Clusters Of Binding Sites Research Articles

Polymerization of ZBTB transcription factors regulates chromatin occupancy

BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.

Three families of CD4-induced antibodies are associated with the capacity of plasma from people living with HIV to mediate ADCC in presence of CD4-mimetics.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation which occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in presence of CD4mc.

Abstract 1695: Genomic clustering tendency of transcription factors reflects phase-separated transcriptional condensates at cancer super-enhancers

Abstract Many transcription factors (TFs) have been shown to bind to super-enhancers, forming transcriptional condensates to activate transcription in various cellular systems including cancer. However, the genomic and epigenomic determinants of phase-separated transcriptional condensate formation remain poorly understood. Questions regarding which TFs tend to associate with transcriptional condensates and what factors influence their association are largely unanswered. Here we systematically analyzed 528 DNA sequence motifs across the human genome and 6,650 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 non-clustered TF binding, densely clustered TF binding sites are more enriched at cell-type-specific super-enhancers with higher chromatin accessibility, elevated chromatin interaction, and 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 at cancer super-enhancers. Citation Format: Zhenjia Wang, Shengyuan Wang, Chongzhi Zang. Genomic clustering tendency of transcription factors reflects phase-separated transcriptional condensates at cancer super-enhancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1695.

BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites

BackgroundPervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters.ResultsHere we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements.ConclusionsThus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

Detection of new pioneer transcription factors as cell-type-specific nucleosome binders.

Wrapping of DNA into nucleosomes restricts accessibility to DNA and may affect the recognition of binding motifs by transcription factors. A certain class of transcription factors, the pioneer transcription factors, can specifically recognize their DNA binding sites on nucleosomes, initiate local chromatin opening, and facilitate the binding of co-factors in a cell-type-specific manner. For the majority of human pioneer transcription factors, the locations of their binding sites, mechanisms of binding, and regulation remain unknown. We have developed a computational method to predict the cell-type-specific ability of transcription factors to bind nucleosomes by integrating ChIP-seq, MNase-seq, and DNase-seq data with details of nucleosome structure. We have demonstrated the ability of our approach in discriminating pioneer from canonical transcription factors and predicted new potential pioneer transcription factors in H1, K562, HepG2, and HeLa-S3 cell lines. Last, we systematically analyzed the interaction modes between various pioneer transcription factors and detected several clusters of distinctive binding sites on nucleosomal DNA.

Detection of new pioneer transcription factors as cell-type-specific nucleosome binders

Wrapping of DNA into nucleosomes restricts accessibility to DNA and may affect the recognition of binding motifs by transcription factors. A certain class of transcription factors, the pioneer transcription factors, can specifically recognize their DNA binding sites on nucleosomes, initiate local chromatin opening, and facilitate the binding of co-factors in a cell-type-specific manner. For the majority of human pioneer transcription factors, the locations of their binding sites, mechanisms of binding, and regulation remain unknown. We have developed a computational method to predict the cell-type-specific ability of transcription factors to bind nucleosomes by integrating ChIP-seq, MNase-seq, and DNase-seq data with details of nucleosome structure. We have demonstrated the ability of our approach in discriminating pioneer from canonical transcription factors and predicted new potential pioneer transcription factors in H1, K562, HepG2, and HeLa-S3 cell lines. Last, we systematically analyzed the interaction modes between various pioneer transcription factors and detected several clusters of distinctive binding sites on nucleosomal DNA.

Multi-feature clustering of CTCF binding creates robustness for loop extrusion blocking and Topologically Associating Domain boundaries

Topologically Associating Domains (TADs) separate vertebrate genomes into insulated regulatory neighborhoods that focus genome-associated processes. TADs are formed by Cohesin-mediated loop extrusion, with many TAD boundaries consisting of clustered binding sites of the CTCF insulator protein. Here we determine how this clustering of CTCF binding contributes to the blocking of loop extrusion and the insulation between TADs. We identify enrichment of three features of CTCF binding at strong TAD boundaries, consisting of strongly bound and closely spaced CTCF binding peaks, with a further enrichment of DNA-binding motifs within these peaks. Using multi-contact Nano-C analysis in cells with normal and perturbed CTCF binding, we establish that individual CTCF binding sites contribute to the blocking of loop extrusion, but in an incomplete manner. When clustered, individual CTCF binding sites thus create a stepwise insulation between neighboring TADs. Based on these results, we propose a model whereby multiple instances of temporal loop extrusion blocking create strong insulation between TADs.

