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Science (New York, N.Y.) Jul 2023Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of...
Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of chromatin regulatory factors during this universal process remains unclear. In this work, we developed chromatin expansion microscopy (ChromExM) to visualize chromatin, transcription, and transcription factors in vivo. ChromExM of embryos during ZGA revealed how the pioneer factor Nanog interacts with nucleosomes and RNA polymerase II (Pol II), providing direct visualization of transcriptional elongation as string-like nanostructures. Blocking elongation led to more Pol II particles clustered around Nanog, with Pol II stalled at promoters and Nanog-bound enhancers. This led to a new model termed "kiss and kick", in which enhancer-promoter contacts are transient and released by transcriptional elongation. Our results demonstrate that ChromExM is broadly applicable to study nanoscale nuclear organization.
Topics: Chromatin; Nucleosomes; RNA Polymerase II; Transcription, Genetic; Microscopy, Fluorescence; Animals; Zebrafish; Embryo, Nonmammalian; Zygote; Nanog Homeobox Protein
PubMed: 37410825
DOI: 10.1126/science.ade5308 -
Trends in Genetics : TIG Sep 2023Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated... (Review)
Review
Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated together. Splicing is catalyzed by a dynamic ribonucleoprotein complex called the spliceosome. Recent evidence has shown that a large fraction of splicing occurs cotranscriptionally as the RNA chain is extruded from Pol II at speeds of up to 5 kb/minute. Splicing is more efficient when it is tethered to the transcription elongation complex, and this linkage permits functional coupling of splicing with transcription. We discuss recent progress that has uncovered a network of connections that link splicing to transcript elongation and other cotranscriptional RNA processing events.
Topics: RNA Precursors; Transcription, Genetic; RNA Splicing; Spliceosomes; Introns
PubMed: 37236814
DOI: 10.1016/j.tig.2023.04.008 -
Journal of Extracellular Vesicles Feb 2024Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These...
Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.
Topics: Extracellular Vesicles; Exosomes; Biological Transport; Biomarkers; Phenotype
PubMed: 38326288
DOI: 10.1002/jev2.12404 -
Molecular Cell Nov 2023The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental... (Review)
Review
The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging.
Topics: Animals; Humans; RNA Polymerase II; Transcriptional Elongation Factors; Neurodegenerative Diseases; Transcription, Genetic; Gene Expression Regulation; Aging; Genes, Developmental
PubMed: 37922911
DOI: 10.1016/j.molcel.2023.10.004 -
Journal of Clinical Microbiology Aug 2023Bacterial vaginosis (BV) is the most common cause of vaginal discharge among reproductive-age women. It is associated with multiple adverse health outcomes, including... (Review)
Review
Bacterial vaginosis (BV) is the most common cause of vaginal discharge among reproductive-age women. It is associated with multiple adverse health outcomes, including increased risk of acquisition of HIV and other sexually transmitted infections (STIs), in addition to adverse birth outcomes. While it is known that BV is a vaginal dysbiosis characterized by a shift in the vaginal microbiota from protective species to an increase in facultative and strict anaerobic bacteria, its exact etiology remains unknown. The purpose of this minireview is to provide an updated overview of the range of tests currently used for the diagnosis of BV in both clinical and research settings. This article is divided into two primary sections: traditional BV diagnostics and molecular diagnostics. Molecular diagnostic assays, particularly 16S rRNA gene sequencing, shotgun metagenomic sequencing, and fluorescence hybridization (FISH), are specifically highlighted, in addition to multiplex nucleic acid amplification tests (NAATs), given their increasing use in clinical practice (NAATs) and research studies (16S rRNA gene sequencing, shotgun metagenomic sequencing, and FISH) regarding the vaginal microbiota and BV pathogenesis. We also provide a discussion of the strengths and weaknesses of current BV diagnostic tests and discuss future challenges in this field of research.
Topics: Humans; Female; Vaginosis, Bacterial; RNA, Ribosomal, 16S; In Situ Hybridization, Fluorescence; Vagina; Sexually Transmitted Diseases
PubMed: 37199636
DOI: 10.1128/jcm.00837-22 -
Nature Communications Aug 2023Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low...
Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (Polϴ). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.
Topics: Gene Editing; CRISPR-Cas Systems; Protein Kinases; DNA Repair; DNA
PubMed: 37580318
DOI: 10.1038/s41467-023-40344-4 -
Nature Aug 2023The transcriptional machinery is thought to dissociate from DNA during replication. Certain proteins, termed epigenetic marks, must be transferred from parent to...
