3D Chromatin Research Articles

Abstract 2735: The role of ca2 in regulating 3d chromatin structure in tamoxifen resistant breast cancer

Abstract Background: Estrogen receptor-positive breast cancer frequently develops resistance to tamoxifen in one third of patients, driven by various mechanisms. Our work has linked 3D chromatin alterations to this resistance. However, most of the work in 3D chromatin regulation was mostly conducted in in vitro cell culture system. It is becoming very clear that to expand the characterization of 3D genome organization in patient samples is an emerging task. Hypothesis: We hypothesize that 3D chromatin alterations, including compartment switching, TAD reorganization, and dysregulated chromatin looping, drive tamoxifen resistance (TR) in breast cancer. Looping-mediated genes may play critical roles in resistance and serve as potential therapeutic targets. Methods and Results: To investigate the role of 3D chromatin changes in tamoxifen resistance, we performed Hi-C profiling on normal tissues (NTs), primary tumors (PTs), and tamoxifen-treated recurrent tumors (RTs), uncovering significant chromatin alterations. These included 46.8% A-to-B and 32.8% B-to-A compartment switching from NTs to PTs, and 58.3% B-to-A flipping during PT-to-RT progression. TAD analysis identified RT-specific Neo and Fuse alterations as well as tumor-specific chromatin loops. CA2 was identified as a enhancer-promoter looping (EPL)-mediated gene with its higher expression linked to poor relapse-free survival in endocrine-treated patients. Functional studies using the CA2 inhibitor brinzolamide demonstrated its ability to reduce cell viability, loop intensity, and CA2 expression in MCF7TR and T47DTR cells. In vivo, brinzolamide significantly reduced tumor growth in tamoxifen-resistant cell-derived xenograft (CDX) models. Additionally, CRISPR deletion of CA2 EPL enhancer region in MCF7TR cells resulted in a marked reduction in cellular viability, gene expression and protein expression. Conclusion: Our findings suggest CA2 as a key looping-mediated gene play an oncogenic role in driving tamoxifen resistance in breast cancer. Our study yields significant mechanistic insights into the role and clinical relevance of 3D chromatin architecture in tamoxifen resistant breast cancer. Citation Format: Lavanya Choppavarapu, Kun Fang, Tianxiang Liu, Aigbe G. Ohihoin, Victor X. Jin. The role of ca2 in regulating 3d chromatin structure in tamoxifen resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2735.

Abstract 207: snNOMeHiC: joint profiling of 3D genome, DNA methylation, and chromatin accessibility from the same nuclei at flash-frozen tissue and primary tumor sample

Abstract Epigenetic marks, including DNA methylation, chromatin accessibility, and three-dimensional (3D) genome organization, play instrumental roles in gene regulation in mammals. Gene activation or repression potential, however, cannot be entirely predicted by looking at a single modality. Accurate predictive models require multiple modalities simultaneously. We previously developed single-cell Methyl-HiC technology to jointly profile DNA methylation and 3D genome from the same cells. Further, we incorporated an additional footprint of GpC methyltransferase activity (i.e., chromatin accessibility) into the assay, named NOMe-HiC, but at the bulk level. Here, we extended NOMe-HiC technology to the single-cell level to enable the joint profiling of the 3D genome, DNA methylation, and chromatin accessibility from the same nuclei. We performed snNOMe-HiC at IMR-90 cell lines and found high concordance across all three modalities with the bulk NOMe-HiC by pooling the single cells. We further prepared a mixture of GM12878 and IMR-90 cells and processed the cell mixture with snNOMe-HiC. snNOMe-HiC can distinguish the two cell types using each of the three epigenetic features, respectively. To benchmark the performance of snNOMe-HiC at flash-frozen heterogeneous tissues, we generated snNOMe-HiC data from ∼2000 nuclei at flash-frozen mice cortex. We identified all the major known cell types in the previous studies across three modalities and showed reproducible results across replicates. Finally, we applied snNOMe-HiC to a primary meningioma sample. We identified previously known copy number alterations in tumor-related cell clusters and found concordant changes of epigenetic heterogeneity across cell populations, which can potentially reveal the crosstalk between genetics and multiple epigenetic marks during the clonal evolution in cancer progression. In summary, snNOMe-HiC is a robust technology that can profile three epigenetic modalities and genetic aberrations in the same nuclei from flash-frozen tissues and primary tumors. Citation Format: Hailu Fu, Jiayan Ma, Eric Tong, Mark Youngblood, Harrshavasan Congivaram, Mateo Gomez, Juan Wang, Feng Yue, Li Wang, Yaping Liu. snNOMeHiC: joint profiling of 3D genome, DNA methylation, and chromatin accessibility from the same nuclei at flash-frozen tissue and primary tumor sample [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 207.