In-silico identification and comparison of transcription factor binding sites cluster in anterior-posterior patterning genes in Drosophila melanogaster and Tribolium castaneum.

The cis-regulatory data that help in transcriptional regulation is arranged into modular pieces of a few hundred base pairs called CRMs (cis-regulatory modules) and numerous binding sites for multiple transcription factors are prominent characteristics of these cis-regulatory modules. The present study was designed to localize transcription factor binding site (TFBS) clusters on twelve Anterior-posterior (A-P) genes in Tribolium castaneum and compare them to their orthologous gene enhancers in Drosophila melanogaster. Out of the twelve A-P patterning genes, six were gap genes (Kruppel, Knirps, Tailless, Hunchback, Giant, and Caudal) and six were pair rule genes (Hairy, Runt, Even-skipped, Fushi-tarazu, Paired, and Odd-skipped). The genes along with 20 kb upstream and downstream regions were scanned for TFBS clusters using the Motif Cluster Alignment Search Tool (MCAST), a bioinformatics tool that looks for set of nucleotide sequences for statistically significant clusters of non-overlapping occurrence of a given set of motifs. The motifs used in the current study were Hunchback, Caudal, Giant, Kruppel, Knirps, and Even-skipped. The results of the MCAST analysis revealed the maximum number of TFBS for Hunchback, Knirps, Caudal, and Kruppel in both D. melanogaster and T. castaneum, while Bicoid TFBS clusters were found only in D. melanogaster. The size of all the predicted TFBS clusters was less than 1kb in both insect species. These sequences revealed more transversional sites (Tv) than transitional sites (Ti) and the average Ti/Tv ratio was 0.75.

Interactions of piRNAs with the mRNA of Candidate Genes in Esophageal Squamous Cell Carcinoma.

Recently, a database of human piRNAs (piwi-interacting RNAs) was created, which allows the study of the binding of many piRNAs to the mRNAs of genes involved in many diseases, including cancer. In the present work, we identified the piRNAs that can interact with candidate esophageal squamous cell carcinoma (ESCC) genes. The binding of 480 thousand piRNAs with the mRNAs of 66 candidate ESCC genes was studied. Bioinformatic studies found that piRNAs bind only to the mRNAs of nine candidate genes: AURKA, BMP7, GCOM1, ERCC1, MTHFR, SASH1, SIX4, SULT1A1, and TP53. It has been shown that piRNAs can bind to mRNA by overlapping nucleotide sequences in limited 3'UTR and 5'UTR regions called clusters of binding sites (BSs). The existence of clusters of piRNA BSs significantly reduces the proportion of the nucleotide sequences of these sites in the mRNA of target genes. Competition between piRNAs occurs for binding to the mRNA of target genes. Individual piRNAs and groups of piRNAs that have separate BSs and clusters of BSs in the mRNAs of two or more candidate genes have been identified in the mRNAs of these genes. This organization of piRNAs BSs indicates the interdependence of the expression of candidate genes through piRNAs. Significant differences in the ability of genes to interact with piRNAs prevent the side effects of piRNAs on genes with a lack of the ability to bind such piRNAs. Individual piRNAs and sets of piRNAs are proposed and recommended for the diagnosis and therapy of ESCC.

