Transcription Factor Binding Sites Research Articles

Characterization of a heat shock factor 1 (HSF1) gene and the association of its single nucleotide polymorphisms with susceptibility/resistance of Magallana gigas to Halomonas sp. 7T.

Heat shock transcription factor 1 (HSF1) is an important activator of innate immunity in the response of invertebrates to pathogen invasion. In the present study, a HSF1 was characterized in Pacific oyster Magallana gigas (MgHSF1), and its association of single nucleotide polymorphisms with susceptibility/resistance of oyster to Halomonas sp. 7T were investigated. MgHSF1 shared highly conserved DNA binding and acetylation sites with other organisms. The MgHSF1 mRNA showed high expression in haemocytes, and was significantly induced by lipopolysaccharide stimulation. The single nucleotide polymorphisms (SNPs) within promoter region of MgHSF1 gene from two oyster populations (the bacterial-resistant population and the common population) were investigated to analyze their association with bacterial resistance. Nine out of 14 SNPs including -543 A/G, -494 C/T, -488 T/A, -476 G/A, -336 C/T, -335 T/C, -334 C/T, -307 T/C and -268 T/C were found to be associated with bacterial resistance. Moreover, haplotypes TCTCGA was associated with bacterial resistance. The results threw lights on the molecular mechanisms of different oyster populations' resistance to bacterial diseases which suggested that the increased bacterial resistance of bacterial-resistant population was associated with the higher expression of MgHSF1. Meanwhile, the nine genotypes (-543 G/G, -494 T/T, -488 A/A, -476 A/A, -336 T/T, -335 C/C, -334 T/T, -307 T/T, and -268 C/C) and one haplotype (TCTCGA) could be used as potential markers for oyster selection breeding with higher bacterial resistance.

Comprehensive Single-Cell Chromatin and Transcriptomic Profiling of Peripheral Immune Cells in Non-Segmental Vitiligo.

Non-segmental vitiligo (NSV) is an autoimmune condition characterized by melanocyte loss. While skin-specific mechanisms are well-studied, systemic immune dysregulation contributing to NSV pathogenesis remains unclear. This study employs a multi-omic single-cell approach to investigate circulating immune cells in NSV, integrating transcriptional and chromatin accessibility data. An integrative scRNA-seq/scATAC-seq analysis was conducted on PBMCs from NSV patients (n=11) and controls (n=5), identifying transcriptional markers, cell-cell interactions, chromatin accessibility, and transcription factor (TF) dynamics. Key findings were validated in an expanded cohort (NSV, n=16; controls, n=9) using spectral flow cytometry, with additional stratification by sex, age, disease activity, severity, and duration. Analysis of 59,192 PBMCs identified 8,204 gene expression markers and 13,925 ATAC peaks across 25 immune cell subtypes. A broadly activated immune response was observed, characterized by cytotoxicity, antigen presentation, cell exhaustion, and stress, predominantly in monocytes, NK cells, CD8+ T cells, and dendritic cells (DCs). Multi-omic integration revealed Th1/Th17 polarization and dysfunctional regulatory T cell (Treg/mTreg) responses. Chromatin accessibility highlighted enriched TF binding sites for FOXO3, SP1, AP1, STAT1/STAT3, IRF1, and IRF4, regulating pathways linked to cytotoxicity, antigen processing, NF-κB, Toll-like receptor, and JAK-STAT signaling.Flow cytometry validated these findings, showing that disease activity and shorter duration were associated with heightened immune dysregulation. Robust TCR activation drove Th1/Th17 polarization and elevated IFN-γ and TNF-α production in CD4+ and CD8+ T cells. CLA+ skin-homing Th1/Th17-polarized CD4+ T cells, CD8+ T cells, and mTregs exhibited persistent activation, marked by basal PD1+ expression. OX40/OX40L-mediated interactions between monocytes and effector T cells amplified inflammation. Regulatory dysfunction, including reduced IL-4 and IL-13 production in mTregs, was prominent in moderate-severe and active disease. This is the first multi-omic single-cell study in PBMCs of NSV patients, revealing systemic immune dysregulation driven by cytotoxicity, antigen presentation, exhaustion, and regulatory failure. Disease severity, activity, and evolution influence these pathways, highlighting the OX40/OX40L axis as a potential therapeutic target to mitigate immune dysregulation and relapse risk.

