Transcription Factor Binding Sequences Research Articles

Abstract We137: PGC-1α-dependent transcriptional regulatory mechanisms for tricarboxylic acid cycle genes in the heart.

Many metabolic genes, including tricarboxylic acid (TCA) cycle genes, are downregulated in heart failure, which may promote the pathology due to insufficient energy production. To clarify how TCA cycle genes are downregulated in the failing heart, it is a prerequisite to clarify how these genes are transcribed in the normal heart. PGC-1α is a transcriptional co-activator that broadly induces metabolic genes through activation of transcription factors, such as ERR, Nrf1, Gabpa and YY1. Here we show that PGC-1α transcribes 13 among 14 TCA cycle genes partly through ERR, Nrf1, Gabpa and YY1. Thirteen TCA cycle genes, except for Suclg2, were downregulated in cardiac specific PGC-1α knockout (KO) mice (Relative mRNA levels in KO mice compared to wild type (WT) were CS 0.66*, Aco2 0.64*, Idh3a 0.62*, Idh3b 0.54*, Idh3g 0.54*, Ogdh 0.64*, Suclg1 0.45*, Suclg2: 1.1, Sdha 0.57*, Sdhb 0.53*, Sdhc 0.72*, Sdhd 0.57*, Fh1 0.32* and Mdh2 0.54*,p<0.05 vs. WT). ChIP-sequencing with PGC-1α antibody demonstrated that PGC-1α is localized at the promoter of thirteen TCA cycle genes, besides Suclg2. Bioinformatics analyses identified consensus binding sequences of ERR, Nrf1, Gabpa and YY1 in these promoters. Gene expression analyses with knockdown of these transcription factors demonstrated that a transcription factor whose binding sequence is found proximal to the PGC-1α localization peak transcribes the downstream genes in general. These transcription factor binding sequences were well conserved between mice and humans. ERR-, Nrf1- and Gabpa-dependent PGC-1α recruitment was verified with in vitro DNA binding assays using endogenous promoter sequences of Idh3a, Idh3g and Sdha. Taken together, this study clarifies a precise transcriptional mechanism for TCA cycle genes in the normal heart, which could be useful for understanding how those genes are dysregulated in the failing heart.

Genomic Variations Explorer (GenVarX): a toolset for annotating promoter and CNV regions using genotypic and phenotypic differences

The rapid growth of sequencing technology and its increasing popularity in biology-related research over the years has made whole genome re-sequencing (WGRS) data become widely available. A large amount of WGRS data can unlock the knowledge gap between genomics and phenomics through gaining an understanding of the genomic variations that can lead to phenotype changes. These genomic variations are usually comprised of allele and structural changes in DNA, and these changes can affect the regulatory mechanisms causing changes in gene expression and altering the phenotypes of organisms. In this research work, we created the GenVarX toolset, that is backed by transcription factor binding sequence data in promoter regions, the copy number variations data, SNPs and Indels data, and phenotypes data which can potentially provide insights about phenotypic differences and solve compelling questions in plant research. Analytics-wise, we have developed strategies to better utilize the WGRS data and mine the data using efficient data processing scripts, libraries, tools, and frameworks to create the interactive and visualization-enhanced GenVarX toolset that encompasses both promoter regions and copy number variation analysis components. The main capabilities of the GenVarX toolset are to provide easy-to-use interfaces for users to perform queries, visualize data, and interact with the data. Based on different input windows on the user interface, users can provide inputs corresponding to each field and submit the information as a query. The data returned on the results page is usually displayed in a tabular fashion. In addition, interactive figures are also included in the toolset to facilitate the visualization of statistical results or tool outputs. Currently, the GenVarX toolset supports soybean, rice, and Arabidopsis. The researchers can access the soybean GenVarX toolset from SoyKB via https://soykb.org/SoybeanGenVarX/, rice GenVarX toolset, and Arabidopsis GenVarX toolset from KBCommons web portal with links https://kbcommons.org/system/tools/GenVarX/Osativa and https://kbcommons.org/system/tools/GenVarX/Athaliana, respectively.

Hippocampal and peripheral blood DNA methylation signatures correlate at the gene and pathway level in a mouse model of autism.

