for quantification. The pool of libraries was denatured prior to seeding on a flowcell at a density of 2.2 pM, where clusters were formed and sequenced using a “NextSeq™ 500 High Output Kit” (Illumina Inc.).t 38 patch release 5 (GRCm38.p5), adding the EGFP sequence with the Bowtie tool [p < 0.05 was used as a criterion for differential expression since the FAD/EGFP vs FAD/E2F4DN comparison yielded no gene tagged as significantly expressed by Cufflinks. As described in the main text, differential expression in several genes was confirmed by qPCR.RNA-Seq libraries were single-end sequenced in a 1 × 75 format using an Illumina NextSeq500 sequencer at the Genomic Unit of the Scientific Park of Madrid, Spain. The raw reads  passed the quality analysis conducted with the FastQC tool. Trimmed reads were subsequently mapped to the Genome Reference Consortium Mouse Buildtie tool  includedRps18 . ΔCt for treatment and control was calculated, and subsequently, the statistical significance was evaluated by a post hoc Student’s t test in genes where two-way ANOVA analysis was found to be significant. No statistically significant changes were observed among the average Ct values of Rps18 for the different genotypes ; n = 3; Student’s t-test).Reverse transcriptase (RT)-qPCR was performed with the 7500 real-time PCR equipment (Applied Biosystems), using specific primers from PrimePCR SYBR Green Assay (BioRad) and the house keeping gene After anesthetizing the mice with intraperitoneal sodium pentobarbital , administered at 50 mg/kg (body weight), they were transcardially perfused with PBS, and then with 4% paraformaldehyde (PFA). Brains were finally postfixed overnight at 4 °C with 4% PFA and cryoprotected by sinking in 30% sucrose in PBS at 4°C. The brains were then embedded in either Tissue-Tek (Sakura) followed by freezing in dry ice to get cryosections (12–15 μm), or in 3% agarose gels prepared in 0.1 phosphate buffer, pH 7.37, before cutting them with a vibratome (50 μm).The vibratome sections were washed three times with phosphate-buffered saline (PBS) containing 0.4% Triton X-100 (Sigma-Aldrich) (0.4% PBTx), and then incubated for 30 min in the dark with 0.05% Thioflavin S (Sigma-Aldrich) in 50% ethanol (Merck). Finally, the sections were washed twice with 50% ethanol, and once with distilled water. Then, the sections were subjected to immunohistochemistry as described below.The cryosections were permeabilized and blocked for 1 h at RT in 0.1% PBTx and 10% fetal calf serum , and incubated overnight (O/N) at 4 °C with the primary antibodies in 0.1% PBTx plus 1% FCS. After washing with 0.1% PBTx, the sections were incubated for 1 h at RT in 0.1% PBTx plus 1% FCS with the secondary antibodies. The sections were washed in 0.1% PBTx, and then incubated with 100 ng/ml 4′,6-diamidine-2′-phenylindole dihydrochloride  in PBS before mounting with ImmunoSelect antifading mounting medium (Dianova). The vibratome sections were permeabilized and blocked in 0.4% PBTx containing 10% FCS for 3 h. They were then incubated overnight at 4 °C with the primary antibodies in 0.1% PBTx containing 1% FCS. After five washes of 20 min with 0.1% PBTx, the sections were incubated with the secondary antibodies plus 100 ng/ml DAPI in 0.1% PBTx for 3 h at room temperature (RT). The sections were then washed five times with 0.1% PBTx, and mounted with ImmunoSelect antifading mounting medium.Lipofuscin present in the brains of 6-month-old mice was quenched with TrueBlackTM Lipofuscin Autofluorescence Quencher (Biotium). Briefly, the vibratome sections were washed once with PBS and treated for 30 s with TrueBlack 1x prepared in 70% ethanol. Finally, the sections were washed three times with PBS, and then immunostained as described above.PLA was performed in cryostat sections (4–6 μm) obtained from frozen samples of human parietal cortex (one Braak I patient and one Braak VI patient) using the Duolink In Situ Detection Reagents Brightfield system (Sigma-Aldrich). This method monitors the coincidence of specific epitopes and has been previously used for monitoring protein phosphorylation . The cryxy) across all stack images (z). In order to analyze the area occupied by GFAP and Iba1, a threshold was set to highlight the area to be quantified. In order to analyze the number and size of Aβ deposits, the region of interest (ROI) tool was used to delimitate the different cortical layers, and a threshold was then set to highlight the area to be quantified. An average of 100 plaques per individual were analyzed. The MorphoLibJ (v1.3.1) integrated library (https://imagej.net/plugins/morpholibj) and plugin was used to analize the soma size and roundness (R), as previously described [R was A is the area of the microglia soma and M is the length of the major axis, derived from the longest axis of an ellipse fit to each microglia soma.Confocal images were acquired at 20× magnification with a Leica SP5 confocal microscope. Image analysis was performed using ImageJ (Fiji). The images used for the analysis  were maximum intensity projections, created as output images whose pixels correspond to the maximum value of each pixel position , 1 mM EDTA (Merck), 1% Triton X-100, 1% SDS (Sigma-Aldrich)) including 1x cOmplete Mini, EDTA-free, protease inhibitor