Transcription factors (TFs) regulate transcription by binding to the specific sequences at the promoter region. However, the mechanisms and functions of TFs binding within the coding sequences (CDS) remain largely elusive in prokaryotes. To this end, we collected 409 data sets for bacterial TFs, including 104 chromatin immunoprecipitation sequencing (ChIP-seq) assays and 305 data sets from the systematic evolution of ligands by exponential enrichment (SELEX) in seven model bacteria. Interestingly, these TFs displayed the same binding capabilities for both coding and intergenic regions. Subsequent biochemical and genetic experiments demonstrated that several TFs bound to the coding regions and regulated the transcription of the binding or adjacent genes. Strand-specific RNA sequencing revealed that these CDS-binding TFs regulated the activity of the cryptic promoters, resulting in the altered transcription of the corresponding antisense RNA. TF RhpR hindered the transcriptional elongation of a subgenic transcript within a CDS. A ChIP-seq and Ribo-seq coanalysis revealed that RhpR influenced the translational efficiency of binding genes. Taken together, the present study reveals three regulatory mechanisms of CDS-bound TFs within individual genes, operons, and antisense RNAs, which demonstrate the variability of the regulatory mechanisms of TFs and expand upon the complexity of bacterial transcriptomes. The fast development of the chromatin immunoprecipitation sequencing (ChIP-seq) approach has revealed the genome-wide occupation of transcription factors (TFs) –4. Previ–Pseudomonas aeruginosa and the plant pathogen Pseudomonas syringae, respectively  , 22. TheE. coli , while 1ifficile . It has ifficile , resultiifficile . For insectively , 29. Theectively , 30.The protein-coding regions account for more than 90% of the bacterial genomes, but their regulatory roles in transcription remain mostly unclear. To date, hundreds of ChIP-seq studies have indicated that TF-binding sites (TFBSs) are extensively scattered across the coding areas, suggesting that TFs can regulate the transcription of coding regions , 14. To in vivo, we collected 104 ChIP-seq data sets from six model bacterial species: M. tuberculosis H37Rv  Total number of peaks for each TF from seven different strains (detail added to the figure). (B) AlgR bound to the coding region of Copyright © 2022 Hua et al.2022Hua et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa and P. syringae as models in the following study. The TF-binding peaks generated by the model-based analysis of ChIP-seq (MACS) have several descriptive characteristics, including the q value and the fold enrichment   . The Rhpl (dadX) . These fTFs regulate various biological processes by recognizing and interacting with specific DNA sequences . We then10.1128/mbio.01643-22.2FIG S2FIG S2, TIF file, 1.1 MB.Comparison of the motif sites between the coding and intergenic regions. (A and B) Comparison of the FIMO scores and the q values of the peaks around motif sites (AlgR and RhpR) located in the coding and intergenic regions. (C and D) Comparison of the peak occupancy profiling around motif sites (AlgR and RhpR) distributed in the coding and intergenic regions. Download Copyright © 2022 Hua et al.2022Hua et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo, the DNA-binding specificity of TFs can be influenced by protein-protein interactions, which are avoided in the systematic evolution of ligands by exponential enrichment (SELEX) approaches  The total binding number for each TF from the HT-SELEX data from P. aeruginosa and P. syringae. (B) Percentage of the TFs’ binding sites located in the coding region, based on genomic-SELEX. (C) The total binding number for each TF, based on E. coli genomic-SELEX data. Download FIG S3, PDF file, 0.7 MB.In total, there were 12,293 predicted TFBS from the SELEX data from Copyright © 2022 Hua et al.2022Hua et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the q values, across the coding and intergenic regions  leads to a 10-fold increase in rhpR expression compared to that observed in the wild-type (WT) strain  in ΔalgR than in the WT strain  to visualize the binding peaks of RhpR, AlgR, and VqsM from their ChIP-seq data , 11, 45.) strain , 38, 46.T strain . SimilarT strain . These r10.1128/mbio.01643-22.4FIG S4algR strain than in the WT strain. Download FIG S4, TIF file, 1.2 MB.AlgR influenced the expression of PA0324 by binding to the PA0323-6 operon. (A) Negative-control EMSA result for RhpR. (B) Negative-control EMSA results for VqsM. (C) AlgR bound to the coding region of PA0324 and PA0325, and the transcription level of PA0324 was higher in the ΔCopyright © 2022 Hua et al.2022Hua et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pta, which encodes phosphate acetyltransferase. Furthermore, the mRNA level of pta was 3-fold higher in the ΔalgR strain than in the WT strain , and its expression was higher in the ΔvqsM strain than in the WT strain . Notably, the expression level of the entire operon was higher in the ΔT strain , suggestopdD-dppA4B operon, AlgR bound to the coding region of dppA4, resulting in higher transcriptional levels of dppA4 and dppB in the ΔalgR strain than in the WT strain  was higher in the ΔrhpS strain than in the WT strain, but the transcription of the two downstream genes did not change  was performed. We found that 20.1% and 36.1% of all reads were mapped to antisense transcripts in P. aeruginosa and P. syringae, respectively  . Interes strains . IGV was strains . TFBSs l strains . In contT strain  and E. OalgR strain, the RNAP occupancy inside PA0930 was lower than that observed in the WT, which correlated with the expression level of the corresponding aRNA in both strains  was inserted at the 3′ end of the fragments to promote translation by preventing rho-mediated transcription termination and stabilizing the RNA . Si. Si41).  the RNA . Accordi the RNA . We did  the RNA . To furt the RNA  to E. As the RNA . To bett the RNA . In concrhpS strain, both reporters were derived from the promoter region of PSPPH 3675, with the 3′ end positioned either before or after the RhpR motif  Lux activities of different reporters located before or after the RhpR motif in the WT strain and the ΔrhpS strain. (C) Agarose gel showed PCR products from the genome and cDNA. D. The EMSA results were obtained using 1 μg DIDC in each reaction. (E) The ChIP-qPCR results of RNAP occupancy at the AlgR binding sites. (F) The ChIP-qPCR results of RNAP occupancy at the RhpR binding sites. Download FIG S5, TIF file, 0.5 MB.The subgenic transcript overlapped with PSPPH_3675, and RhpR had no effect on PSPPH_3675 expression. (A) The diagram shows the position of the PSPPH_3674 and the PSPPH_3675 in the Copyright © 2022 Hua et al.2022Hua et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. syringae WT strain and the ΔrhpS strain  of 286 genes were altered significantly . RhpR impacted the TEs of the CDS binding genes, such as PSPPH_1269, which encodes lytic murein transglycosylase . RhpR-biThe findings of the present study provide a systematic view of binding loci for bacterial TFs, which demonstrates that bacterial CDS-binding TFs influence transcription. Important functions of CDS-binding TFs have been reported in eukaryotes , 50. ForThe present study shows that bacterial CDS-binding TFs regulate the transcription of antisense RNA by regulating cryptic promoters, suggesting an understudied function of bacterial TFs. The abundance of cryptic transcripts has been underestimated in both eukaryotes and prokaryotes. In yeast, TFs interact with CDS to activate cryptic promoters, resulting in unannotated cryptic transcripts, most of which are unstable . The traS. cerevisiae DNA  (dppA4 and dppB)  The transcription of a target gene is induced by TFs  by binding within the coding region of an adjacent gene (PSPPH_4418) . CDS-boupta) . (ii) Thnd dppB) . We propnd dppB) . We propP. aeruginosa input, as well as RhpR ChIP and P. syringae input) from GEO and then mapped them to the P. aeruginosa (NC_002516) and P. syringae (NC_005773.3) genomes using Bowtie2, respectively , with 16S rRNA as the reference. All the reactions were conducted with three biological repeats.The primer sequence can be found in ched 0.6 , and the10.1128/mbio.01643-22.6TABLE S1Table S1, DOCX file, 0.03 MB.Strains, plasmids and primers used in this study. Download Copyright © 2022 Hua et al.2022Hua et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the −(ΔΔCt) with 16S rRNA as the reference. All of the reactions were conducted in three biological repeats.For the antisense RNA RT-qPCR, the specific forward primers were designed to do reverse transcription using a FastKing RT Kit (Tiangen Biotech). Coupling with the corresponding reverse primer, RT-qPCR was performed using the SuperReal Premix Plus (SYBR green) Kit (Tiangen Biotech). We calculated the relative fold changes using 22, 10% glycerol) with a DNA probe and target protein for 30 min. Before running the reaction for 60 min at 100 V, we pre-ran the gel for 30 min. We used GelRed nucleic acid dye to visualize the DNA. The EMSA results were photographed by using a gel imaging system (Bio-Rad). For the EMSA performed with DIDC-DIDC, the probes were FITC modified, and the results were visualized through florescence.The primer sequence can be found in 600 reached 0.6. Then, the strain was pelleted and washed before RNA extraction using a bacteria total RNA isolation kit (Sangon Biotech). Subsequently, the mRNA was enriched by MICROBExpress (Thermo Fisher) kit. The fragmentation and reverse transcription were done using the Clontech Smart Scribe Kit (TaKaRa). The RNA was removed using RNase H and RNase A before cleanup using VAHTS DNA Clean Beads (Vazyme). Phusion was used in the final PCR before the adapter was ligated to the cDNA by T4 RNA ligase 1 (NEB).The LM-seq procedure was done following a previous study with slight modifications . In brieP. aeruginosa (NC_002516) and P. syringae (NC_005773.3) genomes by Bowtie2  and fast-frozen in liquid nitrogen. Sodium deoxycholate was added after thawing the lysate on ice. The supernatant was transferred into a new tube and digested with MNase in MNase buffer . Sephacryl S400 MicroSpin columns were used to purify the MNase-digested products. The sRNA was separated using a Zymo RNA kit, and the rRNA was removed using a Ribo-Zero-rRNA Removal Kit. The final library was constructed using the NEBNext Small RNA Library Prep Set. The library was analyzed using the MetaRiboSeq pipeline (https://github.com/bhattlab/bhattlab_workflows/tree/master/metariboseq). The translational efficiency was calculated by dividing the normalized Ribo-seq counts by the normalized RNA counts.The construction of the library followed a previous protocol. In brief, overnight cultures of 600 = 0.6) was reached. The cross-link was performed by adding formaldehyde to a 1% concentration for 10 min and quenching with glycine. Then, the bacteria were pelleted and washed with a Tris buffer (20 mM Tris-HCl [pH 7.5] and 150 mM NaCl). Prior to sonication, the bacteria were resuspended in in IP buffer . The cell lysis was centrifuged, and the supernatant was incubated with RNA Polymerase ImmunoAffinity Resin (number 673601). The RNAP binding DNA fragments were eluted after proteinase K digestion. The purified DNA fragments were used to perform downstream qPCR.The overnight bacteria cultures were transferred to a fresh medium containing the appropriate antibiotics until the mid-log-phase (ODt tests were performed to analyze the RT-qPCR results in R. The Wilcoxon test was used to analyze the FIMO score differences in R. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Results are presented as means ± standard deviations. All experiments were repeated at least three times.Student's GSE175852. Codes are available upon reasonable request.The data generated in this study were downloaded from public databases –19, 31. –


The cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood.The genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast.Our findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.The online version contains supplementary material available at 10.1186/s13072-022-00469-0. This coLa cells , 7. TherLa cells –13. Elimethality , 15. Mutn cancer , 17. UndThe role of cohesin in genome architecture centers on its ability to generate chromatin loops , 19, 7. Recent studies have described the genome-wide distribution of cohesin variants STAG1 and STAG2 , 12, 13.Stag1 KO MEFs, for SMC1 in WT, Stag2 KO, Pds5A KO, Pds5B KO, Pds5A and Pds5B (Pds5 DKO) MEFs and for CTCF in WT MEFs. Stag1 KO, Pds5A KO and Pds5B KO embryos from which MEFs were obtained are homozygous for constitutive KO alleles and thus the corresponding protein is completely absent [Stag2 KO and Pds5 DKO embryos carry “floxed” alleles that are inactivated by an inducible Cre recombinase and residual amounts of the proteins may be left in the MEFs at the time of cell collection [Stag1 KO and Stag2 KO MEFs revealed that non-CTCF cohesin positions are preferentially lost in the latter  was carried out for STAG2 in WT and y absent , 15. Stallection , 14. ImmWe reanalyzed ChIP-seq data of CTCF and cohesin subunits from other studies to identify non-CTCF cohesin positions in different human cell lines. In immortalized mammary epithelial cells MCF10A) and primary epithelial and endothelial cells , the CTCF cohesin sites were robust and could be occupied by STAG1 or STAG2 while non-CTCF sites had lower occupancies and there was a predominance of STAG2  in MEFs expressing GFP-tagged versions of STAG1, STAG2 and RAD21. To ensure physiological expression of the tagged proteins, the GFP tag was added to the C-terminal regions of the three genes using genome editing with CRISPR–Cas9. Successful targeting and incorporation of the tagged subunits into functional cohesin complexes was assessed by immunofluorescence in fixed cells, immunoblotting of total cell extracts and chromatin fractions and immunoprecipitation . Immunoblot analyses revealed a decrease of CTCF below 20% of its levels in WT MEFs  were cultured in medium with 1 μM 4-hydroxy tamoxifen for 4 and 5 days, respectively. For CTCF elimination, a clone of immortalized Ctcf f/f MEFs was infected with Adeno-Cre viruses (University of Iowa) at 250 pfu/cell in DMEM supplemented with 2% FBS. Medium was replaced after 24 h and cells were collected 9 days later for immunoblot and chromatin immunoprecipitation analyses.MEFs of the following genotypes were used in this study: tag1 −/− , Stag2 fCre-ERT2 , Pds5A ±Cre-ERT2  and CtcfCtcf f/f . Mice weWhole cell extracts for immunoblot were prepared by resuspension in Laemmli buffer at 10,000 cells/µl, sonication and boiling for 5 min at 95 ºC, fractionated in SDS-polyacrylamide gels and transferred to nitrocellulose membranes for 1 h at 100 V in transfer buffer I. Membranes were blocked in 5% skimmed milk in TBST, incubated with antibodies for 1–2 h in 1% BSA-TBST. Antibodies are listed in Additional file Whole cell extracts for immunoprecipitation Fig. : Fig. S42, 0.2 mM EDTA, 20% glycerol, 0.5 mM PMSF, 1 mM 2-mercaptoethanol) as described [For immunoprecipitation reactions shown in Fig. escribed . For the7 cells/mL in buffer A , and incubated on ice for 5 min in the presence of 0.1% Triton X-100. Low-speed centrifugation (4 min/600 g/4 °C) allowed the separation of the cytosolic fraction (supernatant) and nuclei (pellet). Nuclei were washed and subjected to hypotonic lysis in buffer B  30 min on ice. Nucleoplasmic and chromatin fractions were separated after centrifugation (4 min/600 g/4 °C). Chromatin was resuspended in Laemmli buffer and sonicated twice for 15 s at 20% amplitude. For salt extraction experiments, chromatin fractions were either left untreated or treated with 0.5 M NaCl in modified buffer A  for 30 min on ice. Solubilized proteins were separated from insoluble chromatin by low-speed centrifugation (4 min/600 g/4 °C) and prepared for immunoblotting.Chromatin fractionation was performed as described . Cells w2 and 300 mM sucrose) for 5 min before fixation in 2% paraformaldehyde for 15 min at room temperature. Coverslips were blocked with 3% BSA, 0.05% Tween-20 in PBS for 30 min. Primary and secondary antibodies were diluted in blocking solution and incubated for 1 h each. DNA was counterstained with 1 µg/ml DAPI. A Leica DM6000 microscope was used to obtain grayscale images, which were later analyzed using FIJI software.Cells grown on coverslips were pre-extracted with 0.5% Triton X-100 in CSK buffer  confocal microscope from Germany Leica Microsystems using a 40x/1.2 NA HCX PL APO objective with immersion oil. Cells were kept in a climate chamber at 37 ºC with 5% CO2 during the experiment. Image acquisition used the HCSA software in LAS AF 2.7. Cells were photobleached with an argon laser and the recovery was monitored by live-cell imaging, Pictures were taken immediately before and after photobleaching as well as every 30 s during recovery. Videos were analyzed using FIJI software using the plug-in Turboreg (http://bigwww.wpfl.ch/thevenaz/turboreg) for image alignment. For each timepoint, the difference in intensity between the bleached and unbleached areas of the cell nucleus is calculated after background subtraction, normalization to initial fluorescence  and to total cell intensity. Statistical analysis and curve fit (non-linear regression) were carried out with GraphPad Prism.One wild-type MEF clone was immortalized using SV40 large T antigen and used to generate RAD21-GFP, STAG1-GFP and STAG2-GFP expressing cell lines by CRISPR/Cas9-mediated homologous recombination, as described . Donor pCtcf f/f ± Cre, MEFs arrested in G0 were used and around 5% of sonicated chromatin from MCF10A cells was mixed with the mouse chromatin before addition of antibodies for calibration purposes. Around 5 ng of immunoprecipitated chromatin in each sample were used for library preparation. DNA libraries were applied to an Illumina flow cell for cluster generation and sequenced on an Illumina HiSeq2000. Alignment of reads to the reference mouse genome (mm10) was performed using ‘Bowtie2’ (version 2.4.2) under default settings [q value (FDR) to 0.05 and using the ‘–extsize’ argument with the values obtained in the ‘macs2 predictd’ step [P-value cut-off  < 10−10.Chromatin immunoprecipitation was performed in asynchronously growing MEFs as described  with antsettings . Duplicatd’ step . “CTCF” td’ step  as well td’ step , 44. “CTtd’ step . We conshIPm)/(WmIPh), where Wm and IPm are the number of reads mapped to the mouse genome from input (W) and immunoprecipitated (IP) fractions, and Wh and IPh are reads mapped to the human genome from the input and IP fractions used for calibrating [For analysis of calibrated ChIP-seq, profiles for each antibody were normalized by coverage and then multiplied by the occupancy ratio (OR) =  was used and reactions were performed in triplicate. Fold enrichment of cohesin-binding at a given position was calculated over the binding at a nearby position showing few reads in the browser (negative region). Chromosome coordinates of the validated peaks and the corresponding primers are listed in Additional file For ChIP-qPCR, SYBR Green PCR Master Mix and an ABI Prism# 15031047 Rev. D). Adapter-ligated library was completed by limited-cycle PCR with Illumina PE primers and applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on HiSeq2000 following manufacturer’s protocols. Fastq files with 86-nt single-end sequenced reads were quality-checked with FastQC  and aligned to the mouse genome with Nextpresso executing TopHat-2.0.0 using Bowtie 0.12.7 and Samtools 0.1.16 allowing two mismatches and five multi-hits [Asynchronous MEFs (3 clones) were harvested and RNA was extracted using RNeasy kit from Qiagen. PolyA  + RNA was purified with the Dynabeads mRNA purification kit (Invitrogen), randomly fragmented and converted to double-stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing and ligation to adapters as in Illumina’s ‘TruSeq RNA Sample Preparation Guide’ (Part lti-hits . Reads wlti-hits .Genomic data generated in this study have been deposited in GEO database (accession number GSE212151). A list with these and additional datasets used appears in Additional file Additional file 1: Figure. S1 Characterization of conditional KO MEFs. a-c. Immunoblot analyses of whole cell extracts of WT and Stag2 KO (a), Pds5 DKO (b) and Ctcf KO (c) MEFs used for ChIP analyses. Increasing number of WT cells (expressed as %) were loaded to quantitate the extent of depletions in the KOs. In b, the same membrane was incubated first with anti-PDS5B and then with anti-PDS5A. The asterisk indicates previous signal from PDS5B. In c, a scheme of the experimental set up used for CTCF depletion in quiescent MEFs is also included. Figure. S2 Distribution of cohesin subunits and regulators along the genome. Snapshots of the UCSC genome browser at several loci that contain neighboring CTCF and non-CTCF cohesin sites. Examples of non-CTCF cohesin positions that are bound by RNA polymerase II or not are shown in top and bottom rows, respectively. A track showing SMC1 in Stag2 KO cells is also included (in red). Figure. S3 Transcription factor motifs at non-CTCF cohesin sites. STREME analysis showing the most statistically significant motifs enriched in non-CTCF cohesin positions in different cell lines. Percentages next to p-values represent the fraction of positions containing each motif. Logos on the right correspond to transcription factor binding sites statistically associated with those motifs that are shared among different cell lines. Figure. S4 GFP-tagged cohesin subunits form complexes and bind to chromatin. a. Polyclonal populations of iMEFs expressing GFP-tagged versions of RAD21, STAG1 or STAG2 were pre-extracted before fixation and stained with SMC1 (red) and DAPI (blue). Cells showing the corresponding GFP-tagged protein bound to chromatin are encircled. These cells were used in iFRAP analyses shown in Fig. 3c. b. Immunoblot analyses of whole cell extracts of these iMEFs. c. Chromatin fractionation analyses confirm that the tagged proteins are bound to chromatin. d. Immunoprecipitation reactions with SMC1 antibodies (or IgG as negative control) show the incorporation of GFP-tagged proteins in to cohesin complexes. For RAD21-GFP iMEFs, see Morales et al. [Figure. S5 PDS5 proteins and cohesin distribution. a. Matrix showing the correlation between the genome-wide distributions of the indicated proteins . b. Heatmaps showing the distribution of SMC1 in MEFs lacking PDS5A or PDS5B. c. Snapshots of the genome browser showing ChIP-seq data for SMC1 in WT and Pds5 DKO MEFs in regions that were validated by ChIP-qPCR. Graphs represent fold enrichment of the ChIP signal in each region (r1 to r14) over a neighbor negative region (neg). Figure. S6 Distribution of cohesin subunits in non-CTCF and CTCF positions after depletion of regulators. Read density plots for the heatmaps shown in Fig. 5, separating the four clusters defined in Fig. 4. Cluster 1, non-CTCF cohesin positions; cluster 2, CTCF cohesin sites with high NIPBL and low PDS5; cluster 3, CTCF cohesin sites with high NIPBL, PDS5 and CTCF; cluster 4, CTCF cohesin with low NIPBLAdditional file 2:Table S1. Genomic datasets. Table S2. Antibodies. Table S3. Primers for CRISPR. Table S4. Primers for ChIP-qPCR.Additional file 3: Movie S1. STAG1-GFP iFRAP.Additional file 4: Movie S2. STAG2-GFP iFRAP.Additional file 5: Movie S3. RAD21-GFP iFRAP.


