Transcription Factor Binding Sites Research Articles

Identification and functional validation of variants in the promoter region of HAND1 gene in sporadic tetralogy of Fallot.

Tetralogy of Fallot (TOF) is a common congenital heart disease (CHD) but the impact of the variants of the HAND1 gene promoter region has not been explored. DNA from blood samples of 612 subjects (300 sporadic TOF patients and 312 healthy controls) was sequenced to identify variants in the HAND1 gene promoter region that were further tested by cellular function experiments including dual-luciferase reporter gene assays, electrophoretic mobility shift analysis (EMSA), and bioinformatics analysis using JASPAR, a transcription factor binding site database. Eight variants in HAND1 gene promoter region were identified with 3 only found in TOF patients including one novel g.3639 G > T. All 3 variants significantly reduced the transcriptional activity of HAND1 gene promoter in an in vitro reporter assay (p < 0.05). JASPAR analysis indicated that these variants may alter the binding sites of transcription factors, potentially associated with TOF formation. In the promoter region of HAND1gene of TOF patients, 3 variants were found only in the patients including one found for the first time. These variants decreased transcription factor activity. Therefore, the present study implies the role of HAND1 gene in the pathogenesis of TOF and further provides new insights into the genetic basis of TOF formation. Sequencing of DNA from 612 human subjects (300 TOF patients and 312 healthy controls) identified 8 variants in the promoter region of HAND1 gene with 3 found only in TOF including 1 newly identified. Reduced transcriptional activity at these 3 variants was confirmed by dual-luciferase reporter gene assay and EMSA assay. Predictions from the JASPAR database suggest altered binding sites of transcription factors by the variants. We for the first time demonstrate variants in the HAND1 promoter and that cause cellular dysfunction. The variants identified may have a pathological role in the formation of TOF.

Plasticity and environment-specific relationships between gene expression and fitness in Saccharomyces cerevisiae.

The environment influences how an organism's genotype determines its phenotype and how this phenotype affects its fitness. Here, to better understand this dual role of environment in the production and selection of phenotypic variation, we determined genotype-phenotype-fitness relationships for mutant strains of Saccharomyces cerevisiae in four environments. Specifically, we measured how promoter mutations of the metabolic gene TDH3 modified expression level and affected growth for four different carbon sources. In each environment, we observed a clear relationship between TDH3 expression level and fitness, but this relationship differed among environments. Mutations with similar effects on expression in different environments often had different effects on fitness and vice versa. Such environment-specific relationships between phenotype and fitness can shape the evolution of phenotypic plasticity. We also found that mutations disrupting binding sites for transcription factors had more variable effects on expression among environments than those disrupting the TATA box, which is part of the core promoter. However, mutations with the most environmentally variable effects on fitness were located in the TATA box, because of both the lack of plasticity of TATA box mutations and environment-specific fitness functions. This observation suggests that mutations affecting different molecular mechanisms contribute unequally to regulatory sequence evolution in changing environments.

Abstract PR011: Advance prostate cancer detection through epigenomic profiling of cell-free DNA

Abstract Introduction: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease which can be classified into clinically relevant subtypes based on the expression of genes, such as the androgen receptor (AR) and neuroendocrine markers. Neuroendocrine prostate cancer (NEPC), characterized by gain of stem-like and neuroendocrine features and lack of AR expression is a clinically aggressive variant. Due to the lack of adequate biomarkers, NEPC is usually detected at a very advanced stage. There is mounting evidence that molecular subtype changes seen in NEPC are enforced by widespread epigenetic alterations, in particular DNA methylation changes. In this study, we aim to devise a novel DNA methylation-based assay for molecular subtyping and disease monitoring from cell-free DNA (cfDNA). Methods: We analyzed genome wide methylation patterns in 56 prostate cancer patient-derived xenograft (PDX) and 128 mCRPC tumors using array- and sequencing-based assays. We integrated DNA methylation at promoters, gene bodies and transcription factor binding site (TFBS) to determine the landscape of methylation alterations at key lineage specific genes. Using whole genome methylation derived from tissue with matched expression data we developed a deep learning framework to predict gene expression directly from tissue or cfDNA. Using key marker genes, the model was used to discern tumor molecular phenotypes from tissue and cfDNA in three independent cohorts of mCRPC patients using whole genome bisulfite sequencing and low-pass Enzymatic Methyl-Seq (EM-seq). Results: We observed a tight association between promoter, gene body and TFBS methylation with gene expression. Inferring gene expression from methylation for lineage specific markers such as AR, KLK3, ASCL1, INSM1, SRRM4 and DLL3 we classified molecular subtypes from both tissue and cfDNA. Additionally, for AR and ASCL1, we identified core sets of TFBSs whose differential methylation allowed for accurate assay-independent molecular subtype quantification. Applying the optimized quantitative model to mCRPC patients who underwent comprehensive tissue sampling by rapid autopsy we observed accurate subtype classification from both tissue samples and cfDNA for all cases. A similar analytical performance was observed in additional clinical mCRPC cohorts with cfDNA. Conclusion: Whole-genome methylation analysis of cfDNA allows for the prediction of gene expression patterns in tumor tissues, enabling non-invasive tumor subclassification and assessment of therapeutic targets. Citation Format: Mohamed Adil, Brian Hanratty, Pallabi Mustafi, Chitvan Mittal, Helen Richards, Ilsa Coleman, Radhika Patel, Anna-Lisa Doebley, Robert Patton, Eden Cruikshank, Patricia Galipeau, Ruth Dumpit, Martine Roudier, Jin-Yih Low, Navonil Sarkar, Robert Montgomery, Eva Corey, Colm Morrissey, Peter Nelson, Gavin Ha, Michael Haffner. Advance prostate cancer detection through epigenomic profiling of cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR011.

