Cbx3 maintains lineage specificity during neural differentiation

Huang, Chi‐Chun; Su, Trent; Xue, Yong; Cheng, Chen; Lay, Fides D.; McKee, Robin; Li, Meiyang; Vashisht, Ajay A.; Wohlschlegel, James; Novitch, Bennett G.; Plath, Kathrin; Kurdistani, Siavash K.; Carey, Michael

doi:10.1101/gad.292169.116

Cited by 40 publications

(41 citation statements)

References 40 publications

(46 reference statements)

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“…4j-k, left). We also evaluated the RNA expression levels of genes co-marked with H4K20me3/H3K4me3 relative to H4K20me3, H3K9me3, or H3K4me3 regions in MEFs (see methods) and NPs (GSE89574) [28]. Our findings demonstrate that genes co-marked by H4K20me3/ H3K4me3 in ES cells is significantly lower relative to genes marked by H3K4me3 (Fig.…”

Section: H4k20me3/h3k4me3 Marks Transcriptionally Dynamic Genes In Esmentioning

confidence: 83%

“…k Empirical cumulative distribution function (ECDF) for RNAPII density in mouse fibroblasts (left) and neural progenitors deactivation of RNAPII binding upon downregulation of OCT4. Likewise, to investigate whether H4K20me3/ H3K4me3 poises genes for deactivation of RNAPII binding during lineage-specific differentiation, we evaluated RNAPII binding using public ChIP-Seq data from mouse fibroblasts (MEFs) (GSE71507) and neural progenitor cells (NP) (GSE89573) [28]. Results from these analyses demonstrate that RNAPII binding was significantly lower at H4K20me3/H3K4me3 regions relative to H3K4me3 regions ( Fig.…”

Section: H4k20me3/h3k4me3 Marks Transcriptionally Dynamic Genes In Esmentioning

confidence: 99%

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H4K20me3 co-localizes with activating histone modifications at transcriptionally dynamic regions in embryonic stem cells

Kidder

2018

BMC Genomics

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BackgroundBivalent chromatin domains consisting of the activating histone 3 lysine 4 trimethylation (H3K4me3) and repressive histone 3 lysine 27 trimethylation (H3K27me3) histone modifications are enriched at developmental genes that are repressed in embryonic stem cells but active during differentiation. However, it is unknown whether another repressive histone modification, histone 4 lysine 20 trimethylation (H4K20me3), co-localizes with activating histone marks in ES cells.ResultsHere, we describe the previously uncharacterized coupling of the repressive H4K20me3 heterochromatin mark with the activating histone modifications H3K4me3 and histone 3 lysine 36 trimethylation (H3K36me3), and transcriptional machinery (RNA polymerase II; RNAPII), in ES cells. These newly described bivalent domains consisting of H3K4me3/H4K20me3 are predominantly located in intergenic regions and near transcriptional start sites of active genes, while H3K36me3/H4K20me3 are located in intergenic regions and within gene body regions of active genes. Global sequential ChIP, also termed reChIP-Seq, confirmed the simultaneous presence of H3K4me3 and H4K20me3 at the same genomic regions in ES cells. Genes containing H3K4me3/H4K20me3 exhibit decreased RNAPII pausing and are poised for deactivation of RNAPII binding during differentiation relative to H3K4me3 marked genes. An evaluation of transcription factor (TF) binding motif enrichment revealed that DNA sequence may play a role in shaping the landscape of these novel bivalent domains. Moreover, H3K4me3/H4K20me3 and H3K36me3/H4K20me3 bound regions are enriched with repetitive LINE and LTR elements.ConclusionsOverall, these findings highlight a previously undescribed subnetwork of ES cell transcriptional circuitry that utilizes dual marking of the repressive H4K20me3 mark with activating histone modifications.