Autism spectrum disorders (ASD) are polygenic multifactorial disorders influenced by environmental factors. ASD-related differential DNA methylation has been found in human peripheral tissues, such as placenta, paternal sperm, buccal epithelium, and blood. However, these data lack direct comparison of DNA methylation levels with brain tissue from the same individual to determine the extent that peripheral tissues are surrogates for behavior-related disorders. Here, whole genome methylation profiling at all the possible sites throughout the mouse genome (>25 million) from both brain and blood tissues revealed novel insights into the systemic contributions of DNA methylation to ASD. Sixty-six differentially methylated regions (DMRs) share the same genomic coordinates in these two tissues, many of which are linked to risk genes for neurodevelopmental disorders and intellectual disabilities (e.g. Prkch, Ptn, Hcfc1, Mid1, and Nfia). Gene ontological pathways revealed a significant number of common terms between brain and blood (N = 65 terms), and nearly half (30/65) were associated with brain/neuronal development. Furthermore, seven DMR-associated genes among these terms contain methyl-sensitive transcription factor sequence motifs within the DMRs of both tissues; four of them (Cux2, Kcnip2, Fgf13, and Mrtfa) contain the same methyl-sensitive transcription factor binding sequence motifs (HES1/2/5, TBX2 and TFAP2C), suggesting DNA methylation influences the binding of common transcription factors required for gene expression. Together, these findings suggest that peripheral blood is a good surrogate tissue for brain and support that DNA methylation contributes to altered gene regulation in the pathogenesis of ASD.

Dynamic Nanostructures for Conditional Activation and Deactivation of Biological Pathways.

Nucleic acid nanotechnology utilizes natural and synthetic structural motifs to build versatile nucleic acid nanoparticles (NANPs). These rationally designed assemblies can be further equipped with functional nucleic acids and other molecules such as peptides, fluorescent dyes, etc. In addition to nucleic acids that directly interact with the regulated target gene transcripts, NANPs can display decoys, wherein the oligonucleotide stretches with transcription factor binding sequences, preventing transcription initiation. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a group of five crucial transcription factors regulating the pathogenesis of inflammatory diseases and cancer; as such, they are relevant targets for therapy. One therapeutic approach involves interdependent self-recognizing hybridized DNA/RNA fibers designed to bind NF-κB and prevent its interaction with the promotor region of NF-κB-dependent genes involved in inflammatory responses. Decoying NF-κB results in the inability to initiate transcription of regulated genes, showing a promising approach to gene regulation and gene therapy. The protocol described herein provides detailed steps for the synthesis of NF-κB decoy fibers, as well as their characterization using polyacrylamide gel electrophoresis (to confirm desired physicochemical properties and purity) and functional bioassays (to confirm desired biological activity).

Upregulation of IL-8, osteonectin, and myonectin mRNAs by intermittent hypoxia via OCT1- and NRF2-mediated mechanisms in skeletal muscle cells.

Sleep apnoea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/Type 2 diabetes. The induction of insulin resistance in skeletal muscle is a key phenomenon to develop diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human RD and mouse C2C12 muscle cells to normoxia or IH and measured their mRNA levels by real-time RT-PCR. We found that IH significantly increased the mRNA and protein levels of muscle-derived insulin resistance-factors (myokines) such as IL-8, osteonectin (ON), and myonectin (MN) in muscle cells. We further analysed the IH-induced expression mechanisms of IL-8, ON, and MN genes in muscle cells. Deletion analyses of the human myokine promoter(s) revealed that the regions -152 to -151 in IL-8, -105 to -99 in ON, and - 3741 to -3738 in MN promoters were responsible for the activation by IH in RD cells. The promoters contain consensus transcription factor binding sequences for OCT1 in IL-8 and MN promoters, and for NRF2 in ON promoter, respectively. The introduction of siRNA for OCT1 abolished the IH-induced expression(s) of IL-8 and MN and siRNA for NRF2 abolished the IH-induced expression of ON.

Cyclobutane Pyrimidine Dimer Hyperhotspots as Sensitive Indicators of Keratinocyte UV Exposure†.

The dominant DNA damage generated by UV exposure is the cyclobutane pyrimidine dimer (CPD), which alters skin cell physiology and induces cell death and mutation. Genome-wide nucleotide-resolution analysis of CPDs in melanocytes and fibroblasts has identified "CPD hyperhotspots", pyrimidine-pyrimidine sites hundreds of fold more susceptible to the generation of CPDs than the genomic average. Identifying hyperhotspots in keratinocytes could enable measuring individual past UV exposure in small skin samples and predicting future skin cancer risk. We therefore exposed neonatal human epidermal keratinocytes to narrowband UVB and quantified CPDs using the adductSeq high-throughput DNA sequencing method. Keratinocytes contained thousands of CPD hyperhotspots, with a UVB-sensitivity up to 550 fold greater than the genomic average. As with melanocytes, the most sensitive sites were located in promoter regions at ETS-family transcription factor binding sequence motifs, near RNA processing genes. Moreover, they lay at sequence motifs bound to ETS1 in CpG islands. These genes were specifically upregulated in skin and the CPD hyperhotspots were mutated in a fraction of keratinocyte cancers. Crucially for their biological importance and practical application, CPD hyperhotspot locations and UV-sensitivity ranking demonstrated high reproducibility across experiments and across skin donors. CPD hyperhotspots are therefore sensitive indicators of UV exposure.

