Prion-like domain mediated phase separation of ARID1A promotes oncogenic potential of Ewing's sarcoma.

Liquid-liquid phase separation (LLPS) facilitates the formation of membraneless organelles within cells, with implications in various biological processes and disease states. AT-rich interactive domain-containing protein 1A (ARID1A) is a chromatin remodeling factor frequently associated with cancer mutations, yet its functional mechanism remains largely unknown. Here, we find that ARID1A harbors a prion-like domain (PrLD), which facilitates the formation of liquid condensates through PrLD-mediated LLPS. The nuclear condensates formed by ARID1A LLPS are significantly elevated in Ewing's sarcoma patient specimen. Disruption of ARID1A LLPS results in diminished proliferative and invasive abilities in Ewing's sarcoma cells. Through genome-wide chromatin structure and transcription profiling, we identify that the ARID1A condensate localizes to EWS/FLI1 target enhancers and induces long-range chromatin architectural changes by forming functional chromatin remodeling hubs at oncogenic target genes. Collectively, our findings demonstrate that ARID1A promotes oncogenic potential through PrLD-mediated LLPS, offering a potential therapeutic approach for treating Ewing's sarcoma.

Z-DNA formation in promoters conserved between human and mouse are associated with increased transcription reinitiation rates.

A long-standing question concerns the role of Z-DNA in transcription. Here we use a deep learning approach DeepZ that predicts Z-flipons based on DNA sequence, structural properties of nucleotides and omics data. We examined Z-flipons that are conserved between human and mouse genomes after generating whole-genome Z-flipon maps and then validated them by orthogonal approaches based on high resolution chemical mapping of Z-DNA and the transformer algorithm Z-DNABERT. For human and mouse, we revealed similar pattern of transcription factors, chromatin remodelers, and histone marks associated with conserved Z-flipons. We found significant enrichment of Z-flipons in alternative and bidirectional promoters associated with neurogenesis genes. We show that conserved Z-flipons are associated with increased experimentally determined transcription reinitiation rates compared to promoters without Z-flipons, but without affecting elongation or pausing. Our findings support a model where Z-flipons engage Transcription Factor E and impact phenotype by enabling the reset of preinitiation complexes when active, and the suppression of gene expression when engaged by repressive chromatin complexes.

Chromosome structure in Drosophila is determined by boundary pairing not loop extrusion.

Two different models have been proposed to explain how the endpoints of chromatin looped domains ('TADs') in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the 'loop-extrusion' and the 'boundary-pairing' models. Our findings are incompatible with the loop-extrusion model, and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.

A DNA condensation code for linker histones.

Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.

Modeling properties of chromosome territories using polymer filaments in diverse confinement geometries.

Interphase chromosomes reside within distinct nuclear regions known as chromosome territories (CTs). Recent observations from Hi-C analyses, a method mapping chromosomal interactions, have revealed varied decay in contact probabilities among different chromosomes. Our study explores the relationship between this contact decay and the particular shapes of the chromosome territories they occupy. For this, we employed molecular dynamics (MD) simulations to examine how confined polymers, resembling chromosomes, behave within different confinement geometries similar to chromosome territory boundaries. Our simulations unveil so far unreported relationships between contact probabilities and end-to-end distances varying based on different confinement geometries. These findings highlight the crucial impact of chromosome territories on shaping the larger-scale properties of 3D genome organization. They emphasize the intrinsic connection between the shapes of these territories and the contact behaviors exhibited by chromosomes. Understanding these correlations is key to accurately interpret Hi-C and microscopy data, and offers vital insights into the foundational principles governing genomic organization.

HMGA1 orchestrates chromatin compartmentalization and sequesters genes into 3D networks coordinating senescence heterogeneity.

