Chromatin Compaction Research Articles

Chromatin compaction during confined cell migration induces and reshapes nuclear condensates

AbstractCell migration through small constrictions during cancer metastasis requires significant deformation of the nucleus, with associated mechanical stress on the nuclear lamina and chromatin. However, how mechanical deformation impacts various subnuclear structures, including protein and nucleic acid-rich biomolecular condensates, is largely unknown. Here, we find that cell migration through confined spaces gives rise to mechanical deformations of the chromatin network, which cause embedded nuclear condensates, including nucleoli and nuclear speckles, to deform and coalesce. Chromatin deformations exhibit differential behavior in the advancing vs. trailing region of the nucleus, with the trailing half being more permissive for de novo condensate formation. We show that this results from increased chromatin heterogeneity, which gives rise to a shift in the binodal phase boundary. Taken together, our findings show how chromatin deformation impacts condensate assembly and properties, which can potentially contribute to cellular mechanosensing.

The role of histone post-translational modifications in cancer and cancer immunity: functions, mechanisms and therapeutic implications

Histones play crucial roles in both promoting and repressing gene expression, primarily regulated through post-translational modifications (PTMs) at specific amino acid residues. Histone PTMs, including methylation, acetylation, ubiquitination, phosphorylation, lactylation, butyrylation, and propionylation, act as important epigenetic markers. These modifications influence not only chromatin compaction but also gene expression. Their importance extends to the treatment and prevention of various human diseases, particularly cancer, due to their involvement in key cellular processes. Abnormal histone modifications and the enzymes responsible for these alterations often serve as critical drivers in tumor cell proliferation, invasion, apoptosis, and stemness. This review introduces key histone PTMs and the enzymes responsible for these modifications, examining their impact on tumorigenesis and cancer progression. Furthermore, it explores therapeutic strategies targeting histone PTMs and offers recommendations for identifying new potential therapeutic targets.

FOXA1 enhances antitumor immunity via repressing interferon-induced PD-L1 expression in nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a distinct subtype of head and neck cancer which is prevalent in south of China and southeastern of Asia. Consistent activation of interferon (IFN) signaling, and...

DNAR-02. M33 CONDENSES CHROMATIN THROUGH NUCLEAR BODY FORMATION AND METHYLATION OF BOTH HISTONE H3 LYSINE 9 AND LYSINE 27

Abstract Heterochromatin, a type of condensed DNA in eukaryotic cells, has two main categories: Constitutive heterochromatin, which contains H3K9 methylation, and facultative heterochromatin, which contains H3K27 methylation. Methylated H3K9 and H3K27 serve as docking sites for chromodomain-containing proteins that compact chromatin. M33 (also known as CBX2) is a chromodomain-containing protein that binds H3K27me3 and compacts chromatin in vitro. However, whether M33 mediates chromatin compaction in cellulo remains unknown. Here we show that M33 compacts chromatin into DAPI-intense heterochromatin domains in cells. The formation of these heterochromatin domains requires H3K27me3, which recruits M33 to form nuclear bodies. G9a and SUV39H1 are sequentially recruited into M33 nuclear bodies to create H3K9 methylated chromatin in a process that is independent of HP1α. Finally, M33 decreases progerin-induced nuclear envelope disruption caused by loss of heterochromatin. Our findings demonstrate that M33 mediates the formation of condensed chromatin by forming nuclear bodies containing both H3K27me3 and H3K9me3. Our model of M33-dependent chromatin condensation suggests H3K27 methylation corroborates with H3K9 methylation during the formation of facultative heterochromatin and provides the theoretical basis for developing novel therapies to treat heterochromatin-related diseases. Keywords: H3K27 methylation; H3K9 methylation; Heterochromatin; M33 (CBX2); Nuclear body (NB); Progerin.

