Sequence-specific Transcription Factors Research Articles

Physiological, biochemical, and transcriptional regulation in a leguminous forage Trifolium pratense L. responding to silver ions

Trifolium pratense L. (red clover) is an important leguminous crop with great potential for Ag-contaminated environment remediation. Whereas, the molecular mechanisms of Ag tolerance in red clover are largely unknown. Red clover seedlings were used for physiological and transcriptomic investigation under 0, 20, 50, and 100 mg/L Ag+ stress in our research to reveal potential molecular resistance mechanism. Research showed that red clover possessed fairly strong Ag absorbance capacity, the Ag level reached 0.14 and 2.35 mg/g·FW in the leaves and roots under 100 mg/L AgNO3 stress condition. Root fresh weight, root dry weight, root water content, and photosynthetic pigments contents were significantly decreased with elevating AgNO3 concentration. Obvious withered plant tissue, microstructure disorder, and disrupted organelles were observed. In vitro evaluations (e.g., PI and DCFH-DA staining) represented that AgNO3 at high concentration (100 mg/L) exhibited obvious inhibition on cell viability, which was due possibly to the induction of reactive oxygen species (ROS) accumulation. A total of 44643 differentially expressed genes (DEGs) were identified under Ag stress, covering 27155 upregulated and 17488 downregulated genes. 12 stress-responsive DEGs was authenticated utilizing real-time quantitative PCR (qRT-PCR). Gene ontology (GO) analysis revealed that the DEGs were mostly related to metal ion binding (molecular function), nucleus (cellular component), and defense response (biological process). Involved DEGs in sequence-specific DNA binding transcription factor activity, response to various hormones (e.g., abscisic acid, IAA/Auxin, salicylic acid, and etc), calcium signal transduction, and protein ubiquitination were concluded to play crucial roles in Ag tolerance of red clover. On the other hand, Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotated several stress responsive pathways such as plant-pathogen interaction, phenylpropanoid biosynthesis, ubiquitin mediated proteolysis, hormone signal transduction, and autophagy. Several down-regulated genes (e.g., RSF2, RCD1, DOX1, and etc) were identified indicating possible metabolic disturbance. Besides, protein-protein interaction network (PPI) identified several pivotal genes such as ribosomal proteins, TIR, and ZAT.

A high-resolution protein architecture of the budding yeast genome.

The genome-wide protein architecture of chromatin that maintains chromosome integrity and gene regulation is ill-defined. Here we use ChIP-exo/seq1,2 to define this structure in Saccharomyces. We identified 21 ensembles consisting of ~400 different proteins related to DNA replication, centromeres, subtelomeres, transposons, and RNA polymerase (Pol) I, II, and III transcription. Replication proteins engulfed a nucleosome, centromeres lacked a nucleosome, and repressive proteins encompassed three nucleosomes at subtelomeric X-elements. We find that most Pol II promoters evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprised a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bound TFIID to form a preinitiation complex (PIC). Positioned insulators protected core promoters from upstream events. A small fraction of promoters were architected for inducibility, wherein sequence-specific transcription factors (TFs) create a nucleosome-depleted region (NDR) that is distinct from NFRs. We describe TF structural interactions with the genome and cognate cofactors, including nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator, and SWI-SNF. Surprisingly, we do not detect TF-TFIID interactions, suggesting that they do not stably occur. Our model for gene induction involves TFs, cofactors, and general factors like TBP and TFIIB, but not TFIID. However, constitutive transcription involves TFIID but not TFs and cofactors. From this we define a highly integrated network of TF-regulated transcription.

Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs.

