Cnp1 chromatin assembly at the centromere in a redundant manner with another chromatin-remodeling factor Chd1Hrp1. CENP-ACnp1 chromatin actively recruits the Ino80 complex to centromeres to elicit eviction of histone H3-containing nucleosomes. Artificial targeting of Ino80 subunits to a non-centromeric DNA sequence placed in a native centromere enhances the spreading of CENP-ACnp1 chromatin into the non-centromeric DNA. Based on these results, we propose that CENP-ACnp1 chromatin employs the Ino80 complex to mediate the replacement of histone H3 with CENP-ACnp1, and thereby reinforces itself.The centromere is the chromosomal locus at which the kinetochore is assembled to direct chromosome segregation. The histone H3 variant, centromere protein A (CENP-A), is known to epigenetically mark active centromeres, but the mechanism by which CENP-A propagates at the centromere, replacing histone H3, remains poorly understood. Using fission yeast, here we show that the Ino80 adenosine triphosphate (ATP)-dependent chromatin-remodeling complex, which removes histone H3-containing nucleosomes from associated chromatin, promotes CENP-A The histone variant CENP-A marks active centromeres and replaces H3 at centromeres through a poorly understood mechanism. Here, the authors provide evidence that the chromatin remodeller Ino80 promotes CENP-A chromatin assembly at the centromere in fission yeast. At human centromeres, CENP-A replenishment occurs during G1 phase6 and is preceded by the replication-independent loss of histone H3.3 incorporated during S phase7. This suggests that centromeres possess a mechanism that actively removes histone H3.3 to make room for CENP-A deposition. The fission yeast, Schizosaccharomyces pombe, has epigenetically defined “regional centromeres” whose chromatin and protein compositions are similar to those of their human counterparts10. In fission yeast, CENP-ACnp1 replenishment at centromeres occurs during G2 phase in a DNA replication-independent manner11, and histone H3 competes with CENP-ACnp1 for incorporation into centromeres12. This suggests that active removal of histone H3 may be a conserved mechanism required for CENP-A deposition at fission yeast centromeres, and makes the fission yeast an attractive genetic model for studying how histone H3 is replaced by CENP-A at regional centromeres.In most eukaryotes, centromeres are epigenetically specified by chromatin containing the centromere-specific histone H3 variant, centromere protein A (CENP-A). A model of self-propagating centromeres suggests that pre-established CENP-A chromatin at the centromeres is sufficient to direct replenishment of new CENP-A during the cell cycle17. Thus, they are good candidates to be factors through which centromeres induce the removal of histone H3. In support of this possibility, ATP-dependent chromatin-remodeling factors have been implicated in the assembly or maintenance of centromeric chromatin. In Saccharomyces cerevisiae, which has genetically defined “point centromeres” consisting of a single CENP-ACse4 nucleosome per centromere, the Ino80 (inositol-requiring mutant 80) ATP-dependent chromatin-remodeling complex acts to maintain normal chromatin structure at centromeres18. However, the Ino80 complex in S. cerevisiae contributes to the integrity of pericentric chromatin rather than the assembly of CENP-ACse4 nucleosome18. In humans, RSF1 (remodeling and spacing factor 1) localizes to centromeres and promotes the stable association of CENP-A with centromeres19. A recent study showed that RSF1 can stimulate histone exchange accompanied by CENP-A deposition when artificially targeted to ectopically inserted α-satellite DNA20. In addition to RSF1, the conserved ATP-dependent chromatin-remodeling factor, CHD1 (chromodomain helicase DNA binding protein 1), was found to stimulate the deposition of CENP-ACnp1 at centromeres in human and fission yeast22. In these studies, however, RSF1 and CHD1 were found to be only partially involved in the deposition of CENP-A at the centromeres. This raises the possibility that other chromatin-remodeling factor(s) are also responsible for regulating CENP-A chromatin assembly at regional centromeres. Also, it remains unclear whether the critical role played by chromatin-remodeling factors at the centromeres is related to the exchange of histone H3 with CENP-A.Adenosine triphosphate (ATP)-dependent chromatin-remodeling factors can disassemble nucleosomes and, in some cases, induce the exchange of histone variantsCnp1 chromatin assembly at the fission yeast centromeres in a redundant manner with Chd1Hrp1, and the role of the Ino80 complex is linked to removal of histone H3-containing nucleosomes. Consistent with a direct role in CENP-ACnp1 chromatin assembly, the Ino80 complex is associated with centromeric regions in a CENP-ACnp1-dependent manner and when tethered to a non-centromeric DNA inserted in an endogenous centromere promotes spreading of CENP-ACnp1 chromatin into the non-centromeric DNA. Thus, CENP-ACnp1 chromatin utilizes the Ino80 complex together with Chd1Hrp1 to mediate replacement of histone H3 with CENP-ACnp1 at regional centromeres of fission yeast.To address these questions, here we systematically analyze the roles of the ATP-dependent chromatin-remodeling factors in the centromeric chromatin assembly of fission yeast. We find that the Ino80 complex plays an important role in CENP-ACnp1 at centromeres, we first examined whether any of the fission yeast chromatin-remodeling factors could affect CENP-ACnp1 chromatin at centromeres. The silencing of ura4+ inserted into a central core region of centromere 1 (cnt1:ura4+) was previously used to assess the integrity of CENP-ACnp1 chromatin in fission yeast24. Utilizing this assay, we screened a pool of fission yeast strains carrying mutations in chromatin remodeling factors for defective central core silencing. As previously shown25, the loss of Chd1Hrp1 disrupted central core silencing , as well as with specific regions of pericentromeric heterochromatin (otr) and proximal euchromatin  where the Ino80 complex and Chd1Hrp1 act to remove histone H3 , but it plays a weaker role in evicting H3 from euchromatin  cells. Consistent with the notion that the Ino80 complex plays a role in transcription, we identified hundreds of genes whose mRNA levels are significantly down- or up-regulated in ies6Δ cells  chromatin-remodeling complex40 did not produce this additive defect in silencing  cause any silencing defect when combined with ies2Δ . When we combined the silencing-defective ino80-11 with hrp1Δ, we observed a cumulative loss of both silencing and cell viability  compared to the wt , the overall levels of Iec1 and Ies6 bound to the central domain  were diminished and became constrained to more specific regions, including the transfer RNA genes  harboring low levels of CENP-ACnp1  was completely abolished in cnp1-1 cells  without any DNA sequence change42  fusion to tetO sites in a 4.5 kb non-centromeric DNA (bighyg) sequence inserted at centromere 1, and examined whether tethered TetR-Iec1 alters CENP-ACnp1 chromatin assembly at bighyg  treatment as negative controls. We used ChIP analysis to confirm that TetR and TetR-Iec1 were enriched at tetO insertions and that TetR-Iec1 was released after the addition of AHT , clearly implicating the ATPase activity of Ino80 in CENP-ACnp1 chromatin assembly. Moreover, we found that tethered Ino80 subunits exert a stronger effect on CENP-ACnp1 assembly when placed closer to the native centromere  to PMG media. Where indicated, we added TBZ  dissolved in dimethyl sulfoxide to the YES plates and AHT  dissolved in DMSO to the YES or PMG media. We incubated all fission yeast cells at 30 °C unless otherwise indicated. We used PCR-based gene targeting or genetic crosses followed by random spore analysis to create the deletion and Flag-tagged strains. We were able to infer the synthetic lethality of ies6Δ hrp1Δ by crossing ies6Δ and hrp1Δ and performing a random spore analysis; we were unable to find any ies6Δ::natMX6 hrp1Δ::kanMX6 (natRkanR) spores despite selecting over 300 ies6Δ::natMX6 (natR) spores. For more details regarding the yeast strains used in each experiment, see Supplementary Table Standard genetic and molecular techniques were used. YES (Yeast Extract with Supplements) was used as a rich medium and PMG  or EMM  was used as a minimal medium. For the selective and non-selective media in the silencing assays, we used PMG supplemented with amino acids. For 5-fluoroorotic acid (FOA) selection, we added FOA  and included equal numbers of wt cells (lacking any tag) in parallel as controls. We performed the ChIP experiments for CENP-ACnp1 and H3 with only minor modifications to conventional ChIP protocols. We prepared the cell extracts by disrupting the cells in lysis buffer  using a standard bead-beating protocol. Following immunoprecipitation (IP), we washed the DNA-bound protein Aagarose beads and eluted the DNA in IP columns  instead of microfuge tubes. As the external spike-in control for the H3 ChIP-Seq experiments, formaldehyde-fixed wild-type S. cerevisiae (W303a) cells were added to the S. pombe cells at a 1:8 ratio before cell disruption. For the Flag ChIP experiments, we used a modified lysis buffer  and Tris-buffered saline (TBS) -based wash buffers (TBS and TBS with 0.05% Tween-20) to avoid denaturation of the M2 antibody by sodium deoxycholate. As the external spike-in control for the H2A.ZPht1-5×Flag ChIP-Seq experiments, formaldehyde-fixed S. cerevisiae cells expressing Sua7-5×Flag were added to S. pombe cells at a 1:9 ratio before cell disruption. We recovered the de-crosslinked DNA using a Qiagen PCR purification kit. We constructed the ChIP-Seq libraries with 5 to 10 ng of input DNA or 1 to 10 ng of immunoprecipitated DNA, using a ChIP-Seq kit  according to the manufacturer’s protocol. For multiplexed libraries, we sequenced 50 nt single-end reads on an Illumina HiSeq 2500 (Theragen and Macrogen). After removing the adapter sequences, we aligned the processed reads to the S. pombe ASM294v2 genome assembly using Novoalign. We randomly assigned single locations to reads that mapped to multiple locations. For reads obtained from duplicate samples, we confirmed their experimental reproducibility using the bamCorrelate tool from the deepTools package51. For quantitative analysis of the data obtained from the H3 and H2A.ZPht1ChIP-Seq experiments, we calculated the IP/input ratio for each ChIP normalized to that of the spike-in control, which in principle should be constant in all ChIP samples. The normalized ratio of IP/input (EIP) was derived as:Cells were fixed with 1% formaldehyde for 10 min (for cells grown at 36 °C), 15 min (for cells grown at 30 °C), or 40 min (for cells grown at 20 °C). ChIP-Seq required more cells than conventional ChIP, as we needed to ensure that there was sufficient immunoprecipitated DNA for high-throughput sequencing. We used 1.6 × 10IP=(read count of IP mapped to S. pombe genome/read count of input mapped to S. pombe genome)/(read count of IP mapped to S. cerevisiae genome/read count of input mapped to S. cerevisiae genome).EN) for the IP/input in each ChIP-Seq experiment was calculated as:We computed the fold change of IP versus the input using the deepToolsbamCompare tool. The normalization factor (N×read count of IP/read count of input=EIP:1.N) for IPmutant/IPwt was calculated as:When we directly computed the fold change of IP in the mutant versus wild type, the normalization factor  analysis and the profiler tool from deepTools to visualize the average gene profiles. We generated heatmaps using the deepToolsHeatmapper. We performed all further downstream bioinformatic analyses of the ChIP-Seq data using the R software