A Genome-Wide Screen for Genetic Variants That Modify the Recruitment of REST to Its Target Genes

Increasing numbers of human diseases are being linked to genetic variants, but our understanding of the mechanistic links leading from DNA sequence to disease phenotype is limited. The majority of disease-causing nucleotide variants fall within the non-protein-coding portion of the genome, making it likely that they act by altering gene regulatory sequences. We hypothesised that SNPs within the binding sites of the transcriptional repressor REST alter the degree of repression of target genes. Given that changes in the effective concentration of REST contribute to several pathologies—various cancers, Huntington's disease, cardiac hypertrophy, vascular smooth muscle proliferation—these SNPs should alter disease-susceptibility in carriers. We devised a strategy to identify SNPs that affect the recruitment of REST to target genes through the alteration of its DNA recognition element, the RE1. A multi-step screen combining genetic, genomic, and experimental filters yielded 56 polymorphic RE1 sequences with robust and statistically significant differences of affinity between alleles. These SNPs have a considerable effect on the the functional recruitment of REST to DNA in a range of in vitro, reporter gene, and in vivo analyses. Furthermore, we observe allele-specific biases in deeply sequenced chromatin immunoprecipitation data, consistent with predicted differenes in RE1 affinity. Amongst the targets of polymorphic RE1 elements are important disease genes including NPPA, PTPRT, and CDH4. Thus, considerable genetic variation exists in the DNA motifs that connect gene regulatory networks. Recently available ChIP–seq data allow the annotation of human genetic polymorphisms with regulatory information to generate prior hypotheses about their disease-causing mechanism

GATA-1 is essential in EGF-mediated induction of nucleotide excision repair activity and ERCC1 expression through ERK2 in human hepatoma cells.

International audienceThe nucleotide excision repair (NER) pathway and its leading gene excision-repair cross-complementary 1 (ERCC1) have been shown to be up-regulated in hepatocellular carcinomas even in the absence of treatment with chemotherapeutics. The aim of this study was to determine the mechanism involved in NER regulation during the liver cell growth observed in hepatocellular carcinoma. Both NER activity and ERCC1 expression were increased after exposure to the epidermal growth factor (EGF) in cultured normal and tumoral human hepatocytes. These increases correlated with the activation of the kinase signaling pathway mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK that is known to be a key regulator in the G(1) phase of the hepatocyte cell cycle. Moreover, EGF-mediated activation of ERCC1 was specifically inhibited by either the addition of U0126, a MEK/ERK inhibitor or small interfering RNA-mediated knockdown of ERK2. Basal expression of ERCC1 was decreased in the presence of the phosphoinositide-3-kinase (PI3K) inhibitor and small hairpin RNA (shRNA) against the PI3K pathway kinase FKBP12-rapamycin-associated protein or mammalian target of rapamycin. Transient transfection of human hepatocytes with constructs containing different sizes of the 5'-flanking region of the ERCC1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in EGF-treated cells, which correlated with the presence of the nuclear transcription factor GATA-1 recognition sequence. The recruitment of GATA-1 was confirmed by chromatin immunoprecipitation assay. In conclusion, these results represent the first demonstration of an up-regulation of NER and ERCC1 in EGF-stimulated proliferating hepatocytes. The transcription factor GATA-1 plays an essential role in the induction of ERCC1 through the mitogen-activated protein kinase (MAPK) pathway, whereas the PI3K signaling pathway contributes to ERCC1 basal expression

Differentiation driven changes in the dynamic organization of basal transcription initiation

Studies based on cell-free systems and on in vitro-cultured living cells support the concept that many cellular processes, such as transcription initiation, are highly dynamic: individual proteins stochastically bind to their substrates and disassemble after reaction completion. This dynamic nature allows quick adaptation of transcription to changing conditions. However, it is unknown to what extent this dynamic transcription organization holds for postmitotic cells embedded in mammalian tissue. To allow analysis of transcription initiation dynamics directly into living mammalian tissues, we created a knock-in mouse model expressing fluorescently tagged TFIIH. Surprisingly and in contrast to what has been observed in cultured and proliferating cells, postmitotic murine cells embedded in their tissue exhibit a strong and long-lasting transcription-dependent i

SNPs may increase or decrease affinity of RE1s.