HMGA1 is an abundant non-histone chromatin protein that has been implicated in embryonic development, cancer, and cellular senescence, but its specific role remains elusive. Here, we combine functional genomics approaches with graph theory to investigate how HMGA1 genomic deposition controls high-order chromatin networks in an oncogene-induced senescence model. While the direct role of HMGA1 in gene activation has been described previously, we find little evidence to support this. Instead, we show that the heterogeneous linear distribution of HMGA1 drives a specific 3D chromatin organization. HMGA1-dense loci form highly interactive networks, similar to, but independent of, constitutive heterochromatic loci. This, coupled with the exclusion of HMGA1-poor chromatin regions, leads to coordinated gene regulation through the repositioning of genes. In the absence of HMGA1, the whole process is largely reversed, but many regulatory interactions also emerge, amplifying the inflammatory senescence-associated secretory phenotype. Such HMGA1-mediated fine-tuning of gene expression contributes to the heterogeneous nature of senescence at the single-cell level. A similar 'buffer' effect of HMGA1 on inflammatory signalling is also detected in lung cancer cells. Our study reveals a mechanism through which HMGA1 modulates chromatin compartmentalization and gene regulation in senescence and beyond.

Decoding the genomic landscape of chromatin-associated biomolecular condensates.

Biomolecular condensates play a significant role in chromatin activities, primarily by concentrating and compartmentalizing proteins and/or nucleic acids. However, their genomic landscapes and compositions remain largely unexplored due to a lack of dedicated computational tools for systematic identification in vivo. To address this, we develop CondSigDetector, a computational framework designed to detect condensate-like chromatin-associated protein co-occupancy signatures (CondSigs), to predict genomic loci and component proteins of distinct chromatin-associated biomolecular condensates. Applying this framework to mouse embryonic stem cells (mESC) and human K562 cells enable us to depict the high-resolution genomic landscape of chromatin-associated biomolecular condensates, and uncover both known and potentially unknown biomolecular condensates. Multi-omics analysis and experimental validation further verify the condensation properties of CondSigs. Additionally, our investigation sheds light on the impact of chromatin-associated biomolecular condensates on chromatin activities. Collectively, CondSigDetector provides an approach to decode the genomic landscape of chromatin-associated condensates, facilitating a deeper understanding of their biological functions and underlying mechanisms in cells.

DEAD-box ATPase Dbp2 is the key enzyme in an mRNP assembly checkpoint at the 3'-end of genes and involved in the recycling of cleavage factors.

mRNA biogenesis in the eukaryotic nucleus is a highly complex process. The numerous RNA processing steps are tightly coordinated to ensure that only fully processed transcripts are released from chromatin for export from the nucleus. Here, we present the hypothesis that fission yeast Dbp2, a ribonucleoprotein complex (RNP) remodelling ATPase of the DEAD-box family, is the key enzyme in an RNP assembly checkpoint at the 3'-end of genes. We show that Dbp2 interacts with the cleavage and polyadenylation complex (CPAC) and localises to cleavage bodies, which are enriched for 3'-end processing factors and proteins involved in nuclear RNA surveillance. Upon loss of Dbp2, 3'-processed, polyadenylated RNAs accumulate on chromatin and in cleavage bodies, and CPAC components are depleted from the soluble pool. Under these conditions, cells display an increased likelihood to skip polyadenylation sites and a delayed transcription termination, suggesting that levels of free CPAC components are insufficient to maintain normal levels of 3'-end processing. Our data support a model in which Dbp2 is the active component of an mRNP remodelling checkpoint that licenses RNA export and is coupled to CPAC release.

Quantitative analysis of cis-regulatory elements in transcription with KAS-ATAC-seq.

Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation networks during cellular processes. In this study, we develop Kethoxal-Assisted Single-stranded DNA Assay for Transposase-Accessible Chromatin with Sequencing (KAS-ATAC-seq) to quantitatively analyze the transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks, enabling the identification of transcriptional regulatory sequences. This feature is particularly adept at defining Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNAs and specific transcription factors (TFs) binding sites that define cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes. Our analysis of CREs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to retinoic acid (RA) treatment. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes.

Loss of cold tolerance is conferred by absence of the WRKY34 promoter fragment during tomato evolution.