package.The normalization of ChIP-Seq for CENP-A52 using 20 µl of anti-CENP-ACnp1 antibody , 8 µl of anti-H3 antibody , 2 µl of anti-Flag M2 antibody , and 2 µl of anti-GFP tag antibody . We used the Bio-Rad CFX96 system to analyze the input or IP DNA by quantitative PCR. See Supplementary Table We performed the Fast ChIP protocolies6Δ cells using poly(A)beads (NEXTflex). The stranded libraries were generated from biological replicates of each strain using NEXTflex Rapid Directional RNA-Seq Kit according to the manufacturer’s manual and sequenced as single end. The sequence reads were aligned using STAR aligner53. Cufflinks tools were used to assemble the transcripts, to calculate their abundance in FPKM, and to determine list of genes that are differentially expressed in ies6Δ cells compared to wt. Genes whose expressions were increased or decreased more than twofold in ies6Δ cells relative to wt with q-value  of ˂0.05 were defined as significantly upregulated or downregulated genes, respectively  to mutagenize a DNA fragment containing the terminal 1.4 kb of the ino80+ open reading frame (ORF), which covered a significant portion of the catalytic domain. We then used fusion PCR to fuse the mutagenized fragments to a kanR marker gene and sequences downstream of the ino80+ ORF. We selected transformants on G418-containing plates by screening for slow growth at 35.5 °C. Then, we further screened these mutants for defective silencing of cnt1:ura4+ at different temperatures . This technique allowed us to isolate several cold-sensitive silencing-defective mutant alleles, including ino80-11. We identified the respective causative mutations in selected each strain using standard Sanger sequencing.To identify mutant alleles of S. cerevisiae LEU2 gene, which allows it to complement leu1-32. We cultured wt and mutant cells containing Ch16 in minimal medium lacking leucine (PMG-Leu) to maintain the minichromosome. We plated these cells on non-selective (YES) plates with or without allowing them to divide 10 to 12 times in non-selective medium (YES). We then replica-plated the resulting colonies onto selective medium lacking leucine (PMG-Leu) and counted those that could and could not grow on selective medium (PMG-Leu). We calculated the rate of minichromosome loss using the following formula: loss rate per division (%)=100 × (1−(F/I)N1/), where F is the final percentage of minichromosome-containing cells, I is the initial percentage of minichromosome-containing cells, and N is the number of generations54. We tested three independent colonies for each strain. Data are presented as the mean loss rate±s.d. (error bars).We used genetic crossing to introduce a non-essential minichromosome (chromosome 16 (Ch16)). Ch16 bears the 2 HPO4, 4 mM NaH2PO4, 2 mM PMSF, protease inhibitors) and lysed by bead-beating. Extracts were immunoprecipitated with 25 µl anti-Flag (M2)-agarose (Sigma). Immunoprecipitates were subjected to western blotting for the presence of Ies6-5×Flag or CENP-ACnp1 using horseradish peroxidase-conjugated anti-Flag (M2) antibody  or affinity-purified anti-CENP-ACnp1 antibody , respectively.Cell pellets from log-phase cultures were resuspended in NP40 lysis buffer , which is flanked by 2×tetO, into the center of the arg3+ insertion. Finally, we deleted the endogenous ura4+ and arg3+ genes of the resulting cnt1:bighyg strain. The ura4+ terminator sequence at the end of cnt1:bighyg was excluded from the analysis because it is also present at the end of the endogenous ura4+ locus. We generated the TetR-2×Flag-Iec1 fusion strain by replacing the start codon (ATG) of the endogenous iec1+ gene with a DNA fragment encoding TetR-2×Flag. We generated the TetR-2×Flag strain without the Iec1 fusion by fusing the iec1+ promoter and the TetR-2×Flag sequence and integrating them into an ectopic ura4+ locus to replace the promoter and coding region of ura4+. For the Ino80-tethering experiments, we replaced the promoter and coding region of ura4+ with the thiamine-repressible nmt81 promoter and a DNA sequence encoding TetR-2×Flag-Ino80, TetR-2×Flag-Ino80K873A, or TetR-2×Flag. We induced expression of the fusion proteins by growing the strains in PMG medium lacking thiamine. We generated the TetR fusion gene-containing strains and cnt1:bighyg by genetic crosses and verified them using diagnostic PCR.We generated the http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE99589. The authors declare that all the data supporting the findings of this study are available within the article and its The raw and processed sequencing data from this publication have been submitted to the NCBI Gene Expression Omnibus (GEO; Supplementary InformationPeer Review Files


Our study demonstrates a new paradigm of DNA methylation-mediated gene activation and chromatin remodeling, and provides a general framework to dissect the biological functions of DNA methylation readers and effectors.Altered DNA methylation status is associated with human diseases and cancer; however, the underlying molecular mechanisms remain elusive. We previously identified many human transcription factors, including Krüppel-like factor 4 (KLF4), as sequence-specific DNA methylation readers that preferentially recognize methylated CpG (mCpG), here we report the biological function of mCpG-dependent gene regulation by KLF4 in glioblastoma cells. We show that KLF4 promotes cell adhesion, migration, and morphological changes, all of which are abolished by R458A mutation. Surprisingly, 116 genes are directly activated via mCpG-dependent KLF4 binding activity. In-depth mechanistic studies reveal that recruitment of KLF4 to the methylated DOI:http://dx.doi.org/10.7554/eLife.20068.001 This doependent . For exaependent . In addiependent . Intriguependent . The cryependent . HoweverKLF4 plays multiple roles in normal physiology and disease. It is one of the Yamanaka factors that induce pluripotency in somatic cells . KLF4 alRHOC. We further demonstrate that recruitment of KLF4 to methylated cis-regulatory elements results in chromatin remodeling and activation of gene transcription in a genome-wide scale.In this study, we dissected the biological function of KLF4 binding to methylated DNA in malignant brain tumor cells by taking advantage of the R458A mutant lacking the ability to bind to methylated DNA. Our study showed that KLF4-mCpG interaction promotes brain tumor cell migration via the transactivation of genes involved in cell motility pathways, including the small GTPase We chose two human GBM cell lines, namely U87 and U373, with physiologically relevant genetic backgrounds to dissect the function of mCpG-dependent KLF4 binding activity, and because these two cell lines have low endogenous KLF4 expression. To facilitate in vivo studies of mCpG-dependent binding activity of KLF4, we engineered two stable cell lines that, upon doxycycline (Dox) treatment, each expressed KLF4 WT or KLF4 R458A. Cells without doxycycline treatment served as a negative control. Western blot analysis and immunocytochemistry staining confirmed that the endogenous KLF4 level in non-transfected U87 cells was barely detectable; after 48 hr Dox induction, both KLF4 WT and R458A proteins showed a dose-dependent increase without an impact on cell proliferation . For theWe observed that induction of KLF4 WT significantly increased cell adhesion  and promTo confirm that the observed phenotypes were DNA methylation dependent, we pre-treated the cells with a DNA methytransferase inhibitor, 5-aza-2′-deoxycytidine , which eliminates DNA methylation on a genome-wide scale . As illuThe distinct phenotypes induced specifically by KLF4 WT suggested that mCpG-dependent KLF4 binding events regulate the expression of genes involved in these biological processes. To elucidate the molecular mechanisms by which KLF4 regulates gene transcription via its mCpG-dependent activity, we carried out a series of genome-wide analyses to identify the direct gene targets that are regulated by KLF4 WT but not R458A.RHOC, RAC1, LMO7, and MIDN was up-regulated by induction of KLF4 WT but not R458A  and after (48 hr) induction of KLF4 WT and R458A, respectively. Approximately 86% of the sequencing reads were uniquely mapped to the human genome ; compariot R458A . This reRHOC, as well as mapped RHOC RNA-seq, are shown in To determine which genes were directly activated by mCpG-dependent KLF4 binding events, we next performed genome-wide chromatin immunoprecipitation-sequencing (ChIP-seq) in KLF4 WT and R458A-expressing cells . At least 70% of the ChIP-seq reads were mapped to the human genome . A totalTo fully examine the DNA methylation status of the WT and R458A ChIP-seq peaks, we performed whole genome bisulfite sequencing to decode the methylome of U87 cells and combined the DNA methylome data separately with the KLF4 WT and R458A ChIP-seq datasets. We found that 66% of the KLF4 WT-specific ChIP-seq peaks showed a high methylation level  at CpG sites, while only 36% of the ChIP-seq peaks shared by KLF4 WT and R458A reached a similar CpG methylation level (p=3.7e-223). Different cutoffs for defining high methylation levels did not alter this observation . TherefoNext, we carried out motif analysis to identify enriched and highly methylated 6-mer DNA motifs in WT-specific ChIP-seq peaks, as well as in shared ChIP-seq peaks. At a cutoff of β >60% CpG methylation we found 10 methylated 6-mer motifs  that werTo identify genes that were directly activated via mCpG-mediated KLF4 binding activity, we first searched the 2,733 KLF4 WT-specific ChIP-seq peaks against the genomic locations on those proximal regulatory regions, which were classified into three categories: upstream (~10 kb upstream to transcription start sites), 5’-UTRs, and exons. The proximal regulatory regions of 65 KLF4 WT up-regulated genes were found to be occupied by KLF4 WT-specific ChIP-seq peaks, indicating that they were direct targets of KLF4-mCpG interactions .cis-regulatory elements in the proximal regulatory regions (56%) and distal enhancers (44%)  . Using ars (44%) .10.7554/We next examined whether this association was statistically significant. We first focused on 12,824 genes that were expressed (FPKM >0.5) in U87 cells, among which 308 (2.4%) genes were found up-regulated only in KLF4 WT-induced cells. Meanwhile, we observed that KLF4 mCpG-dependent binding peaks were associated with 2518 expressed genes, of which 116 (4.6%) were up-regulated genes that is significantly higher than the ratio of expressed genes not associated with KLF4 WT binding sites .cis-regulatory elements of the 116 direct target genes, we integrated the methylome dataset and found that the majority (72%) of the 116 genes were indeed associated with highly methylated (β > 0.6) cis-regulatory elements  and RAC1 (−922 bp to TSS).To firmly establish the causality between mCpG-dependent KLF4 binding to ed above . For exarognosis . An examRHOC, we first showed that induction of KLF4 WT, but not the R458A mutant, could induce RHOC at both mRNA and protein levels using RT-PCR and immunoblot analysis, respectively  and two repressive (H3K27me3 and H3K9me3) histone marks at the RHOC promoter regions. We also monitored histone mark changes before (0 hr) and after (48 hr) KLF4 induction. We observed that upon KLF4 binding to DNA (48 hr post Dox), the active mark H3K27ac was significantly increased by ~2.5 fold. On the other hand, both repressive marks, H3K9me3 and H3K27me3, were decreased by ~3.5 and~6 fold, respectively  and two repressive (H3K9me3 and H3K27me3) histone marks before (0 hr) and after (48 hr) KLF4 WT induction. For all of the three marks, we observed remarkable dynamic changes, centered around the 162 KLF4 WT ChIP-seq peaks associated with the 116 direct targets