Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites

Follows, George; Dhami, Pawan; Göttgens, Berthold; Bruce, Alexander W.; Campbell, Peter J.; Dillon, Shane C.; Smith, Adam; Koch, Christof; Donaldson, Ian J.; Scott, Mike; Dunham, Ian; Janes, Mary E.; Vetrie, David; Green, Anthony

doi:10.1101/gr.5373606

Cited by 34 publications

(44 citation statements)

References 47 publications

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“…15 Four hundred fifty nanograms of ChIP DNA, obtained for each antibody from 2 independent IP reactions, and 450 ng of input DNA were labeled with Cy3-2Ј-deoxycytosine 5Ј-triphosphate (dCTP; Amersham, Buckinghamshire, United Kingdom) and Cy5-dCTP (Amersham), respectively, with Bioprime Labeling system (Invitrogen, Carlsbad, CA).…”

Section: Chip-chip Analysismentioning

confidence: 99%

Tissue-specific histone modification and transcription factor binding in α globin gene expression

Gobbi

Anguita

Hughes

et al. 2007

Blood

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To address the mechanism by which the human globin genes are activated during erythropoiesis, we have used a tiled microarray to analyze the pattern of transcription factor binding and associated histone modifications across the telomeric region of human chromosome 16 in primary erythroid and nonerythroid cells. This 220-kb region includes the ␣ globin genes and 9 widely expressed genes flanking the ␣ globin locus. This unbiased, comprehensive analysis of transcription factor binding and histone modifications (acetylation and methylation) described here not only identified all known cis-acting regulatory elements in the human ␣ globin cluster but also demonstrated that there are no additional erythroid-specific regulatory elements in the 220-kb region tested. In addition, the pattern of histone modification distinguished promoter elements from potential enhancer elements across this region. Finally, comparison of the human and mouse orthologous regions in a unique mouse model, with both regions coexpressed in the same animal, showed significant differences that may explain how these 2 clusters are regulated differently in vivo. IntroductionGlobin gene expression plays a central role in normal erythropoiesis, and abnormalities of globin synthesis underlie the clinically important thalassaemia syndromes. A long-term goal in the treatment of thalassaemia has been to identify ways in which to manipulate gene expression to redress the imbalance in ␣ and ␤ globin chain synthesis. Progress in this area would be enhanced by understanding in detail how these genes are normally regulated. To address this, it is important to identify all the key cis-acting sequences controlling ␣ and ␤ globin expression, the transcription factors that bind them, and the accompanying modifications of chromatin at different stages of differentiation.To identify and characterize the cis-acting elements controlling ␣ globin expression we have previously used a variety of approaches, including comparative genomics, 1,2 a wide range of chromatin analyses, 3,4 transgenic experiments, 5,6 and the analysis of expression in interspecific hybrids. [7][8][9] Upstream of the 4 ␣-like globin promoters (, ␣D, ␣2, and ␣1) lie 4 multispecies conserved sequences (MCSs) associated with erythroid-specific DNase1 hypersensitive sites (HS), referred to, in human, as HS-48, HS-40, HS-33, and HS-10 ( Figure 1A). The globin genes, the upstream MCSs, and several widely expressed flanking genes all lie within a region of conserved synteny spanning 130 kb of the telomeric region of human chromosome 16 (16p13.3) ( Figure 1A). Functional analyses suggest that HS-40 is the most important of the MCSs. 9 Recently, to develop a relevant experimental system for studying human ␣ globin expression and to evaluate the roles of individual elements, we established a humanized mouse model in which the 117-kb region containing the human ␣-like globin genes and all of their known regulatory elements (the ␣ globin regulatory domain) completely replaces the corresponding, orthologou...

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Section: Chip-chip Analysismentioning

confidence: 99%

Tissue-specific histone modification and transcription factor binding in α globin gene expression

Gobbi

Anguita

Hughes

et al. 2007

Blood

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“…Preparation of DNase I-treated template, chromatin immunoprecipitation, and real-time PCR analysis DNase I-digested material was prepared from the 416B cell line as previously described (Follows et al 2006. ChIP assays were performed as previously described .…”

Section: Methodsmentioning

confidence: 99%

A novel mode of enhancer evolution: The Tal1 stem cell enhancer recruited a MIR element to specifically boost its activity

Smith

Sánchez²,

Follows³

et al. 2008

Genome Res.

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Altered cis-regulation is thought to underpin much of metazoan evolution, yet the underlying mechanisms remain largely obscure. The stem cell leukemia TAL1 (also known as SCL) transcription factor is essential for the normal development of blood stem cells and we have previously shown that the Tal1 +19 enhancer directs expression to hematopoietic stem cells, hematopoietic progenitors, and to endothelium. Here we demonstrate that an adjacent region 1 kb upstream (+18 element) is in an open chromatin configuration and carries active histone marks but does not function as an enhancer in transgenic mice. Instead, it boosts activity of the +19 enhancer both in stable transfection assays and during differentiation of embryonic stem (ES) cells carrying single-copy reporter constructs targeted to the Hprt locus. The +18 element contains a mammalian interspersed repeat (MIR) which is essential for the +18 function and which was transposed to the Tal1 locus ∼160 million years ago at the time of the mammalian/marsupial branchpoint. Our data demonstrate a previously unrecognized mechanism whereby enhancer activity is modulated by a transposon exerting a "booster" function which would go undetected by conventional transgenic approaches.

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“…For ChIP-chip assays, histone 3 lysine 9 acetylation (H3K9ac) precipitated material was labeled with Cy3 and Cy5 fluorochromes, hybridized, and analyzed as described. 22 …”

Section: Chip-quantitative Pcr and Chip-chip Assaysmentioning

confidence: 99%

Two distinct auto-regulatory loops operate at the PU.1 locus in B cells and myeloid cells

Leddin

Perrod

Hoogenkamp

et al. 2011

Blood

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130

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The transcription factor PU.1 occupies a central role in controlling myeloid and early B-cell development, and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis element whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the upstream regulatory cis element alone is insufficient to confer physiologic PU.1 expression in mice but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays, and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross talk between different cis elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell type-specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements. (Blood. 2011;117(10):2827-2838)

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Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites

Cited by 34 publications

References 47 publications

Tissue-specific histone modification and transcription factor binding in α globin gene expression

Tissue-specific histone modification and transcription factor binding in α globin gene expression

A novel mode of enhancer evolution: The Tal1 stem cell enhancer recruited a MIR element to specifically boost its activity

Two distinct auto-regulatory loops operate at the PU.1 locus in B cells and myeloid cells

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