Studying the structure and function of nucleosomes by atomic force microscopy

Chromatin is an epigenetic platform for the implementation of DNA-dependent processes. The nucleosome, as the basic level of chromatin compaction, largely determines its properties and structure. When studying the structure and functions of nucleosomes, physicochemical tools are actively used, such as magnetic and optical “tweezers,” “DNA curtains,” nuclear magnetic resonance, X-ray diffraction analysis and cryoelectron microscopy, as well as optical methods based on FRET. Despite the fact that these approaches make it possible to determine a wide range of structural and functional characteristics of chromatin and nucleosomes with high spatial and temporal resolution, atomic-force microscopy (AFM) complements the capabilities of these methods. This review presents the results of structural studies of nucleosomes in view of the development of the AFM method. The capabilities of AFM are considered in the context of the use of other physicochemical approaches.

Lamins and chromatin join forces

The intricate interplay between lamins and chromatin underpins the structural integrity and functional organization of the eukaryotic nucleus. Lamins, type V intermediate filament proteins, form a robust meshwork beneath the inner nuclear membrane that is crucial for sustaining nuclear architecture through interactions with lamin-associated domains (LADs). LADs are predominantly heterochromatic regions in which compacted chromatin is enriched at the nuclear periphery, interacting with lamins and lamin-associated proteins. Disruptions of these interactions are implicated in a spectrum of diseases, including laminopathies, cancer, and age-related pathologies, highlighting the importance of lamin-LAD interactions. Thus, a detailed understanding of lamin-chromatin interactions may provide new insights into chromatin organization and shed light on the mechanism behind certain disease states. Here, we discuss the current state of knowledge of lamin-chromatin interactions from a biochemical and structural point of view.

Mediator Subunit Med4 Enforces Metastatic Dormancy in Breast Cancer.

Long term survival of breast cancer patients is limited due to recurrence from metastatic dormant cancer cells. However, the mechanisms by which these dormant breast cancer cells survive and awaken remain poorly understood. Our unbiased genome-scale genetic screen in mice identified Med4 as a novel cancer-cell intrinsic gatekeeper in metastatic reactivation. MED4 haploinsufficiency is prevalent in metastatic breast cancer patients and correlates with poorer prognosis. Syngeneic xenograft models revealed that Med4 enforces breast cancer dormancy. Contrary to the canonical function of the Mediator complex in activating gene expression, Med4 maintains 3D chromatin compaction and enhancer landscape, by preventing enhancer priming or activation through the suppression of H3K4me1 deposition. Med4 haploinsufficiency disrupts enhancer poise and reprograms the enhancer dynamics to facilitate extracellular matrix (ECM) gene expression and integrin-mediated mechano-transduction, driving metastatic growth. Our findings establish Med4 as a key regulator of cellular dormancy and a potential biomarker for high-risk metastatic relapse.

Characterization of Medusavirus encoded histones reveals nucleosome-like structures and a unique linker histone

The organization of DNA into nucleosomes is a ubiquitous and ancestral feature that was once thought to be exclusive to the eukaryotic domain of life. Intriguingly, several representatives of the Nucleocytoplasmic Large DNA Viruses (NCLDV) encode histone-like proteins that in Melbournevirus were shown to form nucleosome-like particles. Medusavirus medusae (MM), a distantly related giant virus, encodes all four core histone proteins and, unique amongst most giant viruses, a putative acidic protein with two domains resembling eukaryotic linker histone H1. Here, we report the structure of nucleosomes assembled with MM histones and highlight similarities and differences with eukaryotic and Melbournevirus nucleosomes. Our structure provides insight into how variations in histone tail and loop lengths are accommodated within the context of the nucleosome. We show that MM-histones assemble into tri-nucleosome arrays, and that the putative linker histone H1 does not function in chromatin compaction. These findings expand our limited understanding of chromatin organization by virus-encoded histones.

Phosphorylation of the HMGN1 Nucleosome Binding Domain Decreases Helicity and Interactions with the Acidic Patch.