BackgroundNF-κB is a sequence-specific DNA-binding transcription factor that plays key roles in inflammation and cancer. It is well known that NF-κB is over-activated in these diseases. NF-κB inhibitors are therefore developed as promising drugs for these diseases. However, finding NF-κB inhibitors is dependent on effective screening platforms.MethodsFor providing an easy and visualizable tool for screening NF-κB inhibitors, and other NF-κB-related studies, this study edited all five genes of NF-κB family (RELA, RELB, CREL, NF-κB1, NF-κB2) in three different cell lines (293T, HepG2, and PANC1) with both TALEN and CRISPR. The edited NF-κB genes were repaired by homology-dependent repair using a linear homologous donor containing ZsGreen coding sequence. The edit efficiency was thus directly evaluated by detecting cellular fluorescence. The editing efficiency was also confirmed by PCR detection of NF-κB-ZsGreen fused genes.ResultsIt was found that all genes were more efficiently edited by TALEN in all cells than CRISPR. The positive cells were then isolated from the TALEN-edited cell pool by flow cytometry. The purified positive cells were finally evaluated by regulating NF-κB activity with a known NF-κB inhibitor, BAY 11–7082, and an NF-κB-targeting artificial microRNA, miR533. The results revealed that all the labeled NF-κB genes responded well to the two kinds of NF-κB activity regulators in all cell lines.ConclusionThis study thus obtained 15 cell lines with NF-κB-ZsGreen fused genes, which provide an easy and visualizable tool for screening NF-κB inhibitors and other NF-κB-related studies.

Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis.

The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.

Remodeling the chromatin landscape in T lymphocytes by a division of labor among transcription factors

An extraordinary degree of condensation is required to fit the eukaryotic genome inside the nucleus. This compaction is attained by first coiling the DNA around structures called nucleosomes. Mammalian genomes are further folded into sophisticated three-dimensional (3D) configurations, enabling the genetic code to dictate a diverse range of cell fates. Recent advances in molecular and computational technologies have enabled the query of higher-order chromatin architecture at an unprecedented resolution and scale. In T lymphocytes, similar to other developmental programs, the hierarchical genome organization is shaped by a highly coordinated division of labor among different classes of sequence-specific transcription factors. In this review, we will summarize the general principles of 1D and 3D genome organization, introduce the common experimental and computational techniques to measure the multilayer chromatin organization, and discuss the pervasive role of transcription factors on chromatin organization in T lymphocytes.

Genome-Wide DNA Methylation Analysis of Soybean Curled-Cotyledons Mutant and Functional Evaluation of a Homeodomain-Leucine Zipper (HD-Zip) I Gene GmHDZ20.

DNA methylation is a major, conserved epigenetic modification that influences many biological processes. Cotyledons are specialized tissues that provide nutrition for seedlings at the early developmental stage. To investigate the patterns of genomic DNA methylation of germinated cotyledons in soybean (Glycine max) and its effect on cotyledon development, we performed a genome-wide comparative analysis of DNA methylation between the soybean curled-cotyledons (cco) mutant, which has abnormal cotyledons, and its corresponding wild type (WT) by whole-genome bisulfite sequencing. The cco mutant was methylated at more sites but at a slightly lower level overall than the WT on the whole-genome level. A total of 46 CG-, 92 CHG-, and 9723 CHH- (H = A, C, or T) differentially methylated genes (DMGs) were identified in cotyledons. Notably, hypomethylated CHH-DMGs were enriched in the gene ontology term “sequence-specific DNA binding transcription factor activity.” We selected a DMG encoding a homeodomain-leucine zipper (HD-Zip) I subgroup transcription factor (GmHDZ20) for further functional characterization. GmHDZ20 localized to the nucleus and was highly expressed in leaf and cotyledon tissues. Constitutive expression of GmHDZ20 in Arabidopsis thaliana led to serrated rosette leaves, shorter siliques, and reduced seed number per silique. A yeast two-hybrid assay revealed that GmHDZ20 physically interacted with three proteins associated with multiple aspects of plant growth. Collectively, our results provide a comprehensive study of soybean DNA methylation in normal and aberrant cotyledons, which will be useful for the identification of specific DMGs that participate in cotyledon development, and also provide a foundation for future in-depth functional study of GmHDZ20 in soybean.

Uncovering Sequence-Specific Transcription Factors Interacting with TET2.

Ten-eleven Translocation (TET) enzymes are methylcytosine dioxygenases that are involved in multiple cellular processes, including cellular differentiation and forced cell fate conversions. However, deciphering the molecular mechanisms underlying epigenetic control exerted by these proteins has been hampered by technical limitations, which prevent the identification of essential partners that work in concert with these enzymes to modulate gene expression. In this chapter, we provide a comprehensive description of cutting-edge methods designed to assess physical interactions between sequence-specific transcription factors and the TET2 enzyme.