Comparative genomic analysis of the human genome and six bat genomes using unsupervised machine learning: Mb-level CpG and TFBS islands

BackgroundEmerging infectious disease-causing RNA viruses, such as the SARS-CoV-2 and Ebola viruses, are thought to rely on bats as natural reservoir hosts. Since these zoonotic viruses pose a great threat to humans, it is important to characterize the bat genome from multiple perspectives. Unsupervised machine learning methods for extracting novel information from big sequence data without prior knowledge or particular models are highly desirable for obtaining unexpected insights. We previously established a batch-learning self-organizing map (BLSOM) of the oligonucleotide composition that reveals novel genome characteristics from big sequence data.ResultsIn this study, using the oligonucleotide BLSOM, we conducted a comparative genomic study of humans and six bat species. BLSOM is an explainable-type machine learning algorithm that reveals the diagnostic oligonucleotides contributing to sequence clustering (self-organization). When unsupervised machine learning reveals unexpected and/or characteristic features, these features can be studied in more detail via the much simpler and more direct standard distribution map method. Based on this combined strategy, we identified the Mb-level enrichment of CG dinucleotide (Mb-level CpG islands) around the termini of bat long-scaffold sequences. In addition, a class of CG-containing oligonucleotides were enriched in the centromeric and pericentromeric regions of human chromosomes. Oligonucleotides longer than tetranucleotides often represent binding motifs for a wide variety of proteins (e.g., transcription factor binding sequences (TFBSs)). By analyzing the penta- and hexanucleotide composition, we observed the evident enrichment of a wide range of hexanucleotide TFBSs in centromeric and pericentromeric heterochromatin regions on all human chromosomes.ConclusionFunction of transcription factors (TFs) beyond their known regulation of gene expression (e.g., TF-mediated looping interactions between two different genomic regions) has received wide attention. The Mb-level TFBS and CpG islands are thought to be involved in the large-scale nuclear organization, such as centromere and telomere clustering. TFBSs, which are enriched in centromeric and pericentromeric heterochromatin regions, are thought to play an important role in the formation of nuclear 3D structures. Our machine learning-based analysis will help us to understand the differential features of nuclear 3D structures in the human and bat genomes.

YY1 and NFYA: Potential tr-KIT Specific Transcription Factors in Prostate Cancer

Objective: Via the use of an alternative promoter, a truncated c-KIT protein (tr-KIT) of 30-50 kDa is generated, lacking extracellular and transmembrane domains. Moreover, over-expression of tr-KIT, a stronger activator than c-KIT, appears to be specific to prostate cancer (PCa). Also, Imatinib, a tyrosine kinase inhibitor, blocks the activity of full-length c-KIT but has no effect on tr-KIT in PCa. Tr-KIT has its own nuclear factor binding site. However, the transcription factors (TFs) binding to this region specific to tr-KIT are not known yet. This study was conducted to define the most potential TFs specific for tr-KIT via in silico analysis.Methods: Tr-KIT potential TF binding sequence was uploaded into Tfsitescan database. Five TFs with the highest potential binding to this sequence were selected. Transcriptomic data of LNCaP (PCa expressing tr-KIT), PC3 (PCa not expressing tr-KIT) and RWPE-1 (normal prostate) cell lines (GSM1647378, GSE36022 and GSM738189, respectively) from Gene Expression Omnibus (GEO) database were compared for gene expression levels of pre-defined potential tr-KIT specific TFs using DESeq package of R-program. Finally, two TFs having higher expression levels in both LNCaP and PC3 compared to RWPE-1 and higher expression levels in LNCaP compared to PC3 were detected.Results: Five TFs having the highest potential were selected as: YY1, c-MYB, IL8, NFYA and TCF3. Via in silico analysis performed, it was found that YY1 and NFYA have the highest potential to be tr-KIT specific TFs in PCa, among them.Conclusion: YY1 and NFYA TFs may take a role in formation of tr-KIT in PCa.

