Chromatin Decompaction Research Articles

A Molecular View into the Structure and Dynamics of Phase-Separated Chromatin.

The organization of chromatin is critical for gene expression, yet the underlying mechanisms responsible for this organization remain unclear. Recent work has suggested that phase separation might play an important role in chromatin organization, yet the molecular forces that drive chromatin phase separation are poorly understood. In this work we interrogate a molecular model of chromatin to quantify the driving forces and thermodynamics of chromatin phase separation. By leveraging a multiscale approach, our molecular model is able to reproduce chromatin's chemical and structural details at the level of a few nanometers, yet remain efficient enough to simulate chromatin phase separation across 100 nm length scales. We first demonstrate that our model can reproduce key experiments of phase separating nucleosomal arrays, and then apply our model to quantify the interactions that drive their formation into chromatin condensates with either liquid- or solid-like material properties. We next use our model to characterize the molecular structure within chromatin condensates and find that this structure is irregularly ordered and is inconsistent with existing 30 nm fiber models. Lastly we examine how post-translational modifications can modulate chromatin phase separation and how the acetylation of chromatin can lead to chromatin decompaction while still preserving phase separation. Taken together, our work provides a molecular view into the structure and dynamics of phase-separated chromatin and provides new insights into how phase separation might manifest in the nucleus of living cells.

Transient chromatin decompaction at the start of D. melanogaster male embryonic germline development.

Embryonic germ cells develop rapidly to establish the foundation for future developmental trajectories, and in this process, they make critical lineage choices including the configuration of their unique identity and a decision on sex. Here, we use single-cell genomics patterns for the entire embryonic germline in Drosophila melanogaster along with the somatic gonadal precursors after embryonic gonad coalescence to investigate molecular mechanisms involved in the setting up and regulation of the germline program. Profiling of the early germline chromatin landscape revealed sex- and stage-specific features. In the male germline immediately after zygotic activation, the chromatin structure underwent a brief remodeling phase during which nucleosome density was lower and deconcentrated from promoter regions. These findings echoed enrichment analysis results of our genomics data in which top candidates were factors with the ability to mediate large-scale chromatin reorganization. Together, they point to the importance of chromatin regulation in the early germline and raise the possibility of a conserved epigenetic reprogramming-like process required for proper initiation of germline development.

Aqueous Extract of Wolfberry Alleviates Aging-Related Skeletal Muscle Dysfunction by Modulating PRRs Signaling Pathways and Enhancing DNA Repair.

Aging can lead to a series of degenerative changes in skeletal muscle, which would negatively impact physical activity and the quality of life of the elderly. Wolfberry contains numerous bioactive substances. It's vital to further explore the mechanisms underlying its healthy effects on skeletal muscle function during aging progress. This study discusses the benefits and mechanisms of aqueous extract of wolfberry (AEW) to protect skeletal muscle from aging-related persistent DNA damage based on its anti-inflammatory activity. It is found that AEW improves muscle mass, strength, and endurance, modulates the expression of Atrogin-1, MyH, and MuRF-1, and decreases oxidative stress and inflammation levels in aging mice, which is consistent with the in vitro results. Mechanistically, AEW inhibits the pattern recognition receptors (PRRs) pathway induced by inflammatory gene activation, suggesting its potential in response to DNA damage. AEW is also observed to mitigate chromatin decompaction. Network pharmacology is conducted to analyze the potential targets of AEW in promoting DNA repair. In conclusion, the study shows the anti-aging effects of AEW on skeletal muscle by promoting DNA repair and reducing the transcriptional activity of inflammatory factors. AEW intake may become a potential strategy for strengthening skeletal muscle function in the elderly.

Super-resolution imaging of T lymphocyte activation reveals chromatin decondensation and disrupted nuclear envelope

T lymphocyte activation plays a pivotal role in adaptive immune response and alters the spatial organization of nuclear architecture that subsequently impacts transcription activities. Here, using stochastic optical reconstruction microscopy (STORM), we observe dramatic de-condensation of chromatin and the disruption of nuclear envelope at a nanoscale resolution upon T lymphocyte activation. Super-resolution imaging reveals that such alterations in nuclear architecture are accompanied by the release of nuclear DNA into the cytoplasm, correlating with the degree of chromatin decompaction within the nucleus. The authors show that under the influence of metabolism, T lymphocyte activation de-condenses chromatin, disrupts the nuclear envelope, and releases DNA into the cytoplasm. Taken together, this result provides a direct, molecular-scale insight into the alteration in nuclear architecture. It suggests the release of nuclear DNA into the cytoplasm as a general consequence of chromatin decompaction after lymphocyte activation.

