Topologically Associating Domains Research Articles

Single-molecule imaging reveals a direct role of CTCF's zinc fingers in SA interaction and cluster-dependent RNA recruitment.

CTCF is a zinc finger protein associated with transcription regulation that also acts as a barrier factor for topologically associated domains (TADs) generated by cohesin via loop extrusion. These processes require different properties of CTCF-DNA interaction, and it is still unclear how CTCF's structural features may modulate its diverse roles. Here, we employ single-molecule imaging to study both full-length CTCF and truncation mutants. We show that CTCF enriches at CTCF binding sites (CBSs), displaying a longer lifetime than observed previously. We demonstrate that the zinc finger domains mediate CTCF clustering and that clustering enables RNA recruitment, possibly creating a scaffold for interaction with RNA-binding proteins like cohesin's subunit SA. We further reveal a direct recruitment and an increase of SA residence time by CTCF bound at CBSs, suggesting that CTCF-SA interactions are crucial for cohesin stability on chromatin at TAD borders. Furthermore, we establish a single-molecule T7 transcription assay and show that although a transcribing polymerase can remove CTCF from CBSs, transcription is impaired. Our study shows that context-dependent nucleic acid binding determines the multifaceted CTCF roles in genome organization and transcription regulation.

Inter3D: Capture of TAD Reorganization Endows Variant Patterns of Gene Transcription

Abstract Topologically associating domain (TAD) reorganization commonly occurs in the cell nucleus and contributes to gene activation and inhibition through the separation or fusion of adjacent TADs. However, identification of functional genes impacted by TAD alteration and the mechanism of TAD reorganization underlying gene transcription remain to be fully elucidated. Here, we first developed a novel approach termed Inter3D to specifically identify genes regulated by TAD reorganization. Our study revealed that the segregation of TADs led to the disruption of intrachromosomal looping at the myosin light chain 12B (MYL12B) locus, via the meticulous reorganization of TADs mediating epigenomic landscapes within tumor cells, thereby exhibiting a significant correlation with the downregulation of its transcriptional activity. Conversely, the fusion of TADs facilitated intrachromosomal interactions, suggesting a potential association with the activation of cytochrome P450 family 27 subfamily B member 1 (CYP27B1). Our study provides comprehensive insight into the capture of TAD rearrangement-mediated gene loci and moves toward understanding the functional role of TAD reorganization in gene transcription. The Inter3D pipeline used in this study is freely available at https://github.com/bm2-lab/inter3D.

The probability of chromatin to be at the nuclear lamina has no systematic effect on its transcription level in fruit flies

BackgroundMultiple studies have demonstrated a negative correlation between gene expression and positioning of genes at the nuclear envelope (NE) lined by nuclear lamina, but the exact relationship remains unclear, especially in light of the highly stochastic, transient nature of the gene association with the NE.ResultsIn this paper, we ask whether there is a causal, systematic, genome-wide relationship between the expression levels of the groups of genes in topologically associating domains (TADs) of Drosophila nuclei and the probabilities of TADs to be found at the NE. To investigate the nature of this possible relationship, we combine a coarse-grained dynamic model of the entire Drosophila nucleus with genome-wide gene expression data; we analyze the TAD averaged transcription levels of genes against the probabilities of individual TADs to be in contact with the NE in the control and lamins-depleted nuclei. Our findings demonstrate that, within the statistical error margin, the stochastic positioning of Drosophila melanogaster TADs at the NE does not, by itself, systematically affect the mean level of gene expression in these TADs, while the expected negative correlation is confirmed. The correlation is weak and disappears completely for TADs not containing lamina-associated domains (LADs) or TADs containing LADs, considered separately. Verifiable hypotheses regarding the underlying mechanism for the presence of the correlation without causality are discussed. These include the possibility that the epigenetic marks and affinity to the NE of a TAD are determined by various non-mutually exclusive mechanisms and remain relatively stable during interphase.ConclusionsAt the level of TADs, the probability of chromatin being in contact with the nuclear envelope has no systematic, causal effect on the transcription level in Drosophila. The conclusion is reached by combining model-derived time-evolution of TAD locations within the nucleus with their experimental gene expression levels.

The Genetic Pathophysiology and Clinical Management of the TADopathy, X-Linked Acrogigantism.