Abstract 5100: Mediator subunit Med4 enforces metastatic dormancy in breast cancer

Long term survival of breast cancer patients is limited due to recurrence from metastatic dormant cancer cells. However, the mechanisms by which these dormant breast cancer cells survive and awaken remain poorly understood. Our unbiased genome-scale genetic screen in mice identified Med4 as a novel cancer-cell intrinsic gatekeeper in metastatic reactivation. MED4 haploinsufficiency is prevalent in metastatic breast cancer patients and correlates with poorer prognosis. Syngeneic xenograft models revealed that Med4 enforces breast cancer dormancy. Contrary to the canonical function of the Mediator complex in activating gene expression, Med4 maintains 3D chromatin compaction and enhancer landscape, by preventing enhancer priming or activation through the suppression of H3K4me1 deposition. Med4 haploinsufficiency disrupts enhancer poise and reprograms the enhancer dynamics to facilitate extracellular matrix (ECM) gene expression and integrin-mediated mechano-transduction, driving metastatic growth. Our findings establish Med4 as a key regulator of cellular dormancy and a potential biomarker for high-risk metastatic relapse. Citation Format: Seong-Yeon Bae, Hsiang-Hsi Ling, Yi Chen, Hong Chen, Dhiraj Kumar, Jiankang Zhang, Aaron Viny, Ronald A. DePinho, Filippo G. Giancotti. Mediator subunit Med4 enforces metastatic dormancy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5100.

Abstract 2748: Integrative analysis of 3D chromatin organization revealed regulation mechanisms in angioimmunoblastic T-cell lymphoma

Abstract Molecular studies have highlighted recurrent alterations in epigenetic modifier genes, including TET2, DNMT3A, and IDH2, in angioimmunoblastic T-cell lymphoma (AITL). However, the direct impact of gene regulatory elements on gene expression changes leading to T-cell lymphomagenesis remains poorly understood. We focus on 3D chromatin organization. Hi-C and RNA sequencing was performed on diagnostic FFPE tumor samples from seventeen patients with AITL. Two CD4+ T cells from individual donors and one follicular helper T cells (TFH) from four donors were used as controls. To annotate AITL-specific loops in association with enhancer status, we identified H3K27ac histone marks in TFH cells using CUT&Tag. Promoter-super enhancer (P-SE) loops or stripes were defined as those with non-promoter anchors containing SE predicted by ROSE. In comparison to normal CD4+ T-cell controls, recurrent AITL-specific alterations in A/B (active/inactive) compartments, topologically associating domains (TADs), and chromatin loops were identified. Principal-component analysis (PCA) of genome-wide compartment scores indicated AITL samples and normal CD4+ T cells clearly separated by the first two components. On average, there was 3.5% (range: 2.0-5.3) A-to-B compartment switch and 1.9% (range: 0.8-4.8) B-to-A switch when comparing AITL samples with normal CD4+ T cell controls (p=5.17 × 10-5, two-sided paired t-test). Alterations in TADs were recurrently observed in 434 of 743 cancer census genes in COSMIC, including MYC, TCL1A, and RHOA. On average, 60.1% of loops include promoters in at least one anchor. Genes within AITL-specific loops exhibited significantly higher expression than those outside of loop regions (p=7.39 × 10-88, Kruskal-Wallis test). Genes within P-SE loops/stripes showed significantly higher expression than those within promoter-inactive enhancer (P-IE) loops/stripes, which were defined by ENCODE as enhancers without TFH SE (loops: p = 5.17 × 10-5, stripes: p=1.55e-15, Kruskal-Wallis test). Based on the differential loop analysis using diffpeakachu, significant chromatin loops were observed in the MYC region and genes associated with pathways related to MYC, such as the Wnt signaling pathway. AITL-specific loops in the MYC region were observed in eight of seventeen patients. Recurrent MYC promoter-TFH SE loop (chr8:127730000-127740000, chr8:127960000-127970000) was located between ME1 and ME2 of MYC enhancer in AML (Pulikkan et al., 2018). Our findings indicate that AITL exhibits unique chromatin conformations, suggesting the formation of a lymphoma-specific gene regulatory landscape. Recurrent changes in chromatin conformation involve MYC. Distinct gene regulatory elements associated with enhancer regions in AITL is implicated. Correlation of the regulatory candidates with their mutational signature is underway and will be reported at the meeting. Citation Format: Makoto Iwasaki, Caroline MacVicar, Anna Baird Rider, Aliyah Sohani, Valentina Nardi, Josie Ford, Kusha Chopra, Eric Jacobsen, Jeffrey Barnes, Salvia Jain. Integrative analysis of 3D chromatin organization revealed regulation mechanisms in angioimmunoblastic T-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2748.