A concept of natural genome reconstruction. Part 2. Effect of extracellular double-stranded DNA fragments on hematopoietic stem cells

In this part of the study, the first component of the concept of “natural genome reconstruction” is being proven. It was shown with mouse and human model organisms that CD34+ hematopoietic bone marrow progenitors take up fragments of extracellular double-stranded DNA through a natural mechanism. It is known that the process of internalization of extracellular DNA fragments involves glycocalyx structures, which include glycoproteins/protein glycans, glycosylphosphatidylinositol-anchored proteins and scavenger receptors. The bioinformatic analysis conducted indicates that the main surface marker proteins of hematopoietic stem cells belong to the indicated groups of factors and contain specific DNA binding sites, including a heparin-binding domain and clusters of positively charged amino acid residues. A direct interaction of CD34 and CD84 (SLAMF5) glycoproteins, markers of hematopoietic stem cells, with double-stranded DNA fragments was demonstrated using an electrophoretic mobility shift assay system. In cells negative for CD34, which also internalize fragments, concatemerization of the fragments delivered into the cell occurs. In this case, up to five oligonucleotide monomers containing 9 telomeric TTAGGG repeats are stitched together into one structure. Extracellular fragments delivered to hematopoietic stem cells initiate division of the original hematopoietic stem cell in such a way that one of the daughter cells becomes committed to terminal differentiation, and the second retains its low-differentiated status. After treatment of bone marrow cells with hDNAgr, the number of CD34+ cells in the colonies increases to 3 % (humans as the model organism). At the same time, treatment with hDNAgr induces proliferation of blood stem cells and their immediate descendants and stimulates colony formation (mouse, rat and humans as the model organisms). Most often, the granulocyte-macrophage lineage of hematopoiesis is activated as a result of processing extracellular double-stranded DNA. The commitment process is manifested by the appearance and repair of pangenomic single-strand breaks. The transition time in the direction of differentiation (the time it takes for pangenomic single-strand breaks to appear and to be repaired) is about 7 days. It is assumed that at the moment of initiation of pangenomic single-strand breaks, a “recombinogenic situation” ensues in the cell and molecular repair and recombination mechanisms are activated. In all experiments with individual molecules, recombinant human angiogenin was used as a comparison factor. In all other experiments, one of the experimental groups consisted of hematopoietic stem cells treated with angiogenin.

MetArea: a software package for analysis of the mutually exclusive occurrence in pairs of motifs of transcription factor binding sites based on ChIP-seq data

ChIP-seq technology, which is based on chromatin immunoprecipitation (ChIP), allows mapping a set of genomic loci (peaks) containing binding sites (BS) for the investigated (target) transcription factor (TF). A TF may recognize several structurally different BS motifs. The multiprotein complex mapped in a ChIP-seq experiment includes target and other “partner” TFs linked by protein-protein interactions. Not all these TFs bind to DNA directly. Therefore, both target and partner TFs recognize enriched BS motifs in peaks. A de novo search approach is used to search for enriched TF BS motifs in ChIP-seq data. For a pair of enriched BS motifs of TFs, the co-occurrence or mutually exclusive occurrence can be detected from a set of peaks: the co-occurrence reflects a more frequent occurrence of two motifs in the same peaks, while the mutually exclusive means their more frequent detection in different peaks. We propose the MetArea software package to identify pairs of TF BS motifs with the mutually exclusive occurrence in ChIP-seq data. MetArea was designed to predict the structural diversity of BS motifs of the same TFs, and the functional relation of BS motifs of different TFs. The functional relation of the motifs of the two distinct TFs presumes that they are interchangeable as part of a multiprotein complex that uses the BS of these TFs to bind directly to DNA in different peaks. MetArea calculates the estimates of recognition performance pAUPRC (partial area under the Precision–Recall curve) for each of the two input single motifs, identifies the “joint” motif, and computes the performance for it too. The goal of the analysis is to find pairs of single motifs A and B for which the accuracy of the joint A&B motif is higher than those of both single motifs.