Gene cloning and transcriptional regulation of the alkaline and acid phosphatase genes in Scylla paramamosain.

In crustaceans, innate immunity serves as the frontline of defense against microbes. Alkaline phosphatases (ALPs) and acid phosphatases (ACPs) are essential enzymes that play a significant role in crustaceans' immune defenses. However, the function and transcriptional regulation of the alp and acp genes in the Scylla paramamosain, an important aquaculture species in China, have not been elucidated. In this study, the full-length cDNAs of Spalp and Spacp were identified, which consist of 2,718bp and 3,768bp, encoding 579 and 452 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis showed that these two genes were conserved among different species and shared high homology with crustaceans. The mRNA expression of Spalp and Spacp were examined in eight tested tissues, with the highest levels in the hepatopancreas. The 5'-flanking regions of Spalp and Spacp were cloned and sequenced. The core promoter region of the Spalp and Spacp was -39bp∼+8 bp and -39bp∼+10bp, respectively. Potential binding sequences for SOX-2, c-fos, SP1, NF-κB, GATA-1, YY1, and AP-1 transcription factors were found in the 5'-flanking regions of Spalp and Spacp. The NF-κB binding site located between -1,223bp and -972bp in Spalp while SP1 and AP-1 binding sites located between -1,249bp and -514bp in Spacp. Mutation analysis confirmed that NF-κB negatively regulated the expression of Spalp gene, and SP1 and AP-1 positively regulated Spacp gene expression. These results provide us with essential information to elucidate the function of the Spalp and Spacp in S. paramamosain. This study is the first one to analyze the activity of Spalp and Spacp promoters.

Identification and characterization of the promoter and transcription factors regulating the expression of cerebral sodium/calcium exchanger 2 (NCX2) gene

The isoform 2 of sodium-calcium exchanger family (NCX2) is selectively expressed in neuronal and glial cells where it participates in Ca2+-clearance following neuronal depolarization, synaptic plasticity, hippocampal-dependent learning and memory consolidation processes. On the other hand, NCX2 is also involved in a neuroprotective effect following stroke. Despite the relevance of this antiporter under physiological and pathophysiological conditions, no studies have been reported on its genetic/epigenetic regulation. Therefore, we identified, cloned, and characterized a transcriptional regulatory region (R3) of rat Slc8a2 gene encoding for NCX2. In particular, R3 sequence displayed a promoter activity in PC12, SH-SY5Y and U87MG cell lines consistent with their endogenous NCX2 expression levels. On the other hand, R3 failed to induce detectable luciferase activity in BHK cell line that does not express NCX2 under control conditions. These data support the hypothesis that R3 represents the promoter region of NCX2. Moreover, among several conserved binding sequences for transcription factors identified by in-silico analysis, we evaluated the transcriptional regulation and the binding sites of Sp1, Sp4, NFkB1, GATA2 and CREB1 on R3 sequence by using site-direct mutagenesis and ChIP assays. In particular, transfection of Sp1, Sp4, and CREB1 enhanced both R3 promoter activity and NCX2 transcription in PC12 cell line. More important, CREB1 transfection also enhanced NCX2 protein levels and NCX reverse mode activity in PC12 cells. Altogether, these data suggested that: (i) the identified region contained the regulatory promoter of the antiporter; (ii) NCX2 might represent a downstream effector of transcription factors involved in synaptic plasticity and neuronal survival.

Distal regulation, silencers, and a shared combinatorial syntax are hallmarks of animal embryogenesis.

The chromatin environment plays a central role in regulating developmental gene expression in metazoans. Yet, the ancestral regulatory landscape of metazoan embryogenesis is unknown. Here, we generate chromatin accessibility profiles for six embryonic, plus larval and adult stages in the sponge Amphimedon queenslandica. These profiles are reproducible within stages, reflect histone modifications, and identify transcription factor (TF) binding sequence motifs predictive of cis-regulatory elements operating during embryogenesis in other metazoans, but not the unicellular relative Capsaspora. Motif analysis of chromatin accessibility profiles across Amphimedon embryogenesis identifies three major developmental periods. As in bilaterian embryogenesis, early development in Amphimedon involves activating and repressive chromatin in regions both proximal and distal to transcription start sites. Transcriptionally repressive elements (“silencers”) are prominent during late embryogenesis. They coincide with an increase in cis-regulatory regions harboring metazoan TF binding motifs, as well as an increase in the expression of metazoan-specific genes. Changes in chromatin state and gene expression in Amphimedon suggest the conservation of distal enhancers, dynamically silenced chromatin, and TF-DNA binding specificity in animal embryogenesis.