Abstract 3674: Predicting chromatin organization from cell-free DNA fragmentation patterns

Abstract Epigenetic regulation, including the three-dimensional (3D) architecture of the genome, plays a critical role in gene expression and cellular function. Aberrations in chromatin organization are implicated in various diseases, particularly cancer. While Hi-C is the gold standard for mapping chromosomal conformations, it has significant limitations: it requires fixed cells, limiting its capacity for longitudinal monitoring of dynamic chromatin changes. Cell-free DNA (cfDNA), which consists of short DNA fragments released into circulation through apoptosis and necrosis, offers a promising alternative. CfDNA fragmentation patterns are non-random and influenced by underlying epigenetic features, providing a window into chromatin organization in vivo. Here, we hypothesize that the correlation of cfDNA fragmentation patterns between two genomic regions can potentially reflect the 3D chromatin architecture. Since cfDNA fragments in vivo are released from multiple cell types, we generated in situ Hi-C and matched cfDNA whole-genome sequencing data from a xenograft mice model as the training dataset. We trained a contrastive deep learning model to infer Hi-C contact maps directly from cfDNA fragmentation data. The model demonstrated high accuracy in predicting chromatin compartments and topologically associating domains (TADs) from cfDNA fragmentation patterns, outperforming conventional machine learning approaches. These findings suggest that cfDNA fragmentation can enable the non-invasive and longitudinal monitoring of 3D genome aberrations in vivo. Citation Format: Ravi Bandaru, Zhihan Zhou, Hailu Fu, Weimin Wu, Haizi Zheng, Rashmi S. Hegde, Li Wang, Han Liu, Yaping Liu. Predicting chromatin organization from cell-free DNA fragmentation patterns [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3674.

Abstract 2740: Mechanisms of phase-separation driven chromatin looping in acute myeloid leukemia

Abstract New therapies are desperately needed for NUP98-fusion-driven Acute Myeloid Leukemia (AML) patients as they exhibit a poor prognosis on existing therapies, with a 5-year overall survival rate of ∼30%. We and others have shown that one fusion, NUP98-HOXA9 drives leukemogenesis through the formation of liquid-liquid phase separated (LLPS) condensates. Specifically, NUP98-HOXA9 binds DNA and pulls bound regions into the nuclear condensates, forming 3D chromatin loops that activate oncogenes' transcription. Mutation of the phenylalanine-glycine (FG) repeats responsible for NUP98-HOXA9 phase separation abolishes the formation of these condensates and inhibits malignant transformation. These findings suggest a new class of chromatin loops, that form via a mechanism distinct from loop extrusion and drive oncogene expression. We set out to characterize the necessary components for the formation and maintenance of these aberrant loops. Using an Auxin Inducible Degron (AID) system in HCT116 cells, we rapidly degraded loop extrusion proteins, CTCF and RAD21, followed by in situ Hi-C to observe impact on loop formation. We observe approximately 8, 000 canonical loops, which are bound by CTCF and lost upon auxin treatment. Additionally, we identify approximately 60 phase-separation driven loops, bound by NUP98-HOXA9 which do not require CTCF. We additionally explore the role of ATP, BRD4, and p300 as requirements for the formation of these loops. Future work includes further characterization of loop maintenance and screening for compounds to disrupt condensate formation with the goal of using a deeper understanding of phase-separation driven loops to create new promising therapeutic avenues for NUP98-fusion-driven AMLs. Citation Format: Tooba Rashid, Zachary Drum, Doris Cruz Alonso, Gelila Petros, Yoseli Quiroga, Douglas Phanstiel. Mechanisms of phase-separation driven chromatin looping in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2740.