cocktail (Roche) (one hemicortex in 500 μl extraction buffer). The extracts were centrifuged for 10 min at 14,000 ×g (at 4 °C), and supernatants were then boiled for 5 min in Laemli buffer. The extracts were fractionated by SDS PAGE on 10% acrylamide gels and transferred to Immobilon-FL membranes (Millipore). The membranes were incubated for 1 h with Odyssey Blocking Buffer TBS (LI-COR) (OBB), and then incubated ON at 4 °C with the appropriate antibody in OBB containing 0.1% Tween 20. For Aβ analysis, hippocampal extracts prepared as described above were boiled for 5 min in 50 mM Tris-HCl pH 8.0 containing 12% Glicerol (Merck), 4% SDS, 0,01% Coomasie Brilliant Blue G-250 (Sigma-Aldrich), and 2% β-mercaptoethanol. Extracts were fractionated on 16.5% Mini-PROTEAN Tris-Tricine gels (Bio-Rad) (30 V for the first hour and then 125 V) using 1x Tris/Tricine/SDS Running Buffer (Bio-Rad) (cathode) and 200 mM Tris-HCl pH 9.0 (anode), and then transferred to Pierce Low-Fluorescence PVDF Transfer Membranes (0.2 μm) (ThermoFisher Scientific). The membranes were incubated for 1 h with Intercept Blocking Buffer TBS (LI-COR) (IBB), and then incubated ON at 4 °C with the rabbit anti-β-Amyloid pAb (1:1000) in IBB containing 0.1% Tween 20. After washing the membranes five times in TBS containing 0.1% Tween 20 (TBS-T), they were incubated for 1 h at RT with a 1:15,000 dilution of secondary antibodies in OBB  containing 0.1% Tween 20. Finally, they were washed again with TBS-T as described above, and the protein bands were visualized using the Odyssey CLx Infrared Imaging System (LI-COR).Cell nuclei isolation was performed as described by . BrieflyFlow cytometry was conducted as described by . Cell nuExploratory locomotor activity was recorded using a VersaMax Animal Activity Monitoring System  in an open field (40 cm × 40 cm) over a 10-min period. Infrared beams automatically record horizontal movements and rearing in the open field. The task analyzes the activity behavior by measuring the number of beams that are broken during the designated period of time. Ten trials repeated within two consecutive days  were performed for every animal and the results were expressed as average number of broken beams per trial.Motor coordination was evaluated in a Rotarod apparatus (Ugo Basile) with increasing acceleration. The apparatus consisted of a horizontal motor-driven rotating rod in which the animals were placed perpendicular to the long axis of the rod, with the head placed against the direction of rotation so that the mouse must move ahead in order to avoid falling. The trial was stopped when the animal fell down or after a maximum of 5 min. The time spent in the rotating rod was recorded for each animal and trial. Animals received a pretraining session to familiarize them with the procedure prior to evaluation. Thereafter, a total of six consecutive trials were conducted done for each animal. The data are presented as the average time spent before falling from the apparatus.Each mouse was placed within the center of a Y-maze apparatus (Panlab) and then allowed to freely explore the different arms during an 8-min session. The sequence of arms entered was recorded, and working memory was measured as the percentage of alternation (p.a.), which was calculated as the number of triads containing entries in all three arms divided by all the triads and then multiplied by 100.The MWM test was used to evaluate spatial learning. The apparatus was a circular tank 100 cm in diameter, filled with water (at 21–22 °C) made opaque. A platform was hidden inside the tank 2 cm below the water level. The experiment consisted of five sessions of four trials 30–60 min apart, performed under constant illumination conditions (7–15 lux). Each of the four starting positions  was used randomly in every daily session. Each trial was terminated when the mouse located the platform or when 60 s had elapsed, followed by a period of 15–20 s in which the animal was allowed to stay on the platform. Several fixed extra maze cues were constantly visible from the pool. All trials were recorded by a video camera located 2 m above the water level. Mice trajectories were analyzed using the Ethovision 3.1 computerized tracking system (Noldus), to measure escape latency for each animal in each trial.http://amp.pharm.mssm.edu/Enrichr).The DAVID bioinformatics platform was used for Gene Ontology (GO) functional annotation of gene sets (GOTERM_BP_ALL). MGI Mammalian Phenotype (Level 4 2019) was browsed using the Enrichr bioinformatics platform  or Dixon’s Q test (qPCR), were eliminated from the analysis.The quantitative data are represented as the mean ± SEM. Statistical differences in experiments performed with transgenic mice on either EGFP or E2F4DN background were analyzed using two-tailed Student’s Mapt), as previously described by Yves-A. Barde’s laboratory for EGFP knock-in mice (EGFP mice) [E2F4 is mainly expressed by cortical neurons   Fig. 1aMapt, as FP mice) , the homMapt locus resulted in reduced expression of tau protein, as compared with mice carrying the WT Mapt locus  with those genes whose expression is modulated in the cerebral cortex of APP/PS2 mice , another1a1 Fig. . FunctioThe hypothesis that neuronal expression of E2F4DN favors a non-cytotoxic immune response was further supported by browsing the MGI Mammalian Phenotype database against the protein-encoding genes that are modulated by E2F4DN in the cerebral cortex of 5xFAD mice and are absent in the study by Srinivasan et al.   , recentld Lilrb4  were lard Lilrb4 , there wApoe. Therefore, the capacity of ApoE to prevent the expression of the anti-inflammatory factor TGFβ and to favor cytotoxicity by microglial cells [Tgfb was upregulated in the cerebral cortex of 5xFAD/E2F4DN mice , is also upregulated in the cerebral cortex of 5xFAD mice expressing neuronal E2F4DN  to become upregulated in the cerebral cortex of 5xFAD/E2F4DN mice , we focused on ed in AD . We confice Fig. . Nnt encrganelle , a cruciice Fig. . Aldh1a1nditions . This gentegrity , was alsogenesis , was conarvation , and Stby marker , were aled to AD . We conffication , is upreAdi1, Barx2, Cfap46, and Cfap54. Adi1, which is upregulated in the cerebral cortex of 5xFAD/E2F4DN mice . This hypothesis would be consistent with the decreased Iba1-positive area observed in h5xFAD mice expressing neuronal E2F4DN. A reduced clearance of fibrillar Aβ by microglia could explain the accumulation of Aβ dense core deposits, as evidenced by Thioflavin S labeling, which is observed at 3 months in 5xFAD/E2F4DN mice, and whose deleterious effects are likely attenuated by the expression of a repertoire of genes that prevent Aβ processing, accumulation and toxicity. Interestingly, Aβ deposition was attenuated by E2F4DN in 6 month-old 5xFAD/E2F4DN mice. This observation may be explained by the lack of strong Apoe overexpression since ApoE potentiates fibrillation and compactation of Aβ at later pathological stages, when amyloid plaque growth becomes independent of TREM-2 [ApoE is mainly upregulated by microglia in response to Aβ deposition, and plays a critical role in the ability of microglia to take up and degrade Aβ . Therefof TREM-2 .−/− mice correlates with enhanced cognition [Other pathological mechanisms could also be attenuated by E2F4DN in 5xFAD mice, as relevant genes involved in the complex etiology of AD were found to be positively modulated. This includes regulators of synaptic function, glucose metabolism, oxidative stress, and endothelial cell function. The demonstration that NT is prevented by neuronal E2F4DN expression is also relevant. Cell cycle reentry has been proposed as a major driver of AD , likely ognition .E2F4DN expression in neurons was able to reverse the body weight loss phenotype observed in 5xFAD mice . Weight In this study, we have demonstrated that E2F4DN expression prevents spatial learning deficits observed in 5xFAD mice, evaluated with the spontaneous alternation Y-maze test . This efSince Aβ deposition was not fully prevented by neuronal expression of E2F4DN, cognitive recovery in 5xFAD mice cannot simply result from Aβ accumulation blockage. The latter has been claimed as the reason why therapeutic approaches aimed at reducing amyloid burden that work in transgenic mice are not effective when translated to AD patients . ConversAD is a cruel and devastating disease for which no effective therapies are currently available, possibly due to its multifactorial etiology. The findings of this study provide proof of concept that E2F4DN could be used as a therapeutic agent for the remediation of molecular, cellular and behavioral alterations underlining the pathophysiology of this disease. We propose E2F4DN-based gene therapy as a promising multifactorial approach against AD.ESM 1(DOCX 3.41 MB)ESM 2(pptx 1.55 MB)ESM 3(xlsx 58.0 KB)ESM 4(xlsx 145 KB)ESM 5(xlsx 11.4 KB)ESM 6(xlsx 83.6 KB)


These hydrophilic, neutral lanthanide coordination complexes are shown to bind chloride with apparent association constants of up to 105 M−1 in water and in buffered systems. Hydroxide bridging was observed in these complexes at basic pH, which was proven to be overcome by chloride. Thus, these lanthanide complexes show promise towards chloride recognition in biology and beyond. The results described here have clearly identified a new area of anion coordination chemistry that is ripe for detailed exploration.Halide recognition by supramolecular receptors and coordination complexes in water is a long-standing challenge. In this work, we report chloride binding in water and in competing media by pre-organised binuclear kinetically inert lanthanide complexes, bridged by flexible –(CH A series of binuclear lanthanide complexes address the long-standing challenge of binding chloride ions in aqueous solution. Binding of both chloride and fluoride is signalled by changes in the luminescence and NMR behaviour of the complexes. In solution, solvent molecules can also bind to both guest and host, meaning that anion binding can be hard to achieve in water.1,5 While a host can almost invariably bind to a variety of guests, selectivity for a single type of anion is difficult to achieve.1,3–5 Selective anion recognition requires careful design to match host and guest, so some of these supramolecular systems can be challenging to synthesise and often involve intricate architectures of considerable structural complexity, yet minimal practical applicability.4Anions, including DNA, halides, carbonates, phosphates, sulfates, and