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Molecular Cell Jun 2024Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase...
Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.
Topics: Cyclin-Dependent Kinase-Activating Kinase; Humans; Cyclin-Dependent Kinases; RNA Polymerase II; Promoter Regions, Genetic; Phosphorylation; Transcription Initiation, Genetic; Protein Kinase Inhibitors; Mediator Complex; HeLa Cells; Transcription Factor TFIIH; HEK293 Cells
PubMed: 38821049
DOI: 10.1016/j.molcel.2024.05.007 -
Nature Communications May 2024Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a...
Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.
Topics: Humans; Glioma; Animals; Positive Transcriptional Elongation Factor B; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Mice; RNA Polymerase II; Transcription, Genetic; Apoptosis; Brain Neoplasms; DNA Repair; Xenograft Model Antitumor Assays
PubMed: 38816355
DOI: 10.1038/s41467-024-48214-3 -
The Journal of Biological Chemistry May 2024Autophagy is a pivotal regulatory and catabolic process, induced under various stressful conditions, including hypoxia. However, little is known about alternative...
Autophagy is a pivotal regulatory and catabolic process, induced under various stressful conditions, including hypoxia. However, little is known about alternative splicing of autophagy genes in the hypoxic landscape in breast cancer. Our research unravels the hitherto unreported alternative splicing of BNIP3L, a crucial hypoxia-induced autophagic gene. We showed that BNIP3L, under hypoxic condition, forms two isoforms, a full-length isoform (BNIP3L-F) and a shorter isoform lacking exon 1 (BNIP3L-Δ1). The hypoxia-induced BNIP3L-F promotes autophagy, while under normoxia, the BNIP3L-Δ1 inhibits autophagy. We discovered a novel dimension of hypoxia-mediated epigenetic modification that regulates the alternative splicing of BNIP3L. Here, we showed differential DNA methylation of BNIP3L intron 1, causing reciprocal binding of epigenetic factor CCCTC-binding factor (CTCF) and its paralog BORIS. Additionally, we highlighted the role of CTCF and BORIS impacting autophagy in breast cancer. The differential binding of CTCF and BORIS results in alternative splicing of BNIP3L forming BNIP3L-F and BNIP3L-Δ1, respectively. The binding of CTCF on unmethylated BNIP3L intron 1 under hypoxia results in RNA Pol-II pause and inclusion of exon 1, promoting BNIP3L-F and autophagy. Interestingly, the binding of BORIS on methylated BNIP3L intron 1 under normoxia also results in RNA Pol-II pause but leads to the exclusion of exon 1 from BNIP3L mRNA. Finally, we reported the critical role of BORIS-mediated RNA Pol-II pause, which subsequently recruits SRSF6, redirecting the proximal splice-site selection, promoting BNIP3L-Δ1, and inhibiting autophagy. Our study provides novel insights into the potential avenues for breast cancer therapy by targeting autophagy regulation, specifically under hypoxic condition.
PubMed: 38810696
DOI: 10.1016/j.jbc.2024.107416 -
Polish Journal of Microbiology Jun 2024is a non-fermentative Gram-negative bacterium that can cause nosocomial infections in critically ill patients. Carbapenem-resistant (CRAB) has spread rapidly in...
is a non-fermentative Gram-negative bacterium that can cause nosocomial infections in critically ill patients. Carbapenem-resistant (CRAB) has spread rapidly in clinical settings and has become a key concern. The main objective of this study was to identify the distribution of integrons and biofilm-formation-related virulence genes in CRAB isolates. A total of 269 isolates (219 isolates of CRAB and 50 isolates of carbapenem-sensitive (CSAB)) were collected. Carbapenemase genes ( , , , , and ) and biofilm-formation-related virulence genes (, , , and ) were screened with PCR. Class 1 integron was screened with PCR, and common promoters and gene cassette arrays were determined with restriction pattern analysis combined with primer walking sequencing. Whole-genome sequencing was conducted, and data were analyzed for a -negative isolate. All 219 CRAB isolates were negative for , , , and , while was detected in 218 isolates. The detection rates for , , , and in 219 CRAB were 93.15%, 63.93%, 88.13%, and 77.63%, respectively. Class 1 integron was detected in 75 CRAB (34.25%) and in 3 CSAB. The single gene cassette array with relatively strong PcH2 promoter was detected in class 1 integrons. The -negative CRAB isolate was revealed to be a new sequence type (Oxford 3272, Pasteur 2520) carrying , , and . In conclusion, was the main reason for CRAB's resistance to carbapenems. A new (Oxford 3272, Pasteur 2520) CRAB sequence type carrying the , , and was reported.
