Higher-order Chromatin Structure Research Articles

Exploring the Molecular Impact of Cancer-associated Histone Mutations on Nucleosome Stability, Interactions and Higher-Order Structures

Nucleosomes, as key players in epigenetic regulation, interact with various chromatin factors and enzymes. Recently, a variety of studies have highlighted the significant roles that histone mutations play in the progression of various cancers. Cancer-associated mutations can disrupt nucleosome stability, dynamics and interactions, leading to profound effects on higher-order chromatin structure and epigenetic processes. In this review, we summarize the latest research that provides a comprehensive analysis of how histone mutations in cancers influence nucleosome dynamics, stability and interactions by altering their physicochemical properties. Moreover, we briefly explore the molecular 20 mechanisms 20 by which these mutations disrupt higher-order chromatin organization and epigenetic landscape.

Epigenomic landscapes during prefrontal cortex development and aging in rhesus.

The prefrontal cortex (PFC) is essential for higher-level cognitive functions. How epigenetic dynamics participates in PFC development and aging is largely unknown. Here, we profiled epigenomic landscapes of rhesus monkey PFCs from prenatal to aging stages. The dynamics of chromatin states, including higher-order chromatin structure, chromatin interaction and histone modifications are coordinated to regulate stage-specific gene transcription, participating in distinct processes of neurodevelopment. Dramatic changes of epigenetic signals occur around the birth stage. Notably, genes involved in neuronal cell differentiation and layer specification are pre-configured by bivalent promoters. We identified a cis-regulatory module and the transcription factors (TFs) associated with basal radial glia development, which was associated with large brain size in primates. These TFs include GLI3, CREB5 and SOX9. Interestingly, the genes associated with the basal radial glia (bRG)-associated cis-element module, such as SRY and SOX9, are enriched in sex differentiation. Schizophrenia-associated single nucleotide polymorphisms are more enriched in super enhancers (SEs) than typical enhancers, suggesting that SEs play an important role in neural network wiring. A cis-regulatory element of DBN1 is identified, which is critical for neuronal cell proliferation and synaptic neuron differentiation. Notably, the loss of distal chromatin interaction and H3K27me3 signal are hallmarks of PFC aging, which are associated with abnormal expression of aging-related genes and transposon activation, respectively. Collectively, our findings shed light on epigenetic mechanisms underlying primate brain development and aging.

Research progress on the effect of sperm chromatin integrity on function and its detection methods.

Sperm chromatin not only carries genetic information such as paternal DNA, but also carries structural proteins, epigenetic information, and higher-order chromatin structures (such as matrix attachment regions and telomeres), etc. These information play an important role in embryonic development. This article mainly reviews the effects of these different information carried by sperm chromatin on sperm function and embryonic development and the research progress of related detection methods, in order to provide a theoretical basis and scientific diagnosis and treatment strategies for the etiology screening of clinical infertility, embryo arrest and recurrent miscarriage, so as to improve the pregnancy outcomes of natural conception and assisted reproduction. Keywords: sperm chromatin; epigenetics; sperm DNA damage; sperm function; higher-order chromatin structures.

METTL3-mediated chromatin contacts promote stress granule phase separation through metabolic reprogramming during senescence

METTL3 is the catalytic subunit of the methyltransferase complex, which mediates m6A modification to regulate gene expression. In addition, METTL3 regulates transcription in an enzymatic activity-independent manner by driving changes in high-order chromatin structure. However, how these functions of the methyltransferase complex are coordinated remains unknown. Here we show that the methyltransferase complex coordinates its enzymatic activity-dependent and independent functions to regulate cellular senescence, a state of stable cell growth arrest. Specifically, METTL3-mediated chromatin loops induce Hexokinase 2 expression through the three-dimensional chromatin organization during senescence. Elevated Hexokinase 2 expression subsequently promotes liquid-liquid phase separation, manifesting as stress granule phase separation, by driving metabolic reprogramming. This correlates with an impairment of translation of cell-cycle related mRNAs harboring polymethylated m6A sites. In summary, our results report a coordination of m6A-dependent and -independent function of the methyltransferase complex in regulating senescence through phase separation driven by metabolic reprogramming.

