OR2801 Novel Cis-Regulatory Mechanisms Of Lhb Transcription By Non-Coding RNAs And Non-Canonical DNA Structures

Disclosure: T. Refael: None. H. Gruber: None. G. Golan: None. L. Pnueli: None. P. Melamed: None. Expression of the Lhb gene was shown previously to be directed by synergistic actions of lineage-specific transcription factors at conserved regions of the proximal promoter. However extensive genomic analysis during the last few decades has expanded the repertoire of factors we now know to be involved in transcriptional regulation, revealing roles for distal transcriptional enhancers and long non-coding RNAs (lncRNAs), whose modes of action are only beginning to be elucidated(1). Recently it has also become evident that regulatory regions of the genome are enriched with non-canonical DNA structures. These include G-quadruplexes (G4s) which form on guanine-rich stretches of single-stranded DNA and iMotifs (iMs) on the complementary strand, and are reported to bind various proteins which presumably regulate and mediate their actions. We identified in available mice pituitary scATAC-seq data a region of gonadotrope-specific open chromatin upstream of the Lhb gene, and chromatin conformation capture (3C) in murine gonadotrope cell lines indicated the distal sites that are in physical proximity to the Lhb promoter. Further, we show by chromatin immunoprecipitation (ChIP) a peak of H3K4 monomethylation (H3K4me1) ∼3 kbp upstream of the Lhb TSS, and transcription of two bi-directional non-coding enhancer RNAs (eRNAs), both of which are characteristic of transcriptional enhancers. One of the eRNAs was found to direct open chromatin at the Lhb promoter and is required for Lhb transcription, while guide RNA-mediated recruitment of dCas9- KRAB or dCas9-VP64 to this locus altered Lhb mRNA levels accordingly, confirming its functional enhancer activity. However this enhancer activity is not sufficient for optimal Lhb transcription, and we found that splicing in situ of a distinct downstream lncRNA is also required. The DNA at the central non-coding region of the enhancer, and at the promoter of this lncRNA, forms G4 and iM structures, seen by circular dichroism and ChIP. These DNA structures are affected differentially by local conditions including changes in pH or hormonal treatments, and appear to have diverse effects. HMGB2, which is known to bind structured DNA, was found to bind the enhancer iM in cells and in vitro. Further, HMGB2 knockdown reduced the iM signal and Lhb mRNA, eRNA and lncRNA expression levels quite dramatically. We thus present novel elements involved in the regulation of Lhb transcription through various classes of non-coding RNAs and DNA structures, while emphasizing the complexity of transcriptional regulation via context-dependent cis-acting elements. References: 1. Refael T, Melamed P. (2021). Enhancing gonadotrope gene expression through regulatory lncrnas. Endocrinol. (United States) 162, bqab116. Presentation: Sunday, June 18, 2023