Topics: Acinetobacter baumannii; beta-Lactamases; Integrons; Biofilms; Bacterial Proteins; Acinetobacter Infections; Humans; Anti-Bacterial Agents; Carbapenems; Microbial Sensitivity Tests
PubMed: 38808771
DOI: 10.33073/pjm-2024-017 -
The Plant Genome Jun 2024The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating...
The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type-V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1-RRA variant did not perform better than the wild-type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants.
Topics: Oryza; Solanum lycopersicum; Gene Editing; CRISPR-Cas Systems; Protoplasts; Genome, Plant
PubMed: 38807445
DOI: 10.1002/tpg2.20465 -
Microbial Ecology May 2024Water-filled sinkholes known locally as cenotes, found on the Yucatán Peninsula, have remarkable biodiversity. The primary objective of this study was to explore the...
Water-filled sinkholes known locally as cenotes, found on the Yucatán Peninsula, have remarkable biodiversity. The primary objective of this study was to explore the biotechnological potential of Gram-positive cultivable bacteria obtained from sediment samples collected at the coastal cenote Pol-Ac in Yucatán, Mexico. Specifically, the investigation aimed to assess production of hydrolytic enzymes and antimicrobial compounds. 16 S rRNA gene sequencing led to the identification of 49 Gram-positive bacterial isolates belonging to the phyla Bacillota (n = 29) and Actinomycetota (n = 20) divided into the common genera Bacillus and Streptomyces, as well as the genera Virgibacillus, Halobacillus, Metabacillus, Solibacillus, Neobacillus, Rossellomorea, Nocardiopsis and Corynebacterium. With growth at 55ºC, 21 of the 49 strains were classified as moderately thermotolerant. All strains were classified as halotolerant and 24 were dependent on marine water for growth. Screening for six extracellular hydrolytic enzymes revealed gelatinase, amylase, lipase, cellulase, protease and chitinase activities in 93.9%, 67.3%, 63.3%, 59.2%, 59.2% and 38.8%, of isolated strains, respectively. The genes for polyketide synthases type I, were detected in 24 of the strains. Of 18 strains that achieved > 25% inhibition of growth in the bacterial pathogen Staphylococcus aureus ATCC 6538, 4 also inhibited growth in Escherichia coli ATCC 35,218. Isolates Streptomyces sp. NCA_378 and Bacillus sp. NCA_374 demonstrated 50-75% growth inhibition against at least one of the two pathogens tested, along with significant enzymatic activity across all six extracellular enzymes. This is the first comprehensive report on the biotechnological potential of Gram-positive bacteria isolated from sediments in the cenotes of the Yucatán Peninsula.
Topics: Geologic Sediments; Mexico; Biodiversity; Gram-Positive Bacteria; RNA, Ribosomal, 16S; Bioprospecting; Phylogeny; Anti-Bacterial Agents; Seawater
PubMed: 38806738
DOI: 10.1007/s00248-024-02392-1 -
Biochemistry Jun 2024Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of...
Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of vital cellular processes, encompassing proliferation, apoptosis, migration, tumor suppression, and differentiation. It has emerged as a key player in neuronal differentiation by orchestrating the expression of neuronal lineage-committed genes. The present study uncovers the role of ZMYND8 in regulating the Sonic Hedgehog (SHH) signaling axis, which is crucial for neuronal differentiation. Genetic deletion of ZMYND8 leads to a significant reduction in SHH pathway genes, , and expression during all-trans-retinoic acid (ATRA)-induced differentiation. ZMYND8 and RNA pol II S5P are found to co-occupy the and gene promoters, positively impacting their gene transcription upon ATRA treatment. Interestingly, ZMYND8 is found to counteract the inhibitory effects of Cyclopamine that block the upstream SHH pathway protein SMO, resulting in enhanced neurite formation in neuroblastoma cells following their treatment with ATRA. These results indicate that ZMYND8 is an epigenetic regulator of the SHH signaling pathway and has tremendous therapeutic potential in ATRA-mediated differentiation of neuroblastoma.
Topics: Hedgehog Proteins; Humans; Cell Differentiation; Tretinoin; Signal Transduction; Neuroblastoma; Cell Line, Tumor; Patched-1 Receptor; Transcription Factors; Zinc Finger Protein GLI1; Gene Expression Regulation, Neoplastic; Mice; Animals; Tumor Suppressor Proteins
PubMed: 38804064
DOI: 10.1021/acs.biochem.4c00145 -
IScience Jun 2024RNA polymerase II (Pol II) has a C-terminal domain (CTD) that is unstructured, consisting of a large number of heptad repeats, and whose precise function remains...
