Mapping Chromatin Occupancy of Ppp1r1b-lncRNA Genome-Wide Using Chromatin Isolation by RNA Purification (ChIRP)-seq.

Long non-coding RNA (lncRNA) mediated transcriptional regulation is increasingly recognized as an important gene regulatory mechanism during development and disease. LncRNAs are emerging as critical regulators of chromatin state; yet the nature and the extent of their interactions with chromatin remain to be fully revealed. We have previously identified Ppp1r1b-lncRNA as an essential epigenetic regulator of myogenic differentiation in cardiac and skeletal myocytes in mice and humans. We further demonstrated that Ppp1r1b-lncRNA function is mediated by the interaction with the chromatin-modifying complex polycomb repressive complex 2 (PRC2) at the promoter of myogenic differentiation transcription factors, TBX5 and MyoD1. Herein, we employed unbiased chromatin isolation by RNA purification (ChIRP) and high throughput sequencing to map the repertoire of Ppp1r1b-lncRNA chromatin occupancy genome-wide in the mouse muscle myoblast cell line. We uncovered a total of 99732 true peaks corresponding to Ppp1r1b-lncRNA binding sites at high confidence (p-value < 1E-5) and enrichment score ≥ 10). The Ppp1r1b-lncRNA-binding sites averaged 558 bp in length and were distributed widely within the coding and non-coding regions of the genome. Approximately 46% of these true peaks were mapped to gene elements, of which 1180 were mapped to experimentally validated promoter sequences. Importantly, the promoter-mapped binding sites were enriched in myogenic transcription factors and heart development while exhibiting focal interactions with known motifs of proximal promoters and transcription initiation by RNA Pol-II, including TATA-box, transcription initiator motif, CCAAT-box, and GC-box, supporting Ppp1r1b-lncRNA role in transcription initiation of myogenic regulators. Remarkably, nearly 40% of Ppp1r1b-lncRNA-binding sites mapped to gene introns were enriched with the Homeobox family of transcription factors and exhibited TA-rich motif sequences, suggesting potential motif-specific Ppp1r1b-lncRNA-bound introns. Lastly, more than 136521 enhancer sequences were detected in Ppp1r1b-lncRNA-occupancy sites at high confidence. Among these enhancers, 3390 (12%) exhibited cell type/tissue-specific enrichment in fetal heart and muscles. Together, our findings provide further insights into the genome-wide Ppp1r1b-lncRNA: Chromatin interactome that may dictate its function in myogenic differentiation and potentially other cellular and biological processes.

Mapping Chromatin Occupancy of Ppp1r1b-lncRNA Genome-Wide Using Chromatin Isolation by RNA Purification (ChIRP)-seq.

Long non-coding RNA (lncRNA) mediated transcriptional regulation is increasingly recognized as an important gene regulatory mechanism during development and disease. LncRNAs are emerging as critical regulators of chromatin state; yet the nature and the extent of their interactions with chromatin remain to be fully revealed. We have previously identified Ppp1r1b-lncRNA as an essential epigenetic regulator of myogenic differentiation in cardiac and skeletal myocytes in mice and humans. We further demonstrated that Ppp1r1b-lncRNA function is mediated by the interaction with the chromatin-modifying complex polycomb repressive complex 2 (PRC2) at the promoter of myogenic differentiation transcription factors, TBX5 and MyoD1. Herein, we employed an unbiased chromatin isolation by RNA purification (ChIRP) and high throughput sequencing to map the repertoire of Ppp1r1b-lncRNA chromatin occupancy genome-wide in the mouse muscle myoblast cell line. We uncovered a total of 99732 true peaks corresponding to Ppp1r1b-lncRNA binding sites at high confidence (P-value < 1e-5 and enrichment score ≥ 10). The Ppp1r1b-lncRNA-binding sites averaged 558 bp in length and were distributed widely within the coding and non-coding regions of the genome. Approximately 46% of these true peaks were mapped to gene elements, of which 1180 were mapped to experimentally validated promoter sequences. Importantly, the promoter-mapped binding sites were enriched in myogenic transcription factors and heart development while exhibiting focal interactions with known motifs of proximal promoters and transcription initiation by RNA polII, including TATA, transcription initiator, CCAAT-box, and GC-box, supporting Ppp1r1b-lncRNA role in transcription initiation of myogenic regulators. Remarkably, nearly 40% of Ppp1r1b-lncRNA-binding sites mapped to gene introns, were enriched with the Homeobox family of transcription factors, and exhibited TA-rich motif sequences, suggesting potential motif specific Ppp1r1b-lncRNA-bound introns. Lastly, more than 136521enhancer sequences were detected in Ppp1r1b-lncRNA-occupancy sites at high confidence. Among these enhancers,12% exhibited cell type/tissue-specific enrichment in fetal heart and muscles. Together, our findings provide further insights into the genome-wide Ppp1r1b-lncRNA: Chromatin interactome that may potentially dictate its function in myogenic differentiation and potentially other cellular and biological processes.

Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation.

