enforced-activation-enhancer-rnas-situ-through-dcas9-synergistic

Enforced Activation of Enhancer RNAs In Situ through the dCas9 Synergistic Activation Mediator System

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence ...

University of Texas Health Science Center

University of Texas MD Anderson Cancer Center and UTHealth

The molecular mechanisms underlying the opposing functions of glucocorticoid receptors (GRs) and estrogen receptor α (ERα) in breast cancer development remain poorly understood. Here we report that, in breast cancer cells, liganded GR represses a large ERα-activated transcriptional program by binding, in trans, to ERα-occupied enhancers. This abolishes effective activation of these enhancers and their cognate target genes, and it leads to the inhibition of ERα-dependent binding of components of the MegaTrans complex. Consistent with the effects of SUMOylation on other classes of nuclear receptors, dexamethasone (Dex)-induced trans-repression of the estrogen E program appears to depend on GR SUMOylation, which leads to stable trans-recruitment of the GR-N-CoR/SMRT-HDAC3 corepressor complex on these enhancers. Together, these results uncover a mechanism by which competitive recruitment of DNA-binding nuclear receptors/transcription factors in trans to hot spot enhancers serves as an effective biological strategy for trans-repression, with clear implications for breast cancer and other diseases.

Glucocorticoid Receptor:MegaTrans Switching Mediates the Repression of an ERα-Regulated Transcriptional Program.

Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here, we report that a somite-derived prohematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch up-regulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing (RNA-seq), and assay for transposase-accessible chromatin using sequencing (ATAC-seq) indicate that Srebp2 transregulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.

AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate.

Enhancer RNA (eRNA) is a type of noncoding RNA transcribed from the enhancer. Although critical roles of eRNA in gene transcription control have been increasingly realized, the systemic landscape and potential function of eRNAs in cancer remains largely unexplored. Here, we report the integration of multi-omics and pharmacogenomics data across large-scale patient samples and cancer cell lines. We observe a cancer-/lineage-specificity of eRNAs, which may be largely driven by tissue-specific TFs. eRNAs are involved in multiple cancer signaling pathways through putatively regulating their target genes, including clinically actionable genes and immune checkpoints. They may also affect drug response by within-pathway or cross-pathway means. We characterize the oncogenic potential and therapeutic liability of one eRNA, NET1e, supporting the clinical feasibility of eRNA-targeted therapy. We identify a panel of clinically relevant eRNAs and developed a user-friendly data portal. Our study reveals the transcriptional landscape and clinical utility of eRNAs in cancer.

Transcriptional landscape and clinical utility of enhancer RNAs for eRNA-targeted therapy in cancer.

Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely 'hijacked' by cytoplasmic Malat1 to the cytoplasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention, potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HNRNPK. Losing these RNA-protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases.

The SINEB1 element in the long non-coding RNA Malat1 is necessary for TDP-43 proteostasis.

Enhancers are distal genomic elements critical for gene regulation and cell identify control during development and diseases. Many human cancers were found to associate with enhancer malfunction, due to genetic and epigenetic alterations, which in some cases directly drive tumour growth. Conventionally, enhancers are known to provide DNA binding motifs to recruit transcription factors (TFs) and to control target genes. However, recent progress found that most, if not all, active enhancers pervasively transcribe noncoding RNAs that are referred to as enhancer RNAs (eRNAs). Increasing evidence points to functional roles of at least a subset of eRNAs in gene regulation in both normal and cancer cells, adding new insights into the action mechanisms of enhancers. eRNA expression was observed to be widespread but also specific to tumour types and individual patients, serving as opportunities to exploit them as potential diagnosis markers or therapeutic targets. In this review, we discuss the brief history of eRNA research, their functional mechanisms and importance in cancer gene regulation, as well as their therapeutic and diagnostic values in cancer. We propose that further studies of eRNAs in cancer will offer a promising 'eRNA targeted therapy' for human cancer intervention.

Enhancer RNAs in cancer: regulation, mechanisms and therapeutic potential.

Three-dimensional chromatin structures undergo dynamic reorganization during mammalian spermatogenesis; however, their impacts on gene regulation remain unclear. Here, we focused on understanding the structure-function regulation of meiotic chromosomes by Hi-C and other omics techniques in mouse spermatogenesis across five stages. Beyond confirming recent reports regarding changes in compartmentalization and reorganization of topologically associating domains (TADs), we further demonstrated that chromatin loops are present prior to and after, but not at, the pachytene stage. By integrating Hi-C and RNA-seq data, we showed that the switching of A/B compartments between spermatogenic stages is tightly associated with meiosis-specific mRNAs and piRNAs expression. Moreover, our ATAC-seq data indicated that chromatin accessibility per se is not responsible for the TAD and loop diminishment at pachytene. Additionally, our ChIP-seq data demonstrated that CTCF and cohesin remain bound at TAD boundary regions throughout meiosis, suggesting that dynamic reorganization of TADs does not require CTCF and cohesin clearance.

Article	Year
Glucocorticoid Receptor:MegaTrans Switching Mediates the Repression of an ERα-Regulated Transcriptional Program. Molecular cell\| PubMed ID: 28475868	2017
AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate. Science (New York, N.Y.)\| PubMed ID: 30705153	2019
Transcriptional landscape and clinical utility of enhancer RNAs for eRNA-targeted therapy in cancer. Nature communications\| PubMed ID: 31594934	2019
The SINEB1 element in the long non-coding RNA Malat1 is necessary for TDP-43 proteostasis. Nucleic acids research\| PubMed ID: 31863590	2020
Enhancer RNAs in cancer: regulation, mechanisms and therapeutic potential. RNA biology\| PubMed ID: 31916476	2020
Reorganized 3D Genome Structures Support Transcriptional Regulation in Mouse Spermatogenesis. iScience\| PubMed ID: 32315832	2020

Wenbo Li

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