nitrates are everywhere in the natural world, and play a key role in defining the biology and the environment.via a network of hydrogen bonds from neutral donors. In these proteins, anion affinity is increased by a hydrophobic environment providing complementarity of charge and a shape that minimises the receptor desolvation energy.6 These interactions have inspired biomimetic receptors that integrate additional binding sites within the synthetic host, creating a structured binding pocket with high geometric complementarity.1,3–5 A range of interactions such as the macrocyclic effect,1,2 and conventional ion-pairing have been exploited in anion recognition;5 the latter based on electrostatics,1 and supported by hydrogen bonding,2 halogen bonding,7 anion–π interactions,8 and coordinate bond using Lewis acids such as metals.3,4Nature provides inspiration towards anion binding: protein-based interactions include bacterial sulfate binding protein and phosphatase enzymes, which selectively recognise sulfate and phosphate under physiological conditions 9 and plays important roles in neuronal growth (104–115 mM in extracellular fluid),10 and development of the central nervous system  through the functions of chloride channels. The intracellular chloride concentration (up to 70 mM in eukaryotic cell types) is crucial in moderating neuronal excitability and neurotransmission.11 Tracking chloride in living systems is vital if we are to understand diseases related to chloride transport defects such as cystic fibrosis and epilepsy.9,11,12 Apart from its importance as the counterion of life,9,12 sequestering salt from sea water (559 mM of chloride in surface sea water)13 for clean drinking water is an ongoing challenge.14 However, developing suitably selective receptors for chloride in water continues to be a challenge.1,4,5,15Here, we focus on chloride binding. Chloride is the most abundant physiological anion16 Halide anions exhibit different levels of hydration in different solvents as reflected by the Hofmeister series of anion hydrophobicity.17 Across the series,§§32− > SO42− > S2O32− > H2PO4− > HO− > F− > HCO2− > CH3CO2− > Cl− > Br− > NO3− > I− > CIO4− > SCN−.Hofmeister series of anion hydrophobicity  better than chloride (Ka = 55 M−1) and bromide in water.21 Other chloride binding supramolecular agents have been extensively reviewed elsewhere.1,5,15We previously reported a lanthanide tethered rotaxane that binds to chloride in apolar media . In thisld steel .20 Beer 22 The luminescence applications use lanthanide complexes as luminescent tags, exploiting stable complexes in which their luminescence signal is enhanced via the antenna principle where chromophores sensitise lanthanide luminescence and overcome the inherently low molar absorption coefficients associated with f–f transitions.22,23 Furthermore, the necessity for kinetic stability in clinical use ensures that macrocyclic ligands related to DOTA are well understood in competitive media with high anion concentrations.4,22,23 In such systems, anion chelation is achievable on the metal site without the dissociation of the lanthanide complex.4Lanthanide complexes are an attractive choice for developing anion receptors due to their ability to report anion binding by MRI, luminescence, and optical imaging.24 or anion binding.4 While luminescence can be modulated both by collision and anion binding, the latter generates a ‘turn-off’ event in MRI since the functioning of MRI contrast agents requires the presence of water molecules in the inner-coordination sphere which exchange with the bulk water.24 Thus, isostructural MRI and luminescent probes can be constructed with suitable lanthanide ions for targeted anion binding thereby allowing one design platform to exploit bio-imaging combining paramagnetism and luminescence, an exclusive feature offered by the lanthanides.4,22 Halides are strong Lewis bases which coordinate to metals that are hard Lewis acids.25 Using this HSAB approach, several mononuclear Ln(iii) complexes comprising cryptands,26 and cyclen derived C2  complexes of two heptadentate DO3A-based binding domains tethered by aryl spacers were studied for binding organic dicarboxylate anions at physiological pH;30 sensing biologically relevant anions such as phosphate, methylphosphate, double-stranded DNA and a DNA hairpin loop;31 and as a pH sensor.32 We have explored this binuclear approach by incorporating kinetically inert charge neutral lanthanide DO3A centres bridged by different m-xylyl scaffolds to achieve reversible binding of dinicotinate and isophthalate guests with high affinity at physiological conditions, but this requires methanol to improve the solvation of these guests.33Binuclear lanthanide complexes of DO3A ligands bridged by ethane and propane linkers ([Ln2(DO3A)2C-2] and [Ln2(DO3A)2C-3]) would create a pre-organised pocket between the metal centres capable of hosting halides coordinated to the lanthanide metal centres. Furthermore, having a short and flexible spacer between the coordinatively unsaturated hard Lewis acids, which act as acceptors would increase the steric strain thereby limiting their conformational freedom,35 where subsequent halide binding would lead to a smaller entropy loss in the host. Both binuclear