RNA polymerase II (Pol II) has a C-terminal domain (CTD) that is unstructured, consisting of a large number of heptad repeats, and whose precise function remains unclear. Here, we investigate how altering the CTD's length and fusing it with protein tags affects transcriptional output on a genome-wide scale in mammalian cells at single-cell resolution. While transcription generally appears to occur in burst-like fashion, where RNA is predominantly made during short bursts of activity that are interspersed with periods of transcriptional silence, the CTD's role in shaping these dynamics seems gene-dependent; global patterns of bursting appear mostly robust to CTD alterations. Introducing protein tags with defined structures to the N terminus cause transcriptome-wide effects, however. We find the type of tag to dominate characteristics of the resulting transcriptomes. This is possibly due to Pol II-interacting factors, including non-coding RNAs, whose expression correlates with the tags. Proteins involved in liquid-liquid phase separation appear prominently.
PubMed: 38799575
DOI: 10.1016/j.isci.2024.109914 -
BioRxiv : the Preprint Server For... May 2024serovar Typhimurium causes acute diarrhea upon oral infection in humans. The harsh and proteolytic environment found in the gastrointestinal tract is the first obstacle...
UNLABELLED
serovar Typhimurium causes acute diarrhea upon oral infection in humans. The harsh and proteolytic environment found in the gastrointestinal tract is the first obstacle that these bacteria face after infection. However, the mechanisms that allow to survive the hostile conditions of the gut are poorly understood. The gene is found in an extensive range of known phyla of bacteria and it encodes a protein that has been shown to inhibit serine proteases. Thus, in the present work we studied the role of of Typhimurium in host-pathogen interactions. We found that Typhimurium Δ strain exhibited lower inflammation in a murine model of induced colitis. The Δ mutant was more susceptible to the action of pancreatin and purified pancreatic elastase. In addition, the lack of led to impaired adhesion to Caco-2 and HT-29 cell lines, related to the proteolytic activity of brush border enzymes. Besides, Δ showed higher susceptibility to lysosomal proteolytic content and intracellular replication defects in macrophages. In addition, we found Ecotin to have a crucial role in against the microbicide action of granules released and neutrophil extracellular traps from human polymorphonuclear leukocytes. Thus, the work presented here highlights the importance of in as countermeasures against the host proteolytic defense system.
IMPORTANCE
The gastrointestinal tract is a very complex and harsh environment. is a successful food borne pathogen, but little is known about its capacity to survive against the proteolysis of the gut lumen and intracellular proteases. Here, we show that Ecotin, a serine protease inhibitor, plays an important role in protecting against proteases present at different sites encountered during oral infection. Our results indicate that Ecotin is an important virulence factor in , adding another tool to the wide range of features this pathogen uses during oral infection.
PubMed: 38798423
DOI: 10.1101/2024.05.15.594389 -
Nature Communications May 2024In plants, the plant-specific RNA polymerase V (Pol V) transcripts non-coding RNAs and provides a docking platform for the association of accessory proteins in the...
In plants, the plant-specific RNA polymerase V (Pol V) transcripts non-coding RNAs and provides a docking platform for the association of accessory proteins in the RNA-directed DNA methylation (RdDM) pathway. Various components have been uncovered that are involved in the process of DNA methylation, but it is still not clear how the transcription of Pol V is regulated. Here, we report that the conserved RNA polymerase II (Pol II) elongator, SPT6L, binds to thousands of intergenic regions in a Pol II-independent manner. The intergenic enrichment of SPT6L, interestingly, co-occupies with the largest subunit of Pol V (NRPE1) and mutation of SPT6L leads to the reduction of DNA methylation but not Pol V enrichment. Furthermore, the association of SPT6L at Pol V loci is dependent on the Pol V associated factor, SPT5L, rather than the presence of Pol V, and the interaction between SPT6L and NRPE1 is compromised in spt5l. Finally, Pol V RIP-seq reveals that SPT6L is required to maintain the amount and length of Pol V transcripts. Our findings thus uncover the critical role of a Pol II conserved elongator in Pol V mediated DNA methylation and transcription, and shed light on the mutual regulation between Pol V and II in plants.
Topics: Arabidopsis; Arabidopsis Proteins; DNA Methylation; DNA-Directed RNA Polymerases; Gene Expression Regulation, Plant; Mutation; RNA Polymerase II; RNA, Plant; Transcription, Genetic; Transcriptional Elongation Factors
PubMed: 38796517
DOI: 10.1038/s41467-024-48940-8