Dissecting gene activation and chromatin remodeling dynamics in single human cells undergoing reprogramming

During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here, we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that, following heterokaryon formation, the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48h after cell fusion but, strikingly, exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent.

Reinforcement of repressive marks in the chicken primordial germ cell epigenetic signature: divergence from basal state resetting in mammals

BackgroundIn mammals, primordial germ cells (PGCs), the embryonic precursors of the germline, arise from embryonic or extra-embryonic cells upon induction by the surrounding tissues during gastrulation, according to mechanisms which are elucidated in mice but remain controversial in primates. They undergo genome-wide epigenetic reprogramming, consisting of extensive DNA demethylation and histone post-translational modification (PTM) changes, toward a basal, euchromatinized state. In contrast, chicken PGCs are specified by preformation before gastrulation based on maternally-inherited factors. They can be isolated from the bloodstream during their migration to the genital ridges. Our prior research highlighted differences in the global epigenetic profile of cultured chicken PGCs compared with chicken somatic cells and mammalian PGCs. This study investigates the acquisition and evolution of this profile during development.ResultsQuantitative analysis of global DNA methylation and histone PTMs, including their distribution, during key stages of chicken early development revealed divergent PGC epigenetic changes compared with mammals. Unlike mammalian PGCs, chicken PGCs do not undergo genome-wide DNA demethylation or exhibit a decrease in histone H3 lysine 9 dimethylation. However, chicken PGCs show 5‑hydroxymethylcytosine loss, macroH2A redistribution, and chromatin decompaction, mirroring mammalian processes. Chicken PGCs initiate their epigenetic signature during migration, progressively accumulating high global levels of H3K9me3, with preferential enrichment in inactive genome regions. Despite apparent global chromatin decompaction, abundant heterochromatin marks, including repressive histone PTMs, HP1 variants, and DNA methylation, persists in chicken PGCs, contrasting with mammalian PGCs.ConclusionsChicken PGCs’ epigenetic signature does not align with the basal chromatin state observed in mammals, suggesting a departure from extensive epigenetic reprogramming. Despite disparities in early PGC development, the persistence of several epigenetic features shared with mammals implies their involvement in chromatin-regulated germ cell properties, with the distinctive elevation of chicken-specific H3K9me3 potentially participating in these processes.

Reverse Mechanotransduction: Driving Chromatin Compaction to Decompaction Increases Cell Adhesion Strength and Contractility.

Mechanical extracellular signals elicit chromatin remodeling via the mechanotransduction pathway, thus determining cellular function. However, the reverse pathway is an open question: does chromatin remodeling shape cells, regulating their adhesion strength? With fluidic force microscopy, we can directly measure the adhesion strength of epithelial cells by driving chromatin compaction to decompaction with chromatin remodelers. We observe that chromatin compaction, induced by performing histone acetyltransferase inhibition or ATP depletion, leads to a reduction in nuclear volume, disrupting actin cytoskeleton and focal adhesion assembly, and ultimately decreases in cell adhesion strength and traction force. Conversely, when chromatin decompaction is drived by removing the remodelers, cells recover their original shape, adhesion strength, and traction force. During chromatin decompaction, cells use depolymerized proteins to restore focal adhesion assemblies rather than neo-synthesized cytoskeletal proteins. We conclude that chromatin remodeling shapes cells, regulating adhesion strength through a reverse mechanotransduction pathway from the nucleus to the cell surface involving RhoA activation.

Imaging analysis of six human histone H1 variants reveals universal enrichment of H1.2, H1.3, and H1.5 at the nuclear periphery and nucleolar H1X presence