The glycolytic enzyme, pyruvate kinase Pyk1 maintains telomere heterochromatin by phosphorylating histone H3T11 (H3pT11), which promotes SIR (silent information regulator) complex binding at telomeres and prevents autophagy-mediated Sir2 degradation. However, the exact mechanism of action for H3pT11 is poorly understood. Here, we report that H3pT11 directly inhibits Dot1-catalyzed H3K79 tri-methylation (H3K79me3) and uncover how this histone crosstalk regulates autophagy and telomere silencing. Mechanistically, Pyk1-catalyzed H3pT11 directly reduces the binding of Dot1 to chromatin and inhibits Dot1-catalyzed H3K79me3, which leads to transcriptional repression of autophagy genes and reduced autophagy. Despite the antagonism between H3pT11 and H3K79me3, they work together to promote the binding of SIR complex at telomeres to maintain telomere silencing. Furthermore, we identify Reb1 as a telomere-associated factor that recruits Pyk1-containing SESAME (Serine-responsive SAM-containing Metabolic Enzyme) complex to telomere regions to phosphorylate H3T11 and prevent the invasion of H3K79me3 from euchromatin into heterochromatin to maintain telomere silencing. Together, these results uncover a histone crosstalk and provide insights into dynamic regulation of silent heterochromatin and autophagy in response to cell metabolism. How H3T11 phosphorylation exerts biological functions remains poorly understood. Here, authors show that H3pT11 directly inhibits Dot1-catalyzed H3K79 tri-methylation (H3K79me3) and uncover how this histone crosstalk regulates autophagy and telomere silencing. The transcription of genes located proximal to these heterochromatic regions is repressed or silenced, a phenomenon known as position effect variegation (PEV)3. Maintenance of the integrity of silent heterochromatin is required for chromosome stability, including telomere protection, chromosome segregation fidelity, transposon repression, and DNA damage repair6.In the nucleus, the eukaryotic genome is organized into actively transcribed gene-rich euchromatin and condensed gene-poor silent heterochromatin. In budding yeast, heterochromatin is localized at three genome locations, including telomeres, silent mating-type loci, and ribosomal DNA (rDNA) repeats, which comprise ~10% of the yeast genome+-dependent histone deacetylase with preference for H4K16ac. Telomere heterochromatin formation is initiated by recruitment of Sir2 and Sir4 by Rap1 and the yeast Ku (yKu) complex to cis-acting silencer elements, where Sir2 deacetylates H4K16 on nucleosomes7. Deacetylated H4K16 is bound by Sir3, which in turn recruits more Sir2 and Sir48. The propagation of SIR complex along the telomeres protects the underlying genes from transcription machinery, leading to their repression, so called telomere silencing11. The spread of SIR complex to transcriptionally active chromatin is antagonized by active histone markers, including Sas2-catalyzed H4K16ac, Dot1-catalyzed H3K79me3, and Set1-catalyzed H3K4me3, which prevent SIR complex titration away from heterochromatin into euchromatin regions and keep the concentration of SIR complex high at telomeres15. Notably, these modifications occur predominantly in euchromatin regions and are absent from telomere heterochromatin regions, suggesting that they indirectly regulate heterochromatin structure16. Although Sir2 deacetylates H4K16 and Sir3 competes with Dot1 for nucleosomes18, the mechanism to prevent the occurrence of these histone markers, especially H3K79me3, at silent heterochromatin is not completely understood.The silent heterochromatin at yeast telomeres is formed by assembly and spread of a silent information regulator (SIR) complex, which composed of Sir2, Sir3, and Sir4 at chromatin, among which Sir2 is a NAD20. We have previously reported that the glycolytic enzyme, pyruvate kinase (Pyk1) can translocate into the nucleus, where it phosphorylates histone H3T11 (H3pT11) as a catalytic subunit of SESAME complex (Serine-responsive SAM-containing Metabolic Enzyme)21. The activity of SESAME is stimulated by glycolysis and serine metabolism to repress gene expression and confer cell resistance to oxidative stress22. Moreover, SESAME phosphorylates H3T11 at telomere regions to maintain telomere silencing by promoting SIR complex binding at telomeres and preventing autophagy-mediated Sir2 degradation23. This study also suggests that autophagy may affect telomere silencing by degrading telomere-associated proteins. However, little is known about the proteins and/or protein domains that specifically bind H3pT11 and the exact mechanism of action for H3pT11 remains poorly understood. For example, how H3pT11 represses autophagy (ATG) gene expression remains unknown. Moreover, it remains unclear how SESAME is recruited to telomere regions. In this work, we identify SESAME-catalyzed H3pT11 as an upstream inhibitor of Dot1-catalyzed H3K79me3. By inhibiting Dot1-catalyzed H3K79me3, H3pT11 represses the transcription of ATG genes and prevents autophagy. Moreover, H3pT11 and H3K79me3 work together to enhance the binding of SIR complex at telomeres to maintain telomere silencing. In addition, SESAME is recruited by Reb1 to phosphorylate H3T11 at telomeres and restrict H3K79me3 at the heterochromatin-euchromatin boundary to maintain telomere silencing.Some metabolic enzymes have been reported to translocate into the nucleus, where they regulate telomere silencing. For example, glyceraldehyde 3-phosphate dehydrogenase (Tdh3) and glutamate dehydrogenase 1 (Gdh1) can translocate into the nucleus and maintain telomere silencing25. As shown in Fig. To identify histone modifications regulated by H3T11 phosphorylation (H3pT11), we compared the ChIP-seq data for H3pT11 and the ChIP-seq datasets for other known histone modifications23. When cells were grown in glucose starvation (SD-C) medium, the global H3pT11 was gradually reduced and H3K79me3 was gradually increased -triggered DNA damage response, hydroxyurea (HU)-caused DNA replication block, and different cell cycle phases. However, H3pT11 was not significantly changed in these processes and no anti-correlation was observed between H3pT11 and H3K79me3 30. H2Bub and H4K16ac were not increased in H3T11A mutant  H3 (1-23) peptides. Less Dot1 was pulled down by H3pT11 (1-23) peptide Fig. . We thenTo directly show that H3pT11 inhibits the binding of Dot1 to nucleosomes, we prepared nucleosomes that were phosphorylated by Pyk1 in vitro and then performed the in vitro Co-IP Fig. . SpecifiWe then performed ChIP-seq to examine the effect of H3T11 phosphorylation on the genome-wide occupancy of Dot1 at chromatin. The overall binding intensity of Dot1 at chromatin was significantly increased in H3T11A mutant and significantly reduced in H3T11D mutant when compared with WT Fig. , consistTo examine the effect of H3pT11 on the enzymatic activity of Dot1, we performed in vitro histone methyltransferase (HMT) assay with Dot1 and nucleosomes  purified from yeast cells. Dot1 showed higher H3K79me3 activity towards H3T11A nucleosomes and lower H3K79me3 activity towards H3T11D nucleosomes when compared with WT nucleosomes Fig. . Dot1 shATG) genes23. The expression of ATG genes correlates with the formation of autophagosome and transcriptional regulation of ATG genes represents an important way to control autophagy33. However, it remains unclear how H3pT11 represses ATG gene transcription. By analyzing the RNA-seq data for H3T11A and H3K79A mutants, we found that the overall transcription of ATG genes was increased in H3T11A but reduced in H3K79A mutant  and deleted FPR1 (Δfpr1)28. In contrast, H3K79A and dot1Δ mutants had reduced autophagy flux as indicated by decreased ratio of free GFP/GFP-Atg8  in the vacuoleATG genes. By analyzing our ChIP-seq data, we found that H3K79me3 was increased at ATG genes in H3T11A mutant and decreased in H3T11D mutant  Fig. , loss of21. Indeed, loss of Set1 led to reduced Pyk1 and H3pT11 at ATG genes, which results in increased Dot1 and H3K79me3 at ATG genes  conditions, the occupancy of Pyk1 and H3pT11 at ATG genes was significantly reduced, while the occupancy of Dot1 and H3K79me3 at ATG genes was significantly increased  medium. Nitrogen starvation significantly increased autophagy in WT and mutation of H3T11A further increased autophagy; however, mutation of H3K79A significantly reduced the increased autophagy in H3T11A mutant 36. Cells with repressed URA3 were indicated by their ability to growth on medium containing 5-fluoroorotic acid (5-FOA), which can be converted into toxic 5-fluorouracil (5FU) by Ura3. Interestingly, although H3pT11 antagonizes H3K79me3, the growth of both H3T11A and H3K79A mutants on 5-FOA plate was reduced when compared to their WT counterpart  by anti-FLAG agarose beads from cell extracts. The bound nucleosomes were detected by immunoblots with anti-H3 and anti-H4 antibodies. The results showed that the Co-IPed nucleosomes were reduced in H3T11A mutant and slightly increased in H3T11D mutant  with biotinylated unmodified, H3T11 phosphorylated, or H3K79 trimethylated H3 tail peptides. The SIR complex showed a binding preference for H3pT11 peptide  to the left telomere of chromosome XIII (TEL XIII-L) and H3K79me3 primarily localizes distal  from TEL XIII-L Fig. . Loss ofnts Fig. . Similarnts Fig. .SIR2 under the constitutive strong TEF1 promoter (pTEFpro-SIR2). The transcription of telomere-proximal genes was significantly increased in H3T11A H3K79A mutant; however, overexpression of SIR2 abrogated the increased transcription of these genes in H3T11A mutant  that regulates H3pT11 at telomeres. Although Set1 is required to recruit SESAME to phosphorylate H3T11 at active genes and 47. We first examined H3pT11 in TetO7-REB1 mutant, where the REB1 promoter was replaced with TetO7 and REB1 transcription can be shut off by doxycycline treatment26  analysis revealed the presence of SESAME subunits, Pyk1, Shm2, Sam1, and Acs2 Fig. . We alsoREB1 significantly reduced the occupancy of Pyk1 and H3pT11 at telomeres 29, which is conserved in mammals, where ubiquitination of H2B by RNF20/40 promotes H3K79 methylation by DOT1L48. In addition, the activity of Dot1 is positively regulated by histone acetylation. The histone deacetylase Rpd3 reduces Dot1-catalyzed H3K79me3 and loss of Rpd3 or HDAC1 increases H3K79me324. A more recent study showed that acetylation of H4K16 (H4K16ac) directly stimulates the catalytic activity of Dot1 on nucleosomes30. H4K16ac, together with H2Bub, allosterically enhance methylation of H3K79 by Dot130. H3K79me3 is a very stable histone mark and can exist and accumulate on old histones50. To date, no H3K79 demethylase has been identified in yeast and how H3K79 methylation is negatively regulated remains a mystery. Here, we identify SESAME-catalyzed H3pT11 as a negative regulator of Dot1-catalyzed H3K79me3. The inhibitory effect of H3pT11 is direct, resulting in a reduced substrate binding and low enzymatic activity of Dot1. Therefore, H3pT11 is an important mechanism to counteract Dot1 activity in the absence of H3K79 demethylase. PKM2, the homolog of Pyk1 in mammals, has been reported to phosphorylate H3T11 and play critical role in Mixed-lineage leukemia (MLL) fusions-induced leukemia52. It is possible that the crosstalk between