Promoter DNA Research Articles

Selective Occupation by E2F and RB of Loci Expressed by RNA Polymerase III

In all cases tested, TFIIIB is responsible for recruiting pol III to its genetic templates. In mammalian cells, RB binds TFIIIB and prevents its interactions with both promoter DNA and pol III, thereby suppressing transcription. As TFIIIB is not recruited to its target genes when bound by RB, the mechanism predicts that pol III-dependent templates will not be occupied by RB; this contrasts with the situation at most genes controlled by RB, where it can be tethered by promoter-bound sequence-specific DNA-binding factors such as E2F. Contrary to this prediction, however, ChIP-seq data reveal the presence of RB in multiple cell types and the related protein p130 at many loci that rely on pol III for their expression, including RMRP, RN7SL, and a variety of tRNA genes. The sets of genes targeted varies according to cell type and growth state. In such cases, recruitment of RB and p130 can be explained by binding of E2F1, E2F4 and/or E2F5. Genes transcribed by pol III had not previously been identified as common targets of E2F family members. The data provide evidence that E2F may allow for the selective regulation of specific non-coding RNAs by RB, in addition to its influence on overall pol III output through its interaction with TFIIIB.

An antihypertensive drug-AT1 inhibitor attenuated BRCA development promoted by chronic psychological stress via Ang II/PARP1/FN1 pathway

Chronic psychological stress contributes to the occurrence of cancer and activates the renin-angiotensin system (RAS). However, the mechanisms by which RAS promotes the progression of breast cancer (BRCA) under chronic psychological stress are remain unknown. In this study, we observed elevated levels of Angiotensin II (Ang II) in both serum and BRCA tissue under chronic stress, leading to accelerated BRCA growth in a mouse model. An antihypertensive drug, candesartan (an AT1 inhibitor), effectively attenuated Ang II-induced cell proliferation and metastasis. Utilizing mass spectrometry and weighted gene co-expression network analysis (WGCNA), we identified fibronectin 1 (FN1) as the hub protein involved in chronic stress-Ang II/AT1 axis. Focal adhesion pathway was identified as a downstream signaling pathway activated during the progression of chronic stress. Depletion of FN1 significantly attenuated Ang II-induced proliferation and metastasis of BRCA cells. Poly (ADP-ribose) polymerase 1 (PARP1) was found to bind to the DNA promoter of FN1, leading to the transcription of FN1. Ang II upregulated PARP1 expression, resulting in increased FN1 levels. Recombinant FN1 partially restored the progress of BRCA malignancy induced by the Ang II/PARP1 pathway. In vivo, candesartan reversed the progressive effect of chronic psychological stress on BRCA. In clinical samples, Ang II levels in both serum and tumor tissues are higher in stressed patients compared to control patients. Serum Ang II levels were positively correlated with chronic stress indicators. In conclusion, our study demonstrated that chronic psychological stress accelerates the malignancy of BRCA, and the AT1 inhibitor candesartan counteracts these effects by suppressing the Ang II-AT1 axis and the downstream PARP1/FN1/focal adhesion pathway.

Dietary Folate and Cofactors Accelerate Age-dependent p16 Epimutation to Promote Intestinal Tumorigenesis.

Our study demonstrates that dietary folate and cofactors modulate tumor-suppressor gene methylation to increase intestinal tumorigenesis. Our findings highlight the need for monitoring the long-term safety of folate fortification in high-risk individuals.

TERT promoter methylation is associated with high expression of TERT and poor prognosis in papillary thyroid cancer.

The telomerase reverse transcriptase (TERT) is overexpressed and associated with poor prognosis in papillary thyroid cancer (PTC), the most common subtype of thyroid cancer. The overexpression of TERT in PTC was partially attributed to transcriptional activation by two hotspot mutations in the core promoter region of this gene. As one of the major epigenetic mechanisms of gene expression regulation, DNA methylation has been proved to regulate several tumor-related genes in PTC. However, the association of TERT promoter DNA methylation with TERT expression and PTC progression is still unclear. By treating PTC cell lines with demethylating agent decitabine, we found that the TERT promoter methylation and the genes' expression were remarkably decreased. Consistently, PTC patients with TERT hypermethylation had significantly higher TERT expression than patients with TERT hypomethylation. Moreover, TERT hypermethylated patients showed significant higher rates of poor clinical outcomes than patients with TERT hypomethylation. Results from the cox regression analysis showed that the hazard ratios (HRs) of TERT hypermethylation for overall survival, disease-specific survival, disease-free interval (DFI) and progression-free interval (PFI) were 4.81 (95% CI, 1.61-14.41), 8.28 (95% CI, 2.14-32.13), 3.56 (95% CI, 1.24-10.17) and 3.32 (95% CI, 1.64-6.71), respectively. The HRs for DFI and PFI remained significant after adjustment for clinical risk factors. These data suggest that promoter DNA methylation upregulates TERT expression and associates with poor clinical outcomes of PTC, thus holds the potential to be a valuable prognostic marker for PTC risk stratification.