Caenorhabditis elegans transposable elements harbor diverse transcription factor DNA-binding sites.

Transposable elements are powerful agents of evolution that can diversify transcriptional programs by distributing transcription factor DNA-binding sites throughout genomes. To investigate the extent that transposable elements provide transcription factor-binding motifs in Caenorhabditis elegans, we determined the genomic positions of DNA-binding motifs for 201 different transcription factors. Surprisingly, we found that almost all examined transcription factors have binding motifs that reside within transposable elements, and all types of transposable elements have at least 1 instance of a transcription factor motif, demonstrating that transposable elements provide previously unappreciated numbers of transcription factor-binding motifs to the C. elegans genome. After determining the occurrence of transcription factor motifs in transposable elements relative to the rest of the genome, we identified DNA-binding motifs for 45 different transcription factors that are greater than 20-fold enriched within transposable elements compared to what would be expected by chance. Consistent with potential functional roles for these transposable element-enriched transcription factor-binding sequences, we determined that all transcription factor motif types found in transposable elements have instances of residing within accessible chromatin sites associated with transcription factor binding. The overwhelming majority of transcription factor-binding motifs located within transposable elements associate with their cognate transcription factors, suggesting extensive binding of transcription factors to sequences within transposable elements. In addition, transposable elements with accessible or transcription factor-bound motifs reside in the putative promoter regions of approximately 12% of all protein-coding genes, providing widespread possibilities for influencing gene expression. This work represents the first comprehensive analysis of transposable element–transcription factor interactions in C. elegans and demonstrates that transposable element-provided transcription factor-binding sites are prevalent in this important model organism.

Both Binding Strength and Evolutionary Accessibility Affect the Population Frequency of Transcription Factor Binding Sequences in Arabidopsis thaliana.

Mutations in DNA sequences that bind transcription factors and thus modulate gene expression are a source of adaptive variation in gene expression. To understand how transcription factor binding sequences evolve in natural populations of the thale cress Arabidopsis thaliana, we integrated genomic polymorphism data for loci bound by transcription factors with in vitro data on binding affinity for these transcription factors. Specifically, we studied 19 different transcription factors, and the allele frequencies of 8,333 genomic loci bound in vivo by these transcription factors in 1,135 A. thaliana accessions. We find that transcription factor binding sequences show very low genetic diversity, suggesting that they are subject to purifying selection. High frequency alleles of such binding sequences tend to bind transcription factors strongly. Conversely, alleles that are absent from the population tend to bind them weakly. In addition, alleles with high frequencies also tend to be the endpoints of many accessible evolutionary paths leading to these alleles. We show that both high affinity and high evolutionary accessibility contribute to high allele frequency for at least some transcription factors. Although binding sequences with stronger affinity are more frequent, we did not find them to be associated with higher gene expression levels. Epistatic interactions among individual mutations that alter binding affinity are pervasive and can help explain variation in accessibility among binding sequences. In summary, combining in vitro binding affinity data with in vivo binding sequence data can help understand the forces that affect the evolution of transcription factor binding sequences in natural populations.

Abstract P132: Heterogeneously Expressed Bcl6 Controls Endothelial Inflammatory Response

We recently reported that BCL6 , a DNA binding protein important in germinal center responses and lymphoncogenesis, was heterogeneously expressed across human endothelial cells (EC). Its expression correlated with more intense inflammatory responses. Here, we asked what role BCL6 might play in endothelial inflammation. We examined public expression and epigenomic data and tested BCL6 function in vitro . BCL6 was significantly enriched in cardiac microvascular and endocardial EC, associated with cardiovascular disease in GWAS studies, and was elevated in rejecting cardiac transplant biopsies. The chromatin landscape around BCL6 reflected that of a superenhancer in EC, with high H3K27ac and H3K4me3 signal indicative of active/poised transcription. EC treated with TNFα, IFNγ or IL-1β upregulated BCL6 within 2-3hr, maintained through 24hr. The BCL6 gene had putative NFκB/REL transcription factor binding sequences, and p65/RelA binding to the BCL6 gene increased more than 8-fold under TNFα or IL-1β stimulation. BCL6 DNA binding sites were found in EC adhesion molecule and chemokine genes, including VCAM1 , CXCL2 , CX3CL1 , SELE , CCL5 ; as well as NFKB genes themselves. Perturbation of BCL6 complexes with inhibitors (FX1, BI-3812) led to dramatic changes in the TNFα-induced transcriptome and at the protein level in endothelial cells. For example, FX1 and BI-3812 significantly blocked TNFα-induction of VCAM1 and CXCL2 /GROβ, but upregulated CCL5 /RANTES and CXCL10 /IP-10. Interestingly, TNFα-induced expression of miR155, a pro-inflammatory microRNA important in atherosclerosis, was also suppressed. We hypothesize that BCL6 is a novel NFκB-inducible regulator of vascular inflammation. Surprisingly, BCL6 corepressor antagonism both enhanced and suppressed TNFα mediated EC activation. Therefore BCL6 performs transcriptional modification that is context and cell-type dependent. Its role in vascular inflammation merits further investigation.