T Cells Spatially Regulate B Cell Receptor Signaling in Lymphomas through H3K9me3 Modifications.

Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is a subtype associated with poor survival outcomes. Despite identifying therapeutic targets through molecular characterization, targeted therapies have limited success. New strategies using immune-competent tissue models are needed to understand how DLBCL cells evade treatment. Here, synthetic hydrogel-based lymphoma organoids are used to demonstrate how signals in the lymphoid tumor microenvironment (Ly-TME) can alter B cell receptor (BCR) signaling and specific histone modifications, tri-methylation of histone 3 at lysine 9 (H3K9me3), dampening the effects of BCR pathway inhibition. Using imaging modalities, T cells increase DNA methyltransferase 3A expression and cytoskeleton formation in proximal ABC-DLBCL cells, regulated by H3K9me3. Expansion microscopy on lymphoma organoids reveals T cells increase the size and quantity of segregated H3K9me3 clusters in ABC-DLBCL cells. Findings suggest the re-organization of higher-order chromatin structures that may contribute to evasion or resistance to therapy via the emergence of novel transcriptional states. Treating ABC-DLBCL cells with a G9α histone methyltransferase inhibitor reverses T cell-mediated modulation of H3K9me3 and overcomes T cell-mediated attenuation of treatment response to BCR pathway inhibition. This study emphasizes the Ly-TME's role in altering DLBCL fate and suggests targeting aberrant signaling and microenvironmental cross-talk that can benefit high-risk patients.

Statistical modeling and significance estimation of multi-way chromatin contacts with HyperloopFinder.

Recent advances in chromatin conformation capture technologies, such as SPRITE and Pore-C, have enabled the detection of simultaneous contacts among multiple chromatin loci. This has made it possible to investigate the cooperative transcriptional regulation involving multiple genes and regulatory elements at the resolution of a single molecule. However, these technologies are unavoidably subject to the random polymer looping effect and technical biases, making it challenging to distinguish genuine regulatory relationships directly from random polymer interactions. Here, we present HyperloopFinder, a method for identifying regulatory multi-way chromatin contacts (hyperloops) by jointly modeling the random polymer looping effect and technical biases to estimate the statistical significance of multi-way contacts. The results show that our model can accurately estimate the expected interaction frequency of multi-way contacts based on the distance distribution of pairwise contacts, revealing that most multi-way contacts can be formed by randomly linking the pairwise contacts adjacent to each other. Moreover, we observed the spatial colocalization of the interaction sites of hyperloops from image-based data. Our results also revealed that hyperloops can function as scaffolds for the cooperation among multiple genes and regulatory elements. In summary, our work contributes novel insights into higher-order chromatin structures and functions and has the potential to enhance our understanding of transcriptional regulation and other cellular processes.

Light control ofthree-dimensional chromatin organization in soybean.

Higher-order chromatin structure is critical for regulation of gene expression. In plants, light profoundly affects the morphogenesis of emerging seedlings as well as global gene expression to ensure optimal adaptation to environmental conditions. However, the changes and functional significance of chromatin organization in response to light during seedling development are not well documented. We constructed Hi-C contact maps for the cotyledon, apical hook and hypocotyl of soybean subjected to dark and light conditions. The resulting high-resolution Hi-C contact maps identified chromosome territories, A/B compartments, A/B sub-compartments, TADs (Topologically Associated Domains) and chromatin loops in each organ. We observed increased chromatin compaction under light and we found that domains that switched from B sub-compartments in darkness to A sub-compartments under light contained genes that were activated during photomorphogenesis. At the local scale, we identified a group of TADs constructed by gene clusters consisting of different numbers of Small Auxin-Upregulated RNAs (SAURs), which exhibited strict co-expression in the hook and hypocotyl in response to light stimulation. In the hypocotyl, RNA polymerase II (RNAPII) regulated the transcription of a SAURs cluster under light via TAD condensation. Our results suggest that the 3D genome is involved in the regulation of light-related gene expression in a tissue-specific manner.

Individual transcription factors modulate both the micromovement of chromatin and its long-range structure

The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization.