Abstract IA025: A DNA methylation atlas of normal human cell types

Abstract DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies. Citation Format: Tommy Kaplan. A DNA methylation atlas of normal human cell types [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA025.

An Optimized Method for Reconstruction of Transcriptional Regulatory Networks in Bacteria Using ChIP-exo and RNA-seq Datasets.

Transcriptional regulatory networks (TRNs) in bacteria are crucial for elucidating the mechanisms that regulate gene expression and cellular responses to environmental stimuli. These networks delineate the interactions between transcription factors (TFs) and their target genes, thereby uncovering the regulatory processes that modulate gene expression under varying environmental conditions. Analyzing TRNs offers valuable insights into bacterial adaptation, stress responses, and metabolic optimization from an evolutionary standpoint. Additionally, understanding TRNs can drive the development of novel antimicrobial therapies and the engineering of microbial strains for biofuel and bioproduct production. This protocol integrates advanced data analysis pipelines, including ChEAP, DEOCSU, and DESeq2, to analyze omics datasets that encompass genome-wide TF binding sites and transcriptome profiles derived from ChIP-exo and RNA-seq experiments. This approach minimizes both the time required and the risk of bias, making it accessible to non-expert users. Key steps in the protocol include preprocessing and peak calling from ChIP-exo data, differential expression analysis of RNA-seq data, and motif and regulon analysis. This method offers a comprehensive and efficient framework for TRN reconstruction across various bacterial strains, enhancing both the accuracy and reliability of the analysis while providing valuable insights for basic and applied research.

Identification of a DNA methylation signature in whole blood of newborn guinea pigs and human neonates following antenatal betamethasone exposure

Antenatal corticosteroids (ACS) are administered where there is risk of preterm birth to promote fetal lung development and improve perinatal survival. However, treatment may be associated with increased risk of developing neurobehavioural disorders. We have recently identified that ACS results in significant changes to DNA methylation patterns in the newborn and juvenile prefrontal cortex (PFC) of exposed guinea pig offspring. Methylation changes at transcription factor binding sites (TFBS) for PLAGL1, TFAP2C, ZNF263, and SP1 were consistently noted at both post-natal stages, suggesting a long-lasting signature of ACS exposure. In this study, we determined if comparable methylation changes are also present in the newborn blood of ACS-exposed guinea pig offspring, as this would determine whether blood methylation patterns may be used as a peripheral biomarker of changes in the brain. Pregnant guinea pigs were treated with saline or betamethasone (1 mg/kg) on gestational days 50/51. gDNA from whole blood of term-born offspring on post-natal day (PND) 1 was used for reduced representation bisulfite sequencing. Overall, 1677 differentially methylated CpG sites (DMCs) were identified in response to ACS. While no specific DMCs identified in the blood overlapped with those previously reported in the PFC of PND1 offspring, significant differential methylation at TFBSs for PLAGL1, TFAP2C, EGR1, ZNF263, and SP1 was persistently observed. Furthermore, re-examination of our previously reported data of DMCs in human neonatal blood following ACS identified the presence of this same TFBS signature in human infants, suggesting the potential for clinical translation of our epigenomic data.