of KLF4-mCpG . SpecifiTo further confirm that mCpG-mediated KLF4 binding caused chromatin remodeling on a genome-wide scale, we compared the H3K27ac ChIP-seq data obtained before (0 hr) and after (48 hr) KLF4 WT and R458A induction. An example was shown in Overall, in KLF4 WT-induced cells, 3593 novel H3K27ac peaks were generated, while only a negligible number of H3K27ac peaks (41) disappeared at 48 hr of KLF4 WT induction . Among tcis-regulatory elements resulted in a global chromatin remodeling , which contradicts the classic view that CpG methylation is a result of chromatin remodeling .Abnormal DNA methylation has been found in many cancer types including glioblastoma. Contrary to traditional view of DNA methylation in gene silencing, recent survey of methylome and gene expression profiling demonstrated that the promoters of as many as 20% highly expressed genes in tumors are methylated at CpG sites, suggesting that at least in some cases, DNA methylation positively correlates with gene expression , 2007. Acis-regulatory elements (RHOC promoter region) indicated that methylation is not a consequence of KLF4 binding (data not shown). Other forms of cytosine modification, including 5-hydroxymethyl-cytosine (5-hmC), 5-formylcytosine, and/or 5-carboxylcytosine also exist in cells  Comparison of ChIP-seq between KLF4 WT and R458A showed that WT-specific binding sites are enriched for highly methylated sites. Given the phenotypic differences between KLF4 WT and R458A, the result suggested that the observed phenotypes in WT are likely due to specific interactions between KLF4 and DNA methylation sites. (4) Our new RNA-seq data in the presence of 5-Aza also indicated that 5-Aza globally reversed the up-regulation of many KLF4-mCpG targets.We are fully aware that 5-Aza is only a sub-optimal approach to examine the effect of specific methylation sites, because it induces global demethylation and may have some non-specific effects . HoweverIn our study, we placed a higher priority on KLF4-mCpG-mediated gene transactivating effect, as this will be most paradigm-shifting based on current understandings that CpG methylation results in gene silencing. Here we found that KLF4 recognizes mCpG at proximal and enhancer regions to activate gene expression. Many questions remain regarding how DNA methylation, a repressive epigenetic mark, activates gene expression. In cells, opening the chromatin structure is often the first step for gene transcription . KLF4 isIn summary, TF binding to methylated regions of the genome to activate gene transcription is a new paradigm supported by several studies . Our worCVCL_0022) and U373  were originally purchased from ATCC  and cultured in our laboratory. Both cell lines are free from mycoplasma and authenticated with short tandem repeat (STR) profiling by Johns Hopkins Genetic Resources Core facility using Promega GenePrint 10 system . U87 cells were cultured in Minimum Essential Media  supplemented with sodium pyruvate (1%), sodium bicarbonate (2%), non-essential amino acid (1%) and 10% fetal calf serum . U373 cells were cultured in Dulbecco’s Modified eagle medium  supplemented with 2% (4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid (HEPES) and 10% FCS. Cells were incubated in a humidified incubator containing 5% CO2/95% air at 37°C, and passaged every 4–5 days.All reagents were purchased from Sigma Chemical Co.  unless otherwise stated. The human glioblastoma (GBM) cell lines U87  . Virus wg for 10 min. Thirty micrograms of protein were separated using 4–20% SDS-PAGE gels  and blotted onto nitrocellulose membranes  containing protease and phosphatase inhibitors  and sonicated for 15 s; the suspensions were centrifuged at 3000 embranes . Membranembranes . AntibodFor staining, GBM cells grown on glass slides were fixed with 4% paraformaldehyde for 30 min at 4°C and permeabilized with PBS containing 0.1% Triton X-100 for 10 min. The cells were then incubated with primary antibodies at 4°C overnight and then incubated with appropriate corresponding secondary antibodies conjugated with Alex Flourescent 488 or cy3 for 30 min at room temperature. For double staining with F-actin, Alex Flourecent 647-conjugated phalloindin  was used to incubate with the cells. Slides were mounted with Vectashield Antifade solution containing DAPI  and observed under fluorescent microscopy. Immunofluorescent images were taken and analyzed using Axiovision software .Medium (DMEM) containing 0.5% bovine serum albumin (BSA) for 1 hr at 37°C followed by plating GBM cells at a density of 4 × 105 cells/well. After 1 hr incubation at 37°C, plates were shaken at 2000 rpm for 15 s, washed twice with pre-warmed DMEM medium containing 0.1% BSA, and the number of remaining adherent cells were measured with MTT assays , GBM cells were suspended at 1 × 10e medium . Six hune medium .For wound healing assays, GBM cells were grown under 10% FCS medium in 35 mm dishes until confluent. Cell proliferation was inhibited by mitomycin C (1 µg/ml) for half hour. Several scratches were created using a 10 μl pipette tip through the confluent cells. Dishes were washed with PBS for three times and cells were grown in 0.1% FCS medium for 24–48 hr. Phase contrast pictures were taken at different time points. The width of the scratch was measured and quantified as previously described .Total RNA (1 µg) was reverse-transcribed using the oligo (dT)12–18 primer and Superscript II (Thermo Fisher Scientific) according to the manufacturer's instructions. The thermal cycling conditions were as follows: 95°C, 5 min, followed by 30 cycles of 95°C for 10 s, 55°C for 10 s, and ended with 72°C for 30 s.Data were analyzed using parametric statistics with one-way ANOVA. Post-hoc tests included the Students T-Test and the Tukey multiple comparison tests as appropriate using Prism . All experiments reported here represent at least three independent replications. All data are represented as mean value ± standard error of mean (S.E.) significance was set at p<0.05.RNAs from KLF4 WT and R458A-expressing cells (0 hr and 48 hr +Dox) was subjected to Illumina HiSeq next generation sequencing following the standard amplification and library construction protocol provided by the Johns Hopkins Deep Sequencing and Microarray Core Facility. Sequencing was performed using 76-base single-end reads, with 23- to 33 million reads generated from each sample. We first used Tophat2 to map all reads to human genome (hg19) then employed Cufflink to summarize the gene/transcript expression based on mapped reads. An R package, DEGseq, was taken to identify DEGs for p<0.001 between 0 hr and 48 hr in KLF4 WT and R458A cells, respectively.We employed the software package bismark  to perfoTo identify methylated motifs enriched in KLF4 WT-specific peaks, we first used WGBS information to selected all 6-mers including mCpG (β >0.6), then enumerated these 6-mers to compare their occurrence in 2733 KLF4 WT-specific peaks and all KLF4 binding peaks. The p-values were calculated based on hypergeometric model to represent the significance of methylated 6-mers’ frequencies in KLF4 WT-specific peaks compared to all, followed by multiple-test Bonferroni correction. The 6-mers with p<0.01 were selected to construct the motif logo. The package, MEME (Multiple EM for Motif Elicitation) , was useGene Ontology analysis  was perfA commercial ChIP-grade anti-KLF4 antibody  recognizing the N-terminal region of KLF4 (DNA-binding domains of KLF4 are located to the very C-terminus) was used for ChIP. Tet-on KLF4 WT and R458A cells were treated with Dox for 48 hr followed by ChIP using the anti-KLF4 antibody and Dynabeads Protein A/G (Thermo Fisher Scientific) according to a protocol described previously . DNA libKLF4 ChIP-Seq data were mapped by Bowtie2, followed by MACS 1.4 being used to call peaks with cutoff of p<1E-5. We first obtained binding peaks for KLF4 WT and R458A, respectively. The peaks identified for both KLF4 WT and R458A at the same locus were referred as shared KLF4 binding peaks. Then we used KLF4 WT as foreground and R458A as background control to call peaks again. The new peaks were marked as KLF4 WT-specific ChIP peaks, only if they were not overlapped with shared ones which had been already identified. Same approach was used to obtain R458A specific binding peaks for which the foreground was KLF4 R458A ChIP-Seq data compared to the background of KLF4 WT ChIP-seq data.We utilized MACS2 to recognize broad peaks of H3K27ac based on their ChIP-Seq data mapped by Bowtie2. The cutoff of broad peak call was q < 0.1. The same procedure as that for KLF4 ChIP-Seq was taken to distinguish H3K27ac peaks at 0 hr only, at 48 hr only, or shared at both 0 hr and 48 hr, for KLF4 WT and R458A, respectively.We chose two sets of genomic regions to quantitatively study dynamic changes of histone modification between 48 hr and 0 hr. The first group is regions within ±1 kb from the middle points of KLF4 binding peaks. As negative control, we selected other 7780 regions, which locate 4 kb to 6 kb away from the boundaries of 3890 KLF4 binding peaks, then removed 558 out of 7780 regions in the negative control group which overlap with at least one of KLF4 binding peaks. The signal of the histone modification within the region was represented by RPM (Reads per Million) in logarithmic scale base 2. The difference of signals between 48 hr and 0 hr was further normalized so that the difference for 7222 random regions was 50% up and 50% down.All the raw data for our large-scale studies have been deposited in GEO (GSE97632).Sanger bisulfite sequencing was performed as previously described . Purifie In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom, Bing Ren (Reviewer #1), is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Kevin Struhl as the Senior Editor.Thank you for submitting your article "Methylated cis-regulatory elements mediate KLF4-dependent gene transactivation and cell migration" for consideration by Your work has been considered by the editors and three reviewers. The comments from the reviewers are attached. The reviewers recognize the potential significance of your finding of the function of DNA methylation-dependent binding of Klf4 in cell adhesion and migration, but raised considerable concerns regarding experimental evidence and the conclusions. In light of these concerns we cannot recommend publication of this manuscript. However, if you are able to address fully the main concerns as listed below then we would be happy to consider a much-revised version of the manuscript.Essential revisions:1) New experimental evidence is required to fully establish the DNA methylation dependent binding of wild type and the mutant form. Control experiments with Klf4 mutant lacking DNA binding potential altogether needs to be conducted; DNA methylation and motif analysis of the cell systems need to be performed.2) Causality of DNA methylation dependent binding of Klf4 on the genome and induction of some target genes needs to be more fully established. As pointed by reviewer #3, the presence of the binding within 10kb of the promoter does not prove direct targeting relationship. New experimental data should be provided to support the notion that a target gene is directly regulated by Klf4's binding to the methylated DNA near the gene. This could be CRISPR/cas9 mediated genome editing or other means.3) The treatment of cells by 5-Aza is inducing global DNA demethylation, and the observed phenotypes in the cell system can be attributed to non-specific effects. The authors need to discuss this aspect of the experiments to avoid drawing un-substantiated conclusions.4) Statistical methods need to be better described and experimental data need to be more rigorously