Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with patients having unresectable or metastatic disease at diagnosis, with poor prognosis and very short survival. Given that genetic variation within autophagy-related genes influences autophagic flux and susceptibility to solid cancers, we decided to investigate whether 55,583 single nucleotide polymorphisms (SNPs) within 234 autophagy-related genes could influence the risk of developing PDAC in three large independent cohorts of European ancestry including 12,754 PDAC cases and 324,926 controls. The meta-analysis of these populations identified, for the first time, the association of the BIDrs9604789 variant with an increased risk of developing the disease (ORMeta = 1.31, p = 9.67 × 10-6). We also confirmed the association of TP63rs1515496 and TP63rs35389543 variants with PDAC risk (OR = 0.89, p = 6.27 × 10-8 and OR = 1.16, p = 2.74 × 10-5). Although it is known that BID induces autophagy and TP63 promotes cell growth, cell motility and invasion, we also found that carriers of the TP63rs1515496G allele had increased numbers of FOXP3+ Helios+ T regulatory cells and CD45RA+ T regulatory cells (p = 7.67 × 10-4 and p = 1.56 × 10-3), but also decreased levels of CD4+ T regulatory cells (p = 7.86 × 10-4). These results were in agreement with research suggesting that the TP63rs1515496 variant alters binding sites for FOXA1 and CTCF, which are transcription factors involved in modulating specific subsets of regulatory T cells. In conclusion, this study identifies BID as new susceptibility locus for PDAC and confirms previous studies suggesting that the TP63 gene is involved in the development of PDAC. This study also suggests new pathogenic mechanisms of the TP63 locus in PDAC.
- MeSH
- Autophagy * genetics MeSH
- White People genetics MeSH
- Carcinoma, Pancreatic Ductal * genetics pathology MeSH
- Forkhead Transcription Factors MeSH
- Genetic Predisposition to Disease * MeSH
- Hepatocyte Nuclear Factor 3-alpha genetics metabolism MeSH
- Polymorphism, Single Nucleotide * MeSH
- Cohort Studies MeSH
- Humans MeSH
- Biomarkers, Tumor * genetics MeSH
- Tumor Suppressor Proteins * genetics MeSH
- Pancreatic Neoplasms * genetics pathology MeSH
- Case-Control Studies MeSH
- Transcription Factors genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Meta-Analysis MeSH
The ABCB1 gene, encoding the ATP-dependent translocase ABCB1, plays a crucial role in the clearance of amyloid-beta (Aβ) peptides and the transport of cholesterol, implicating it in the pathogenesis of Alzheimer's disease. The study aims to investigate the association between polymorphisms in the ABCB1 gene and cognitive decline in individuals with mild cognitive impairment (MCI), particularly focusing on language function. A longitudinal cohort study involving 1 005 participants from the Czech Brain Aging Study was conducted. Participants included individuals with Alzheimer's disease, amnestic MCI, non-amnestic MCI, subjective cognitive decline, and healthy controls. Next-generation sequencing was utilized to analyze the entire ABCB1 gene. Cognitive performance was assessed using a comprehensive battery of neuropsychological tests, including the Boston Naming Test and the semantic verbal fluency test. Ten ABCB1 polymorphisms (rs55912869, rs56243536, rs10225473, rs10274587, rs2235040, rs12720067, rs12334183, rs10260862, rs201620488, and rs28718458) were significantly associated with cognitive performance, particularly in language decline among amnestic MCI patients. In silico analyses revealed that some of these polymorphisms may affect the binding sites for transcription factors (HNF-3alpha, C/EBPβ, GR-alpha) and the generation of novel exonic splicing enhancers. Additionally, polymorphism rs55912869 was identified as a potential binding site for the microRNA hsa-mir-3163. Our findings highlight the significant role of ABCB1 polymorphisms in cognitive decline, particularly in language function, among individuals with amnestic MCI. These polymorphisms may influence gene expression and function through interactions with miRNAs, transcription factors, and alternative splicing mechanisms.