Pituitary gigantism is a rare manifestation of chronic growth hormone (GH) excess that begins before closure of the growth plates. Nearly half of pituitary gigantism patients have an identifiable genetic cause. X-linked acrogigantism (X-LAG; 10% of pituitary gigantism) typically begins during infancy and can lead to the tallest individuals described. In the 10 years since its discovery, about 40 patients have been identified. Patients with X-LAG usually develop mixed GH and prolactin macroadenomas with occasional hyperplasia that secrete copious amounts of GH, and frequently prolactin. Circulating GH releasing hormone (GHRH) is also elevated in a proportion of patients. X-LAG is caused by constitutive or sporadic mosaic duplications at chromosome Xq26.3 that disrupt the normal chromatin architecture of a topologically associating domain (TAD) around the orphan G protein coupled receptor (GPCR), GPR101. This leads to the formation of a neoTAD in which GPR101 over-expression is driven by ectopic enhancers ("TADopathy"). X-LAG has been seen in three families due to transmission of the duplication from affected mothers to sons. GPR101 is a constitutively active receptor with an unknown natural ligand that signals via multiple G proteins and protein kinases A and C to promote GH/prolactin hypersecretion. Treatment of X-LAG is challenging due to the young patient population and resistance to somatostatin analogs; the GH receptor antagonist pegvisomant is often an effective option. GH, insulin-like growth factor 1 (IGF-1) and prolactin hypersecretion and physical overgrowth can be controlled before definitive adult gigantism occurs, often at the cost of permanent hypopituitarism.

Abstract PO3-24-04: Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer

Abstract Title Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer Background Secondary trastuzumab resistance seriously affects the treatment of HER2-positive breast cancer. Although studies have demonstrated several key cellular properties that are closely accompanied with trastuzumab resistance formation, we still have poor knowledge on the genetic and epigenetic variations that promote such transformation. Here we suggested that the epigenetic modification changes on histone H3 together with altered chromatin architecture promote lipid metabolism reprogramming during secondary trastuzumab formation. Materials and methods Induced secondary trastuzumab-resistant SKBR3_HR cell line together with the original trastuzumab-sensitive SKBR3 cell line were applied in this study. Total RNA was collected for transcriptome analysis. Anti-H3K4me3 and K27me3 antibodies were chosen for CUT&Tag sequencing library preparation. Total genome DNA was prepared for Micro-C sequencing library preparation. Activity score of metabolism pathway was calculated as relative gene expression value averaged over all genes in this pathway in certain cell types. Results Upregulation of arachidonic acid metabolism and downregulation of unsaturated fatty acid synthesis, which are mainly characterized by the activation of PTGS1 and PTGES genes and the repression of FASN and SCD genes, respectively, were observed during the formation of secondary trastuzumab resistance from SKBR3 cell to SKBR3_HR cells. Variations of H3k4me3 instead of H3k27me3 regulate the expression of these 4 genes. The accumulation of H3k4me3 was detected at the promoters of PTGS1 and PTGES genes while they were removed from the promoters of FASN and SCD genes in SKBR3_HR cells. More intra-chromosomal interactions were constituted during resistance formation. In detail, 4626 and 4394 topological associated domains, 3125 and 5824 DNA loops were founded in SKBR3 and SKBR3_HR cells, respectively. Furthermore, the lost and gained DNA loops between SCD and PTGS1 genes and distant genome regions may indicate the weaken and strengthen interactions with transcriptional regulators located there. Conclusions During trastuzumab resistance formation, altered histone modifications as well as higher genome structure could regulate the expression of key genes in lipid metabolism pathways, which may further affect cell properties and interactions with cells in tumor microenvironment. Citation Format: Ningjun Duan, Yijia Hua, Yongmei Yin. Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-04.

A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification

Cytokine expression during Tcell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both invitro and invivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

TAD boundary deletion causes PITX2-related cardiac electrical and structural defects

While 3D chromatin organization in topologically associating domains (TADs) and loops mediating regulatory element-promoter interactions is crucial for tissue-specific gene regulation, the extent of their involvement in human Mendelian disease is largely unknown. Here, we identify 7 families presenting a new cardiac entity associated with a heterozygous deletion of 2 CTCF binding sites on 4q25, inducing TAD fusion and chromatin conformation remodeling. The CTCF binding sites are located in a gene desert at 1 Mb from the Paired-like homeodomain transcription factor 2 gene (PITX2). By introducing the ortholog of the human deletion in the mouse genome, we recapitulate the patient phenotype and characterize an opposite dysregulation of PITX2 expression in the sinoatrial node (ectopic activation) and ventricle (reduction), respectively. Chromatin conformation assay performed in human induced pluripotent stem cell-derived cardiomyocytes harboring the minimal deletion identified in family#1 reveals a conformation remodeling and fusion of TADs. We conclude that TAD remodeling mediated by deletion of CTCF binding sites causes a new autosomal dominant Mendelian cardiac disorder.