Genomic clustering tendency of transcription factors reflects phase-separated transcriptional condensates at super-enhancers.

Many transcription factors (TFs) have been shown to bind to super-enhancers, forming transcriptional condensates to activate transcription in various cellular systems. However, the genomic and epigenomic determinants of phase-separated transcriptional condensate formation remain poorly understood. Questions regardingwhich TFs tend to associate with transcriptional condensates and what factors influence their association are largely unanswered. Here we systematically analyzed 571 DNA sequence motifs across the human genome and 6650 TF binding profiles across different cell types to identify the molecular features contributing to the formation of transcriptional condensates. We found that the genomic distributions of sequence motifs for different TFs exhibit distinct clustering tendencies. Notably, TF motifs with a high genomic clustering tendency are significantly associated with super-enhancers. TF binding profiles showing a high genomic clustering tendency are further enriched at cell-type-specific super-enhancers. TFs with a high binding clustering tendency also possess high liquid-liquid phase separation abilities. Compared to nonclustered TF binding, densely clustered TF binding sites are more enriched at cell-type-specific super-enhancers with higher chromatin accessibility, elevated chromatin interactionand stronger association with cancer outcomes. Our results indicate that the clustered genomic binding patterns and the phase separation properties of TFs collectively contribute to the formation of transcriptional condensates.

DOP135 Patient-specific regulatory network rewiring in Inflammatory Bowel Disease: How genetic polymorphisms divert incoming signals and contribute to disease pathogenesis

Abstract Background Inflammatory bowel disease (IBD), comprising ulcerative colitis (UC) and Crohn’s disease (CD), arises due to complex interactions between multiple environmental factors and genetic risk loci in the form of single nucleotide polymorphisms (SNPs)1. Many IBD-associated SNPs are located within non-coding genomic regions containing transcription factor (TF) binding sites, where they may cause regulatory shifts by altering TF binding affinity. TFs may lose or gain binding sites, causing a rewiring of the incoming signals acting on the genome2. Characterising these incoming signals could unveil critical insights into the upstream signalling mechanisms driving IBD which via non-coding genetic risk-loci impact downstream cellular processes. The identification of such incoming signals has been elusive with most studies to date evaluating the downstream impact of IBD-associated SNPs3. Methods We developed a novel systems genomics pipeline that individually analysed genotype data from 2636 IBD patients to infer the rewiring of incoming signals affecting gene regulatory networks. Our in silico approach predicted changes to the repertoire of TFs binding to genomic loci due to IBD-associated non-coding SNPs in each patient compared to the healthy state, where these SNPs may be absent. By functionally annotating the TFs in the disease and healthy states using the Gene Ontology database, we inferred the rewiring of upstream signalling pathways (Figure 1). Aggregating these patient-specific findings across the entire cohort allowed us to capture overarching shifts in the incoming signals in IBD that are likely to arise in the context of non-coding SNPs. Results This analysis revealed that 144 and 138 signals were gained or lost in UC patients and CD patients, respectively, compared to the healthy state. Among these, 95 signals were shared between UC and CD, indicating a significant overlap in the upstream signalling mechanisms underpinning both types of IBD. Notably, both diseases showed a net loss of incoming signals, emphasising a major rewiring and decrease of regulation by upstream signalling pathways. These signals belonged to broader categories representing immune response, cell stress response, epithelial barrier function, wound healing, and response to viral/bacterial infection (Figure 2). Conclusion This study highlights that IBD-associated SNPs contribute to disease pathogenesis not only through their downstream effects, but by rewiring the incoming signals which regulate gene expression. Our findings underscore the importance of investigating signalling processes upstream of genetic polymorphisms to gain a more comprehensive understanding of IBD pathogenesis.