Histone H1 participates in chromatin condensation and regulates nuclear processes. Human somatic cells may contain up to seven histone H1 variants, although their functional heterogeneity is not fully understood. Here, we have profiled the differential nuclear distribution of the somatic H1 repertoire in human cells through imaging techniques including super-resolution microscopy. H1 variants exhibit characteristic distribution patterns in both interphase and mitosis. H1.2, H1.3, and H1.5 are universally enriched at the nuclear periphery in all cell lines analyzed and co-localize with compacted DNA. H1.0 shows a less pronounced peripheral localization, with apparent variability among different cell lines. On the other hand, H1.4 and H1X are distributed throughout the nucleus, being H1X universally enriched in high-GC regions and abundant in the nucleoli. Interestingly, H1.4 and H1.0 show a more peripheral distribution in cell lines lacking H1.3 and H1.5. The differential distribution patterns of H1 suggest specific functionalities in organizing lamina-associated domains or nucleolar activity, which is further supported by a distinct response of H1X or phosphorylated H1.4 to the inhibition of ribosomal DNA transcription. Moreover, H1 variants depletion affects chromatin structure in a variant-specific manner. Concretely, H1.2 knock-down, either alone or combined, triggers a global chromatin decompaction. Overall, imaging has allowed us to distinguish H1 variants distribution beyond the segregation in two groups denoted by previous ChIP-Seq determinations. Our results support H1 variants heterogeneity and suggest that variant-specific functionality can be shared between different cell types.

Imaging analysis of six human histone H1 variants reveals universal enrichment of H1.2, H1.3, and H1.5 at the nuclear periphery and nucleolar H1X presence.

Histone H1 participates in chromatin condensation and regulates nuclear processes. Human somatic cells may contain up to seven histone H1 variants, although their functional heterogeneity is not fully understood. Here, we have profiled the differential nuclear distribution of the somatic H1 repertoire in human cells through imaging techniques including super-resolution microscopy. H1 variants exhibit characteristic distribution patterns in both interphase and mitosis. H1.2, H1.3, and H1.5 are universally enriched at the nuclear periphery in all cell lines analyzed and co-localize with compacted DNA. H1.0 shows a less pronounced peripheral localization, with apparent variability among different cell lines. On the other hand, H1.4 and H1X are distributed throughout the nucleus, being H1X universally enriched in high-GC regions and abundant in the nucleoli. Interestingly, H1.4 and H1.0 show a more peripheral distribution in cell lines lacking H1.3 and H1.5. The differential distribution patterns of H1 suggest specific functionalities in organizing lamina-associated domains or nucleolar activity, which is further supported by a distinct response of H1X or phosphorylated H1.4 to the inhibition of ribosomal DNA transcription. Moreover, H1 variants depletion affects chromatin structure in a variant-specific manner. Concretely, H1.2 knock-down, either alone or combined, triggers a global chromatin decompaction. Overall, imaging has allowed us to distinguish H1 variants distribution beyond the segregation in two groups denoted by previous ChIP-Seq determinations. Our results support H1 variants heterogeneity and suggest that variant-specific functionality can be shared between different cell types.

Actin contraction controls nuclear blebbing and rupture independent of actin confinement.

The nucleus is a mechanically stable compartment of the cell that contains the genome and performs many essential functions. Nuclear mechanical components chromatin and lamins maintain nuclear shape, compartmentalization, and function by resisting antagonistic actin contraction and confinement. Studies have yet to compare chromatin and lamins perturbations side-by-side as well as modulated actin contraction while holding confinement constant. To accomplish this, we used nuclear localization signal green fluorescent protein to measure nuclear shape and rupture in live cells with chromatin and lamin perturbations. We then modulated actin contraction while maintaining actin confinement measured by nuclear height. Wild type, chromatin decompaction, and lamin B1 null present bleb-based nuclear deformations and ruptures dependent on actin contraction and independent of actin confinement. Actin contraction inhibition by Y27632 decreased nuclear blebbing and ruptures while activation by CN03 increased rupture frequency. Lamin A/C null results in overall abnormal shape also reliant on actin contraction, but similar blebs and ruptures as wild type. Increased DNA damage is caused by nuclear blebbing or abnormal shape which can be relieved by inhibition of actin contraction which rescues nuclear shape and decreases DNA damage levels in all perturbations. Thus, actin contraction drives nuclear blebbing, bleb-based ruptures, and abnormal shape independent of changes in actin confinement.