PKM2-catalyzed H3pT11 and DOT1L-catalyzed H3K79me3 plays a role in leukemia.H3K79 is methylated by Dot1 in yeast and DOT1L in mammals, among which DOT1L plays an important role in a subset of leukemias that express an MLL fusion protein caused by rearrangements of the MLL gene (MLL-r)ATG genes represents an important way to control autophagy32. Autophagy is regulated by histone modifications. Under starvation conditions, reduced H2B monoubiquitination (H2Bub) results in the activation of autophagy by controlling the transcription of autophagy regulatory genes53. The histone deacetylase Rpd3 represses the transcription of autophagy genes to inhibit autophagy54. The acetyltransferase hMOF-catalyzed H4K16ac is involved in autophagy regulation55. Shin et al. identified co-activator-associated arginine methyltransferase 1 (CARM1) as a crucial regulator of autophagy in mammals56. Increased histone H3R17 dimethylation by CARM1 is required for proper autophagy activity56. Although we have previously shown that H3pT11 inhibits autophagy by repressing the transcription of autophagy genes, it remains unclear about how H3pT11 represses the transcription of autophagy genes as no H3pT11 reader or effector has been characterized so far. Here, we identify Dot1-catalyzed H3K79me3 as a positive regulator of autophagy by facilitating the transcription of autophagy genes. By inhibiting Dot1-catalyzed H3K79me3, H3pT11 represses the transcription of autophagy genes and reduces autophagy. Since loss of Set2, which catalyzes H3K36 methylation, has no significant effect on autophagy, it is unlikely that H3pT11 represses the transcription of autophagy genes by inhibiting Set2-catalyzed H3K36 methylation. As most autophagy genes are responsible for autophagosome formation and down-regulation of these autophagy genes may lead to reduced autophagic activities, it remains to be determined whether there is a subset of autophagy genes that specifically accounts for H3pT11-mediated repression of autophagy.Autophagy is an evolutionarily conserved cellular process that primarily participates in lysosome-mediated protein degradation. Transcriptional regulation of 54, Dot1-catalyzed H3K79me320, Set1-catalyzed H3K4me321, play important roles in regulation of telomere silencing by preventing the SIR complex from spreading from heterochromatin to euchromatin regions. However, it is unclear about the mechanism to prevent euchromatin invading into heterochromatin regions. Our previous work identified SESAME-catalyzed H3pT11 as a histone mark that is enriched in telomere heterochromatin regions and maintains Sir2 homeostasis23. Here, we show that SESAME-catalyzed H3pT11 promotes the binding of SIR complex to telomere regions, which is consistent with the reports that H3 N-termini 1-23 can be bound by Sir3 and Sir457. Moreover, H3pT11 prevents the mislocalization of H3K79me3 at telomere heterochromatin, which facilitates the establishment of boundary between heterochromatin and euchromatin. Kitada and colleagues reported that the presence or absence of H3K79me3 is the critical determinant of whether the subtelomeric loci are active or silenced3. However, due to lack of H3K79 demethylase, H3K79me3 is not actively demethylated and therefore only diluted by rounds of cell division, which makes it difficult for selective exclusion of H3K79me3 from silent domains58. Here, we find an antagonistic relationship between H3pT11 and H3K79me3. The presence of H3pT11 inhibits the promiscuous occurrence of H3K79me3 at silent chromatin, which compensates for the absence of H3K79 demethylase. Moreover, H3pT11 enhances SIR complex binding at silent chromatin and H3K79me3 prevents SIR binding at active chromatin, which makes SIR complex highly concentrated and restricted in certain areas. As a consequence, H3pT11, inhibition of Dot1 activity and replicative dilution of H3K79me3 synergistically promote SIR complex binding at telomeres and enhance telomeric silencing. The binding of Dot1 to chromatin can positively affect local gene expression by inducing chromatin rearrangements59. Moreover, Dot1 has been reported to regulate nucleosome dynamics60. We cannot exclude the possibility that the combined effect of H3T11A and H3K79A on gene silencing could be due to a destabilization of nucleosomes by these mutations.It has been established that histone modifications, including Sas2-catalyzed H4K16acset1Δ, and H3T11A set1Δ mutants, there was no significant difference between set1Δ and H3T11A set1Δ mutants and no additive effect was observed for H3T11A and set1Δ mutants  were purchased from Yeast Tet-promoters Hughes Collection in Open Biosystems (GE Dharmacon)26. The gene-deletion mutants and genomic integration of C-terminal epitope tags were constructed by homologous recombination of PCR fragments according to standard protocols62. All yeast strains were verified by colony PCR, DNA sequencing, qRT-PCR, and/or immunoblots before being used in experiments. The effect of epitope tags on protein functions was checked by growth assays and immunoblots for histone modifications. The primers used for qPCR are listed in Supplementary Table All yeast strains used in this study are described in Supplementary Table 600 of 0.7. For nitrogen starvation treatment, cells were grown in YPD until OD600 of 0.7 and then harvested at 2000 × g for 5 min. After being washed with sterile double-distilled water (ddH2O) twice, cells were grown in SD-N medium  for 0–1 h. For glucose starvation treatment, cells were grown in YPD until OD600 of 0.7 and then pelleted at 2000 × g for 5 min. After being washed with sterile ddH2O twice, cells were grown in SD-C medium  for 0–6 h. For promoter-shutoff mutants , cells were grown in YPD medium at 30 °C until OD600 of 0.5 and then treated with 40–120 μg/ml doxycycline for 3 h. The knockdown efficiency was determined by RT-qPCR  medium at 30 °C until OD600 of 0.7–1.0 or conditions indicated. Cells were harvested by centrifugation at 2000 × g and lysed in alkaline lysis buffer  2-mercaptoethanol) on ice for 15 min. After centrifugation at 15,000 × g, the protein pellet was resuspended in 100-150 μl 2×SDS-sample buffer. Protein samples were separated by 8–15% SDS-PAGE and transferred to Immobilon-P PVDF membrane (Bio-Rad). The blots were probed with primary antibodies followed by horseradish peroxidase-labeled IgG secondary antibodies. The protein bands were visualized using the ECL Chemiluminescence Detection Kit  and quantified with ImageJ.Cells were grown in 5 ml YPD or selective medium as indicated until OD31.Antibodies against anti-H3 , anti-H4  and H3pT11  were purchased from Abcam; antibodies against H3K79me1 , H3K79me2 , H3K79me3 , anti-His  and anti-FLAG  were purchased from Abclonal; antibody against Sir2  was purchased from Santa Cruz Biotechnology; antibodies against GAPDH , GFP , Myc , goat polyclonal anti-mouse IgG  and goat polyclonal anti-rabbit IgG  were obtained from Proteintech; antibody against FLAG M2  was obtained from Sigma-Aldrich; antibody against H4K16ac  was obtained from EMD Millipore; antibody against CBP  was purchased from Absin Bioscience Inc. The custom-made antibodies against Acs2, Pyk1, Sam1 and Shm2 were produced by Covance Inc. and their specificity was verified by immunoblots of cell lysates of the corresponding histone point mutants, gene deletion or knockdown mutants600 of 1.0–1.5. The samples were crosslinked with 1% formaldehyde and the crosslinking was quenched by adding 10 ml of 2.5 M glycine. After centrifugation, cells were washed with cold washing buffer (TBS + 1 mM PMSF), lysed with glass beads in FA-SDS lysis buffer . Chromatin was sonicated to an average size of ~500 bp and then subjected to immunoprecipitation with anti-FLAG M2 antibody , anti-H3 , anti-H3pT11 , anti-H3K79me3  pre-bound to Protein G Dynabeads (Invitrogen) at 4 °C overnight. The beads were then washed successively with FA lysis buffer, FA buffer + 1 M NaCl, FA buffer + 0.5 M NaCl, TEL buffer  and TE . The eluted DNA/protein complexes were treated with 2 µl Proteinase K (10 µg/µl) to remove proteins at 55 °C for 1 h and then treated at 65 °C overnight. The RNA was removed by digestion with RNase , purified with ethanol precipitation and quantitated by qPCR with primers listed in Supplementary Table Yeast cells were grown in 200 ml YPD media at 30 °C until OD33. The libraries were constructed and sequenced on an Illumina platform33. Reads were aligned to yeast genome sacCer3 from UCSC using bowtie2 version 2.1.0 with parameter -k 1. Telomere repeated sequences were excluded for further analysis. Data was read into R (3.1.0) for further analysis. Peaks were called using MACS2  with parameter -t -c -g 1.2e7 -n -B -q 0.01 -nomodel. Peaks annotation was performed on a website service (https://manticore.niehs.nih.gov/pavis2/). Tracks were smoothed by deepTools2 (v.2.0) and visualized by IGV software (v.2.0) with a reference genome of S. cerevisiae (sacCer3).ChIP-seq was performed with anti-H3K79me3, anti-H3, and anti-FLAG M2 antibodiesFor correlation analyses of Fig. 600 of 0.5–0.7 by standard phenol-chloroform extraction procedures. DNA was removed by digestion with DNase I  for 0.5 h, quantified by Nanodrop 2000 (Thermo scientific) and its integrity was assessed by agarose gel electrophoresis. Generally, 500 ng total RNA was used for reverse transcription PCR (RT-PCR) in a 10 μl reaction volume with Reverse Transcriptase Kit (M-MLV) (ZOMANBIO)33. qPCR was performed with iTaq™ Universal SYBR® Green Supermix  on a real-time PCR machine (Bio-Rad). Primers used for qPCR are described in Supplementary Table 63.Total RNA was isolated from yeast cells grown to an OD64. The whole cell extract was prepared by vortexing with glass beads and then digested with Micrococcal Nuclease (MNase). For TAP-tagged strains, the whole cell extract was incubated with 30 μl of calmodulin beads  for 4 h at 4 °C in calmodulin binding buffer . For other IPs, the whole cell extract was mixed with 30 μl anti-Myc agarose beads (Proteintech), or anti-FLAG affinity gel (Gescript) and incubated for 1 hr to overnight at 4 °C in IP binding buffer . The beads were washed three times with 1 ml calmodulin binding buffer or IP binding buffer. The beads were boiled and the supernatant was subject to immunoblot analysis.Immunoprecipitation was performed as described with minor modifications21. Purified Dot1-FLAG was incubated with 0.2 µg in vitro-assembled recombinant nucleosomes. Dot1-FLAG was then immunoprecipitated with 10 µl anti-FLAG affinity gel (Gescript) and washed with 3 × 1 ml IP washing buffer. Supernatant from the boiled beads was subject to immunoblot analysis.For in vitro immunoprecipitation, Dot1-FLAG was affinity purified by anti-FLAG affinity gel (Gescript) from whole cell lysateXenopus laevis histones H2A, H2B, H3 and H4 were expressed in Escherichia coli BL21 (DE3) and purified by SP ion exchange column. Octamers and nucleosomes were assembled using the salt-dialysis method55.The recombinant 600 of ~0.7, harvested and resuspended in a 500 μl lysis buffer . 