Pervasive and programmed nucleosome distortion patterns on single mammalian chromatin fibers.

We present a genome-scale method to map the single-molecule co-occupancy of structurally distinct nucleosomes, subnucleosomes, and other protein-DNA interactions via long-read high-resolution adenine methyltransferase footprinting. I teratively D efined L engths of Inaccessibility (IDLI) classifies nucleosomes on the basis of shared patterns of intranucleosomal accessibility, into: i.) minimally-accessible chromatosomes; ii.) octasomes with stereotyped DNA accessibility from superhelical locations (SHLs) ±1 through ±7; iii.) highly-accessible unwrapped nucleosomes; and iv.) subnucleosomal species, such as hexasomes, tetrasomes, and other short DNA protections. Applying IDLI to mouse embryonic stem cell (mESC) chromatin, we discover widespread nucleosomal distortion on individual mammalian chromatin fibers, with >85% of nucleosomes surveyed displaying degrees of intranucleosomally accessible DNA. We observe epigenomic-domain-specific patterns of distorted nucleosome co-occupancy and positioning, including at enhancers, promoters, and mouse satellite repeat sequences. Nucleosome distortion is programmed by the presence of bound transcription factors (TFs) at cognate motifs; occupied TF binding sites are differentially decorated by distorted nucleosomes compared to unbound sites, and degradation experiments establish direct roles for TFs in structuring binding-site proximal nucleosomes. Finally, we apply IDLI in the context of primary mouse hepatocytes, observing evidence for pervasive nucleosomal distortion in vivo. Further genetic experiments reveal a role for the hepatocyte master regulator FOXA2 in directly impacting nucleosome distortion at hepatocyte-specific regulatory elements in vivo . Our work suggests extreme-but regulated-plasticity in nucleosomal DNA accessibility at the single-molecule level. Further, our study offers an essential new framework to model transcription factor binding, nucleosome remodeling, and cell-type specific gene regulation across biological contexts.

Anti-correlation of LacI association and dissociation rates observed in living cells

The rate at which transcription factors (TFs) bind their cognate sites has long been assumed to be limited by diffusion, and thus independent of binding site sequence. Here, we systematically test this assumption using cell-to-cell variability in gene expression as a window into the in vivo association and dissociation kinetics of the model transcription factor LacI. Using a stochastic model of the relationship between gene expression variability and binding kinetics, we performed single-cell gene expression measurements to infer association and dissociation rates for a set of 35 different LacI binding sites. We found that both association and dissociation rates differed significantly between binding sites, and moreover observed a clear anticorrelation between these rates across varying binding site strengths. These results contradict the long-standing hypothesis that TF binding site strength is primarily dictated by the dissociation rate, but may confer the evolutionary advantage that TFs do not get stuck in near-operator sequences while searching.

Protein target search diffusion-association/dissociation free energy landscape around DNA binding site with flanking sequences.