Analysis of long-range chromatin contacts, compartments and looping between mouse embryonic stem cells, lens epithelium and lens fibers

BackgroundNuclear organization of interphase chromosomes involves individual chromosome territories, “open” and “closed” chromatin compartments, topologically associated domains (TADs) and chromatin loops. The DNA- and RNA-binding transcription factor CTCF together with the cohesin complex serve as major organizers of chromatin architecture. Cellular differentiation is driven by temporally and spatially coordinated gene expression that requires chromatin changes of individual loci of various complexities. Lens differentiation represents an advantageous system to probe transcriptional mechanisms underlying tissue-specific gene expression including high transcriptional outputs of individual crystallin genes until the mature lens fiber cells degrade their nuclei.ResultsChromatin organization between mouse embryonic stem (ES) cells, newborn (P0.5) lens epithelium and fiber cells were analyzed using Hi-C. Localization of CTCF in both lens chromatins was determined by ChIP-seq and compared with ES cells. Quantitative analyses show major differences between number and size of TADs and chromatin loop size between these three cell types. In depth analyses show similarities between lens samples exemplified by overlaps between compartments A and B. Lens epithelium-specific CTCF peaks are found in mostly methylated genomic regions while lens fiber-specific and shared peaks occur mostly within unmethylated DNA regions. Major differences in TADs and loops are illustrated at the ~ 500 kb Pax6 locus, encoding the critical lens regulatory transcription factor and within a larger ~ 15 Mb WAGR locus, containing Pax6 and other loci linked to human congenital diseases. Lens and ES cell Hi-C data (TADs and loops) together with ATAC-seq, CTCF, H3K27ac, H3K27me3 and ENCODE cis-regulatory sites are shown in detail for the Pax6, Sox1 and Hif1a loci, multiple crystallin genes and other important loci required for lens morphogenesis. The majority of crystallin loci are marked by unexpectedly high CTCF-binding across their transcribed regions.ConclusionsOur study has generated the first data on 3-dimensional (3D) nuclear organization in lens epithelium and lens fibers and directly compared these data with ES cells. These findings generate novel insights into lens-specific transcriptional gene control, open new research avenues to study transcriptional condensates in lens fiber cells, and enable studies of non-coding genetic variants linked to cataract and other lens and ocular abnormalities.

A method for chromatin domain partitioning based on hypergraph clustering

For many years, multi-scale models of chromatin domains, such as A/B compartments, sub-compartments, topologically associated domains (TADs), sub-TADs, and loops have been popular. However, existing methods can only identify structures at a single scale and cannot partition multi-scale structures. In this paper, we proposed a method (TORNADOES) for chromatin domain partitioning based on hypergraph clustering. First, we use a density clustering algorithm to identify TADs at different scales based on Hi-C data with different resolutions. Then, by combining ChIP-seq data features and TAD results at different scales, we generate a hypergraph based on these TADs. Finally, we partition the chromatin domain structure at different scales, including A/B, A1, A2, B1, B2, and B3 based on the Laplacian matrix feature of the hypergraph. Similarity comparison experiments and ChIP-seq signal enrichment analysis are performed on the A/B region and sub-TAD levels, respectively, demonstrating that our method can identify chromatin domains with distinct features and provide a deeper understanding of the organizational patterns and functional differences in TADs at the genomic hierarchical structure. Comparative analysis of multiple cell line data shows that TORNADOES can better classify different numbers and types of compartments by changing the factors ChIP-seq data and clustering number used to characterize TAD compared to other methods. Source code for the TORNADOES method can be found at https://github.com/ghaiyan/TORNADOES.

The long non-coding RNA Meg3 mediates imprinted gene expression during stem cell differentiation.

The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.