<p>Examples of SNPs that decrease (A) and increase (B) the affinity of an RE1 sequence. On the left are diagrams of the genomic location of polymorphic RE1s, their target genes and the REST ChIPseq read density taken from ENCODE data. On the right is corresponding quantitative EMSA data. In (A), the well-studied RE1 that lies within the 3′ UTR of the <i>NPPA</i> gene contains the SNP rs12565 that strongly decreases its affinity for REST. In (B), the RE1 lying distally upstream of the <i>CDH4</i> gene contains the SNP rs6093022 that strongly increases its affinity. (C) The SNP rs1040480 within an intron of <i>PTPRT</i> reduces the affinity of REST. B = Bound complex of REST with probe; U = Unbound probe; D = Degradation product. The latter band represents a fraction of purified REST protein that is partially degraded, as was observed previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002624#pgen.1002624-Johnson4" target="_blank">[20]</a>.</p

Electrophoretic mobility shift assay to measure affinity differences between RE1 alleles.

<p>To measure RE1 affinity in vitro, we employed a competition EMSA method. We tested the ability of unlabelled competitor sequences to compete for REST binding with a fluorescently labelled DNA probe. (A) Various control oligonucleotides were used to validate the sensitivity and selectivity of the comparative EMSA assay. The Ideal RE1 motif is a high affinity synthetic sequence we used previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002624#pgen.1002624-Johnson4" target="_blank">[20]</a>. By swapping two highly conserved dinucleotides in the sequence, the affinity of the Ideal RE1 can be completely abolished (Mutated RE1). We also designed four pairs of RE1 alleles (N1-4), where SNPs lie outside the RE1 half sites, and thus would not be expected to alter binding affinity. (B) The results of control EMSAs are shown, where replicate EMSA gels have been quantitated and plotted. The data are displayed in units of Fraction Bound (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002624#s4" target="_blank">Materials and Methods</a>), where a low Fraction Bound value indicates high binding affinity, and vice versa. Example raw data are shown in panels: (C) Ideal/Mutated RE1s and (D) N1 RE1s. (E) Summary results of EMSA for all pRE1s in this study. The y-axis plots the difference in Fraction Bound between Major and Minor alleles, where the arrow begins at the value for Major, and ends at Minor. All RE1s are ranked by their change in Fraction Bound.</p

Allele-specific recruitment to pRE1s in GM12878 cells.

<p>The data shown corresponds to the 5 heterozygous SNPs discovered in GM12878 cells. In all cases blue indicates the Major allele, and red the Minor allele. EMSA data is shown in left panel (Note that Fraction Bound units correlate inversely with binding affinity), allele-specific ChIPseq read density from the ENCODE project is shown in the central panel, and allele-specific ChIP enrichment (where available) is shown in the right panel. Statistical significance was calculated using Student's <i>t</i> test (EMSA, allele-specific ChIP) and Binomial statistics (ChIPseq).</p

Polymorphic RE1s where the minor allele has increased affinity.

<p>FB: Change in Fraction Bound value, FB−FB; Frequency indicates Hapmap populations where the Minor SNP allele occurs at 5% (ND-Not determined, -5% in all populations)(Note: genotype data come from Hapmap, except for genotyping carried out on Hapmap CEU set in this study, denoted CEU*); Dist: Distance from RE1 to gene transcriptional start site (negative indicate upstream). Known REST target genes are underlined - see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002624#pgen.1002624.s005" target="_blank">File S1</a> for more information.</p

Experimental pipeline to discover SNPs that affect gene repression by REST.

<p>(A) The structure of the RE1 motif, illustrating its two strongly constrained half sites and weakly constrained spacer and 3′ regions. The spacer region may have “canonical” size of two nucleotides, or other “non-canonical” sizes. (B) Cartoon illustrating the hypothetical effect of a SNP in an RE1 element. In the upper panel, the Major (ie more frequent) allele contains a high-affinity RE1 sequence that strongly recruits REST, resulting in target gene repression. The presence of the SNP reduces REST binding affinity, and results in an increase in target gene transcription. (C) The flowchart illustrates the pipeline employed in this study to discover pRE1 SNPs.</p