Interaction with B-type lamin reveals the function of Drosophila Keap1 xenobiotic response factor in nuclear architecture.

The Keap1-Nrf2 pathway serves as a central regulator that mediates transcriptional responses to xenobiotic and oxidative stimuli. Recent studies have shown that Keap1 and Nrf2 can regulate transcripts beyond antioxidant and detoxifying genes, yet the underlying mechanisms remain unclear. Our research has uncovered that Drosophila Keap1 (dKeap1) and Nrf2 (CncC) proteins can control high-order chromatin structure, including heterochromatin. In this study, we identified the molecular interaction between dKeap1 and lamin Dm0, the Drosophila B-type lamin responsible for the architecture of nuclear lamina and chromatin. Ectopic expression of dKeap1 led to an ectopic localization of lamin to the intra-nuclear area, corelated with the spreading of the heterochromatin marker H3K9me2 into euchromatin regions. Additionally, mis-regulated dKeap1 disrupted the morphology of the nuclear lamina. Knocking down of dKeap1 partially rescued the lethality induced by lamin overexpression, suggesting their genetic interaction during development. The discovered dKeap1-lamin interaction suggests a novel role for the Keap1 oxidative/xenobiotic response factor in regulating chromatin architecture.

TEAD2 initiates ground-state pluripotency by mediating chromatin looping

The transition of mouse embryonic stem cells (ESCs) between serum/LIF and 2i(MEK and GSK3 kinase inhibitor)/LIF culture conditions serves as a valuable model for exploring the mechanisms underlying ground and confused pluripotent states. Regulatory networks comprising core and ancillary pluripotency factors drive the gene expression programs defining stable naïve pluripotency. In our study, we systematically screened factors essential for ESC pluripotency, identifying TEAD2 as an ancillary factor maintaining ground-state pluripotency in 2i/LIF ESCs and facilitating the transition from serum/LIF to 2i/LIF ESCs. TEAD2 exhibits increased binding to chromatin in 2i/LIF ESCs, targeting active chromatin regions to regulate the expression of 2i-specific genes. In addition, TEAD2 facilitates the expression of 2i-specific genes by mediating enhancer-promoter interactions during the serum/LIF to 2i/LIF transition. Notably, deletion of Tead2 results in reduction of a specific set of enhancer-promoter interactions without significantly affecting binding of chromatin architecture proteins, CCCTC-binding factor (CTCF), and Yin Yang 1 (YY1). In summary, our findings highlight a novel prominent role of TEAD2 in orchestrating higher-order chromatin structures of 2i-specific genes to sustain ground-state pluripotency.

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 6048: Targeting AR positive prostate cancer cells by the novel P300/CBP oral available PROTAC degrader

Abstract Background The Androgen Receptor (AR) serves as the core lineage-specific transcription factor for prostatic epithelial cells. Targeting AR is currently the most promising therapeutic strategy. However, the inevitable development of acquired resistance to anti-androgen treatments underscores the urgent need to devise new therapeutic strategies. Recent studies have revealed that in prostate cancer cells, AR is hijacked by oncogenic transcription factors, such as FOXA1 and HOXB13, which promiscuously bind to distinct enhancer sites, driving the progression of prostate cancer. This process leads to the formation of a substantially rewired AR cancer specific enhanceosome, requiring a variety of coregulators to exhibit oncogenic driver characteristics, which can be potential therapeutic targets for prostate cancer. Histone acetyltransferases (HATs) P300 and CBP are paralogs which play dominant roles in orchestrating high-order chromatin structures and transcription factor complexes by acetylating histone proteins at multiple residuals. Although accumulating evidence suggests that P300/CBP function as AR coregulator and targeting P300/CBP can markedly suppress AR-positive prostate cancer cell growth, including growth of castration-resistant prostate cancer (CRPC), most of the P300/CBP inhibitors have performed poorly in clinical trials. Methods We developed a novel P300/CBP dual oral available PROTAC degrader, namely CBPD-409. The on-target effects of CBPD-409 were assessed by quantitative proteomics (TMT-mass spectrometry) in prostate cancer cell lines. The RNA-seq analysis was conducted to investigate CBPD-409 effects on global transcriptome in prostate cancer cells. The cytotoxicity of CBPD-409 in multiple prostate cancer cell lines was examined by cell-titer-glo and incucyte assays. Conclusion In multiple cell lines, CBPD-409 exhibits remarked potency to degrade both P300 and CBP proteins in time and does dependent manner, without evident off-target effects. By employing CBPD-409, we demonstrate that degrading P300/CBP preferentially suppress AR+ prostate cancer cell growth. The global transcriptomic profiling in CBPD-409 treated prostate cancer cells revealed that loss of P300/CBP results in remarkedly downregulation of AR, Myc, FOXA1 and ERG signaling. Assessment of CBPD-409 cytotoxicity in a panel prostate cancer cell lines suggests that AR positive prostate cancer cells display highest sensitivity to CBPD-409. Citation Format: Jie luo, Abhijit Prolia, Yuanyuan Qiao, Jean Tian, Rahul Mannan, Somnath Mahapatra, Zhixiang Chen, Rithvik Seri, Shaomeng Wang, Arul Chinnaiyan. Targeting AR positive prostate cancer cells by the novel P300/CBP oral available PROTAC degrader [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 6048.