NCAPD3 Prompts Diffuse Large B-Cell Lymphoma Progression through Modulating SIRT1 Expression in H3K9 Monomethylation-Dependent Manner

Chromosomal instability, including number and structure abnormality, is the critical molecular basis in lymphoid oncogenesis. Condensin, a family of structural maintenance of chromosome complexes, has been shown to regulate chromosome compaction and segregation during mitosis. NCAPD3, a HEAT-repeat subunit of condensin II, plays a dominant role in condensin-mediated chromosome dynamics, whose abnormalities are related to tumorigenesis and developmental disorders but remain unexplored in lymphoma. This study aims to unravel the expression, molecular function and mechanism of NCAPD3 in diffuse large B-cell lymphoma (DLBCL). We first elucidated the expression of NCAPD3 in DLBCL. Lymph node tissues from 70 DLBCL patients and 35 reactive hyperplasia cases were collected with informed consent approved by the Medical Ethical Committee of Shandong Provincial Hospital. Higher NCAPD3 expression was identified in DLBCL tissues and correlated with advanced Ann Arbor stage and higher IPI score. The survival analysis presented patients with higher NCAPD3 suffered shorter survival (P=0.045). Meanwhile, upregulation of NCAPD3 was validated in DLBCL cells compared with normal CD19+ B-cell. We further explored the biological functions of NCAPD3 in DLBCL and constructed NCAPD3-knockdown, knockout and overexpression models. Deficiency of NCAPD3 impeded cell proliferation, induced apoptosis and increased the sensitivity to doxorubicin and ibrutinib. Instead, NCAPD3 overexpression facilitated cell proliferation and therapeutic resistance. In vivo experiments further indicated that xenograft DLBCL mice with deficient NCAPD3 presented delayed tumor growth. These suggested NCAPD3 promoted DLBCL progression. Strikingly, we found knocking down NCAPD3 disturbed the mitosis, triggering the formation of aneuploidy, due to its function in chromatin structure and dynamics. Hence, we further portrayed the transformation of chromosome architecture under different NCAPD3 expression. Chromosome spreads exhibited chromosomes became slim and elongated upon NCAPD3 overexpression, instead, NCAPD3 knockout triggered short and highly-curled transformation with the global upregulation of H3K4me3, an indication of transcriptional activation and chromatin decompaction (Figure 1). This suggested NCAPD3 could modulate transcriptional activity. Therewith, we investigated how NCAPD3 regulated gene transcription. We examined the interacting proteins of NCAPD3 by mass spectrometry and Clusters of Orthologous Groups analysis revealed they were mainly enriched in RNA processing and modification, chromatin dynamics and cell cycle control. Of them, transcription factor, GTF2I, may be responsible for NCAPD3-mediated transcription regulation. Coimmunoprecipitation (co-IP) verified their interaction and chromatin immunoprecipitation (ChIP) presented they were both located on the promoter of target gene, SIRT1. We further examined SIRT1 expression after regulating NCAPD3 expression, showing a significantly positive correlation. This suggested NCAPD3 could support SIRT1 transcription via being anchored by GTF2I on the promoter of SIRT1. We further elucidated the potential mechanism how NCAPD3 regulated SIRT1 expression. Previous study found that NCAPD3 could interact with histone methylation markers in mammalian genome, thus, we explored whether NCAPD3 could modulate the SIRT1 expression via recognizing the histone methylation marker on SIRT1 promoter. Co-IP assay identified the interaction between NCAPD3 and monomethylation of lysine residue K9 of histone H3 (H3K9me1), an active promoter tag, in DLBCL and ChIP assay verified the location of H3k9me1 on SIRT1 promoter. When NCAPD3 downregulated, H3K9me1 on SIRT promoter diminished, ruining the transcription of SIRT. To clarify whether the oncogenic effects of NCAPD3 was mediated by SIRT1, we conducted functional reversion assays, showing regulating SIRT1 partially reversed the effects of NCAPD3. The above suggested NCAPD3 prompted DLBCL progression through modulating SIRT expression in H3k9me1-mediated manner. In conclusion, this study unraveled that dysregulation of NCAPD3 could disturb chromosome compaction and segregation, induce genome instability and alter the transcription of SIRT1 via modulating H3k9 monomethylation, which provided novel insights into NCAPD3-based targeting strategy for DLBCL.

