Recruitment Of RNA Polymerase Research Articles

TAF2, within the TFIID complex, regulates the expression of a subset of protein-coding genes

TFIID, one of the general transcription factor (GTF), regulates transcriptional initiation of protein-coding genes through direct binding to promoter elements and subsequent recruitment of other GTFs and RNA polymerase II. Although generally required for most protein-coding genes, accumulated studies have also demonstrated promoter-specific functions for several TFIID subunits in gene activation. Here, we report that TBP-associated factor 2 (TAF2) specifically regulates TFIID binding to a small subset of protein-coding genes and is essential for cell growth of multiple cancer lines. Co-immunoprecipitation assays revealed that TAF2 may be sub-stoichiometrically associated with the TFIID complex, thus indicating a minor fraction of TAF2-containing TFIID in cells. Consistently, integrated genome-wide profiles show that TAF2 binds to and regulates only a small subset of protein-coding genes. Furthermore, through the use of an inducible TAF2 degradation system, our results reveal a reduction of TBP/TFIID binding to several ribosomal genes upon selective ablation of TAF2. In addition, depletion of TAF2, as well as the TAF2-regulated ribosomal protein genes RPL30 and RPL39, decreases ribosome assembly and global protein translation. Collectively, this study suggests that TAF2 within the TFIID complex is of functional importance for TBP/TFIID binding to and expression of a small subset of protein-coding genes, thus establishing a previously unappreciated promoter-selective function for TAF2.

Structure of the SigF1-dependent pilA1 gene promoter and characterization of the light-activated response in the cyanobacterium Synechococcus elongatus PCC 7942.

In cyanobacteria that perform oxygenic photosynthesis, alternative sigma factors can play critical roles in environmental acclimation at the transcriptional initiation step. Here, we found in Synechococcus elongatus PCC 7942 that transcription of the pilA1 gene, encoding the type IV pilin, is dependent on one of the group 3 sigma factors, SigF1. We analyzed the promoter sequence determinants and proposed herein that the -10 and -35 boxes upstream of the transcriptional start site are critical for transcription. Interestingly, while the pilA1 promoter is activated by illumination, RNA polymerase containing SigF1 is already located on the promoter region under dark conditions, prior to illumination. This strongly suggests that promoter activation by light follows the recruitment of RNA polymerase during transcriptional initiation.

Transcription directionality is licensed by Integrator at active human promoters.

A universal characteristic of eukaryotic transcription is that the promoter recruits RNA polymerase II (RNAPII) to produce both precursor mRNAs (pre-mRNAs) and short unstable promoter upstream transcripts (PROMPTs) toward the opposite direction. However, how the transcription machinery selects the correct direction to produce pre-mRNAs is largely unknown. Here, through multiple acute auxin-inducible degradation systems, we show that rapid depletion of an RNAPII-binding protein complex, Integrator, results in robust PROMPT accumulation throughout the genome. Interestingly, the accumulation of PROMPTs is compensated by the reduction of pre-mRNA transcripts in actively transcribed genes. Consistently, Integrator depletion alters the distribution of polymerase between the sense and antisense directions, which is marked by increased RNAPII-carboxy-terminal domain Tyr1 phosphorylation at PROMPT regions and a reduced Ser2 phosphorylation level at transcription start sites. Mechanistically, the endonuclease activity of Integrator is critical to suppress PROMPT production. Furthermore, our data indicate that the presence of U1 binding sites on nascent transcripts could counteract the cleavage activity of Integrator. In this process, the absence of robust U1 signal at most PROMPTs allows Integrator to suppress the antisense transcription and shift the transcriptional balance in favor of the sense direction.

Comprehensive Characterization of fucAO Operon Activation in Escherichia coli.