Deep learning of immune cell differentiation

Although we know many sequence-specific transcription factors (TFs), how the DNA sequence of cis-regulatory elements is decoded and orchestrated on the genome scale to determine immune cell differentiation is beyond our grasp. Leveraging a granular atlas of chromatin accessibility across 81 immune cell types, we asked if a convolutional neural network (CNN) could learn to infer cell type-specific chromatin accessibility solely from regulatory DNA sequences. With a tailored architecture and an ensemble approach to CNN parameter interpretation, we show that our trained network ("AI-TAC") does so by rediscovering ab initio the binding motifs for known regulators and some unknown ones. Motifs whose importance is learned virtually as functionally important overlap strikingly well with positions determined by chromatin immunoprecipitation for several TFs. AI-TAC establishes a hierarchy of TFs and their interactions that drives lineage specification and also identifies stage-specific interactions, like Pax5/Ebf1 vs. Pax5/Prdm1, or the role of different NF-κB dimers in different cell types. AI-TAC assigns Spi1/Cebp and Pax5/Ebf1 as the drivers necessary for myeloid and B lineage fates, respectively, but no factors seemed as dominantly required for T cell differentiation, which may represent a fall-back pathway. Mouse-trained AI-TAC can parse human DNA, revealing a strikingly similar ranking of influential TFs and providing additional support that AI-TAC is a generalizable regulatory sequence decoder. Thus, deep learning can reveal the regulatory syntax predictive of the full differentiative complexity of the immune system.

VGLL4 with low YAP expression is associated with favorable prognosis in colorectal cancer.

The Hippo pathway is a tumor suppressive pathway regulating Yes-associated protein-TEA domain-containing sequence-specific transcription factor (YAP-TEAD) complex. VGLL (Vestigial-like) proteins are transcriptional cofactors competing with YAP for TEAD binding and interfering oncogenic activity of YAP-TEAD complex. We evaluated the expression of VGLL4, YAP, and TEAD4 and assessed their correlations with clinicopathologic factors and prognostic effects in 295 colorectal cancers. VGLL4 was positive in 164 (55.6%) cases and correlated with small tumor size, low pT classification, and absence of lymph node metastasis. YAP and TEAD4 were highly expressed in 138 (46.8%) cases and 144 (48.8%) cases, respectively, and high expressions were associated with presence of lymphovascular invasion and lymph node metastasis, or distant metastasis. VGLL4 expression was significantly correlated with low YAP expression (p<0.001) and had significantly better overall survival than negative expression (p<0.001). High YAP (HR, 2.108; 95% confidence interval, 1.239-3.584; p=0.006) and TEAD4 (1.724; 1.021-2.912; p=0.042) expressions were associated with poor overall survivals. The combined VGLL4pos YAPlow expression showed the best overall survival than other groups (p<0.001). VGLL4 expression (0.381; 0.212-0.683; p=0.001) and combined VGLL4pos YAPlow expression (0.227; 0.108-0.475; p<0.001) were independent good prognostic factors in colorectal cancers. The expressions of VGLL4, YAP, and TEAD4 can be used as prognostic markers in colorectal cancer patients.

Non-coding RNAs, guardians of the p53 galaxy.

The TP53 gene is arguably the most important tumor suppressor gene known, contributing multifaceted roles to the process of tumor development. Its protein product p53, is a crucial sequence-specific transcription factor which regulates the expression of a large network of protein-coding genes, as well as thousands of noncoding RNAs (ncRNAs), notably microRNAs and long ncRNAs (lncRNAs). Through a variety of direct and indirect mechanisms, ncRNAs in turn modulate p53 levels and activity. Here the numbers of studies are steadily building which link the contributions of dysregulated ncRNAs to tumorigenesis via their participation throughout the p53 regulatory network. In this review, we will examine how the principal forms of ncRNAs, namely microRNAs, lncRNAs and circular RNAs (circRNAs) function as either effectors or regulators amongst the diversity of p53's cellular responses. We first discuss the more recently discovered connections between miRNAs and p53 signaling before focusing on the remarkable diversity of crosstalk evident between lncRNAs and p53, and subsequently, developing reports linking circRNAs to p53. Highlighted throughout the review are the mechanistic impacts of dysregulated ncRNAs on p53 functions as well as the possible prognostic implications of these interactions. We also describe the emerging connections between ncRNAs and the often-perplexing functions of mutant p53. Finally, in the context of p53 therapeutic approaches, we describe some of the challenges in ncRNA research and their potential for translation.