Super-resolution microscopy reveals the nanoscale cluster architecture of the DEK protein cancer biomarker

DEK protein, a key chromatin regulator, is strongly overexpressed in various forms of cancer. While conventional microscopy revealed DEK as uniformly distributed within the cell nucleus, advanced super-resolution techniques uncovered cluster-like structures. However, a comprehensive understanding of DEK's cellular distribution and its implications in cancer and cell growth remained elusive. To bridge this gap, we employed single-molecule localization microscopy (SMLM) to dissect DEK's nanoscale organization in both normal-like and aggressive breast cancer cell lines. Our investigation included characteristics such as localizations per cluster, cluster areas, and intra-cluster localization densities (ICLDs). We elucidated how cluster features align with different breast cell types and how chromatin decompaction influences DEK clusters in these contexts. Our results indicate that DEK's intra-cluster localization density and nano-organization remain preserved and not significantly influenced by protein overexpression or chromatin compaction changes. This study advances the understanding of DEK's role in cancer and underscores its stable nanoscale behavior.

Mutant FUS induces chromatin reorganization in the hippocampus and alters memory processes

Cytoplasmic mislocalization of the nuclear Fused in Sarcoma (FUS) protein is associated to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS accumulation is recapitulated in the frontal cortex and spinal cord of heterozygous Fus∆NLS/+ mice. Yet, the mechanisms linking FUS mislocalization to hippocampal function and memory formation are still not characterized. Herein, we show that in these mice, the hippocampus paradoxically displays nuclear FUS accumulation. Multi-omic analyses showed that FUS binds to a set of genes characterized by the presence of an ETS/ELK-binding motifs, and involved in RNA metabolism, transcription, ribosome/mitochondria and chromatin organization. Importantly, hippocampal nuclei showed a decompaction of the neuronal chromatin at highly expressed genes and an inappropriate transcriptomic response was observed after spatial training of Fus∆NLS/+ mice. Furthermore, these mice lacked precision in a hippocampal-dependent spatial memory task and displayed decreased dendritic spine density. These studies shows that mutated FUS affects epigenetic regulation of the chromatin landscape in hippocampal neurons, which could participate in FTD/ALS pathogenic events. These data call for further investigation in the neurological phenotype of FUS-related diseases and open therapeutic strategies towards epigenetic drugs.

UV-DDB as a General Sensor of DNA Damage in Chromatin: Multifaceted Approaches to Assess Its Direct Role in Base Excision Repair.

Base excision repair (BER) is a cellular process that removes damaged bases arising from exogenous and endogenous sources including reactive oxygen species, alkylation agents, and ionizing radiation. BER is mediated by the actions of multiple proteins which work in a highly concerted manner to resolve DNA damage efficiently to prevent toxic repair intermediates. During the initiation of BER, the damaged base is removed by one of 11 mammalian DNA glycosylases, resulting in abasic sites. Many DNA glycosylases are product-inhibited by binding to the abasic site more avidly than the damaged base. Traditionally, apurinic/apyrimidinic endonuclease 1, APE1, was believed to help turn over the glycosylases to undergo multiple rounds of damaged base removal. However, in a series of papers from our laboratory, we have demonstrated that UV-damaged DNA binding protein (UV-DDB) stimulates the glycosylase activities of human 8-oxoguanine glycosylase (OGG1), MUTY DNA glycosylase (MUTYH), alkyladenine glycosylase/N-methylpurine DNA glycosylase (AAG/MPG), and single-strand selective monofunctional glycosylase (SMUG1), between three- and five-fold. Moreover, we have shown that UV-DDB can assist chromatin decompaction, facilitating access of OGG1 to 8-oxoguanine damage in telomeres. This review summarizes the biochemistry, single-molecule, and cell biology approaches that our group used to directly demonstrate the essential role of UV-DDB in BER.

Histone modifications regulate pioneer transcription factor cooperativity

Pioneer transcription factors have the ability to access DNA in compacted chromatin1. Multiple transcription factors can bind together to a regulatory element in a cooperative way, and cooperation between the pioneer transcription factors OCT4 (also known as POU5F1) and SOX2 is important for pluripotency and reprogramming2–4. However, the molecular mechanisms by which pioneer transcription factors function and cooperate on chromatin remain unclear. Here we present cryo-electron microscopy structures of human OCT4 bound to a nucleosome containing human LIN28B or nMATN1 DNA sequences, both of which bear multiple binding sites for OCT4. Our structural and biochemistry data reveal that binding of OCT4 induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional OCT4 and of SOX2 to their internal binding sites. The flexible activation domain of OCT4 contacts the N-terminal tail of histone H4, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA-binding domain of OCT4 engages with the N-terminal tail of histone H3, and post-translational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our findings suggest that the epigenetic landscape could regulate OCT4 activity to ensure proper cell programming.

