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.

The Upstream Sequence Transcription Complex dictates nucleosome positioning and promoter accessibility at piRNA genes in the C. elegans germ line.

The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization.

Single gene analysis in yeast suggests nonequilibrium regulatory dynamics for transcription.

Fluctuations in the initiation rate of transcription, the first step in gene expression, ensue from the stochastic behavior of the molecular process that controls transcription. In steady state, the regulatory process is often assumed to operate reversibly, i.e., in equilibrium. However, reversibility imposes fundamental limits to information processing. For instance, the assumption of equilibrium is difficult to square with the precision with which the regulatory process executes its task in eukaryotes. Here we provide evidence - from microscopic analyses of the transcription dynamics at a single gene copy of yeast - that the regulatory process for transcription is cyclic and irreversible (out of equilibrium). The necessary coupling to reservoirs of free energy occurs via sequence-specific transcriptional activators and the recruitment, in part, of ATP-dependent chromatin remodelers. Our findings may help explain how eukaryotic cells reconcile the dual but opposing requirements for fast regulatory kinetics and high regulatory specificity.

Reader-Effectors as Actuators of Epigenome Editing.

Epigenome editing applications are gaining broader use for targeted transcriptional control as more enzymes with diverse chromatin-modifying functions are being incorporated into fusion proteins. Development of these fusion proteins, called epigenome editors, has outpaced the study of proteins that interact with edited chromatin. One type of protein that acts downstream of chromatin editing is the reader-effector, which bridges epigenetic marks with biological effects like gene regulation. As the name suggests, a reader-effector protein is generally composed of a reader domain and an effector domain. Reader domains directly bind epigenetic marks, while effector domains often recruit protein complexes that mediate transcription, chromatin remodeling, and DNA repair. In this chapter, we discuss the role of reader-effectors in driving the outputs of epigenome editing and highlight instances where abnormal and context-specific reader-effectors might impair the effects of epigenome editing. Lastly, we discuss how engineered reader-effectors may complement the epigenome editing toolbox to achieve robust and reliable gene regulation.

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.

Molecular and structural basis of the chromatin remodeling activity by Arabidopsis DDM1.

The histone H2A variant H2A.W occupies transposons and thus prevents access to them in Arabidopsis thaliana. H2A.W is deposited by the chromatin remodeler DDM1, which also promotes the accessibility of chromatin writers to heterochromatin by an unknown mechanism. To shed light on this question, we solve the cryo-EM structures of nucleosomes containing H2A and H2A.W, and the DDM1-H2A.W nucleosome complex. These structures show that the DNA end flexibility of the H2A nucleosome is higher than that of the H2A.W nucleosome. In the DDM1-H2A.W nucleosome complex, DDM1 binds to the N-terminal tail of H4 and the nucleosomal DNA and increases the DNA end flexibility of H2A.W nucleosomes. Based on these biochemical and structural results, we propose that DDM1 counters the low accessibility caused by nucleosomes containing H2A.W to enable the maintenance of repressive epigenetic marks on transposons and prevent their activity.

The Function of E2A in B-Cell Development.

Helix-loop-helix (HLH) transcription factors (TFs) play a key role in various cellular differentiation and function through the regulation of enhancer activity. E2A, a member of the mammalian E-protein family (class I HLH protein), is well known to play an important role in hematopoiesis, especially in adaptive lymphocyte development. E2A instructs B- and T-cell lineage development through the regulation of enhancer activity for B- or T-cell signature gene expression, including Rag1 and Rag2 (Rag1/2) genes. In this chapter, we mainly focus on the function of E2A in B-cell development and on the roles of E2A in establishing the enhancer landscape through the recruitment of EP300/KAT3B, chromatin remodeling complex, mediator, cohesion, and TET proteins. Finally, we demonstrate how E2A orchestrates the assembly of the Rag1/2 gene super-enhancer (SE) formation by changing the chromatin conformation across the Rag gene locus.