In this work we present a minimal structure-based model of protein diffusional search along local DNA amid protein binding and unbinding events on the DNA, taking into account protein-DNA electrostatic interactions and hydrogen-bonding (HB) interactions or contacts at the interface. We accordingly constructed the protein diffusion-association/dissociation free energy surface and mapped it to 1D as the protein slides along DNA, maintaining the protein-DNA interfacial HB contacts that presumably dictate the DNA sequence information detection. Upon DNA helical path correction, the protein 1D diffusion rates along local DNA can be physically derived to be consistent with experimental measurements. We also show that the sequence-dependent protein sliding or stepping patterns along DNA are regulated by collective interfacial HB dynamics, which also determines the ruggedness of the protein diffusion free energy landscape on the local DNA. In comparison, protein association or binding with DNA are generically dictated by the protein-DNA electrostatic interactions, with an interaction zone of nanometers around DNA. Extra degrees of freedom (DOFs) of the protein such as rotations and conformational fluctuations can be well accommodated within the protein-DNA electrostatic interaction zone. As such we demonstrate that the protein binding or association free energy profiling along DNA smoothens over the 1D diffusion free energy landscape, which leads to population variations for an order of magnitude upon a marginal free energetic smoothening around the specific or consensus sites. We further show that the protein unbinding or dissociation from a comparatively high-binding affinity DNA site is dominated by lateral diffusion to the flanking low-affinity sites. The results predict that experimental characterizations on the relative protein-DNA binding affinities or population profiling on the DNA are systematically and physically impacted by the extra DOFs of protein motions aside from 1D translation or helical tracking, as well as from flanking DNA sequences due to protein 1D diffusion and nonspecific binding/unbinding.

Uncovering the whole genome silencers of human cells via Ss-STARR-seq

Silencers, the yin to enhancers’ yang, play a pivotal role in fine-tuning gene expression throughout the genome. However, despite their recognized importance, comprehensive identification of these regulatory elements in the genome is still in its early stages. We developed a method called Ss-STARR-seq to directly determine the activity of silencers in the whole genome. In this study, we applied Ss-STARR-seq to human cell lines K562, LNCaP, and 293 T, and identified 134,171, 137,753, and 125,307 silencers on a genome-wide scale, respectively, these silencers function in various cells in a cell-specific manner. Silencers exhibited a substantial enrichment of transcriptional-inhibitory motifs, including REST, and demonstrated overlap with the binding sites of repressor transcription factors within the endogenous environment. Interestingly, H3K27me3 did not reflect silencer activity but facilitated the silencer’s inhibitory role on gene expression. Additionally, the silencer did not have any significant histone markers at the genome-wide level. Our findings unveil that aspect-silencers not only transition into enhancers throughout diverse cell lines but also achieve functional conversion with insulators. Regarding to biological effects, knockout experiments underscored the functional redundancy and specificity of silencers in regulating gene expression and cell proliferation. In summary, this study pioneers the elucidation of the genome-wide silencer landscape in human cells, delineates their global regulatory features, and identifies specific silencers influencing cancer cell proliferation.

Negative DNA supercoiling enhances DARS2 binding of DNA-bending protein IHF in the activation of Fis-dependent ATP-DnaA production.

Oscillation of the active form of the initiator protein DnaA (ATP-DnaA) allows for the timely regulation for chromosome replication. After initiation, DnaA-bound ATP is hydrolyzed, producing inactive ADP-DnaA. For the next round of initiation, ADP-DnaA interacts with the chromosomal locus DARS2 bearing binding sites for DnaA, a DNA-bending protein IHF, and a transcription activator Fis. The IHF binding site is about equidistant between the DnaA and Fis binding sites within DARS2. The DARS2-IHF-Fis complex promotes ADP dissociation from DnaA and furnishes ATP-DnaA at the pre-initiation stage, which dissociates Fis in a negative-feedback manner. However, regulation for IHF binding as well as mechanistic roles of Fis and specific DNA structure at DARS2 remain largely unknown. We have discovered that negative DNA supercoiling of DARS2 is required for stimulating IHF binding and ADP dissociation from DnaA in vitro. Consistent with these, novobiocin, a DNA gyrase inhibitor, inhibits DARS2 function in vivo. Fis Gln68, an RNA polymerase-interaction site, is suggested to be required for interaction with DnaA and full DARS2 activation. Based on these and other results, we propose that DNA supercoiling activates DARS2 function by stimulating stable IHF binding and DNA loop formation, thereby directing specific Fis-DnaA interaction.

Design and Optimization of a Two-Component TorRST-Based Biosensor for Detection and Degradation of Trimethylamine N-Oxide.