Hidden origami in Trypanosoma cruzi nuclei highlights its non-random 3D genomic organization.

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease and is known for its polycistronic transcription, with about 50% of its genome consisting of repetitive sequences, including coding (primarily multigenic families) and non-coding regions (such as ribosomal DNA, spliced leader [SL], and retroelements, etc). Here, we evaluated the genomic features associated with higher-order chromatin organization in T. cruzi (Brazil A4 strain) by extensive computational processing of high-throughput chromosome conformation capture (Hi-C). Through the mHi-C pipeline, designed to handle multimapping reads, we demonstrated that applying canonical Hi-C processing, which overlooks repetitive DNA sequences, results in a loss of DNA-DNA contacts, misidentifying them as chromatin-folding (CF) boundaries. Our analysis revealed that loci encoding multigenic families of virulence factors are enriched in chromatin loops and form shorter and tighter CF domains than the loci encoding core genes. We uncovered a non-random three-dimensional (3D) genomic organization in which nonprotein-coding RNA loci (transfer RNAs [tRNAs], small nuclear RNAs, and small nucleolar RNAs) and transcription termination sites are preferentially located at the boundaries of the CF domains. Our data indicate 3D clustering of tRNA loci, likely optimizing transcription by RNA polymerase III, and a complex interaction between spliced leader RNA and 18S rRNA loci, suggesting a link between RNA polymerase I and II machineries. Finally, we highlighted a group of genes encoding virulence factors that interact with SL-RNA loci, suggesting a potential regulatory role. Our findings provide insights into 3D genome organization in T. cruzi, contributing to the understanding of supranucleosomal-level chromatin organization and suggesting possible links between 3D architecture and gene expression.IMPORTANCEDespite the knowledge about the linear genome sequence and the identification of numerous virulence factors in the protozoan parasite Trypanosoma cruzi, there has been a limited understanding of how these genomic features are spatially organized within the nucleus and how this organization impacts gene regulation and pathogenicity. By providing a detailed analysis of the three-dimensional (3D) chromatin architecture in T. cruzi, our study contributed to narrowing this gap. We deciphered part of the origami structure hidden in the T. cruzi nucleus, showing the unidimensional genomic features are non-randomly 3D organized in the nuclear organelle. We uncovered the role of nonprotein-coding RNA loci (e.g., transfer RNAs, spliced leader RNA, and 18S RNA) in shaping genomic architecture, offering insights into an additional epigenetic layer that may influence gene expression.

Tensor-FLAMINGO unravels the complexity of single-cell spatial architectures of genomes at high-resolution

The dynamic three-dimensional spatial conformations of chromosomes demonstrate complex structural variations across single cells, which plays pivotal roles in modulating single-cell specific transcription and epigenetics landscapes. The high rates of missing contacts in single-cell chromatin contact maps impose significant challenges to reconstruct high-resolution spatial chromatin configurations. We develop a data-driven algorithm, Tensor-FLAMINGO, based on a low-rank tensor completion strategy. Implemented on a diverse panel of single-cell chromatin datasets, Tensor-FLAMINGO generates 10kb- and 30kb-resolution spatial chromosomal architectures across individual cells. Tensor-FLAMINGO achieves superior accuracy in reconstructing 3D chromatin structures, recovering missing contacts, and delineating cell clusters. The unprecedented high-resolution characterization of single-cell genome folding enables expanded identification of single-cell specific long-range chromatin interactions, multi-way spatial hubs, and the mechanisms of disease-associated GWAS variants. Beyond the sparse 2D contact maps, the complete 3D chromatin conformations promote an avenue to understand the dynamics of spatially coordinated molecular processes across different cells.