- MeSH
- Alzheimer Disease genetics MeSH
- Polymorphism, Single Nucleotide * MeSH
- Cognitive Dysfunction * genetics MeSH
- Humans MeSH
- Longitudinal Studies MeSH
- Neuropsychological Tests MeSH
- ATP Binding Cassette Transporter, Subfamily B genetics MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
Somatic hypermutation (SHM) and class switch recombination (CSR) diversify immunoglobulin (Ig) genes and are initiated by the activation-induced deaminase (AID), a single-stranded DNA cytidine deaminase thought to engage its substrate during RNA polymerase II (RNAPII) transcription. Through a genetic screen, we identified numerous potential factors involved in SHM, including elongation factor 1 homolog (ELOF1), a component of the RNAPII elongation complex that functions in transcription-coupled nucleotide excision repair (TC-NER) and transcription elongation. Loss of ELOF1 compromises SHM, CSR, and AID action in mammalian B cells and alters RNAPII transcription by reducing RNAPII pausing downstream of transcription start sites and levels of serine 5 but not serine 2 phosphorylated RNAPII throughout transcribed genes. ELOF1 must bind to RNAPII to be a proximity partner for AID and to function in SHM and CSR, and TC-NER is not required for SHM. We propose that ELOF1 helps create the appropriate stalled RNAPII substrate on which AID acts.
- MeSH
- AICDA (Activation-Induced Cytidine Deaminase) MeSH
- B-Lymphocytes * immunology metabolism MeSH
- Cytidine Deaminase metabolism genetics MeSH
- Phosphoproteins * genetics metabolism MeSH
- Phosphorylation MeSH
- Transcription, Genetic MeSH
- Humans MeSH
- Mice, Knockout MeSH
- Mice MeSH
- DNA Repair MeSH
- Immunoglobulin Class Switching * MeSH
- RNA Polymerase II metabolism genetics MeSH
- Somatic Hypermutation, Immunoglobulin * MeSH
- Transcriptional Elongation Factors * genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Single-cell RNA-seq methods can be used to delineate cell types and states at unprecedented resolution but do little to explain why certain genes are expressed. Single-cell ATAC-seq and multiome (ATAC + RNA) have emerged to give a complementary view of the cell state. It is however unclear what additional information can be extracted from ATAC-seq data besides transcription factor binding sites. Here, we show that ATAC-seq telomere-like reads counter-inituively cannot be used to infer telomere length, as they mostly originate from the subtelomere, but can be used as a biomarker for chromatin condensation. Using long-read sequencing, we further show that modern hyperactive Tn5 does not duplicate 9 bp of its target sequence, contrary to common belief. We provide a new tool, Telomemore, which can quantify nonaligning subtelomeric reads. By analyzing several public datasets and generating new multiome fibroblast and B-cell atlases, we show how this new readout can aid single-cell data interpretation. We show how drivers of condensation processes can be inferred, and how it complements common RNA-seq-based cell cycle inference, which fails for monocytes. Telomemore-based analysis of the condensation state is thus a valuable complement to the single-cell analysis toolbox.
- MeSH
- Single-Cell Analysis * methods MeSH
- B-Lymphocytes metabolism cytology MeSH
- Cell Cycle * genetics MeSH
- Chromatin Immunoprecipitation Sequencing methods MeSH
- Chromatin * metabolism chemistry genetics MeSH
- Fibroblasts metabolism cytology MeSH
- Humans MeSH
- RNA-Seq methods MeSH
- Telomere * genetics MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which plays numerous and pivotal roles in human physiology and pathophysiology. Therefore, pharmacotherapeutic targeting of the AhR is a highly pertinent issue. The identification of new AhR ligands and the characterization of the interactions between the AhR ligands and AhR protein requires appropriate methodology. In spite the AhR is monomeric intracellular soluble receptor, the full-length human AhR protein has not been crystallized so far, and its isolation in a form applicable in the binding assays is highly challenging. Recent advances, including crystallization of AhR fragments, recombinant protein technologies, and cryogenic electron microscopy, allowed for exploitation of diverse experimental techniques for studying interactions between ligands and the AhR. In the current paper, we review existing AhR ligand binding assays, including their description, applicability and limitations.