BACKGROUND: The IRE1a-XBP1 pathway is a conserved adaptive mediator of the unfolded protein response. The pathway is indispensable for the development of secretory cells by facilitating protein folding and enhancing secretory capacity. In the immune system, it is known to function in dendritic cells, plasma cells, and eosinophil development and differentiation, while its role in T helper cell is unexplored. Here, we investigated the role of the IRE1a-XBP1 pathway in regulating activation and differentiation of type-2 T helper cell (Th2), a major T helper cell type involved in allergy, asthma, helminth infection, pregnancy, and tumor immunosuppression. METHODS: We perturbed the IRE1a-XBP1 pathway and interrogated its role in Th2 cell differentiation. We performed genome-wide transcriptomic analysis of differential gene expression to reveal IRE1a-XBP1 pathway-regulated genes and predict their biological role. To identify direct target genes of XBP1 and define XBP1's regulatory network, we performed XBP1 ChIPmentation (ChIP-seq). We validated our predictions by flow cytometry, ELISA, and qPCR. We also used a fluorescent ubiquitin cell cycle indicator mouse to demonstrate the role of XBP1 in the cell cycle. RESULTS: We show that Th2 lymphocytes induce the IRE1a-XBP1 pathway during in vitro and in vivo activation. Genome-wide transcriptomic analysis of differential gene expression by perturbing the IRE1a-XBP1 pathway reveals XBP1-controlled genes and biological pathways. Performing XBP1 ChIPmentation (ChIP-seq) and integrating with transcriptomic data, we identify XBP1-controlled direct target genes and its transcriptional regulatory network. We observed that the IRE1a-XBP1 pathway controls cytokine secretion and the expression of two Th2 signature cytokines, IL13 and IL5. We also discovered that the IRE1a-XBP1 pathway facilitates activation-dependent Th2 cell proliferation by facilitating cell cycle progression through S and G2/M phase. CONCLUSIONS: We confirm and detail the critical role of the IRE1a-XBP1 pathway during Th2 lymphocyte activation in regulating cytokine expression, secretion, and cell proliferation. Our high-quality genome-wide XBP1 ChIP and gene expression data provide a rich resource for investigating XBP1-regulated genes. We provide a browsable online database available at http://data.teichlab.org .

Collaborative repressive action of the antagonistic ETS transcription factors Pointed and Yan fine-tunes gene expression to confer robustness in Drosophila.

The acquisition of cellular identity during development depends on precise spatiotemporal regulation of gene expression, with combinatorial interactions between transcription factors, accessory proteins and the basal transcription machinery together translating complex signaling inputs into appropriate gene expression outputs. The opposing repressive and activating inputs of the Drosophila ETS family transcription factors Yan and Pointed orchestrate numerous cell fate transitions downstream of receptor tyrosine kinase signaling, providing one of the premier systems for studying this process. Current models describe the differentiative transition as a switch from Yan-mediated repression to Pointed-mediated activation of common target genes. We describe here a new layer of regulation whereby Yan and Pointed co-occupy regulatory elements to repress gene expression in a coordinated manner, with Pointed being unexpectedly required for the genome-wide occupancy of both Yan and the co-repressor Groucho. Using even skipped as a test-case, synergistic genetic interactions between Pointed, Groucho, Yan and components of the RNA polymerase II pausing machinery suggest that Pointed integrates multiple scales of repressive regulation to confer robustness. We speculate that this mechanism may be used broadly to fine-tune the expression of many genes crucial for development.

GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of histone H3, lysine 27.

GATA4 is expressed in the proximal 85% of small intestine where it promotes a proximal intestinal ('jejunal') identity while repressing a distal intestinal ('ileal') identity, but its molecular mechanisms are unclear. Here, we tested the hypothesis that GATA4 promotes a jejunal versus ileal identity in mouse intestine by directly activating and repressing specific subsets of absorptive enterocyte genes by modulating the acetylation of histone H3, lysine 27 (H3K27), a mark of active chromatin, at sites of GATA4 occupancy. Global analysis of mouse jejunal epithelium showed a statistically significant association of GATA4 occupancy with GATA4-regulated genes. Occupancy was equally distributed between down- and up-regulated targets, and occupancy sites showed a dichotomy of unique motif over-representation at down- versus up-regulated genes. H3K27ac enrichment at GATA4-binding loci that mapped to down-regulated genes (activation targets) was elevated, changed little upon conditional Gata4 deletion, and was similar to control ileum, whereas H3K27ac enrichment at GATA4-binding loci that mapped to up-regulated genes (repression targets) was depleted, increased upon conditional Gata4 deletion, and approached H3K27ac enrichment in wild-type control ileum. These data support the hypothesis that GATA4 both activates and represses intestinal genes, and show that GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of H3K27.

Genome wide chromatin occupancy of mrhl RNA and its role in gene regulation in mouse spermatogonial cells.

Mrhl RNA is a nuclear lncRNA encoded in the mouse genome and negatively regulates Wnt signaling in spermatogonial cells through p68/Ddx5 RNA helicase. Mrhl RNA is present in the chromatin fraction of mouse spermatogonial Gc1-Spg cells and genome wide chromatin occupancy of mrhl RNA by ChOP (Chromatin oligo affinity precipitation) technique identified 1370 statistically significant genomic loci. Among these, genes at 37 genomic loci also showed altered expression pattern upon mrhl RNA down regulation which are referred to as GRPAM (Genes Regulated by Physical Association of Mrhl RNA). p68 interacted with mrhl RNA in chromatin at these GRPAM loci. p68 silencing drastically reduced mrhl RNA occupancy at 27 GRPAM loci and also perturbed the expression of GRPAM suggesting a role for p68 mediated mrhl RNA occupancy in regulating GRPAM expression. Wnt3a ligand treatment of Gc1-Spg cells down regulated mrhl RNA expression and also perturbed expression of these 27 GRPAM genes that included genes regulating Wnt signaling pathway and spermatogenesis, one of them being Sox8, a developmentally important transcription factor. We also identified interacting proteins of mrhl RNA associated chromatin fraction which included Pc4, a chromatin organizer protein and hnRNP A/B and hnRNP A2/B1 which have been shown to be associated with lincRNA-Cox2 function in gene regulation. Our findings in the Gc1-Spg cell line also correlate with the results from analysis of mouse testicular tissue which further highlights the in vivo physiological significance of mrhl RNA in the context of gene regulation during mammalian spermatogenesis.