In Vitro Genome-Wide Identification of Transcription Factor Binding Sites.

DNA affinity purification followed by sequencing (DAP-seq) profiles transcription factor (TF) binding sites in vitro, in which recombinant TF captures the genomic fragments with TF binding sites. Here, I describe the preparation of pre-PCR DNA-seq libraries for 96 TFs, including the expression and purification of recombinant TF, the affinity purification of TF binding fragments, and the sequencing-ready library preparation. Using this method, up to 96 DAP-seq libraries can be prepared in 2days.

Three-dimensional localization and tracking of chromosomal loci throughout the Escherichia coli cell cycle.

The intracellular position of genes may impact their expression, but it has not been possible to accurately measure the 3D position of chromosomal loci. In 2D, loci can be tracked using arrays of DNA-binding sites for transcription factors (TFs) fused with fluorescent proteins. However, the same 2D data can result from different 3D trajectories. Here, we have developed a deep learning method for super-resolved astigmatism-based 3D localization of chromosomal loci in live E. coli cells which enables a precision better than 61 nm at a signal-to-background ratio of ~4 on a heterogeneous cell background. Determining the spatial localization of chromosomal loci, we find that some loci are at the periphery of the nucleoid for large parts of the cell cycle. Analyses of individual trajectories reveal that these loci are subdiffusive both longitudinally (x) and radially (r), but that individual loci explore the full radial width on a minute time scale.

A Foxf1-Wnt-Nr2f1 cascade promotes atrial cardiomyocyte differentiation in zebrafish.

Nr2f transcription factors (TFs) are conserved regulators of vertebrate atrial cardiomyocyte (AC) differentiation. However, little is known about the mechanisms directing Nr2f expression in ACs. Here, we identified a conserved enhancer 3' to the nr2f1a locus, which we call 3'reg1-nr2f1a (3'reg1), that can promote Nr2f1a expression in ACs. Sequence analysis of the enhancer identified putative Lef/Tcf and Foxf TF binding sites. Mutation of the Lef/Tcf sites within the 3'reg1 reporter, knockdown of Tcf7l1a, and manipulation of canonical Wnt signaling support that Tcf7l1a is derepressed via Wnt signaling to activate the transgenic enhancer and promote AC differentiation. Similarly, mutation of the Foxf binding sites in the 3'reg1 reporter, coupled with gain- and loss-of-function analysis supported that Foxf1 promotes expression of the enhancer and AC differentiation. Functionally, we find that Wnt signaling acts downstream of Foxf1 to promote expression of the 3'reg1 reporter within ACs and, importantly, both Foxf1 and Wnt signaling require Nr2f1a to promote a surplus of differentiated ACs. CRISPR-mediated deletion of the endogenous 3'reg1 abrogates the ability of Foxf1 and Wnt signaling to produce surplus ACs in zebrafish embryos. Together, our data support that downstream members of a conserved regulatory network involving Wnt signaling and Foxf1 function on a nr2f1a enhancer to promote AC differentiation in the zebrafish heart.

Pleiotropic Expression Quantitative Trait Loci Are Enriched in Enhancers and Transcription Factor Binding Sites and Impact More of Genes.

Pleiotropic Expression Quantitative Trait Loci Are Enriched in Enhancers and Transcription Factor Binding Sites and Impact More of Genes.

Bioinformatic meta-analysis of transcriptomics of developing Drosophila muscles identifies temporal regulatory transcription factors including a notch effector

The intricate mechanism of gene regulation coordinates the precise control of when, where, and to what extent genes are activated or repressed, directing the complex processes that govern cellular functions and development. Dysregulation of gene expression can lead to diseases such as autoimmune disorders, cancer, and neurodegeneration. Transcriptional regulation, especially involving transcription factors (TFs), plays a major role in controlling gene expression. This study focuses on identifying gene regulatory mechanisms that generate distinct gene expression patterns during Drosophila muscle development. Utilising a bioinformatics approach, we analysed the developmental time-point-specific transcriptomics resource generated by Spletter et al., which includes mRNA sequencing data at eight stages of indirect flight muscle (IFM) development. They had identified 40 distinct genome-wide clusters representing various temporal expression dynamics using “soft” clustering. Promoter sequences of genes in these clusters were analysed to predict novel motifs that act as TF binding sites. Comparative analysis with known motifs revealed significant overlaps, indicating shared transcriptional regulation. The physiological relevance of predicted TFs was confirmed by cross-referencing with experimental ChIP-seq data. We focused on Cluster 36, characterised by a unique bimodal temporal expression profile, and identified candidate genes Rbfox1 and zfh1 for further study. Ectopic overexpression experiments revealed that the TF Enhancer of split m8 helix- loop-helix [E(spl)m8-HLH], part of the Notch signalling pathway, acts as a transcriptional repressor for Rbfox1 and zfh1. Our findings highlight the complexity of transcriptional regulation during myogenesis and identify key TFs that could be targeted for further research in muscle development and related disorders.