The RNA content is crucial for the formation of nuclear compartments, such as nuclear speckles and nucleoli. Phosphatidylinositol 4,5-bisphosphate (PIP2) is found in nuclear speckles, nucleoli, and nuclear lipid islets and is involved in RNA polymerase I/II transcription. Intriguingly, the nuclear localization of PIP2 was also shown to be RNA-dependent. We therefore investigated whether PIP2 and RNA cooperate in the establishment of nuclear architecture. In this study, we unveiled the RNA-dependent PIP2-associated (RDPA) nuclear proteome in human cells by mass spectrometry. We found that intrinsically disordered regions (IDRs) with polybasic PIP2-binding K/R motifs are prevalent features of RDPA proteins. Moreover, these IDRs of RDPA proteins exhibit enrichment for phosphorylation, acetylation, and ubiquitination sites. Our results show for the first time that the RDPA protein Bromodomain-containing protein 4 (BRD4) associates with PIP2 in the RNA-dependent manner via electrostatic interactions, and that altered PIP2 levels affect the number of nuclear foci of BRD4 protein. Thus, we propose that PIP2 spatiotemporally orchestrates nuclear processes through association with RNA and RDPA proteins and affects their ability to form foci presumably via phase separation. This suggests the pivotal role of PIP2 in the establishment of a functional nuclear architecture competent for gene expression.
- MeSH
- Cell Nucleus * metabolism genetics MeSH
- Phosphatidylinositol 4,5-Diphosphate * metabolism MeSH
- Phosphorylation MeSH
- Nuclear Proteins * metabolism genetics MeSH
- Humans MeSH
- Cell Cycle Proteins metabolism genetics MeSH
- Bromodomain Containing Proteins MeSH
- RNA metabolism genetics MeSH
- Transcription Factors * metabolism genetics MeSH
- Protein Binding MeSH
- Intrinsically Disordered Proteins * metabolism genetics chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Many enhancers control gene expression by assembling regulatory factor clusters, also referred to as condensates. This process is vital for facilitating enhancer communication and establishing cellular identity. However, how DNA sequence and transcription factor (TF) binding instruct the formation of high regulatory factor environments remains poorly understood. Here we developed a new approach leveraging enhancer-centric chromatin accessibility quantitative trait loci (caQTLs) to nominate regulatory factor clusters genome-wide. By analyzing TF-binding signatures within the context of caQTLs and comparing episomal versus endogenous enhancer activities, we discovered a class of regulators, 'context-only' TFs, that amplify the activity of cell type-specific caQTL-binding TFs, that is, 'context-initiator' TFs. Similar to super-enhancers, enhancers enriched for context-only TF-binding sites display high coactivator binding and sensitivity to bromodomain-inhibiting molecules. We further show that binding sites for context-only and context-initiator TFs underlie enhancer coordination, providing a mechanistic rationale for how a loose TF syntax confers regulatory specificity.
- MeSH
- Chromatin * genetics metabolism MeSH
- Humans MeSH
- Quantitative Trait Loci * MeSH
- Mice MeSH
- Gene Expression Regulation MeSH
- Transcription Factors * metabolism genetics MeSH
- Protein Binding MeSH
- Binding Sites genetics MeSH
- Enhancer Elements, Genetic * MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Adenoid cystic carcinoma (AdCC) is one of the most common salivary gland malignancies and occurs in all major and minor salivary gland and seromucous gland sites. AdCCs of salivary gland origin have long been categorized as fusion-defined carcinomas owing to the almost consistent presence of fusion genes MYB::NFIB , or less commonly MYBL1::NFIB. We collected a cohort of 95 cases of AdCC, which were largely characterized by canonical fusions MYB::NFIB (49 cases) or MYBL1::NFIB (9 cases). In additional 11 cases of AdCC, rearrangements in MYB or NFIB genes were detected by FISH. In addition, NGS revealed novel noncanonical fusion transcripts EWSR1::MYB ; ACTB::MYB; ESRRG::DNM3, MYB::TULP4 , and ACTN4::MYB , each of them in 1 case. The tumors that showed noncanonical fusions had features of metatypical AdCC with a diverse architecture, lobulated multinodular growth pattern, and hypercellular peripheral palisading of nuclei (2 cases), tubular hypereosinophilia (2 cases), and pale eosinophilic to vacuolated (bubbly) cytoplasm (3 cases). Our study documented 3 cases of AdCC of salivary glands harboring novel gene fusions TULP4::MYB , ACTN4::MYB , and ACTB::MYB , in 1 case each, which have not been described before. A rare EWSR1::MYB fusion was detected in 1 case. Moreover, 1 case of sinonasal metatypical AdCC showed EWSR1 rearrangement detected by FISH. Also, 1 case with an ESRRG::DNM3 fusion of unknown significance is described in this study. These discoveries illustrate how broad molecular profiling will expand understanding of changes in known entities.
