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Nature Genetics Jun 2024Chromatin modifications are linked with regulating patterns of gene expression, but their causal role and context-dependent impact on transcription remains unresolved....
Chromatin modifications are linked with regulating patterns of gene expression, but their causal role and context-dependent impact on transcription remains unresolved. Here we develop a modular epigenome editing platform that programs nine key chromatin modifications, or combinations thereof, to precise loci in living cells. We couple this with single-cell readouts to systematically quantitate the magnitude and heterogeneity of transcriptional responses elicited by each specific chromatin modification. Among these, we show that installing histone H3 lysine 4 trimethylation (H3K4me3) at promoters can causally instruct transcription by hierarchically remodeling the chromatin landscape. We further dissect how DNA sequence motifs influence the transcriptional impact of chromatin marks, identifying switch-like and attenuative effects within distinct cis contexts. Finally, we examine the interplay of combinatorial modifications, revealing that co-targeted H3K27 trimethylation (H3K27me3) and H2AK119 monoubiquitination (H2AK119ub) maximizes silencing penetrance across single cells. Our precision-perturbation strategy unveils the causal principles of how chromatin modification(s) influence transcription and dissects how quantitative responses are calibrated by contextual interactions.
Topics: Chromatin; Histones; Epigenome; Humans; Gene Editing; Epigenesis, Genetic; Promoter Regions, Genetic; Chromatin Assembly and Disassembly; Ubiquitination; Histone Code; Transcription, Genetic; Single-Cell Analysis
PubMed: 38724747
DOI: 10.1038/s41588-024-01706-w -
Cell May 2024Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two...
Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two overlapping reading frames, and during evolution, multiple selective pressures can act on shared nucleotides. This study combines an RNA-based HBV cell culture system with deep mutational scanning (DMS) to uncouple cis- and trans-acting sequence requirements in the HBV genome. The results support a leaky ribosome scanning model for polymerase translation, provide a fitness map of the HBV polymerase at single-nucleotide resolution, and identify conserved prolines adjacent to the HBV polymerase termination codon that stall ribosomes. Further experiments indicated that stalled ribosomes tether the nascent polymerase to its template RNA, ensuring cis-preferential RNA packaging and reverse transcription of the HBV genome.
Topics: Humans; Genome, Viral; Hepatitis B virus; Mutation; Reverse Transcription; Ribosomes; RNA, Viral; Cell Line
PubMed: 38723628
DOI: 10.1016/j.cell.2024.04.008 -
JAMA Dermatology Jun 2024
Topics: Humans; Melanoma; Uveal Neoplasms; Darier Disease; Eyebrows; Chromatin Assembly and Disassembly; Male; Female; Middle Aged; Transcription Factors; Abnormalities, Multiple
PubMed: 38717752
DOI: 10.1001/jamadermatol.2024.1137 -
BioRxiv : the Preprint Server For... Apr 2024Acute inflammation, characterized by a rapid influx of neutrophils, is a protective response that can lead to chronic inflammatory diseases when left unresolved....
Acute inflammation, characterized by a rapid influx of neutrophils, is a protective response that can lead to chronic inflammatory diseases when left unresolved. Secretion of LTB -containing exosomes is required for effective neutrophil infiltration during inflammation. In this study, we show that neutrophils release nuclear DNA in a non-lytic, rapid, and repetitive manner, via a mechanism distinct from suicidal NET release and cell death. The packaging of nuclear DNA occurs in the lumen of nuclear envelope (NE)-derived multivesicular bodies (MVBs) that harbor the LTB synthesizing machinery and is mediated by the lamin B receptor (LBR) and chromatin decondensation. Disruption of secreted exosome-associated DNA (SEAD) in a model of sterile inflammation in mouse skin amplifies and prolongs the presence of neutrophils, impeding the onset of resolution. Together, these findings advance our understanding of neutrophil functions during inflammation and the physiological significance of NETs, with implications for novel treatments for inflammatory disorders.
PubMed: 38712240
DOI: 10.1101/2024.04.21.590456 -
BioRxiv : the Preprint Server For... Apr 2024ICP1, a lytic bacteriophage of , is parasitized by phage satellites, PLEs, which hijack ICP1 proteins for their own horizontal spread. PLEs' dependence on ICP1's DNA...
ICP1, a lytic bacteriophage of , is parasitized by phage satellites, PLEs, which hijack ICP1 proteins for their own horizontal spread. PLEs' dependence on ICP1's DNA replication machinery, and virion components results in inhibition of ICP1's lifecycle. PLEs' are expected to depend on ICP1 factors for genome packaging, but the mechanism(s) PLEs use to inhibit ICP1 genome packaging is currently unknown. Here, we identify and characterize Gpi, PLE's indiscriminate genome packaging inhibitor. Gpi binds to ICP1's large terminase (TerL), the packaging motor, and blocks genome packaging. To overcome Gpi's negative effect on TerL, a component PLE also requires, PLE uses two genome packaging specifiers, GpsA and GpsB, that specifically allow packaging of PLE genomes. Surprisingly, PLE also uses mimicry of ICP1's site as a backup strategy to ensure genome packaging. PLE's site mimicry, however, is only sufficient if PLE can inhibit ICP1 at other stages of its lifecycle, suggesting an advantage to maintaining Gpi, GpsA, and GpsB. Collectively, these results provide mechanistic insights into another stage of ICP1's lifecycle that is inhibited by PLE, which is currently the most inhibitory of the documented phage satellites. More broadly, Gpi represents the first satellite-encoded inhibitor of a phage TerL.
