Analysis Of Transcription Factor Binding Research Articles

Differential expression of lncRNAs during the HIV replication cycle: an underestimated layer in the HIV-host interplay.

Studying the effects of HIV infection on the host transcriptome has typically focused on protein-coding genes. However, recent advances in the field of RNA sequencing revealed that long non-coding RNAs (lncRNAs) add an extensive additional layer to the cell’s molecular network. Here, we performed transcriptome profiling throughout a primary HIV infection in vitro to investigate lncRNA expression at the different HIV replication cycle processes (reverse transcription, integration and particle production). Subsequently, guilt-by-association, transcription factor and co-expression analysis were performed to infer biological roles for the lncRNAs identified in the HIV-host interplay. Many lncRNAs were suggested to play a role in mechanisms relying on proteasomal and ubiquitination pathways, apoptosis, DNA damage responses and cell cycle regulation. Through transcription factor binding analysis, we found that lncRNAs display a distinct transcriptional regulation profile as compared to protein coding mRNAs, suggesting that mRNAs and lncRNAs are independently modulated. In addition, we identified five differentially expressed lncRNA-mRNA pairs with mRNA involvement in HIV pathogenesis with possible cis regulatory lncRNAs that control nearby mRNA expression and function. Altogether, the present study demonstrates that lncRNAs add a new dimension to the HIV-host interplay and should be further investigated as they may represent targets for controlling HIV replication.

Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes.

BackgroundObesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity.ResultsMuscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 ± 0.7 kg/m2) and obese (n = 10; BMI = 32.9 ± 0.7 kg/m2) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P < 0.05) that were altered in the promoter and untranslated (5' and 3'UTR) regions in the obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P < 0.05) altered. Of these, 12 genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change −1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5'UTR (Chr.8:22,423,530–22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity.ConclusionsThese results demonstrate that obesity alters the epigenome through DNA methylation and highlights novel transcriptomic changes in SORBS3 in skeletal muscle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0246-x) contains supplementary material, which is available to authorized users.

Systematic Characterization of Long Noncoding RNAs Reveals the Contrasting Coordination of Cis- and Trans-Molecular Regulation in Human Fetal and Adult Hearts.

The molecular regulation of heart development is regulated by cis- and trans-factors acting on the genome and epigenome. As a class of important regulatory RNAs, the role of long noncoding RNAs (lncRNAs) in human heart development is still poorly understood. Furthermore, factors that interact with lncRNAs in this process are not well characterized. Using RNA sequencing, we systematically define the contrasting lncRNA expression patterns between fetal and adult hearts. We report that lncRNAs upregulated in adult versus fetal heart have different sequence features and distributions. For example, the adult heart expresses more sense lncRNAs compared with fetal heart. We also report the coexpression of lncRNAs and neighboring coding genes that have important functions in heart development. Importantly, the regulation of lncRNA expression during fetal to adult heart development seems to be due, in part, to the coordination of specific developmental epigenetic modifications, such as H3K4me1 and H3k4me3. The expression of promoter-associated lncRNAs in adult and fetal hearts also seems to be related to these epigenetic states. Finally, transcription factor-binding analysis suggests that lncRNAs are directly regulating cardiac gene expression during development. We provide a systematic analysis of lncRNA control of heart development that gives clues to the roles that specific lncRNAs play in fetal and adult hearts.

Cooperative binding of AP-1 and TEAD4 modulates the balance between vascular smooth muscle and hemogenic cell fate.

The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.