- MeSH
- Carcinoma, Adenoid Cystic * genetics pathology MeSH
- Adult MeSH
- Gene Fusion MeSH
- Oncogene Proteins, Fusion * genetics MeSH
- Genetic Predisposition to Disease MeSH
- Gene Rearrangement MeSH
- In Situ Hybridization, Fluorescence MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Biomarkers, Tumor * genetics MeSH
- Salivary Gland Neoplasms * genetics pathology MeSH
- RNA-Binding Protein EWS * genetics MeSH
- Proto-Oncogene Proteins c-myb genetics MeSH
- Proto-Oncogene Proteins MeSH
- Aged MeSH
- Trans-Activators genetics MeSH
- NFI Transcription Factors genetics MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Armadillo repeat-containing proteins (ARMCs) are a large family found throughout eukaryotes, which play prominent roles in cell adhesion, signaling and cytoskeletal regulation. The ARMC6 protein is highly conserved in primates, including humans, but to date does not have a clear function beyond initial hints of a link to cancer and telomerase activity. We report here in vitro experiments showing ARMC6 binding to DNA promoter sequences from several cancer-related genes (e.g., EGFR, VEGF and c-MYC), and also to the telomeric RNA repeat (TERRA). ARMC6 binding activity appears to recognize G-quadruplex motifs, which are being increasingly implicated as structure-based protein binding sites in chromosome maintenance and repair. In vivo investigation of ARMC6 function revealed that when this protein is overexpressed in human cell lines, there is different expression of genes connected with oncogenic pathways and those implicated in downstream non-canonical telomerase pathways (e.g., VEGF, hTERT, c-MYC, ESM1, MMP3). ARMC6 is already known to interact with human shelterin protein TRF2 and telomerase. The protein binds G-quadruplex structures and does so preferentially to RNA over DNA. As such, this protein may be an example of how a non-canonical nucleic acid structural motif allows mediation between gene regulation and telomeric chromatin rearrangement pathways.
- MeSH
- DNA-Binding Proteins MeSH
- G-Quadruplexes * MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Neoplasms genetics metabolism MeSH
- Promoter Regions, Genetic * MeSH
- Armadillo Domain Proteins * metabolism genetics MeSH
- Gene Expression Regulation, Neoplastic MeSH
- RNA metabolism genetics MeSH
- Telomerase metabolism genetics MeSH
- Telomere * metabolism MeSH
- Transcription Factors MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Monogenic diabetes is a gateway to precision medicine through molecular mechanistic insight. Hepatocyte nuclear factor 1A (HNF-1A) and HNF-4A are transcription factors that engage in crossregulatory gene transcription networks to maintain glucose-stimulated insulin secretion in pancreatic β cells. Variants in the HNF1A and HNF4A genes are associated with maturity-onset diabetes of the young (MODY). Here, we explored 4 variants in the P2-HNF4A promoter region: 3 in the HNF-1A binding site and 1 close to the site, which were identified in 63 individuals from 21 families of different MODY disease registries across Europe. Our goal was to study the disease causality for these variants and to investigate diabetes mechanisms on the molecular level. We solved a crystal structure of HNF-1A bound to the P2-HNF4A promoter and established a set of techniques to probe HNF-1A binding and transcriptional activity toward different promoter variants. We used isothermal titration calorimetry, biolayer interferometry, x-ray crystallography, and transactivation assays, which revealed changes in HNF-1A binding or transcriptional activities for all 4 P2-HNF4A variants. Our results suggest distinct disease mechanisms of the promoter variants, which can be correlated with clinical phenotype, such as age of diagnosis of diabetes, and be important tools for clinical utility in precision medicine.
- MeSH
- Diabetes Mellitus, Type 2 * genetics metabolism MeSH
- Hepatocyte Nuclear Factor 1-alpha * genetics metabolism MeSH
- Hepatocyte Nuclear Factor 4 * genetics metabolism MeSH
- Crystallography, X-Ray MeSH
- Humans MeSH
- Promoter Regions, Genetic * genetics MeSH
- Gene Expression Regulation MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