Abstract LB011: Landscape of promoter centric chromatin interactions and co-regulated gene network provide insights for transcriptional regulation in prostate cancer

Abstract Eukaryotic genomes are organized into hierarchical three-dimensional (3D) structure inside the nucleus, forming topological associating domains (TADs) and providing insulated neighborhoods for the highly interacting genome regions. These regions are established by potential chromatin bridging proteins, such as the cohesin complex and CTCF, forming loops. Within the framework of these TADs lie the loops defined by the transcriptional machinery, or RNA polymerase II (RNAPol II). Changes to this 3D genome structure are associated with the development of human diseases, including cancers. Prostate cancer (PCa), associated with transcriptional dysregulation, serves as an optimal model to understand the molecular mechanism behind the formation of dynamic loops between distant regulatory elements and their target genes in 3D space to regulate gene expression and cancer progression. This study integrates two protein-centric ChIA-PET data (RNAPol II and cohesin) with multiple ChIP-Seq data (CTCF, H3K27ac, AR and FOXA1), and RNA-Seq data of PCa cell lines (LNCaP, VCaP, DU145, and RWPE1) to perform promoter (P) centered analysis. Firstly, we investigated the proportions of RNAPol II loops with co-occupancy of cohesin peaks and vice versa to quantify and understand the involvement of these proteins in transcription regulation and validate them using simulations. The results supported the hypothesis that most RNAPol II loops were significantly enriched with cohesin peaks. However, the reverse was not true since inner RNAPol II loops enriched with cohesin regulate transcription, while most of the cohesin loops form long-range interactions, TADs. Further, we compared the correlation between the promoter centric RNAPol II-associated and cohesin-mediated interactions with gene expression. In the case of RNA Pol II mediated interactions, we found a quantitative increase in the strength of interaction between loci, this trend was not observed in cohesin mediated interactions, which were relatively stable. The only exception was in the cell line VCaP, possibly due to its genetic complexity, as the strength of interactions associated with Cohesin as well as RNA Pol II increased. We next examined if the strength of interactions between cohesin loops remains unaffected in the VCaP cell line when treated with dBET inhibitor. However, there was no significant difference observed. This explained that RNAPol II loops are more dynamic, whereas cohesin loops are more stable. However, whether the cohesin loops (internal and TAD boundary) stay stable in the active and inactive regions of the genome remains unexplored. Next, the study inspects how the cluster of regulated genes is organized, preferentially forming multi-gene complexes in PCa for efficient and potential cooperative transcription. We expect a significant role of super-enhancers in identifying interactions between key oncogenes in PCa, owing to cell-type specific biology. Highly co-regulated single- and multi-valent loops will be validated in the patient cohorts to establish significant correlations. Overall, this study enhances the knowledge of promoter-centric molecular mechanisms involved in multi-gene complex formation and coregulated transcription in PCa cell lines. Citation Format: Khyati Raghunath Chandratre, Susmita Ramanad, Ahmed abbas Elmahdi, Yunpeng Gao, Ram Shankar Mani, Michael Zhang. Landscape of promoter centric chromatin interactions and co-regulated gene network provide insights for transcriptional regulation in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB011.

Abstract LB287: VariLink: A rapid, high-resolution proximity ligation method for the detection of structural variants and chromatin topology features in cancer