Targeted Inactivation of the c-Myb Oncogene through Integrated Network Analysis

Major progress has been made in the treatment of Acute Myeloid Leukaemia (AML). However, drugs that have been developed to treat leukaemia still fail in about half the patients. New treatments are urgently needed. In the last couple of years we have been working to understand the exact molecular changes in the blood forming cells that cause leukaemia. We, and others, have identified key proteins that these cells need to survive. These are known as the driving oncogenes. Here we describe a pipeline, implemented by us, to inactivate key driving transcription factors through drug repositioning. In this study we focus on the oncogene c-MYB, a transcription factor that has a critical role in AML.To achieve the goal of discovering drugs that pharmacologically target the driving transcription factors for inactivation we set up a technique for integrated analysis of gene expression datasets and transcription factor binding to DNA and combined this with pattern-matching software to mine the connectivity map database. Briefly, inducible expression/knockdown systems and next generation profiling allowed us to integrate cellular output (RNA-Seq or Genechip) and transcription factor binding (ChIP-Seq or ChIPip-on-Chip) of the driving c-MYB oncoprotein in leukaemia. This profile was used to mine the connectivity database for perturbagens of this profile.The top hit, of this integrated network-based analysis of transcription factor behaviour and perturbing agents, was taken forward for analysis. It was found to cause an acute reduction in the protein levels of MYB, preceding changes in the level of its mRNA. This reduction in c-MYB proteins level could be blocked with proteasome inhibitor MG132. Meaning we induced the proteolysis of the c-MYB oncogene.The reduction in c-MYB levels was accompanied by a significant anti-AML activity in an in vitro colony forming assay of both AML cell lines and primary patient samples. Interestingly, the drug had no significant effect on colony formation of CD34 positive cord blood cells.To investigate the specificity we tried to rescue the function of MYB. Through overexpressing a "stabilized" MYB mutant, lacking the negative regulatory domain we could, in large part, rescue the block in leukemic self-renewal. Meaning, its main effect on the block in AML stem cells is through targeting c-MYB oncogene.It is vital to know if this drug would be active in patients and we therefore required a model that reflected, as closely as possible, the disease progression in vivo. In order to achieve this, we established a robust xenograft model with human AML cell lines. Next, we genetically modified these cells with a luciferase-expressing lentiviral vector, so that they could be detected within the xenograft host using the IVIS III pre-clinical imaging system. Using this approach, we have shown that oral administration, even when used alone as a monotherapy, causes a strong block in the leukaemia progression (over a 300 fold difference in leukemic burden at day 17 of treatment vs control), resulting in prolonged survival of xenograft hosts.Here we describe a pipeline to discover drugs that inactivate key transcription factors important in AML. By combining our knowledge of transcription factor behaviour and drug repositioning, we discovered candidate drugs that inactivate key oncogenes. It's important to note that repositioning candidates have been through several stages of clinical development and therefore have well-known safety and pharmacokinetic profiles. Therefore, we hope that these candidates can be taken forward rapidly into clinical trials to improve the patient outcome in AML. DisclosuresNo relevant conflicts of interest to declare.

Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions.

Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal “fingerprint” as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding.

MORPHEUS, a Webtool for Transcription Factor Binding Analysis Using Position Weight Matrices with Dependency.

Transcriptional networks are central to any biological process and changes affecting transcription factors or their binding sites in the genome are a key factor driving evolution. As more organisms are being sequenced, tools are needed to easily predict transcription factor binding sites (TFBS) presence and affinity from mere inspection of genomic sequences. Although many TFBS discovery algorithms exist, tools for using the DNA binding models they generate are relatively scarce and their use is limited among the biologist community by the lack of flexible and user-friendly tools. We have developed a suite of web tools (called Morpheus) based on the proven Position Weight Matrices (PWM) formalism that can be used without any programing skills and incorporates some unique features such as the presence of dependencies between nucleotides positions or the possibility to compute the predicted occupancy of a large regulatory region using a biophysical model. To illustrate the possibilities and simplicity of Morpheus tools in functional and evolutionary analysis, we have analysed the regulatory link between LEAFY, a key plant transcription factor involved in flower development, and its direct target gene APETALA1 during the divergence of Brassicales clade.

Identifying differential transcription factor binding in ChIP-seq.

ChIP seq is a widely used assay to measure genome-wide protein binding. The decrease in costs associated with sequencing has led to a rise in the number of studies that investigate protein binding across treatment conditions or cell lines. In addition to the identification of binding sites, new studies evaluate the variation in protein binding between conditions. A number of approaches to study differential transcription factor binding have recently been developed. Several of these methods build upon established methods from RNA-seq to quantify differences in read counts. We compare how these new approaches perform on different data sets from the ENCODE project to illustrate the impact of data processing pipelines under different study designs. The performance of normalization methods for differential ChIP-seq depends strongly on the variation in total amount of protein bound between conditions, with total read count outperforming effective library size, or variants thereof, when a large variation in binding was studied. Use of input subtraction to correct for non-specific binding showed a relatively modest impact on the number of differential peaks found and the fold change accuracy to biological validation, however a larger impact might be expected for samples with more extreme copy number variations between them. Still, it did identify a small subset of novel differential regions while excluding some differential peaks in regions with high background signal. These results highlight proper scaling for between-sample data normalization as critical for differential transcription factor binding analysis and suggest bioinformaticians need to know about the variation in level of total protein binding between conditions to select the best analysis method. At the same time, validation using fold-change estimates from qRT-PCR suggests there is still room for further method improvement.