GeoNet enables the accurate prediction of protein-ligand binding sites through interpretable geometric deep learning.

The identification of protein binding residues is essential for understanding their functions invivo. However, it remains a computational challenge to accurately identify binding sites due to the lack of known residue binding patterns. Local residue spatial distribution and its interactive biophysical environment both determine binding patterns. Previous methods could not capture both information simultaneously, resulting in unsatisfactory performance. Here, we present GeoNet, an interpretable geometric deep learning model for predicting DNA, RNA, and protein binding sites by learning the latent residue binding patterns. GeoNet achieves this by introducing a coordinate-free geometric representation to characterize local residue distributions and generating an eigenspace to depict local interactive biophysical environments. Evaluation shows that GeoNet is superior compared to other leading predictors and it shows a strong interpretability of learned representations. We present three test cases, where interaction interfaces were successfully identified with GeoNet.

Cell-state dependent regulation of PPARγ signaling by the transcription factor ZBTB9 in adipocytes.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear hormone receptor that is a master regulator of adipocyte differentiation and function. ZBTB9 is a widely expressed but poorly studied transcription factor that was predicted to interact with PPARγ based on large-scale protein-protein interaction experiments. In addition, genome-wide association studies (GWAS) revealed associations between ZBTB9 and BMI, T2D risk, and HbA1c levels. Here we show that Zbtb9 deficiency in mature adipocytes decreased PPARγ activity and protein level, and thus acts as a positive regulator of PPARγ signaling. In contrast, Zbtb9 deficiency in 3T3-L1 and human preadipocytes increased PPARγ levels and enhanced adipogenesis. Transcriptomic and transcription factor binding site analyses of Zbtb9 deficient preadipocytes revealed that the E2F pathway, controlled by the E2F family of transcription factors that are classically associated with cell cycle regulation, was among the most upregulated pathways. E2F1 positively regulates adipogenesis by promoting Pparg expression, independent of its cell cycle role, via direct binding to the Pparg promoter early during adipogenesis. RB phosphorylation (pRB), which regulates E2F activity, was also upregulated in Zbtb9 deficient preadipocytes. Critically, an E2F1 inhibitor blocked the effects of Zbtb9 deficiency on adipogenesis. Collectively, these results demonstrate that Zbtb9 inhibits adipogenesis as a negative regulator of Pparg expression via pRB-E2F signaling. Our findings reveal cell-state dependent roles of ZBTB9 in adipocytes, identifying a new molecule that regulates adipocyte biology as both a positive and negative regulator of PPARγ signaling depending on the cellular context, and thus may be important in the pathogenesis of obesity and T2D.

A Novel Regulatory Motif at the Hinge Dimer Interface of the MksB Mediates Dimerization and DNA Binding Activity.

The Structural Maintenance of Chromosome (SMC) protein is essential for various cellular processes, including chromosome organization, DNA repair, and genome stability. MksB, an alternative SMC protein present in prokaryotes, comprises a hinge dimerization domain and an ABC ATPase head domain linked by a coiled-coil arm. While hinge dimerization in bacterial and eukaryotic SMCs is attributed to conserved glycines, our study unveils the critical role of a novel KDDR motif located at a loop near the hinge dimer interface in Mycobacterium smegmatis MksB (MsMksB). We demonstrate the regulatory role of this motif in MsMksB dimerization and DNA binding activity. The K600D mutation in the KDDR motif induces MsMksB dimer-to-monomer conversion, highlighting the significance of this motif in MsMksB dimerization. Mass spectrometry-based mapping of the DNA binding site revealed the lysine's involvement in protein-DNA interaction. Monomers of the hinge domain lose DNA binding activity, and MsMksB single-arm mutants exhibit reduced DNA binding and ATPase activity, underscoring the importance of hinge-mediated dimerization in MsMksB function. Notably, the R603D mutant retains dimerization but shows compromised ATPase and DNA binding activities. Mutants with defective ATPase activity exhibit impaired DNA condensation in vivo. These findings provide novel regulatory insight into the mechanism of MksB dimerization and DNA binding, uncovering the fundamental processes of chromosome condensation and segregation.