Abstract Despite numerous technological advances, including the widespread adoption of next-generation sequencing, many clinically relevant cancer driver mutations go undetected. The reason for this gap in understanding is two-fold: 1) Structural variants (SVs) are particularly challenging to detect using shotgun or long read-based approaches, particularly in heterogenous samples; and 2) It is increasingly accepted that epigenetic dysregulation may be a driver of disease state through mechanisms such as enhancer hijacking or changes to 3D genome organization. To address this gap in understanding, we developed VariLink: a dual-purpose proximity ligation library protocol that yields high-quality, high-resolution, unbiased Hi-C libraries capable of sensitive detection of structural variants and chromatin topological features from a single data set. Importantly, the VariLink protocol can be performed in under 8 hours—less than half of the time required for conventional Hi-C protocols. In the current study, we demonstrate the performance of VariLink for the detection of variants within the primary sequence (such as SVs, SNVs/Indels, and CNVs) in addition to high-resolution reporting of 3D chromatin conformation. We performed benchmarking analyses for both genetic variant detection and chromatin topology in two well-characterized lymphoblast cell lines (GM12878 and K562). We demonstrate the capability of VariLink data to detect interchromosomal translocations with 10-fold higher sensitivity over shotgun. Furthermore, using hybridization capture to detect the BCR-ABL1 fusion in K562 cells, we show that fewer than 100k read pairs is sufficient to detect this fusion with high confidence in VariLink libraries. We find that the shotgun-like, uniform coverage of VariLink data also enables detection of SNVs/Indels with improved sensitivity and precision over conventional Hi-C methods. Importantly, we demonstrate that VariLink libraries maintain topological features consistent with biologically relevant topologically-associated domains (TADs) and chromatin loops, providing insight into novel epigenetic cancer drivers. VariLink enables higher-resolution assessments of chromatin topology over conventional multi-RE-based Hi-C approaches, as demonstrated by 50% more TADs and loops called at 5-kb resolution. Taken together, our data demonstrate that VariLink is a next-generation Hi-C method that enables sensitive detection of clinically relevant structural variants and high-resolution chromatin topology in a single, rapid workflow. Citation Format: Jonathan Torchia, Mital Bhakta, Cory Padilla, Meredith L. Carpenter, Philip Uren, Lisa Munding. VariLink: A rapid, high-resolution proximity ligation method for the detection of structural variants and chromatin topology features in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB287.

H3.1/3.2 regulate the initial progression of the gene expression program.

In mice, transcription from the zygotic genome is initiated at the mid-one-cell stage, and occurs promiscuously in many areas of the genome, including intergenic regions. Regulated transcription from selected genes is established during the two-cell stage. This dramatic change in the gene expression pattern marks the initiation of the gene expression program and is essential for early development. We investigated the involvement of the histone variants H3.1/3.2 in the regulation of changes in gene expression pattern during the two-cell stage. Immunocytochemistry analysis showed low nuclear deposition of H3.1/3.2 in the one-cell stage, followed by a rapid increase in the late two-cell stage. Where chromatin structure is normally closed between the one- and two-cell stages, it remained open until the late two-cell stage when H3.1/3.2 were knocked down by small interfering RNA. Hi-C analysis showed that the formation of the topologicallyassociating domain was disrupted in H3.1/3.2 knockdown (KD) embryos. Promiscuous transcription was also maintained in the late two-cell stage in H3.1/3.2 KD embryos. These results demonstrate that H3.1/3.2 are involved in the initial process of the gene expression program after fertilization, through the formation of a closed chromatin structure to execute regulated gene expression during the two-cell stage.

LATS1 controls CTCF chromatin occupancy and hormonal response of 3D-grown breast cancer cells

The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

The effect of trisomic chromosomes on spatial genome organization and global transcription in embryonic stem cells.

Aneuploidy frequently occurs in cancer and developmental diseases such as Down syndrome, with its functional consequences implicated in dosage effects on gene expression and global perturbation of stress response and cell proliferation pathways. However, how aneuploidy affects spatial genome organization remains less understood. In this study, we addressed this question by utilizing the previously established isogenic wild-type (WT) and trisomic mouse embryonic stem cells (mESCs). We employed a combination of Hi-C, RNA-seq, chromosome painting and nascent RNA imaging technologies to compare the spatial genome structures and gene transcription among these cells. We found that trisomy has little effect on spatial genome organization at the level of A/B compartment or topologically associating domain (TAD). Inter-chromosomal interactions are associated with chromosome regions with high gene density, active histone modifications and high transcription levels, which are confirmed by imaging. Imaging also revealed contracted chromosome volume and weakened transcriptional activity for trisomic chromosomes, suggesting potential implications for the transcriptional output of these chromosomes. Our data resources and findings may contribute to a better understanding of the consequences of aneuploidy from the angle of spatial genome organization.