interpreted. Please check their comments carefully and make sure to remove or modify the particular statements in the revised manuscript.Reviewer #1In this manuscript Wan et al. reported the finding that Klf4 can indeed bind methylated DNA to activate transcription of genes in mammalian cells. Work in the laboratory of one of the co-authors (Dr. Zhu) previously showed that Klf4 could recognize DNA motifs with methylated CpG, and a point mutation R458A disrupts its methyl-DNA binding but not affect the binding to unmethylated sequences. In the current work, the authors tried to demonstrate function of the methyl-CG binding of Klf4. They chose two gliobastoma multiforme (GBM) cell lines U87 and U373 expressing either an inducible Wild Type Klf4 or one with R458A mutation. They found that upon induction of WT Klf4, but not MT form, the GBM cells acquire cell adhesion and migratory properties. They further investigated the binding sites of the two forms of proteins, and identified a substantial number of binding sites for the WT form but not the methyl-binding defective mutant. The also carried out RNA-seq analyses to identify genes regulated differentially by the WT versus Mutant form, and linked some of them to the differential binding of the Klf4 proteins. They claimed to identify 116 genes activated by mCpG-dependent KLF4 binding activity. In order to link the differential binding to DNA methylation at DNA, the group picked 15 regions and performed bisulfite sequencing. They showed that 10 of them contain DNA methylation. The authors conclude that Klf4's methyl-CG binding activity is critical for its role in mediating cell adhesion and migration in the GBM cells, and its binding to the methylated sites results in chromatin remodeling and transcription activation.The work represents an in-depth follow up study of a phenomenon that some transcription factors binds to methylated DNA. The authors provided multiple lines of evidence supporting the functional role of methyl-CpG binding of Klf4. However, they also made some over-statement, and some conclusions are not sufficiently established with existing evidence. Additional experiments and analyses are needed to strengthen these conclusions.Major problems:1) Differential binding of WT and R458A Klf4 in GBM cells: Is this due to failure of the mutant protein to bind methylated DNA? Unfortunately, the evidence presented is weak: the authors performed Bisulfite Sequencing on 15 sites and found that 10 of them contain methylated CpG. Are these DNA sequences recognized by WT Klf4 but not mutant klf4? EMSA should be performed to establish this point. Further, 15 is too small a number to generalize to the rest of the differential binding sites. It is important to carry out base-resolution analysis of DNA methylation in these cells and determined if differential DNA binding by WT and R458A Klf4 in the GBM cells is indeed correlated with presence of DNA methylation at the Klf4 binding motifs in these regions.2) The authors claimed that 116 genes are activated by mCpG-dependent KLF4 binding activity. Again, the evidence to support this claim is weak. Besides the reasons outlined in #1, there is also a need to establish the causality of KLF4 binding to methylated DNA in the cells and activation of these genes. Since the authors observed partial loss of cell adhesion and migration after 5-AZA treatment, it is important to show that the induction of these 116 genes is affected by 5-AZA in the direction that is consistent with their being targeted by Klf4.3) The 5-AZA treatment leads to partial loss of migration of cells expressing WT Klf4. Is this due to DNA methylation dependent binding of Klf4? It is necessary to demonstrate loss of DNA methylation and loss of Klf4 binding at some sites after treatment.Reviewer #2:Binding of transcription factors to methylated regions of the genome to activate gene transcription is a new paradigm, and several studies have provided convincing evidence supporting this hypothesis. The manuscript by Wan et al., 'Methylated cis-regulatory elements mediate KLF4-dependent gene transactivation and cell migration' supports this hypothesis. In this study Wan et al. provides the best evidence of transcription factors binding to methylated DNA motifs and mediating gene activation. The experiments support their hypothesis convincingly and the manuscript describes it well. However, there are certain aspects that need to be addressed before it can be accepted.eLife 2013). They used this mutation as a negative control in the examination of KLF4 binding to methylated DNA sequences. However, in the present manuscript, they do not show that KLF4 R458A is able to activate unmethylated motifs in their genomic assays as they showed previously in transfection experiments. They need a negative control with no KLF4 function.Previously, the authors discovered that KLF4 can bind to methylated motifs and that a mutation in KLF4, (R458A) inhibited binding to methylated motifs but not unmethylated motifs  The authors observe KLF4 ChIP-seq in WT and R458A transfected cells. There are some key aspects that need to be included. How many / percentage of canonical and methylated sites are specific or shared between WT and R458A cells. A representation of the motifs needs to be addressed. 2) The R458A mutant binds to ~1200 regions, the authors need to provide details regarding these regions.3) The methods do not indicate whether biological replicates were performed for the sequencing based assays, in that case reproducibility is questionable. The authors can also provide a supplementary summary table for the mapped reads for all the sequencing experiments.4) 5) 6) 7) What is the methylation status of the WT specific KLF4 chip peaks sharing acetylation mark.Reviewer #3:The paper has a single major finding which is that the KLF4 R458 mutation, which presumably affects nothing other than binding to methylated DNA, is severely compromised in its ability to induce both cellular phenotypes and gene expression changes in GBM cell lines. Moreover, these changes induced by WT KLF4 are largely abrogated by removal of DNA methylation. If one assumes that R458 has no other role, and that methylation has no indirect impact on the effects of KLF4 binding, then the core findings do seem to be supported by several lines of evidence.That said, the paper makes a series of claims that are not supported by the data, and contains interpretations that I believe are erroneous. It also seems to contain some redundancy. It strikes me that the paper would be stronger if most of the material presented was removed and only the essence was retained. I have grouped my major concerns into four points, some of which are applicable to multiple sections of the paper.Major points:1) The statistical analyses are incompletely described, interpreted incorrectly, confuse correlation with causality, and contain biases.The RNA-seq, ChIP-seq, and bisulfite sections need to state which overlaps are significant, and what is the predictive power, otherwise there is a strong potential that the manuscript is mainly describing phenomena with little or no significant relationship, and/or is confusing weak correlations or random overlaps with causality, due to unwarranted conditioning on unproven expectation. This is relevant to several sections, starting with this one:Subsection "Identify direct targets of mCpG-dependent KLF4 interactions in GBM cells" is flawed. 613 genes are identified as responsive to KLF4 overexpression (induced more than 2-fold). Then, "To determine which genes were directly activated by mCpG-dependent KLF4 binding events, ChIP-seq identifies 3,890 peaks. 65 of these bind within 10kb of the promoter, or within the UTRs or exons of the induced genes, "indicating that most likely they were direct targets of KLF4-mCpG interactions" . I disagree that this shows that they are direct targets – the null hypothesis should be that there is no relationship, and in fact if there is any overlap it seems to be low. It is entirely possible that there is a random association between the ChIP data and the RNA-seq data, and even if it is nonrandom, the fold enrichment over random should be given.Instead, this section concludes "Taken together, a total of 116 genes were directly [my emphasis] up-regulated via mCpG-dependent KLF4 binding to the cis-regulatory elements in the proximal regulatory regions (56%) and distal enhancers (44%)". There is simply not the evidence in this paper to make that conclusion.Since we don't have the right numbers in the current paper I will explain why the reasoning is flawed using typical numbers seen in other studies (which seem to be similar here). Let's say that there are 10,000 active genes, peaks are found in 900, 600 are up-regulated, and 60 overlap. So, peaks are found in 9% of all promoters, but 10% of up-regulated promoters. 6% of genes are up-regulated, and 7% of genes with a peak are up-regulated. Certainly there is something statistically significant happening, but the relationship between binding sites and expression responses is weak, so even when we see it, we cannot infer causality. We know that many binding sites do nothing (at least 93%), and that many genes are induced with no binding at all (at least 90%). So, it is certainly possible that there could be genes that have a binding site that does nothing and are induced indirectly.By analogy, if the same numbers were used for a high-cholesterol diet and heart attacks, we would be wrong to assume that all 60 people with heart attacks and a high cholesterol diet were caused by the diet, since certainly the diet is not deterministic, and there are multiple causes of heart attacks. Instead, we can conclude that seven of the heart attacks can be attributed to the diet, but we can't tell which seven they were.In addition, two technical modifications of these analyses are needed to further avoid bias. First, the comparisons among binding sites, induced genes, marks etc need to be restricted to genes that are expressed at all in these cells – it is possible to get high overlaps among unrelated regulators only because everything tends to pile up on open chromatin and active promoters. Similarly, the GO analyses later in the paper should be done taking the induced genes as a background. KLF4 overexpression induces migration and morphological changes, which could explain why genes related to this process are expressed. It is possible that KLF4 only directly induces a small number of genes, and the rest of what we are seeing is a secondary effect.eLife asked me to write a short review, but the same issues pervade subsequent analyses of overlaps with histone ChIP data and methylation. It is abundantly clear from ENCODE that there are lots of pervasive marks of all kinds in all cell types, many of which concentrate in active regions. Not every overlap is functionally significant, however, and in fact it is likely that most of them are doing nothing at any given time.2) It seems possible that global DNA demethylation could attenuate the ability of cells to do many things, and thus the results shown in 3) I do not see that 4) The association of active marks with induced promoters and with many of the Klf4 sites is unsurprising – this would be seen even if the induction of the supposed "target genes" was indirect, and the KLF4 binding was coincidental. Indeed, the fact that there are 3,593 novel H3K27ac peaks, but only 274 overlap KLF4, shows that the two are largely dissociated. Thus, the statement in the Discussion that "Our study reveals that mCpG-dependent binding activity of KLF4 serves as the link between methylated CpG and changes in chromatin status" is not supported by the data shown. It is "a" link, but not "the" link. Essential revisions:1) New experimental evidence is required to fully establish the DNA methylation dependent binding of wild type and the mutant form. Control experiments with Klf4 mutant lacking DNA binding potential altogether needs to be conducted; DNA methylation and motif analysis of the cell systems need to be performed.p = 3.7e-223). Different cutoffs for defining high methylation levels did not alter this observation  at CpG sites, while only 36% of the ChIP-seq peaks shared by KLF4 WT and R458A reached a similar CpG methylation level (ervation . Therefop = 6.6e-37). Many of them share sequence similarity to the motif 5’-CCCGCC (eLife 2013). In contrast, the peaks shared by WT and R458A were found to be enriched for different motifs . The new data is now in Next, we carried out motif analysis to identify enriched and highly methylated 6-mer DNA motifs in WT-specific ChIP-seq peaks, as well as in shared ChIP-seq peaks. At a cutoff of β > 60% CpG methylation we found 10 methylated 6-mer motifs that were significantly over-represented in the WT-specific peaks (KLF4ΔC (lacking the entire KLF4 DNA binding domain) in mouse retinal ganglion cells could no longer suppress the axon outgrowth as the WT did :298-301). Similarly, induction of the R458A mutant on the same background showed the same phenotype as KLF4ΔC , suggesting the removal of the entire DNA binding domain of KLF4 did not cause any additional phenotype. Even if a truncated KLF4 mutant might cause additional phenotypes, it is not the focus of this study. In our opinion, the R458A mutant is the most relevant comparison with KLF4 WT because it loses the ability to bind to methylated DNA motifs while maintaining binding activity to KLF4’s canonical motif . Using a truncated KLF4 mutant that lacks the entire DNA binding domains would not help us to identify mCpG-dependent KLF4 targets because we would not be able to distinguish KLF4-mCpG binding from the canonical KLF4 binding activity.We respectfully disagree that “control experiments with Klf4 mutant lacking DNA binding potential altogether needs to be conducted” because it would not further strengthen the conclusion of this study. In a previous study, the Goldberg group nicely showed that induction of a 2) Causality of DNA methylation dependent binding of Klf4 on the genome and induction of some target genes needs to be more fully established. As pointed by reviewer #3, the presence of the binding within 10kb of the promoter does not prove direct targeting relationship. New experimental data should be provided to support the notion that a target gene is directly regulated by Klf4's binding to the methylated DNA near the gene. This could be CRISPR/cas9 mediated genome editing or other means.RAC1 and RHOC, because 1) they are known to play a crucial role in cell migration; 2) we found that they were associated with WT-specific ChIP-seq peaks and genome-wide bisulfite sequencing confirmed that these peaks contained highly methylated CpGs; and 3) these peaks are in close proximity to the coding regions of RAC1 (-922 bp to TSS) and RHOC (-507bp to TSS). In a newly added case of RAC1, we first showed that induction of KLF4 WT, but not the R458A mutant, induced RAC1 expression at both mRNA and protein levels using RT-PCR and immunoblot analysis, respectively  with and without 5-Aza treatment and observed that reduction of methylation in this region also substantially reduced recruitment of KLF4 WT. Taken together, these new results demonstrate that KLF4 WT activated the expression of these two genes via binding to their highly methylated cis-regulatory elements nearby the coding regions. The new data is added as panel In the case of p = 1.2e-14 compared to all expressed genes), suggesting that DNA methylation is responsible for the transcription activation of these 101 genes. The new data is in To examine the impact of DNA demethylation on the global expression level, we performed RNA-seq analysis and compared changes in gene expression profiles with and without 5-Aza treatment. We observed that in the absence of KLF4 WT induction the overall expression levels of the 116 KLF4 WT-specific target genes were not significantly affected by 5-Aza treatment , as expeeLife 2013). In other words, the use of CRISPR/Cas9 to mutate the DNA sequence  of interest would not assist us to de-couple the effects of DNA sequence and DNA methylation on the KLF4 binding activity. Ideally, a CRISPR-guided site-specific DNA demethylation approach would be appropriate; however, this method might not guarantee maintenance of low DNA methylation level due to the endogenous DNA methyltransferase activity.In our opinion, using CRISPR/Cas9-mediated genome editing may not help strengthen the causality because the mCpG-dependent KLF4 DNA binding activity is also sequence-specific  The treatment of cells by 5-Aza is inducing global DNA demethylation, and the observed phenotypes in the cell system can be attributed to non-specific effects. The authors need to discuss this aspect of the experiments to avoid drawing un-substantiated conclusions.We agree with the reviewers and have added the following discussion to address this concern in Discussion section.RHOC and RAC1, we showed that DNA methylation is responsible for KLF4 binding and gene activation. RHOC and RAC1 are major GTPase involved in cell migration and motility. (3) Comparison of ChIP-seq between KLF4 WT and R458A showed that WT-specific binding sites are enriched for highly methylated sites. Given the phenotypic differences between KLF4 WT and R458A, the result suggested that the observed phenotypes in WT are likely due to specific interactions between KLF4 and DNA methylation sites. (4) Our new RNA-seq data in the presence of 5-Aza also indicated that 5-Aza globally reversed the up-regulation of many KLF4-mCpG targets.”“We are fully aware that 5-Aza is only a sub-optimal approach to examine the effect of specific methylation sites, because it induces global demethylation and may have some non-specific effects . However, we have multiple lines of evidence showing that the observed phenotypes are due to specific interactions between KLF4 and methylated sites. (1) In our control experiments when KLF4 R458A cells were treated with 5-Aza, we did not see changes in gene expression and cell migration. (2) In the focused studies of 4) Statistical methods need to be better described and experimental data need to be more rigorously interpreted. Please check their comments carefully and make sure to remove or modify the particular statements in the revised manuscript.We thank reviewers for the constructive suggestion on the statistics analyses. We have added more rigorous statistical analyses throughout the revised manuscript according to the reviewers’ suggestions. Detailed responses can be found in the specific points raised by each reviewer. Please refer to our responses to reviewer #1’s major points 1 and 2; reviewer #2’s major points 1, 2, 3 and 7; reviewer #3’s major points 1 and 2. All of these new analyses have been incorporated into the revised manuscript.Reviewer #1[…] Major problems:1) Differential binding of WT and R458A Klf4 in GBM cells: Is this due to failure of the mutant protein to bind methylated DNA? Unfortunately, the evidence presented is weak: the authors performed Bisulfite Sequencing on 15 sites and found that 10 of them contain methylated CpG. Are these DNA sequences recognized by WT Klf4 but not mutant klf4? EMSA should be performed to establish this point. Further, 15 is too small a number to generalize to the rest of the differential binding sites. It is important to carry out base-resolution analysis of DNA methylation in these cells and determined if differential DNA binding by WT and R458A Klf4 in the GBM cells is indeed correlated with presence of DNA methylation at the Klf4 binding motifs in these regions.cis-regulatory elements in the promoters of two cell migration genes (RAC1 and RHOC) abolished the recruitment of KLF4 WT to these promoters and repressed their gene transcription  WT-specific ChIP-seq peaks contained CpGs of a much higher methylation level than those peaks shared by the WT and R458A mutant  DNA moticription . Taken t2) The authors claimed that 116 genes are activated by mCpG-dependent KLF4 binding activity. Again, the evidence to support this claim is weak. Besides the reasons outlined in #1, there is also a need to establish the causality of KLF4 binding to methylated DNA in the cells and activation of these genes. Since the authors observed partial loss of cell adhesion and migration after 5-AZA treatment, it is important to show that the induction of these 116 genes is affected by 5-AZA in the direction that is consistent with their being targeted by Klf4.p = 1.2e-14 compared to all genes), suggesting that DNA methylation is responsible for the transcription activation of these 101 genes.The reviewer suggested a very important way to strength our findings. Please see our responses to essential revision #2 above for more details. To examine the impact of demethylation on global expression level, we performed RNA-seq analysis and compared changes in gene expression profiles with and without 5-Aza treatment. We observed that in the absence of KLF4 WT induction the overall expression levels of the 116 KLF4 WT-specific target genes were not significantly affected by 5-Aza treatment , as expe3) The 5-AZA treatment leads to partial loss of migration of cells expressing WT Klf4. Is this due to DNA methylation dependent binding of Klf4? It is necessary to demonstrate loss of DNA methylation and loss of Klf4 binding at some sites after treatment.The answer is yes. Please see our response to essential revision #2.RAC1 and RHOC, because 1) they are known to play a crucial role in cell migration; 2) we found that they were associated with WT-specific ChIP-seq peaks and genome-wide bisulfite sequencing confirmed that these peaks contained highly methylated CpGs; and 3) these peaks are in close proximity to the coding regions of RAC1 (-922 bp to TSS) and RHOC (-507 bp to TSS), respectively. In the case of RAC1, we first showed that induction of WT, but not the R458A mutant, could induce RAC1 at both mRNA and protein levels using RT-PCR and immunoblot analysis, respectively  The authors observe KLF4 ChIP-seq in WT and R458A transfected cells. There are some key aspects that need to be included. How many / percentage of canonical and methylated sites are specific or shared between WT and R458A cells. A representation of the motifs needs to be addressed. We appreciate the reviewer’s positive comments and insightful suggestions to strengthen our findings. As discussed in essential point #1 above, we have performed the whole genome bisulfite sequencing and combined this dataset with our ChIP-seq datasets. We observed that 10 methylated 6-mer motifs, each containing one CpG of high methylation level (β>0.6), were significantly enriched in the KLF4 WT-specific peaks, while distinct motifs without CpG sites were found enriched in the shared ChIP-seq peaks. Please see essential point #1 for more details.We calculated numbers of WT-specific and shared peaks, respectively, including one of non-methylated motifs (5’-AAAGGA) and methylated (5’-CCCGCC) sites. We found that the non-methylated motif was significantly overrepresented in shared peaks , whereas the methylated motif was significantly enriched in WT-specific peaks .We have also provided the input track and screenshot of some KLF4-mCpG target genes see . The newWe agree that MACS2 is widely used for calling ChIP-seq peaks. In general, the results by MACS 1.4 were quite similar to those obtained using MACS2. However, in our case some peaks observed obvious to eye-balling  