L-arginine supplementation minimizes aging-induced changes in the sperm chromatin of roosters

Arginine is the main amino acid that constitutes the sperm protamine of roosters, named galline, which complexes with sperm DNA, allowing high compaction of its chromatin. Arginine supplementation has positive effects on semen quality in aged roosters, but this supplementation is not known to limit the progressive worsening of sperm chromatin compaction. This work aimed to verify whether L-arginine supplementation in the feed improve or maintain sperm chromatin quality since aging in roosters is usually accompanied by worsening chromatin quality. Four groups of 52-wk-old Ross AP95 lineage roosters were used, of which 6 semen samples per group were evaluated, totaling 24 samples. Another 24 samples, 6 per group, were evaluated after 6 wk of supplementation when one group was not supplemented (control) and the other 3 were supplemented with 1.15 kg (treatment 1), 2.17 kg (treatment 2), and 3.18 kg (treatment 3) of L-arginine/ton of feed. Computer image analysis of semen smears stained with toluidine blue pH 4.0 was used for sperm chromatin evaluation. Sperm chromatin was evaluated for compaction heterogeneity and compaction intensity by percentage decompaction relative to standard heads and by integrated optical density (IOD), which was used for the first time to identify sperm chromatin changes. Sperm head morphology was also evaluated by means of area and length. The IOD proved to be more efficient in identifying changes in rooster sperm chromatin compaction than the percentual decompaction. In general, chromatin compaction was positively influenced by the supplementation with L-arginine, being better in the supplementation with the highest levels tested. This was corroborated by the smaller average of the variables referring to the size of the spermatozoa heads of the animals that received feed with a higher content of L-arginine, since better compacted heads naturally tend to be smaller. Finally, arginine supplementation was able to limit or even improve sperm chromatin decompaction during the experimental period.

NSD2 E1099K drives relapse in pediatric acute lymphoblastic leukemia by disrupting 3D chromatin organization

BackgroundThe NSD2 p.E1099K (EK) mutation is shown to be enriched in patients with relapsed acute lymphoblastic leukemia (ALL), indicating a role in clonal evolution and drug resistance.ResultsTo uncover 3D chromatin architecture-related mechanisms underlying drug resistance, we perform Hi-C on three B-ALL cell lines heterozygous for NSD2 EK. The NSD2 mutation leads to widespread remodeling of the 3D genome, most dramatically in terms of compartment changes with a strong bias towards A compartment shifts. Systematic integration of the Hi-C data with previously published ATAC-seq, RNA-seq, and ChIP-seq data show an expansion in H3K36me2 and a shrinkage in H3K27me3 within A compartments as well as increased gene expression and chromatin accessibility. These results suggest that NSD2 EK plays a prominent role in chromatin decompaction through enrichment of H3K36me2. In contrast, we identify few changes in intra-topologically associating domain activity. While compartment changes vary across cell lines, a common core of decompacting loci are shared, driving the expression of genes/pathways previously implicated in drug resistance. We further perform RNA sequencing on a cohort of matched diagnosis/relapse ALL patients harboring the relapse-specific NSD2 EK mutation. Changes in patient gene expression upon relapse significantly correlate with core compartment changes, further implicating the role of NSD2 EK in genome decompaction.ConclusionsIn spite of cell-context-dependent changes mediated by EK, there appears to be a shared transcriptional program dependent on compartment shifts which could explain phenotypic differences across EK cell lines. This core program is an attractive target for therapeutic intervention.

Aberrant HMGA2 Expression Sustains Genome Instability That Promotes Metastasis and Therapeutic Resistance in Colorectal Cancer

Colorectal cancer (CRC) is one of the most lethal cancers worldwide, accounting for nearly ~10% of all cancer diagnoses and deaths. Current therapeutic approaches have considerably increased survival for patients diagnosed at early stages; however, ~20% of CRC patients are diagnosed with late-stage, metastatic CRC, where 5-year survival rates drop to 6-13% and treatment options are limited. Genome instability is an enabling hallmark of cancer that confers increased acquisition of genetic alterations, mutations, copy number variations and chromosomal rearrangements. In that regard, research has shown a clear association between genome instability and CRC, as the accumulation of aberrations in cancer-related genes provides subpopulations of cells with several advantages, such as increased proliferation rates, metastatic potential and therapeutic resistance. Although numerous genes have been associated with CRC, few have been validated as predictive biomarkers of metastasis or therapeutic resistance. A growing body of evidence suggests a member of the High-Mobility Group A (HMGA) gene family, HMGA2, is a potential biomarker of metastatic spread and therapeutic resistance. HMGA2 is expressed in embryonic tissues and is frequently upregulated in aggressively growing cancers, including CRC. As an architectural, non-histone chromatin binding factor, it initiates chromatin decompaction to facilitate transcriptional regulation. HMGA2 maintains the capacity for stem cell renewal in embryonic and cancer tissues and is a known promoter of epithelial-to-mesenchymal transition in tumor cells. This review will focus on the known molecular mechanisms by which HMGA2 exerts genome protective functions that contribute to cancer cell survival and chemoresistance in CRC.