Wingless-type MMTV integration site family member 2 (WNT2) gene is associated with resistance to MAP in faecal culture and antibody response in Holstein cattle.

Mycobacterium avium subspecies paratuberculosis (MAP) is a pathogenic bacterium responsible for the lethal Johne's disease in cattle. So far, several genome-wide association studies (GWAS) have been carried out to identify chromosomal regions highly associated with Johne's disease. The aim of this study was to investigate the genetic variability within a pool of seven genes (LAMB1, DLD, WNT2, PRDM1, SOCS5, PTGER4 and IL10) indicated by former GWAS/RNA-Seq studies as putatively associated with MAP infections and to achieve a confirmation study of association with paratuberculosis susceptibility in a population of 324 German Holstein cattle (162 cases MAP positive and 162 controls MAP negative) using ELISA and fecal cultural tests. SNP validation and genotyping information are provided, quick methods for allelic discrimination were set up and transcription factor binding analyses were performed. The rs43390642:G>TSNP in the WNT2 promoter region is associated with paratuberculosis susceptibility (P = 0.013), suggesting a protective role of the T allele (P = 0.043; odds ratio 0.50 [0.25-0.97]). The linkage disequilibrium with the DLD rs134692583:A>T might suggest a combined mechanism of action of these neighboring genes in resistance to MAP infection, which is also supported by a significant effect shown by the haplotype DLD(T) /WNT2(T) (P = 0.047). In silico analysis predicted rs43390642:G>T and rs134692583:A>T as essential parts of binding sites for the transcription factors GR, C/EBPβ and GATA-1, hence suggesting a potential influence on WNT2 and DLD gene expression. This study confirmed the region on BTA 4 (UMD 3.1: 50639460-51397892) as involved in tolerance/resistance to Johne's disease. In addition, this study clarifies the involvement of the investigated genes in MAP infection and contributes to the understanding of genetic variability involved in Johne's disease susceptibility.

A regulatory SNP modifies cystic fibrosis by disrupting NFκB complex binding on FAS

s of the 51st Workshop for Pediatric Research 51st Workshop for Pediatric Research Gottingen, Germany 16-17 April 2015 This supplement has not been sponsored. Meeting abstracts Meeting abstract Cystic Fibrosis (CF) is the most common autosomal recessive disease affecting about 1 in 3200 in Caucasians with increased morbidity, chronic infections, disability and reduced life expectancy. The mutations causing CF are well characterized, but the gene-gene and gene-environment interactions which significantly modify CF disease severity are completely unknown. We report the pathogenetic mechanisms underlying how a regulatory SNP modifies Cystic Fibrosis (CF). Association studies of affected individuals with Cystic Fibrosis identified the causal variant, a regulatory SNP rs7910656 on the non-coding region of FAS gene on human chromosome 10q24.1, which includes the binding site of many transcription factors especially the NFκB complex, but no molecular alterations were detected by conventional approaches. Using a combination of functional in-silico gene expression analysis and computational transcription factor binding analysis together with electrophoretic mobility shift and Supershift assays, we have identified that an allele of the regulatory SNP disrupts the binding of the master transcription factor NFκB on FAS and which likely interferes with normal immune activation and programmed cell death. Thus, our work demonstrates for the first time how a regulatory SNP disrupts the binding of a master transcription factor NFκB and a key signaling pathway FAS in immune regulation and programmed cell death modifying Cystic Fibrosis.

Alteration in basal and depolarization induced transcriptional network in iPSC derived neurons from Timothy syndrome.