Wildtype Escherichia coli cells cannot grow on L-1,2-propanediol, as the fucAO operon within the fucose (fuc) regulon is thought to be silent in the absence of L-fucose. Little information is available concerning the transcriptional regulation of this operon. Here, we first confirm that fucAO operon expression is highly inducible by fucose and is primarily attributable to the upstream operon promoter, while the fucO promoter within the 3'-end of fucA is weak and uninducible. Using 5'RACE, we identify the actual transcriptional start site (TSS) of the main fucAO operon promoter, refuting the originally proposed TSS. Several lines of evidence are provided showing that the fucAO locus is within a transcriptionally repressed region on the chromosome. Operon activation is dependent on FucR and Crp but not SrsR. Two Crp-cAMP binding sites previously found in the regulatory region are validated, where the upstream site plays a more critical role than the downstream site in operon activation. Furthermore, two FucR binding sites are identified, where the downstream site near the first Crp site is more important than the upstream site. Operon transcription relies on Crp-cAMP to a greater degree than on FucR. Our data strongly suggest that FucR mainly functions to facilitate the binding of Crp to its upstream site, which in turn activates the fucAO promoter by efficiently recruiting RNA polymerase.

Abstract 1692: MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal type of pancreatic neoplasm. Unfortunately, most patients are diagnosed with unresectable or metastatic disease, making current treatments insufficient. As a result, the 5-year survival rate for PDAC is below 10%.Pancreatic neoplasms are primarily initiated by the KRAS mutation, accounting for 95% of cases. However, the progression of PDAC to a highly aggressive and therapy-resistant cancer is accelerated by chronic inflammation. The role of mutant KRAS signaling and NF-κB inflammatory signaling in promoting PDAC progression has been extensively studied. Yet, the transcriptional mechanism of their cooperation remains unclear. Additionally, the interaction between PDAC tumor cells and signals in the tumor microenvironment (TME) is not yet fully understood. Our study demonstrates that the simultaneous activation of KRAS/MAPK and NF-κB by epidermal growth factor (EGF) and TNFα, respectively, increases cell migration as shown through live-cell imaging analysis. An integrated analysis of epigenetic and transcriptomic data (ChIP-seq and RNA-seq) was performed after dual stimulation of both pathways. This revealed a high occupancy of the active mark, H3K27ac at transcription start sites of a subset of genes involved in cell migration and subtype switch. Through gain and loss-of-function studies, FOSL1 and RELA were identified as the key transcription factors. The epigenome mapping of FOSL1 and RELA after the stimulation of both pathways revealed their occupancy at RELA-dominant and FOSL1/RELA co-occupied regions. Inhibitor treatments and loss-of-function studies revealed that RELA binding controls RNA polymerase recruitment for transcription initiation and enhances cell migration. Further analysis of RNA-seq data on PDAC cell lines and cells from patient-derived xenografts highlighted that active inflammatory signaling contributes to a subtype switch from the classical to the more migratory basal subtype. Our study, combined with single-cell RNA-seq analysis of published data, shows that macrophages in the tumor microenvironment supply TNFα that activates NF-κB signaling, leading to changes in cell migration. Our study has provided a comprehensive understanding of the mechanism by which mutant KRAS and inflammatory signaling contribute to the progression of PDAC. These findings could offer a new perspective in the development of mechanism-based therapeutic approaches that can improve the efficacy of chemotherapeutic agents and make tumors more amenable to surgical resection. Citation Format: Joana E. Aggrey-Fynn, Meghana Manjunath, Steven A. Johnsen. MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1692.

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

Maternal KLF17 controls zygotic genome activation by acting as a messenger for RNA Pol II recruitment in mouse embryos

Initiation of timely and sufficient zygotic genome activation (ZGA) is crucial for the beginning of life, yet our knowledge of transcription factors (TFs) contributing to ZGA remains limited. Here, we screened the proteome of early mouse embryos after cycloheximide (CHX) treatment and identified maternally derived KLF17 as a potential TF for ZGA genes. Using a conditional knockout (cKO) mouse model, we further investigated the role of maternal KLF17 and found that it promotes embryonic development and full fertility. Mechanistically, KLF17 preferentially binds to promoters and recruits RNA polymerase II (RNA Pol II) in early 2-cell embryos, facilitating the expression of major ZGA genes. Maternal Klf17 knockout resulted in a downregulation of 9% of ZGA genes and aberrant RNA Pol II pre-configuration, which could be partially rescued by introducing exogenous KLF17. Overall, our study provides a strategy for screening essential ZGA factors and identifies KLF17 as a crucial TF in this process.