Perturb-tracing enables high-content screening of multi-scale 3D genome regulators.

Three-dimensional (3D) genome organization becomes altered during development, aging and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multi-scale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screens, a cellular barcode readout method (BARC-FISH) and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their 3D chromatin folding conformations, alongside perturbation-paired barcode readout in the same single cells. We discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop extrusion and A-B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome.

Pooled CRISPR screening with optical sequencing reveals regulators of 3D chromatin organization.

Pooled CRISPR screening with optical sequencing reveals regulators of 3D chromatin organization.

PC-SAC: Amethod for high-resolution 3D genome reconstruction from low-resolution Hi-C data.

The three-dimensional (3D) organization of the genome is crucial for gene regulation, with disruptions linked to various diseases. High-throughput Chromosome Conformation Capture (Hi-C) and related technologies have advanced our understanding of 3Dgenome organization by mapping interactions between distalgenomic regions. However, capturing enhancer-promoter interactions at high resolution remains challenging due to the high sequencing depth required. We introduce pC-SAC (probabilistically Constrained Self-Avoiding Chromatin), a novel computational method for producing accurate high-resolution Hi-C matrices from low-resolution data. pC-SAC uses adaptive importance sampling with sequential Monte Carlo to generate ensembles of 3D chromatin chains that satisfy physical constraints derived from low-resolution Hi-C data. Our method achieves over 95% accuracy in reconstructing high-resolution chromatin maps and identifies novel interactions enriched with candidate cis-regulatory elements (cCREs) and expression quantitative trait loci (eQTLs). Benchmarking against state-of-the-art deep learning models demonstrates pC-SAC'sperformance in both short- and long-range interaction reconstruction. pC-SAC offers a cost-effective solution for enhancing the resolution of Hi-C data, thus enabling deeper insights into 3D genome organization and its role in gene regulation and disease. Our tool can be found at https://github.com/G2Lab/pCSAC.

Active regulatory elements recruit cohesin to establish cell specific chromatin domains

As the 3D structure of the genome is analysed at ever increasing resolution it is clear that there is considerable variation in the 3D chromatin architecture across different cell types. It has been proposed that this may, in part, be due to increased recruitment of cohesin to activated cis-elements (enhancers and promoters) leading to cell-type specific loop extrusion underlying the formation of new sub-TADs. Here we show that cohesin correlates well with the presence of active enhancers and that this varies in an allele-specific manner with the presence or absence of polymorphic enhancers which vary from one individual to another. Using the alpha globin cluster as a model, we show that when all enhancers are removed, peaks of cohesin disappear from these regions and the erythroid specific sub-TAD is no longer formed. Re-insertion of the major alpha globin enhancer (R2) is associated with re-establishment of recruitment and increased interactions. In complementary experiments insertion of the R2 enhancer element into a “neutral” region of the genome recruits cohesin, induces transcription and creates a new large (75 kb) erythroid-specific domain. Together these findings support the proposal that active enhancers recruit cohesin, stimulate loop extrusion and promote the formation of cell specific sub-TADs.

Chromatin as a Coevolutionary Graph: Modeling the Interplay of Replication with Chromatin Dynamics.

Modeling DNA replication poses significant challenges due to the intricate interplay of biophysical processes and the need for precise parameter optimization. In this study, we explore the interactions among three key biophysical factors that influence chromatin folding: replication, loop extrusion, and compartmentalization. Replication forks, known to act as barriers to the motion of loop extrusion factors, also correlate with the phase separation of chromatin into A and B compartments. Our approach integrates three components: (1) a numerical model that takes into advantage single-cell replication timing data to simulate replication fork propagation; (2) a stochastic Monte Carlo simulation that captures the interplay between the biophysical factors, with loop extrusion factors binding, unbinding, and extruding dynamically, while CTCF barriers and replication forks act as static and moving barriers, and a Potts Hamiltonian governs the spreading of epigenetic states driving chromatin compartmentalization; and (3) a 3D OpenMM simulation that reconstructs the chromatin's 3D structure based on the states generated by the stochastic model. To our knowledge, this is the first framework to dynamically integrate and simulate these three biophysical factors, enabling insights into chromatin behavior during replication. Furthermore, we investigate how replication stress alters these dynamics and affects chromatin structure.