BackgroundCommon genetic variation and rare mutations in genes encoding calcium channel subunits have pleiotropic effects on risk for multiple neuropsychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia. To gain further mechanistic insights by extending previous gene expression data, we constructed co-expression networks in Timothy syndrome (TS), a monogenic condition with high penetrance for ASD, caused by mutations in the L-type calcium channel, Cav1.2.MethodsTo identify patient-specific alterations in transcriptome organization, we conducted a genome-wide weighted co-expression network analysis (WGCNA) on neural progenitors and neurons from multiple lines of induced pluripotent stem cells (iPSC) derived from normal and TS (G406R in CACNA1C) individuals. We employed transcription factor binding site enrichment analysis to assess whether TS associated co-expression changes reflect calcium-dependent co-regulation.ResultsWe identified reproducible developmental and activity-dependent gene co-expression modules conserved in patient and control cell lines. By comparing cell lines from case and control subjects, we also identified co-expression modules reflecting distinct aspects of TS, including intellectual disability and ASD-related phenotypes. Moreover, by integrating co-expression with transcription factor binding analysis, we showed the TS-associated transcriptional changes were predicted to be co-regulated by calcium-dependent transcriptional regulators, including NFAT, MEF2, CREB, and FOXO, thus providing a mechanism by which altered Ca2+ signaling in TS patients leads to the observed molecular dysregulation.ConclusionsWe applied WGCNA to construct co-expression networks related to neural development and depolarization in iPSC-derived neural cells from TS and control individuals for the first time. These analyses illustrate how a systems biology approach based on gene networks can yield insights into the molecular mechanisms of neural development and function, and provide clues as to the functional impact of the downstream effects of Ca2+ signaling dysregulation on transcription.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0075-5) contains supplementary material, which is available to authorized users.

Abstract CT308: Pre-surgical statin-treatment in a window-of-opportunity breast cancer trial: Changes in gene expression profiles

Abstract Background Lipophilic statins proposedly exert anti-tumoral effects in breast cancer. Previous studies have shown statin-induced decrease in proliferation and increased apoptosis. The underlying mechanisms are currently unexplored. The MAP kinase pathway is one of the most important pathways for cell proliferation and survival. Dual specific phosphatase 1 (Dusp1) acts as an inhibitor of the MAP kinase pathway. The aim of this study was to explore statin-induced effects on global gene expression (GEX) profiles in primary breast cancer. Study Design and Methods The study was designed as a window-of-opportunity trial, including 50 patients with primary invasive breast cancer. Following core needle biopsies, patients were prescribed high-dose atorvastatin (80 mg/day) during two weeks prior to surgery. For the gene expression analysis, fresh frozen pre- and post treatment tumor samples were analyzed on Illumina's HumanHT-12 v4 microarrays. Significance Analysis of Microarrays (SAM) was performed using the TMEV v4.0 software to identify differentially expressed genes in paired samples before and after treatment. Genes with a q-value = 0 and fold change (FC) ≥ 1.4 were used for further analysis. The selected genes were analyzed by the transcription factor binding analysis program SMART. LitVAn (Literature Vector Analysis) was used for gene module functionality and functional annotation analysis was done using the GeneGo Pathways Software (MetaCore™). Results Fifty patients entered the study with forty-two patients completing all study parts. No serious adverse events were reported. After RNA extraction, 25 pre- and post-treatment tumor pairs with sufficient RNA quality and quantity were available for transcriptional analysis. Comparison of gene expression profiles in paired tumor samples revealed 407 significantly differentially expressed genes. A total of 32 genes were up-regulated, while 375 were down-regulated. Following filtration for LOC-genes and fold change ≥ 1,4, 67 genes were significantly changed; 21 of which were up-regulated and 46 down-regulated. Significantly up-regulated genes included DUSP1, RHOB1 and RGS1. Suggested biological processes were identified with LitVAn and SMART analysis. Finally, results from gene expression, LitVAn and SMART analyses were pooled together, thereby identifying statin-induced effects on the MAP-kinase pathway, including downstream events affecting apoptosis and cell proliferation. Conclusions Herein, we report statin-induced changes in gene expression profiles, indicating MAP-kinase pathway inhibition and pro-apoptotic events. Citation Format: Olof Bjarnadottir, Ida Johansson, Srinivas Veerla, Karin Jirstrom, Dorthe Grabau, Ingrid Hedenfalk, Signe Borgquist. Pre-surgical statin-treatment in a window-of-opportunity breast cancer trial: Changes in gene expression profiles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT308. doi:10.1158/1538-7445.AM2014-CT308

Single-molecule analysis of transcription factor binding at transcription sites in live cells.