Intrinsically disordered proteins are abundant in the nucleus and are prime sites for posttranslational modifications that modulate transcriptional regulation. Lacking a defined three-dimensional structure, intrinsically disordered proteins populate an ensemble of several conformational states, which are dynamic and often altered by posttranslational modifications, or by binding to interaction partners. Although there is growing appreciation for the role that intrinsically disordered regions have in regulating protein-protein interactions, we still have a poor understanding of how to determine conformational population shifts, their causes under various conditions, and how to represent and model conformational ensembles. Here, we study the effects of serine phosphorylation in the nucleosome-binding domain of an intrinsically disordered protein - HMGN1 - using NMR spectroscopy, circular dichroism and modelling of protein complexes. We show that phosphorylation induces local conformational changes in the peptide backbone and decreases the helical propensity of the nucleosome binding domain. Modelling studies using AlphaFold3 suggest that phosphorylation disrupts the interface between HMGN1 and the nucleosome acidic patch, but that the models over-predict helicity in comparison to experimental data. These studies help us to build a picture of how posttranslational modifications might shift the conformational populations of disordered regions, alter access to histones, and regulate chromatin compaction.

Transcription-dependent mobility of single genes and genome-wide motions in live human cells

The human genome is highly dynamic across all scales. At the gene level, chromatin is persistently remodeled and rearranged during active processes such as transcription, replication and DNA repair. At the genome level, chromatin moves in micron-scale domains that break up and re-form over seconds, but the origin of these coherent motions is unknown. Here, we investigate the connection between genomic motions and gene-level activity. Simultaneous mapping of single-gene and genome-wide motions shows that the coupling of gene transcriptional activity to flows of the nearby genome is modulated by chromatin compaction. A motion correlation analysis suggests that a single active gene drives larger-scale motions in low-compaction regions, but high-compaction chromatin drives gene motion regardless of its activity state. By revealing unexpected connections among gene activity, spatial heterogeneities of chromatin and its emergent genome-wide motions, these findings uncover aspects of the genome’s spatiotemporal organization that directly impact gene regulation and expression.

Structural mechanism of HP1⍺-dependent transcriptional repression and chromatin compaction

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. Here, we present the cryoelectron microscopy (cryo-EM) structure of an HP1α dimer bound to an H2A.Z-nucleosome, revealing two distinct HP1α-nucleosome interfaces. The primary HP1α binding site is located at the N terminus of histone H3, specifically at the trimethylated lysine 9(K9me3) region, while a secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1α binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. Our study offers a model for HP1α-mediated heterochromatin maintenance and gene silencing. It also sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

Erythroblast enucleation at a glance.

Erythroid enucleation, the penultimate step in mammalian erythroid terminal differentiation, is a unique cellular process by which red blood cells (erythrocytes) remove their nucleus and accompanying nuclear material. This complex, multi-stage event begins with chromatin compaction and cell cycle arrest and ends with generation of two daughter cells: a pyrenocyte, which contains the expelled nucleus, and an anucleate reticulocyte, which matures into an erythrocyte. Although enucleation has been compared to asymmetric cell division (ACD), many mechanistic hallmarks of ACD appear to be absent. Instead, enucleation appears to rely on mechanisms borrowed from cell migration, endosomal trafficking and apoptosis, as well as unique cellular interactions within the microenvironment. In this Cell Science at a Glance article and the accompanying poster, we summarise current insights into the morphological features and genetic drivers regulating the key intracellular events that culminate in erythroid enucleation and engulfment of pyrenocytes by macrophages within the bone marrow microenvironment.

Dynamics of microcompartment formation at the mitosis-to-G1 transition.