Proteomic Profiling of Fast Neutron-Induced Soybean Mutant Unveiled Pathways Associated with Increased Seed Protein Content.

Mutagenesis through fast neutron (FN) radiation of soybean resulted in a mutant with a 15% increase in seed protein content. A comparative genomic hybridization analysis confirmed that the mutant is lacking 24 genes located at chromosomes 5 and 10. A tandem mass tag-based proteomic profiling of the wild type and the FN mutant revealed 3,502 proteins, of which 206 proteins exhibited increased abundance and 214 proteins showed decreased abundance. Among the abundant proteins, basic 7S globulin increased fourfold, followed by vacuolar-sorting receptor and protein transporters. The differentially expressed proteins were mapped on the global metabolic pathways. It was observed that there was an enrichment of 29 ribosomal proteins, 16 endoplasmic reticular proteins, and several proteins in export metabolic pathways. The deletion of the sequence-specific DNA binding transcription factor along with 23 other genes may have altered the negative regulation of protein syntheses processes, resulting in an increase in the overall protein content of the mutant seed. This mutant is a valuable resource for researchers to understand the metabolic pathways that may affect an increase in seed protein content (the mass spectrometry data files were submitted to massive.ucsd.edu # MassIVE MSV000084228).

Transcriptional Activation of Matricellular Protein Spondin2 (SPON2) by BRG1 in Vascular Endothelial Cells Promotes Macrophage Chemotaxis

The matricellular protein SPON2 plays diverse roles in the development of cardiovascular diseases. SPON2 is expressed in endothelial cells, but its transcription regulation in the context of atherogenesis remains incompletely appreciated. Here we report that SPON2 expression was up-regulated by pro-atherogenic stimuli (oxLDL and TNF-α) in vascular endothelia cells. In addition, endothelial SPON2 was elevated in Apoe–/– mice fed on a Western diet compared to the control mice. Induction of SPON2 in endothelial cells by pro-atherogenic stimuli was mediated by BRG1, a chromatin remodeling protein, both in vitro and in vivo. Further analysis revealed that BRG1 interacted with the sequence-specific transcription factor Egr-1 to activate SPON2 transcription. BRG1 contributed to SPON2 trans-activation by modulating chromatin structure surrounding the SPON2 promoter. Functionally, activation of SPON2 transcription by the Egr-1/BRG1 complex provided chemoattractive cues for macrophage trafficking. SPON2 depletion abrogated the ability of BRG1 or Egr-1 to stimulate endothelial derived chemoattractive cue for macrophage migration. On the contrary, recombinant SPON2 rescued endothelial chemo-attractability in the absence of BRG1 or Egr-1. In conclusion, our data have identified a novel transcriptional cascade in endothelial cells that may potentially promote macrophage recruitment and vascular inflammation leading to atherogenesis.

Global translation during early development depends on the essential transcription factor PRDM10

Members of the PR/SET domain-containing (PRDM) family of zinc finger transcriptional regulators play diverse developmental roles. PRDM10 is a yet uncharacterized family member, and its function in vivo is unknown. Here, we report an essential requirement for PRDM10 in pre-implantation embryos and embryonic stem cells (mESCs), where loss of PRDM10 results in severe cell growth inhibition. Detailed genomic and biochemical analyses reveal that PRDM10 functions as a sequence-specific transcription factor. We identify Eif3b, which encodes a core component of the eukaryotic translation initiation factor 3 (eIF3) complex, as a key downstream target, and demonstrate that growth inhibition in PRDM10-deficient mESCs is in part mediated through EIF3B-dependent effects on global translation. Our work elucidates the molecular function of PRDM10 in maintaining global translation, establishes its essential role in early embryonic development and mESC homeostasis, and offers insights into the functional repertoire of PRDMs as well as the transcriptional mechanisms regulating translation.

SMAD-6, -7 and -9 are potential molecular biomarkers for the prognosis in human lung cancer.