DiffGR: Detecting Differentially Interacting Genomic Regions from Hi-C Contact Maps

Abstract Recent advances in high-throughput chromosome conformation capture (Hi-C) techniques have allowed us to map genome-wide chromatin interactions and uncover higher-order chromatin structures, thereby shedding light on the principles of genome architecture and functions. However, statistical methods for detecting changes in large-scale chromatin organization such as topologically associating domains (TADs) are still lacking. We proposed a new statistical method, DiffGR, for detecting differentially interacting genomic regions at the TAD level between Hi-C contact maps. We utilized the stratum-adjusted correlation coefficient to measure similarity of local TAD regions. We then developed a nonparametric approach to identify statistically significant changes of genomic interacting regions. Through simulation studies, we demonstrated that DiffGR can robustly and effectively discover differential genomic regions under various conditions. Furthermore, we successfully revealed cell type-specific changes in genomic interacting regions in both human and mouse Hi-C datasets, and illustrated that DiffGR yielded consistent and advantageous results compared with state-of-the-art differential TAD detection methods. The DiffGR R package is published under the GNU General Public License (GPL) ≥ 2 license and is publicly available at https://github.com/wmalab/DiffGR.

Abstract 1648: RB represses cohesin-dependent loop formation and activates E2F-independent transcription

Abstract The retinoblastoma protein (RB) suppresses the activity of the E2F transcription factor family, controlling cellular proliferation. Recent studies, however, indicate that RB's role in chromatin organization, which is not yet fully understood, might be distinct from this E2F-dependent regulation. Employing chromosome conformation capture at a single nucleosome resolution, we identified RB as a cell cycle-regulated repressor of cohesin-dependent loop formation at topologically associating domain (TAD) boundaries. RB depletion increased the number and size of cohesin-dependent loops and strengthened topologically associating domains (TADs). This phenomenon was specific to the G1 phase and was not observed in the S phase, indicating that this novel RB function is regulated by the cell cycle. Mechanistically, RB showed extensive colocalization with cohesin in the human genome, and it impacted cohesin’s distribution on the chromatin. Active RB reduced cohesin from RB-bound TAD boundaries and decreased cohesin activity therein, as assessed by the increased K105/106 acetylation of the cohesin subunit SMC3. This led to reduced insulation in chromosome conformation capture assays. Importantly, by weakening the insulation activity of the adjacent insulators, RB non-canonically enhanced the expression of non-E2F target genes salient for cell adhesion and extracellular matrix organization. When RB was lost, cells showed a more rapid cellular detachment rate and an elevated migration rate, indicating that this novel RB function controls specific transcriptional programs rather than arbitrary genes to regulate the process of cell adhesion and migration. Overall, we conclude that RB has a central role in the interplay between cell cycle and chromatin organization, by repressing the cohesin-dependent loop formation at TAD boundaries. This RB function safeguards E2F-independent transcriptional programs driven by active enhancers and helps maintain cellular adhesion to the extracellular matrix. Citation Format: Hanjun Lee, Ioanna-Maria Gkotinakou, Badri Krishnan, Nicholas J. Dyson, Michael S. Lawrence, Ioannis Sanidas. RB represses cohesin-dependent loop formation and activates E2F-independent transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1648.

Abstract 7355: Integration analysis of lncRNAs and R-loops in 3D genome organization and gene regulation in the cancer genome

Abstract Existing evidence shows that some long non-coding RNAs (lncRNAs) can recruit chromatin regulators and form R-loops to mediate CCTCC-binding factor (CTCF) binding at topologically associated domain (TAD) boundaries to activate transcription of target genes. However, it remains unclear to what extent such lncRNA-dependent chromatin organization events occur genome-wide in normal and cancer cells. By curated public data of lncRNA, CTCF, RAD21 profiles and integrative analysis of multi-omics data using innovative computational approaches, we investigated global associations of lncRNAs and R-loops with activities of transcription factors and chromatin regulators in gene expression regulations across multiple human cell types. We developed a comprehensive computational pipeline for cross-cell-type analysis of lncRNA and protein-coding gene expression profiles and transcription factor binding profiles. We identified a total of 542 candidate genomic regions where lncRNAs play a pivotal role in regulating target genes by facilitating CTCF/cohesin binding. R-loops are enriched at CTCF/cohesin binding sites, suggesting their potential contribution to CTCF binding. Additionally, our findings underscore the significant cell-type specificity of CTCF binding patterns across the genome. This work adds knowledge to cancer genome organization and gene regulation, and builds foundations for further functional and mechanistic studies of lncRNAs and R-loops in the human genome. Citation Format: Shengyuan Wang, Zhenjia Wang, Yuh-Hwa Wang, Hui Li, Peter Todd Stukenburg, Chongzhi Zang. Integration analysis of lncRNAs and R-loops in 3D genome organization and gene regulation in the cancer genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7355.