1.5 μl zymolyase 20 T was added to digest cell wall at 37 °C for 30 min. MNase was then added to digest chromatin into soluble nucleosomes. After centrifugation, the nucleosome-containing supernatant was collected, quantitated with Super-Bradford Quantification Kit  and stored with aliquots at −80 °C.For purification of nucleosomes from yeast cells, cells were grown in 100 ml YPD medium at 30 °C until ODE. coli and purified by Ni-NTA Agarose (QIAGEN). The TAP-tagged Dot1 (Dot1-TAP) was purified from yeast cells by TAP purification66. For FLAG affinity purification of SIR complex, cells were cultured in 12 L YPD medium and collected at OD600 of 1.5. Cells were resuspended in 10 ml H350 buffer  and then homogenized by Biospec bead beater. After centrifugation at 17,000 × g for 2 h, the cell lysate was incubated with 500 μl pre-washed anti-FLAG Affinity Gel (Gescript) at 4 °C for 4 h. The beads were washed successively with 5 ml H350 buffer and 3 ml E100 buffer . The SIR complex was eluted with E100 buffer containing 0.25 mg/ml FLAG peptide and concentrated using Amicon Ultra-0.5 ml Centrifugal Filters (Millipore).The recombinant 6×His-tagged Dot1 (Dot1-His6) and Pyk1 (Pyk1-His6) proteins were expressed in 2 and 1 mM DTT) with 10 mM SAM at 30 °C for 0–3 h. The reactions were quenched by adding 5×SDS buffer and incubated at 95 °C for 5 min. The products were resolved in a 10% or 18% SDS-PAGE gel and the histone methylation level was determined by immunoblotting with indicated antibodies.The HMT reactions were performed by mixing purified 20 μg Dot1-TAP and 0.5 mg nucleosomes in the reaction buffer , cells were fixed with 4% formaldehyde in PBS for 0.5 h and treated with DAPI for 15 min. Cells were then washed with cold PBS and visualized by fluorescent microscopy using a ZEISS LSM710 microscope (Germany) with a 100× oil immersion objective. Images were acquired using ZEN Imaging Software ZEN 2.1 (ZEISS). The nucleus was indicated by DAPI blue staining. The merged color Images were generated by ImageJ.Yeast cells were cultured in YPD or selective medium until OD67. In brief, 1.0 µg of biotinylated histone peptides were incubated with 1 μg of protein or 0.5 ml whole cell extract in binding buffer  overnight at 4 °C. After 1 h incubation with Streptavidin beads (Amersham), the beads were washed with the binding buffer and boiled for 5 min. The supernatant from boiled beads was subject to immunoblot analysis. The peptides used are the following:The peptide pull-down assays were performed in accordance with the protocol describedUnmodified H3 (1–23): ARTKQTARKSTGGKAPRKQIASK;H3K4me3 (1–23): ARTK(me3)QTARKSTGGKAPRKQIASK;H3pT11 (1–23): ARTKQTARKST(p)GGKAPRKQIASK;Unmodified H3 (72–85): REIAQDFKTDLRFQ;H3K79me3 (72–85): REIAQDFK(me3)TDLRFQ.67. The peptides were eluted with a 5–20% gradient of acetonitrile with 0.1% formic acid over 70 min. The flow rate was 300 nl min−1. The MS1 scans were performed at a resolution of 60,000 over a mass range of 380–1560 m/z, with a maximum injection time of 120 ms and an AGC target of 1 × 106. The MS2 scans were performed at a resolution of 15,000 with a normalized collision energy of 24, a maximum injection time of 50 ms and an AGC target of 2 × 105. The raw mass spectrometry data were searched against the Saccharomyces cerevisiae proteome database from Uniport (https://www.uniprot.org/proteomes/UP000002311) using Sequest HT, MS Amanda and ptmRS algorithms in Proteome Discoverer 2.3 (Thermo Fisher). The precursor ion mass tolerance was set to 10 ppm and the fragment ion mass was 0.02 Da. Dynamic modifications were set for methionine oxidation and phosphorylation on serine, threonine and tyrosine. Only fully tryptic peptides with up to two mis-cleavages and a false-detection rate of 1% using the percolator validator algorithms were accepted. One biological replicate was performed for Reb1-FLAG IP sample.The immunoprecipitated proteins were disulfide-reduced by 25 mM DTT at 37 °C for 40 min. Cysteines were alkylated by adding iodoacetamide to a final concentration of 50 mM. The immunoprecipitated proteins were digested with sequencing-grade trypsin (Promega) at 37 °C overnight and the supernatant was then desalted using C18 columns (Thermo Fisher) and lyophilized. The dried peptides were reconstituted in 0.1% formic acid (FA) and loaded onto an Acclaim PepMap 100 C18 LC column (Thermo Fisher) utilizing a Thermo Easy nLC 1000 LC system (Thermo Fisher) connected to Q Exactive HF mass spectrometer (Thermo Fisher)2O and then resuspended in 900 μl of cold buffer SB . After washing, cell pellets were resuspended in buffer SB plus 2 mM 2-mercaptoethanol and 2 mg/ml Zymolase 20 T and incubated at 30 °C for 0.5 hr. The pellet was washed with sorbitol-containing buffer SB solution and resuspended in 400 μl of lysis buffer  at 4 °C for 1 h. Half of each sample was stored at −20 °C as the whole cell extract. The other half was centrifuged at 12,000 × g for 5 min at 4 °C. The pellet was washed with lysis buffer and resuspended in 200 μl of lysis buffer as the chromatin fraction. Protein distribution in each fraction was detected by immunoblotting with the antibodies against GAPDH (Proteintech) and H3 (Abcam).Log phase cells were harvested, washed with sterile cold Ht-test was used for comparison between two groups and a P value < 0.05 was considered statistically significant. For all error bars, data are mean ±  standard error (SEM). Microsoft Excel  for basic statistical analysis.Representative results of at least two biologically independent experiments were performed in all of the figure panels. The two-sided Student’s Further information on research design is available in the Supplemental informationReporting Summary


Transcription pause-release is an important, highly regulated step in the control of gene expression. Modulated by various factors, it enables signal integration and fine-tuning of transcriptional responses. Mutations in regulators of pause-release have been identified in a range of neurodevelopmental disorders that have several common features affecting multiple organ systems. This review summarizes current knowledge on this novel subclass of disorders, including an overview of clinical features, mechanistic details, and insight into the relevant neurodevelopmental processes. Gene transcription is a highly regulated process that ultimately determines cellular identity and response to external stimuli. Transcription often occurs in bursts  and is pEstablishment and in particular release from pausing is a highly regulated process involving multiple factors that often also act in other phases of the transcription cycle. Central to pause-release is P-TEFb recruitment to paused RNApol2 as part of a complex with BRD4 (bromodomain containing protein 4) or the SEC (super elongation complex), assisted by Mediator and the PAF1 (polymerase-associated factor 1) complex . MoreoveAFF4, encoding a subunit of the SEC, result in the CdLS-related CHOPS (OMIM# 616368)  syndrome , including CdLS (Cornelia de Lange Syndrome) and CdLS-like disorders . Some ofsyndrome . Heterozsyndrome  and NIPB complex . Mutatio complex . These oIn this review we will present current insights into the mechanism and gene regulatory implications of RNApol2 pausing and discuss in detail the involvement of specific pausing regulators in the aetiology of several NDDs.Transcription initiation involves establishment of the preinitiation complex (PIC), comprised of general transcription factors (GTFs) and RNApol2. TFIIH, the last GTF to be recruited to the PIC, is required both for initiation and establishment of promoter proximal pausing (reviewed in RNApol2 pausing is stabilized and shielded from premature termination by the association of DSIF (DRB-sensitivity inducing factor) and NELF (negative elongation factor). Structural studies on the paused elongation complex have shown that NELF stabilizes the paused state by limiting RNApol2 intramolecular mobility and nucleotide triphosphate (NTP) active site entry and by interfering with binding to elongation factors, including TFIIS . DiversiRelease from pausing requires the kinase activity of the P-TEFb complex, consisting of CDK9 and (in most cases) Cyclin T1. CDK9 activity is regulated in a multistep process. First, activation of CDK9 requires phosphorylation of a conserved threonine residue (Thr286) in its T-loop region, primarily catalyzed by TFIIH-subunit CDK7 . Most ofIn vitro, RNApol2 escape from the paused state requires P-TEFb, PAF1c (PAF1 complex) and the elongation factor SPT6  followed by RNApol2 tracking over time determined median pause durations of between 5 and 20 min in se cells . By intrse cells  or on anse cells . InteresThe TFIID complex consists of TATA box-binding protein TBP and TBP-associated factors (TAFs). It is mostly known for its function as a general transcription factor (GTF) during transcription initiation, where it recognizes several core promoter motifs  and, upon promoter binding, recruits RNApol2. Together with other GTFs and RNApol2, the preinitiation complex (PIC) is formed [as reviewed by in vitro assays indicate that the presence of TBP is sufficient for PIC formation  been associated with neurodevelopmental disorders, some have been further studied for their impact in neuronal development. r RanBPM . Taf4a k defects . Moreoveon genes . Taf9b i in vivo .In summary, the high number of TFIID components found associated with neuronal defects upon mutation or loss indicates that misregulation of TFIID broadly impacts neuronal differentiation. Moreover, the CdLS diagnosis for mutations in TAFs implicated in the regulation of pausing warrants further investigation into this link, as will be discussed below.CDK9 resulting in CHARGE -like syndrome (OMIM#214800)  . Five ofin vitro, suggesting that loss of function of CDK9 causes the phenotype  family, together with BRD2, BRD3 and the testis specific BRDT . The broThe BET inhibitors such as JQ1 and I-BET result in chromatin dissociation of BRD4 and subsequent deregulation of global gene expression . Rapid BBRD4 contains a unique interaction domain for P-TEFb  and intein vitro  . SimilarBrd4 is predominantly expressed in neurons, where it regulates immediate early gene (IEG) expression , caused by loss of function of the X-linked gene encoding MeCP2 (methyl-CpG binding protein 2). Increased chromatin binding of BRD4 in in vitro differentiated human RTT interneurons, and in MGE  and cortex mimicking RTT organoids, caused extensive transcriptional dysregulation that was reverted upon exposure to JQ1. Importantly, JQ1 treatment of RTT modeling –/YMeCP2 mouse pups improved short term survival and slowed down phenotypic progression.Dysregulation of BRD4 has been causally linked to Rett Syndrome  and fragCollectively, these studies highlight the importance of BRD4 dosage during neurodevelopment. They also underscore the feasibility of postnatal phenotypic reversal of some aspects of NDDs and suggest that at least part of the pathology results from aberrant gene regulation in fully differentiated postmitotic neurons. Rebalancing of transcription pause regulation and elongation can thus be used as a therapeutic strategy to ameliorate symptoms related to NDDs.NIPBL (Nipped-B-like) encodes for the protein delangin, the human homolog of fly Nipped-B protein and fungal sister chromatid cohesion protein 2 (SCC2), which together with SCC4 forms a complex that is necessary for cohesin loading onto chromosomes. Recent studies physically and functionally linked NIPBL to the regulation of transcriptional pausing . Here, tGene variants in cohesin core components and regulatory proteins are identified as the cause of CdLS , a dominant and genetically heterogeneous neurodevelopmental disorder with physical, cognitive and behavioral characteristics . CdLS prNIPBL allele is frequently observed and a reduction of ∼15% in expression is enough to observe a clinical phenotype classic cases with overlapping and often milder phenotypes . Heterozhenotype . Furtherpatients .