Biology subjects

Goloborodko, A.

Publications and source records attributed to Goloborodko, A..

16 recordsLinked to original sources

bioRxiv2026

Nested TAD hierarchy defines cohesion zones on replicated chromosomes

During each cell cycle, cells must not only duplicate DNA sequence but also preserve the three-dimensional genome architecture required for gene regulation and DNA repair. After replication, this requires coordination between cohesin-mediated loop extrusion and sister-chromatid cohesion for homology-directed DNA repair. Because extrusion promotes sister resolution whereas cohesion can impede extrusion, how both activities coexist on replicated chromosomes is unclear. Here we show that cohesin function is partitioned within the nested TAD hierarchy: cohesive cohesin accumulates at high-level boundaries to form "cohesion zones," while loop-extruding cohesin occupies boundaries across all hierarchical levels. Our data support a model in which this segregation emerges from dynamic interplay between cohesin pools and semi-permeable CTCF barriers, which act in cis and in trans to constrain sister-sister misalignment. Thus, a CTCF-defined boundary framework enables sister tethering while preserving dynamic loop folding, allowing replicated genomes to simultaneously support gene regulation and faithful DNA repair.

genomics↗

bioRxiv2025

Conformational asymmetry of replicated human chromosomes

DNA replication creates two sister chromatids that must acquire specific three-dimensional conformations to support genome function and stability. This organization is largely mediated by cohesin complexes, which extrude intra-chromosomal loops and link two chromatids, thus forming "chromatid cohesion". Although sister chromatids are genetically identical, the replication process is intrinsically asymmetric: each chromatid inherits a different parental DNA strand, while the new strands are synthesized using distinct "leading" and "lagging" mechanisms of the replication fork. Whether and how this molecular asymmetry impacts higher-order chromatin organization remains unknown. Using sister-chromatid-sensitive Hi-C, strand-specific FISH, and polymer modeling, we reveal a consistent, genome-wide shift in sister chromatid alignment, biased along the 5'-3' direction of the inherited strands. This shift persists without loop extrusion but is lost upon disruption of cohesion, implicating cohesive cohesins in maintaining the displacement. Polymer simulations indicate that a modest ([~]100 kb) misalignment of "cohesive" cohesins is responsible for the observed asymmetry. We propose two mechanistic models that explain how this displacement arises from replication fork asymmetry: either through the dislocation of cohesin during replication or through the asymmetric anchoring and subsequent random sliding of cohesin pairs. These findings reveal a previously unrecognized chromosome-scale asymmetry in sister chromatid organization, which has implications for homology search during DNA repair.

molecular biology↗

bioRxiv2024

Rules of engagement for condensins and cohesins guide mitotic chromosome formation

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at [~]1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

molecular biology↗

bioRxiv2023

Pairtools: from sequencing data to chromosome contacts

The field of 3D genome organization produces large amounts of sequencing data from Hi-C and a rapidly-expanding set of other chromosome conformation protocols (3C+). Massive and heterogeneous 3C+ data require high-performance and flexible processing of sequenced reads into contact pairs. To meet these challenges, we present pairtools - a flexible suite of tools for contact extraction from sequencing data. Pairtools provides modular command-line interface (CLI) tools that can be flexibly chained into data processing pipelines. Pairtools provides both crucial core tools as well as auxiliary tools for building feature-rich 3C+ pipelines, including contact pair manipulation, filtration, and quality control. Benchmarking pairtools against popular 3C+ data pipelines shows advantages of pairtools for high-performance and flexible 3C+ analysis. Finally, pairtools provides protocol-specific tools for multi-way contacts, haplotype-resolved contacts, and single-cell Hi-C. The combination of CLI tools and tight integration with Python data analysis libraries makes pairtools a versatile foundation for a broad range of 3C+ pipelines.

