An i-motif-regulated enhancer, eRNA and adjacent lncRNA affect Lhb expression through distinct mechanisms in a sex-specific context.

Genome-wide studies have demonstrated regulatory roles for diverse non-coding elements, but their precise and interrelated functions have often remained enigmatic. Addressing the need for mechanistic insight, we studied their roles in expression of Lhb which encodes the pituitary gonadotropic hormone that controls reproduction. We identified a bi-directional enhancer in gonadotrope-specific open chromatin, whose functional eRNA (eRNA2) supports permissive chromatin at the Lhb locus. The central untranscribed region of the enhancer contains an iMotif (iM), and is bound by Hmgb2 which stabilizes the iM and directs transcription specifically towards the functional eRNA2. A distinct downstream lncRNA, associated with an inducible G-quadruplex (G4) and iM, also facilitates Lhb expression, following its splicing in situ. GnRH activates Lhb transcription and increased levels of all three RNAs, eRNA2 showing the highest response, while estradiol, which inhibits Lhb, repressed levels of eRNA2 and the lncRNA. The levels of these regulatory RNAs and Lhb mRNA correlate highly in female mice, though strikingly not in males, suggesting a female-specific function. Our findings, which shed new light on the workings of non-coding elements and non-canonical DNA structures, reveal novel mechanisms regulating transcription which have implications not only in the central control of reproduction but also for other inducible genes.

Communication between DNA and nucleotide binding sites facilitates stepping by the RecBCD helicase.

Double-strand DNA breaks are the severest type of genomic damage, requiring rapid response to ensure survival. RecBCD helicase in prokaryotes initiates processive and rapid DNA unzipping, essential for break repair. The energetics of RecBCD during translocation along the DNA track are quantitatively not defined. Specifically, it's essential to understand the mechanism by which RecBCD switches between its binding states to enable its translocation. Here, we determine, by systematic affinity measurements, the degree of coupling between DNA and nucleotide binding to RecBCD. In the presence of ADP, RecBCD binds weakly to DNA that harbors a double overhang mimicking an unwinding intermediate. Consistently, RecBCD binds weakly to ADP in the presence of the same DNA. We did not observe coupling between DNA and nucleotide binding for DNA molecules having only a single overhang, suggesting that RecBCD subunits must both bind DNA to 'sense' the nucleotide state. On the contrary, AMPpNp shows weak coupling as RecBCD remains strongly bound to DNA in its presence. Detailed thermodynamic analysis of the RecBCD reaction mechanism suggests an 'energetic compensation' between RecB and RecD, which may be essential for rapid unwinding. Our findings provide the basis for a plausible stepping mechanism' during the processive translocation of RecBCD.