AI for the collective analysis of a massive number of genome sequences: various examples from the small genome of pandemic SARS-CoV-2 to the human genome.

In genetics and related fields, huge amounts of data, such as genome sequences, are accumulating, and the use of artificial intelligence (AI) suitable for big data analysis has become increasingly important. Unsupervised AI that can reveal novel knowledge from big data without prior knowledge or particular models is highly desirable for analyses of genome sequences, particularly for obtaining unexpected insights. We have developed a batch-learning self-organizing map (BLSOM) for oligonucleotide compositions that can reveal various novel genome characteristics. Here, we explain the data mining by the BLSOM: an unsupervised AI. As a specific target, we first selected SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) because a large number of viral genome sequences have been accumulated via worldwide efforts. We analyzed more than 0.6 million sequences collected primarily in the first year of the pandemic. BLSOMs for short oligonucleotides (e.g., 4-6-mers) allowed separation into known clades, but longer oligonucleotides further increased the separation ability and revealed subgrouping within known clades. In the case of 15-mers, there is mostly one copy in the genome; thus, 15-mers that appeared after the epidemic started could be connected to mutations, and the BLSOM for 15-mers revealed the mutations that contributed to separation into known clades and their subgroups. After introducing the detailed methodological strategies, we explain BLSOMs for various topics, such as the tetranucleotide BLSOM for over 5 million 5-kb fragment sequences derived from almost all microorganisms currently available and its use in metagenome studies. We also explain BLSOMs for various eukaryotes, including fishes, frogs and Drosophila species, and found a high separation ability among closely related species. When analyzing the human genome, we found enrichments in transcription factor-binding sequences in centromeric and pericentromeric heterochromatin regions. The tDNAs (tRNA genes) could be separated according to their corresponding amino acid.

Transcriptional-regulatory convergence across functional MDD risk variants identified by massively parallel reporter assays

Family and population studies indicate clear heritability of major depressive disorder (MDD), though its underlying biology remains unclear. The majority of single-nucleotide polymorphism (SNP) linkage blocks associated with MDD by genome-wide association studies (GWASes) are believed to alter transcriptional regulators (e.g., enhancers, promoters) based on enrichment of marks correlated with these functions. A key to understanding MDD pathophysiology will be elucidation of which SNPs are functional and how such functional variants biologically converge to elicit the disease. Furthermore, retinoids can elicit MDD in patients and promote depressive-like behaviors in rodent models, acting via a regulatory system of retinoid receptor transcription factors (TFs). We therefore sought to simultaneously identify functional genetic variants and assess retinoid pathway regulation of MDD risk loci. Using Massively Parallel Reporter Assays (MPRAs), we functionally screened over 1000 SNPs prioritized from 39 neuropsychiatric trait/disease GWAS loci, selecting SNPs based on overlap with predicted regulatory features—including expression quantitative trait loci (eQTL) and histone marks—from human brains and cell cultures. We identified >100 SNPs with allelic effects on expression in a retinoid-responsive model system. Functional SNPs were enriched for binding sequences of retinoic acid-receptive transcription factors (TFs), with additional allelic differences unmasked by treatment with all-trans retinoic acid (ATRA). Finally, motifs overrepresented across functional SNPs corresponded to TFs highly specific to serotonergic neurons, suggesting an in vivo site of action. Our application of MPRAs to screen MDD-associated SNPs suggests a shared transcriptional-regulatory program across loci, a component of which is unmasked by retinoids.

Arid4b physically interacts with Tfap2c in mouse embryonic stem cells.