G-quadruplex DNA structure is a positive regulator of MYC transcription

DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human MYC oncogene. G4 loss leads to suppression of MYC transcription from the P1 promoter that is mediated by the deposition of a de novo nucleosome alongside alterations in RNA polymerase recruitment. We also show that replacement of the endogenous MYC G4 with a different G4 structure from the KRAS oncogene restores G4 folding and MYC transcription. Moreover, we demonstrate that the MYC G4 structure itself, rather than its sequence, recruits transcription factors and histone modifiers. Overall, our work establishes that G4 structures are important features of transcriptional regulation that coordinate recruitment of key chromatin proteins and the transcriptional machinery through interactions with DNA secondary structure, rather than primary sequence.

Lsr2: A Nucleoid Associated Protein (NAP) and a transcription factor in mycobacteria

Lsr2, a small protein mainly found in actinobacteria, plays a crucial role in the virulence and adaptation of mycobacteria to environmental conditions. As a member of the nucleoid-associated protein (NAPs) superfamily, Lsr2 influences DNA organization by facilitating the formation of chromosomal loops in vitro and, therefore, may be a major player in the three-dimensional folding of the genome. Additionally, Lsr2 also acts as a transcription factor, regulating the expression of numerous genes responsible for coordinating a myriad of cellular and molecular processes essential for the actinobacteria. Similar to the H-NS protein, its ortholog in enterobacteria, its role in transcriptional repression likely relies on oligomerization, rigidifying, and bridging of DNA, thereby disrupting RNA polymerase recruitment as well as the elongation of RNA transcripts.

Histone deacetylases maintain expression of the pluripotent gene network via recruitment of RNA polymerase II to coding and noncoding loci.

Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening, and therefore, HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of Hdac1 and Hdac2 in embryonic stem cells (ESCs) reduces expression of the pluripotency-associated transcription factors Pou5f1, Sox2, and Nanog (PSN). By shaping global histone acetylation patterns, HDACs indirectly regulate the activity of acetyl-lysine readers, such as the transcriptional activator BRD4. Here, we use inhibitors of HDACs and BRD4 (LBH589 and JQ1, respectively) in combination with precision nuclear run-on and sequencing (PRO-seq) to examine their roles in defining the ESC transcriptome. Both LBH589 and JQ1 cause a marked reduction in the pluripotent gene network. However, although JQ1 treatment induces widespread transcriptional pausing, HDAC inhibition causes a reduction in both paused and elongating polymerase, suggesting an overall reduction in polymerase recruitment. Using enhancer RNA (eRNA) expression to measure enhancer activity, we find that LBH589-sensitive eRNAs are preferentially associated with superenhancers and PSN binding sites. These findings suggest that HDAC activity is required to maintain pluripotency by regulating the PSN enhancer network via the recruitment of RNA polymerase II.

A Potential Link between hnRNP K and Cebpα: Implications for Ribosomal Dysfunction in Acute Myeloid Leukemia (AML)