bioinformatics↗

bioRxiv2022

Cooltools: enabling high-resolution Hi-C analysis in Python

Chromosome conformation capture (3C) technologies reveal the incredible complexity of genome organization. Maps of increasing size, depth, and resolution are now used to probe genome architecture across cell states, types, and organisms. Larger datasets add challenges at each step of computational analysis, from storage and memory constraints to researchers time; however, analysis tools that meet these increased resource demands have not kept pace. Furthermore, existing tools offer limited support for customizing analysis for specific use cases or new biology. Here we introduce cooltools (https://github.com/open2c/cooltools), a suite of computational tools that enables flexible, scalable, and reproducible analysis of high-resolution contact frequency data. Cooltools leverages the widely-adopted cooler format which handles storage and access for high-resolution datasets. Cooltools provides a paired command line interface (CLI) and Python application programming interface (API), which respectively facilitate workflows on high-performance computing clusters and in interactive analysis environments. In short, cooltools enables the effective use of the latest and largest genome folding datasets.

bioinformatics↗

bioRxiv2022

A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination

Conflicts between transcription and cohesin-mediated loop extrusion can majorly influence 3D chromatin architecture but whether these structural changes affect biological function is unknown. Here, we show that a critical step in antibody class switch recombination (CSR) in activated B cells, namely, the juxtaposition (synapsis) of donor and acceptor switch (S) recombination sequences at the immunoglobulin heavy chain locus (Igh), occurs at the interface of a de novo topologically associating domain (TAD) boundary formed via transcriptional activity at acceptor S regions. Using Tri-C to capture higher-order multiway chromatin conformations, we find that synapsis occurs predominantly in the proximity of distal 3 CTCF-binding sites and that this multiway conformation is abolished upon downregulation of transcription and loss of the TAD boundary at the acceptor S region. Thus, an insulating de novo TAD boundary created by the conflict between transcription and loop extrusion plays a direct role in the mechanism of CSR.

molecular biology↗

bioRxiv2022

Bioframe: Operations on Genomic Intervals in Pandas Dataframes

MotivationGenomic intervals are one of the most prevalent data structures in computational genome biology, and used to represent features ranging from genes, to DNA binding sites, to disease variants. Operations on genomic intervals provide a language for asking questions about relationships between features. While there are excellent interval arithmetic tools for the command line, they are not smoothly integrated into Python, one of the most popular general-purpose computational and visualization environments. ResultsBioframe is a library to enable flexible and performant operations on genomic interval dataframes in Python. Bioframe extends the Python data science stack to use cases for computational genome biology by building directly on top of two of the most commonly-used Python libraries, numpy and pandas. The bioframe API enables flexible name and column orders, and decouples operations from data formats to avoid unnecessary conversions, a common scourge for bioinformaticians. Bioframe achieves these goals while maintaining high performance and a rich set of features. Availability and implementationBioframe is open-source under MIT license, cross-platform, and can be installed from the Python package index. The source code is maintained by Open2C on Github at https://github.com/open2c/bioframe.

bioinformatics↗

bioRxiv2021

Germ plasm anchors at tight junctions in the early zebrafish embryo

The zebrafish germline is specified during early embryogenesis by inherited maternal RNAs and proteins collectively called germ plasm. Only the cells containing germ plasm will become part of the germline, whereas other cells will commit to somatic cell fates. Therefore, proper localization of germ plasm is key for germ cell specification and its removal is critical for the development of soma. The molecular mechanism underlying this process in vertebrates is largely unknown. Here we show that germ plasm localization in zebrafish is similar to Xenopus and amniotes but distinct from Drosophila. We identified non muscle myosin II (NMII) and tight junction (TJ) components as interaction candidates of Bucky ball (Buc), which is the germ plasm organizer in zebrafish. Remarkably, we also found that TJ protein ZO1 colocalizes with germ plasm and electron microscopy (EM) of zebrafish embryos uncovered TJ like structures at early cleavage furrows. In addition, injection of the TJ-receptor Claudin-d (Cldn-d) produced extra germ plasm aggregates. Our findings discover for the first time a role of TJs in germ plasm localization.

developmental biology↗

bioRxiv2020

Sister-chromatid-sensitive Hi-C reveals the conformation of replicated human chromosomes