SMADs, a family of proteins that function as signal transducers and transcriptional regulators to regulate various signaling pathways, including the transforming growth factor-β signaling pathway, are similar to the mothers against decapentaplegic family of genes and the sma gene family in Caenorhabditis elegans. SMADs generate context-dependent modulation by interacting with various sequence-specific transcription factors, such as E2F4/5, c-Fos, GATA3, YY1 and SRF, which have been found to serve a key role in lung carcinoma oncogenesis and progression. However, the prognostic values of the eight SMADs in lung cancer have not been fully understood. In the present study, the expression levels and survival data of SMADs in patients with lung carcinoma from the Oncomine, Gene Expression Profiling Interactive Analysis, Kaplan-Meier plotter and cBioPortal databases were downloaded and analyzed. It was found that the mRNA expression levels of SMAD-6, −7 and −9 were decreased in lung adenocarcinoma and squamous cell carcinoma compared with that in adjacent normal tissues, while there was no significant difference in SMADs 1–5. Survival analysis revealed that not only were low transcriptional levels of SMAD-6, −7 and −9 associated with low overall survival but they also had prognostic role for progression-free survival and post-progression survival (P<0.05) in patients with lung carcinoma. In conclusion, the present study demonstrated that SMAD-6, −7 and −9 are potential biomarkers for the prognosis of patients with lung carcinoma.

SRF Potentiates Colon Cancer Metastasis and Progression in a microRNA-214/PTK6-Dependent Manner.

ObjectiveSerum response factor (SRF), a sequence-specific transcription factor, is closely related to metastasis of gastric cancer, a digestive tract cancer. Herein, we probed the effect of SRF on metastasis and progression of colon cancer (CC), another digestive tract disorder, and the detailed mechanism.MethodsMicroarray analysis was conducted on tumor and adjacent tissues to filter differentially expressed miRNA, followed by RT-qPCR validation in CC cell lines. The transcription factor and the target gene of microRNA-214 (miR-214) were predicted, and their binding relationships were tested by luciferase reporter assays and ChIP assays. Subsequently, SRF and protein tyrosine kinase 6 (PTK6) expression in CC patients and cells was evaluated by RT-qPCR, while JAK2 and STAT3 expression in cells by Western blot analysis. To further explore functions of miR-214, PTK6 and SRF on CC, CC cells were delivered with si-PTK6, miR-214 mimic and/or SRF overexpression.ResultsmiR-214 expressed poorly in CC tissues and cell lines, which related to advanced TNM staging and survival. miR-214 mimic inhibited proliferation, migration, invasion, xenograft tumor growth and metastasis of CC cells. SRF, overexpressed in CC samples and cells, suppressed the transcription of miR-214. Meanwhile, SRF upregulation counteracted the inhibitory role of miR-214 mimic in CC cell growth. miR-214 negatively regulated PTK6 expression to impair the JAK2/STAT3 pathway activation, thereby halting CC cell proliferation, migration, invasion, xenograft tumor growth and metastasis.ConclusionAltogether, miR-214 may perform as a tumor suppressor in CC, and the SRF/miR-214/PTK6/JAK2/STAT3 axis could be applied as a biomarker and potential therapeutic target.

LIN28B regulates transcription and potentiates MYCN-induced neuroblastoma through binding to ZNF143 at target gene promotors.

LIN28B is highly expressed in neuroblastoma and promotes tumorigenesis, at least, in part, through inhibition of let-7 microRNA biogenesis. Here, we report that overexpression of either wild-type (WT) LIN28B or a LIN28B mutant that is unable to inhibit let-7 processing increases the penetrance of MYCN-induced neuroblastoma, potentiates the invasion and migration of transformed sympathetic neuroblasts, and drives distant metastases in vivo. Genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and coimmunoprecipitation experiments show that LIN28B binds active gene promoters in neuroblastoma cells through protein-protein interaction with the sequence-specific zinc-finger transcription factor ZNF143 and activates the expression of downstream targets, including transcription factors forming the adrenergic core regulatory circuitry that controls the malignant cell state in neuroblastoma as well as GSK3B and L1CAM that are involved in neuronal cell adhesion and migration. These findings reveal an unexpected let-7-independent function of LIN28B in transcriptional regulation during neuroblastoma pathogenesis.