Mesoscale chromatin organization is regulated by active transcription and epigenetic regulation.

Epigenetically regulated post-translational modifications of histone tails via acetylation or methylation have been established to play a role in determining the transcriptional status of genes. Beyond epigenetic regulation, chromatin loop extrusion through cohesin rings, driven by transcription-generated DNA supercoiling, can also alter spatial chromatin organization. Here, we present a mesoscale phase-field model to explain the underlying physics of chromatin reorganization in the presence of transcriptionally driven chromatin loop extrusion. The model incorporates energetic considerations of chromatin-chromatin and chromatin-lamina interactions leading to phase separation in the interior and periphery of the nucleus. We incorporate the kinetics of the diffusion of nucleoplasm and epigenetic marks. In addition, a non-conservative interconversion of euchromatin and heterochromatin is captured by incorporating first-order reaction kinetics of histone methylation and acetylation. Lastly, and vitally, the extrusion of genetically expressive chromatin enhancer-promoter loops is included via a kinetic conversion of heterochromatin into euchromatin in the presence of RNAPII-mediated transcriptional activity. Our theoretical analysis predicts that the size of the stable heterochromatin domains is determined solely by the kinetics of acetylation, methylation, and chromatin loop extrusion. Our numerical simulations, in conjunction with the super-resolution in-vitro nucleus imaging techniques, reveal that the absence of transcription increases chromatin compaction and heterochromatin domain sizes in the nucleus's interior and periphery. We also predict that enhanced chromatin loop extrusion would result in reduced sizes of heterochromatin domains - which is validated by super-resolution imaging of WAPL-deficient nuclei. Lastly, we reveal the synergistic effects of WAPL deficiency and transcriptional abrogation such that chromatin decompaction in WAPL-deficient nuclei is blocked by inhibiting transcription. By uncovering the physical mechanisms of mesoscale chromatin organization in the nucleus, our model presents a new step in understanding how cell fate is affected by its chemo-mechanical environment.

High-content image-based CRISPR screening reveals regulators of 3D genome architectures.

The spatial organization of chromatin critically impacts many essential genomic functions, from the regulation of gene expression to the replication of the genome. Changes in chromatin organization are associated with aging and a wide range of diseases including cancer. Despite the fundamental importance of correct chromatin organization and its effects on cellular behavior, very little is known regarding the precise folding arrangement of chromatin in various cell states, nor the molecular mechanisms that control chromatin organization in the cell nucleus. Current approaches are severely limited by a lack of experimental tools to directly trace chromatin folding, and to efficiently screen for molecular regulators of chromatin organization in the genome. To address these needs, we previously developed an image-based 3D genomics technique termed chromatin tracing, which enables direct 3D tracing of chromatin folding along individual chromosomes in single cells. Recently, we further developed Multiplexed Imaging of Nucleome Architectures (MINA), which enables simultaneous measurements of multiscale chromatin folding, associations of genomic regions with nuclear lamina and nucleoli, and copy numbers of numerous RNA species in the same cells in mammalian tissue. Here we report our latest technology development - an image-based high-content CRISPR screening platform - to systematically uncover new regulators of 3D genome organization. We performed a pooled loss-of-function screen of hundreds of selected genes in human cells, identified target genes by inventing a cellular barcoding technique termed BARC-FISH, and visualized genome organization by chromatin tracing in the same single cells. Using 1.4 million imaged 3D-positions along chromosome traces, we identified tens of novel regulators of chromatin architectures at different length scales. Loss of the ATP-dependent helicase CHD7, which causes the congenital syndrome CHARGE, promotes multi-scale chromatin decompaction. This method enables scalable, high-throughput identification of chromatin topology regulators in diverse contexts.