RNA fine-tunes estrogen receptor-alpha binding on low-affinity DNA motifs for transcriptional regulation.

Transcription factors (TFs) regulate gene expression by binding with varying strengths to DNA via their DNA-binding domain. Additionally, some TFs also interact with RNA, which modulates transcription factor binding to chromatin. However, whether RNA-mediated TF binding results in differential transcriptional outcomes remains unknown. In this study, we demonstrate that estrogen receptor α (ERα), a ligand-activated TF, interacts with RNA in a ligand-dependent manner. Defects in RNA binding lead to genome-wide loss of ERα recruitment, particularly at weaker ERα-motifs. Furthermore, ERα mobility in the nucleus increases in the absence of its RNA-binding capacity. Unexpectedly, this increased mobility coincides with robust polymerase loading and transcription of ERα-regulated genes that harbor low-strength motifs. However, highly stable binding of ERα on chromatin negatively impacts ligand-dependent transcription. Collectively, our results suggest that RNA interactions spatially confine ERα on low-affinity sites to fine-tune gene transcription.

Active enhancers strengthen insulation by RNA-mediated CTCF binding at chromatin domain boundaries.

Vertebrate genomes are partitioned into chromatin domains or topologically associating domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at domain boundaries correlates with better insulation; however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF and MYC TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer-promoter interactions, and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters, we show that active TAD enhancers, but not promoters, induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

Ligand dependent gene regulation by transient ERα clustered enhancers.

Unliganded Estrogen receptor alpha (ERα) has been implicated in ligand-dependent gene regulation. Upon ligand exposure, ERα binds to several EREs relatively proximal to the pre-marked, unliganded ERα-bound sites and affects transient but robust gene expression. However, the underlying mechanisms are not fully understood. Here we demonstrate that upon ligand stimulation, persistent sites interact extensively, via chromatin looping, with the proximal transiently ERα-bound sites, forming Ligand Dependent ERα Enhancer Cluster in 3D (LDEC). The E2-target genes are regulated by these clustered enhancers but not by the H3K27Ac super-enhancers. Further, CRISPR-based deletion of TFF1 persistent site disrupts the formation of its LDEC resulting in the loss of E2-dependent expression of TFF1 and its neighboring genes within the same TAD. The LDEC overlap with nuclear ERα condensates that coalesce in a ligand and persistent site dependent manner. Furthermore, formation of clustered enhancers, as well as condensates, coincide with the active phase of signaling and their later disappearance results in the loss of gene expression even though persistent sites remain bound by ERα. Our results establish, at TFF1 and NRIP1 locus, a direct link between ERα condensates, ERα enhancer clusters, and transient, but robust, gene expression in a ligand-dependent fashion.

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of ß-hemoglobinopathies.

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate ß-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to ß-globin exon-1 (ßE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-ßE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells in vivo. Furthermore, PRR-ßE1 gene editing is feasible without ex vivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and ß-thalassemia major. These findings imply that PRR-ßE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for ß-hemoglobinopathy gene therapy.

An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus.

The INK4a/ARF locus encodes important cell-cycle regulators p14ARF, p15INK4b, and p16INK4a. The neighboring gene desert to this locus is the most reproducible GWAS hotspot that harbors one of the densest enhancer clusters in the genome. However, how multiple enhancers that overlap with GWAS variants regulate the INK4a/ARF locus is unknown, which is an important step in linking genetic variation with associated diseases. Here, we show that INK4a/ARF promoters interact with a subset of enhancers in the cluster, independent of their H3K27ac and eRNA levels. Interacting enhancers transcriptionally control each other and INK4a/ARF promoters over long distances as an interdependent single unit. The deletion of even a single interacting enhancer results in an unexpected collapse of the entire enhancer cluster and leads to EZH2 enrichment on promoters in an ANRIL-independent manner. Dysregulated genes genome-wide mimic 9p21-associated diseases under these scenarios. Our results highlight intricate dependencies of promoter-interacting enhancers on each other.

A rare variant of African ancestry activates 8q24 lncRNA hub by modulating cancer associated enhancer.

Genetic variation at the 8q24 locus is linked with the greater susceptibility to prostate cancer in men of African ancestry. One such African ancestry specific rare variant, rs72725854 (A>G/T) (~6% allele frequency) has been associated with a ~2-fold increase in prostate cancer risk. However, the functional relevance of this variant is unknown. Here we show that the variant rs72725854 is present in a prostate cancer-specific enhancer at 8q24 locus. Chromatin-conformation capture and dCas9 mediated enhancer blocking establish a direct regulatory link between this enhancer and lncRNAs PCAT1, PRNCR1 and PVT1. The risk allele ('T') is associated with higher expression of PCAT1, PVT1 and c-myc in prostate tumors. Further, enhancer with the risk allele gains response to androgen stimulation by recruiting the transcription factor SPDEF whereas, non-risk alleles remain non-responsive. Elevated expression of these lncRNAs and c-myc in risk allele carriers may explain their greater susceptibility to prostate cancer.