Regulated repression governs the cell fate promoter controlling yeast meiosis

Intrinsic signals and external cues from the environment drive cell fate decisions. In budding yeast, the decision to enter meiosis is controlled by nutrient and mating-type signals that regulate expression of the master transcription factor for meiotic entry, IME1. How nutrient signals control IME1 expression remains poorly understood. Here, we show that IME1 transcription is regulated by multiple sequence-specific transcription factors (TFs) that mediate association of Tup1-Cyc8 co-repressor to its promoter. We find that at least eight TFs bind the IME1 promoter when nutrients are ample. Remarkably, association of these TFs is highly regulated by different nutrient cues. Mutant cells lacking three TFs (Sok2/Phd1/Yap6) displayed reduced Tup1-Cyc8 association, increased IME1 expression, and earlier onset of meiosis. Our data demonstrate that the promoter of a master regulator is primed for rapid activation while repression by multiple TFs mediating Tup1-Cyc8 recruitment dictates the fate decision to enter meiosis.

Family-specific analysis of variant pathogenicity prediction tools.

Using the presently available datasets of annotated missense variants, we ran a protein family-specific benchmarking of tools for predicting the pathogenicity of single amino acid variants. We find that despite the high overall accuracy of all tested methods, each tool has its Achilles heel, i.e. protein families in which its predictions prove unreliable (expected accuracy does not exceed 51% in any method). As a proof of principle, we show that choosing the optimal tool and pathogenicity threshold at a protein family-individual level allows obtaining reliable predictions in all Pfam domains (accuracy no less than 68%). A functional analysis of the sets of protein domains annotated exclusively by neutral or pathogenic mutations indicates that specific protein functions can be associated with a high or low sensitivity to mutations, respectively. The highly sensitive sets of protein domains are involved in the regulation of transcription and DNA sequence-specific transcription factor binding, while the domains that do not result in disease when mutated are responsible for mediating immune and stress responses. These results suggest that future predictors of pathogenicity and especially variant prioritization tools may benefit from considering functional annotation.

Interactome of vertebrate GAF/ThPOK reveals its diverse functions in gene regulation and DNA repair

GAGA associated factor (GAF) is a sequence-specific DNA binding transcription factor that is evolutionarily conserved from flies to humans. Emerging evidence shows a context-dependent function of vertebrate GAF (vGAF, a.k.a. ThPOK) in multiple processes like gene activation, repression, and enhancer-blocking. We hypothesize that context-dependent interaction of vGAF with a diverse set of proteins forms the basis for the multifunctional nature of vGAF. To this end, we deciphered the protein-protein interactome of vGAF and show that vGAF interacts with chromatin remodelers, RNA metabolic machinery, transcriptional activators/ repressors, and components of DNA repair machinery. We further validated the biological significance of our protein-protein interaction data with functional studies and established a novel role of vGAF in DNA repair and cell-survival after UV-induced DNA damage. One of the major risk factors for skin cutaneous melanoma is prolonged exposure of UV and subsequent DNA damage. vGAF is highly expressed in normal skin tissue. Interestingly, our analysis of high-throughput RNA-sequencing data shows that vGAF is heavily downregulated across all major stages of skin cutaneous melanoma suggesting its potential as a diagnostic biomarker. Taken together, our study provides a plausible explanation for the diverse gene regulatory functions of vGAF and unravels its novel role in DNA repair.

ChExMix: A Method for Identifying and Classifying Protein-DNA Interaction Subtypes.

Regulatory proteins can employ multiple direct and indirect modes of interaction with the genome. The ChIP-exo mixture model (ChExMix) provides a principled approach to detecting multiple protein-DNA interaction modes in a single ChIP-exo experiment. ChExMix discovers and characterizes binding event subtypes in ChIP-exo data by leveraging both protein-DNA cross-linking signatures and DNA motifs. In this study, we present a summary of the major features and applications of ChExMix. We demonstrate that ChExMix does not require high-resolution protein-DNA binding assay data to detect binding event subtypes. Specifically, we apply ChExMix to analyze 393 ChIP-seq data profiles in K562 cells. Similar binding event subtypes are discovered across multiple proteins, suggesting the existence of colocalized regulatory protein modules that are recruited to DNA through a particular sequence-specific transcription factor. Our results thus suggest that ChExMix can characterize protein-DNA binding interaction modes using data from multiple types of protein-DNA interaction assays.