The R458A mutant binds to ~1200 regions, the authors need to provide details regarding these regions.We now provide the chromosomal locations of the WT and R458A shared peaks listed in Figure 3—source data 33.3) The methods do not indicate whether biological replicates were performed for the sequencing based assays, in that case reproducibility is questionable. The authors can also provide a supplementary summary table for the mapped reads for all the sequencing experiments.We appreciate the reviewer’s comments. We have added the summary tables in 4) Results section). Moreover, as the reviewer suggested, we did cell migration assays in the non-transfected U87 cells (control) and found that non-transfected U87 cells behave the same as the non-Dox treated U87 KLF4 WT cells. Please refer to our response to reviewer #2, major point 8 later.We apologize for the poor explanation of the cell system we used in our study. The purpose of using tet-on system, i.e., doxycycline (Dox)-inducible cell lines for KLF4 functional study, was to have an internally-controlled system, such that when the cells are not treated with Dox, they behave like the parental cells, therefore serves as a control cell line to study the biological function of KLF4. Compared to their own control cells, the induction level of KLF4 R458A was a bit higher than that of KLF4 WT, consistent with as our more quantitative RNA-seq data indicating the fold change of KLF4 WT and KLF4 R458A are 20 and 27 fold, respectively. However, the differences in expression level did not affect the ability of KLF4’s binding to methylated CpGs, hence cellular phenotype changes. In the revised version, we specifically explained the tet-on cell system we used (5) In the revised version, we have chosen more representative photographs to replace 6) RHOC expression  What is the methylation status of the WT specific KLF4 chip peaks sharing acetylation mark.The reviewer raised a very good point. Of 1,773 WT-specific KLF4 chip peaks sharing acetylation mark, 978 (55.2%) contain CpG sites of >60% methylation level.Reviewer #3:[…] Major points:1) […] In addition, two technical modifications of these analyses are needed to further avoid bias. First, the comparisons among binding sites, induced genes, marks etc need to be restricted to genes that are expressed at all in these cells – it is possible to get high overlaps among unrelated regulators only because everything tends to pile up on open chromatin and active promoters. Similarly, the GO analyses later in the paper should be done taking the induced genes as a background. KLF4 overexpression induces migration and morphological changes, which could explain why genes related to this process are expressed. It is possible that KLF4 only directly induces a small number of genes, and the rest of what we are seeing is a secondary effect.p = 1.2e-12). Adding KLF4 mCpG binding sites in enhancer region, we finally identified 116 targets out of 2,601 genes, indicating significantly over-representation . In the revised manuscript, we added this new analysis in the text (subsection “Identification of direct targets of mCpG-dependent KLF4 interactions in GBM cells”). In addition, we performed whole genome bisulfite sequencing, integrated it with the ChIP-seq datasets, and re-analyzed the data . We also obtained RNA-seq data after 5-Aza treatment and re-analyzed the entire dataset . To firmly establish the causality between mCpG-dependent KLF4 binding and target gene activation, we performed in-depth assays to examine impact of de-methylation on the recruitment of KLF4 WT to the promoter regions of two target genes in conjunction with changes in their RNA level .We appreciate the points raised by the reviewer and performed additional statistical analyses according to the reviewer’s suggestions. We have calculated the statistical significance of the overlapped genes. We focused on 12,824 genes that were expressed (FPKM > 0.5) in U87 cells. Among them, 308 (2.4%) up-regulated genes were identified. In the meantime, we observed that KLF4 mCpG-dependent binding peaks were associated with 1,072 expressed genes, of which 65 were up-regulated genes. The ratio of 6.1% is significantly higher than the ratio of expressed genes not associated with KLF4 binding sites  It seems possible that global DNA demethylation could attenuate the ability of cells to do many things, and thus the results shown in Please see our response to essential point #3. As far as we know, 5-Aza treatment is the only effective means to reduce global DNA methylation level. In response to essential point #3, we have added discussion of 5-Aza treatment to the manuscript. To examine the impact of DNA demethylation on the global expression level, we performed RNA-seq analysis and compared changes in gene expression profiles with and without 5-Aza treatment. We observed that in the absence of KLF4 WT induction the overall expression levels of the 116 KLF4 WT-specific target genes were not significantly affected by 5-Aza treatment .3) I do not see that We respectfully disagree with the reviewer. 4) The association of active marks with induced promoters and with many of the Klf4 sites is unsurprising – this would be seen even if the induction of the supposed "target genes" was indirect, and the KLF4 binding was coincidental. Indeed, the fact that there are 3,593 novel H3K27ac peaks, but only 274 overlap KLF4, shows that the two are largely dissociated. Thus, the statement in the Discussion that "Our study reveals that mCpG-dependent binding activity of KLF4 serves as the link between methylated CpG and changes in chromatin status" is not supported by the data shown. It is "a" link, but not "the" link.serves as a link between methylated CpG and changes in chromatin status" .We have changed this sentence to "Our study reveals that mCpG-dependent binding activity of KLF4


Escherichia coli (EPEC) to form attaching and effacing (A/E) lesions. A/E lesions are characterized by intimate bacterial adherence to intestinal cells and destruction of microvilli, which leads to diarrhea. Therefore, studies interrogating the regulation of the locus of enterocyte effacement (LEE) are critical for understanding the molecular epidemiology of EPEC infections and developing interventional strategies. Hitherto, most studies have centered on protein-based regulators, whereas the role of small regulatory RNAs remains underappreciated. Previously, we identified the first sRNAs—MgrR, RyhB, and McaS—that regulate the LEE of EPEC. This study was undertaken to identify additional sRNAs that impact the LEE. Our results suggest that the catabolite-responsive sRNA, Spot42, indirectly controls the LEE by inhibiting synthesis of its inducer, indole. Spot42 base-pairs with the tnaCAB mRNA and presumably destabilizes the transcript, thereby preventing expression of the regulatory and structural proteins that are involved in the import and hydrolysis of tryptophan into indole. The absence of intracellular indole leads to reduced transcription of the LEE1-encoded master transcriptional activator Ler, thereby maintaining the LEE in its silenced state and delaying A/E lesion morphogenesis. Our results highlight the importance of riboregulators that synchronize metabolic and virulence pathways in bacterial infection.The locus of enterocyte effacement is necessary for enteropathogenic  Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli (EHEC), genome-wide approaches have been undertaken to identify the full complement of sRNAs involved in virulence  (1 mM), and arabinose (0.02%). Strains, plasmids, and oligonucleotides used are listed in Bacteria were routinely propagated in Luria-Bertani (LB) broth or LB agar. The medium was supplemented with appropriate antibiotics and/or inducers when necessary. The following supplements were used at appropriate concentrations: Streptomycin (100 µg/mL), chloramphenicol (12.5–25 µg/mL), kanamycin (50 µg/mL), tetracycline (15 µg/mL), ampicillin (100 µg/mL), Isopropyl β-E. coli were engineered by lambda red-mediated recombineering essentially as described in our previous paper . O. OtnaCABy 4-fold C, while ng motif . Howeversequence A. The SpZ fusion C, confirLEE1 promoter in EPEC [LEE1 operon encodes Ler, which is the master regulator that spatiotemporally synchronizes transcription from the other LEE operons (LEE2–5) to stimulate morphogenesis of A/E lesions. Because indole levels were down-regulated in the Spot42 overproducer, we hypothesized that transcriptional activity from LEE1 would be diminished. Consistent with this prediction, overexpression of Spot42 negatively regulated β-galactosidase activity by 3-fold in a reporter E. coli strain harboring a chromosomal LEE1-ler’-lacZP+ transcriptional fusion (LEE1 promoter is reduced, one would anticipate a correlative reduction in expression of Ler-activated targets and A/E lesion biogenesis.The metabolite indole activates transcription from the  in EPEC . The firl fusion . BecausetnaCAB mRNA to globally silence gene expression from the entire transcript presumably by destabilizing it. The reduction in mRNA levels, in turn, leads to reduced expression and steady-state levels of the structural and regulatory proteins involved in indole biosynthesis (LEE1 promoter is not optimally active and Ler transcription is reduced (In summary, we have demonstrated that the Hfq-dependent sRNA Spot42 base-pairs to the ynthesis . In the  reduced , which, spf gene, is a well-studied riboregulator that is present in both pathogenic and nonpathogenic strains of E. coli [spf, thereby derepressing mRNAs involved in metabolism of secondary carbon sources [E. coli [The nutritional status of a bacterium can impact its virulence potential. For instance, the quality and/or quantity of a carbon source, such as sugars, dictate the molecular epidemiology of a bacterial infection ,43. Reci E. coli ,46. The  E. coli ,47. Pref E. coli . Spot42, E. coli . Upon de E. coli ,50. The  sources ,48. Thus sources . Whereas[E. coli , Spot42 [E. coli ,52.tnaCAB catabolic operon houses the regulatory and structural gene products that enables its host bacterium to utilize tryptophan as a carbon and energy source [LEE1 promoter, thereby affecting bacterial virulence. Thus, the gene products encoded within the tnaCAB operon bridge cellular metabolism with virulence in EPEC. As expected, in E. coli, tnaCAB is transcriptionally activated by CRP [tnaCAB operon is preserved between E. coli and EPEC, suggesting that the CRP-dependent transcriptional regulation is intact in this A/E pathogen [tnaCAB mRNA is also subject to antisense regulation by Spot42 suggests that CCR exerts a multi-tiered regulatory control to fine-tune gene expression from the tnaCAB operon. Based on our results, it is reasonable to hypothesize that Spot42 levels are likely to be higher in EPEC when the bacterium is in an environment where the expression from the LEE is not needed, such as outside of the mammalian host. Upon entry into the small intestine, Spot42 levels are likely to be down-regulated. This down-regulation would lead to derepression of the tnaCAB mRNA and stimulate indole biosynthesis. Indole production, in turn, would activate ler and prime the LEE regulatory cascade to culminate with the biogenesis of A/E lesions. Experiments to test this hypothesis are underway in our lab. The y source ,53. Addid by CRP ,55. The pathogen . Our disIn summary, the presented results expand the repertoire of genes, co-regulated by both CRP and Spot42, that bridge bacterial metabolism and virulence. Moreover, these results also highlight a recurring, but underappreciated, theme in the growing body of work on A/E pathogens, about the importance of regulatory RNAs in modulating pathogenetic pathways.


