Biology subjects

Gibcus, J. H.

Publications and source records attributed to Gibcus, J. H..

11 recordsLinked to original sources

bioRxiv2026

Stepwise reorganization of chromosome conformation and nuclear organization during stem cell differentiation

Nuclear organization is a fundamental feature of cell identity and cell fate determination. Compared with differentiated cells, pluripotent stem cells exhibit markedly distinct nuclear architecture at multiple levels, including chromosome folding, lamina association, histone modification landscapes and nuclear body organization. Although individual aspects of this reorganization have been characterized, how and in what order these features are remodeled as cells commit to a lineage remains poorly understood. Here, we temporally map nuclear reorganization across five stages of an in vitro differentiation system of human embryonic stem cells through definitive endoderm and hepatoblast intermediates into hepatocyte-like cells. By integrating Hi-C, RNA-seq, ATAC-seq, ChIP-seq, and CUT&RUN data, we establish a genome-wide framework linking structural, epigenetic, and transcriptional changes across differentiation. Combined with immunofluorescence imaging, chromosome painting, and liquid chromatin Hi-C (LC-Hi-C) we show that nuclear reorganization proceeds in a stepwise and temporally ordered manner through three major transitions. In the first transition, chromosomes condense into defined territories concurrent with transient anchoring of centromere-proximal regions to the nuclear periphery. In a second transition, active and inactive chromatin segregate, leading to stronger compartmentalization. This coincides with deposition and peripheralization of H3K9me2-marked chromatin and morphological changes in nuclear speckles, while chromatin conformation at speckle-associated regions stabilizes. In a third transition, after lineage-specific genes are activated, chromatin interactions globally stabilize, establishing a more stable nuclear architecture on top of these earlier large-scale structural rearrangements. Together, our results define a stepwise framework for nuclear reorganization during human embryonic stem cell differentiation and reveal that the transition from pluripotency to a differentiated state proceeds through coordinated, temporally ordered structural events.

genomics↗

bioRxiv2026

UnionLoops: a workflow for calling chromatin loops across related Hi-C datasets with improved specificity, precision, and sensitivity

Chromatin loop calling from chromatin interaction data often exhibits substantial variability across related samples. We present UnionLoops, a computational workflow for chromatin loop calling across multiple related samples. UnionLoops integrates information across datasets to determine positions and dataset-specificity of looping interactions. It constructs a unified candidate loop set, applies consistent filtering and aggregation, and evaluates loop support across samples. We demonstrate that UnionLoops increases sensitivity for detecting shared chromatin loops, reduces spurious sample-specific calls, and improves concordance with independent genomic features, including CTCF and cohesin occupancy. UnionLoops enables improved biological interpretation of chromatin loop organization and dynamics across related conditions.

genomics↗

bioRxiv2024

An integrated view of the structure and function of the human 4D nucleome

The dynamic three-dimensional (3D) organization of the human genome (the "4D Nucleome") is closely linked to genome function. Here, we integrate a wide variety of genomic data generated by the 4D Nucleome Project to provide a detailed view of human 3D genome organization in widely used embryonic stem cells (H1-hESCs) and immortalized fibroblasts (HFFc6). We provide extensive benchmarking of 3D genome mapping assays and integrate these diverse datasets to annotate spatial genomic features across scales. The data reveal a rich complexity of chromatin domains and their sub-nuclear positions, and over one hundred thousand structural loops and promoter-enhancer interactions. We developed 3D models of population-based and individual cell-to-cell variation in genome structure, establishing connections between chromosome folding, nuclear organization, chromatin looping, gene transcription, and DNA replication. We demonstrate the use of computational methods to predict genome folding from DNA sequence, uncovering potential effects of genetic variants on genome structure and function. Together, this comprehensive analysis contributes insights into human genome organization and enhances our understanding of connections between the regulation of genome function and 3D genome organization in general.

genomics↗

bioRxiv2024

Polymer physics models reveal structural folding features of single-molecule gene chromatin conformations

Here, we employ polymer physics models of chromatin to investigate the 3D folding of a 2Mb wide genomic region encompassing the human LTN1 gene, a crucial DNA locus involved in key cellular functions. Through extensive Molecular Dynamics simulations, we reconstruct in-silico the ensemble of single-molecule LTN1 3D structures, which we benchmark against recent in-situ Hi-C 2.0 data. The model-derived single molecules are then used to predict structural folding features at the single-cell level, providing testable predictions for super-resolution microscopy experiments.