TRUHiC: A TRansformer-embedded U-2 Net to enhance Hi-C data for 3D chromatin structure characterization.

High-throughput chromosome conformation capture sequencing (Hi-C) is a key technology for studying the three-dimensional (3D) structure of genomes and chromatin folding. Hi-C data reveals important patterns of genome organization such as topologically associating domains (TADs) and chromatin loops with critical roles in transcriptional regulation and disease etiology and progression. However, the relatively low resolution of existing Hi-C data often hinders robust and reliable inference of 3D structures. Hence, we propose TRUHiC, a new computational method that leverages recent state-of-the-art deep generative modeling to augment low-resolution Hi-C data for the characterization of 3D chromatin structures. Applying TRUHiC to publically available Hi-C data for human and mice, we demonstrate that the augmented data significantly improves the characterization of TADs and loops across diverse cell lines and species. We further present a pre-trained TRUHiC on human lymphoblastoid cell lines that can be adaptable and transferable to improve chromatin characterization of various cell lines, tissues, and species.

Single-cell analysis of the epigenome and 3D chromatin architecture in the human retina.

Most genetic risk variants linked to ocular diseases are non-protein coding and presumably contribute to disease through dysregulation of gene expression, however, deeper understanding of their mechanisms of action has been impeded by an incomplete annotation of the transcriptional regulatory elements across different retinal cell types. To address this knowledge gap, we carried out single-cell multiomics assays to investigate gene expression, chromatin accessibility, DNA methylome and 3D chromatin architecture in human retina, macula, and etinal pigment epithelium (RPE)/choroid. We identified 420,824 unique candidate regulatory elements and characterized their chromatin states in 23 sub-classes of retinal cells. Comparative analysis of chromatin landscapes between human and mouse retina cells further revealed both evolutionarily conserved and divergent retinal gene-regulatory programs. Leveraging the rapid advancements in deep-learning techniques, we developed sequence-based predictors to interpret non-coding risk variants of retina diseases. Our study establishes retina-wide, single-cell transcriptome, epigenome, and 3D genome atlases, and provides a resource for studying the gene regulatory programs of the human retina and relevant diseases.

CHD7 binds distinct regions in the Sox11 locus to regulate neuronal differentiation.

The chromodomain helicase DNA binding protein 7 (CHD7) is a nucleosome repositioner implicated in multiple cellular processes, including neuronal differentiation. We identified CHD7 genome-wide binding sites that regulate neuronal differentiation in an otic stem cell line. We identified CHD7 enrichment at the Sox11 promoter and 3' untranslated region (UTR). Sox11 is a transcription factor essential for neuronal differentiation. CRISPRi of Sox11 promoter or 3'UTR displayed decreased neurite lengths and reduced neuronal marker expression TUBB3 expression. We showed that the Sox11 locus resides at TAD boundaries, and CTCF marks the 3'UTR. We propose that CHD7 modulates chromatin accessibility of the Sox11 promoter and CTCF-marked insulators in the 3'UTR to facilitate neuronal differentiation. CRISPRi of the insulator site alters 3D chromatin organization, affects gene expression and ultimately perturbs cellular processes. Our results implicate a general mechanism of CHD7 in facilitating neuronal differentiation and provide insight into CHD7 dysfunction in CHARGE syndrome, a congenital disorder associated with hearing loss.

Hi-C profiling in tissues reveals 3D chromatin-regulated breast tumor heterogeneity informing a looping-mediated therapeutic avenue.

Hi-C profiling in tissues reveals 3D chromatin-regulated breast tumor heterogeneity informing a looping-mediated therapeutic avenue.

Integrated promoter-capture Hi-C and Hi-C analysis reveals fine-tuned regulation of the 3D chromatin architecture in colorectal cancer.