Longitudinal analysis of influenza vaccination implicates regulation of RIG-I signaling by DNA methylation

Influenza virus infection alters the promoter DNA methylation of key immune response-related genes, including type-1 interferons and proinflammatory cytokines. However, less is known about the effect of the influenza vaccine on the epigenome. We utilized a targeted DNA methylation approach to study the longitudinal effects (day 0 pre-vaccination and day 28 post-vaccination) on influenza vaccination responses in peripheral blood mononuclear cells. We found that baseline, pre-vaccination methylation profiles are associated with pre-existing, protective serological immunity. Additionally, we identified 481 sites that were differentially methylated between baseline and day 28 post-vaccination. These were enriched for genes involved in the regulation of the RIG-I signaling pathway, an important regulator of viral responses. Our results suggest that DNA methylation changes to components of the RIG-I pathway are associated with vaccine effectiveness. Therefore, immunization strategies that target this pathway may improve serological responses to influenza vaccination.

Multimodal epigenetic sequencing analysis (MESA) of cell-free DNA for non-invasive colorectal cancer detection

BackgroundDetecting human cancers through cell-free DNA (cfDNA) in blood is a sensitive and non-invasive option. However, capturing multiple forms of epigenetic information remains a technical and financial challenge.MethodsTo address this, we developed multimodal epigenetic sequencing analysis (MESA), a flexible and sensitive approach to capturing and integrating a diverse range of epigenetic features in cfDNA using a single experimental assay, i.e., non-disruptive bisulfite-free methylation sequencing, such as Enzymatic Methyl-seq. MESA enables simultaneous inference of four epigenetic modalities: cfDNA methylation, nucleosome occupancy, nucleosome fuzziness, and windowed protection score for regions surrounding gene promoters and polyadenylation sites.ResultsWhen applied to 690 cfDNA samples from 3 colorectal cancer clinical cohorts, MESA’s novel modalities, which include nucleosome fuzziness, and genomic features, including polyadenylation sites, improve cancer detection beyond the traditional epigenetic markers of promoter DNA methylation.ConclusionsTogether, MESA stands as a major advancement in the field by utilizing comprehensive and complementary epigenetic profiles of cfDNA for effective non-invasive cancer detection.

Transcription factor FoxO1 regulates myoepithelial cell diversity and growth

Salivary gland myoepithelial cells regulate saliva secretion and have been implicated in the histological diversity of salivary gland tumors. However, detailed functional analysis of myoepithelial cells has not been determined owing to the few of the specific marker to isolate them. We isolated myoepithelial cells from the submandibular glands of adult mice using the epithelial marker EpCAM and the cell adhesion molecule CD49f as indicators and found predominant expression of the transcription factor FoxO1 in these cells. RNA-sequence analysis revealed that the expression of cell cycle regulators was negatively regulated in FoxO1-overexpressing cells. Chromatin immunoprecipitation analysis showed that FoxO1 bound to the p21/p27 promoter DNA, indicating that FoxO1 suppresses cell proliferation through these factors. In addition, FoxO1 induced the expression of ectodysplasin A (Eda) and its receptor Eda2r, which are known to be associated with X-linked hypohidrotic ectodermal dysplasia and are involved in salivary gland development in myoepithelial cells. FoxO1 inhibitors suppressed Eda/Eda2r expression and salivary gland development in primordial organ cultures after mesenchymal removal. Although mesenchymal cells are considered a source of Eda, myoepithelial cells might be one of the resources of Eda. These results suggest that FoxO1 regulates myoepithelial cell proliferation and Eda secretion during salivary gland development in myoepithelial cells.

Structural basis for transcription activation by the nitrate-responsive regulator NarL.

Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a nitrate-responsive global transcription factor that controls the expression of nearly 100 genes. However, the molecular mechanism of NarL-mediated transcription activation is not well defined. Here we present a cryo-EM structure of NarL-dependent transcription activation complex (TAC) assembled on the yeaR promoter at 3.2 Å resolution. Our structure shows that the NarL dimer binds at the -43.5 site of the promoter DNA with its C-terminal domain (CTD) not only binding to the DNA but also making interactions with RNA polymerase subunit alpha CTD (αCTD). The key role of these NarL-mediated interactions in transcription activation was further confirmed by in vivo and in vitro transcription assays. Additionally, the NarL dimer binds DNA in a different plane from that observed in the structure of class II TACs. Unlike the canonical class II activation mechanism, NarL does not interact with σ4, while RNAP αCTD is bound to DNA on the opposite side of NarL. Our findings provide a structural basis for detailed mechanistic understanding of NarL-dependent transcription activation on yeaR promoter and reveal a potentially novel mechanism of transcription activation.

Alteration of the -35 and -10 sequences and deletion the upstream sequence of the -35 region of the promoter A1 of the phage T7 in dsDNA confirm the contribution of non-specific interactions with E. coli RNA polymerase to the transcription initiation process.

Transcription initiation is a multi-step process, in which the RNA polymerase holoenzyme binds to the specific promoter sequences to form a closed complex, which, through intermediate stages, isomerizes into an open complex capable of initiating the productive phase of transcription. The aim of this work was to determine the contribution of the -10 and -35 regions of the promoter, as well as the role of non-specific interactions, in the binding of RNA polymerase and the formation of an active initiation complex capable of transcription. Therefore, fragments of promoter DNA, derived from the strong promoter A1 of the phage T7, containing completely and partially altered elements -35 and -10, and devoid of an upstream region, were constructed using genetic engineering methods. Functional analyses of modified promoter fragments were carried out, checking their ability to form binary complexes with Escherichia coli RNA polymerase (RNAP) and the efficiency of converting binary complexes into triple complexes characteristic of the productive phase of transcription. The obtained results suggest that, in relation to the A1 promoter of the T7 phage, the most important role of the -35 region is carrying the open complex through the next phases of transcription initiation. The weakening of specific impacts within the region -35 is a reason for the defect associated with the transformation of the open complex, formed by a DNA fragment containing the completely altered -35 region, into elongation and the impairment of RNA synthesis. This leads to breaking contacts with the RNA polymerase holoenzyme, and destabilization and disintegration of the complex in the initial phase of productive transcription. This confirms the hypothesis of the so-called stressed intermediate state associated with the stage of transition from the open complex to the elongation complex. The experiments carried out in this work confirm also that the process of promoter localization and recognition, as well as the formation of binary complexes, is sequential in nature, and that the region located upstream of the -35 hexamer, and the hexamer itself, plays here an additive role.

Promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions are associated with tumor differentiation, nodal involvement and survival.

Differentially methylated regions (DMRs) can be used as head and neck squamous cell carcinoma (HNSCC) diagnostic, prognostic and therapeutic targets in precision medicine workflows. DNA from 21 HNSCC and 10 healthy oral tissue samples was hybridized to a genome-wide tiling array to identify DMRs in a discovery cohort. Downstream analyses identified differences in promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions associated with tumor differentiation, nodal involvement and survival. Genome-wide DMR analysis showed 2,565 DMRs common to the three subsites. A total of 738 DMRs were unique to laryngeal cancer (n=7), 889 DMRs were unique to oral cavity cancer (n=10) and 363 DMRs were unique to pharyngeal cancer (n=6). Based on the genome-wide analysis and a Gene Ontology analysis, 10 candidate genes were selected to test for prognostic value and association with clinicopathological features. TIMP3 was associated with tumor differentiation in oral cavity cancer (P=0.039), DAPK1 was associated with nodal involvement in pharyngeal cancer (P=0.017) and PAX1 was associated with tumor differentiation in laryngeal cancer (P=0.040). A total of five candidate genes were selected, DAPK1, CDH1, PAX1, CALCA and TIMP3, for a prevalence study in a larger validation cohort: Oral cavity cancer samples (n=42), pharyngeal cancer tissues (n=25) and laryngeal cancer samples (n=52). PAX1 hypermethylation differed across HNSCC anatomic subsites (P=0.029), and was predominantly detected in laryngeal cancer. Kaplan-Meier survival analysis (P=0.043) and Cox regression analysis of overall survival (P=0.001) showed that DAPK1 methylation is associated with better prognosis in HNSCC. The findings of the present study showed that the HNSCC subsites oral cavity, pharynx and larynx display substantial differences in aberrant DNA methylation patterns, which may serve as prognostic biomarkers and therapeutic targets.