In mammals, Trimethylamine N-oxide (TMAO) is involved in various physiological processes, and is considered a biomarker for multiple diseases. As a natural molecule found in marine organisms, TMAO is also an important indicator of seafood freshness. In this study, a TMAO biosensor was developed in Escherichia coli harnessing TorRST two-component system. By using a cascade amplification circuit based on HrpRS-PhrpL, the biosensor's dynamic range was increased from 4.1- to 10.3-fold. By optimizing the affinity between the regulatory protein TorR and DNA binding sites in promoters, the concentration for 50% of maximal effect (EC50) value was reduced from 1008 to 141 μM. The biosensor was successfully used for aquatic sample detection. By introducing an exogenous TMAO degradation pathway into E. coli Nissle 1917, a probiotic chassis capable of TMAO detection, transportation, and degradation was constructed, providing an effective tool for rapid detection of TMAO and prevention of multiple diseases.

Effect of Epstein-Barr Virus infection on gene regulation in immune cells of patients with Immune-Mediated Diseases.

It has been known that Epstein-Barr virus (EBV) can latently infect immune cells after the initial infection, and epidemiological studies have suggested its association with the onset of immune-mediated diseases (IMDs). However, the specific impact of EBV infection on IMDs pathology remains unclear. We quantified EBV load of B cell subsets (Naïve B cells, Unswitched memory B cells, Switched memory B cells, Double negative B cells, and Plasmablasts) in IMD patients as well as healthy control (HC) using bulk RNA sequencing data of 504 donors. The EBV load was clearly higher in IMD patients compared to HC. Furthermore, the wide range of EBV load in this dataset enabled us to assess the impact of EBV load on gene regulation. We found many examples of expression quantitative trait loci (eQTL) whose effects were associated with EBV load. Expression QTLs that exhibited larger effects with increasing EBV load were significantly overlapped with binding sites of transcription factors derived from the EBV genome. These EBV load-associated eQTLs exhibited high enrichment of systemic lupus erythematosus (SLE) GWAS signals, suggesting the mechanical link of EBV infection and the onset of the disease via gene regulation. These findings provide the first evidence of the influence of EBV infection on gene regulation in human primary cells and its association with the SLE onset and/or progression.

ATF6 enables pathogen infection in ticks by inducing stomatin and altering cholesterol dynamics.

How tick-borne pathogens interact with their hosts has been primarily studied in vertebrates where disease is observed. Comparatively less is known about pathogen interactions within the tick. Here, we report that Ixodes scapularis ticks infected with either Anaplasma phagocytophilum (causative agent of anaplasmosis) or Borrelia burgdorferi (causative agent of Lyme disease) show activation of the ATF6 branch of the unfolded protein response (UPR). Disabling ATF6 functionally restricts pathogen survival in ticks. When stimulated, ATF6 functions as a transcription factor, but is the least understood out of the three UPR pathways. To interrogate the Ixodes ATF6 transcriptional network, we developed a custom R script to query tick promoter sequences. This revealed stomatin as a potential gene target, which has roles in lipid homeostasis and vesical transport. Ixodes stomatin was experimentally validated as a bona fide ATF6-regulated gene through luciferase reporter assays, pharmacological activators, and RNAi transcriptional repression. Silencing stomatin decreased A. phagocytophilum colonization in Ixodes and disrupted cholesterol dynamics in tick cells. Furthermore, blocking stomatin restricted cholesterol availability to the bacterium, thereby inhibiting growth and survival. Taken together, we have identified the Ixodes ATF6 pathway as a novel contributor to vector competence through Stomatin-regulated cholesterol homeostasis. Moreover, our custom, web-based transcription factor binding site search tool "ArthroQuest" revealed that the ATF6-regulated nature of stomatin is unique to blood-feeding arthropods. Collectively, these findings highlight the importance of studying fundamental processes in non-model organisms.

Discovery of Diverse CRISPR Leader Motifs, Putative Functions, and Applications for Enhanced CRISPR Detection and Subtype Annotation.