Deletion of the long arm of chromosome 9, del(9q), is a recurrent genetic abnormality involving the haploinsufficient loss of the RNA binding protein hnRNPK and is often associated with poor treatment responses in AML. In addition to reduced hnRNP K expression, other critical proteins, not localized to this locus, are also aberrantly expressed in del9q AML. One protein of critical interest is CEBPα, a well-established myeloid differentiation transcription factor. Recently, CEBPα was discovered to have a novel role in ribosomal RNA (rRNA) synthesis. However, as opposed to CEBPα, hnRNP K's role in RNA stability, rRNA processing, and ribosomal functions is already firmly rooted. Collectively, these results suggest that del(9q) AML dysregulations in hnRNP K and CEBPα expression may have a multi-faceted influence on ribosomal biogenesis and protein translation. Previously, we used animal models and patient samples to demonstrate that Hnrnpk haploinsufficiency alters CEBPa expression, suggesting that reduced hnRNP K expression contributes to decreased CEBPα levels and activity. These findings led us to hypothesize that these distinct, yet interconnected proteins, play a cooperative role in regulating rRNA synthesis in two major ways. Firstly, hnRNP K directly governs rRNA processing and ribosomal loading. And secondly, via hnRNP K's mediated regulation of the nucleolar-localized isoform of CEBPα (p42 isoform), which was recently shown to enhance recruitment of RNA polymerase I to rRNA genes. To explore these relationships, we used AML patient samples, Hnrnpk haploinsufficient animal models, si- and sh- HNRNPK AML cell lines, and biochemical analyses. Initially, RPPA, CyTOF, and western blot analyses were used to investigate how reduced hnRNP K levels affect proteins involved in ribogenesis and protein translation. Here, we observed a direct correlation between hnRNP K expression and defects in proteins and pathways involved in protein translation, such as mTOR, phos-S6, and eEF- and eIF-family members, and CEBPa (log 10pAdj < .0001). Subsequently, we performed polysome assays that revealed 40S and 60S subunit formation was impaired in AML cell lines with reduced hnRNP K levels. Across multiple cell lines, reduced hnRNP K expression directly led to a significant increase in monosomes (unbound/inactive ribosomes; p < 0.01) and a significant decrease in polysome-bound RNA (p < 0.05) . Critically, HNRNPK haploinsufficiency and si- and sh- HNRNPK knock down experiments disrupted the abundance and processing of the 47S transcript (p < 0.05). To understand the impact that hnRNP K has on CEBPα in the context of rRNA biology, we performed co-immunoprecipitation of hnRNP K followed by RNA-seq (RIP-Seq) in human AML cell lines KG-1 and KG-1a. 1076 interacting RNAs were identified, which were strongly enriched in transcription factors implicated in myeloid biology, including CEBPA (log 2pAdj < .0001). Biochemical analyses of sequence-specific motifs conferred direct and functional interactions between hnRNP K and the 5‘ and 3‘ UTRs of the human CEBPA. These interactions support our in vivo findings that del9q AML and Hnrnpk haploinsufficiency lead to reduced CEBPA expression, specifically in the p42 isoform, potentially impacting its role in nucleolar localization and rRNA transcription regulation. Currently, we are evaluating the significance of these interactions at both the biological and translational levels. Using biochemical and cell-based assays, we are evaluating the direct effect that Hnrnpk haploinsufficiency has on rRNA abundance and on the processing of ITS products in the context of CEBPA. Through the use of viability screens, we are examining the efficacy of inhibitors of protein translation, translation initiation, and RNA Pol I on leukemic cells with 9q deletions of reduced hnRNP K expression. Altogether, these findings identify an uncharacterized relationship between del9qAML ( HNRNPK haploinsufficiency), CEBPα expression, and ribogenesis, providing valuable insight towards mechanisms driving this subtype of leukemia. Collectively, these data also suggest that del9q AML represents a novel and currently uncharacterized ribosomopathy.

SETD5 regulates the OGT-catalyzed O-GlcNAcylation of RNA polymerase II, which is involved in the stemness of colorectal cancer cells

The dosage-dependent recruitment of RNA polymerase II (Pol II) at the promoters of genes related to neurodevelopment and stem cell maintenance is required for transcription by the fine-tuned expression of SET-domain-containing protein 5 (SETD5). Pol II O-GlcNAcylation by O-GlcNAc transferase (OGT) is critical for preinitiation complex formation and transcription cycling. SETD5 dysregulation has been linked to stem cell-like properties in some cancer types; however, the role of SETD5 in cancer cell stemness has not yet been determined. We here show that aberrant SETD5 overexpression induces stemness in colorectal cancer (CRC) cells. SETD5 overexpression causes the upregulation of PI3K-AKT pathway-related genes and cancer stem cell (CSC) markers such as CD133, Kruppel-like factor 4 (KLF4), and estrogen-related receptor beta (ESRRB), leading to the gain of stem cell-like phenotypes. Our findings also revealed a functional relationship between SETD5, OGT, and Pol II. OGT-catalyzed Pol II glycosylation depends on SETD5, and the SETD5-Pol II interaction weakens in OGT-depleted cells, suggesting a SETD5-OGT-Pol II interdependence. SETD5 deficiency reduces Pol II occupancy at PI3K-AKT pathway-related genes and CD133 promoters, suggesting a role for SETD5-mediated Pol II recruitment in gene regulation. Moreover, the SETD5 depletion nullified the SETD5-induced stemness of CRC cells and Pol II O-GlcNAcylation. These findings support the hypothesis that SETD5 mediates OGT-catalyzed O-GlcNAcylation of RNA Pol II, which is involved in cancer cell stemness gain via CSC marker gene upregulation.

