An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability.

Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes.Research in the authors’ laboratories was supported by Bloodwise, The Wellcome Trust, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, the National Institute of Health Research, the Medical Research Council, the MRC Molecular Haematology Unit (Oxford) core award, a Weizmann-UK “Making Connections” grant (Oxford) and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research (100140) and Wellcome Trust–MRC Cambridge Stem Cell Institute (097922).This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.1146

A GWAS sequence variant for platelet volume marks an alternative DNM3 promoter in megakaryocytes near a MEIS1 binding site

We recently identified 68 genomic loci where common sequence variants are associated with platelet count and volume. Platelets are formed in the bone marrow by megakaryocytes, which are derived from hematopoietic stem cells by a process mainly controlled by transcription factors. The homeobox transcription factor MEIS1 is uniquely transcribed in megakaryocytes and not in the other lineage-committed blood cells. By ChIP-seq, we show that 5 of the 68 loci pinpoint a MEIS1 binding event within a group of 252 MK-overexpressed genes. In one such locus in DNM3, regulating platelet volume, the MEIS1 binding site falls within a region acting as an alternative promoter that is solely used in megakaryocytes, where allelic variation dictates different levels of a shorter transcript. The importance of dynamin activity to the latter stages of thrombopoiesis was confirmed by the observation that the inhibitor Dynasore reduced murine proplatelet for-mation in vitro

Disease-Related Growth Factor and Embryonic Signaling Pathways Modulate an Enhancer of <i>TCF21</i> Expression at the 6q23.2 Coronary Heart Disease Locus

<div><p>Coronary heart disease (CHD) is the leading cause of mortality in both developed and developing countries worldwide. Genome-wide association studies (GWAS) have now identified 46 independent susceptibility loci for CHD, however, the biological and disease-relevant mechanisms for these associations remain elusive. The large-scale meta-analysis of GWAS recently identified in Caucasians a CHD-associated locus at chromosome 6q23.2, a region containing the transcription factor <i>TCF21</i> gene. TCF21 (Capsulin/Pod1/Epicardin) is a member of the basic-helix-loop-helix (bHLH) transcription factor family, and regulates cell fate decisions and differentiation in the developing coronary vasculature. Herein, we characterize a <i>cis</i>-regulatory mechanism by which the lead polymorphism rs12190287 disrupts an atypical activator protein 1 (AP-1) element, as demonstrated by allele-specific transcriptional regulation, transcription factor binding, and chromatin organization, leading to altered <i>TCF21</i> expression. Further, this element is shown to mediate signaling through platelet-derived growth factor receptor beta (PDGFR-β) and Wilms tumor 1 (WT1) pathways. A second disease allele identified in East Asians also appears to disrupt an AP-1-like element. Thus, both disease-related growth factor and embryonic signaling pathways may regulate CHD risk through two independent alleles at <i>TCF21</i>.</p></div

AP-1 mediated regulation at rs12190287 <i>in vivo</i>.

<p>(<b>a</b>) Total enrichment of c-Jun, JunD, and ATF3 at rs12190287 determined by chromatin immunoprecipitation (ChIP) in heterozygous HCASMC treated with PDGF-BB (20 ng/ml) or vehicle (Control) for 6 hrs. (<b>b</b>) Allele-specific enrichment of c-Jun, JunD and ATF3 at rs12190287 determined by HaploChIP in heterozygous HCASMC treated with PDGF-BB, shown as normalized allelic-ratio C/G. (<b>c</b>–<b>d</b>) AP-1 positive and negative control regions for c-Jun, JunD, and ATF3 enrichment at <i>FOSB</i> (<b>c</b>) and <i>MYOG</i> (<b>d</b>) promoters, respectively. (<b>e</b>) Total enrichment of histone modifications H3K4me1, H3K4me3, H3K27ac, and H3K27me1 at rs12190287 determined by ChIP in heterozygous HCASMC treated with PDGF-BB. (<b>f</b>) Allele-specific enrichment of H3K4me1, H3K4me3, H3K27ac, and H3K27me1 at rs12190287 determined by HaploChIP in heterozygous HCASMC treated with PDGF-BB, shown as normalized allelic ratio C/G. *P<0.001 versus Control for each condition. Values are mean ± SD from triplicates. Similar results were observed from 3–4 independent experiments. For HaploChIP experiments, separate heterozygous individual HCASMCs were used for each replicate experiment.</p

Haplotype and regulatory analysis of <i>TCF21</i> locus at 6q23.2.

<p>(<b>a</b>) Regional association plot of <i>TCF21</i> locus showing results from CARDIoGRAM meta-analysis in Caucasians, identifying rs12190287, and also depicting rs12524865. (<b>b</b>) Integrated workflow to identify CHD risk-associated mechanisms, with emphasis of approaches used in the study (blue boxes). CEU: Caucasians of European descent from UT, USA; EA: East Asians; eQTL: Expression quantitative trait loci; ASE: Allele-specific expression, TFBS: Transcription factor binding site. (<b>c</b>) Linkage disequilibrium (LD) plot of the <i>TCF21</i> locus at 6q23.2 from 1568 Metabochip genotyped Europeans (ADVANCE replication cohort) with imputation from HapMap Phase II and III CEU, showing eSNPs for <i>TCF21</i>, and rs2327429. White to black squares represent increasing r² values. (<b>d</b>) The risk haplotype block containing all of the eSNPS (in red) at the <i>TCF21</i> locus has a combined frequency of 0.366. r² values in LD with rs12190287 shown as a percentage in parentheses. Risk and protective alleles determined from CARDIoGRAM meta-analysis phenotypic data. (<b>e</b>) ENCODE ChIP-seq active chromatin histone modification data from 7 cell lines including promoter and enhancer marks, H3K4me1, H3K4me3, and H3K27ac. ENCODE tracks for DNase hypersensitivity, RNA-seq, and TF binding ChIP-seq data. Green box surrounds peaks overlapping rs12190287.</p

Allele-specific transcriptional activity at rs12190287.