PubMed: 38712175
DOI: 10.1101/2024.04.22.590561 -
The Journal of Cell Biology Aug 2024Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in...
Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.
Topics: Animals; Humans; Mice; Adenosine Triphosphatases; Bromodomain Containing Proteins; Centromere; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Heterochromatin; Histone-Lysine N-Methyltransferase; Histones; Methylation; Repressor Proteins; Transcription Factors
PubMed: 38709169
DOI: 10.1083/jcb.202310084 -
Biological Research May 2024Chromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at...
BACKGROUND
Chromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at gene regulatory regions are general regulatory factors (GRFs). These factors contribute to establishment and maintenance of nucleosome-depleted regions (NDRs). These regions are populated by nucleosomes through histone deposition and nucleosome sliding, the latter catalyzed by a number of ATP-dependent chromatin remodeling complexes, including ISW1a. It has been observed that GRFs can act as barriers against nucleosome sliding towards NDRs. However, the relative ability of the different GRFs to hinder sliding activity is currently unknown.
RESULTS
Considering this, we performed a comparative analysis for the main GRFs, with focus in their ability to modulate nucleosome sliding mediated by ISW1a. Among the GRFs tested in nucleosome remodeling assays, Rap1 was the only factor displaying the ability to hinder the activity of ISW1a. This effect requires location of the Rap1 cognate sequence on linker that becomes entry DNA in the nucleosome remodeling process. In addition, Rap1 was able to hinder nucleosome assembly in octamer transfer assays. Concurrently, Rap1 displayed the highest affinity for and longest dwell time from its target sequence, compared to the other GRFs tested. Consistently, through bioinformatics analyses of publicly available genome-wide data, we found that nucleosome occupancy and histone deposition in vivo are inversely correlated with the affinity of Rap1 for its target sequences in the genome.
CONCLUSIONS
Our findings point to DNA binding affinity, residence time and location at particular translational positions relative to the nucleosome core as the key features of GRFs underlying their roles played in nucleosome sliding and assembly.
Topics: Nucleosomes; Chromatin Assembly and Disassembly; Adenosine Triphosphatases; Saccharomyces cerevisiae Proteins; Transcription Factors; Saccharomyces cerevisiae; Histones; DNA-Binding Proteins
PubMed: 38704609
DOI: 10.1186/s40659-024-00500-6 -
Current Opinion in Genetics &... Jun 2024Polycomb-associated chromatin and pericentromeric heterochromatin form genomic domains important for the epigenetic regulation of gene expression. Both Polycomb... (Review)
Review
Polycomb-associated chromatin and pericentromeric heterochromatin form genomic domains important for the epigenetic regulation of gene expression. Both Polycomb complexes and heterochromatin factors rely on 'read and write' mechanisms, which, on their own, are not sufficient to explain the formation and the maintenance of these epigenetic domains. Microscopy has revealed that they form specific nuclear compartments separated from the rest of the genome. Recently, some subunits of these molecular machineries have been shown to undergo phase separation, both in vitro and in vivo, suggesting that phase separation might play important roles in the formation and the function of these two kinds of repressive chromatin. In this review, we will present the recent advances in the field of facultative and constitutive heterochromatin formation and maintenance through phase separation.
Topics: Heterochromatin; Epigenesis, Genetic; Polycomb-Group Proteins; Chromatin; Animals; Humans; Histones; Chromatin Assembly and Disassembly; Phase Separation
PubMed: 38701672
DOI: 10.1016/j.gde.2024.102201 -
Cell Reports May 2024During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and...
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 24 h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48 h 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.
Topics: Humans; Cellular Reprogramming; Chromatin Assembly and Disassembly; Histones; Single-Cell Analysis; Transcriptional Activation; Octamer Transcription Factor-3; Chromatin; Nanog Homeobox Protein; Induced Pluripotent Stem Cells
PubMed: 38700983
DOI: 10.1016/j.celrep.2024.114170 -
Open Biology May 2024The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells,...
The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells, those in totipotent cells, such as murine zygotes or two-cell embryos, have a unique nucleolar structure known as nucleolus precursor bodies (NPBs). Previously, it was widely accepted that NPBs in zygotes are simply passive repositories of materials that will be gradually used to construct a fully functional nucleolus after zygotic genome activation (ZGA). However, recent research studies have challenged this simplistic view and demonstrated that functions of the NPBs go beyond ribosome biogenesis. In this review, we provide a snapshot of the functions of NPBs in zygotes and early two-cell embryos in mice. We propose that these membrane-less organelles function as a regulatory hub for chromatin organization. On the one hand, NPBs provide the structural platform for centric and pericentric chromatin remodelling. On the other hand, the dynamic changes in nucleolar structure control the release of the pioneer factors (i.e. double homeobox (Dux)). It appears that during transition from totipotency to pluripotency, decline of totipotency and initiation of fully functional nucleolus formation are not independent events but are interconnected. Consequently, it is reasonable to hypothesize that dissecting more unknown functions of NPBs may shed more light on the enigmas of early embryonic development and may ultimately provide novel approaches to improve reprogramming efficiency.
Topics: Animals; Humans; Mice; Cell Nucleolus; Chromatin; Chromatin Assembly and Disassembly; Embryonic Development; Gene Expression Regulation, Developmental; Zygote
PubMed: 38689555
DOI: 10.1098/rsob.230358