Crystal structures of Streptomyces tsukubaensis sigma factor SigG1 and anti-sigma RsfG

Transcription of specific genes in bacteria under environmental stress is frequently initiated by extracytoplasmic function (ECF) σ factors. ECFs σ factors harbour two conserved domains, σ2 and σ4, for transcription initiation by recognition of the promoter region and recruitment of RNA polymerase (RNAP). The crystal structure of Streptomyces tsukubaensis SigG1, an ECF56-family σ factor, was determined revealing σ2, σ4 and the additional carboxi-terminal domain SnoaL_2 tightly packed in a compact conformation. The structure of anti-sigma RsfG was also determined by X-ray crystallography and shows a rare β-barrel fold. Analysis of the metal binding motifs inside the protein barrel are consistent with Fe(III) binding, which is in agreement with previous findings that the Streptomyces tsukubaensis ECF56 SigG1-RsfG system is involved in metal-ion homeostasis.

The role of the Mediator complex in fungal pathogenesis and response to antifungal agents.

Mediator is a complex of polypeptides that plays a central role in the recruitment of RNA polymerase II to promoters and subsequent transcriptional activation in eukaryotic organisms. Studies have now shown that Mediator has a role in regulating expression of genes implicated in virulence and antifungal drug resistance in pathogenic fungi. The roles of specific Mediator subunits have been investigated in several species of pathogenic fungi, particularly in the most pathogenic yeast Candida albicans. Uniquely, pathogenic yeast also present several interesting examples of divergence in Mediator structure and function, most notably in C. glabrata, which possesses two orthologues of Med15, and in C. albicans, which has a massively expanded family of Med2 orthologues known as the TLO gene family. This review highlights specific examples of recent progress in characterizing the role of Mediator in pathogenic fungi.

Targeting BRF2: insights from in silico screening and molecular dynamic simulations

The Transcription factor II B (TFIIB)‑related factor 2 (BRF2) containing TFIIIB complex recruits RNA polymerase III multi-subunit complex to selective gene promoters that altogether are responsible for synthesizing a variety of small non-coding RNAs, including a special type of selenocysteine tRNA (tRNASec), micro-RNA (miRNA), and other regulatory RNAs. BRF2 has been identified as a potential oncogene that promotes cancer cell survival under oxidative stress through its genetic activation. The structure of the BRF2 protein was modeled using the Robetta server, refined, and validated using the Ramachandran plot. A virtual approach utilizing molecular docking was used to screen a natural compound library to determine potential compounds that can interact with the molecular pin motif of the BRF2 protein using Maestro (Schrodinger). Subsequent molecular dynamics simulation studies of the top four ligands that exhibited low glide scores were performed using GROMACS. The findings derived from the simulations, in conjunction with the exploration of hydrogen bonding patterns, evaluation of the free energy landscape, and thorough analysis of residue decomposition, collectively converged to emphasize the robust interaction characteristics exhibited by Ligand 366 (Deacetyl lanatoside C) and ligand 336 (Neogitogenin)—with the BRF2 protein. These natural compounds may be potential inhibitors of BRF2, which could modulate the regulation of selenoprotein synthesis in cancer cells. Targeting BRF2 using these promising compounds may offer a new therapeutic approach to sensitize cancer cells to ferroptosis and apoptosis. Communicated by Ramaswamy H. Sarma

RNA polymerase II associates with active genes during DNA replication.

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 states1,2. 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 rebuilt3,4. 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.

Structural insights into human TFIIIC promoter recognition.

Transcription factor (TF) IIIC recruits RNA polymerase (Pol) III to most of its target genes. Recognition of intragenic A- and B-box motifs in transfer RNA (tRNA) genes by TFIIIC modules τA and τB is the first critical step for tRNA synthesis but is mechanistically poorly understood. Here, we report cryo-electron microscopy structures of the six-subunit human TFIIIC complex unbound and bound to a tRNA gene. The τB module recognizes the B-box via DNA shape and sequence readout through the assembly of multiple winged-helix domains. TFIIIC220 forms an integral part of both τA and τB connecting the two subcomplexes via a ~550-amino acid residue flexible linker. Our data provide a structural mechanism by which high-affinity B-box recognition anchors TFIIIC to promoter DNA and permits scanning for low-affinity A-boxes and TFIIIB for Pol III activation.

A homozygous mutation in the human selenocysteine tRNA gene impairs UGA recoding activity and selenoproteome regulation by selenium.