<p>(<b>a</b>) Transcriptional activity of rs12190287-C and G variants were determined in heterozygous primary human coronary artery smooth muscle (HCASM), rat aortic smooth muscle cells (RASM) and HEK, HepG2, and A7r5 cell lines. pLuc-MCS vector containing the putative enhancer region for each rs12190287 variant (C-Luc and G-Luc) was transfected for 24 hours and the ratio of firefly and <i>Renilla</i> luciferase activities were normalized to empty reporter (pLuc). *P<0.01 versus G-Luc for each condition. (<b>b</b>) Dual-luciferase assay of wildtype rs12190287 enhancer or mutants, DelT or T/A (as shown), transfected in A7r5 as described above. *P<0.001 versus G-Luc for each condition. (<b>c</b>) Electrophoretic mobility shift assays (EMSA) showing protein binding to [γ³²P]ATP-labeled rs12190287 C/G probes incubated with nuclear extract (NE) from various cell types, along with 100× excess C, G, or negative control (C, G, or Ctrl comp) unlabeled probe as competitor. Arrows and bar-headed lines represent specific and non-specific shifted complexes, respectively. (<b>d</b>) Dual-luciferase assay of rs12190287 C/G enhancer co-transfected with empty vector (Empty) or constitutively active protein kinase A (PKA) or mitogen-activated protein kinase (MEKK) in A7r5 using consensus CRE and AP-1 reporters as positive controls. *P<0.001 versus C-Luc+Empty or CRE-Luc+Veh or AP1-Luc+Veh where indicated. (<b>e</b>) EMSA of rs12190287 C/G with respective competition showing protein binding using control A7r5 or PMA treated A7r5 NE. Values are mean ± SD from triplicates. Similar results were observed from three independent experiments.</p

Predicted model of AP-1 dependent regulation of <i>TCF21</i> at rs12190287 and rs12524865.

<p>Individuals carrying risk alleles for rs12190287 or rs12524865 at 6q23.2 are expected to have increased <i>TCF21</i> expression upon stimulation of PDGFR-β by PDGF-BB in coronary artery smooth muscle cells, due to increased enrichment of active histone modifications (represented by closed and open diamonds) leading to an open chromatin conformation, allowing binding of an active AP-1 TF complex containing various combinations of c-Jun, JunD, and ATF3. WT1 functions as a transrepressor of this active complex at rs12190287, whereby WT1 may fine-tune the spatial and temporal activation of <i>TCF21</i> expression. Multiple kinases other than mitogen-activated kinase (MEKK) may be involved in the activation and recruitment of AP-1 complexes to these binding sites.</p

AP-1 regulation at rs12524865 and haplotype structure in East Asians.

<p>(<b>a</b>) Linkage disequilibrium (LD) plot from ∼2400 Metabochip genotyped East Asian samples from the TAICHI study (HALST cohort) with imputation from HapMap Phase II and III Han Chinese (CHB), showing eSNPs for <i>TCF21</i> and distinct haplotype blocks containing rs12524865 and rs12190287. White to black squares represent increasing r² values, also shown in blocks. (<b>b</b>) Dual-luciferase assay of rs12524865 C/A enhancer transfected in A7r5 and treated with adenylyl cyclase activator, forskolin (Fsk) or PKC activator, phorbol-12-myristate-13-acetate (PMA) for 4 hours. Relative luciferase activities measured after 24 hours. *P<0.01 versus C-Luc+Veh. (<b>c</b>) Total enrichment of c-Jun, JunB, JunD, and ATF3 at rs12524865 determined by chromatin immunoprecipitation (ChIP) in HCASMC treated with PDGF-BB or vehicle (Control) for 6 hours. *P<0.005 versus Control for each condition. (<b>d</b>) Total enrichment of histone modifications H3K4me1, H3K4me3, H3K27ac, and H3K27me1 at rs12524865 determined by ChIP in HCASMC treated with PDGF-BB. *P<0.001 versus Control for each condition. Values are mean ± SD from triplicates. Similar results were observed from three independent experiments.</p

<i>In silico</i> allele-specific transcription factor binding to rs12524865.

<p>Predicted transcription factor binding site searches were performed using five programs: TRANSFAC, PROMO, MatInspector, TFSearch, and JASPAR. The SNP is shown in boldface within the predicted binding sequence. Core binding sequence shown in capital letters for TRANSFAC and MatInspector generated sequences. The scores represent matrix sequence similarity for each program. A minimum threshold of 85.0 was used for each program, and lowered to 75.0 for allelic comparison if necessary.</p