ARID1A safeguards the canalization of the cell fate decision during osteoclastogenesis.

Chromatin remodeler ARID1A regulates gene transcription by modulating nucleosome positioning and chromatin accessibility. While ARID1A-mediated stage and lineage-restricted gene regulation during cell fate canalization remains unresolved. Using osteoclastogenesis as a model, we show that ARID1A transcriptionally safeguards the osteoclast (OC) fate canalization during proliferation-differentiation switching at single-cell resolution. Notably, ARID1A is indispensable for the transcriptional apparatus condensates formation with coactivator BRD4/lineage-specifying transcription factor (TF) PU.1 at Nfatc1 super-enhancer during safeguarding the OC fate canalization. Besides, the antagonist function between ARID1A-cBAF and BRD9-ncBAF complex during osteoclastogenesis has been validated with in vitro assay and compound mutant mouse model. Furthermore, the antagonistic function of ARID1A-"accelerator" and BRD9-"brake" both depend on coactivator BRD4-"clutch" during osteoclastogenesis. Overall, these results uncover sophisticated cooperation between chromatin remodeler ARID1A, coactivator, and lineage-specifying TF at super-enhancer of lineage master TF in a condensate manner, and antagonist between distinct BAF complexes in the proper and balanced cell fate canalization.

Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1.

Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.

nucMACC: An MNase-seq pipeline to identify structurally altered nucleosomes in the genome.

Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.

Transitions in chromatin conformation shaped by fatty acids and the circadian clock underlie hepatic transcriptional reorganization in obese mice.

The circadian clock system coordinates metabolic, physiological, and behavioral functions across a 24-h cycle, crucial for adapting to environmental changes. Disruptions in circadian rhythms contribute to major metabolic pathologies like obesity and Type 2 diabetes. Understanding the regulatory mechanisms governing circadian control is vital for identifying therapeutic targets. It is well characterized that chromatin remodeling and 3D structure at genome regulatory elements contributes to circadian transcriptional cycles; yet the impact of rhythmic chromatin topology in metabolic disease is largely unexplored. In this study, we explore how the spatial configuration of the genome adapts to diet, rewiring circadian transcription and contributing to dysfunctional metabolism. We describe daily fluctuations in chromatin contacts between distal regulatory elements of metabolic control genes in livers from lean and obese mice and identify specific lipid-responsive regions recruiting the clock molecular machinery. Interestingly, under high-fat feeding, a distinct interactome for the clock-controlled gene Dbp strategically promotes the expression of distal metabolic genes including Fgf21. Alongside, new chromatin loops between regulatory elements from genes involved in lipid metabolism control contribute to their transcriptional activation. These enhancers are responsive to lipids through CEBPß, counteracting the circadian repressor REVERBa. Our findings highlight the intricate coupling of circadian gene expression to a dynamic nuclear environment under high-fat feeding, supporting a temporally regulated program of gene expression and transcriptional adaptation to diet.

SWI/SNF chromatin remodeling factor BAF60b restrains inflammatory diseases by affecting regulatory T cell migration.

Regulatory T (Treg) cells play a critical regulatory role in the immune system by suppressing excessive immune responses and maintaining immune balance. The effective migration of Treg cells is crucial for controlling the development and progression of inflammatory diseases. However, the mechanisms responsible for directing Treg cells into the inflammatory tissue remain incompletely elucidated. In this study, we identified BAF60b, a subunit of switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complexes, as a positive regulator of Treg cell migration that inhibits the progression of inflammation in experimental autoimmune encephalomyelitis (EAE) and colitis animal models. Mechanistically, transcriptome and genome-wide chromatin-landscaped analyses demonstrated that BAF60b interacts with the transcription factor RUNX1 to promote the expression of CCR9 on Treg cells, which in turn affects their ability to migrate to inflammatory tissues. Our work provides insights into the essential role of BAF60b in regulating Treg cell migration and its impact on inflammatory diseases.