+/– lymphoblastoid cells (LCLs), +/Nipbl– mouse embryonic fibroblasts (MEFs), and fetal liver cells exhibit reduced global or local cohesin binding and defective 3D genome organization  and consequently for normal cortical neuron migration in vivo. Defects in neuronal migration and subsequent aberrant neuronal positioning disrupt neural circuit formation and have been causally linked to intellectual disability and seizures, both features of CdLS  comprises various elongation factors and incorporates active P-TEFb. The AF4/FRM2 family (AFF) forms the scaffold; the canonical SEC contains either AFF1 or AFF4, whereas SEC-like complex 2 and 3 contain AFF2 or AFF3, respectively. These scaffolding proteins interact with 11—19 lysine-rich leukemia (ELL) 1, ELL2 or EEL3, the ELL-associated factor EAF1 or EAF2 and eleven-nineteen leukemia (ENL) or AF9 .Initially, many of the SEC components were identified as translocation partner of the mixed lineage leukemia (MLL) gene . Upon MLHOX genes  (AF4/FRM2 family member 4 (AFF4) is essential for SEC stability and proper transcription in metazoans . Initial 616368) .AFF4 leading to CHOPS syndrome , also known as AFF3, is also an MLL fusion partner . Like thXIST . Aff3 ovXIST . DysreguXIST  and breaXIST .AFF3 gene promoter , encompassing a CGG repeat expansion called FRA2A, in the promoter . Hypermepromoter . FRA2A he skills .To date, AFF3 deletions (2) and missense (16) variants have been identified in 18 individuals with developmental delay and intellectual disability . InteresAFF3 gene silencing. This could be explained by a lack of AFF3 inactivation in the first few weeks after fertilization , often referred to as FMR2, is an X-linked gene and known to encode a transcription activator  of exon 1. CGG repeat expansion at this site leads to AFF2 silencing and can result in FRAXE intellectual disability . FRAXE IAFF2 can also cause a similar phenotype  and cortical plate of the mouse cortex  (e cortex . Aff2 knpocampus . Furtheractivity  and sile and FOS . Dysregutations) , suggestAF9, also known as MLLT3, is one of the most common fusion partners of the MLL gene, and is often associated with leukemia  were identified to cause a disruption of the AF9 gene, resulting in neurodevelopmental delay with intellectual disability, growth delay, seizures and ataxia  in the subventricular zone (SVZ)  . In humane (SVZ) .Taken together, these studies indicate that various SEC components have an important role in neural development, albeit not exclusively in the context of the SEC. Comparing patient phenotypes and commonly deregulated pathways should reveal the contribution of pausing dysregulation to the neurological symptoms.The Integrator complex is a > 1 MDa protein complex that is conserved across metazoans and consists of 14 subunits . It can Drosophila and human cells. Furthermore, association with protein phosphatase 2A (PP2A) provides Integrator with an alternative catalytic function to dampen transcriptional output through dephosphorylation of the RNApol2 CTD and DSIF-subunit SPT5  and 1B (ARID1B) are one of the main, mutually exclusive subunits of the switch/sucrose non-fermentable (SWI/SNF)-like brahma-associated factor (BAF) complex, a multiprotein ATP-dependent chromatin remodeling complex composed of conserved core- and variant subunits . SWI/SNFARID1A and ARID1B are also an important cause of Coffin-Siris Syndrome , a rare autosomal-dominant neurodevelopmental syndrome (ARID1A microduplications have been described (ARID1B mutations have been found in up to 62% of (often milder) CSS cases and also explain a significant fraction  of idiopathic ID cases that are often accompanied by speech impairment and agenesis of the corpus callosum hallmarks  is a multifunctional and highly conserved protein complex that regulates all stages of the RNA transcription cycle [recently reviewed in ears ago , and foupression . PAF1c ipression . In highpression .In recent years, the role of PAF1c in regulation of RNApol2 pause-release has been studied in detail. PAF1c strongly associates with P-TEFb and both factors show interdependent recruitment to target gene promoters . RNAi-mePathogenic variants in genes encoding subunits or interactors of PAF1c have been identified in various neurodevelopmental processes and ID disorders. Most well-described are mutations in the X-linked gene encoding the PHD-Like Zinc Finger Protein 6 (PHF6) that associates with PAF1c and causes the ID disorder Börjeson–Forssman–Lehmann syndrome  . BesidesPhf6 is highly expressed in embryonic and early postnatal stages of mouse brain development   . SETD5 h#602118) . Setd5 d#602118) . SETD5 h and ASD  and have and ASD . Setd5 h and ASD .The Mediator complex interacts with different TFs and is implicated in almost every aspect of transcription regulation [reviewed by In vitro, Mediator overcomes the inhibitory activity of Gdown1 , Ohdo syndrome (OMIM# 300895), and Lujan–Fryns syndrome (OMIM# 309520)  . These t 309520) , show sy 309520) . These eMed12 null mice , MED23, MED25, and MED27 (tail) have also been found to cause neurodevelopmental disorders upon mutation. In seven individuals, biallelic mutations of MED23 were identified to cause ID . A homoz and FOS .MED25 missense mutations  now provide an excellent opportunity to uncover the disease-relevant neurodevelopmental pathways typically affected by dysregulated pause-release. We envision that this knowledge can directly translate into improved diagnostics in the clinic, by providing evidence for gene variant causality and through transcriptome-based diagnostics. Finally, as transcription pause-release is a process amenable to drug intervention, it forms a potentially promising target for drug-mediated therapeutic intervention in NDDs.Shared clinical features and diagnosis (notably the frequent occurrence of CdLS-like characteristics) suggest a common underlying mechanism. In this respect it will be interesting to investigate a possible role in the regulation of transcription pause-release for additional factors that, when mutated, result in a CdLS-like appearance . Indeed, a link between KMT2A and transcriptional pausing has been suggested in HIV latency reversal, where KMT2A displaces PAF1c and facilitates SEC recruitment . FollowiAll authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The dynamic changes in chromatin conformation alter the organization and structure of the genome and further regulate gene transcription. Basically, the chromatin structure is controlled by reversible, enzyme-catalyzed covalent modifications to chromatin components and by noncovalent ATP-dependent modifications via chromatin remodeling complexes, including switch/sucrose nonfermentable (SWI/SNF), inositol-requiring 80 (INO80), imitation switch (ISWI) and chromodomain-helicase DNA-binding protein (CHD) complexes. Recent studies have shown that chromatin remodeling is essential in different stages of postnatal and adult neurogenesis. Chromatin deregulation, which leads to defects in epigenetic gene regulation and further pathological gene expression programs, often causes a wide range of pathologies. This review first gives an overview of the regulatory mechanisms of chromatin remodeling. We then focus mainly on discussing the physiological functions of chromatin remodeling, particularly histone and DNA modifications and the four classes of ATP-dependent chromatin-remodeling enzymes, in the central and peripheral nervous systems under healthy and pathological conditions, that is, in neurodegenerative disorders. Finally, we provide an update on the development of potent and selective small molecule modulators targeting various chromatin-modifying proteins commonly associated with neurodegenerative diseases and their potential clinical applications. They are further packaged into higher-order chromatin structures. The location and structure of nucleosomes affect the function of chromatin. In most eukaryotic cells, chromatin exists in a highly compacted form, which is referred to as heterochromatin. This highly folded chromatin structure is necessary for its packing into the nucleus but limits access of gene promoter regions to the binding of various proteins, such as transcriptional machinery. Chromatin may also adopt a more relaxed state, referred to as euchromatin, which is open to modifications and transcriptional processes. Chromatin remodeling involves a dynamic interchange of chromatin between a condensed state and a transcriptionally accessible state, allowing the regulatory transcription machinery proteins to access condensed genomic DNA and control gene expression. Therefore, it is a vital process to regulate important physiological functions and maintain cellular homeostasis, and impairment of chromatin remodeling machinery leads to the progression of various diseases due to accumulated epigenetic abnormalities.The nucleosome, consisting of 147 bp of double helix DNA wound around a histone octamer core that possesses one (H3-H4)Eukaryotes have evolved a large family of chromatin remodeling enzymes and related protein factors that alter the location and structure of chromatin. These various chromatin regulatory factors are thus critical determinants of access to genomic loci by the transcriptional machinery, and they function diversely in normal tissues and disease contexts. Consequently, chromatin remodeling is essential for the basal maintenance of the structural homeostasis of chromatin as well as for establishing gene expression patterns , 2. In aA better understanding of the mechanisms of chromatin remodeling will foster the development of new drug targets for improved pharmaceutical interventions. Basically, the mechanisms underlying chromatin remodeling can be divided into two categories: covalent modification of chromatin components  and noncovalent modifications of ATP-dependent chromatin remodeling complexes (CRCs) –10. The Histones, predominantly globular proteins with flexible N- and C-terminal tails, are once considered to be static and nonparticipating structural elements, but the bias is clarified as research reveals that histones are dynamic regulators responsible for gene transcription. The electrostatic interactions between the positively charged histone tails and negatively charged DNA make chromatin in a highly condensed state. Diverse posttranslational modifications (PTMs) occur in the tail region of histones. Epigenetic modifications to histone proteins, including but not limited to acetylation, methylation, phosphorylation, ubiquitination, sumoylation, crotonylation and glycosylation, influence transcriptional access to genes by changing chromatin structure or recruiting histone modifiers. When histones are modified in any way mentioned above, they endure changes in