complexes respond to halides under physiological conditions and the binding can be followed by luminescence and NMR spectroscopy. A neutral mononuclear lanthanide propargyl DO3A complex [Ln(pDO3A)] was23 used to compare the relative effectiveness of mononuclear complexes.In this work, we report how binuclear complexes can be tailored to bind chloride ions in water under conditions where mononuclear complexes22.1N-alkylated using t-butyl bromoacetate to form the triester, DO3A(t-BuO)3. Subsequent alkylation with 1,2-dibromoethane and 1,3-dibromopropane yielded the ethane and propane bridged bis-macrocycles, (DO3A(t-BuO)3)2C-2 or (DO3A(t-BuO)3)2C-3. Following purification and deprotection with trifluoroacetic acid, the pro-ligands (DO3A)2C-2 and (DO3A)2C-3 were obtained. These were reacted with the appropriate lanthanide triflate salt in methanolic solution under reflux and worked-up to afford the complexes [Ln2(DO3A)2C-2] and [Ln2(DO3A)2C-3]. Single crystal X-ray structures of (DO3A(t-BuO)3)2C-2 3 2C-3] and [Ln(pDO3A)] (Ln = Eu(iii) and Yb(iii)) were obtained. The binuclear complexes crystallised as a cluster containing 12 metal centres via μ-oxo bridges from the carbonyl oxygens, maintaining the connectivity and neutrality of the complexes ] (Ln = Eu(iii) and Yb(iii)) crystallised without any anion bound to the metal centre  and Yb(iii) complexes consist of broad lines; however, at pD 10.12, the lines are sharp and well defined 2C-2] and [Eu2(DO3A)2C-3] at pH 4 and 7 showed no significant change  complexes have been observed to bind chloride at the metal centre.26–29 Since the binuclear complexes in this work are neutral species, a neutral monometallic Ln(iii) complex [Ln(pDO3A)] was used as a model system to study halide binding. Steady-state luminescence titration of [Eu(pDO3A)] with potassium chloride (KCl) salt in deionised water did not result in any binding event  complexes were then explored. Steady-state luminescence titrations were performed in deionised water, phosphate buffer at pH 7.4, and in CHES buffer at pH 9.9. In the case of deionised water and in phosphate buffer, the emission intensity decreased with increasing KCl addition, but the opposite was the case in CHES buffer  was observed in all three cases and the binding strength increased as we move into buffered systems ]− complexes give well-resolved 35Cl NMR spectra that can be interpreted as evidence of interaction with chloride.36 By contrast, when [Tb(pDO3A)] was added to a solution of KCl in deuterium oxide, no new peaks were observed in 35Cl NMR, and the chemical shift of the chloride resonance was unchanged, suggesting that chloride does not bind in our model system 2C-3] in deuterium oxide display chemical shift upon the addition of KCl (35Cl NMR titrations were pursued with [Tb2(DO3A)2C-3] against KCl in D2O. The resulting chemical shifts were fitted to generate a binding isotherm. A strong first binding event followed by a very weak second binding event was observed 2C-3] 2C-3] (35Cl NMR titrations were performed with [Tb2(DO3A)2C-2] against KCl in D2O at 4 different temperatures. Although chlorine bound chemical shifts were observed, the resulting binding isotherms were sigmoidal in shape which was difficult in deducing meaningful binding events. However, they suggest the likely competition between chloride and hydroxide in occupying the binding site between the metal centres in [Tb2(DO3A)2C-2].The n of KCl . This sh Fig. S45,36 this 3A)2C-3] . Similariii) and Tb(iii) complexes in water, the association was further evaluated by high resolution ESI-Mass spectrometry. The mass spectral peak of a chloride bound to the Eu(iii) binuclear complexes in deionised water were found at m/z 1051.1486 and m/z 1065.1640 for C-2 and C-3 complexes, respectively, and confirmed by the calculated isotopic distribution pattern 2C-2(μ-Cl)]− and [Ln2(DO3A)2C-3(μ-Cl)]−.After establishing chloride binding by the binuclear Eu. However, we can use the fluoride interaction to further investigate the mode of chloride binding.To investigate the mechanism of halide binding in binuclear lanthanide complexes, fluoride binding was explored by titrating the complexes with solutions of potassium fluoride (KF) salt. Fluoride is known to bind strongly to a range of complexes,39 The binding isotherms were fitted to three binding events; all three were needed to generate an acceptable fit 2C-2]; Fig. S84, S85, S92, and S932(DO3A)2C-3]), it can be hypothesised that the speciation involves binding of first fluoride (K1) by chelation between the metal centres. Excess addition of KF generates the second binding event (K2) which involves chelation of fluoride to each metal, while the third binding event (K3) forms a bridging fluoride in addition to the fluoride chelated to each metal  complexes and KF were performed in competing , non-competing (Tris–HCl buffer at pH 7.4), and basic (CHES buffer at pH 9.98) media, the binding strength as well as the number of fluoride bound to the binuclear Eu(iii) complexes were lowered and no more than two binding events were observed 2C-2] and Fig. S84–S912(DO3A)2C-3]). A single crystal X-ray structure of [Yb2(DO3A)2C-3(2F)]2− was obtained  complexes, upon increasing the concentration of fluoride in deionised