Biology subjects

Melamed, P.

Publications and source records attributed to Melamed, P..

7 recordsLinked to original sources

bioRxiv2026

Tunable kinetic destabilization governs RNA polymerase passage through a DNA-bound transcription factor

Eukaryotic transcription factors recognize short motifs, creating abundant binding sites within gene bodies and potential collisions with elongating RNA polymerases, yet how such encounters are resolved remains unclear. Here, we use optical tweezers to monitor RNA polymerase transcription through DNA-bound Egr-1, a zinc-finger transcription factor. Using DNA-fluctuation suppression as a readout of polymerase arrival, we show that Egr-1 delays elongation in an orientation- and rNTP-dependent manner, whereas force measurements indicate that RNAP does not bypass the TF by mechanical eviction. Instead, RNAP destabilizes the Egr-1-DNA complex over a short, structured interaction zone, increasing TF dissociation non-monotonically with distance. Monte Carlo simulations incorporating these kinetic changes recapitulate passage-time distributions. CpG methylation shortens Egr-1 residence time and largely eliminates the TF-dependent delay, suggesting a role for gene-body methylation in reducing kinetic barriers to elongation. These results reveal DNA-bound TFs as tunable barriers that locally shape transcription elongation.

biophysics↗

bioRxiv2024

Gonadotrophs have a dual origin, with most derived from pituitary stem cells during minipuberty.

Gonadotrophs are the essential pituitary endocrine cells for reproduction. They produce both luteinizing (LH) and follicle-stimulating (FSH) hormones that act on the gonads. Gonadotrophs first appear in the embryonic pituitary, along with other endocrine cell types, and all expand after birth. We show here that most gonadotrophs originate from a population of postnatal pituitary stem cells during minipuberty, while those generated in the embryo are maintained, revealing an unsuspected dual origin of the adult population. This has implications for our understanding of the establishment and regulation of reproductive functions, both in health and in disease.

developmental biology↗

bioRxiv2021

Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin, to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a proteins binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

biophysics↗

bioRxiv2020

Extended and dynamic linker histone-DNA interactions control chromatosome compaction

Chromatosomes play a fundamental role in chromatin regulation, but a detailed understanding of their structure is lacking, partially due to their complex dynamics. Using single-molecule DNA unzipping with optical tweezers, we reveal that linker histone interactions with DNA are remarkably extended, with the C-terminal domain binding both DNA linkers as far as ~ {+/-}140 bp from the dyad. In addition to a symmetrical compaction of the nucleosome core governed by globular domain contacts at the dyad, the C-terminal domain compacts the nucleosomes entry and exit. These interactions are dynamic, exhibiting rapid binding and dissociation, sensitive to phosphorylation of a specific residue, and crucial to determining the symmetry of the chromatosomes core. Extensive unzipping of the linker DNA, which mimics its invasion by motor proteins, shifts H1 into an asymmetric, off-dyad configuration and triggers nucleosome decompaction, highlighting the plasticity of the chromatosome structure and its potential regulatory role.

biophysics↗

bioRxiv2020

Childhood location correlates with epigenetic age and methylation stability in British-Bangladeshi migrants

BackgroundMigration from one environment to another often causes marked changes in developmental conditions. Here we compare epigenetic ageing and stability of the epigenetic maintenance system among British-Bangladeshi women who grew up in Bangladesh (adult migrants), where there are higher pathogen loads and poorer health care, to second-generation Bangladeshis who grew up in the UK. In our previous studies of these migrants, those who spent their childhoods in Bangladesh also had lower levels of reproductive hormones and a shorter reproductive lifespan compared to those who grew up in the UK, suggesting life history trade-offs during development. In the present study, we hypothesised that women who grew up in Bangladesh would have i) an older epigenetic/biological age compared to the women with a childhood in the UK and ii) that differences in the pace of epigenetic ageing might also be reflected by altered stability of DNA methylation marks. ResultsIllumina EPIC array methylation data from buccal tissue was used to establish epigenetic age estimates from 15 adult migrants and 11 second-generation migrants, aged 18-35 years. Using residuals from linear regression of DNA methylation-based biological age (DNAm age) on the chronological age, the results showed significant differences (p=0.016) in epigenetic age estimates: women whose childhood was in Bangladesh are on average 6.02 ({+/-} 2.34) years older, than those who grew up in London. We further investigated the efficiency of the epigenetic maintenance system which purportedly is reflected by epigenetic clocks. Methylation states of CpGs at the LHCGR/LHR locus, which contributes to Horvaths multi tissue epigenetic clock were evaluated. Based on the Ratio of Concordance Preference (RCP) approach that uses double-stranded methylation data, we find that maintenance of epigenetic information is more stable in women who grew up in Bangladesh. ConclusionsThe work supports earlier findings that adverse childhood environments lead to phenotypic life history trade-offs. The data indicate that childhood environments can induce subtle changes to the epigenetic maintenance system that are detectable long after exposure occurred. The implication of such a finding warrants further investigation as it implies that a less flexible epigenetic memory system established early in life could reduce the capacity to respond to different environmental conditions in adult life.

genetics↗

bioRxiv2020

Early-life environment programs reproductive strategies through epigenetic regulation of SRD5A1

Reproductive function and duration of the reproductive life span are phenotypically plastic and programmed in response to the early-life environment. Such adaptive responses are described and rationalized in life history theory in the context of resource availability, but the molecular mechanisms responsible have remained enigmatic. In this study, we hypothesized that epigenetic modifications underlie adaptive reproductive strategies, and found distinct methylation patterns in buccal DNA of Bangladeshi women who grew up in Bangladesh or the UK. The later pubertal onset and lower ovarian reserve associated with Bangladeshi childhood was seen to correlate with more numerous childhood infections, so we adopted a mouse model of pre-pubertal colitis to mimic these conditions. These mice have a similarly-altered reproductive phenotype, which enabled us to determine its mechanistic basis. Several genes encoding proteins with known functions in follicle recruitment were differentially expressed in the mice ovaries, and were also differentially methylated in the womens buccal DNA. One of these, SRD5A1 which encodes the steroidogenic enzyme 5 reductase-1, was down-regulated in the mice ovaries and hyper methylated at the same putative transcriptional enhancer as in the womens DNA; the levels of methylation correlating with gene expression levels. Srd5a1 expression was down-regulated also in the hypothalamus where 5 reductase-1 catalyzes production of neurosteroids that regulate gonadotropin releasing hormone (GnRH). Chemical inhibition of this enzyme affected both GnRH synthesis and release, and resulted in delayed pubertal onset in vivo. The activity of 5 reductase-1 in hypothalamus and ovary and the sensitivity of SRD5A1 to epigenetic regulation attest to its role in directing long-term physiological strategies in response to environmental conditions. In the reproductive axis, this includes timing of pubertal onset, adult reproductive function and duration of the reproductive lifespan.

developmental biology↗

bioRxiv2017

Single-molecule DNA unzipping reveals asymmetric modulation of the transcription factor Egr-1 by its binding site sequence and context

Most functional transcription factor (TF) binding sites deviate from their \"consensus\" recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harbouring 3 zinc fingers (ZF1,ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 base pairs bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.

biophysics↗