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1-3 has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells4-11 yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

cell biology↗

bioRxiv2018

The genome-wide, multi-layered architecture of chromosome pairing in early Drosophila embryos

Genome organization involves cis and trans chromosomal interactions, both implicated in gene regulation, development, and disease. Here, we focused on trans interactions in Drosophila, where homologous chromosomes are paired in somatic cells from embryogenesis through adulthood. We first addressed the long-standing question of whether pairing extends genome-wide and, to this end, developed a haplotype-resolved Hi-C approach that uses a new strategy to minimize homolog misassignment and thus robustly distinguish trans-homolog from cis contacts. This approach revealed striking genome-wide pairing in Drosophila embryos. Moreover, we discovered pairing to be surprisingly structured, with trans-homolog domains and interaction peaks, many coinciding with the positions of analogous cis features. We also found a significant correlation between pairing and the chromatin accessibility mediated by the pioneer factor Zelda. Our findings reveal a complex, highly structured organization underlying homolog pairing, first discovered more than a century ago.\n\nOne Sentence SummaryA robust approach for haplotype-resolved Hi-C reveals highly-structured homolog pairing in early stage Drosophila embryos.

genetics↗

bioRxiv2018

Highly Structured Homolog Pairing Reflects Functional Organization of the Drosophila Genome

Trans-homolog interactions encompass potent regulatory functions, which have been studied extensively in Drosophila, where homologs are paired in somatic cells and pairing-dependent gene regulation, or transvection, is well-documented. Nevertheless, the structure of pairing and whether its functional impact is genome-wide have eluded analysis. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, discovering that homologs pair relatively precisely genome-wide in addition to establishing trans-homolog domains and compartments. We also elucidated the structure of pairing with unprecedented detail, documenting significant variation across the genome. In particular, we characterized two forms: tight pairing, consisting of contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional role genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, we show that disruption of pairing-promoting factors results in global changes in pairing.\n\nOne Sentence SummaryHaplotype-resolved Hi-C reveals structures of homolog pairing and global implications for gene activity in hybrid PnM cells.

genetics↗

bioRxiv2018

Emerging Evidence of Chromosome Folding by Loop Extrusion

Chromosome organization poses a remarkable physical problem with many biological consequences: how can molecular interactions between proteins at the nanometer scale organize micron-long chromatinized DNA molecules, insulating or facilitating interactions between specific genomic elements? The mechanism of active loop extrusion holds great promise for explaining interphase and mitotic chromosome folding, yet remains difficult to assay directly. We discuss predictions from our polymer models of loop extrusion with barrier elements, and review recent experimental studies that provide strong support for loop extrusion, focusing on perturbations to CTCF and cohesin assayed via Hi-C in interphase. Finally, we discuss a likely molecular mechanism of loop extrusion by SMC complexes.

genomics↗

bioRxiv2017

Mitotic chromosomes fold by condensin-dependent helical winding of chromatin loop arrays

During mitosis, chromosomes fold into compacted rod shaped structures. We combined imaging and Hi-C of synchronous DT40 cell cultures with polymer simulations to determine how interphase chromosomes are converted into compressed arrays of loops characteristic of mitotic chromosomes. We found that the interphase organization is disassembled within minutes of prophase entry and by late prophase chromosomes are already folded as arrays of consecutive loops. During prometaphase, this array reorganizes to form a helical arrangement of nested loops. Polymer simulations reveal that Hi-C data are inconsistent with solenoidal coiling of the entire chromatid, but instead suggest a centrally located helically twisted axis from which consecutive loops emanate as in a spiral staircase. Chromosomes subsequently shorten through progressive helical winding, with the numbers of loops per turn increasing so that the size of a helical turn grows from around 3 Mb (~40 loops) to ~12 Mb (~150 loops) in fully condensed metaphase chromosomes. Condensin is essential to disassemble the interphase chromatin conformation. Analysis of mutants revealed differing roles for condensin I and II during these processes. Either condensin can mediate formation of loop arrays. However, condensin II was required for helical winding during prometaphase, whereas condensin I modulated the size and arrangement of loops inside the helical turns. These observations identify a mitotic chromosome morphogenesis pathway in which folding of linear loop arrays produces long thin chromosomes during prophase that then shorten by progressive growth of loops and helical winding during prometaphase.\n\nOne Sentence SummaryMitotic chromosome morphogenesis occurs through condensin-mediated disassembly of the interphase conformation and formation of extended prophase loop arrays that then shorten by loop growth and condensin-dependent helical winding.

