Enhancer RNA

Enhancers are gene regulatory sequences present at both upstream and downstream of the transcription unit. They are cis-acting elements (50-1000bp) regulating both spatial and temporal control of gene expression e.g. activating in specific cells. Enhancers as non-coding sequences can also be a transcription unit i.e. generating non-coding enhancer RNA (eRNA) regulating the transcription of the gene. eRNA is divided into Class 1, Class 2 and Class 3 (involved in binding with TFs, and controlling gene expression). Due to chromatin folding, these enhancers are spatially close to the promoter while being far linearly. The enhancer is bound by activator proteins which shows specific binding with transcription mediator complex and chromatin regulators thus recruiting RNA Polymerase2. Enhancer units in the genome could be discovered by DNase sensitivity assay, H3K4 methylation pattern (monomethylation - activation, trimethylation - repression), H3K27 acetylation, ChIP analysis of coactivator binding with enhancer sequences. Other tools to detect eRNAs are RT-PCR (high sensitivity, cost-effective but low throughput to study single locus region), RNA-FISH (to study spatial and molecular interaction in cells, less stable). The functionality of the enhancers particularly in specific cells could be annotated as epi-genomic markers that could be useful for finding issues or cell-specific gene expression. The enhancers also work as transcriptional units (intergenic/extragenic) i.e. their product eRNA regulate the other transcriptional units away from their corresponding promoter and transcription unit. It is not necessary that only one enhancer works at a time, the phenomenon of multiple enhancers coordinating with other TFs could be understood by studying shadow enhancers, super-enhancers, regulatory archipelagos, locus control regions, HOT regions, TF collective enhancers which control important genes required for the development. Thus eRNAs play a functional role in transcription and markers for enhancing gene expression activity. Comparatively, eRNAs producing enhancers show higher binding with co-activators, forming a stable enhancer-promoter loop, high acetylation, low methylation than non-transcribing enhancers. Enhancers regulating promoters work through two mechanisms: a) looping mechanism (more valid) b) RNAP2 tracking. The structures formed are 3D chromatin architecture supported by lncRNA, eRNA and chromatin architectural proteins. Some of the TFs are also associated with enhancers besides binding to promoters e.g FOXA1, ERalpha, PU.1 and these interactions are mediated through chromatin state/chromatin accessibility, ncRNA, cooperative binding b/w TFs present on enhancers and promoter. Significant similarities in mRNA and eRNA transcripts are nucleosome spacing, poly A-cleavage sites. Besides this, the differences are in low trimethylation of H3K36 and low pSer2 of RNAP2 and lack of U1 splicing sites in eRNA. Transcription of both mRNA and eRNA is bidirectional yet eRNA is localised only to nuclear space. pTyr1 on RNAP2 and 5-cytosine methylation affect eRNA transcription and stability respectively. There is an unresolved understanding of how TFs are loaded at promoters and enhancers at different times to which it is assumed that activated enhancers bound to TFs further activate promoters or induce loading of TFs at promoter sequence. PTF, CTF collectively recruit CoF and HMT, HAT which further recruit GTF and RNAP2 to initiate bidirectional transcription. Thus eRNA may act as cis- and trans-transcriptional activator, chromatin remodeler. The activity of enhancers over long distances is due to the stability of enhancer-promoter looping. Depletion of specific eRNAs (KLK3 and DRR eRNA) affects the expression of genes (KLK3 and MyoD1) present on other chromosomes, this could mean that Spatio-temporal position has its role in eRNA stability. The function of eRNA is reported in cell cycle, growth (cancer cells) and RBC maturation, R-loop formation (misregulation results in malignancy).

Unliganded ERα could bind to specific genomic locations and later these locations are marked as functional enhancers/persistent sites. After ligand exposure, these clustered sites interact with enhancers via chromatin looping. Ligand-dependent enhancers clusters formed upon E2 treatment and disappeared after the active phase of signalling along with drop-in eRNA levels. ERα has a higher affinity for persistent sites than transient sites (sites other than premarked). Besides similar ERE present on persistent sites, FOXA1 was uniquely preferred for binding on these sites. These sites are acetylated at H3K27 and ERα super-enhancer marked for enhancer activity for their closely related genes (E2 dependent genes). Persistent sites show a higher degree of interaction with ERα than transient sites. Persistent and transient interaction with each other and TFF1 promoter shows extensive chromatin looping to form a 3D complex. Deleting persistent sites completely blocks the ERα binding on transient sites, thus suggesting their inevitable role in ERα recruitment on transient sites. Upon ligand treatment formation of ER puncta (spatial crowding of ERα and DNA) on LDEC sites was observed. E2 ligand treatment results in increased target gene expression closer to clustered enhancers and higher levels of eRNA at persistent sites and the formation of genomic clusters through interaction between mediator complex and ligand-binding domain. These unliganded ERα drive the formation of an enhancer cluster that is a functional unit of gene expression during signalling.

Statements:

1. Analysing eRNA levels could tell us about the activity of enhancers, chromatin accessibility and indirectly the gene expression.
2. Studying the eRNA enhancing the expression of oncogene could give us clues about molecular signalling behind the growth, cell division of tumour cells. Assuming these onco-eRNA could be associated with the oncogenes and these eRNA transcripts could be targeted. Also, identifying miRNA against the eRNA as tumour suppressors as anti-cancer therapeutics.