Biology subjects

Vouzas, A.

Publications and source records attributed to Vouzas, A..

4 recordsLinked to original sources

bioRxiv2025

Autosomal Allelic Inactivation: Variable Replication and Dosage Sensitivity

Autosomal monoallelic gene expression and asynchronous replication between alleles are established features of imprinted genes and genes regulated by allelic exclusion. Inactivation/Stability Centers (I/SCs) are recently described autosomal loci that exhibit epigenetic regulation of allelic expression and replication timing, with differences that can be comparable to those observed between the active and inactive X chromosomes1. Here we characterize >100 autosomal loci with allele-specific epigenetic regulation of replication timing and gene expression, defining them as I/SCs. I/SCs are approximately 1 megabase in size and can contain both protein-coding and noncoding genes. In different single cell derived clones, these genes may be expressed from a single allele, the opposite allele, both alleles, or not expressed at all. This stochastic, yet mitotically stable, pattern indicates that the choice of which allele is expressed is independent of parent of origin and independent of the expression status of the other allele. Similarly, alleles within I/SCs show varying replication timing, either earlier or later, that is also independent of the other allele. Additionally, we identify syntenic loci in the mouse genome that display epigenetic regulation of allelic replication timing, highlighting the genomic organization and conservation of I/SC-associated regulation between human and mouse genomes. The allele-restricted regulation described here creates extensive cellular mosaicism through a stable epigenetic mechanism. This mosaicism impacts numerous dosage-sensitive genes associated with human diseases such as Alzheimer, Parkinson, epilepsy, deafness, and impaired intellectual development.

genomics↗

bioRxiv2025

Transcription can be sufficient, but is not necessary, to advance replication timing

DNA replication timing (RT) is correlated with transcription during cell fate changes but there are many exceptions and our understanding of this relationship suffers from a paucity of reductionist approaches. Here, we manipulated length and strength of transcription in hybrid-genome mouse embryonic stem cells (mESCs) at a single locus upstream of the silent, late replicating, Pleiotrophin (Ptn) gene, directly comparing RT to nascent transcription rates at engineered vs. wild-type alleles. First, we inserted four reporter genes that differ only in their promoter. Two promoters transcribed the reporter gene at high rates and advanced RT. The other two transcribed at lower rates and did not advance RT. Since these promoters may prove useful in applications where effects on RT are undesirable, we confirmed the inability of one of them to advance RT at numerous ectopic sites. We next juxtaposed these same four promoters upstream of the Ptn transcription start site where they all transcribed the 96kb Ptn gene and advanced RT to different extents correlated with transcription rates. Indeed, a doxycycline-responsive promoter, which could not advance RT when induced as a small reporter gene, elicited a rapid and reversible RT advance proportional to the rate of transcription, providing direct evidence that transcription itself can advance RT. However, deletion of the Ptn promoter and enhancer, followed by directed differentiation to neural precursors, eliminated induction of transcription throughout the entire Ptn replication domain, without preventing the switch to early replication. Our results provide a solid empirical base with which to re-evaluate many decades of literature, demonstrating that length and strength of transcription is sufficient but not necessary to advance RT. Our results also provide a robust system in which to rapidly effect an RT change, permitting mechanistic studies of the role of transcription in RT and the consequences of RT changes to epigenomic remodeling.

genomics↗

bioRxiv2023

Master transcription factor binding sites are necessary for early replication control element activity

Eukaryotic genomes replicate in a defined temporal order called the replication timing (RT) program. RT is developmentally regulated with potential to drive cell fate transitions, but mechanisms controlling RT remain elusive. We previously identified "Early Replication Control Elements" (ERCEs) necessary for early RT, domain-wide transcription, 3D chromatin architecture and compartmentalization in mouse embryonic stem cells (mESCs) but, deletions identifying ERCEs were large and encompassed many putative regulatory elements. Here, we show that ERCEs are compound elements whose RT activity can largely be accounted for by multiple sites of diverse master transcription factor binding (subERCEs), distinguished from other such sites by their long-range interactions. While deletion of subERCEs had large effects on both transcription and RT, deleting transcription start sites eliminated nearly all transcription with moderate effects on RT. Our results suggest a model in which subERCEs respond to diverse master transcription factors by functioning both as transcription enhancers and as elements that organize chromatin domains structurally and support early RT, potentially providing a feed-forward loop to drive robust epigenomic change during cell fate transitions.

molecular biology↗

bioRxiv2023

Transcription-Replication Conflict Shapes DNA Break Dynamics

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA revealed co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery resulted in 60% DSB contribution to the head-on compared to 40% for co-directional. Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability.

genomics↗