EGLN3 attenuates gastric cancer cell malignant characteristics by inhibiting JMJD8/NF-κB signalling activation independent of hydroxylase activity.

The expression of Egl-9 family hypoxia-inducible factor 3 (EGLN3) is notably decreased in various malignancies, including gastric cancer (GC). While the predominant focus has been on the hydroxylase activity of EGLN3 for its antitumour effects, recent findings have suggested nonenzymatic roles for EGLN3. This study assessed the clinical significance of EGLN3 expression in GC and explored the connection between EGLN3 DNA promoter methylation and transcriptional silencing. To investigate the effect of EGLN3 on GC cells, a gain-of-function strategy was adopted. RNA sequencing was conducted to identify the key effector molecules and signalling pathways associated with EGLN3. EGLN3 expression was significantly reduced in GC tissues, correlating with poorer patient prognosis. EGLN3 hypermethylation disrupts transcriptional equilibrium, contributing to deeper tumour invasion and lymph node metastasis, thus exacerbating GC progression. Conversely, restoration of EGLN3 expression in GC cells substantially inhibited cell proliferation and metastasis. EGLN3 was also found to impede the malignant progression of GC cells by downregulating Jumonji C domain-containing protein 8-mediated activation of the NF-κB pathway, independent of its hydroxylase activity. EGLN3 has the potential to hinder the spread of GC cells through a nonenzymatic mechanism, thereby shedding light on the complex nature of GC progression.

IL4I1 in M2-like macrophage promotes glioma progression and is a promising target for immunotherapy.

Glioma is the prevailing malignant intracranial tumor, characterized by an abundance of macrophages. Specifically, the infiltrating macrophages often display the M2 subtype and are known as tumor-associated macrophages (TAMs). They have a critical role in promoting the oncogenic properties of tumor cells. Interleukin-4-induced-1 (IL4I1) functions as an L-phenylalanine oxidase, playing a key part in regulating immune responses and the progression of various tumors. However, there is limited understanding of the IL4I1-mediated cross-talk function between TAMs and glioma cell in the glioma microenvironment. TCGA, GTEx, and HPA databases were applied to assess the IL4I1 expression, clinical characteristics, and prognostic value of pan-cancer. The link between IL4I1 levels and the prognosis, methylation, and immune checkpoints (ICs) in gliomas were explored through Kaplan-Meier curve, Cox regression, and Spearman correlation analyses. The IL4I1 levels and their distribution were investigated by single-cell analysis and the TIMER 2 database. Additionally, validation of IL4I1 expression was performed by WB, RT-qPCR, IHC, and IF. Co-culture models between glioma cells and M2-like macrophages were used to explore the IL4I1-mediated effects on tumor growth, invasion, and migration of glioma cells. Moreover, the function of IL4I1 on macrophage polarization was evaluated by ELISA, RT-qPCR, WB, and siRNA transfection. Both transcriptome and protein levels of IL4I1 were increased obviously in various tumor types, and correlated with a dismal prognosis. Specifically, IL4I1 was implicated in aggressive progression and a dismal prognosis for patients with glioma. A negative association was noticed between the glioma grade and DNA promoter methylation of IL4I1. Enrichment analyses in glioma patients suggested that IL4I1 was linked to cytokine and immune responses, and was positively correlated with ICs. Single-cell analysis, molecular experiments, and in vitro assays showed that IL4I1 was significantly expressed in TAMs. Importantly, co-culture models proved that IL4I1 significantly promoted the invasion and migration of glioma cells, and induced the polarization of M2-like macrophages. IL4I1 could be a promising immunotherapy target for selective modulation of TAMs and stands as a novel macrophage-related prognostic biomarker in glioma.

DNA looping mediates cooperative transcription activation.

Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here we report two cryo-electron microscopy structures of Anabaena transcription activation complexes (TACs): NtcA-TAC composed of RNAP holoenzyme, promoter and a global activator NtcA, and NtcA-NtcB-TAC comprising an extra context-specific regulator, NtcB. Structural analysis showed that NtcA binding makes the promoter DNA bend by ∼50°, which facilitates RNAP to contact NtcB at the distal upstream NtcB box. The sequential binding of NtcA and NtcB induces looping back of promoter DNA towards RNAP, enabling the assembly of a fully activated TAC bound with two activators. Together with biochemical assays, we propose a 'DNA looping' mechanism of cooperative transcription activation in bacteria.