As cells exit mitosis and enter G1, mitotic chromosomes decompact and transcription is reestablished. Previously, Hi-C studies showed that essentially all interphase 3D genome features including A/B-compartments, TADs, and CTCF loops, are lost during mitosis. However, Hi-C remains insensitive to features such as microcompartments, nested focal interactions between cis-regulatory elements (CREs). We therefore applied Region Capture Micro-C to cells from mitosis to G1. Unexpectedly, we observe microcompartments in prometaphase, which further strengthen in ana/telophase before gradually weakening in G1. Loss of loop extrusion through condensin depletion differentially impacts microcompartments and large A/B-compartments, suggesting that they are partially distinct. Using polymer modeling, we show that microcompartment formation is favored by chromatin compaction and disfavored by loop extrusion activity, explaining why ana/telophase likely provides a particularly favorable environment. Our results suggest that CREs exhibit intrinsic homotypic affinity leading to microcompartment formation, which may explain transient transcriptional spiking observed upon mitotic exit.

Nutrient starvation activates ECM remodeling gene enhancers associated with inflammatory bowel disease risk in fibroblasts.

Nutrient deprivation induces a reversible cell cycle arrest state termed quiescence, which often accompanies transcriptional silencing and chromatin compaction. Paradoxically, nutrient deprivation is associated with activated fibroblast states in pathological microenvironments in which fibroblasts drive extracellular matrix (ECM) remodeling to alter tissue environments. The relationship between nutrient deprivation and fibroblast activation remains unclear. Here, we report that serum deprivation extensively activates transcription of ECM remodeling genes in cultured fibroblasts, despite the induction of quiescence. Starvation-induced transcriptional activation accompanied large-scale histone acetylation of putative distal enhancers, but not promoters. The starvation-activated putative enhancers were enriched for non-coding genetic risk variants associated with inflammatory bowel disease (IBD), suggesting that the starvation-activated gene regulatory network may contribute to fibroblast activation in IBD. Indeed, the starvation-activated gene PLAU, encoding uPA serine protease for plasminogen and ECM, was upregulated in inflammatory fibroblasts in the intestines of IBD patients. Furthermore, the starvation-activated putative enhancer at PLAU, which harbors an IBD risk variant, gained chromatin accessibility in IBD patient fibroblasts. This study implicates nutrient deprivation in transcriptional activation of ECM remodeling genes in fibroblasts and suggests nutrient deprivation as a potential mechanism for pathological fibroblast activation in IBD.

Histone H3 mutations and their impact on genome stability maintenance.

Histones are essential for maintaining chromatin structure and function. Histone mutations lead to changes in chromatin compaction, gene expression, and the recruitment of DNA repair proteins to the DNA lesion. These disruptions can impair critical DNA repair pathways, such as homologous recombination and non-homologous end joining, resulting in increased genomic instability, which promotes an environment favorable to tumor development and progression. Understanding these mechanisms underscores the potential of targeting DNA repair pathways in cancers harboring mutated histones, offering novel therapeutic strategies to exploit their inherent genomic instability for better treatment outcomes. Here, we examine how mutations in histone H3 disrupt normal chromatin function and DNA damage repair processes and how these mechanisms can be exploited for therapeutic interventions.

Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 μg/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin.

Abstract 75: CTCF and Rad21 Mediated Regulation of Gene Expression in Human-Induced Pluripotent Stem Cells-derived Endothelial Cells