Pharmacological blockade of a pioneer transcription factor.

Cancers frequently co-opt lineage-specific transcription factors (TFs) utilized in normal development to sustain proliferation. However, the effects of these TFs on tumor development depend considerably on where in the genome they bind. A new paper by Taylor and colleagues expands on previously developed diamidine compounds that obstruct the DNA binding sites of the pioneer TF PU.1 (SPI1) in acute myeloid leukemia (AML). Immobilization and sequencing of genomic DNA targeted by these compounds revealed that these inhibitors alter the genomic binding patterns of PU.1. The authors report that their strategy constrains the genomic binding preferences of PU.1, leading to redistribution of PU.1 to promoters and other gene-proximal regions with elevated G/C content. Here we discuss recent developments for targeting PU.1 in hematologic malignancies. We also explore the shared functional roles of PU.1 and SWI/SNF ATP-dependent chromatin remodeling complexes, which work together to sustain the enhancer landscape needed for tumor cell proliferation but also have key roles in non-tumor settings.

Comprehensive analysis of transcription factor binding sites and expression profiling of rice pathogenesis related genes (OsPR1).

Pathogenesis-related (PR) proteins, found in plants, play a crucial role in responding to both biotic and abiotic stresses and are categorized into 17 distinct families based on their properties and functions. We have conducted a phylogenetic analysis of OsPR1 genes (rice PR1 genes) in conjunction with 58 putative PR1 genes identified in Brachypodium distachyon, Hordeum vulgare, Brassica rapa, and Zea mays through BLASTP predictions. We extensively investigated the responses of the remaining 11 rice PR1 genes, using OsPR1a as a reference, under various stress conditions, including phytohormone treatments (salicylic acid and brassinosteroid [BR]), wounding, and heat stress (HS). In rice, of the 32 predicted OsPR1 genes, 12 have been well-characterized for their roles in disease resistance, while the functions of the remaining genes have not been studied extensively. In our study, we selected an additional 11 OsPR1 genes for further analysis and constructed a phylogenetic tree based on the presence of a 10-amino-acid-long conserved motif within these proteins. The phylogenetic analysis revealed that both OsPR1a from earlier studies and OsPR1-74 from our current study belong to the same clade. These genes consistently exhibit upregulation in response to diverse stress treatments such as biotic stress and abiotic stresses such as heat, drought, and salinity, indicating their potential roles in enhancing stress tolerance in rice. Significantly, this study delves into the previously unexplored role of OsPR1 genes in responding to Brassinosteroid (BR) and heat stress (HS) treatments, confirming their involvement in stress responses through qRT-PCR analysis. We found that seven genes were upregulated by EBR treatment. During heat stress (HS), six and seven genes were upregulated at 1hand 4h HS, respectively. The remaining genes OsPR1-22 and OsPR1-75 were upregulated at 1h but downregulated at 4h HS and under EBR treatment. In contrast, OsPR1-76 was upregulated at both 1h and 4h HS, but downregulated under EBR treatment. Promoters of PR1 genes in rice and other crops are rich in transcription factor binding sites (TFBSs) and feature a conserved Cysteine-rich secretory protein (SCP or CAP) motif. This study advances our understanding of PR1 gene regulation and its potential to enhance stress tolerance in rice.

Binding profiles for 961 Drosophila and C. elegans transcription factors reveal tissue-specific regulatory relationships.

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here we present the culmination of the efforts of the Model Organism ENCyclopedia Of DNA Elements (modENCODE) and the model organism Encyclopedia of Regulatory Networks (modERN) consortia to systematically assay TF binding events in vivo in two major model organisms, Drosophila melanogaster (fly) and Caenorhabditis elegans (worm). These datasets comprise 605 TFs identifying 3.6M sites in the fly and 356 TFs identifying 0.9 M sites in the worm and represent the majority of the regulatory space in each genome. We demonstrate that TFs associate with chromatin in clusters termed "metapeaks", that larger metapeaks have characteristics of high occupancy target (HOT) regions, and that the importance of consensus sequence motifs bound by TFs depends on metapeak size and complexity. Combining ChIP-seq data with single cell RNA-seq data in a machine learning model identifies TFs with a prominent role in promoting target gene expression in specific cell types, even differentiating between parent-daughter cells during embryogenesis. These data are a rich resource for the community that should fuel and guide future investigations into TF function. To facilitate data accessibility and utility, all strains expressing GFP-tagged TFs are available at the stock centers for each organism. The chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center, GEO, and through a direct interface that provides rapid access to processed data sets and summary analyses, as well as widgets to probe the cell type-specific TF-target relationships.

