RNA matchmaking in chromatin regulation.

Beyond being the product of gene expression, RNA can also influence the regulation of chromatin. The majority of the human genome has the capacity to be transcribed and the majority of the non-protein-coding transcripts made by RNA Polymerase II are enriched in the nucleus. Many chromatin regulators can bind to these ncRNAs in the nucleus; in some cases, there are clear examples of direct RNA-mediated chromatin regulation mechanisms stemming from these interactions, while others have yet to be determined. Recent studies have highlighted examples of chromatin regulation via RNA matchmaking, a term we use broadly here to describe intermolecular base-pairing interactions between one RNA molecule and an RNA or DNA match. This review provides examples of RNA matchmaking that regulates chromatin processes and summarizes the technical approaches used to capture these events.

SILAC-MS Profiling of Reconstituted Human Chromatin Platforms for the Study of Transcription and RNA Regulation.

DNA packaged into chromatin is the core structure of the human genome. Nearly all eukaryotic genome regulation must interface with this genomic structure, and modification of the chromatin can influence molecular mechanisms that regulate the underlying DNA. Many processes are governed by regulated stepwise assembly mechanisms that build complex machinery on chromatin to license a specific activity such as transcription. Transcriptional activators drive the initial steps of gene expression, regulated in part by chromatin. Here we describe tools to study the stepwise assembly of protein complexes on chromatin in a highly controlled manner using reconstituted human chromatin platforms and quantitative proteomic profiling. We profile the early steps in transcriptional activation and highlight the potential for understanding the multiple ways chromatin can influence transcriptional regulation. We also describe modifications of this approach to study the activity of a long noncoding RNA to act as a dynamic scaffold for proteins to be recruited to chromatin. This approach has the potential to provide a more comprehensive understanding of important macromolecular complex assembly that occurs on the human genome. The reconstituted nature of the chromatin substrate offers a tunable system that can be trapped at specific substeps to understand how chromatin interfaces with genome regulation machinery.

An RNA matchmaker protein regulates the activity of the long noncoding RNA HOTAIR.

The human long noncoding RNA (lncRNA) HOTAIR acts in trans to recruit the Polycomb repressive complex 2 (PRC2) to the HOXD gene cluster and to promote gene silencing during development. In breast cancers, overexpression of HOTAIR increases metastatic potential via the repression of many additional genes. It has remained unclear what factors determine HOTAIR-dependent PRC2 activity at specific genomic loci, particularly when high levels of HOTAIR result in aberrant gene silencing. To identify additional proteins that contribute to the specific action of HOTAIR, we performed a quantitative proteomic analysis of the HOTAIR interactome. We found that the most specific interaction was between HOTAIR and the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, a member of a family of proteins involved in nascent mRNA processing and RNA matchmaking. Our data suggest that A2/B1 are key contributors to HOTAIR-mediated chromatin regulation in breast cancer cells: A2/B1 knockdown reduces HOTAIR-dependent breast cancer cell invasion and decreases PRC2 activity at the majority of HOTAIR-dependent loci. We found that the B1 isoform, which differs from A2 by 12 additional amino acids, binds with highest specificity to HOTAIR. B1 also binds chromatin and associates preferentially with RNA transcripts of HOTAIR gene targets. We furthermore demonstrate a direct RNA-RNA interaction between HOTAIR and a target transcript that is enhanced by B1 binding. Together, these results suggest a model in which B1 matches HOTAIR with transcripts of target genes on chromatin, leading to repression by PRC2.

Global profiling of hnRNP A2/B1-RNA binding on chromatin highlights LncRNA interactions.

Long noncoding RNAs (lncRNAs) often carry out their functions through associations with adaptor proteins. We recently identified heterogeneous ribonucleoprotein (hnRNP) A2/B1 as an adaptor of the human HOTAIR lncRNA. hnRNP A2 and B1 are splice isoforms of the same gene. The spliced version of HOTAIR preferentially associates with the B1 isoform, which we hypothesize contributes to RNA-RNA matching between HOTAIR and transcripts of target genes in breast cancer. Here we used enhanced cross-linking immunoprecipitation (eCLIP) to map the direct interactions between A2/B1 and RNA in breast cancer cells. Despite differing by only twelve amino acids, the A2 and B1 splice isoforms associate preferentially with distinct populations of RNA in vivo. Through cellular fractionation experiments we characterize the pattern of RNA association in chromatin, nucleoplasm, and cytoplasm. We find that a majority of interactions occur on chromatin, even those that do not contribute to co-transcriptional splicing. A2/B1 binding site locations on multiple RNAs hint at a contribution to the regulation and function of lncRNAs. Surprisingly, the strongest A2/B1 binding site occurs in a retained intron of HOTAIR, which interrupts an RNA-RNA interaction hotspot. In vitro eCLIP experiments highlight additional exonic B1 binding sites in HOTAIR which also surround the RNA-RNA interaction hotspot. Interestingly, a version of HOTAIR with the intron retained is still capable of making RNA-RNA interactions in vitro through the hotspot region. Our data further characterize the multiple functions of a repurposed splicing factor with isoform-biased interactions, and highlight that the majority of these functions occur on chromatin-associated RNA.

Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

Establishing RNA-RNA interactions remodels lncRNA structure and promotes PRC2 activity.

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

Exploring the mechanisms behind long noncoding RNAs and cancer.

Over the past decade, long noncoding RNAs (lncRNAs) have been identified as significant players in gene regulation. They are often differentially expressed and widely-associated with a majority of cancer types. The aberrant expression of these transcripts has been linked to tumorigenesis, metastasis, cancer stage progression and patient survival. Despite their apparent link to cancer, it has been challenging to gain a mechanistic understanding of how they contribute to cancer, partially due the difficulty in discriminating functional RNAs from other noncoding transcription events. However, there are several well-studied lncRNAs where specific mechanisms have been more clearly defined, leading to new discoveries into how these RNAs function. One major observation that has come to light is the context-dependence of lncRNA mechanisms, where they often have unique function in specific cell types and environment. Here, we review the molecular mechanisms of lncRNAs with a focus on cancer pathways, illustrating a few informative examples. Together, this type of detailed insight will lead to a greater understanding of the potential for the application of lncRNAs as targets of cancer therapies and diagnostics.