genomics↗

bioRxiv2016

Targeted degradation of CTCF decouples local insulation of chromosome domains from higher-order genomic compartmentalization

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and segmental organization into topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding, and CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Further, our data supports that CTCF mediates transcriptional insulator function through enhancer-blocking but not direct chromatin barrier activity. These results define the functions of CTCF in chromosome folding, and provide new fundamental insights into the rules governing mammalian genome organization.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC=\"FIGDIR/small/095802_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (49K):\norg.highwire.dtl.DTLVardef@79c6c5org.highwire.dtl.DTLVardef@e8e61forg.highwire.dtl.DTLVardef@5c2eedorg.highwire.dtl.DTLVardef@cf7f3_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2016

Cohesin dependent compaction of mitotic chromosomes

Structural Maintenance of Chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modelling, we study how cohesin and condensin, two deeply-conserved SMC complexes, organize chromosomes in budding yeast. The canonical role of cohesins is to co-align sister chromatids whilst condensins generally compact mitotic chromosomes. We find strikingly different roles in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosomes arms, independent of and in addition to its role in sister-chromatid cohesion. Cohesin dependent mitotic chromosome compaction can be fully accounted for through cis-looping of chromatin by loop extrusion. Second, condensin is dispensable for compaction along chromosomal arms and instead plays a specialized role, structuring rDNA and peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that SMC-dependent looping is readily deployed in a range of contexts to functionally organize chromosomes.\n\nHighlightsO_LICohesin compacts mitotic chromosomes independently of sister chromatid cohesion.\nC_LIO_LIFormation of cis-loops by loop extrusion fully accounts for cohesin-mediated compaction.\nC_LIO_LICondensin is not required for mitotic chromosome compaction of yeast chromosome arms\nC_LIO_LICondensin has a focused pre-anaphase role at centromeres and rDNA in yeast\nC_LI

genomics↗

bioRxiv2016

Two independent modes of chromosome organization are revealed by cohesin removal

The three-dimensional organization of chromosomes is tightly related to their biological function 1. Both imaging and chromosome conformation capture studies have revealed several layers of organization 2-4: segregation into active and inactive compartments at the megabase scale 5, and partitioning into domains (TADs) 6,7 and associated loops 8 at the sub-megabase scale. Yet, it remains unclear how these layers of genome organization form, interact with one another, and contribute to or result from genome activities. TADs seem to have critical roles in regulating gene expression by promoting or preventing interactions between promoters and distant cis-acting regulatory elements 9-14, and different architectural proteins, including cohesin, have been proposed to play central roles in their formation 15,16. However, experimental depletions of these proteins have resulted in marginal changes in chromosome organization 17-19. Here, we show that deletion of the cohesin-loading factor, Nipbl, leads to loss of chromosome-associated cohesin and results in dramatic genome reorganization. TADs and associated loops vanish globally, even in the absence of transcriptional changes. In contrast, segregation into compartments is preserved and even reinforced. Strikingly, the disappearance of TADs unmasks a finer compartment structure that accurately reflects the underlying epigenetic landscape. These observations demonstrate that the 3D organization of the genome results from the independent action of two distinct mechanisms: 1) cohesin-independent segregation of the genome into fine-scale compartment regions, defined by the underlying chromatin state; and 2) cohes-dependent formation of TADs possibly by the recently proposed loop extrusion mechanism 20,21, enabling long-range and target-specific activity of promiscuous enhancers. The interplay between these mechanisms creates an architecture that is more complex than a simple hierarchy of layers and can be central to guiding normal development.

genomics↗