Somatic mosaicism in schizophrenia brains reveals prenatal mutational processes.

Germline mutations modulate the risk of developing schizophrenia (SCZ). Much less is known about the role of mosaic somatic mutations in the context of SCZ. Deep (239×) whole-genome sequencing (WGS) of brain neurons from 61 SCZ cases and 25 controls postmortem identified mutations occurring during prenatal neurogenesis. SCZ cases showed increased somatic variants in open chromatin, with increased mosaic CpG transversions (CpG>GpG) and T>G mutations at transcription factor binding sites (TFBSs) overlapping open chromatin, a result not seen in controls. Some of these variants alter gene expression, including SCZ risk genes and genes involved in neurodevelopment. Although these mutational processes can reflect a difference in factors indirectly involved in disease, increased somatic mutations at developmental TFBSs could also potentially contribute to SCZ.

A Rare Noncoding Enhancer Variant in SCN5A Contributes to the High Prevalence of Brugada Syndrome in Thailand.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder that causes sudden death in young adults. Rare genetic variants in the SCN5A gene encoding the Nav1.5 sodium channel and common noncoding variants at this locus are robustly associated with the condition. BrS is particularly prevalent in Southeast Asia but the underlying ancestry-specific factors remain largely unknown. Genome sequencing of BrS probands and population-matched controls from Thailand was performed to identify rare noncoding variants at the SCN5A-SCN10A locus that were enriched in patients with BrS. A likely causal variant was prioritized by computational methods and introduced into human induced pluripotent stem cell (hiPSC) lines using CRISPR-Cas9. The effect of the variant on SCN5A expression and Nav1.5 sodium channel current was then assessed in hiPSC-derived cardiomyocytes (hiPSC-CMs). A rare noncoding variant in an SCN5A intronic enhancer region was highly enriched in patients with BrS (detected in 3.9% of cases with a case-control odds ratio of 45.2). The variant affects a nucleotide conserved across all mammalian species and predicted to disrupt a Mef2 transcription factor binding site. Heterozygous introduction of the enhancer variant in hiPSC-CMs caused significantly reduced SCN5A expression from the variant-containing allele and a 30% reduction in Nav1.5-mediated sodium current density compared with isogenic controls, confirming its pathogenicity. Patients with the variant had severe phenotypes, with 89% experiencing cardiac arrest. This is the first example of a functionally validated rare noncoding variant at the SCN5A locus and highlights how genome sequencing in understudied populations can identify novel disease mechanisms. The variant partly explains the increased prevalence of BrS in this region and enables the identification of at-risk variant carriers to reduce the burden of sudden cardiac death in Thailand.

Targeting circadian transcriptional programs through a cis-regulatory mechanism in triple negative breast cancer.

Circadian clock genes are emerging targets in many types of cancer, but their mechanistic contributions to tumor progression are still largely unknown. This makes it challenging to stratify patient populations and develop corresponding treatments. In this work, we show that in breast cancer, the disrupted expression of circadian genes has the potential to serve as biomarkers. We also show that the master circadian transcription factors (TFs) BMAL1 and CLOCK are required for the proliferation of metastatic mesenchymal stem-like (mMSL) triple-negative breast cancer (TNBC) cells. Using currently available small molecule modulators, we found that a stabilizer of cryptochrome 2 (CRY2), the direct repressor of BMAL1 and CLOCK transcriptional activity, synergizes with inhibitors of proteasome, which is required for BMAL1 and CLOCK function, to repress a transcriptional program comprising circadian cycling genes in mMSL TNBC cells. Omics analyses on drug-treated cells implied that this repression of transcription is mediated by the transcription factor binding sites (TFBSs) features in the cis-regulatory elements (CRE) of clock-controlled genes. Through a massive parallel reporter assay, we defined a set of CRE features that are potentially repressed by the specific drug combination. The identification of cis -element enrichment might serve as a new concept of defining and targeting tumor types through the modulation of cis -regulatory programs, and ultimately provide a new paradigm of therapy design for cancer types with unclear drivers like TNBC.