Identification and prognostic biomarkers among ZDHHC4/12/18/24, and APT2 in lung adenocarcinoma

S-palmitoylases and S-depalmitoylases are differentially expressed in various cancers and several malignant tumors and show a strong prognostic ability. Notwithstanding, the potential clinical impact of S-palmitoylases and S-depalmitoylases, particularly in the prognosis and progression of lung adenocarcinoma (LUAD), has not been clarified. Expression levels of S-palmitoylases and S-depalmitoylases in LUAD were investigated using TCGA. GEPIA was used to evaluate the mRNA levels of S-palmitoylases and S-depalmitoylases at different pathological stages. Metascape was used to investigate the biological significance of S-palmitoylases and S-depalmitoylases. The Kaplan–Meier plotter was used to analyze the prognostic value of S-palmitoylases and S-depalmitoylases. CBioportal was used to analyze gene alterations in S-palmitoylases and S-depalmitoylases. UALCAN was used to examine DNA promoter methylation levels of S-palmitoylases and S-depalmitoylases. Finally, we investigated the relationship between S-palmitoylases, S-depalmitoylases, and tumor-infiltrating immune cells using TIMER. Correlations with immune checkpoint-related genes were determined using the R packages reshape2, ggpubr, ggplot2, and corrplot. PCR was also performed to assess the degree of ZDHHC4/12/18/24 and APT2 transcript expression in lung adenocarcinoma and adjacent normal lung tissues. HPA was utilized to investigate protein levels of S-palmitoylases and S-depalmitoylases in LUAD and normal lung tissue. Our study found that ZDHHC2/3/4/5/6/7/9/12/13/16/18/20/21/23/24, APT1/2, PPT1, LYPLAL1, ABHD4/10/11/12/13 and ABHD17C mRNA expression was significantly upregulated in LUAD, whereas ZDHHC1/8/11/11B/14/15/17/19/22, ABHD6/16A and ABHD17A mRNA expression was significantly downregulated. The functions of the differentially expressed S-palmitoylases and S-depalmitoylases were mainly associated with protein-cysteine S-palmitoyltransferase and protein-cysteine S-acyltransferase activities. Patients with high expression of ZDHHC4/12/18/24, APT2, ABHD4, ABHD11 and ABHD12 had a shorter overall survival. Infiltration of six immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells) was closely associated with the expression of ZDHHC4/12/18/24 and APT2. ZDHHC4/12/18/24 and APT2 positively correlated with the immune checkpoint-related gene CD276. We assessed the mRNA levels of ZDHHC4/12/18/24 and APT2 using qRT-PCR and found increased expression of ZDHHC4/12/18/24 in LUAD compared with healty control lung tissues. ZDHHC4/12/18/24, and APT2 are potential prognostic biomarkers of LUAD. Their expression levels could be related to the tumor microenvironment in LUAD.

Structural basis of σ54 displacement and promoter escape in bacterial transcription.

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ70 class, represented by the σ70 that regulates housekeeping genes. σ54 forms a class on its own and regulates stress response genes. Extensive studies on σ70 have revealed the molecular mechanisms of the σ70 dependent process while how σ54 transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ54 initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ54 and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ54 and upstream DNA, enabling the transition to elongation.

Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

Background and AimsEfforts to decode pancreatic ductal adenocarcinoma (PDAC) heterogeneity and the consequent therapeutic selection remains a challenge. We aimed to characterize epigenetically regulated pathways involved in PDAC progression. MethodsGlobal DNA methylation analysis in pancreatic cancer patient tissues and cell lines was performed to identify differentially methylated genes. Targeted bisulfite sequencing and in vitro methylation reporter assays were employed to investigate the direct link between site-specific methylation and transcriptional regulation. A series of in vitro loss- and gain-of function studies, and in vivo xenograft and the KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre) mouse models were used to assess pancreatic cancer cell properties. Gene and protein expression analyses were performed in three different cohorts of pancreatic cancer patients and correlated to clinicopathological parameters. ResultsWe identify Hepatocyte Nuclear Factor 4A (HNF4A) as a novel target of hypermethylation in pancreatic cancer and demonstrate that site-specific proximal promoter methylation drives HNF4A transcriptional repression. Expression analyses in patients, indicate the methylation-associated suppression of HNF4A expression in pancreatic cancer tissues. In vitro and in vivo studies reveal that HNF4A is a novel tumor suppressor in pancreatic cancer, regulating cancer growth and aggressiveness. As evidenced in both the KPC mouse model and human pancreatic cancer tissues, HNF4A expression declines significantly in the early stages of the disease. Most importantly, HNF4 loss correlates with poor overall patient survival. ConclusionsHNF4A silencing, mediated by promoter DNA methylation, drives pancreatic cancer development and aggressiveness leading to poor patient survival.