Natural evolution has resulted in reduced cold tolerance in cultivated tomato (Solanum lycopersicum). Herein, we perform a combined analysis of ATAC-Seq and RNA-Seq in cold-sensitive cultivated tomato and cold-tolerant wild tomato (S. habrochaites). We identify that WRKY34 has the most significant association with differential chromatin accessibility and expression patterns under cold stress. We find that a 60 bp InDel in the WRKY34 promoter causes differences in its transcription and cold tolerance among 376 tomato accessions. This 60 bp fragment contains a GATA cis-regulatory element that binds to SWIBs and GATA29, which synergistically suppress WRKY34 expression under cold stress. Moreover, WRKY34 interferes with the CBF cold response pathway through regulating transcription and protein levels. Our findings emphasize the importance of polymorphisms in cis-regulatory regions and their effects on chromatin structure and gene expression during crop evolution.

3D chromatin architecture, BRD4, and Mediator have distinct roles in regulating genome-wide transcriptional bursting and gene network.

Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Despite comparable effects on RNA levels among these perturbations, acute depletion of MED26 had the most profound impact on the entire gene regulatory network, acting downstream of chromatin spatial architecture and without affecting TATA box-binding protein (TBP) recruitment. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells.

Meet the authors: Emily R. Feierman and Erica Korb.

We talk to first author Emily R. Feierman and corresponding author Erica Korb about the journey toward their paper "Histone variant H2BE enhances chromatin accessibility in neurons to promote synaptic gene expression and long-term memory" (this issue of Molecular Cell) and changes smoothing the road for women in science.

Efficient Enrichment of Synchronized Mouse Spermatocytes Suitable for Genome-Wide Analysis.

Chromatin undergoes extensive remodeling during meiosis, leading to specific patterns of gene expression and chromosome organization, which ultimately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies we adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin-binding sites by ChIP-seq. High-quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin-binding sites.

Canine hyper-sociability structural variants associated with altered three-dimensional chromatin state.

Strong selection on complex traits can lead to skewed trait means and reduced trait variability in populations. An example of this phenomenon can be evidenced in allele frequency changes and skewed trait distributions driven by persistent human-directed selective pressures in domesticated species. Dog domestication is linked to several genomic variants; however, the functional impacts of these variants may not always be straightforward when found in non-coding regions of the genome. Four polymorphic transposable elements (TE) found within non-coding sites along a 5 Mb region on canine CFA6 have evolved due to directional selection associated with heightened human-directed hyper-sociability in domesticated dogs. We found that the polymorphic TE in intron 17 of the canine GTF2I gene, which was previously reported to be negatively correlated with canid human-directed hyper-sociability, is associated with altered chromatin looping and hence distinct cis-regulatory landscapes. We reported supporting evidence of an E2F1-DNA binding peak concordant with the altered loop and higher expression of GTF2I exon 18, indicative of alternative splicing. Globally, we discovered differences in pathways regulating the extra-cellular matrix with respect to TE copy number. Overall, we reported evidence suggesting an intriguing molecular convergence between the emergence of hypersocial behaviors in dogs and the same genes that, when hemizygous, produce human Williams Beuren Syndrome characterized by cranio-facial defects and heightened social behaviors. Our results additionally emphasize the often-overlooked potential role of chromatin architecture in social evolution.

Essential gene screening identifies the bromodomain-containing protein BRPF1 as a new actionable target for endocrine therapy-resistant breast cancers.

Identifying master epigenetic factors controlling proliferation and survival of cancer cells allows to discover new molecular targets exploitable to overcome resistance to current pharmacological regimens. In breast cancer (BC), resistance to endocrine therapy (ET) arises from aberrant Estrogen Receptor alpha (ERα) signaling caused by genetic and epigenetic events still mainly unknown. Targeting key upstream components of the ERα pathway provides a way to interfere with estrogen signaling in cancer cells independently from any other downstream event. By combining computational analysis of genome-wide 'drop-out' screenings with siRNA-mediated gene knock-down (kd), we identified a set of essential genes in luminal-like, ERα + BC that includes BRPF1, encoding a bromodomain-containing protein belonging to a family of epigenetic readers that act as chromatin remodelers to control gene transcription. To gather mechanistic insights into the role of BRPF1 in BC and ERα signaling, we applied chromatin and transcriptome profiling, gene ablation and targeted pharmacological inhibition coupled to cellular and functional assays. Results indicate that BRPF1 associates with ERα onto BC cell chromatin and its blockade inhibits cell cycle progression, reduces cell proliferation and mediates transcriptome changes through the modulation of chromatin accessibility. This effect is elicited by a widespread inhibition of estrogen signaling, consequent to ERα gene silencing, in antiestrogen (AE) -sensitive and -resistant BC cells and pre-clinical patient-derived models (PDOs). Characterization of the functional interplay of BRPF1 with ERα reveals a new regulator of estrogen-responsive BC cell survival and suggests that this epigenetic factor is a potential new target for treatment of these tumors.