A neuron is unique in its ability to dynamically modify its transcriptional output in response to synaptic activity while maintaining a core gene expression program that preserves cellular identity throughout a lifetime that is longer than almost every other cell type in the body. A contributing factor to the immense adaptability of a neuron is its unique epigenetic landscape that elicits locus-specific alterations in chromatin architecture, which in turn influences gene expression. One such epigenetic modification that is sensitive to changes in synaptic activity, as well as essential for maintaining cellular identity, is DNA methylation. The focus of this article is on the importance of DNA methylation in neuronal function, summarizing recent studies on critical players in the establishment of (the “writing”), the modification or erasure of (the “editing”), and the mediation of (the “reading”) DNA methylation in neurodevelopment and neuroplasticity. One “reader” of DNA methylation in particular, methyl-CpG-binding protein 2 (MeCP2), is highlighted, given its undisputed importance in neuronal function.  Neuroplasticity is the ability of the brain to alter neuronal function in response to environmental input. This adaptability, expressed as long-lasting plasticity, is the basis of learning and memory formation. Two neural mechanisms that are essential for long-lasting plasticity are long-term depression (LTD) and long-term potentiation (LTP), which decrease and increase synaptic strength, respectively, in response to activity ,2. Here 2+) through NMDA receptors (NMDARs). Ca2+ entry in turn triggers a biochemical cascade starting with the activation (via phosphorylation) of calcium/calmodulin-dependent protein kinase II (CaMKII), which translocates to active synapses, where it is thought to bind to NMDARs, leading to the activation (via phosphorylation) of AMPA receptors (AMPARs). CaMKII is also thought to play a role in the activation of the RAS-extracellular signal-regulated kinase (ERK) (RAS-ERK) pathway, which subsequently leads to an increase in the insertion of AMPARs into the synapse. Together, these CaMKII-dependent actions increase the conductance of Ca2+ through AMPARs in stimulated spines, which triggers a molecular cascade that strengthens potentiated spines [LTP is generally broken down into two phases: a transient “early” phase 30–60 min) that is dependent upon the activation of pre-existing proteins and a stable “late” phase  that is contingent upon new gene transcription and translation . The indd spines ,3. An im0–60 min d spines ,3,4,5. Arc), a gene important in cytoskeleton organization at activated synapses [Similarly to the CaMKII cascade, many of the other early phase LTP signaling cascades converge upon the activation of ERK, which in turn activates various biochemical pathways that are essential for late phase LTP. One group of proteins activated by ERK are transcription factors, such as cAMP response element binding protein (CREB), FBJ osteosarcoma oncogene B (FOSB), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which subsequently induce gene expression changes essential for the potentiation of active synapses, such as immediate early response genes, ion channels, structural proteins, and neurotrophins ,6. One nsynapses . It is dde novo DNMTs, DNMT3A and DNMT3B, which establish methylation patterns on unmethylated DNA; and the maintenance DNMT, DNMT1, which preserves existing methylation patterns by methylating hemimethylated DNA [Dnmt3a and Dnmt1 are expressed in both the embryonic and adult stages of the brain, Dnmt3b is detectable only during early neurogenesis [DNA methylation is an epigenetic mechanism that allows for sustained adaptability of gene expression in response to developmental or environmental factors; it plays an essential role in various biological functions, such as regulation of gene transcription and the establishment and maintenance of cellular identity ,9,10. DNated DNA . Whereasogenesis ,17, sugg5mCG in all somatic cell types, including neurons, correlates with the repression of genes and repetitive DNA regions given whole genome bisulfite sequencing (WGBS) findings, which is a method used to determine genome-wide methylated cytosine sites, that 5mCG is enriched at intergenic regions, silenced genes, and repetitive DNA regions and is depleted at enhancers, promoters, and actively expressed gene bodies ,13,18,19Historically, DNA methylation was thought to be a stable, repressive covalent modification, existing predominantly, if not exclusively, as 5mCG. However, this view has dramatically changed over the past few years with the discovery that neurons, unlike other adult somatic cells, accumulate two other forms of DNA methylation over development, 5-hydroxymethylcystosine (5hmC) and non-CG methylation . 5hmC levels in neurons are up to 10-fold higher than other cell types, reaching almost 20% of all DNA methylation in the mouse frontal cortex ,25,26,27Tet1–3), with Tet2 and Tet3 mRNA levels being considerably higher than Tet1 postnatally [5mC is oxidized by the ten-eleven translocation (TET) methylcytosine dioxygenase family of enzymes to form 5hmC . Three Ttnatally ,31. Recetnatally . 5hmC istnatally ,32,33  and levels remain unchanged during development , 5mCH ise period ,29. Furtovo 5mCH . Additio context . Similar context ,29  of mice also results in neuronal dysfunction, such as hypoactivity, motor abnormalities, decreased grip strength, a reduction in motor neuron number, and a shortened lifespan [DNMT1 have been linked to hereditary sensory and neuropathy with dementia and hearing loss [DNMT3A with an overgrowth syndrome with intellectual disability [DNMT3B with immunodeficiency, centromere instability, and facial anomalies (ICF) syndrome, in which a large percentage of patients have intellectual disability [The importance of DNA methylation in neurodevelopment has been demonstrated by studies in which the loss of key enzymes that regulate DNA methylation, DNMTs and TETs, leads to deficits in neuronal function. Embryonic deletion of neration ,10. Similifespan . Human ging loss , DNMT3A sability , and DNMsability ,43,44.Tet1 constitutive knockout mice or mice lacking the catalytic dioxygenase domain of Tet1 display memory deficits, a reduction in the expression of genes associated with neurogenesis and neuronal activity , LTP defects, increased LTD, and a reduction in the proliferating potential of neural progenitor cells (NPCs) [Tet2 and Tet3 via electroporation of short hairpin RNAs (shRNAs) into the cortex of mice leads to defects in the progression of differentiated neurons from the subventricular zone [Similarly to DNMTs, various studies have also demonstrated the importance of TETs in brain development. s (NPCs) ,46. The lar zone . Given that these are embryonic perturbations of DNMTs and TETS, these findings highlight the importance of these enzymes and DNA methylation dynamics during neuronal development. Studies specifically focusing on these enzymes in postmitotic neurons have also highlighted the importance of DNA methylation in neuronal maturation and neuroplasticity, as discussed below.Dnmt1 and de novo enzyme Dnmt3a in adult postmitotic neurons [Since DNMTs are usually expressed in frequently dividing cells for the establishment and maintenance of DNA methylation patterns, it was originally perplexing to find considerable levels of maintenance enzyme  neurons ,48. HowePp1 to become rapidly methylated, resulting in the repression of this gene. This response is dependent upon DNMT activity given that chemical inhibition of this family of enzymes abolishes this effect [Bdnf, which undergoes promoter-specific methylation that corresponds with isoform-specific changes in gene expression [Bdnf is DNMT-dependent since chemical inhibition of DNMTs results in alterations in promoter methylation and isoform expression [Dnmt3a and Dnmt3b expression in the hippocampus in response to fear conditioning in both mice and rats [Learning and memory are associated with alterations in the methylation status of various plasticity-related genes. Fear conditioning causes the memory suppressor gene s effect . Anotherpression ,51. Thisand rats ,52, and and rats . Dnmt1 and Dnmt3a in 2-3 week postnatal forebrain excitatory neurons results in a decrease in DNA methylation, the misexpression of genes, a reduction in soma size, alterations in synaptic function, and deficits in learning and memory [Dnmt1 single conditional knockout in 2-3 week postnatal forebrain excitatory neurons does not appear to affect learning and memory nor a variety of cellular and molecular characteristics in the hippocampus or cortex, including methylation levels, neuronal survival, gene expression, soma size, or synaptic function (LTP and LTD) [Dnmt1 does result in an anxiolytic and antidepressant-like phenotype, suggesting that cell types, brain regions, or neuronal circuits that underlie specific behaviors can be differentially vulnerable to the loss of certain DNMTs [Dnmt3a loss in postmitotic forebrain excitatory neurons remains to be determined, as one group reports no changes in any parameters assessed , while another group finds deficits in LTP in conjunction with associative and episodic memory dysfunction [Double conditional knockout of d memory . Althougand LTD) ,53,54, tin DNMTs . A consefunction ,53.Tet1 in the dentate gyrus (DG) abolishes electroconvulsive stimulation (ECS)-induced demethylation of a Bdnf promoter [Tet1 knockdown in hippocampal neurons leads to the hypermethylation of a Bdnf promoter and subsequent repression of transcription from this promoter [Tet3 mRNA in the cortex, and when Tet3 is knocked down via lentiviral plasmids in the infralimbic prefrontal cortex (ILPFC), mice display impaired fear memory extinction. Furthermore, inhibiting NMDAR activity blocks the increase in Tet3 mRNA levels during fear memory extinction, suggesting that the rise in Tet3 expression occurs via an activity-dependent, NMDAR-mediated pathway. Notably, fear acquisition or fear extinction alters 5hmC levels at genomic locations that contain CA or CT dinucleotides more so than regions that contain CG dinucleotides [Tet3 expression levels correlate with neuronal activity as well; an increase in synaptic transmission correlates with an increase in Tet3, but not Tet1 or Tet2, mRNA and protein levels. When Tet3 is knocked down from hippocampal neurons in culture, miniature excitatory postsynaptic current (mEPSC) amplitudes are significantly larger than controls, and the reciprocal effect occurs when Tet3 is overexpressed. Tet3 is also essential for the maintenance of homeostatic synaptic plasticity; when Tet3 is knocked down in hippocampal cultured neurons, promoter IV of Bdnf is hypermethylated, resulting in a decrease in expression from this promoter [Tet2 knockout mice, it was found that this TET family member is responsible for the oxidation of a large fraction (~20%) of CG genomic regions that gain hydroxymethylation status over