Blood pressure (BP)-relevant non-coding SNPs identified by GWAS may influence the expression of distal protein-coding genes. Our previous study detected 195 SNPs (out of 26585) in CTCF-binding sites. CTCF and Cohesin are known to modulate cell-type-specific gene expression from a distance by forming chromatin loops. We hypothesized that both may regulate gene expression in BP-relevant cell types. To test this, we depleted CTCF and Rad21 (Cohesin subunit) in human induced pluripotent stem cells-derived endothelial cells (iEC) and performed genome-wide transcriptomic analysis. We detected 366 and 4184 differentially expressed genes (DEGs) in CTCF and Rad21 depleted iEC (CTCF-/Rad21-KD-iEC) compared to iEC control, respectively. Among them, 111 DEGs are common between the CTCF-KD and Rad21-KD-iEC, though majority showed opposite regulatory effects (Figure 1A). We found 5619 DEGs in the CTCF-KD vs Rad21-KD-iEC (Figure 1B). Pathway analysis detected elevated cell cycle (G2M checkpoint), DNA replication (E2F targets), and repair pathways in Rad21-KD compared to the CTCF-KD-iEC. Rad21 plays an important role in chromosome partitioning and repair, so its depletion may cause dysregulation of these pathways. While the CTCF-KD-iEC showed enrichment of the Epithelial-to-mesenchymal transition (EMT), inflammatory (IFN-gamma, IFN-alpha), and hypoxia pathways (Figure 1C). As CTCF maintains the chromosome boundaries, CTCF-KD may affect chromatin compactness and trigger abnormal gene expression related to these pathways. In conclusion, our study detected different effects of depletion of CTCF and Rad21 on gene expression in a BP-relevant cell type. The findings provide a basis to explore the role of chromatin looping in BP-relevant gene regulation.

Ubiquitinated histone H2B as gatekeeper of the nucleosome acidic patch.

Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.

Deciphering the dynamics: Exploring the impact of mechanical forces on histone acetylation.

Living cells navigate a complex landscape of mechanical cues that influence their behavior and fate, originating from both internal and external sources. At the molecular level, the translation of these physical stimuli into cellular responses relies on the intricate coordination of mechanosensors and transducers, ultimately impacting chromatin compaction and gene expression. Notably, epigenetic modifications on histone tails govern the accessibility of gene-regulatory sites, thereby regulating gene expression. Among these modifications, histone acetylation emerges as particularly responsive to the mechanical microenvironment, exerting significant control over cellular activities. However, the precise role of histone acetylation in mechanosensing and transduction remains elusive due to the complexity of the acetylation network. To address this gap, our aim is to systematically explore the key regulators of histone acetylation and their multifaceted roles in response to biomechanical stimuli. In this review, we initially introduce the ubiquitous force experienced by cells and then explore the dynamic alterations in histone acetylation and its associated co-factors, including HDACs, HATs, and acetyl-CoA, in response to these biomechanical cues. Furthermore, we delve into the intricate interactions between histone acetylation and mechanosensors/mechanotransducers, offering a comprehensive analysis. Ultimately, this review aims to provide a holistic understanding of the nuanced interplay between histone acetylation and mechanical forces within an academic framework.

Chromatin remodeling-driven autophagy activation induces cisplatin resistance in oral squamous cell carcinoma

It is still challenging to predict the efficacy of cisplatin-based therapy, particularly in relation to the activation of macroautophagy/autophagy in oral squamous cell carcinoma (OSCC). We studied the effect of selected chromatin remodeling genes on the cisplatin resistance and their interplay with autophagy in 3-dimensional tumor model and xenografts. We analyzed gene expression patterns in the cisplatin-sensitive UMSCC1, and a paired cisplatin-resistant UM-Cis cells. Many histone protein gene clusters involved in nucleosome assembly showed significant difference of expression. Gain- and loss-of-function analyses revealed an inverse correlation between cisplatin resistance and HIST1H3D expression, while a positive correlation was observed with HIST3H2A or HIST3H2B expression. In UM-Cis, HIST3H2A- and HIST3H2B-mediated chromatin remodeling upregulates autophagy status, which results in cisplatin resistance. Additionally, knockdown of HIST3H2A or HIST3H2B downregulated autophagy-activating genes via chromatin compaction of their promoter regions. MiTF, one of the key autophagy regulators upregulated in UM-Cis, negatively regulated transcription of HIST1H3D, suggesting an interplay between chromatin remodeling-dependent cisplatin resistance and autophagy. On comparing the staining intensity between cisplatin-sensitive and –insensitive tissues from OSCC patients, protein expression pattern of the selected histone protein genes were matched with the in vitro data. By examining the relationship between autophagy and chromatin remodeling genes, we identified a set of candidate genes with potential use as markers predicting chemoresistance in OSCC biopsy samples.