The transcriptional machinery is thought to dissociate from DNA during replication. Certain proteins, termed epigenetic marks, must be transferred from parent to daughter DNA strands in order to maintain the memory of transcriptional states. These proteins are believed to re-initiate rebuilding of chromatin structure, which ultimately recruits RNA polymerase II (Pol II) to the newly replicated daughter strands. It is believed that Pol II is recruited back to active genes only after chromatin is rebuilt. However, there is little experimental evidence addressing the central questions of when and how Pol II is recruited back to the daughter strands and resumes transcription. Here we show that immediately after passage of the replication fork, Pol II in complex with other general transcription proteins and immature RNA re-associates with active genes on both leading and lagging strands of nascent DNA, and rapidly resumes transcription. This suggests that the transcriptionally active Pol II complex is retained in close proximity to DNA, with a Pol II-PCNA interaction potentially underlying this retention. These findings indicate that the Pol II machinery may not require epigenetic marks to be recruited to the newly synthesized DNA during the transition from DNA replication to resumption of transcription.
Topics: Chromatin; DNA; DNA Polymerase II; DNA Replication; Epigenesis, Genetic; Genes; Proliferating Cell Nuclear Antigen; RNA Polymerase II; Transcription, Genetic; Transcription Factors, General; RNA
PubMed: 37468626
DOI: 10.1038/s41586-023-06341-9 -
Nature Genetics Aug 2023How enhancers control target gene expression over long genomic distances remains an important unsolved problem. Here we investigated enhancer-promoter communication by...
How enhancers control target gene expression over long genomic distances remains an important unsolved problem. Here we investigated enhancer-promoter communication by integrating data from nucleosome-resolution genomic contact maps, nascent transcription and perturbations affecting either RNA polymerase II (Pol II) dynamics or the activity of thousands of candidate enhancers. Integration of new Micro-C experiments with published CRISPRi data demonstrated that enhancers spend more time in close proximity to their target promoters in functional enhancer-promoter pairs compared to nonfunctional pairs, which can be attributed in part to factors unrelated to genomic position. Manipulation of the transcription cycle demonstrated a key role for Pol II in enhancer-promoter interactions. Notably, promoter-proximal paused Pol II itself partially stabilized interactions. We propose an updated model in which elements of transcriptional dynamics shape the duration or frequency of interactions to facilitate enhancer-promoter communication.
Topics: RNA Polymerase II; Enhancer Elements, Genetic; Promoter Regions, Genetic; Transcription, Genetic
PubMed: 37430091
DOI: 10.1038/s41588-023-01442-7 -
Distinct layers of BRD4-PTEFb reveal bromodomain-independent function in transcriptional regulation.Molecular Cell Aug 2023The BET family protein BRD4, which forms the CDK9-containing BRD4-PTEFb complex, is considered to be a master regulator of RNA polymerase II (Pol II) pause release....
The BET family protein BRD4, which forms the CDK9-containing BRD4-PTEFb complex, is considered to be a master regulator of RNA polymerase II (Pol II) pause release. Because its tandem bromodomains interact with acetylated histone lysine residues, it has long been thought that BRD4 requires these bromodomains for its recruitment to chromatin and transcriptional regulatory function. Here, using rapid depletion and genetic complementation with domain deletion mutants, we demonstrate that BRD4 bromodomains are dispensable for Pol II pause release. A minimal, bromodomain-less C-terminal BRD4 fragment containing the PTEFb-interacting C-terminal motif (CTM) is instead both necessary and sufficient to mediate Pol II pause release in the absence of full-length BRD4. Although BRD4-PTEFb can associate with chromatin through acetyl recognition, our results indicate that a distinct, active BRD4-PTEFb population functions to regulate transcription independently of bromodomain-mediated chromatin association. These findings may enable more effective pharmaceutical modulation of BRD4-PTEFb activity.
Topics: Nuclear Proteins; Transcription Factors; Histones; Gene Expression Regulation; Chromatin; Cell Cycle Proteins
PubMed: 37442129
DOI: 10.1016/j.molcel.2023.06.032 -
Cell Chemical Biology Oct 2023KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including...
KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.
Topics: Humans; Female; Breast Neoplasms; Histones; Histone Acetyltransferases; Signal Transduction; Cell Line, Tumor
PubMed: 37557181
DOI: 10.1016/j.chembiol.2023.07.005