Although numerous live-cell measurements have shown that transcription factors bind chromatin transiently, no measurements of transient binding have been reported at the endogenous response elements (REs) where transcription is normally induced. Here we show that at endogenous REs, the transcriptionally productive specific binding of p53 and of the glucocorticoid receptor (GR) is transient. We also find residence times roughly comparable to that of endogenous GR REs at an artificial, multi-copy array of gene regulatory sites, supporting the use of multi-copy arrays for live-cell analysis of transcription. Finally, we find that at any moment only a small fraction of TF molecules are engaged in transcriptionally productive binding at endogenous REs. The small fraction of bound factors provides one explanation for gene bursting and it also indicates that REs may often be unoccupied, resulting in partial responses to transcriptional signals.

Impact of artifact removal on ChIP quality metrics in ChIP-seq and ChIP-exo data.

With the advent of ChIP-seq multiplexing technologies and the subsequent increase in ChIP-seq throughput, the development of working standards for the quality assessment of ChIP-seq studies has received significant attention. The ENCODE consortium's large scale analysis of transcription factor binding and epigenetic marks as well as concordant work on ChIP-seq by other laboratories has established a new generation of ChIP-seq quality control measures. The use of these metrics alongside common processing steps has however not been evaluated. In this study, we investigate the effects of blacklisting and removal of duplicated reads on established metrics of ChIP-seq quality and show that the interpretation of these metrics is highly dependent on the ChIP-seq preprocessing steps applied. Further to this we perform the first investigation of the use of these metrics for ChIP-exo data and make recommendations for the adaptation of the NSC statistic to allow for the assessment of ChIP-exo efficiency.

A Force-Based, Parallel Assay for the Quantification of Protein-DNA Interactions

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

CCAAT Enhancer Binding Protein and Nuclear Factor of Activated T Cells Regulate HIV-1 LTR via a Novel Conserved Downstream Site in Cells of the Monocyte-Macrophage Lineage

Transcriptional control of the human immunodeficiency virus type 1 (HIV-1) promoter, the long terminal repeat (LTR), is achieved by interactions with cis-acting elements present both upstream and downstream of the start site. In silico transcription factor binding analysis of the HIV-1 subtype B LTR sequences revealed a potential downstream CCAAT enhancer binding protein (C/EBP) binding site. This binding site (+158 to+172), designated DS3, was found to be conserved in 67% of 3,858 unique subtype B LTR sequences analyzed in terms of nucleotide sequence as well as physical location in the LTR. DS3 was found to be well represented in other subtypes as well. Interestingly, DS3 overlaps with a previously identified region that bind members of the nuclear factor of activated T cells (NFAT) family of proteins. NFATc2 exhibited a higher relative affinity for DS3 as compared with members of the C/EBP family (C/EBP α and β). DS3 was able to compete efficiently with the low-affinity upstream C/EBP binding site I with respect to C/EBP binding, suggesting utilization of both NFAT and C/EBP. Moreover, cyclosporine A treatment, which has been shown to prevent dephosphorylation and nuclear translocation of NFAT isoforms, resulted in enhanced C/EBPα binding. The interactions at DS3 were also validated in an integrated HIV-1 LTR in chronically infected U1 cells. A binding knockout of DS3 demonstrated reduced HIV-1 LTR-directed transcription under both basal and interleukin-6-stimulated conditions only in cells of the monocyte-macrophage lineage cells and not in cells of T-cell origin. Thus, the events at DS3 positively regulate the HIV-1 promoter in cells of the monocyte-macrophage lineage.

Dynamic Analysis of Gene Expression and Genome-wide Transcription Factor Binding during Lineage Specification of Multipotent Progenitors

SummaryWe used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.