How Histone Acetyltransferases Shape Plant Photomorphogenesis and UV Response.

Higher plants have developed complex mechanisms to adapt to fluctuating environmental conditions with light playing a vital role in photosynthesis and influencing various developmental processes, including photomorphogenesis. Exposure to ultraviolet (UV) radiation can cause cellular damage, necessitating effective DNA repair mechanisms. Histone acetyltransferases (HATs) play a crucial role in regulating chromatin structure and gene expression, thereby contributing to the repair mechanisms. HATs facilitate chromatin relaxation, enabling transcriptional activation necessary for plant development and stress responses. The intricate relationship between HATs, light signaling pathways and chromatin dynamics has been increasingly understood, providing valuable insights into plant adaptability. This review explores the role of HATs in plant photomorphogenesis, chromatin remodeling and gene regulation, highlighting the importance of chromatin modifications in plant responses to light and various stressors. It emphasizes the need for further research on individual HAT family members and their interactions with other epigenetic factors. Advanced genomic approaches and genome-editing technologies offer promising avenues for enhancing crop resilience and productivity through targeted manipulation of HAT activities. Understanding these mechanisms is essential for developing strategies to improve plant growth and stress tolerance, contributing to sustainable agriculture in the face of a changing climate.

Expression Proteomics and Histone Analysis Reveal Extensive Chromatin Network Changes and a Role for Histone Tail Trimming during Cellular Differentiation.

In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification.

The relationship between nucleosome positioning and higher-order genome folding.

Eukaryotic genomes are organized into 3D structures, which range from small-scale nucleosome arrays to large-scale chromatin domains. These structures have an important role in the regulation of transcription and other nuclear processes. Despite advances in our understanding of the properties, functions, and underlying mechanisms of genome structures, there are many open questions about the interplay between these structures across scales. In particular, it is not well understood if and how 1D features of nucleosome arrays influence large-scale 3D genome folding patterns. In this review, we discuss recent studies that address these questions and summarize our current understanding of the relationship between nucleosome positioning and higher-order genome folding.

Remodeling of perturbed chromatin can initiate de novo transcriptional and post-transcriptional silencing.

In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin.

Monitoring and quantifying replication fork dynamics with high-throughput methods.

Before each cell division, eukaryotic cells must replicate their chromosomes to ensure the accurate transmission of genetic information. Chromosome replication involves more than just DNA duplication; it also includes chromatin assembly, inheritance of epigenetic marks, and faithful resumption of all genomic functions after replication. Recent progress in quantitative technologies has revolutionized our understanding of the complexity and dynamics of DNA replication forks at both molecular and genomic scales. Here, we highlight the pivotal role of these novel methods in uncovering the principles and mechanisms of chromosome replication. These technologies have illuminated the regulation of genome replication programs, quantified the impact of DNA replication on genomic mutations and evolutionary processes, and elucidated the mechanisms of replication-coupled chromatin assembly and epigenome maintenance.

A high-throughput synthetic biology approach for studying combinatorial chromatin-based transcriptional regulation.

The construction of synthetic gene circuits requires the rational combination of multiple regulatory components, but predicting their behavior can be challenging due to poorly understood component interactions and unexpected emergent behaviors. In eukaryotes, chromatin regulators (CRs) are essential regulatory components that orchestrate gene expression. Here, we develop a screening platform to investigate the impact of CR pairs on transcriptional activity in yeast. We construct a combinatorial library consisting of over 1,900 CR pairs and use a high-throughput workflow to characterize the impact of CR co-recruitment on gene expression. We recapitulate known interactions and discover several instances of CR pairs with emergent behaviors. We also demonstrate that supervised machine learning models trained with low-dimensional amino acid embeddings accurately predict the impact of CR co-recruitment on transcriptional activity. This work introduces a scalable platform and machine learning approach that can be used to study how networks of regulatory components impact gene expression.