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A long non-coding RNA tunes PRC1 function during human and zebrafish development

ABSTRACT: Long non-coding RNAs (lncRNAs) are emerging as functional regulators of gene expression in many species1,2. However, aligning lncRNA-dependent phenotypes with their underlying molecular mechanisms remains challenging. The Polycomb Repressive Complex 1 (PRC1) family of the Polycomb Group (PcG) complexes interacts with lncRNAs and is essential for gene silencing during development. Despite the functional importance of PRC1, few specific lncRNAs that guide PRC1 activity are known3-5. We screened for human RNAs which co-precipitate with the PRC1-component BMI1, from a chromatin fraction. Knockdown of several of the lncRNA candidates perturbed PcG-regulated gene expression in vivo. In particular, we found that the PRC1-associated lncRNA CAT7 tunes PRC1 function during zebrafish and human development. During neuronal differentiation of human embryonic stem cells, CAT7 impacts expression and PcG-binding of the MNX1 locus, which encodes a master regulator of motor neuron development. During zebrafish development, human CAT7 functionally rescues defects caused by interference of the non-syntenic analog, zebrafish cat7 (zcat7.) Further, zcat7 genetically interacts with bmi1a/b during zebrafish embryogenesis. We propose that PRC1 acts in concert with specific lncRNAs, and that CAT7/zcat7 represent lncRNAs that convergently evolved to tune PRC1 repression at individual loci. We immunoprecipitated PRC1-interacting RNAs from a chromatin fraction using a technology called GRIP. We identified candidates in HeLa cells containing a 25% overexpression of FLAG-tagged murine Bmi1 (ectopic). Candidates were selected which were enriched in both Bmi1 and FLAG-Bmi1 GRIP versus input. Knockdown of 17 of these candidates was performed (vs a scramble control) and total RNA was sequenced 48 hours after knockdown. One candidate, CAT7 was also knocked down in developing (motor) neural cells (grown from human embryonic stem cells). We also performed ChIP targeting Polycomb proteins in the HeLa cells depleted for CAT7 or scramble controls.

ORGANISM(S): Homo sapiens

PROVIDER: GSE61549 | GEO | 2016/07/07

SECONDARY ACCESSION(S): PRJNA261439

REPOSITORIES: GEO

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Project description:Polycomb group (PcG) proteins control organism development by regulating the expression of developmental genes. Transcriptional regulation by PcG proteins is achieved at least partly through the PRC2-mediated methylation on lysine 27 of histone H3 (H3K27) and PRC1-mediated ubiquitylation on lysine 119 of histone H2A (uH2A). As an integral component of PRC1, Bmi1 has been demonstrated to be critical for H2A ubiquitylation. Although recent studies have revealed the genome wide binding patterns of some of the PRC1 and PRC2 components, as well as the H3K27me3 mark, there have been no reports describing genome wide localization of uH2A. Using the recently developed ChIP-Seq technology, here we report genome wide localization of the Bmi1-dependent uH2A mark in MEF cells. Gene promoter averaging analysis indicates a peak of uH2A just inside the transcription start site (TSS) of well annotated genes. This peak is enriched at promoters containing the H3K27me3 mark and represents the least expressed genes in WT MEF cells. In addition, peak finding reveals regions of local uH2A enrichment throughout the mouse genome, including almost 700 gene promoters. Genes with promoter peaks of uH2A exhibit lower level expression when compared to genes that do not contain promoter peaks of uH2A. Moreover, we demonstrate that genes with uH2A peaks have increased expression upon Bmi1 knockout. Importantly, local enrichment of uH2A is not limited to regions containing the H3K27me3 mark. We provide evidence to suggest that DNA methylation is tightly linked to H2A ubiquitylation in high density CpG promoters. Thus, our work not only reveals Bmi1-dependent H2A ubiquitylation but also suggests that uH2A targeting in differentiated cells may employ a different mechanism from that in ES cells. Experiment Overall Design: RNA was extracted from wild-type MEF cells which were immortalized through TBX2 overexpression, amplified, labeled, and hybridized to an Affymetrix 430 2 microarray. In parallel, RNA was prepared from TBX2-immortalized Bmi1 null MEF cells and hybridized to an Affymetrix 430 2 microarray.

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A long non-coding RNA tunes PRC1 function during human and zebrafish development

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