water, methanol and their deuterated counterparts, a decrease in the hydration number accounting for the displacement of inner-sphere water by the coordination of fluoride ions was observed  complexes which we hypothesise due to a counteracting interference between Photoinduced electron transfer (PeT) from chloride and intermetallic luminescence quenching.As expected, in luminescence lifetime measurements on the binuclear Eu(1H NMR spectra of Eu(iii) and Yb(iii) binuclear complexes sharpen with increasing addition of fluoride due to its chelation involving both the metal centres which slows the rate of interconversion between the SAP and TSAP isomers  complexes in deuterium oxide contain a small peak at around −450 ppm for the fluoride bound to the complexes ], no chloride chelation was observed  complexes against bromide and iodide solutions in deuterium oxide and deionised water gave broadened NMR spectra and no observable changes to the fine structure in the luminescence spectra 2C-2]; Fig. S100–S1032(DO3A)2C-3]) indicating that these ions bind very weakly, if at all. Thus, we have a highly selective receptor for chloride.In contrast to fluoride, significant interference for a chloride receptor can be envisioned from the heavier halides. Titration of the binuclear Eu3)2C-2, CCDC 2201929 for pDO3A(t-BuO)3, CCDC 2201930 for [Eu2(DO3A)2C-3], CCDC 2201931 for [Yb2(DO3A)2C-3], CCDC 2201932 for [Yb2(DO3A)2C-3(2F)]2−, CCDC 2201933 for [Eu(pDO3A)], and CCDC 2201934 for [Yb(pDO3A)].ESI is available which contains detailed methods and materials, detailed synthesis and characterization, titrations (NMR and luminescence), speciation models, luminescence lifetimes and single crystal X-ray data. Crystallographic information for the structures reported in this article have been deposited at the Cambridge Crystallographic Data Centre, under deposition numbers CCDC Conceptualisation, S. F.; supervision, S. F., A. M. K. and T. J. S.; methodology, C. A., S. F., A. M. K., T. J. S. and K. E. C.; investigation, C. A., K. E. C. and J. A. T.; validation, C. A.; formal analysis, C. A., T. J. S., K. E. C. and S. F.; data curation, C. A. and K. E. C.; visualisation, C. A.; writing – original draft, C. A., T. J. S. and S. F.; writing – review & editing, C. A., T. J. S., A. M. K., S. F., K. E. C. and J. A. T.; resources, S. F. and K. E. C.; funding acquisition, S. F. and C. A.There are no conflicts to declare.SC-014-D2SC05417E-s001SC-014-D2SC05417E-s002


The panzootic caused by A/goose/Guangdong/1/96‐lineage highly pathogenic avian influenza (HPAI) A(H5) viruses has occurred in multiple waves since 1996. From 2013 onwards, clade 2.3.4.4 viruses of subtypes A(H5N2), A(H5N6), and A(H5N8) emerged to cause panzootic waves of unprecedented magnitude among avian species accompanied by severe losses to the poultry industry around the world. Clade 2.3.4.4 A(H5) viruses have expanded in distinct geographical and evolutionary pathways likely via long distance migratory bird dispersal onto several continents and by poultry trade among neighboring countries. Coupled with regional circulation, the viruses have evolved further by reassorting with local viruses. As of February 2019, there have been 23 cases of humans infected with clade 2.3.4.4 H5N6 viruses, 16 (70%) of which had fatal outcomes. To date, no HPAI A(H5) virus has caused sustainable human‐to‐human transmission. However, due to the lack of population immunity in humans and ongoing evolution of the virus, there is a continuing risk that clade 2.3.4.4 A(H5) viruses could cause an influenza pandemic if the ability to transmit efficiently among humans was gained. Therefore, multisectoral collaborations among the animal, environmental, and public health sectors are essential to conduct risk assessments and develop countermeasures to prevent disease and to control spread. In this article, we describe an assessment of the likelihood of clade 2.3.4.4 A(H5) viruses gaining human‐to‐human transmissibility and impact on human health should such human‐to‐human transmission occur. This structured analysis assessed properties of the virus, attributes of the human population, and ecology and epidemiology of these viruses in animal hosts. HPAIHighly Pathogenic Avian InfluenzaLPAILow Pathogenicity Avian InfluenzaWHOWorld Health OrganizationGIPGlobal Influenza ProgrammeTIPRATool for Influenza Pandemic Risk AssessmentIRATInfluenza Risk Assessment ToolCDCUnited States Centers for Disease Control and PreventionGISRSWHO Global Influenza Surveillance and Response SystemOIEWorld Organization for Animal HealthFAOFood and Agriculture OrganizationHAHemagglutininROKRepublic of KoreaCVVWHO Candidate Vaccine VirusUSDAUnited States Department of AgricultureARDSAcute Respiratory Distress SyndromeMOFMultiple Organ FailureSia‐α2,6GalSialic Acid Linked to Galactose by an α2,6 linkage3′SLeXSialyl Lewis XGOFGain‐Of‐FunctionGISAIDGlobal Initiative on Sharing All Influenza DataGSDGenetic Sequence Data1Influenza A viruses infect a wide spectrum of animal species precluding global eradication. Genetically diverse viruses circulate among wild aquatic birds, which are considered to be their natural reservoir and experience no or only mild signs of disease when infected. In birds, the viruses typically replicate in