Precise regulation of gene expression is required for embryonic stem cell (ESC) differentiation. Transcription factor (TF) networks coordinate the balance of pluripotency and differentiation in response to extracellular and intracellular signals. Chromatin factors work alongside TFs to achieve timely regulation of gene expression for differentiation process. Our previous studies showed that a member of the Sin3a corepressor complex, Arid4b, is critical for proper mouse ESC differentiation into mesoderm and endoderm. We found elevated histone 3 lysine 27 acetylation (H3K27Ac) in a subset of genomic loci in meso/endoderm directed arid4bΔ cells, coincident with their derepression. We reasoned that Sin3a complex may be required for the suppression of these genes during differentiation. To identify TFs that might cooperate with Arid4b for this function, we found consensus TF binding sequences enriched in H3K27Ac elevated regions in arid4bΔ cells. Of these candidate TFs, we validated expression of Bach1, Ddit3, Prrx2, Znf354c and Tfap2c in mESCs. We then demonstrated a physical interaction between Arid4b and Tfap2c in mESCs using endogenous coimmunoprecipitation and proximity ligation assay experiments. Our results point to a role of Arid4b in the Sin3a complex in repression of a subset of Tfap2c-regulated genes during meso/endoderm differentiation.

Neofunctionalization of a polyploidization-activated cotton long intergenic non-coding RNA DAN1 during drought stress regulation.

The genomic shock of whole-genome duplication (WGD) and hybridization introduces great variation into transcriptomes, for both coding and noncoding genes. An altered transcriptome provides a molecular basis for improving adaptation during the evolution of new species. The allotetraploid cotton, together with the putative diploid ancestor species compose a fine model for study the rapid gene neofunctionalization over the genome shock. Here we report on Drought-Associated Non-coding gene 1 (DAN1), a long intergenic noncoding RNA (lincRNA) that arose from the cotton progenitor A-diploid genome after hybridization and WGD events during cotton evolution. DAN1 in allotetraploid upland cotton (Gossypium hirsutum) is a drought-responsive lincRNA predominantly expressed in the nucleoplasm. Chromatin isolation by RNA purification profiling and electrophoretic mobility shift assay analysis demonstrated that GhDAN1 RNA can bind with DNA fragments containing AAAG motifs, similar to DNA binding with one zinc finger transcription factor binding sequences. The suppression of GhDAN1 mainly regulates genes with AAAG motifs in auxin-response pathways, which are associated with drought stress regulation. As a result, GhDAN1-silenced plants exhibit improved tolerance to drought stress. This phenotype resembles the drought-tolerant phenotype of the A-diploid cotton ancestor species, which has an undetectable expression of DAN1. The role of DAN1 in cotton evolution and drought tolerance regulation suggests that the genomic shock of interspecific hybridization and WGD stimulated neofunctionalization of non-coding genes during the natural evolutionary process.

