Multi-species, multi-transcription factor binding highlights conserved control of tissue-specific biological pathways

Ballester, Benoît; Medina-Rivera, Alejandra; Schmidt, Dominic; Gonzàlez-Porta, Mar; Carlucci, Matthew; Rothenberg, Marc E.; Chessman, Kyle; Faure, Aline; Funnell, Alister P. W.; Gonçalves, Ângela; Kutter, Claudia; Lukk, Margus; Menon, Suraj; McLaren, William; Stefflova, Klara; Watt, Stephen; Weirauch, Matthew T.; Crossley, Merlin; Marioni, John C.; Odom, Duncan T.; Flicek, Paul; Wilson, Michael D.

doi:10.7554/elife.02626

Cited by 94 publications

(118 citation statements)

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“…We first enumerated all CREs occupied by GATA1 in primary human EBs that are proximal to the ∼20 known pathogenic MED genes. Within these CREs, we identified 176 core GATA sites, and investigated the evolutionary conservation of these four nucleotides as a proxy for functionality (32,44,47,48). Surprisingly, although we observed only slight conservation for the GATA site within all ∼20,000 GATA1 chromatin occupancy sites across the genome, GATA sites within MED CREs were substantially conserved (Fig.…”

Section: Isogenic Cellular Models Phenocopy Cell-intrinsic Effects Ofmentioning

confidence: 95%

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Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

Wakabayashi

Ulirsch

Ludwig

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptional cis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.GATA1 | cis-regulatory elements | noncoding mutations | Mendelian erythroid disorders W hole-exome sequencing (WES) and targeted sequencing approaches have greatly accelerated our ability to identify causal genetic lesions in both previously implicated and novel genes underlying monogenic disorders (1, 2). In hematology, WES has been extremely useful for identifying unknown genetic etiologies for various disorders, such as those affecting red blood cell (RBC) production, including Diamond-Blackfan anemia and congenital dyserythropoietic anemia (3-5), disorders of RBC structure and function (6, 7), and disorders affecting other aspects of hematologic function (2, 8). Despite this considerable success, however, more than 50% of cases of presumed monogenic diseases are refractory to current WES approaches (9). Although resolving these remaining cases will benefit from improvements in exome capture (10), read alignment (11), and variant annotation methodologies (11), the importance of genetic variation occurring within regulatory elements (REs) outside of the traditionally investigated coding sequences in hematologic and other diseases is being increasingly appreciated (12).Whole-genome sequencing (WGS) approaches are becoming progressively more available and affordable, but separating pathogenic genetic variation from benign or unrelated mutations remains especially difficult outside of protein-coding gen...

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Section: Isogenic Cellular Models Phenocopy Cell-intrinsic Effects Ofmentioning

confidence: 95%

“…3D) (40). (9,44,47,48). This suggests the possibility that an important minority of causal genetic lesions may instead be located within NC regulatory regions.…”

Section: Isogenic Cellular Models Phenocopy Cell-intrinsic Effects Ofmentioning

confidence: 99%

Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

Wakabayashi

Ulirsch

Ludwig

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Among these master regulators, HNF4A is essential for the differentiation of human hepatic progenitor cells (DeLaForest et al 2011). Likewise, CEBPA is required for the liver cell specification and gene function, and the associated TFBSs are highly conserved across mammals (Ballester et al 2014). Both CEBPA and HNF4A have conserved cis-regulatory activity and a large number of shared TF binding events across distant vertebrates (Schmidt et al 2010).…”

Section: Specific Transcription Factor Motifs Are Associated With Regmentioning

confidence: 99%

Transposable elements are the primary source of novelty in primate gene regulation

et al. 2017

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Gene regulation shapes the evolution of phenotypic diversity. We investigated the evolution of liver promoters and enhancers in six primate species using ChIP-seq (H3K27ac and H3K4me1) to profile cis-regulatory elements (CREs) and using RNAseq to characterize gene expression in the same individuals. To quantify regulatory divergence, we compared CRE activity across species by testing differential ChIP-seq read depths directly measured for orthologous sequences. We show that the primate regulatory landscape is largely conserved across the lineage, with 63% of the tested human liver CREs showing similar activity across species. Conserved CRE function is associated with sequence conservation, proximity to coding genes, cell-type specificity, and transcription factor binding. Newly evolved CREs are enriched in immune response and neurodevelopmental functions. We further demonstrate that conserved CREs bind master regulators, suggesting that while CREs contribute to species adaptation to the environment, core functions remain intact. Newly evolved CREs are enriched in young transposable elements (TEs), including Long-Terminal-Repeats (LTRs) and SINE-VNTR-Alus (SVAs), that significantly affect gene expression. Conversely, only 16% of conserved CREs overlap TEs. We tested the cis-regulatory activity of 69 TE subfamilies by luciferase reporter assays, spanning all major TE classes, and showed that 95.6% of tested TEs can function as either transcriptional activators or repressors. In conclusion, we demonstrated the critical role of TEs in primate gene regulation and illustrated potential mechanisms underlying evolutionary divergence among the primate species through the noncoding genome.

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“…We previously found that conserved orthologous transcription factor binding can reveal functional enhancers (Ballester et al, 2014). To identify evolutionarily conserved, epigenetically modified enhancers for further functional analyses, we performed ERG ChIP-seq experiments in both human (HUVECs) and bovine (BAECs) ECs cultured in complete media (i.e.…”

Section: Erg Regulates a Network Of Constitutive And Vegf-inducible Gmentioning

confidence: 99%

“…Approximately 20 million cells were used for the ERG and ∼3 million cells for the H3K27ac ChIPs. ChIP experiments were conducted as previously described (Ballester et al, 2014). Antibodies used for ChIP were mouse anti-H3K27ac (Millipore, 05-1334 monoclonal) and rabbit anti-ERG 1/2/3 (Santa Cruz Biotechnology, sc353 polyclonal).…”

Section: Chip-seq Experiments and Analysismentioning

confidence: 99%

Dynamic regulation of VEGF-inducible genes by an ERK-ERG-p300 transcriptional network

et al. 2017

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The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGFmediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis.

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Multi-species, multi-transcription factor binding highlights conserved control of tissue-specific biological pathways

Cited by 94 publications

References 90 publications

Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

Transposable elements are the primary source of novelty in primate gene regulation

Dynamic regulation of VEGF-inducible genes by an ERK-ERG-p300 transcriptional network

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