the intestinal and respiratory tracts and are shed in the environment where other hosts become infected. Viruses from the aquatic wild bird reservoir may infect other avian species including terrestrial poultry, such as chickens and quail, and domesticated waterfowl, such as ducks and geese. Following circulation in these densely populated host species, avian influenza viruses may then transmit to mammalian hosts, including humans, pigs, horses, dogs, and marine mammals.Globalization and industrialization over the past decades have contributed to the emergence of novel influenza viruses that threaten animal and human health. Once they emerge and become transmissible between humans, influenza viruses can rapidly spread worldwide. Current vaccines which take 6 months to distribute from strain selection in the current influenza manufacturing cycle are unlikely to be available to contain the first wave of human infections of a pandemic. Therefore, it is strategically important to risk‐assess and prioritize animal influenza viruses with pandemic potential to initiate possible responses, including preparatory development of vaccines, and antiviral drug efficacy testing. The World Health Organization (WHO) Global Influenza Programme (GIP) developed a Tool for Influenza Pandemic Risk Assessment (TIPRA)Because the emergence of the highly pathogenic avian influenza (HPAI) A(H5) viruses of the A/goose/Guangdong/1/96 (gs/GD) hemagglutinin (HA) lineage, there have been 883 officially reported human infections by viruses of this lineage: 860 by A(H5N1) and 23 by A(H5N6) viruses.2The clade 2.3.4.4 A(H5N8) viruses were first reported in migratory ducks and curlews in Shanghai, China in 2013 by retrospective surveillance,Although the A/gyrfalcon/Washington/41088‐6/2014‐group viruses were disseminated from Asia to other continents, a group of viruses represented by A/Fujian‐Sanyuan/21099/2017 (hereby A/Fujian‐Sanyuan/21099/2017 group) did not initially appear to spread outside Asia.In contrast to the A/gyrfalcon/Washington/41088‐6/2014‐group and A/Fujian‐Sanyuan/21099/2017‐group viruses, which were characterized by global spread, other genetic groups of clade 2.3.4.4 A(H5) viruses have to date remained more limited in geographic range. One group represented by the WHO CVVs, A/Hubei/29578/2016, A/chicken/Vietnam/NCVD‐15A59/2015, and A/duck/Hyogo/1/2016 , AH5N6), and AH5N8) with genetic groups have been identified from both poultry and wild birds., and A virus shed virus for 7 days with no mortality, similar to what was observed with a control HPAI A(H5N1) virus.Several studies have documented the enhanced virulence of some clade 2.3.4.4 A(H5) viruses in experimentally infected mammals.4The first human infection caused by a clade 2.3.4.4 A(H5N6) virus was reported by China in April 2014.Although virologic surveillance is typically not designed to detect cases that are not severe such as influenza‐like illness, serologic studies can estimate the frequencies of less severe and mild infections. Two studies have looked for evidence of seroconversion to clade 2.3.4.4 A(H5) viruses in poultry farmers.The 22 A(H5N6) viruses from human cases for which genetic sequence data (GSD) are available in the EpiFlu database of GISAID were all classified as clade 2.3.4.4 A(H5) viruses. Subgroups within clade 2.3.4.4 to which the human viruses belong have changed over time: a virus collected in February 2014 belonged to the A/Sichuan/26221/2014‐like group, 20 viruses collected between April 2014 and November 2017 belonged to the A/Hubei/29578/2016 group, and a virus in the A/Fujian‐Sanyuan/21099/2017 group was detected in 2017 , which is abundantly displayed in the upper respiratory tract of humans.Among 1994 clade 2.3.4.4 A(H5) viruses isolated between January 2013 and October 2018 with GSD available in the EpiFlu database of GISAID, 1988 (99.7%) had an HA‐160A (H3 numbering) amino acid residue and 1295 (64.8%) had HA‐227R amino acid residue  viruses. These included assessing direct contact and respiratory droplet transmission using multiple animal models, namely ferrets, pigs, guinea pigs, and dogsReceptor binding affinity is a prerequisite, but insufficient alone to promote airborne transmission of A(H5) avian viruses. Several studies have shown that compensatory mutations in HA are required to counteract the HA instability caused by human‐type receptor‐binding mutations.7The experimental data generated to date has not detected differences in receptor binding specificity and transmission capability among mammals between clade 2.3.4.4 A(H5N2), A(H5N6), and A(H5N8) viruses despite that only the A(H5N6) subtype of the clade 2.3.4.4 A(H5) viruses among three has been found in humans. Dual‐receptor binding specificity, viruses that show equal binding in vitro to both human and avian receptor analogues, has been observed in viruses of all three subtypes, and some of the viruses were transmitted via direct contact among ferrets or guinea pigs. However, no studies have identified receptor binding profiles showing a preference for binding to human receptor analogues, or animal model transmission patterns, showing spread via the aerosol route, consistent with a virus adapted to transmit in humans. What is less clear is precisely which