In This Issue

HomeCirculation ResearchVol. 127, No. 12In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published3 Dec 2020https://doi.org/10.1161/RES.0000000000000452Circulation Research. 2020;127:1469is related toArterial Stiffness Preceding DiabetesTamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle CellsATAC-Seq Reveals an Isl1 Enhancer That Regulates Sinoatrial Node Development and FunctionTamoxifen and E2 Effects on Reendothelialization (p 1473)Zahreddine et al investigate Tamoxifen’s vasculoprotective effects.Download figureDownload PowerPointFor breast cancers that contain high levels of estrogen receptor, a standard treatment is to give drugs that block either estrogen production, or the receptor itself, such as Tamoxifen. Eliminating the vasculoprotective effects of estrogen might increase the risk of cardiovascular disease. But because Tamoxifen can antagonize or activate the receptor depending on the tissue, its role in such risk is unclear. Some evidence even suggests it might have protective effects, such as promoting vascular endothelial healing. Zahreddine and colleagues now show that while mice suffering endovascular injury—damage to the inside of a blood vessel—have improved healing when treated with Tamoxifen or with estrogen, those suffering pervascular injury—which also affects the surrounding smooth muscle—heal only in response to estrogen. This suggested Tamoxifen’s healing effect might require smooth muscle cells (SMCs). Sure enough, mice lacking the estrogen receptor in SMCs, healed endovascular injuries in response to the hormone but not Tamoxifen, while mice lacking the receptor in endothelial cells, healed such injuries in response to Tamoxifen but not estrogen. The work reveals nuance in the molecular actions of Tamoxifen that should inform future assessments of its risks and benefits, say the authors.Arterial Stiffness Preceding Diabetes (p 1491)Having stiffer arteries may increase a person’s risk for diabetes, say Zheng et al.Download figureDownload PowerPointAs a person ages, their risk of developing diabetes and cardiovascular disease increases. Aging is also linked to increasing arterial stiffness and high blood pressure, but how all these individual conditions affect and influence each other is not entirely clear. For example, while arterial stiffness and diabetes tend to correlate, whether one increases the risk for the other, or the risk relationship is bidirectional, is not known. To find out, Zheng and colleagues studied diabetes and arterial stiffness in a cohort of 8,956 Chinese people between 2010 and 2015, none of whom had diabetes or cardiovascular disease at the outset. With repeated measures of fasting glucose levels—as an indicator of diabetes—and pulse wave velocity—as a measure of arterial stiffness—the team found that participants with a higher baseline arterial stiffness were more likely to develop diabetes during the five-year period than those with lower stiffness. A total of 979 individuals developed diabetes during the study. Higher baseline glucose levels did not predict future arterial stiffness, however, suggesting the risk relationship is one-way. While the results require confirmation in further cohorts, they also set the stage for investigations into pathological mechanisms linking arterial stiffness to diabetes.ATAC-seq Identifies Novel Isl1 SAN Enhancer (p 1502)Galang et al discover a novel sinoatrial node enhancer element.Download figureDownload PowerPointPacemaker cells (PCs) of the sinoatrial node establish and control the rhythmic contractions of the heart. These cells differ from regular cardiomyocytes in their transcription profiles, but how this transcriptional fate is established and maintained is not fully understood. To investigate the epigenetic landscape defining PC fate, Galang and colleagues have employed a technique called ATAC-seq, which identifies areas of the genome with accessible, open chromatin structures—an indication of transcriptional activity. The team compared the genomes of PCs and atrial cardiomyocytes and found a number of PC-specific accessible loci that had both large numbers of transcription factor binding sequences and enhancer activity when assayed in mice. The team went on to specifically characterize one novel enhancer upstream of the gene encoding Isl1—a key transcription factor for PC identity. They showed that deleting the enhancer caused underdevelopment of the sinoatrial node and arrhythmias in mice. They also noted that single nucleotide polymorphisms at the equivalent locus in humans had been linked to variations in resting heart rate. The report verifies ATAC-seq as an effective tool for identifying PC enhancers, and will launch further studies into how such enhancers function in heart development and disease. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesArterial Stiffness Preceding DiabetesMengyi Zheng, et al. Circulation Research. 2020;127:1491-1498Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle CellsRana Zahreddine, et al. Circulation Research. 2020;127:1473-1487ATAC-Seq Reveals an Isl1 Enhancer That Regulates Sinoatrial Node Development and FunctionGiselle Galang, et al. Circulation Research. 2020;127:1502-1518 December 4, 2020Vol 127, Issue 12Article InformationMetrics Download: 308 © 2020 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000452 Originally publishedDecember 3, 2020 PDF download

Functional analysis of low-grade glioma genetic variants predicts key target genes and transcription factors.

Large-scale genome-wide association studies (GWAS) have implicated thousands of germline genetic variants in modulating individuals' risk to various diseases, including cancer. At least 25 risk loci have been identified for low-grade gliomas (LGGs), but their molecular functions remain largely unknown. We hypothesized that GWAS loci contain causal single nucleotide polymorphisms (SNPs) that reside in accessible open chromatin regions and modulate the expression of target genes by perturbing the binding affinity of transcription factors (TFs). We performed an integrative analysis of genomic and epigenomic data from The Cancer Genome Atlas and other public repositories to identify candidate causal SNPs within linkage disequilibrium blocks of LGG GWAS loci. We assessed their potential regulatory role via in silico TF binding sequence perturbations, convolutional neural network trained on TF binding data, and simulated annealing-based interpretation methods. We built an interactive website (http://education.knoweng.org/alg3/) summarizing the functional footprinting of 280 variants in 25 LGG GWAS regions, providing rich information for further computational and experimental scrutiny. We identified as case studies PHLDB1 and SLC25A26 as candidate target genes of rs12803321 and rs11706832, respectively, and predicted the GWAS variant rs648044 to be the causal SNP modulating ZBTB16, a known tumor suppressor in multiple cancers. We showed that rs648044 likely perturbed the binding affinity of the TF MAFF, as supported by RNA interference and in vitro MAFF binding experiments. The identified candidate (causal SNP, target gene, TF) triplets and the accompanying resource will help accelerate our understanding of the molecular mechanisms underlying genetic risk factors for gliomas.