Bacteria and archaea acquire resistance to genetic parasites by preferentially integrating short fragments of foreign DNA at one end of a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). "Leader" DNA upstream of CRISPR loci regulates transcription and foreign DNA integration into the CRISPR. Here, we analyze 37,477 CRISPRs from 39,277 bacterial and 556 archaeal genomes to identify conserved sequence motifs in CRISPR leaders. A global analysis of all leader sequences fails to identify universally conserved motifs. However, an analysis of leader sequences that have been grouped by 16S rRNA-based taxonomy and CRISPR subtype reveals 87 specific motifs in type I, II, III, and V CRISPR leaders. Fourteen of these leader motifs have biochemically demonstrated roles in CRISPR biology including integration, transcription, and CRISPR RNA processing. Another 28 motifs are related to DNA binding sites for proteins with functions that are consistent with regulating CRISPR activity. In addition, we show that these leader motifs can be used to improve existing CRISPR detection methods and enhance the accuracy of CRISPR classification.

High hydrostatic pressure promotes gene transcription via a cystathionine-β-synthase domain-containing protein in the hyperthermophilic archaeon Pyrococcus yayanosii.

Cystathionine-β-synthase (CBS) domains are ubiquitously prevalent in all kingdoms of life. Remarkably, in archaea, proteins consisting of solely CBS domains are widespread. However, the biological functions of CBS proteins in archaea are still unknown. Here, we identified a high hydrostatic pressure regulator (HhpR) that comprises four CBS domains serving as a transcriptional activator via specifically binding to the UAS (upstream activating sequence) motif situated within the promoter region of an operon in a hyperthermophilic archaeon Pyrococcus yayanosii under high hydrostatic pressure (HHP). By combining molecular dynamics simulations,in vitro and in vivo assays, we revealed the potential binding interfaces between HhpR and its specific DNA binding site. Particularly, one stem-loop region in HhpR (termed as 'Arm') was found to play a critical role in regulating the transcription activity,and the 192 position in the Arm region is an essential site in dictating the conformational changes of HhpR at HHP condition. Our work provides novel insights into the structure-function relationship of CBS-containing proteins that participate in archaeal gene regulation as general transcriptional activators.

Machine and Deep Learning Methods for Predicting 3D Genome Organization.

Three-dimensional (3D) chromatin interactions, such as enhancer-promoter interactions (EPIs), loops, topologically associating domains (TADs), and A/B compartments, play critical roles in a wide range of cellular processes by regulating gene expression. Recent development of chromatin conformation capture technologies has enabled genome-wide profiling of various 3D structures, even with single cells. However, current catalogs of 3D structures remain incomplete and unreliable due to differences in technology, tools, and low data resolution. Machine learning methods have emerged as an alternative to obtain missing 3D interactions and/or improve resolution. Such methods frequently use genome annotation data (ChIP-seq, DNAse-seq, etc.), DNA sequencing information (k-mers and transcription factor binding site (TFBS) motifs), and other genomic properties to learn the associations between genomic features and chromatin interactions. In this review, we discuss computational tools for predicting three types of 3D interactions (EPIs, chromatin interactions, and TAD boundaries) and analyze their pros and cons. We also point out obstacles to the computational prediction of 3D interactions and suggest future research directions.

ERβ-regulated circATP2B1/miR-204-3p/TWIST1 positive feedback loop facilitates epithelial to mesenchymal transition in clear cell renal cell carcinoma.

