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Project description:Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts. We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. Our results provide an evolutionary perspective on H3.3's conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.
| S-EPMC10080907 | biostudies-literature
Project description:Through epigenetic modifications and selective incorporation of histone variants into chromatin, eukaryotic cells can adjust their transcriptional profile in response to molecular needs. We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, as a model system to investigate the dynamics of H3 variant function. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggests that both Cac2 and Hir1 might be dispensable for the replication-dependent and -independent deposition of H3.1 and H3.3, respectively. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over promoters, genes bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. Our results provide an evolutionary perspective on H3.3’s conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.
2023-04-03 | GSE210902 | GEO
Project description:Through epigenetic modifications and selective incorporation of histone variants into chromatin, eukaryotic cells can adjust their transcriptional profile in response to molecular needs. We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, as a model system to investigate the dynamics of H3 variant function. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggests that both Cac2 and Hir1 might be dispensable for the replication-dependent and -independent deposition of H3.1 and H3.3, respectively. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over promoters, genes bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. Our results provide an evolutionary perspective on H3.3’s conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.
2023-04-03 | GSE210901 | GEO
Project description:Multilevel interrogation of H3.3 dynamics reveals a primordial role in transcription regulation in Tetrahymena.
| PRJNA868288 | ENA
Project description:Multilevel interrogation of H3.3 dynamics reveals a primordial role in transcription regulation in Tetrahymena [ChIP-seq]
| PRJNA868292 | ENA
Project description:Multilevel interrogation of H3.3 dynamics reveals a primordial role in transcription regulation in Tetrahymena [RNA-seq]
| PRJNA868291 | ENA
Project description:In Escherichia coli, translocation of RNA polymerase (RNAP) during transcription introduces supercoiling to DNA, which influences the initiation and elongation behaviors of RNAP. To quantify the role of supercoiling in transcription regulation, we developed a spatially resolved supercoiling model of transcription. The integrated model describes how RNAP activity feeds back with the local DNA supercoiling and how this mechanochemical feedback controls transcription, subject to topoisomerase activities and stochastic topological domain formation. This model establishes that transcription-induced supercoiling mediates the cooperation of co-transcribing RNAP molecules in highly expressed genes, and this cooperation is achieved under moderate supercoiling diffusion and high topoisomerase unbinding rates. It predicts that a topological domain could serve as a transcription regulator, generating substantial transcriptional noise. It also shows the relative orientation of two closely arranged genes plays an important role in regulating their transcription. The model provides a quantitative platform for investigating how genome organization impacts transcription.
| S-EPMC9522292 | biostudies-literature
Project description:Complex organisms can rapidly induce select genes in response to diverse environmental cues. This regulation occurs in the context of large genomes condensed by histone proteins into chromatin. The sensing of pathogens by macrophages engages conserved signalling pathways and transcription factors to coordinate the induction of inflammatory genes1-3. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a general feature of dynamically regulated chromatin and transcription4-7. However, how chromatin is regulated at induced genes, and what features of H3.3 might enable rapid and high-level transcription, are unknown. The amino terminus of H3.3 contains a unique serine residue (Ser31) that is absent in 'canonical' H3.1 and H3.2. Here we show that this residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along rapidly induced genes in mouse macrophages. This selective mark of stimulation-responsive genes directly engages the histone methyltransferase SETD2, a component of the active transcription machinery, and 'ejects' the elongation corepressor ZMYND118,9. We propose that features of H3.3 at stimulation-induced genes, including H3.3S31ph, provide preferential access to the transcription apparatus. Our results indicate dedicated mechanisms that enable rapid transcription involving the histone variant H3.3, its phosphorylation, and both the recruitment and the ejection of chromatin regulators.
| S-EPMC7517595 | biostudies-literature
Project description:Liver fibrosis is a common feature of progressive liver disease and is manifested as a dynamic series of alterations in both the biochemical and biophysical properties of the liver. Hepatic stellate cells (HSCs) reside within the perisinusoidal space of the liver sinusoid and are one of the main drivers of liver fibrosis, yet it remains unclear how changes to the sinusoidal microenvironment impact HSC phenotype in the context of liver fibrosis. Cellular microarrays were used to examine and deconstruct the impacts of bio-chemo-mechanical changes on activated HSCs in vitro. Extracellular matrix (ECM) composition and stiffness were found to act individually and in combination to regulate HSC fibrogenic phenotype and proliferation. Hyaluronic acid and collagen III promoted elevated collagen I expression while collagen IV mediated a decrease. Previously activated HSCs exhibited reduced lysyl oxidase (Lox) expression as array substrate stiffness increased, with less dependence on ECM composition. Collagens III and IV increased HSC proliferation, whereas hyaluronic acid had the opposite effect. Meta-analysis performed on these data revealed distinct phenotypic clusters (e.g. low fibrogenesis/high proliferation) as a direct function of their microenvironmental composition. Notably, soft microenvironments mimicking healthy tissue (1 kPa), promoted higher levels of intracellular collagen I and Lox expression in activated HSCs, compared to stiff microenvironments mimicking fibrotic tissue (25 kPa). Collectively, these data suggest potential HSC functional adaptations in response to specific bio-chemo-mechanical changes relevant towards the development of therapeutic interventions. These findings also underscore the importance of the microenvironment when interrogating HSC behavior in healthy, disease, and treatment settings. STATEMENT OF SIGNIFICANCE: In this work we utilized high-throughput cellular microarray technology to systematically interrogate the complex interactions between HSCs and their microenvironment in the context of liver fibrosis. We observed that HSC phenotype is regulated by ECM composition and stiffness, and that these phenotypes can be classified into distinct clusters based on their microenvironmental context. Moreover, the range of these phenotypic responses to microenvironmental stimuli is substantial and a direct consequence of the combinatorial pairing of ECM protein and stiffness signals. We also observed a novel role for microenvironmental context in affecting HSC responses to potential fibrosis therapeutics.
| S-EPMC8738161 | biostudies-literature
Project description:All three genes encoding histone H3 proteins were cloned and sequenced from Tetrahymena thermophila. Two of these genes encode a major H3 protein identical to that of T. pyriformis and 87% identical to the major H3 of vertebrates. The third gene encodes hv2, a quantitatively minor replication independent (replacement) variant. The sequence of hv2 is only 85% identical to the animal replacement variant H3.3 and is the most divergent H3 replacement variant described. Phylogenetic analysis of 73 H3 protein sequences suggests that hv2, H3.3, and the plant replacement variant H3.III evolved independently, and that H3.3 is not the ancestral H3 gene, as was previously suggested (Wells, D., Bains, W., and Kedes, L. 1986, J. Mol. Evol., 23: 224-241). These results suggest it is the replication independence and not the particular protein sequence that is important in the function of H3 replacement variants.
| S-EPMC307769 | biostudies-other