development and aging [The importance of TETs in postmitotic neurons has also been ascertained. TET1 is thought to be involved in neuronal activity-induced DNA demethylation and gene expression changes since shRNA-mediated knockdown of endogenous promoter . These fpromoter . Studiesleotides . Tet3 expromoter . And finnd aging . Taken tMethylation is thought to influence transcription by altering DNA-protein interactions, such as the binding of transcription factors  or the rMECP2 cause Rett Syndrome (RTT), a severe neurological disorder that affects 1 in 10,000 live female births, making it one of the most common causes of intellectual disability in females [MECP2 is an X-linked gene, RTT is almost exclusive found in females, as hemizygous loss of MeCP2 function in males leads to severe neonatal encephalopathy and death [MeCP2 is a nuclear protein found in various tissues in mammals ,72, with females . Given tnd death ,82,83,84RTT is characterized by normal development for the first 6 months of life followed by regression and symptomatic presentation, which includes loss of previously acquired purposeful hand skills and spoken language, gait abnormalities, and hand stereotypies ,86. PostMecp2 or carrying RTT-associated mutations in Mecp2 have been developed, and the majority of clinical features are observed in these whole-body Mecp2-mutant mice, such as normal development during early life followed by symptomatic presentation of altered gait, motor incoordination, hindlimb clasping, and cognitive deficits [Mecp2 from only the CNS either embryonically  or postnatally  results in phenotypes that are indistinguishable from the loss of Mecp2 from the whole-body [Mecp2-mutant mice also display pathological and histological features found in RTT patients, such as a smaller brain size [Mecp2-null mice are reduced in number and altered in morphology, resembling immature spines [Mecp2-null mice, and in fact, electrophysiological experiments corroborate this. In Mecp2-null mice, a reduction in excitatory synapses, as well as the quantal excitatory transmission at those synapses, contributes to a reduction in the spontaneous firing rate of cortical neurons [MECP2 mutations or loss have been intensely studied in order to understand the biological function of this protein, as will be discussed below.To understand the molecular underpinnings of RTT, mice lacking deficits ,92,93. Lole-body , emphasiain size ,92,93 anain size ,95. Furte spines ,95,96,97 neurons ,99,100. This year marks the 25th anniversary of the discovery of MeCP2 . Over thA screen for proteins that bind methylated DNA led to the discovery of MeCP2, given its high affinity for DNA methylation, as it is capable of binding to DNA with a symmetrically methylated 5mCG  through Mecp2 knockout mice are enriched for MeCP2 binding and 5mCA in the wild type setting [Mecp2 in cortical tissue, upregulated genes tend to be long, implicating MeCP2 as a transcriptional repressor [Mecp2 knockout mice, this gene group is upregulated [Mecp2-null hypothalamus tissue are enriched for MeCP2 binding and 5mCH in the wild type setting, suggesting a more complex model in which MeCP2 acts as both an activator and repressor [Various studies support the idea that MeCP2 binds globally across the genome at methylated cytosine sites ,73,110.  setting ,37,69,70Mecp2-null ESCs, there is a significant reduction of overall RNA synthesis, which was detected with the use of a radioactive ribonucleotide incorporation assay [MECP2-null ESCs, per-cell total RNA levels are significantly reduced, and this phenotype gets worse as time passes [Mecp2-null mouse neurons in vivo using array tomography (AT) imaging [Mecp2-null mice have found that more genes are repressed than activated, supporting MeCP2’s role as an activator [bona fide transcriptional activator or repressor.In direct opposition of the repressor model, MeCP2 has also been proposed to be a transcriptional activator. In mouse neurons differentiated from on assay . Similare passes . These f imaging . Additioctivator ,116,117.ctivator . Moleculctivator . In suppctivator . Howeverctivator ,119,120,ctivator . Taken tBdnf, with transcript and protein levels being significantly reduced in the brains of Mecp2-knockout mice [Bdnf appear to be critically important for the RTT phenotype, since increasing levels of Bdnf in Mecp2-null mice ameliorates electrophysiological dysfunction, improves locomotor deficits, and extends lifespan [Bdnf locus, suggesting that MeCP2 binding to developmentally regulated 5mCH sites is critical for the regulation of genes that are important in the manifestation of RTT phenotypes [Bdnf where it is thought to act as a repressor of transcription through the recruitment of the Sin3a repressor complex, which is found to be present via ChIP [Bdnf promoter, methylation levels decrease at the promoter, and Bdnf mRNA expression levels increase. Furthermore, the neurons subsequently display an increase in dendritic complexity and spine maturation, suggestive of an increase in neuroplasticity [Bdnf promoter is mediated by the phosphorylation of MeCP2 at serine 421 (pS421), given that the loss of this phosphorylation site on MeCP2 results in the loss of activity-dependent Bdnf mRNA expression [In line with MeCP2 acting as a regulator of transcription are findings demonstrating MeCP2’s ability to regulate gene expression in an activity-dependent manner. In this model, at basal conditions, unmodified MeCP2 functions as a repressor when bound to gene regions. However, upon stimulation, post-translational modifications on MeCP2 lead to a reduction in MeCP2 binding and subsequent gene activation. This function of MeCP2 is particularly intriguing given the necessity of gene expression changes for the maintenance of long-lasting neuronal plasticity. One important neuronal plasticity-related gene affected by the loss of MeCP2 function is out mice ,115,121.enotypes . Under bvia ChIP ,122,123.asticity ,122,123.Bdnf. To investigate the importance of MeCP2 phosphorylation at S421 in vivo, knock-in mice have been created in which S421 is converted to alanine (S421A), preventing the phosphorylation of this site. Cortical pyramidal neurons in these mutants have increased dendritic complexity and an increase in the amplitude of miniature inhibitory postsynaptic currents (mIPSCs), highlighting the importance of this phosphorylation site on MeCP2 in neuronal development and function. When MeCP2 binding is assessed in wild type and MeCP2-S421A primary cultures in response to neuronal activity, it was found that the binding patterns are maintained in the mutant, implying that this phosphorylation site is not necessary for the release of MeCP2 from bound sites in response to neuronal activation [In the mouse brain in response to neuronal activity, 10–30% of total MeCP2 becomes phosphorylated at S421 . Howevertivation . Additiotivation . And in tivation . In additivation ,126. TakMecp2-null mice or in mice carrying several RTT-associated point mutations in Mecp2 abolishes ATRX localization to pericentric heterochromatin in the brain [In addition to the MBD, MeCP2 has three additional domains that enable it to bind to DNA. These are three basic clusters containing AT-hook-like domains, which bind to AT-rich sequences of DNA ,127. Thehe brain . Recombinant MeCP2 binds, compacts, and oligomerizes nucleosomal arrays in vitro ,131,132.Mecp2-null CA1 pyramidal neurons of the hippocampus, which is indicative of an increase in condensation of heterochromatin. Additionally, it was found that heterochromatin volume and nuclear volume are negatively correlated, suggesting that chromatin condensation reduces nuclear size. AT imaging of Mecp2-null CA1 pyramidal neurons of the hippocampus also detected a redistribution of trimethylation of histone H4 at lysine 20 (H4K20me3) to pericentromeric heterochromatin, which is a region occupied by MeCP2 in the wild type setting. This redistribution of H4K20me3 in Mecp2-null neurons is dramatic, with 65% of total H4K20me3 changing its localization to dense heterochromatin, while there was a negligible increase (11%) in overall H4K20me3 nuclear levels. Additionally, there is a significant redistribution of trimethylation of histone H3 at lysine 9 (H3K9me3) to pericentromeric heterochromatin, as well as a slight increase (10%) in total H3K9me3 abundance [Mecp2-null neurons, these effects are not found later during development. The importance of chromocenter size is highlighted by a study that found an increase in heterochromatin size after induced depolarization of primary neuronal cultures; however, this effect is absent in Mecp2-deficient primary neurons [However, although few in number, studies examining the effects of MeCP2 loss on heterochromatin compaction in mouse neurons have found an increase, rather than decrease, in heterochromatinization. Using array tomography (AT) imaging in mosaic RTT mouse females, there was found to be an increase of DAPI density in heterochromatin in bundance . This ch neurons . Given tMecp2 knockout neurons from the cortex, H1 protein levels double, reaching the abundance of 1 molecule per nucleosome [Further support for MeCP2’s role in chromatin organization comes from studies that have compared MeCP2 to histone H1 (H1), an essential component of chromatin that binds to linker DNA and organizes nucleosomes into higher order structures. H1 is present at the level of one molecule per nucleosome in most somatic cells, except for neurons, where it is present at the level of one molecule for every two nucleosomes ,138,139.cleosome . H1 levecleosome . This sucleosome . HoweverTwenty-five years of research on MeCP2 has led to many interesting insights into the complex, multifaceted function of this protein, generating numerous hypothesized molecular models. And yet another model is emerging—a synergistic model that treats many of the proposed functions as an integrative piece of MeCP2’s function, instead of treating them as mutually exclusive entities. It is possible that MeCP2 is predominantly an architectural protein that is important for regulating chromatin structural changes that aid in the increase or decrease of gene expression necessary for long-lasting plasticity in response to neuronal activity. Therefore, in this model, the transcriptional activator or repressor effects that have been attributed to MeCP2 would be secondary to its organizational role, similar to the function of CCCTC-binding factor (CTCF) ,142. HowNeurons are unique in that they readily modify their gene expression programs to enable long-lasting plasticity throughout their lifetime. A key contributing factor to this characteristic is a neuron’s unique, adaptable methylome. Two families of enzymes that regulate DNA methylation status, DNMTs and TETs, are essential for normal neuronal function. Additionally, a neuronally enriched “reader” of DNA methylation, MeCP2, plays a particularly important role in mediating the biological functions of the brain’s distinctive methylome.