Endogenous piRNAs Can Interact with the Omicron Variant of the SARS-CoV-2 Genome

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the COVID-19 pandemic, can still infect populations in many countries around the globe. The Omicron strain is the most mutated variant of SARS-CoV-2. The high transmissibility of the strain and its ability to evade immunity necessitate a priority study of its properties in order to quickly create effective means of preventing its spread. The current research aimed to examine the in silico interaction between PIWI-interacting RNAs (piRNAs) and the SARS-CoV-2 genome (gRNA) to identify endogenous piRNAs and propose synthetic piRNAs with strong antiviral activity for drug development. This study used validated bioinformatic approaches regarding the interaction of more than eight million piRNAs with the SARS-CoV-2 genome. The piRNAs' binding sites (BSs) in the 5'UTR were located with overlapping nucleotide sequences termed clusters of BSs. Several BSs clusters have been found in the nsp3, nsp7, RNA-dependent RNA polymerase, endoRNAse, S surface glycoprotein, ORF7a, and nucleocapsid. Sixteen synthetic piRNAs that interact with gRNA have been proposed with free binding energy ranging from -170 kJ/mol to -175 kJ/mol, which can be used to create drugs that suppress the reproduction of SARS-CoV-2.

PiRNA and miRNA Can Suppress the Expression of Multiple Sclerosis Candidate Genes

Multiple sclerosis (MS) is a common inflammatory demyelinating disease with a high mortality rate. MS is caused by many candidate genes whose specific involvement has yet to be established. The aim of our study was to identify endogenous miRNAs and piRNAs involved in the regulation of MS candidate gene expression using bioinformatic methods. A program was used to quantify the interaction of miRNA and piRNA nucleotides with mRNA of the target genes. We used 7310 miRNAs from three databases and 40,000 piRNAs. The mRNAs of the candidate genes revealed miRNA binding sites (BSs), which were located separately or formed clusters of BSs with overlapping nucleotide sequences. The miRNAs from the studied databases were generally bound to mRNAs in different combinations, but miRNAs from only one database were bound to the mRNAs of some genes. For the first time, a direct interaction between the complete sequence of piRNA nucleotides and the nucleotides of their mRNA BSs of target genes was shown. One to several clusters of BSs of miRNA and piRNA were identified in the mRNA of ADAM17, AHI1, CD226, EOMES, EVI5, IL12B, IL2RA, KIF21B, MGAT5, MLANA, SOX8, TNFRSF1A, and ZBTB46 MS candidate genes. These piRNAs form the expression regulation system of the MS candidate genes to coordinate the synthesis of their proteins. Based on these findings, associations of miRNAs, piRNAs, and candidate genes for MS diagnosis are recommended.

Detecting and quantifying liquid–liquid phase separation in living cells by model-free calibrated half-bleaching

Cells contain numerous substructures that have been proposed to form via liquid–liquid phase separation (LLPS). It is currently debated how to reliably distinguish LLPS from other mechanisms. Here, we benchmark different methods using well-controlled model systems in vitro and in living cells. We find that 1,6-hexanediol treatment and classical FRAP fail to distinguish LLPS from the alternative scenario of molecules binding to spatially clustered binding sites without phase-separating. In contrast, the preferential internal mixing seen in half-bleach experiments robustly distinguishes both mechanisms. We introduce a workflow termed model-free calibrated half-FRAP (MOCHA-FRAP) to probe the barrier at the condensate interface that is responsible for preferential internal mixing. We use it to study components of heterochromatin foci, nucleoli, stress granules and nuage granules, and show that the strength of the interfacial barrier increases in this order. We anticipate that MOCHA-FRAP will help uncover the mechanistic basis of biomolecular condensates in living cells.

PiRNAs may regulate expression of candidate genes of esophageal adenocarcinoma.

Elucidation of ways to regulate the expression of candidate cancer genes will contribute to the development of methods for cancer diagnosis and therapy. The aim of the present study was to show the role of piRNAs as efficient regulators of mRNA translation of esophageal adenocarcinoma (EAC) candidate genes. We used bioinformatic methods to study the interaction characteristics of up to 200 thousand piRNAs with mRNAs of 38 candidate EAC genes. The piRNAs capable of binding to mRNA of AR, BTG3, CD55, ERBB3, FKBP5, FOXP1, LEP, SEPP1, SMAD4, and TP53 genes with high free energy by the formation of hydrogen bonds between canonical (G-C, A-U) and noncanonical (G-U, A-C) piRNA and mRNA nucleotide pairs were revealed. The organization of piRNA binding sites (BSs) in the mRNA of candidate genes was found to overlap nucleotide sequences to form clusters. Clusters of piRNA BSs were detected in the 5'-untranslated region, coding domain sequence, and 3'-untranslated region of mRNA. Due to the formation of piRNA binding site clusters, compaction of BSs occurs and competition between piRNAs for binding to mRNA of candidate EAC genes occurs. Associations of piRNA and candidate genes were selected for use as markers for the diagnosis of EAC.

Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance

Binding to binding site clusters has yet to be characterized in depth, and the functional relevance of low-affinity clusters remains uncertain. We characterized transcription factor binding to low-affinity clusters in vitro and found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, small clusters with binding sites an order of magnitude lower in affinity give rise to high mean occupancies at physiologically-relevant transcription factor concentrations. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters in a synthetic and native gene regulatory network in S. cerevisiae. In both systems, clusters of low-affinity binding sites generated transcriptional output comparable to single or even multiple consensus sites. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.

In Silico Study of piRNA Interactions with the SARS-CoV-2 Genome.

A prolonged pandemic with numerous human casualties requires a rapid search for means to control the various strains of SARS-CoV-2. Since only part of the human population is affected by coronaviruses, there are probably endogenous compounds preventing the spread of these viral pathogens. It has been shown that piRNA (PIWI-interacting RNAs) interact with the mRNA of human genes and can block protein synthesis at the stage of translation. Estimated the effects of piRNA on SARS-CoV-2 genomic RNA (gRNA) in silico. A cluster of 13 piRNA binding sites (BS) in the SARS-CoV-2 gRNA region encoding the oligopeptide was identified. The second cluster of BSs 39 piRNAs also encodes the oligopeptide. The third cluster of 24 piRNA BS encodes the oligopeptide. Twelve piRNAs were identified that strongly interact with the gRNA. Based on the identified functionally important endogenous piRNAs, synthetic piRNAs (spiRNAs) are proposed that will suppress the multiplication of the coronavirus even more strongly. These spiRNAs and selected endogenous piRNAs have little effect on human 17494 protein-coding genes, indicating a low probability of side effects. The piRNA and spiRNA selection methodology created for the control of SARS-CoV-2 (NC_045512.2) can be used to control all strains of SARS-CoV-2.

Etv2 regulates enhancer chromatin status to initiate Shh expression in the limb bud

Sonic hedgehog (Shh) is essential for limb development, and the mechanisms that govern the propagation and maintenance of its expression has been well studied; however, the mechanisms that govern the initiation of Shh expression are incomplete. Here we report that ETV2 initiates Shh expression by changing the chromatin status of the developmental limb enhancer, ZRS. Etv2 expression precedes Shh in limb buds, and Etv2 inactivation prevents the opening of limb chromatin, including the ZRS, resulting in an absence of Shh expression. Etv2 overexpression in limb buds causes nucleosomal displacement at the ZRS, ectopic Shh expression, and polydactyly. Areas of nucleosome displacement coincide with ETS binding site clusters. ETV2 also functions as a transcriptional activator of ZRS and is antagonized by ETV4/5 repressors. Known human polydactyl mutations introduce novel ETV2 binding sites in the ZRS, suggesting that ETV2 dosage regulates ZRS activation. These studies identify ETV2 as a pioneer transcription factor (TF) regulating the onset of Shh expression, having both a chromatin regulatory role and a transcriptional activation role.