Hi-C is a widely used technique for mapping chromosomal interactions within a 3D genomic framework, however, its resolution is often constrained by sequencing depth, making it challenging to detect fine-scale interactions. To overcome this limitation, Promoter-Capture Hi-C (PCHi-C), as it selectively enriches for promoter-associated interactions, was employed. This study integrates PCHi-C and Hi-C datasets from colorectal cancer (CRC) models investigate chromosomal interaction dynamics across various regulatory levels, from cis-regulatory elements to topologically associated domains (TADs). The primary goal is to examine how genomic structural alterations shape the epigenomic landscape in CRC and to assess their potential role in colorectal cancer susceptibility. PCHi-C and Hi-C datasets from multiple colorectal cancer (CRC) studies were integrated to enhance the resolution of chromatin interaction mapping. The analysis focused on identifying fine-scale interactions within topologically associated domains (TADs) while incorporating histone modification landscapes (H3K27ac, H3K4me3) and transcriptomic signatures from CRC cell lines and the TCGA database. For experimental validation, ChIP-quantitative PCR was performed at the promoters of target genes using the highly malignant colorectal cell line HT29 and compared it to an embryonic cell line NT2D1. Our integrated analysis revealed significant genomic structural instability in CRC cells, closely associated with tumor-suppressive transcriptional programs. We identified nine dysregulated genes, including long non-coding RNAs (MALAT1, NEAT1, FTX, and PVT1), small nucleolar RNAs (SNORA26 and SNORA71A), and protein-coding genes (TMPRSS11D, TSPEAR, and DSG4), all of which exhibited a substantial increase in expression in CRC cell lines compared to human embryonic stem cells (hESCs). Additionally, we observed enriched activation-associated histone modifications (H3K27ac and H3K4me3) at the potential enhancer regions of these genes, indicating possible transcriptional activation. ChIP-quantitative PCRs conducted using in the highly malignant CRC cell line HT29, compared to the embryonic cell line NT2D1, further validated these findings, reinforcing the link between altered chromosomal interactions and gene dysregulation in CRC. This study sheds light on the dynamic 3D genome organization in CRC, highlighting critical structural changes associated with disease-associated loci. The identification of nine dysregulated genes points to potential biomarkers for colorectal cancer, with implications for diagnostic and therapeutic strategies. The combination of Hi-C and PCHi-C offers a refined approach for detecting chromosomal interactions at a higher resolution, laying the foundation for future studies on cancer-associated chromatin architecture.

The Impact of Polycomb Group Proteins on 3D Chromatin Structure and Environmental Stresses in Plants.

The two multi-subunit complexes, Polycomb Repressive Complex 1 and 2 (PRC1/2), act synergistically during development to maintain the gene silencing state among different species. In contrast with mammals and Drosophila melanogaster, the enzyme activities and components of the PRC1 complex in plants are not fully conserved. In addition, the mutual recruitment of PRC1 and PRC2 in plants differs from that observed in mammals and Drosophila. Polycomb Group (PcG) proteins and their catalytic activity play an indispensable role in transcriptional regulation, developmental processes, and the maintenance of cellular identity. In plants, PRC1 and PRC2 deposit H2Aub and H3K27me3, respectively, and also play an important role in influencing three-dimensional (3D) chromatin structure. With the development of high-throughput sequencing techniques and computational biology, remarkable progress has been made in the field of plant 3D chromatin structure, and PcG has been found to be involved in the epigenetic regulation of gene expression by mediating the formation of 3D chromatin structures. At the same time, some genetic evidence indicates that PcG enables plants to better adapt to and resist a wide range of stresses by dynamically regulating gene expression. In the following review, we focus on the recruitment relationship between PRC1 and PRC2, the crucial role of PcG enzyme activity, the effect of PcG on 3D chromatin structure, and the vital role of PcG in environmental stress in plants.

Human Body Single-Cell Atlas of 3D Genome Organization and DNA Methylation.

Higher-order chromatin structure and DNA methylation are critical for gene regulation, but how these vary across the human body remains unclear. We performed multi-omic profiling of 3D genome structure and DNA methylation for 86,689 single nuclei across 16 human tissues, identifying 35 major and 206 cell subtypes. We revealed extensive changes in CG and non-CG methylation across almost all cell types and characterized 3D chromatin structure at an unprecedented cellular resolution. Intriguingly, extensive discrepancies exist between cell types delineated by DNA methylation and genome structure, indicating that the role of distinct epigenomic features in maintaining cell identity may vary by lineage. This study expands our understanding of the diversity of DNA methylation and chromatin structure and offers an extensive reference for exploring gene regulation in human health and disease.