Biology subjects

Nocente, M. C.

Publications and source records attributed to Nocente, M. C..

4 recordsLinked to original sources

bioRxiv2025

Poising and connectivity of emergent human developmental enhancers in the transition from naive to primed pluripotency

In primed human pluripotent stem cells (hPSCs) resembling post-implantation epiblast, numerous lineage-specific enhancers assume the poised chromatin state, co-marked by H3K4me1 and Polycomb-associated H3K27me3 histone modifications. In contrast, poised enhancers (PEs) are scarce in naive hPSCs that model pre-implantation epiblast. PEs form abundant chromosomal contacts with developmental genes, but when these contacts emerge, how their formation relates to enhancer poising and their functional significance remains incompletely understood. Here, we devise high-resolution, PE-targeted Capture Hi-C to generate a comprehensive atlas of PE chromosomal contacts in the time course of hPSC transition from the naive to primed state. We find that enhancer poising emerges early in the transition, while the contacts show diverse dynamics that is only partially coupled to poising. PROTAC-induced degradation of Polycomb Repressive Complex 2 (PRC2) early in the transition weakens PE connectivity, while inhibition of its H3K27 methyltransferase activity does not, suggesting a non-catalytic role of Polycomb in supporting PE contacts. Notably, PE contacts persist after developmental activation or ectopic CRISPRa targeting and can mediate long-range gene induction. Together, these findings reveal the temporal and mechanistic principles of PE connectivity, highlighting a potential role of PE contacts in establishing gene expression patterns in human development.

genomics↗

bioRxiv2025

How cBAF and PBAF regulate the nucleosomal and subnucleosomal landscape of promoters, enhancers and REST repressor binding sites

BAF (SWI/SNF) chromatin remodeling complexes belong to three subfamilies, cBAF, PBAF, and ncBAF, that regulate the nucleosomal organization and accessibility of transcription factors (TFs) DNA binding sites at cis-regulatory elements (CREs). However, the functional specificities of these complexes at promoters, enhancers, and other categories of CREs are poorly understood. Here, we examined how the BAF complexes regulate the nucleosomal and subnucleosomal landscape in which transcription initiation occurs in vivo. We first described how these complexes regulate the earliest step of transcription preinitiation complex (PIC) assembly, which is defined by the binding of the general transcription factors TBP and TFIIA. We show that in the presence of a TATA box, PIC formation occurs onto a core promoter or enhancer region still wrapped into a fragile nucleosome or a subnucleosomal particle. Conversely, without a TATA box, PIC assembles onto genomic templates mostly stripped of histones. We establish that cBAF, but not PBAF or ncBAF, regulates this chromatin organization and the first step of PIC formation at promoters and enhancers. We observed that individual subunits of cBAF have specific functions at promoters: the BRG1 ATPase and SMARCB1 regulate the production of fragile nucleosomes and subnucleosomal particles, while ARID1A stimulates the binding of TBP/TFIIA independently of the ATPase subunit. In contrast, all three subunits are systematically required for both chromatin organization and TBP/TFIIA binding at enhancers. Finally, we revealed that a subset of PBAF subunits controls the nucleosomal and subnucleosomal landscape at REST repressor binding sites. Our integrative study identified specific functions of cBAF and PBAF subunits in controlling the binding of TFs and the nucleosomal and subnucleosomal organization of CREs.

genomics↗

bioRxiv2022

Oct4 interacts with subnucleosomal particles generated by Brg1 at enhancers

BRG1, the catalytic subunit of the mammalian SWI/SNF complexes, is essential for chromatin opening at enhancers. However, the nature of the open chromatin remains unclear. Here we show that in addition to producing histone-free DNA, BRG1 generates hemisome-like subnucleosomal particles containing the four core histones associated with 50-80 base pairs of DNA. Our genome-wide analysis indicates that BRG1 makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4-subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude, compared to the region occupied on histone-free DNA. We suggest that BRG1-dependent subnucleosomes orchestrate an epigenetic mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs.

genomics↗

bioRxiv2017

The non-homologous end joining factor Ku orchestrates replication fork resection and fine-tunes Rad51-mediated fork restart

Replication requires Homologous Recombination (HR) to stabilize and restart terminally-arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily Double Strand Breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe. We found that fork-resection is a two-step process coordinated by the non-homologous end joining factor Ku. An initial resection mediated by MRN/Ctp1 removes Ku from terminally-arrested forks, generating ~ 110 bp sized gaps obligatory for subsequent Exo1-mediated long-range resection and replication restart. The lack of Ku results in slower fork restart, excessive resection, and impaired RPA recruitment. We propose that terminally-arrested forks undergo fork reversal, providing a single DNA end for Ku binding which primes RPA-coated ssDNA. We uncover an unprecedented role for Ku in orchestrating resection of unbroken forks and in fine-tuning HR-mediated replication restart.\n\nO_LIKu orchestrates a two-steps DNA end-resection of terminally-arrested and unbroken forks\nC_LIO_LIMRN/Ctp1 removes Ku from terminally-arrested forks to initiate fork-resection\nC_LIO_LIa ~110 bp sized ssDNA gap is sufficient and necessary to promote fork restart.\nC_LIO_LIThe lack of Ku decreases ssDNA RPA-coating, and slows down replication fork restart.\nC_LI

genetics↗