charge characteristics and binding sites . CurrentHistone acetylation is one of the most common and best-studied histone PTMs and involves the addition of an acetyl group to the lysine residues at the amino-terminal tails of histone proteins. The balance between acetylation and deacetylation of histones is reversibly controlled by histone acetyltransferase (HAT) and histone deacetylase (HDAC). Generally, histone acetylation mediated by HATs leads to the relaxation of chromatin structure and facilitates gene activation, whereas deacetylation of lysine residues on histone tails mediated by HDACs results in a more closed chromatin structure and represses gene expression. However, both HATs and HDACs have been found to exist in some large protein complexes, suggesting that a great deal of fine-tuning occurs at the level of DNA and the addition and subtraction of acetyl groups. Most HATs function as transcriptional coactivators. In humans, there are approximately 30 known HATs, which have been classified into two general categories based on their subcellular localization: Type A and Type B. Type A HATs, which contain a bromodomain, are predominantly localized in the nucleus and acetylate nucleosomal histones and other chromatin-associated proteins. Type B HATs, which lack a bromodomain, are mostly found in the cytoplasm and acetylate free newly synthesized histones in the cytoplasm prior to incorporation into chromatin. In mammals, the HDAC family consists of 18 members that can be divided into four classes  and histone demethylases (HDMs). Methylation occurs primarily on at least 11 lysine and arginine residues in histones, with the lysine residues being mono-, di- and trimethylated and the arginine residues being mono-, symmetric di- and asymmetric di-methylated. Thereby, HMTs and HDMs not only have a high degree of specificity for the specific lysine/arginine but also strictly regulate the degree of methylation of their substrates and products. HMTs comprise approximately 59 proteins . There aHistone ubiquitination influences chromatin dynamics and exhibits crosstalk with other histone modifications , 16. H2AHistone phosphorylation, which is controlled by kinases and phosphatases, usually takes place on serine, threonine and tyrosine residues. The majority of histone phosphorylation occurs in the N-terminal tails of H3, followed by H2B, H2A, H4 and H1. H3 S10 phosphorylation, which can be intact with H3 acetylation/methylation, is vital for chromosome condensation and transcriptional activation of eukaryotic genes in various organisms. The phosphorylation of histones also affects the level of ubiquitination of H2B at K120 .DNA methylation, the major form of DNA modification, which involves the covalent addition of a methyl group (-CH3) to the 5´ carbon of cytosine residues (5mC) predominantly in the context of CpG dinucleotides, is generally associated with repressed regions of the genome. DNA methylation is mediated by DNA methyltransferases (DNMTs) that catalyze the transfer of the methyl group from S-adenosylmethionine onto cytosine . DNMT1 aDNA methylation carries out distinct functions in different genomic regions. It is associated with gene repression in mammals by attracting or repelling various DNA-binding proteins. DNA methylation can prevent transcription by inhibiting the binding of transcription factors to target sites. On the other hand, it can suppress gene transcription by recruiting repressor complexes to methylated promoter regions. Recently, it is reported that DNA methylation can also induce gene activation when it replaces H3 lysine 27 trimethylation (H3K27me3), a histone modification mediated by the polycomb repressive complex (PRC) and relates to gene silencing . DNA metDNA demethylation is realized by either an active pathway involving the dioxygenases called ten-eleven translocation (TET) proteins, including TET1, TET2 and TET3, to remove the methyl group from 5mC, or a passive pathway by lack of maintenance methylation (mediated by DNMT1) during cell divisions . In the In addition to covalent modifications, the chromatin architecture and access can also be modulated by noncovalent chromatin modifications involving ATP-dependent CRCs. CRCs are evolutionarily conserved multisubunit assemblies of ATP-dependent remodeling enzymes. Based on the sequence and structure of the ATPase subunit, CRCs have been grouped into four main families: switch/sucrose nonfermentable (SWI/SNF), inositol-requiring 80 (INO80), imitation switch (ISWI) and chromodomain-helicase DNA-binding protein (CHD), each representing a specific remodeling pathway with distinct subunits and functions . Other uMammalian SWI/SNF (mSWI/SNF) complexes, canonical BRG1/BRM-associated factor (BAF) and polybromo-associated BAF (PBAF) and newly defined noncanonical BAF (ncBAF), are composed of up to 15 core subunits, with an interchangeable core ATPase subunit (BRG1 or BRM) and different BAF subunits . Among tThe INO80 remodeler has been identified in yeast, flies and mammals and is present as several complexes. The mammalian complexes are INO80, SRCAP (yeast SWRI or SWR-C) and TRAAP/Tip60 (yeast NuA4). Due to the presence of an insertion in the ATPase domain to which the subsequently recruited helicases (Ruvbl1 and Ruvbl2) could bind , the catSmarca5 and Smarca1   martinostat, a PET tracer that recognizes class I HDACs in the brain, revealed global reduced HDAC I levels in AD-affected brain regions where amyloid and tau are high [The role of HDACs and the change in histone acetylation in AD have been an intense field of research. Dramatically decreased level of brain H4 acetylation has been observed in different mouse models of AD, which can be restored by HDAC inhibitors –168. In are high . HoweverChanges in histone methylation have been reported in postmortem brain tissues from human donors with AD , suggestParkinson's disease (PD) is the second most common age-related neurodegenerative disease and is characterized by involuntary resting tremor, stooped posture, weakness of the limbs and some nonmotor symptoms, such as cognitive impairment and sleep disorders. It is associated with degeneration of dopaminergic neurons due to intracytoplasmic inclusions composed of aggregated α-synuclein, ubiquitin-protein and damaged nerve cells. Most PD cases are sporadic and do not have a clear genetic link. Increasing evidence supports that epigenetic changes are involved in PD progression. However, current studies show large inconsistencies in histone acetylation and HAT/HDAC changes in PD patients. Histone acetylation is disease-dependently altered in PD . α-SynucHuntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine repeat expansion within the huntingtin protein, and transcriptional dysregulation has been proposed to be an early event in HD pathogenesis. Recent studies have demonstrated that mutant huntingtin alters HDAC activity, the dysfunction of which might be an underlying mechanism of transcriptional dysregulation in HD. In addition, other epigenetic modifications, such as histone methylation, ubiquitination, phosphorylation, DNA modifications, and the expression of histone variants, have all been implicated in HD –187. ForALS is the third most common neurodegenerative disorder and is characterized by the selective loss of both upper and lower motor neurons. Approximately 10% of all ALS patients suffer from familial ALS that is associated with a number of genetic variants, including superoxide dismutase 1 (SOD1), fused in sarcoma (FUS), TAR DNA binding protein 43 (TDP-43), and chromosome 9 open reading frame 72 (C9orf72). Recent evidence shows a link between ALS and epigenetics. The global changes in histone acetylation have been characterized in yeast ALS proteinopathy models. Yeast overexpressing human FUS results in hypoacetylation of H3K14 and H3K56, while yeast overexpressing TDP-43 reveals hyperacetylation of H4K12 and H4K16, suggesting that each proteinopathy shows distinct changes in histone modification profiles . In addiDNA methylation, the best-known epigenetic mark, has attracted much attention in the pathogenesis of age-related neurological disorders , 204. DyAside from histone modifications and enzymes targeting histone modifications, some ATP-dependent CRCs have also been associated with neurodegenerative disease. It should be noted that, given the essential roles of CRCs in neurogenesis and neurodevelopment, it is not surprising that abnormal function of subunits of these CRCs will elicit neurodevelopmental diseases. For instance, studies have revealed that heterozygous mutations in distinct subunits of the BAF complex will lead to many neurodevelopmental disorders that show similar intellectual impairment . PathogeAs mentioned above, proper chromatin regulation is essential for gene expression in the brain, and imbalance of neuronal chromatin structure and the ensuring changes in gene expression are detrimental to brain functions. Chromatin remodeling can be accomplished by covalent modification of histones or by the action of ATP-dependent remodeling complexes. Some histone modifications directly contribute to chromatin structure, and enzymes, including those directly affecting histone modifications and ATP-dependent CRCs, can also function in the regulation of chromatin structure. Notably, other factors may also be included, such as PARP1-mediated chromatin remodeling. Since derangement of these various epigenetic mechanisms of chromatin remodeling has been implicated in several neurodegenerative diseases, drugs targeting all these underlying epigenetic defects might be useful as therapeutic agents in various neurodegenerative diseases.In the postgenomics epigenetic era, because epigenetic mechanisms are pharmaceutically accessible and largely reversible, inhibitors of chromatin remodeling factors, such as epigenetic drugs, have made great progress. Currently, 14 types of epigenetic drugs have been approved. Most small molecule inhibitors are in the clinical stage for cancer treatment, and some inhibitors that are used for the treatment of neurodegenerative diseases are in the early clinical stage. Based on the targeted substrates, these small molecule modulators may be divided into different groups. In this section, we will give an overview of the recent progress that has been made in this field Table .Table 3THDAC inhibitors are small molecules that have been identified from natural sources and developed synthetically. They restrain HDAC activity and maintain chromatin in the decondensed state, which further affects transcription by inducing acetylation of histones, transcription factors and other proteins regulating transcription. These HDAC inhibitors (HDACi) vary in structure, specificity and biological activity and can be divided into four classes based on the chemical structure, which include hydroxamic acid derivatives, short-chain fatty acid derivatives, synthetic benzamides, and cyclic peptides. While the majority of HDACi work broadly on all HDAC isoforms but not the SIRT enzymes (pan inhibitors), certain HDACi may selectively inhibit specific HDACs. To date, 20 different HDACi have been evaluated in clinical trials for the treatment of a broad range of cancers , 226. AmAs mentioned above, impairments in HDAC