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↗

bioRxiv2022

The little skate genome and the evolutionary emergence of wing-like fin appendages

Skates are cartilaginous fish whose novel body plan features remarkably enlarged wing-like pectoral fins that allow them to thrive in benthic environments. The molecular underpinnings of this unique trait, however, remain elusive. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins - gene expression, chromatin occupancy and three-dimensional (3D) conformation - we find skate-specific genomic rearrangements that alter the 3D regulatory landscape of genes involved in the planar cell polarity (PCP) pathway. Functional inhibition of PCP signaling resulted in marked reduction of anterior fin size, confirming this pathway as a major contributor of batoid fin morphology. We also identified a fin-specific enhancer that interacts with 3 HOX genes, consistent with the redeployment of Hox gene expression in anterior pectoral fins, and confirmed the potential of this element to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganizations and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

evolutionary biology↗

bioRxiv2022

Multi-omics comparison of malignant and normal uveal melanocytes reveals novel molecular features of uveal melanoma.

Uveal Melanoma (UM) is a rare cancer resulting from transformation of melanocytes residing in the uveal tract. Integrative analysis has identified four molecular and clinical subsets in UM. To improve our molecular understanding of UM we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis by Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with up-regulation of PRAME, an independent prognostic biomarker in UM and potential therapeutic target. Our findings illustrate how multi-omics approaches can improve the understanding of tumorigenesis and reveals two novel mechanisms of gene expression dysregulation in UM.

genomics↗

bioRxiv2021

Cohesin-mediated loop anchors confine the location of human replication origins

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability across dividing cells in metazoans. It is currently unknown how the location of replication origins and the timing of their activation is determined in the human genome. Here, we dissect the role for G1 phase topologically associating domains (TADs), subTADs, and loops in the activation of replication initiation zones (IZs). We identify twelve subtypes of self-interacting chromatin domains distinguished by their degree of nesting, the presence of corner dot structures indicative of loops, and their co-localization with A/B compartments. Early replicating IZs localize to boundaries of nested corner-dot TAD/subTADs anchored by high density arrays of co-occupied CTCF+cohesin binding sites with divergently oriented motifs. By contrast, late replicating IZs localize to weak TADs/subTAD boundaries devoid of corner dots and most often anchored by singlet CTCF+cohesin sites. Upon global knock-down of cohesin-mediated loops in G1, early wave focal IZs replicate later in S phase and convert to diffuse placement along the genome. Moreover, IZs in mid-late S phase are delayed to the final minutes before entry into G2 when cohesin-mediated dot-less boundaries are ablated. We also delete a specific loop anchor and observe a sharp local delay of an early wave IZ to replication in late S phase. Our data demonstrate that cohesin-mediated loops at genetically-encoded TAD/subTAD boundaries in G1 phase are an essential determinant of the precise genomic placement of human replication origins in S phase.

genomics↗

bioRxiv2020

Systematic evaluation of chromosome conformation capture assays

Chromosome conformation capture (3C)-based assays are used to map chromatin interactions genome-wide. Quantitative analyses of chromatin interaction maps can lead to insights into the spatial organization of chromosomes and the mechanisms by which they fold. A number of protocols such as in situ Hi-C and Micro-C are now widely used and these differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of experimental parameters of 3C-based protocols. We find that different protocols capture different 3D genome features with different efficiencies. First, the use of cross-linkers such as DSG in addition to formaldehyde improves signal-to-noise allowing detection of thousands of additional loops and strengthens the compartment signal. Second, fragmenting chromatin to the level of nucleosomes using MNase allows detection of more loops. On the other hand, protocols that generate larger multi-kb fragments produce stronger compartmentalization signals. We confirmed our results for multiple cell types and cell cycle stages. We find that cell type-specific quantitative differences in chromosome folding are not detected or underestimated by some protocols. Based on these insights we developed Hi-C 3.0, a single protocol that can be used to both efficiently detect chromatin loops and to quantify compartmentalization. Finally, this study produced ultra-deeply sequenced reference interaction maps using conventional Hi-C, Micro-C and Hi-C 3.0 for commonly used cell lines in the 4D Nucleome Project.

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

HI-C 2.0: AN OPTIMIZED HI-C PROCEDURE FOR HIGH-RESOLUTION GENOME-WIDE MAPPING OF CHROMOSOME CONFORMATION

Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions). Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (Kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra-chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and TADs, as well as high-resolution conformational features such as DNA loops.

genomics↗