molecular changes would lead to such adaptation.A/Hubei/29578/2016‐group clade 2.3.4.4 A(H5) viruses, which are primarily A(H5N6) viruses, have been confined to Asia. In contrast, A/gyrfalcon/Washington/41088‐6/2014‐group A(H5N2) and A(H5N8) viruses and A/Fujian‐Sanyuan/21099/2017‐group A(H5N8) viruses spread from Asia to North America, Europe, the Middle East, and the African continent and gave rise to numerous outbreaks among poultry and wild birds following reassortment with viruses from local avian species. Despite significant exposure to A(H5N2), A(H5N6) or A(H5N8) infected poultry, so far only the A(H5N6) subtype viruses have caused human infection. What then differentiates the zoonotic potential of the A(H5N6) viruses from that of the other two subtypes?Several possible reasons can be considered here. First, biosecurity systems vary across countries. A/Fujian‐Sanyuan/21099/2017‐group A(H5N8) and A/gyrfalcon/Washington/41088‐6/2014‐group A(H5N2) and A(H5N8) viruses were detected in poultry in multiple places in the United States and in Europe, resulting in severe impacts on the poultry industries. The majority of affected countries executed a systematic stamping‐out strategy.Last, although different exposures to infected poultry may have contributed to human infections with the specific subtype A(H5N6), it is also possible that biological features of A(H5N6) viruses also contributed to occurrence of human infections. In a study conducted by Chen et al., an A(H5N1) reassortant virus with several amino acid substitutions in HA and the NA gene of a human seasonal A(H3N2) virus (A/Brisbane/10/2007) was transmitted via respiratory droplets between ferrets, but an A(H5N1) reassortant virus with the same HA gene and the NA gene of a human seasonal A(H1N1) virus (A/Brisbane/59/2007) was not transmitted.The importance of continued monitoring of the ecology and ongoing evolution of potentially zoonotic avian influenza viruses should not be underestimated. Some of the fundamental and important activities such as surveillance programs in diverse animal reservoirs, including wildlife, are not always a high priority and properly funded. Several studies on poultry outbreaks caused by clade 2.3.4.4 A(H5) viruses in the United States and the Netherlands suggested that the viruses were introduced from wild birds rather than farm‐to‐farm transmissions.8Because their emergence, the clade 2.3.4.4 HPAI A(H5) viruses have evolved through point mutations and reassortment with circulating local viruses following global expansion via distinct pathways. So far, these viruses have caused only sporadic human infections and are unable to transmit efficiently among humans. Studies have shown that some clade 2.3.4.4 A(H5) viruses have dual‐receptor specificity and can transmit between ferrets in direct contact. Furthermore, some A(H5N6) viruses isolated from humans have molecular signatures related to mammalian adaptation. It is uncertain what other changes are necessary for these viruses to become transmissible among humans. Their widespread distribution, ongoing evolution, and periodic infection of mammalian hosts increase the chances that efficient transmissibility is possible to be acquired. This calls for surveillance of influenza viruses in domestic and wild birds to be enhanced to allow for timely development and updating of veterinary and public health countermeasures and to reduce the threats of zoonotic and pandemic influenza.9HA genetic sequence data (GSD) of HPAI viruses isolated from animals, including mammals and avian species, that possessed multibasic amino acids at the HA cleavage site between 2013 and February 2019 and available in the EpiFlu database of Global Initiative on Sharing All Influenza Data (GISAID) were analyzed. Between 2013 and February 2019, 2553 A(H5) HPAI viruses were available. The open reading frame of the HA genes of A(H5) viruses was used for phylogenetic analysis. Multiple sequence alignment of H5 viruses was performed using BioEdit 7.2. A maximum‐likelihood tree was constructed for MEGA 7 with 1000 replicates. The 242 virus GSDs were used as the reference for the nomenclature of A(H5) HA systematized by World Health Organization/World Organization for Animal Health/Food and Agriculture Organization (WHO/OIE/FAO) H5 Evolution Working Group.JSMP has received research funding from Crucell NV and is ad‐hoc consultant for GlaxoSmithKline and Sanofi. YK has received speaker's honoraria from Toyama Chemical and Astellas; grant support from Chugai, Daiichi Sankyo, Toyama Chemicals, Tauns, Tsumura, and Denka Seiken and is a co‐founder of FluGen.RY drafted the manuscript and constructed the tables and figures with contributions from MDS, CTD, DES, DW, FYKW, JWM, JSMP, RJW, RAMF, YK, and WZ in reviewing results, reviewing the manuscript, and providing suggestions. RY, MDS, and WZ managed the project.Supplementary Table 1 A(H5N6) viruses with the A/Fujian‐Sanyuan/21099/2017‐like HA gene for which sequences are available in the EpiFlu database of GISAIDClick here for additional data file.Supplementary Table 2 Amino acid substitutions related to receptor binding specificity among clade 2.3.4.4 A(H5) avian viruses available from GISAID as of February 2019Click here for additional data file.