Abstract 464: Chromatin associated long non-coding RNAs involved in dormancy for late recurrence of ER-positive breast cancer

Abstract Late recurrence is one of the major problems for hormone receptor-positive breast cancer patients. It is suggested that the risk of recurrence may be maintained by cancer cells that have been already disseminated in the body at surgery. They enter long-term dormancy, thus evade immune surveillance and become resistant to therapies. The detailed molecular mechanism for this process is largely unknown.Nuclear long noncoding RNAs, which are not translated to proteins, often function as epigenetic regulators and determine the phenotype of cells and individuals. We found that a cluster of long noncoding RNAs, ELEANORS, is specifically expressed in ER (estrogen receptor)-positive recurrent breast cancer cells, forms a molecular condensate (RNA cloud) in the nucleus, and activates the ESR1 gene that encodes ER. ELEANORS define a large chromatin domain, the 0.7 Mb topologically associated domain (TAD), which contains the ESR1 and three other breast-cancer-associated genes. ELEANORS also regulates the three-dimensional genome architecture. They mediate a long-range chromatin interaction of ESR1 and FOXO3 genes, which are 42.9 Mb apart, and contribute to their cooperatively activate transcription, equilibrating cell proliferation (ESR1) and apoptosis (FOXO3). Upon ELEANOR knockdown, the ELEANOR clouds are disappeared, and ESR1 gene transcription is repressed. Further, the two genes are dissociated and only the FOXO3 gene remains transcriptionally active, resulting in the induction of apoptosis.Further analysis of clinical specimens revealed that ELEANOR expression in primary tumors correlates with recurrences over 5 years after surgeries, suggesting that ELEANORs are involved in late recurrence. We also found that ELEANOR activates transcription of CD44, a breast cancer stem cell marker gene. Cancer stem cell maintenance and equilibration of proliferation and apoptosis by ELEANOR may be involved in long-term tumor dormancy in late recurrence. This study suggests the possibility of ELEANORS as a biomarker to predict late recurrence, as well as a therapeutic target for ER-positive breast cancer. (References)Abdalla, MOA. et al., Nat. Commun, 10, 3778 (2019) Tomita, S. et al., Nat. Commun, 6, 6966 (2015) Citation Format: Noriko Saitoh, Maierdan Palihati, Hiroaki Tachiwana. Chromatin associated long non-coding RNAs involved in dormancy for late recurrence of ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 464.

Abstract 2860: High-resolution mapping of oncogenic structural changes in osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify cancer-specific oncogenic alterations for treatment of osteosarcomas. Osteosarcoma is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. The functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments to affect gene expression. SVs can also lead to the formation of extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers yet they remain poorly understood. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), we studied SVs in osteosarcoma with the goal of elucidating their contribution to cancer development. We used a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies as well as genomically via whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in osteosarcoma, we integrated OGM and WGS data for all 11 cell lines to describe detailed genome-wide SVs including ecDNA formation. PDX lines assayed using OGM exhibited between 106 and 704 translocations each (versus zero detected in germline controls) and we identified dozens of potential ecDNA amplicons, including one amplicon which may explain exceptionally high expression of YAP1 and BIRC3 transcripts. Further, we used HiC to describe the topological environment resulting from these SVs and observed potential enhancer hijacking events affecting dozens of known oncogenes including MYC and CDK4. HiC interaction maps among six cell lines reveal genomic compartments and TAD boundaries in detail, with 4,167 unique TAD boundaries identified among six cell lines using 50kb genomic bins. Despite the observed complexity of these genomes, we note that 71% of TAD boundaries are consistent in at least four of the six HiC assayed cell lines, and 44% are present in all six lines. Changing TAD boundaries are associated with numerous SVs and/or copy number discrepancies, and are associated with genomic regions that contain genes with dysregulated RNA transcript levels. Overall, OGM and HiC modalities add considerably to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew Scott Clugston, Stan Leung, Eunice Fuentes, Leanne Sayles, Megan Ostrowski, Coco Wu, Christina Curtis, Zhicheng Ma, Yanding Zhao, Georgi K. Marinov, Vijay Ramani, Marcus Breese, Alejandro Sweet-Cordero. High-resolution mapping of oncogenic structural changes in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2860.