Transcriptional regulation mechanism of PARP1 and its application in disease treatment.

Poly (ADP-ribose) polymerase 1 (PARP1) is a multifunctional nuclear enzyme that catalyzes poly-ADP ribosylation in eukaryotic cells. In addition to maintaining genomic integrity, this nuclear enzyme is also involved in transcriptional regulation. PARP1 can trigger and maintain changes in the chromatin structure and directly recruit transcription factors. PARP1 also prevents DNA methylation. However, most previous reviews on PARP1 have focused on its involvement in maintaining genome integrity, with less focus on its transcriptional regulatory function. This article comprehensively reviews the transcriptional regulatory function of PARP1 and its application in disease treatment, providing new ideas for targeting PARP1 for the treatment of diseases other than cancer.

Current genomic deep learning models display decreased performance in cell type-specific accessible regions.

BACKGROUND: A number of deep learning models have been developed to predict epigenetic features such as chromatin accessibility from DNA sequence. Model evaluations commonly report performance genome-wide; however, cis regulatory elements (CREs), which play critical roles in gene regulation, make up only a small fraction of the genome. Furthermore, cell type-specific CREs contain a large proportion of complex disease heritability. RESULTS: We evaluate genomic deep learning models in chromatin accessibility regions with varying degrees of cell type specificity. We assess two modeling directions in the field: general purpose models trained across thousands of outputs (cell types and epigenetic marks) and models tailored to specific tissues and tasks. We find that the accuracy of genomic deep learning models, including two state-of-the-art general purpose models-Enformer and Sei-varies across the genome and is reduced in cell type-specific accessible regions. Using accessibility models trained on cell types from specific tissues, we find that increasing model capacity to learn cell type-specific regulatory syntax-through single-task learning or high capacity multi-task models-can improve performance in cell type-specific accessible regions. We also observe that improving reference sequence predictions does not consistently improve variant effect predictions, indicating that novel strategies are needed to improve performance on variants. CONCLUSIONS: Our results provide a new perspective on the performance of genomic deep learning models, showing that performance varies across the genome and is particularly reduced in cell type-specific accessible regions. We also identify strategies to maximize performance in cell type-specific accessible regions.

A pattern emerges in chromatin aging: AP-1 steals the show.

During aging, transcriptional programs of cell identity are partially eroded, reducing cellular fitness and resilience. Patrick et al.1 unveil a general mechanism for this process that consists of the progressive loss of transcription factor AP-1 from cell identity enhancers and its relocation by competition to stress-response elements.

Chromatin Immunoprecipitation in Adipose Tissue and Adipocytes: How to Proceed and Optimize the Protocol for Transcription Factor DNA Binding.

Chromatin immunoprecipitation (ChIP) coupled to qPCR or sequencing is a crucial experiment to determine direct transcriptional regulation under the control of specific transcriptional factors or co-regulators at loci-specific or pan-genomic levels.Here we provide a reliable method for processing ChIP from adipocytes or frozen adipose tissue collection, isolation of nuclei, cross-linking of protein-DNA complexes, chromatin shearing, immunoprecipitation, and DNA purification. We also discuss critical steps for optimizing the experiment to perform a successful ChIP in lipid-rich cells/tissues.

Native ChIP: Studying the Genome-Wide Distribution of Histone Modifications in Cells and Tissue.

For the genome-wide mapping of histone modifications, chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing remains the benchmark method. While crosslinked ChIP can be used for all kinds of targets, native ChIP is predominantly used for strong and direct DNA interactors like histones and their modifications. Here we describe a native ChIP protocol that can be used for cells and tissue material.