The Copper Efflux Regulator (CueR).

The copper efflux regulator (CueR) is a classical member of the MerR family of metalloregulators and is common in gram-negative bacteria. Through its C-terminal effector-binding domain, CueR senses cytoplasmic copper ions to regulate the transcription of genes contributing to copper homeostasis, an essential process for survival of all cells. In this chapter, we review the regulatory roles of CueR in the model organism Escherichia coli and the mechanisms for CueR in copper binding, DNA recognition, and interplay with RNA polymerase in regulating transcription. In light of biochemical and structural analyses, we provide molecular details for how CueR represses transcription in the absence of copper ions, how copper ions mediate CueR conformational change to form holo CueR, and how CueR bends and twists promoter DNA to activate transcription. We also characterize the functional domains and key residues involved in these processes. Since CueR is a representative member of the MerR family, elucidating its regulatory mechanisms could help to understand the CueR-like regulators in other organisms and facilitate the understanding of other metalloregulators in the same family.

Mechanistic and biophysical characterization of polymyxin resistance response regulator PmrA in Acinetobacter baumannii.

Acinetobacter baumannii PmrAB is a crucial two-component regulatory system (TCS) that plays a vital role in conferring resistance to polymyxin. PmrA, a response regulator belonging to the OmpR/PhoB family, is composed of a C-terminal DNA-binding effector domain and an N-terminal receiver domain. The receiver domain can be phosphorylated by PmrB, a transmembrane sensor histidine kinase that interacts with PmrA. Once phosphorylated, PmrA undergoes a conformational change, resulting in the formation of a symmetric dimer in the receiver domain. This conformational change facilitates the recognition of promoter DNA by the DNA-binding domain of PmrA, leading to the activation of adaptive responses. X-ray crystallography was carried out to solve the structure of PmrA receiver domain. Electrophoretic mobility shift assay and Isothermal titration calorimetry were recruited to validate the interaction between the recombinant PmrA protein and target DNA. Field-emission scanning electron microscopy (FE-SEM) was employed to characterize the surface morphology of A. baumannii in both the PmrA knockout and mutation strains. The receiver domain of PmrA follows the canonical α5β5 response regulator assembly, which undergoes dimerization upon phosphorylation and activation. Beryllium trifluoride is utilized as an aspartate phosphorylation mimic in this process. Mutations involved in phosphorylation and dimerization significantly affected the expression of downstream pmrC and naxD genes. This impact resulted in an enhanced cell surface smoothness with fewer modifications, ultimately contributing to a decrease in colistin (polymyxin E) and polymyxin B resistance. Additionally, a conservative direct-repeat DNA PmrA binding sequence TTTAAGNNNNNTTTAAG was identified at the promoter region of the pmrC and naxD gene. These findings provide structural insights into the PmrA receiver domain and reveal the mechanism of polymyxin resistance, suggesting that PmrA could be a potential drug target to reverse polymyxin resistance in Acinetobacter baumannii.

PromSEMBLE: Hard Pattern Mining and Ensemble Learning for Detecting DNA Promoter Sequences.

Accurate identification of DNA promoter sequences is of crucial importance in unraveling the underlying mechanisms that regulate gene transcription. Initiation of transcription is controlled through regulatory transcription factors binding to promoter core regions in the DNA sequence. Detection of promoter regions is necessary if we are to build genetic regulatory networks for biomedical and clinical applications, and for identification of rarely expressed genes. We propose a novel ensemble learning technique using deep recurrent neural networks with convolutional feature extraction and hard negative pattern mining to detect several types of promoter sequences, including promoter sequences with the TATA-box and without the TATA-box, within DNA sequences of four different species. Using extensive independent tests and previously published results, we demonstrate that our method sets a new state-of-the-art of over 98% Matthews correlation coefficient in all eight organism categories for recognizing the stretch of base pairs that code for the promoter region within DNA sequences.