functions and associated pathways as well as transcriptional dysfunctions have been implicated in the pathogenesis of AD, PD, HD, ALS and other neurodegenerative diseases , 229. FoHydroxamic acids are the largest class of HDACi, mainly including SAHA, LBH589, PXD101, trichostatin A (TSA), dacinostat (LAQ824), m-carboxycinnamic acid (CBHA), azelaic bis-hydroxamic acid (ABHA), givinostat (ITF2357) and PCI-34051. Notably, three of the six anticancer HDAC inhibitors approved by the FDA are hydroxamic acids, including SAHA, LBH589 and PXD101.Drosophila models of PD shows that administration of SAHA restores α-synuclein-dependent neurotoxicity due to an interaction between histones and α-synuclein [As a class I/II HDAC inhibitor, SAHA has been reported to ameliorate the progressive neurodegeneration involved in different models  of various neurodegenerative disorders, such as AD , PD 179179, 238,ynuclein . In a phLBH589, as a novel broad-spectrum HADC (class I/II/IV) inhibitor, is a cinnamic hydroxamic acid analog that is approved by the FDA in 2015. As LBH589 is an orally bioavailable HDACi and is at least tenfold more potent than SAHA, its therapeutic potential for neurodegenerative diseases has been assessed. LBH589 has been shown to cross the blood‒brain barrier, induce histone hyperacetylation and ameliorate neuropathological phenotypes in two preclinical mouse models of HD . Two yeaTSA, a natural derivative of dienodydroxamic acid, is an antifungal antibiotic that is reported to selectively inhibit class I/IIb HDAC. TSA has also been shown neuroprotective effects in many studies. TSA treatment in APP/PS1 mice inhibits the reduction of H4 acetylation, leading to an increase in memory formation . TSA shoThe second class of HDACis are short-chain fatty acids, which mainly include sodium butyrate (NaBu), valproic acid (VPA), phenylbutyrate (4-PBA), and AR-42. Compared with hydroxamic acid-based HDACis, short-chain fatty acids have good penetration into the brain, making them potential candidates for treating neurodegenerative diseases. VPA, NaBu and 4-PBA have validated therapeutic utility in the treatment of different neurodegenerative disorders, such as AD, PD, HD, and SMA .NaBu, one of the first discovered HDACis, belongs to the short-chain fatty acid class of HDACis. NaBu is a very potent HDACi of class I/II HDAC enzymes. Because NaBu is capable of crossing the blood‒brain barrier and increasing the level of H3 acetylation in the brain, its potential use in the intervention of neurodegenerative diseases  is of loVPA is a short-chain fatty acid that inhibits both class I and II HDACs. The therapeutic ability of VPA has been demonstrated in different animal models of AD, PD and ALS . Early dAnother short-chain fatty acid drug, 4-PBA, is a derivative of the aromatic fatty acid butyrate. In the Tg2576 AD mouse model, daily injection of 4-PBA is reported to reverse spatial memory impairment by normalizing tau hyperphosphorylation in the hippocampus, with restored hippocampal H4 acetylation and dendritic spine density defects . In a stApart from hydroxamic acids and short-chain fatty acids, benzamide-containing compounds represent another class of HDACi with clinical potential. Entinostat MS-275) is a synthetic benzamide derivative that blocks the activity of HDAC Class I (isoform 1/2/3). Of note, oral administration of entinostat reduces amyloid plaque deposition in the hippocampus and cortical areas of a transgenic mouse model of AD . RGFP966 is a synNicotinamide (NAM), an inhibitor of class III HDACs that readily crosses the blood‒brain barrier, is found to be effective in preclinical Friedreich's ataxia (FA) models and has been tested in clinical trials in FA patients for dose escalation (NCT01589809). NAM is described as a protective agent in PD mainly through the activation of SIRT1 . RecentlThe cyclic peptide-based class represents the most potent and most structurally complex group of HDACi, comprising both epoxyketone- and nonepoxyketone-containing tetrapeptides. Cyclic peptide HDACis mainly include romidepsin (FK228), apicidin, trapoxin, depudesin, CHAP, spiruchostatin A and largazole. FK228, a bicyclic class I selective HDACi, is the first FDA-approved peptide-based drug for the treatment of cutaneous T-cell lymphoma (CTCL) and PTCL. As an HDAC1 and HDAC2 inhibitor, FK228 has been shown to play an essential role in cognitive loss in AD , 282.Other HDACis are miscellaneous based on chemical structure. A novel class II HDACi, mercaptoacetamide-based compound W2, has been found to reduce Aβ levels and improve cognition in 3 xTg AD mice . CM-144 CKD family members have been exploited recently by Chong Kun Dang Pharmaceutical Corp in Korea. Among them, CKD-510 is currently in a phase I trial to treat Charcot-Marie-Tooth disease (NCT04746287), an inherited neurological disease characterized by a slowly progressive degeneration of the muscles in the feet, legs and hands. CKD-504, a specific inhibitor of HDAC6, is currently in a phase I trial to treat HD (NCT03713892). Moreover, CKD-504 can reduce pathological tau protein and rescue memory impairment of AD mice by effectively regulating global acetylation in brain .Resveratrol (RVT), a phytoalexin produced naturally by several plants, modulates several epigenetic targets, including HDAC, DNMT and LSD1 . RVT-medThe mechanism of action of HDACi in the treatment of neurodegenerative disorders is currently unclear. Notably, there have been studies that show contradictory effects of HDACi. For example, Wang et al. find that TSA increases dopaminergic cell death caused by MPP + or rotenone treatment . In anotAlthough clinical evidence shows that most HDACi have an acceptable toxicity profile, they lack specificity and may show substantial adverse effects. Due to limitations associated with pan-HDAC inhibitors, the development of isoform-specific HDAC inhibitors would lead to fewer side effects and more selective inhibition. In addition, to enhance the therapeutic efficacy of HDACis, combination therapy might also be an important direction in the future.A mounting number of studies suggest that histone methylation may have a crucial role in the pathophysiology of many neurodegenerative diseases. Aberrant histone methylations, such as elevated levels of H3K9me2 , 297 andHDMs mainly include the LSD1 and JmjC families. With the participation of FAD, LSD1 not only specifically removes the dimethyl and monomethyl modification of H3K4 in vitro but can also mediate H3K9me demethylation in vivo . Among tIn addition, histone methylation-mediated gene regulation has been recognized to be essential in the pathophysiology of various neurodegenerative diseases, such as the α-synuclein coding gene SNCA in PD and C9ORF72 in ALS. However, how specific histone methylation of the genes regulates gene expression needs further study.Accumulating evidence implicates the important functions of CBP and p300 in the brain. Unfortunately, studies based on their direct pharmacological activation are still missing due to the lack of cell-permeable activators. A previous study shows that TTK21, a small molecule activator of CBP/p300 conjugated to glucose-based carbon nanospheres (CSPs), can cross the blood‒brain barrier and reach different parts of the brain without causing toxicity . CSP-TTKThe first study investigating the potential of HAT activators is conducted in a cellular model of PD . CTPB  inhibitor. Studies of the therapeutic potential of JQ-1 in multiple mouse models of AD have led to conflicting reports , 314. UsApart from AD, the effectiveness of JQ1 in other neurodegenerative diseases has also been explored. For example, chronic JQ1 administration is found to suppress the induction of levodopa-induced dyskinesia in rats, an animal model for PD . Several40 in patients [40 transport from the brain and showing its therapeutic potential for the treatment of AD.RVX-208, a BD2-selective BET inhibitor, is demonstrated to be safe and well tolerated in a phase Ib/IIa study (NCT01058018). An exploratory phase Ia trial is conducted to evaluate RVX 208 for the treatment of AD. Despite only 24 study subjects, it has demonstrated a 12–14% increase in plasma levels of Aβpatients , supportTogether, these studies support the view that BET proteins play important roles in many distinct neurodegenerative diseases and that modulation of BET activity may hold therapeutic promise in the management of these disorders.90, cryo-electron microscopy and quantitative mass spectrometry will be needed to answer such questions. Applying these state-of-the-art techniques would provide new insight into how chromatin remodeling functions during neurodevelopment in health or disease.Chromatin remodeling is an absolute requirement for gene expression and is thus a vital process in regulating many fundamental biological pathways. In addition, chromatin can be regulated by several processes. These mechanisms function individually and in concert to modulate gene expression. Although tremendous progress has been made in the field of chromatin regulation over the past decade, the mechanisms of the remodeling process are far from being understood and will constitute a fascinating area of research in the future. Particularly, new techniques, such as RNA sequencing (RNA-seq), assays for transposase accessible chromatin using sequencing (ATAC-seq) and others, would prompt important discoveries of chromatin modifications and functions as well as mutations in genes involved in chromatin regulation at a genome-wide level. On the other hand, although previous biochemical and structural studies are informative for the chromatin remodeling mechanisms, detailed studies are needed to understand how remodelers recognize and interact with their targets and the underlying structural basis of chromatin remodeling. New probing techniques such as chromosome conformation capture (3C)Mounting evidence has shown that many neurodegenerative diseases are linked to chromatin deregulation, thus sparking significant efforts to develop new small molecules targeting chromatin remodeling. An increasing number of small molecule inhibitors against a variety of epigenetic regulators have proven effective in the treatment of various neurodegenerative diseases. However, there are still many challenges to overcome. For instance, one major disadvantage of these drugs is their broad specificity and potentially adverse side effects. Furthermore, how can the resistance abilities and blood‒brain barrier penetration of some of these epigenetic drugs be improved? What are the mechanisms of certain inhibitors that cause clinical effects? Only with an improved understanding of the mode of action of these inhibitors can we develop selective, safe and efficacious therapeutic epigenetic drugs. For AD, PD, HD, and ALS, the transfer of promising therapeutic approaches into clinical applications will depend on early-stage treatment. In this regard, compounds that are effectively able to restore distinct aspects of adult neurogenesis might be of specific interest for future clinical studies. Specifically, improving neuropsychiatric and severely disabling symptoms is an urgent need and will have a major impact on the quality of life for all patients suffering from these devastating disorders.