Biology subjects

Golov, A. K.

Publications and source records attributed to Golov, A. K..

6 recordsLinked to original sources

bioRxiv2026

Micro-C-TALE: BAC-based targeted high-resolution measurement of 3D chromatin interactions

Chromosome conformation capture methods have greatly advanced our understanding of genome organization, but obtaining high-resolution interaction maps of selected genomic regions remains costly due to the sequencing requirements. Here, we introduce Micro-C-TALE, a new chromosome conformation capture method that combines Micro-C, which uses micrococcal nuclease to improve the resolution and sensitivity of chromatin interaction measurements, and C-TALE, which uses probes generated from bacterial artificial chromosomes (BACs) for targeted enrichment. This combination enables the generation of high-resolution interaction maps without requiring custom synthesis of capture oligonucleotides. We applied Micro-C-TALE to a 214 kb region in human HAP1 cells, obtaining detailed interaction maps with approximately 5-6 million sequenced read pairs. Compared with published genome-wide Micro-C data, Micro-C-TALE yielded a higher amount of informative interactions within the targeted region despite ~550-fold lower sequencing depth. To evaluate the sensitivity of the method to fine-scale changes in genome organization, we introduced a heterozygous CTCF binding site into the targeted region using CRISPR-Cas9. Comparing Micro-C-TALE to the restriction enzyme-based C-TALE, we found Micro-C-TALE yielded a ~5-fold higher fraction of informative reads and detected novel CTCF-mediated point interactions associated with the inserted CTCF site, which were barely discernible using C-TALE. Our results demonstrate that Micro-C-TALE provides a flexible and cost-effective approach for targeted high-resolution measurement of chromatin interactions, facilitating the investigation of fine-scale genome organization and its response to genetic perturbations.

genomics↗

bioRxiv2026

CTCF-mediated cis-regulatory chromatin insulation enforces a central B-cell tolerance checkpoint

The generation of a diverse and self-tolerant B cell repertoire is essential for adaptive immunity and is achieved through V(D)J recombination. In mice, Ig{kappa} is the dominant light chain, whereas Ig{lambda} rearrangement typically occurs in response to nonproductive or autoreactive Ig{kappa} recombination, a process termed receptor editing. Recombination at the RS element deletes the Ig{kappa} constant exon, silencing the locus and enabling Ig{lambda} expression. However, the epigenetic regulatory framework that orchestrates and governs receptor editing remains poorly defined. Here, we identify a CTCF-binding insulator element (CBE) within the 3' Ig{kappa} super-enhancer (3'-SE{kappa}) that regulates receptor editing and directs the {kappa}-to-{lambda} switch required for Ig{lambda} B-cell development. Mechanistically, loss of this CBE activates an insulated enhancer within the 3'-SE{kappa}, causing aberrant V{kappa} rearrangements and altered chromatin interactions through disrupted loop extrusion dynamics. Notably, loss of this CBE in mice leads to increased autoantibody production by ten weeks of age, demonstrating that CBE-mediated chromatin architecture shapes B cell fate by constraining autoreactive potential. Collectively, our findings define a novel CTCF-dependent cis-regulatory insulation checkpoint that connects chromatin loop extrusion to antigen-driven receptor editing, thereby enforcing B-cell tolerance.

immunology↗

bioRxiv2025

SPT5 regulates Pol II pausing and elongation in different ways at early versus late embryonic stages

Transcription involves a cycle of initiation, pausing, elongation, and termination. SPT5 regulates both promoter proximal pausing and elongation, but how it orchestrates both steps during dynamic changes in gene expression remains unclear. Here, using Drosophila embryogenesis, we show that pausing both precedes and follows gene expression, while active transcription is accompanied by pause release. Optogenetic rapid depletion of SPT5 from the nucleus uncovered different sensitivities at different developmental stages. In early embryogenesis, SPT5 depletion caused a downstream shift in Pol II pausing to the +1 nucleosome, resulting in defective elongation and early termination. In late embryogenesis, it led to both up- and downregulation of expression, depending on the genes transcriptional and pausing state - upregulation is caused by pause release while downregulation is due to defective elongation. These changes are intensified when genes are increasing or decreasing their transcriptional state, indicating that SPT5 contributes to fine-tuning dynamic changes in gene expression.

genomics↗

bioRxiv2023

A genome-wide nucleosome-resolution map of promoter-centered interactions in human cells corroborates the enhancer-promoter looping model

The enhancer-promoter looping model, in which enhancers activate their target genes via physical contact, has long dominated the field of gene regulation. However, the ubiquity of this model has been questioned due to evidence of alternative mechanisms and the lack of its systematic validation, primarily owing to the absence of suitable experimental techniques. In this study, we present a new MNase-based proximity ligation method called MChIP-C, allowing for the measurement of protein-mediated chromatin interactions at single-nucleosome resolution on a genome-wide scale. By applying MChIP-C to study H3K4me3 promoter-centered interactions in K562 cells, we found that it had greatly improved resolution and sensitivity compared to restriction endonuclease-based C-methods. This allowed us to identify EP300 histone acetyltransferase and the SWI/SNF remodeling complex as potential candidates for establishing and/or maintaining enhancer-promoter interactions. Finally, leveraging data from published CRISPRi screens, we found that most functionally-verified enhancers do physically interact with their cognate promoters, supporting the enhancer-promoter looping model.

genomics↗

bioRxiv2020

Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells

Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome nanodomains and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, but increased further mixing of the chromatin compartments and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

molecular biology↗

bioRxiv2019

Studying RNA#8211;DNA interactome by Red-C identifies noncoding RNAs associated with repressed chromatin compartment and reveals transcription dynamics

Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA-DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. We found two microRNAs--MIR3648 and MIR3687 transcribed from the rRNA locus--that are associated with inactive chromatin genome wide. These miRNAs favor bulk heterochromatin over Polycomb-repressed chromatin and interact preferentially with late-replicating genomic regions. Analysis of the RNA-DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.

genomics↗