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Section: H4k20me3/h3k4me3 Marks Transcriptionally Dynamic Genes In Esmentioning

confidence: 83%

Section: H4k20me3/h3k4me3 Marks Transcriptionally Dynamic Genes In Esmentioning

confidence: 99%

H4K20me3 co-localizes with activating histone modifications at transcriptionally dynamic regions in embryonic stem cells

Kidder

2018

BMC Genomics

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“…5A). Recent studies have shown that some repressive chromatin modifiers regulate lineage fidelity during neural differentiation of ESCs by enhancing neural gene expression and suppressing genes specific to other lineages (8,9). Furthermore, a previous report has shown that the CHD4/NuRD complex establishes the identities of the two striated muscle types, cardiac and skeletal muscle, by suppressing the alternate lineage gene program (34).…”

Section: Inhibition Of Mesendoderm Differentiation Does Not Affect Immentioning

confidence: 99%

“…Previous studies have uncovered that the intrinsic programs mediated by transcription factors and epigenetic regulators play important roles in the default model of neural fate determination (3)(4)(5)(6)(7). Recent studies have shown that repressive chromatin modifiers, polycomb repressive complex 2 (PRC2) and Chromobox homolog 3, regulate lineage fidelity during neural differentiation of ESCs by enhancing neural gene expression and suppressing the genes specific to other cell lineages (8,9). These results indicate the importance of repressive chromatin modifiers in neural lineage commitment.…”

mentioning

confidence: 98%

The nucleosome remodeling and deacetylase complex protein CHD4 regulates neural differentiation of mouse embryonic stem cells by down-regulating p53

Hirota

Nakajima-Koyama

Ashida

et al. 2019

Journal of Biological Chemistry

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Edited by Xiao-Fan WangLineage specification of the three germ layers occurs during early embryogenesis and is critical for normal development. The nucleosome remodeling and deacetylase (NuRD) complex is a repressive chromatin modifier that plays a role in lineage commitment. However, the role of chromodomain helicase DNAbinding protein 4 (CHD4), one of the core subunits of the NuRD complex, in neural lineage commitment is poorly understood. Here, we report that the CHD4/NuRD complex plays a critical role in neural differentiation of mouse embryonic stem cells (ESCs). We found that RNAi-mediated Chd4 knockdown suppresses neural differentiation, as did knockdown of methyl-CpG-binding domain protein Mbd3, another NuRD subunit. Chd4 and Mbd3 knockdowns similarly affected changes in global gene expression during neural differentiation and up-regulated several mesendodermal genes. However, inhibition of mesendodermal genes by knocking out the master regulators of mesendodermal lineages, Brachyury and Eomes, through a CRISPR/Cas9 approach could not restore the impaired neural differentiation caused by the Chd4 knockdown, suggesting that CHD4 controls neural differentiation by not repressing other lineage differentiation processes. Notably, Chd4 knockdown increased the acetylation levels of p53, resulting in increased protein levels of p53. Double knockdown of Chd4 and p53 restored the neural differentiation rate. Furthermore, overexpression of BCL2, a downstream factor of p53, partially rescued the impaired neural differentiation caused by the Chd4 knockdown. Our findings reveal that the CHD4/NuRD complex regulates neural differentiation of ESCs by down-regulating p53. 3 The abbreviations used are: ESC, embryonic stem cell; NuRD, nucleosome remodeling and deacetylase; CHD4, chromodomain helicase DNA-binding protein 4; qRT, quantitative RT; CpG, cytosine-phosphate-guanine; GO, gene ontology; shRNA, short hairpin RNA; AFP, ␣-fetoprotein; GMEM, Glasgow minimum essential medium; LIF, leukemia inhibitory factor. Figure 6. CHD4 and MBD3 regulate the acetylation and protein levels of p53. A, qRT-PCR analysis. Each mRNA level was normalized to the ␤-actin level, and the value of control shRNA-expressing cells at day 4 was set to 1. The data are shown as the means Ϯ S.D. (n ϭ 3 independent experiments). **, p Ͻ 0.01. The p values were calculated using Student's unpaired two-tailed t tests compared with control cells on the same day. B, immunoblotting analysis for acetyl-p53 (Lys-379), p53, p300, CHD4, HDAC1, Myc, and ␣-tubulin (as a loading control) in lysates from HEK293T cells transfected with the indicated combination of expression vectors (left). The relative acetyl-p53 (Lys-379) signal intensities are shown (right). The data are shown as the means Ϯ S.D. (n ϭ 3 independent experiments). *, p Ͻ 0.05, and **, p Ͻ 0.01. N.S., not significant. The p values were calculated using Student's unpaired two-tailed t tests. C, immunoblotting analysis for p53, acetyl-p53 (Lys-379), and ␣-tubulin (as a loading control) in control shRNA-...