The selenocysteine (Sec) tRNA (tRNA[Ser]Sec) governs Sec insertion into selenoproteins by the recoding of a UGA codon, typically used as a stop codon. A homozygous point mutation (C65G) in the human tRNA[Ser]Sec acceptor arm has been reported by two independent groups and was associated with symptoms such as thyroid dysfunction and low blood selenium levels; however, the extent of altered selenoprotein synthesis resulting from this mutation has yet to be comprehensively investigated. In this study, we used CRISPR/Cas9 technology to engineer homozygous and heterozygous mutant human cells, which we then compared with the parental cell lines. This C65G mutation affected many aspects of tRNA[Ser]Sec integrity and activity. Firstly, the expression level of tRNA[Ser]Sec was significantly reduced due to an altered recruitment of RNA polymerase III at the promoter. Secondly, selenoprotein expression was strongly altered, but, more surprisingly, it was no longer sensitive to selenium supplementation. Mass spectrometry analyses revealed a tRNA isoform with unmodified wobble nucleotide U34 in mutant cells that correlated with reduced UGA recoding activities. Overall, this study demonstrates the pleiotropic effect of a single C65G mutation on both tRNA phenotype and selenoproteome expression.

Unveiling a Virulence-Regulating Mechanism in Aeromonas hydrophila: a Quantitative Exoproteomic Analysis of an AraC-Like Protein.

Bacterial AraC is a transcription factor family that initiates transcription by recruiting RNA polymerase to the promoter and directly regulating various bacterial phenotypes. It also directly regulates various bacterial phenotypes. However, how this transcription factor regulates bacterial virulence and affects host immunity is still largely unknown. In this study, deleting the orf02889 (AraC-like transcription factor) gene in virulent Aeromonas hydrophila LP-2 affected several important phenotypes, such as increasing biofilm formation and siderophore production abilities. Moreover, Δorf02889 also significantly decreased the virulence of A. hydrophila and has promising attenuated vaccine potential. To better understand the effects of orf02889 on biological functions, a data independent acquisition (DIA)-based quantitative proteomics method was performed to compare the differentially expressed proteins between Δorf02889 and the wild-type strain in extracellular fractions. The following bioinformatics analysis suggested that ORF02889 may regulate various metabolic pathways, such as quorum sensing and ATP binding cassette (ABC) transporter metabolism. Moreover, 10 selected genes from the top 10 decreasing abundances in proteomics data were deleted, and their virulence to zebrafish was evaluated, respectively. The results showed that ΔcorC, Δorf00906, and Δorf04042 significantly reduced bacterial virulence. Finally, the following chromatin immunoprecipitation and polymerase chain reaction (ChIP-PCR) assay validated that the promoter of corC was directly regulated by ORF02889. Overall, these results provide insight into the biological function of ORF02889 and demonstrate its inherent regulatory mechanism for the virulence of A. hydrophila.

PTEN-negative endometrial cancer cells protect their genome through enhanced DDB2 expression associated with augmented nucleotide excision repair

BackgroundEndometrial cancer (EC) arises from uterine endometrium tissue and is the most prevalent cancer of the female reproductive tract in developed countries. It has been predicted that the global prevalence of EC will increase in part because of its positive association with economic growth and lifestyle. The majority of EC presented with endometrioid histology and mutations in the tumor suppressor gene PTEN, resulting in its loss of function. PTEN negatively regulates the PI3K/Akt/mTOR axis of cell proliferation and thus serves as a tumorigenesis gatekeeper. Through its chromatin functions, PTEN is also implicated in genome maintenance procedures. However, our comprehension of how DNA repair occurs in the absence of PTEN function in EC is inadequate.MethodsWe utilized The Cancer Genome Atlas (TCGA) data analysis to establish a correlation between PTEN and DNA damage response genes in EC, followed by a series of cellular and biochemical assays to elucidate a molecular mechanism utilizing the AN3CA cell line model for EC.ResultsThe TCGA analyses demonstrated an inverse correlation between the expression of the damage sensor protein of nucleotide excision repair (NER), DDB2, and PTEN in EC. The transcriptional activation of DDB2 is mediated by the recruitment of active RNA polymerase II to the DDB2 promoter in the PTEN-null EC cells, revealing a correlation between increased DDB2 expression and augmented NER activity in the absence of PTEN.ConclusionOur study indicated a causal relationship between NER and EC that may be exploited in disease management.