RBBP4 dysfunction reshapes the genomic landscape of H3K27 methylation and acetylation and disrupts gene expression.

RBBP4 is a subunit of the chromatin remodeling complexes known as Polycomb repressive complex 2 and histone deacetylase 1/2-containing complexes. These complexes are responsible for histone H3 lysine 27 methylation and deacetylation, respectively. How RBBP4 modulates the functions of these complexes remains largely unknown. We generated viable Rbbp4 mutant alleles in mouse embryonic stem cell lines by CRISPR-Cas9. The mutations disrupted Polycomb repressive complex 2 assembly and H3K27me3 establishment on target chromatin and altered histone H3 lysine 27 acetylation genome wide. Moreover, Rbbp4 mutant cells underwent dramatic changes in transcriptional profiles closely tied to the deregulation of H3K27ac. The alteration of H3K27ac due to RBBP4 dysfunction occurred on numerous cis-regulatory elements, especially putative enhancers. These data suggest that RBBP4 plays a central role in regulating histone H3 lysine 27 methylation and acetylation to modulate gene expression.

Long non-coding RNA OIP5-AS1 (Cyrano): A context-specific regulator of normal and disease processes.

Long non-coding (lnc) RNAs have been implicated in a plethora of normal biological functions, and have also emerged as key molecules in various disease processes. OIP5-AS1, also commonly known by the alias Cyrano, is a lncRNA that displays broad expression across multiple tissues, with significant enrichment in particular contexts including within the nervous system and skeletal muscle. Thus far, this multifaceted lncRNA has been found to have regulatory functions in normal cellular processes including cell proliferation and survival, as well as in the development and progression of a myriad disease states. These widespread effects on normal and disease states have been found to be mediated through context-specific intermolecular interactions with dozens of miRNAs and proteins identified to date. This review explores recent studies to highlight OIP5-AS1's contextual yet pleiotropic roles in normal homeostatic functions as well as disease oetiology and progression, which may influence its utility in the generation of future theranostics.

Multimodal Long Noncoding RNA Interaction Networks: Control Panels for Cell Fate Specification.

Lineage specification in early development is the basis for the exquisitely precise body plan of multicellular organisms. It is therefore critical to understand cell fate decisions in early development. Moreover, for regenerative medicine, the accurate specification of cell types to replace damaged/diseased tissue is strongly dependent on identifying determinants of cell identity. Long noncoding RNAs (lncRNAs) have been shown to regulate cellular plasticity, including pluripotency establishment and maintenance, differentiation and development, yet broad phenotypic analysis and the mechanistic basis of their function remains lacking. As components of molecular condensates, lncRNAs interact with almost all classes of cellular biomolecules, including proteins, DNA, mRNAs, and microRNAs. With functions ranging from controlling alternative splicing of mRNAs, to providing scaffolding upon which chromatin modifiers are assembled, it is clear that at least a subset of lncRNAs are far from the transcriptional noise they were once deemed. This review highlights the diversity of lncRNA interactions in the context of cell fate specification, and provides examples of each type of interaction in relevant developmental contexts. Also highlighted are experimental and computational approaches to study lncRNAs.

SWI/SNF remains localized to chromatin in the presence of SCHLAP1.

SCHLAP1 is a long noncoding RNA that is reported to function by depleting the SWI/SNF complex from the genome. We investigated the hypothesis that SCHLAP1 affects only specific compositions of SWI/SNF. Using several assays, we found that SWI/SNF is not depleted from the genome by SCHLAP1 and that SWI/SNF is associated with many coding and noncoding RNAs, suggesting that SCHLAP1 may function in a SWI/SNF-independent manner.

Interactome determination of a Long Noncoding RNA implicated in Embryonic Stem Cell Self-Renewal.

