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7. m6A mediates alternative intron/exon inclusion in the nascent transcriptome.

We investigate the presence of m6A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m6A peaks are located in alternative introns/exons, often close to 5' splice sites. m6A peaks significantly overlap with RBM15 RNA-binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5' splice sites that are proximal to m6A peaks. For terminal or variable-length exons, m6A peaks are generally located on or immediately downstream of a 5' splice site that is suppressed in the presence of m6A, and upstream of a 5' splice site that is promoted in the presence of m6A. Genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function.  High throughput sequencing data of the ChrMeRIP-seq, m6A-seq, RBM15 irCLIP-seq, and dTAG METTL3 4sU-seq, are deposited in GEO under GSE154709.

6. SLAM-seq of Xist half-life, ChrRNA-seq of Xist-mediated chromsome silencing.

The noncoding RNA Xist, which controls the process of X chromosome inactivation in mammals, accumulates and spreads over the chromosome from which it is transcribed. The underlying basis for this unusual behavior is poorly understood. Using a new imaging approach called RNA-SPLIT for time-resolved analysis of Xist RNA molecules at super-resolution, Rodermund et al. analyzed fundamental parameters of Xist RNA behavior in normal cells and after the perturbation of factors implicated in Xist RNA function. The authors provide new insights into the basis of Xist RNA localization and confinement within the territory of a single X chromosome. 

High throughput sequencing data of the SLAM-seq measuring Xist RNA half-life, ChrRNA-seq measuring SPEN SPOC mutant as well as other mutants, ChIP-seq of histone modification along X chromosome, are deposited in GEO under GSE154658.

5. The PWWP2A Histone Deacetylase Complex Represses Intragenic Spurious Transcription Initiation in mESCs

PWWP2A/B is an H3K36me3 reader which forms a stable complex with HDAC1/2. We used CAGE-seq to profile all transcription initiation sites in wildtype mESCs and cells lacking PWWP2A/B. Loss of PWWP2A/B enhances spurious initiation from intragenic sites present in wildtype mESCs, and this effect is associated with increased levels of initiating Pol-II and histone acetylation. Spurious initiation events in Pwwp2a/b DKO mESCs do not overlap in genomic location or chromatin features with spurious sites that arise in Dnmt3b KO mESCs, previously reported to function in the suppression of intragenic transcriptional initiation, suggesting these pathways function cooperatively in maintaining the fidelity of transcription initiation in metazoans. The CAGE-seq data as well as processed bigwig files are deposited in GEO under GSE148382.

4. Systematic Analysis Defines the Interplay of Key Pathways in X Chromosome Inactivation

Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing.  Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/m6A-methyltransferase complex make only a minor contribution to gene silencing. The allelic data of ChrRNA-seq, ChIP-seq(H3K27me3 and H2AK119ub), and m6A-seq are deposited in GEO under GSE119607.

3. PWWP2A/B-NuRD complex or variant NuRD complex

Transcriptional regulation by chromatin is a highly dynamic process directed through the recruitment and coordinated action of epigenetic modifiers and readers of these modifications. Using an unbiased proteomic approach to find interactors of H3K36me3, a modification enriched on active chromatin, here we identify PWWP2A and HDAC2 among the top interactors. PWWP2A and its paralog PWWP2B form a stable complex with NuRD subunits MTA1/2/3:HDAC1/2:RBBP4/7, but not with MBD2/3, p66α/β, and CHD3/4. PWWP2A competes with MBD3 for binding to MTA1, thus defining a new variant NuRD complex that is mutually exclusive with the MBD2/3-containing NuRD. In mESCs, PWWP2A/B is most enriched at highly transcribed genes. Loss of PWWP2A/B leads to increases in histone acetylation predominantly at highly expressed genes, accompanied by decreases in Pol II elongation. Collectively, these findings suggest a role for PWWP2A/B in regulating transcription through the fine-tuning of histone acetylation dynamics at actively transcribed genes.  The high-throughput sequencing data generated in this study can be found at GSE112114.

2. hnRNPK recruits PCGF3/5-PRC1 to the Xist RNA B-repeat to establish Polycomb mediated chromosomal silencing

We define the Xist RNA Polycomb Interaction Domain (XR-PID), a 600 nt sequence encompassing the Xist B-repeat element. Repression Score (RS) analysis were developed to quantitatively compare the silencing ability among different mutated Xist in the autosomal transgene background. Deletion of XR-PID abolishes Xist-dependent Polycomb recruitment, in turn abrogating Xist-mediated gene silencing (measured by allelic 4sU-RNA-seq) and reversing Xist induced chromatin inaccessibility (measured by allelic ATAC-seq). We identify the RNA-binding protein hnRNPK as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1 (Mass-Spec and biochemical assay). Accordingly, synthetically tethering hnRNPK to Xist RNA lacking XR-PID is sufficient for Xist-dependent Polycomb recruitment (allelic 4sU-seq). Our findings define a key pathway for Polycomb recruitment by Xist RNA, providing important insights into mechanisms of chromatin modification by non-coding RNA. The high-throughput sequencing data generated by this study were deposited in GSE103370.

1. Long Noncoding RNA, SNHG1 Regulates Transcription of both Local and Distal Genes

SNHG1 is a nuclear-enriched long noncoding RNA, which displays high expression across almost all the cell types and tissues. Higher expression of SNHG1 in tumor tissues is observed at multiple cancer types. ChIRP-seq against SNHG1 were performed in HCT116 cells. SNHG1 binding signals are concentrated around the 5' one-third and last exon of the SNHG1 gene locus, and spread to the 3' downstream region. This interaction with chromatin is crucial for establishing the chromatin looping with the locus of neighboring gene, SLC3A2, further to promote nearby gene transcription (SLC3A2).  The ChIRP-seq data are deposited on GEO database under accession, GSE85842.