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Nature Cell Biology Apr 2024The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if...
The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how transcription bodies affect gene expression. Here we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-seq and live-cell imaging. We find that the disruption of transcription bodies results in the misregulation of hundreds of genes. Here we focus on genes that are upregulated. These genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9-the kinase that releases paused polymerase II-is highly enriched in the two large transcription bodies. Overexpression of CDK9 in wild-type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription by sequestering machinery, thereby preventing genes elsewhere in the nucleus from being transcribed.
Topics: Animals; Chromatin; Gene Expression; Positive Transcriptional Elongation Factor B; RNA Polymerase II; Transcription, Genetic; Zebrafish
PubMed: 38589534
DOI: 10.1038/s41556-024-01389-9 -
The Journal of Biological Chemistry May 2024DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6...
DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6 complex with DnaB and opens a channel in the DnaB hexamer through which single-stranded DNA is thought to pass. During replication, replisomes frequently encounter DNA damage and nucleoprotein complexes that can lead to replication fork collapse. Such events require DnaB re-loading onto DNA to allow replication to continue. Replication restart proteins mediate this process by recruiting DnaB/DnaC to abandoned DNA replication forks. Several dnaC mutations that bypass the requirement for replication restart proteins or that block replication restart have been identified in E. coli. To better understand how these DnaC variants function, we have purified and characterized the protein products of several such alleles. Unlike wild-type DnaC, three of the variants (DnaC 809, DnaC 809,820, and DnaC 811) can load DnaB onto replication forks bound by single-stranded DNA-binding protein. DnaC 809 can also load DnaB onto double-stranded DNA. These results suggest that structural changes in the variant DnaB/DnaC complexes expand the range of DNA substrates that can be used for DnaB loading, obviating the need for the existing replication restart pathways. The protein product of dnaC1331, which phenocopies deletion of the priB replication restart gene, blocks loading through the major restart pathway in vitro. Overall, the results of our study highlight the utility of bacterial DnaC variants as tools for probing the regulatory mechanisms that govern replicative helicase loading.
Topics: Escherichia coli Proteins; Escherichia coli; DnaB Helicases; DNA Replication; DNA, Bacterial; DNA, Single-Stranded; Mutation
PubMed: 38588814
DOI: 10.1016/j.jbc.2024.107275 -
BioRxiv : the Preprint Server For... May 2024Prostate cancer is an exemplar of an enhancer-binding transcription factor-driven disease. The androgen receptor (AR) enhanceosome complex comprised of chromatin and...
Prostate cancer is an exemplar of an enhancer-binding transcription factor-driven disease. The androgen receptor (AR) enhanceosome complex comprised of chromatin and epigenetic coregulators assembles at enhancer elements to drive disease progression. The paralog lysine acetyltransferases p300 and CBP deposit histone marks that are associated with enhancer activation. Here, we demonstrate that p300/CBP are determinant cofactors of the active AR enhanceosome in prostate cancer. Histone H2B N-terminus multisite lysine acetylation (H2BNTac), which is exclusively reliant on p300/CBP catalytic function, marked active enhancers and was notably elevated in prostate cancer lesions relative to the adjacent benign epithelia. Degradation of p300/CBP rapidly depleted acetylation marks associated with the active AR enhanceosome, which was only partially phenocopied by inhibition of their reader bromodomains. Notably, H2BNTac was effectively abrogated only upon p300/CBP degradation, which led to a stronger suppression of p300/CBP-dependent oncogenic gene programs relative to bromodomain inhibition or the inhibition of its catalytic domain. experiments using an orally active p300/CBP proteolysis targeting chimera (PROTAC) degrader (CBPD-409) showed that p300/CBP degradation potently inhibited tumor growth in preclinical models of castration-resistant prostate cancer and synergized with AR antagonists. While mouse p300/CBP orthologs were effectively degraded in host tissues, prolonged treatment with the PROTAC degrader was well tolerated with no significant signs of toxicity. Taken together, our study highlights the pivotal role of p300/CBP in maintaining the active AR enhanceosome and demonstrates how target degradation may have functionally distinct effects relative to target inhibition, thus supporting the development of p300/CBP degraders for the treatment of advanced prostate cancer.
PubMed: 38586029
DOI: 10.1101/2024.03.29.587346 -
MedRxiv : the Preprint Server For... Mar 2024To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs).
PURPOSE
To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs).
METHODS
We coupled phenotyping with exome or genome sequencing of 467 pedigrees with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations.
RESULTS
Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., ), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., ), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses.
CONCLUSION
This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.
PubMed: 38585811
DOI: 10.1101/2024.03.22.24304594 -
Neurobiology of Disease Jun 2024Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes,...
Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRS mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of Yars mice modelling DI-CMT. We determined that Yars homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRS, but not TyrRS, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.
Topics: Animals; Charcot-Marie-Tooth Disease; Brain-Derived Neurotrophic Factor; Mice; Axonal Transport; Disease Models, Animal; Tyrosine-tRNA Ligase; Humans; Mice, Transgenic; Muscle, Skeletal; Receptor, trkB; Mutation
PubMed: 38583640
DOI: 10.1016/j.nbd.2024.106501 -
The Journal of Investigative Dermatology Apr 2024Chronic non-healing wounds negatively impact quality of life and are a significant financial drain on health systems. The risk of infection that exacerbates...