Epigenetic regulation in cancer.

Epigenetic modifications are defined as heritable changes in gene activity that do not involve changes in the underlying DNA sequence. The oncogenic process is driven by the accumulation of alterations that impact genome's structure and function. Genetic mutations, which directly disrupt the DNA sequence, are complemented by epigenetic modifications that modulate gene expression, thereby facilitating the acquisition of malignant characteristics. Principals among these epigenetic changes are shifts in DNA methylation and histone mark patterns, which promote tumor development and metastasis. Notably, the reversible nature of epigenetic alterations, as opposed to the permanence of genetic changes, positions the epigenetic machinery as a prime target in the discovery of novel therapeutics. Our review delves into the complexities of epigenetic regulation, exploring its profound effects on tumor initiation, metastatic behavior, metabolic pathways, and the tumor microenvironment. We place a particular emphasis on the dysregulation at each level of epigenetic modulation, including but not limited to, the aberrations in enzymes responsible for DNA methylation and histone modification, subunit loss or fusions in chromatin remodeling complexes, and the disturbances in higher-order chromatin structure. Finally, we also evaluate therapeutic approaches that leverage the growing understanding of chromatin dysregulation, offering new avenues for cancer treatment.

ScENCORE: leveraging single-cell epigenetic data to predict chromatin conformation using graph embedding.

Dynamic compartmentalization of eukaryotic DNA into active and repressed states enables diverse transcriptional programs to arise from a single genetic blueprint, whereas its dysregulation can be strongly linked to a broad spectrum of diseases. While single-cell Hi-C experiments allow for chromosome conformation profiling across many cells, they are still expensive and not widely available for most labs. Here, we propose an alternate approach, scENCORE, to computationally reconstruct chromatin compartments from the more affordable and widely accessible single-cell epigenetic data. First, scENCORE constructs a long-range epigenetic correlation graph to mimic chromatin interaction frequencies, where nodes and edges represent genome bins and their correlations. Then, it learns the node embeddings to cluster genome regions into A/B compartments and aligns different graphs to quantify chromatin conformation changes across conditions. Benchmarking using cell-type-matched Hi-C experiments demonstrates that scENCORE can robustly reconstruct A/B compartments in a cell-type-specific manner. Furthermore, our chromatin confirmation switching studies highlight substantial compartment-switching events that may introduce substantial regulatory and transcriptional changes in psychiatric disease. In summary, scENCORE allows accurate and cost-effective A/B compartment reconstruction to delineate higher-order chromatin structure heterogeneity in complex tissues.

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer–promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer–promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.

Super-Resolution Microscopy Unveils Synergistic Structural Changes of Organelles Upon Point Mutation.