Long noncoding RNAs (lncRNAs) constitute a significant fraction of mammalian transcriptomes and they have emerged as intricate regulators of many biological processes. Their broad capacity to adopt diverse structures facilitates their involvement in the transcriptional, translational and signaling processes that are central to embryonic stem (ES) cell self-renewal and pluripotency. While lncRNAs have been implicated in ES cell maintenance, detailed analyses of those that show significant expression in ES cells is largely absent. Moreover, cooperative molecular relationships that facilitate lncRNA action are poorly understood. Cyrano is a developmentally important lncRNA, and in ES cells, it supports gene expression network maintenance, cell adhesion and cell survival. We have interrogated the interactome of Cyrano to identify protein partners and find that Cyrano is involved in multiple protein networks. We identify a developmentally important cell-signaling hub and find STAT3 as a candidate through which Cyrano can function to reinforce self-renewal of ES cells. Based on commonalities between ES cells and cancer cells, we postulate such functional interactions may support cell proliferation, cell identity and adhesion characteristics in rapidly proliferating cell types. The interactome data will therefore provide a resource for further investigations into interactions that regulate Cyrano or mediate its function.

Long Noncoding RNA Moderates MicroRNA Activity to Maintain Self-Renewal in Embryonic Stem Cells.

Of the thousands of long noncoding RNAs expressed in embryonic stem cells (ESCs), few have known roles and fewer have been functionally implicated in the regulation of self-renewal and pluripotency, or the reprogramming of somatic cells to the pluripotent state. In ESCs, Cyrano is a stably expressed long intergenic noncoding RNA with no previously assigned role. We demonstrate that Cyrano contributes to ESC maintenance, as its depletion results in the loss of hallmarks of self-renewal. Delineation of Cyrano's network through transcriptomics revealed widespread effects on signaling pathways and gene expression networks that contribute to ESC maintenance. Cyrano shares unique sequence complementarity with the differentiation-associated microRNA, mir-7, and mir-7 overexpression reduces expression of a key self-renewal factor to a similar extent as Cyrano knockdown. This suggests that Cyrano functions to restrain the action of mir-7. Altogether, we provide a view into the multifaceted function of Cyrano in ESC maintenance.

Myc orchestrates a regulatory network required for the establishment and maintenance of pluripotency.

Pluripotent stem cells (PSCs) are maintained by a complex regulatory network orchestrated by transcription factors, epigenetic modifiers and non-coding RNAs. Central to this regulatory network is the Myc family of transcription factors. Defining roles for Myc in PSCs has been problematic but recently, a number of reports have provided insight into this question. An emerging picture now places Myc as a key regulator of the cell cycle, genomic maintenance and general metabolic activity in PSCs through its ability to directly regulate large numbers of target genes and more indirectly through control of microRNAs. One of Myc's main roles is to repress the activity of genes required for differentiation such as the endoderm master regulator, GATA6. The general mechanism by which Myc activates target genes is well understood but a remaining major challenge is to understand how it represses gene activity. Here we discuss potential mechanisms for how Myc establishes and maintains the pluripotent state and incorporate proteomics data that supports a model where Myc acts as part of a regulatory network with epigenetic modifiers.

Myc represses primitive endoderm differentiation in pluripotent stem cells.

The generation of induced pluripotent stem cells (iPSCs) provides a novel method to facilitate investigations into the mechanisms that control stem cell pluripotency and self-renewal. Myc has previously been shown to be critical for murine embryonic stem cell (mESC) maintenance, while also enhancing directed reprogramming of fibroblasts by effecting widespread changes in gene expression. Despite several studies identifying in vivo target genes, the precise mechanism by which Myc regulates pluripotency remains unknown. Here we report that codeletion of c- and N-MYC in iPSCs and ESCs results in their spontaneous differentiation to primitive endoderm. We show that Myc sustains pluripotency through repression of the primitive endoderm master regulator GATA6, while also contributing to cell cycle control by regulation of the mir-17-92 miRNA cluster. Our findings demonstrate the indispensable requirement for c- or N-myc in pluripotency beyond proliferative and metabolic control.