AntimiR targeting of microRNA-134 reduces seizures in a mouse model of Angelman syndrome

Angelman syndrome (AS) is a severe neurodevelopmental disorder featuring ataxia, cognitive impairment, and drug-resistant epilepsy. AS is caused by mutations or deletion of the maternal copy of the paternally imprinted UBE3A gene, with current precision therapy approaches focusing on re-expression of UBE3A. Certain phenotypes, however, are difficult to rescue beyond early development. Notably, a cluster of microRNA binding sites was reported in the untranslated Ube3a1 transcript, including for miR-134, suggesting that AS may be associated with microRNA dysregulation. Here, we report levels of miR-134 and key targets are normal in the hippocampus of mice carrying a maternal deletion of Ube3a (Ube3am−/p+). Nevertheless, intracerebroventricular injection of an antimiR oligonucleotide inhibitor of miR-134 (Ant-134) reduced audiogenic seizure severity over multiple trials in 21- and 42-day-old AS mice. Interestingly, Ant-134 also improved distance traveled and center crossings of AS mice in the open-field test. Finally, we show that silencing miR-134 can upregulate targets of miR-134 in neurons differentiated from Angelman patient-derived induced pluripotent stem cells. These findings indicate that silencing miR-134 and possibly other microRNAs could be useful to treat clinically relevant phenotypes with a later developmental window in AS.

Protein-PAINT: Superresolution microscopy with signaling proteins.

Superresolution techniques have advanced our understanding of complex cellular structures and processes but require the attachment of fluorophores to targets through tags or antibodies, which can be bulky and result in underlabeling. To overcome these limitations, we developed a technique to visualize the nanoscale binding locations of signaling proteins by taking advantage of their native interaction domains. Here, we demonstrated that pPAINT (protein point accumulation in nanoscale topography) is a new, single-molecule localization microscopy (SMLM) technique and used it to investigate T cell signaling by visualizing the Src homology 2 (SH2) domain, which is common in signaling molecules. When SH2 domain-containing proteins relocate to the plasma membrane, the domains selectively, transiently, and reversibly bind to preferred phosphorylated tyrosine residues on receptors. This transient binding yields the stochastic blinking events necessary for SMLM when observed with total internal reflection microscopy and enables quantification of binding coefficients in intact cells. We used pPAINT to reveal the binding sites of several T cell receptor-proximal signaling molecules, including Zap70, PI3K, Grb2, Syk, Eat2, and SHP2, and showed that the probes could be multiplexed. We showed that the binding half-life of the tandem SH2 domain of PI3K correlated with binding site cluster size at the immunological synapses of T cells, but that longer binding lifetimes were associated with smaller clusters for the monovalent SH2 domain of Eat2. These results demonstrate the potential of pPAINT for investigating phosphotyrosine-mediated signaling processes at the plasma membrane.

Statistical estimates of multiple transcription factors binding in the model plant genomes based on ChIP-seq data.

The development of high-throughput genomic sequencing coupled with chromatin immunoprecipitation technologies allows studying the binding sites of the protein transcription factors (TF) in the genome scale. The growth of data volume on the experimentally determined binding sites raises qualitatively new problems for the analysis of gene expression regulation, prediction of transcription factors target genes, and regulatory gene networks reconstruction. Genome regulation remains an insufficiently studied though plants have complex molecular regulatory mechanisms of gene expression and response to environmental stresses. It is important to develop new software tools for the analysis of the TF binding sites location and their clustering in the plant genomes, visualization, and the following statistical estimates. This study presents application of the analysis of multiple TF binding profiles in three evolutionarily distant model plant organisms. The construction and analysis of non-random ChIP-seq binding clusters of the different TFs in mammalian embryonic stem cells were discussed earlier using similar bioinformatics approaches. Such clusters of TF binding sites may indicate the gene regulatory regions, enhancers and gene transcription regulatory hubs. It can be used for analysis of the gene promoters as well as a background for transcription networks reconstruction. We discuss the statistical estimates of the TF binding sites clusters in the model plant genomes. The distributions of the number of different TFs per binding cluster follow same power law distribution for all the genomes studied. The binding clusters in Arabidopsis thaliana genome were discussed here in detail.

Dissecting CTCF site function in a tense HoxD locus.

In this issue of Genes & Development, Amândio and colleagues (pp. 1490-1509) dissect the function of a cluster of several CTCF binding sites in the HoxD cluster by iterative deletions in mice. They found additive functions for some, and intriguingly found that some sites function as insulators, while others function as anchors for enhancer-promoter interactions. These functions vary depending on developmental context. The work provides new insights into the roles played by CTCF in regulating developmental patterns and 3D chromatin organization.