Ethyl methanesulphonate (EMS), is a widely used chemical mutagen that causes high-frequency germline null mutation by inserting an alkyl group into the nucleotide guanine in eukaryotic cells. The effect of EMS on the dynamics of the aneuploid genome, increased cellular instability, and carcinogenicity in relation to benign and malignant tumors are reported, but the molecular level understanding of morphological changes of higher-order chromatin structure has poorly been understood. This is due to a lack of sufficient resolution in conventional microscopic techniques to see small structures below the diffraction limit. Here, using super-resolution radial fluctuation, a largely fragmented, decompaction, and less dense heterochromatin structure upon EMS treatment to HEK 293A cells without any change in nuclear DNA domains is observed. This result suggests an early stage of carcinogenicity happened due to the point mutation. In addition, the distinct structural changes with an elongated morphology of lysosomes are also observed. On the other hand, fragmented and increased heterogeneous populations with an increased cytoplasmic occupancy of mitochondria are observed.

The Molecular Impacts of Retrotransposons in Development and Diseases.

Retrotransposons are invasive genetic elements that constitute substantial portions of mammalian genomes. They have the potential to influence nearby gene expression through their cis-regulatory sequences, reverse transcription machinery, and the ability to mold higher-order chromatin structures. Due to their multifaceted functions, it is crucial for host fitness to maintain strict regulation of these parasitic sequences to ensure proper growth and development. This review explores how subsets of retrotransposons have undergone evolutionary exaptation to enhance the complexity of mammalian genomes. It also highlights the significance of regulating these elements, drawing on recent studies conducted in human and murine systems.

Allelic chromatin structure precedes imprinted expression of Kcnk9 during neurogenesis.

Differences in chromatin state inherited from the parental gametes influence the regulation of maternal and paternal alleles in offspring. This phenomenon, known as genomic imprinting, results in genes preferentially transcribed from one parental allele. While local epigenetic factors such as DNA methylation are known to be important for the establishment of imprinted gene expression, less is known about the mechanisms by which differentially methylated regions (DMRs) lead to differences in allelic expression across broad stretches of chromatin. Allele-specific higher-order chromatin structure has been observed at multiple imprinted loci, consistent with the observation of allelic binding of the chromatin-organizing factor CTCF at multiple DMRs. However, whether allelic chromatin structure impacts allelic gene expression is not known for most imprinted loci. Here we characterize the mechanisms underlying brain-specific imprinted expression of the Peg13-Kcnk9 locus, an imprinted region associated with intellectual disability. We performed region capture Hi-C on mouse brains from reciprocal hybrid crosses and found imprinted higher-order chromatin structure caused by the allelic binding of CTCF to the Peg13 DMR. Using an in vitro neuron differentiation system, we showed that imprinted chromatin structure precedes imprinted expression at the locus. Additionally, activation of a distal enhancer induced imprinted expression of Kcnk9 in an allelic chromatin structure-dependent manner. This work provides a high-resolution map of imprinted chromatin structure and demonstrates that chromatin state established in early development can promote imprinted expression upon differentiation.

A genetic screen identifies BEND3 as a regulator of bivalent gene expression and global DNA methylation.

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elementsand higher-order chromatin structures. Consequently, these chromatin marks are indispensable for mammalian development and alterations often lead to disease, such as cancer. Bivalent promoters are especially important during differentiation and development. Here we used a genetic screen to identify new regulators of a bivalent repressed gene. We identify BEND3 as a regulator of hundreds of bivalent promoters, some of which it represses, and some of which it activates. We show that BEND3 is recruited to a CpG-containg consensus site that is present in multiple copies in many bivalent promoters. Besides having direct effect on the promoters it binds, the loss of BEND3 leads to genome-wide gains of DNA methylation, which are especially marked at regions normally protected by the TET enzymes. DNA hydroxymethylation is reduced in Bend3 mutant cells, possibly as consequence of altered gene expression leading to diminished alpha-ketoglutarate production, thus lowering TET activity. Our results clarify the direct and indirect roles of an important chromatin regulator, BEND3, and, more broadly, they shed light on the regulation of bivalent promoters.

ScNanoHi-C uncovers single-cell high-order chromatin structures and gene regulation.

scNanoHi-C uncovers single-cell high-order chromatin structures and gene regulation.