Chronic non-healing wounds negatively impact quality of life and are a significant financial drain on health systems. The risk of infection that exacerbates comorbidities in patients necessitates regular application of wound care. Understanding the mechanisms underlying impaired wound healing are therefore a key priority to inform effective new-generation treatments. In this study, we demonstrate that 14-3-3-mediated suppression of signaling through ROCK is a critical mechanism that inhibits the healing of diabetic wounds. Accordingly, pharmacological inhibition of 14-3-3 by topical application of the sphingo-mimetic drug RB-11 to diabetic wounds on a mouse model of type II diabetes accelerated wound closure more than 2-fold than vehicle control, phenocopying our previous observations in 14-3-3ζ-knockout mice. We also demonstrate that accelerated closure of the wounded epidermis by 14-3-3 inhibition causes enhanced signaling through the Rho-ROCK pathway and that the underlying cellular mechanism involves the efficient recruitment of dermal fibroblasts into the wound and the rapid production of extracellular matrix proteins to re-establish the injured dermis. Our observations that the 14-3-3/ROCK inhibitory axis characterizes impaired wound healing and that its suppression facilitates fibroblast recruitment and accelerated re-epithelialization suggest new possibilities for treating diabetic wounds by pharmacologically targeting this axis.
PubMed: 38582367
DOI: 10.1016/j.jid.2024.03.032 -
RSC Chemical Biology Apr 2024Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development...
Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development of cell-free protein synthesis (CFPS) technology. Phenotypic screening identified numerous compounds that enhanced protein production in yeast lysate CFPS reactions. Notably, many of these were competitive ATP kinase inhibitors, with the exploitation of their inherent substrate promiscuity redirecting ATP flux towards heterologous protein expression. Chemoproteomic-guided strain engineering partially phenocopied drug effects, with a 30% increase in protein yield observed upon deletion of the ATP-consuming SSA1 component of the HSP70 chaperone. Moreover, drug-mediated metabolic rewiring coupled with template optimization generated the highest protein yields in yeast CFPS to date using a hitherto less efficient, but more cost-effective glucose energy regeneration system. Our approach highlights the utility of target-agnostic phenotypic screening and target identification to deconvolute cell-lysate complexity, adding to the expanding repertoire of strategies for improving CFPS.
PubMed: 38576719
DOI: 10.1039/d4cb00004h -
BioRxiv : the Preprint Server For... Mar 2024Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes,...
Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRS mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of mice modelling DI-CMT. We determined that homozygotes display a selective, age-dependent impairment in axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRS, but not TyrRS, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.
PubMed: 38559020
DOI: 10.1101/2023.04.09.536152 -
Infection and Immunity May 2024The mouse pathogen is utilized as a model organism for studying infections caused by the human pathogens enteropathogenic (EPEC) and enterohemorrhagic (EHEC) and to...
The mouse pathogen is utilized as a model organism for studying infections caused by the human pathogens enteropathogenic (EPEC) and enterohemorrhagic (EHEC) and to elucidate mechanisms of mucosal immunity. In response to infection, innate lymphoid cells and T cells secrete interleukin (IL)-22, a cytokine that promotes mucosal barrier function. IL-22 plays a pivotal role in enabling mice to survive and recover from infection, although the exact mechanisms involved remain incompletely understood. Here, we investigated whether particular components of the host response downstream of IL-22 contribute to the cytokine's protective effects during infection. In line with previous research, mice lacking the IL-22 gene ( mice) were highly susceptible to infection. To elucidate the role of specific antimicrobial proteins modulated by IL-22, we infected the following knockout mice: (calprotectin), (lipocalin-2), (Reg3β), (Reg3γ), and (C3). All knockout mice tested displayed a considerable level of resistance to infection, and none phenocopied the lethality observed in mice. By investigating another arm of the IL-22 response, we observed that -infected mice exhibited an overall decrease in gene expression related to intestinal barrier integrity as well as significantly elevated colonic inflammation, gut permeability, and pathogen levels in the spleen. Taken together, these results indicate that host resistance to lethal infection may depend on multiple antimicrobial responses acting in concert, or that other IL-22-regulated processes, such as tissue repair and maintenance of epithelial integrity, play crucial roles in host defense to attaching and effacing pathogens.
Topics: Animals; Mice; Citrobacter rodentium; Disease Models, Animal; Enterobacteriaceae Infections; Interleukin-22; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis-Associated Proteins
PubMed: 38557196
DOI: 10.1128/iai.00099-24 -
Nature Communications Mar 2024The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved...
The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5' regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process.
Topics: Animals; Female; Humans; Male; Cell Line; Gonadal Dysgenesis; Mammals; Regulatory Sequences, Nucleic Acid; Sertoli Cells; Sex-Determining Region Y Protein; Steroidogenic Factor 1; Testis
PubMed: 38555298
DOI: 10.1038/s41467-024-47162-2