ChroMoS: an integrated web tool for SNP classification, prioritization and functional interpretation

Summary: Genome-wide association studies and re-sequencing projects are revealing an increasing number of disease-associated SNPs, a large fraction of which are non-coding. Although they could have relevance for disease susceptibility and progression, the lack of information about regulatory regions impedes the assessment of their functionality. Here we present a web server, ChroMoS (Chromatin Modified SNPs), which combines genetic and epigenetic data with the goal of facilitating SNPs' classification, prioritization and prediction of their functional consequences. ChroMoS uses a large database of SNPs and chromatin states, but allows a user to provide his/her own genetic information. Based on the SNP classification and interactive prioritization, a user can compute the functional impact of multiple SNPs using two prediction tools, one for differential analysis of transcription factor binding (sTRAP) and another for SNPs with potential impact on binding of miRNAs (MicroSNiPer).Availability: Web server, ChroMoS, is freely available at http://epicenter.immunbio.mpg.de/services/chromos.Contact: barenboim@ie-freiburg.mpg.de or manke@ie-freiburg.mpg.de

Abstract 745: Role of G-quadruplex structures in the human RET promoter region in the regulation of this gene.

Abstract The medullary thyroid carcinoma (MTC) is a tumor type characterized by germline point mutations of the RET (REarranged during Transfection) proto-oncogene. Since these mutations are known to induce oncogenic activation of RET tyrosine kinase, various therapeutic strategies interfering with the oncogenic function of RET protein have been developed and tested for treating RET-associated cancers in several preclinical and clinical studies. Our previous study has demonstrated that the G-rich strand in the polypurine/polypyrimidine (pPu/pPy) region of the RET proximal promoter is capable of forming intramolecular G-quadruplex structures in vitro in the presence of K+, which can be further stabilized by a G-quadruplex interactive agents. Thus, the detailed mechanism by which G-quadruplex structures regulate RET transcription has been studied using mutation analysis and promoter reporter assays together with a detailed analysis of transcription factor binding to the RET promoter in vivo. To this end, we have explored the Flp-In cell system using homologous recombination, which allows integration of a single copy of a wild type or G-quadruplex-mutant RET promoter–luciferase construct into a specific genomic recombination locus (FRT site) in a host Flp-In the human embryonic kidney 293 (HEK293) cell line. Thus, differences in reporter expression levels from isolated isogenic cell clones can be ascribed more confidently to promoter-specific effects, rather than enhancer/silencer artifacts due to variations in integration sites. Using these isogenic cell lines, we are able to provide strong evidence for G-quadruplex-dependent transcriptional repression of the RET gene in vivo. Citation Format: Daekyu Sun, Yoon-Joo Shin. Role of G-quadruplex structures in the human RET promoter region in the regulation of this gene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 745. doi:10.1158/1538-7445.AM2013-745

Regulatory Elements Associated with Paternally-Expressed Genes in the Imprinted Murine Angelman/Prader-Willi Syndrome Domain

The Angelman/Prader-Willi syndrome (AS/PWS) domain contains at least 8 imprinted genes regulated by a bipartite imprinting center (IC) associated with the SNRPN gene. One component of the IC, the PWS-IC, governs the paternal epigenotype and expression of paternal genes. The mechanisms by which imprinting and expression of paternal genes within the AS/PWS domain – such as MKRN3 and NDN – are regulated by the PWS-IC are unclear. The syntenic region in the mouse is organized and imprinted similarly to the human domain with the murine PWS-IC defined by a 6 kb interval within the Snrpn locus that includes the promoter. To identify regulatory elements that may mediate PWS-IC function, we mapped the location and allele-specificity of DNase I hypersensitive (DH) sites within the PWS-IC in brain cells, then identified transcription factor binding sites within a subset of these DH sites. Six major paternal-specific DH sites were detected in the Snrpn gene, five of which map within the 6 kb PWS-IC. We postulate these five DH sites represent functional components of the murine PWS-IC. Analysis of transcription factor binding within multiple DH sites detected nuclear respiratory factors (NRF's) and YY1 specifically on the paternal allele. NRF's and YY1 were also detected in the paternal promoter region of the murine Mrkn3 and Ndn genes. These results suggest that NRF's and YY1 may facilitate PWS-IC function and coordinately regulate expression of paternal genes. The presence of NRF's also suggests a link between transcriptional regulation within the AS/PWS domain and regulation of respiration. 3C analyses indicated Mkrn3 lies in close proximity to the PWS-IC on the paternal chromosome, evidence that the PWS-IC functions by allele-specific interaction with its distal target genes. This could occur by allele-specific co-localization of the PWS-IC and its target genes to transcription factories containing NRF's and YY1.