Our previous studies have shown that estrogen receptor beta (ERβ) can promote the progression of clear cell renal cell carcinoma (ccRCC) by downregulating the expression of circATP2B1 and miR-204-3p. Here, we found that ERβ might promote the epithelial-mesenchymal transition(EMT) of ccRCC by modulating the circATP2B1/miR-204-3p/TWIST1(Twist family basic helix-loop-helix transcription factor 1) signaling pathway. We utilized bioinformatics analysis to determine the clinical significance of TWIST1 in ccRCC. The expression of TWIST1 in ccRCC tissues and cells was examined using immunohistochemistry, real-time quantitative polymerase chain reaction and western blotting assay. Chromatin Immunoprecipitation assay were conducted to validate the relationship between ERβ and TWIST1. Luciferase reporter gene assays were employed to validate the binding targets of TWIST1 and miR-204-3p. The role of TWIST1 in ccRCC was studied through in vitro and in vivo experiments. Transwell assays and wound healing assays were used to assess the impact of TWIST1 on the invasive and migratory abilities of ccRCC cells. Mechanism analysis revealed that miR-204-3p can inhibit TWIST1 by targeting its 3' untranslated region. Additionally, TWIST1 can promote ERβ transcription by directly binding to transcription factor binding site in the ERβ promoter region, forming a positive feedback loop. These in vitro data were further validated in an in vivo mouse model. Importantly, analysis of data from the TCGA-KIRC database further confirmed the above in vitro/in vivo findings. Together, our results suggest that ERβ/circATP2B1/miR-204-3p/TWIST1 can promote EMT by forming a positive feedback loop, thus promoting the progression of ccRCC. Targeting this newly identified signaling pathway may more effectively control the progression of ccRCC.

Promoter Analysis and Dissection Using Reporter Genes, Comparative Genomics, and Gel Shift Assays in Phytophthora.

Transcriptional regulation allows cells to execute developmental programs, maintain homeostasis, and respond to intra- and extracellular signals. Central to these processes are promoters, which in eukaryotes are sequences upstream of genes that bind transcription factors (TFs) and which recruit RNA polymerase to initiate mRNA synthesis. Valuable tools for studying promoters include reporter genes, which can be used to indicate when and where genes are activated. Moreover, functional regions within promoters (typically TF binding sites) can be identified by integrating reporter assays with promoter mutagenesis. These sites may also be revealed through comparative genomics, or by the DNA-protein binding procedure known as a gel shift or electrophoretic mobility shift assay (EMSA). The latter can also be used to test if a specific TF binds a DNA target or assess the binding kinetics or affinity of the complex. In this chapter, we describe procedures for expressing reporter genes in Phytophthora, assaying reporter activity, identifying functional sites within promoters, and testing purified TFs or proteins within nuclear extracts for DNA binding.

Analyses of the MYBL1 Gene in Triple Negative Breast Cancer: Evidence of Regulation of the VCPIP1 Gene and Identification of a Specific Exon Overexpressed in Tumor Cell Lines.

Previous data show that the knockdown of the MYBL1 gene in the MDA-MB-231 cell line leads to the downregulation of VCPIP1 gene expression. In addition, MYBL1 and VCPIP1 genes are co-expressed and dysregulated in some of the same triple negative breast cancer patient samples. We propose that the co-expression of the two genes is attributed to the MYBL1 transcription factor regulation of the VCPIP1 gene. We identify the MYBL1 transcription factor binding site upstream of the VCPIP1 start site and show that the MYBL1 protein can bind to the sequence identified in the VCPIP1 promoter region. Combined with the results from the knockdown study, these data support the ability of MYBL1 to regulate the VCPIP1 gene. The VCPIP1 gene functions as a deubiquitinating enzyme involved in DNA repair, protein positioning, and the assembly of the Golgi apparatus during mitotic signaling. The transcriptional regulation of VCPIP1 by the MYBL1 gene could implicate MYBL1 in these processes, which might contribute to tumor processes in TNBC. Although both genes are involved in cell cycle regulatory mechanisms, converging signaling mechanisms have not been identified. In a separate study, we performed sequence alignment of the MYBL1 transcript variants and identified an exon unique to the canonical variant. Probes that specifically target the unique MYBL1 exon show that the exon is overexpressed in tumor cell lines compared to non-tumor breast cells. We are classifying this unique MYBL1 exon as a tumor-associated exon.