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“…Beyond their role in structuring heterochromatin, HP1 proteins also functions as regulators of euchromatic transcription. For instance, at the promoters of many inducible genes involved in development or in cellular defense, they participate in the transient silencing of transcription while awaiting stimulation [15][16][17][18][19][20] . But HP1 proteins, in particular HP1g in mammals, are also present inside the coding region of genes 21 , a localization which is not always correlated with H3K9me3 19 .…”

Section: Introductionmentioning

confidence: 99%

HP1γ binding pre-mRNA at intronic repeats increases splicing fidelity and regulates alternative exon usage

Rachez

Legendre

Varet

et al. 2019

Preprint

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HP1 proteins are best known as markers of heterochromatin and gene silencing. Yet, they are also RNA-binding proteins and the HP1g/Cbx3 family member is present on transcribed genes together with RNA polymerase II, where it regulates co-transcriptional processes such as alternative splicing. To gain insight in the role of the RNA binding activity of HP1g in transcriptionally active chromatin, we have captured and analyzed RNAs associated with this protein. We find that HP1g specifically recognizes hexameric RNA motifs and coincidentally transposable elements of the SINE family. As these elements are abundant in introns, while essentially absent from exons, the HP1g RNA binding activity tethers unspliced pre-mRNA to chromatin via the intronic region and limits the usage of intronic cryptic splice sites. Thus, our data unveil novel determinants in the relationship between chromatin and cotranscriptional splicing.showing that its role in mRNA maturation, alike its role in heterochromatin structuring, relies on its ability to associate with repeat-encoded RNAs. RESULTS HP1g associates with chromatin-enriched RNA.To better understand the relationship between HP1g and RNA, beyond its classical role in heterochromatin formation, we used a genome-wide approach to assay the interactions between HP1g and RNA on chromatin (RNAchIP; Fig. 1a). For this, we used a modification of our previously described strategy to solubilize native chromatin and produce chromatinenriched RNA fragments suitable for immunoprecipitation 25 (Supplementary Fig. 1d). HP1g -/-(KO cells) mouse embryonic fibroblast (MEF)-derived cell lines, re-complemented with FLAG-tagged HP1g (HP1g cells) as previously described were used in these assays 16 . These cells expressed ectopic FLAG-tagged HP1g at the same level as the endogenous HP1g in WT MEFs ( Supplementary Fig. 1a). As in our previous studies, the cells were treated or not with the phorbol ester PMA, an activator of the PKC signaling pathway, in order to observe the cellular response to stress on chromatin and transcription. Nuclei were isolated to obtain a chromatin-enriched RNA fraction, ( Supplementary Fig. 1a and b). Our procedure was modified to include a limited cross-linking step in order to stabilize association of HP1g with RNA, together with stringent immunoprecipitation conditions to favor direct protein-RNA associations. Under these conditions, anti-FLAG antibody did not precipitate any of the HP1g-interacting proteins we tested (HP1a, H3, RNA polymerase II) ( Supplementary Fig. 1b). When quantified by RT-qPCR, HP1g was found to associate with chromatin-enriched RNA from the Fosl1 locus with a 20-to 25-fold enrichment compared to KO cells. IP in KO cells consistently showed very low to undetectable RNA levels ( Supplementary Fig. 1c).Genome-wide analysis of the RNAchIP by HP1g (IP) versus RNA detected in the chromatin fraction (input) was then performed using Illumina sequencing on biological triplicates. Reads were then mapped onto the mouse genome. In a first approach, RNAchIPseq dat...

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Cbx3 maintains lineage specificity during neural differentiation

Cited by 40 publications

References 40 publications

H4K20me3 co-localizes with activating histone modifications at transcriptionally dynamic regions in embryonic stem cells

H4K20me3 co-localizes with activating histone modifications at transcriptionally dynamic regions in embryonic stem cells

The nucleosome remodeling and deacetylase complex protein CHD4 regulates neural differentiation of mouse embryonic stem cells by down-regulating p53

HP1γ binding pre-mRNA at intronic repeats increases splicing fidelity and regulates alternative exon usage

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