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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 CTCF and its paralogue 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 May 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.
PubMed: 38808771
DOI: 10.33073/pjm-2024-017 -
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 -
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; Gene Expression Regulation, Plant; RNA Polymerase II; DNA-Directed RNA Polymerases; Transcription, Genetic; Transcriptional Elongation Factors; Mutation; RNA, Plant
PubMed: 38796517
DOI: 10.1038/s41467-024-48940-8 -
Briefings in Bioinformatics May 2024RNA Polymerase II (Pol II) transcriptional elongation pausing is an integral part of the dynamic regulation of gene transcription in the genome of metazoans. It plays a...
RNA Polymerase II (Pol II) transcriptional elongation pausing is an integral part of the dynamic regulation of gene transcription in the genome of metazoans. It plays a pivotal role in many vital biological processes and disease progression. However, experimentally measuring genome-wide Pol II pausing is technically challenging and the precise governing mechanism underlying this process is not fully understood. Here, we develop RP3 (RNA Polymerase II Pausing Prediction), a network regularized logistic regression machine learning method, to predict Pol II pausing events by integrating genome sequence, histone modification, gene expression, chromatin accessibility, and protein-protein interaction data. RP3 can accurately predict Pol II pausing in diverse cellular contexts and unveil the transcription factors that are associated with the Pol II pausing machinery. Furthermore, we utilize a forward feature selection framework to systematically identify the combination of histone modification signals associated with Pol II pausing. RP3 is freely available at https://github.com/AMSSwanglab/RP3.
Topics: RNA Polymerase II; Histone Code; Humans; Transcription Elongation, Genetic; Chromatin; Histones; Machine Learning; Animals
PubMed: 38783706
DOI: 10.1093/bib/bbae246 -
PLoS Pathogens May 2024The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral...
The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5' HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) of the core promoter by host cell pre-initiation complexes of HIV (PICH) has been shown to be necessary for Tat trans-activation, yet the protein composition of PICH has remained obscure. Here, DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo. Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control activated HIV gene expression, and as new drug targets for HIV cure strategies.
Topics: Humans; HIV-1; Gene Expression Regulation, Viral; Promoter Regions, Genetic; Virus Latency; HIV Infections; tat Gene Products, Human Immunodeficiency Virus; Viral Transcription
PubMed: 38781120
DOI: 10.1371/journal.ppat.1011821 -
Retrovirology May 2024Detection of viruses by host pattern recognition receptors induces the expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), which suppress viral...
BACKGROUND
Detection of viruses by host pattern recognition receptors induces the expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), which suppress viral replication. Numerous studies have described HIV-1 as a poor activator of innate immunity in vitro. The exact role that the viral capsid plays in this immune evasion is not fully understood.
RESULTS
To better understand the role of the HIV-1 capsid in sensing we tested the effect of making HIV-1 by co-expressing a truncated Gag that encodes the first 107 amino acids of capsid fused with luciferase or GFP, alongside wild type Gag-pol. We found that unlike wild type HIV-1, viral particles produced with a mixture of wild type and truncated Gag fused to luciferase or GFP induced a potent IFN response in THP-1 cells and macrophages. Innate immune activation by Gag-fusion HIV-1 was dependent on reverse transcription and DNA sensor cGAS, suggesting activation of an IFN response by viral DNA. Further investigation revealed incorporation of the Gag-luciferase/GFP fusion proteins into viral particles that correlated with subtle defects in wild type Gag cleavage and a diminished capacity to saturate restriction factor TRIM5α, likely due to aberrant particle formation. We propose that expression of the Gag fusion protein disturbs the correct cleavage and maturation of wild type Gag, yielding viral particles that are unable to effectively shield viral DNA from detection by innate sensors including cGAS.
CONCLUSIONS
These data highlight the crucial role of capsid in innate evasion and support growing literature that disruption of Gag cleavage and capsid formation induces a viral DNA- and cGAS-dependent innate immune response. Together these data demonstrate a protective role for capsid and suggest that antiviral activity of capsid-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.
Topics: HIV-1; Humans; gag Gene Products, Human Immunodeficiency Virus; Immunity, Innate; Nucleotidyltransferases; Antiviral Restriction Factors; Macrophages; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; THP-1 Cells; Carrier Proteins; Immune Evasion; Capsid; Virus Replication; Virion; Host-Pathogen Interactions; DNA, Viral; Cell Line
PubMed: 38778414
DOI: 10.1186/s12977-024-00643-0 -
Genome Biology May 2024DNA replication progression can be affected by the presence of physical barriers like the RNA polymerases, leading to replication stress and DNA damage. Nonetheless, we...
BACKGROUND
DNA replication progression can be affected by the presence of physical barriers like the RNA polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know how transcription influences overall DNA replication progression.
RESULTS
To characterize sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase to identify replication fork/stalling hotspots as replication progresses through the genome. These sites are typically associated with increased DNA damage, overlapped with fragile sites and with breakpoints of rearrangements identified in cancers but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. Indeed, we find that slowing down transcription elongation slows down directly replication progression through genes. This indicates that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. At the same time, we find instances where transcription activity favors replication progression because it reduces histone density.
CONCLUSIONS
Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.
Topics: DNA Replication; RNA Polymerase II; Humans; Transcription, Genetic; S Phase; DNA Damage; Ataxia Telangiectasia Mutated Proteins; Genome, Human; Replication Origin
PubMed: 38773641
DOI: 10.1186/s13059-024-03278-8