Genome-wide analyses reveal properties of redundant and specific promoter occupancy within the<i>ETS</i>gene family

Hollenhorst, Peter C.; Shah, Atul A.; Hopkins, Chris; Graves, Barbara J.

doi:10.1101/gad.1561707

Cited by 267 publications

(343 citation statements)

References 56 publications

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“…Our data revealed that ELF1, but not GABPα or FLI1 displayed strong enrichment in the MEIS1 promoter region both from EMSA and ChIP experiments in K562 cells. This was consistent with a recent genome wide ChIP study performed in a human T leukemia cell line, Jurkat, where ELF1 but not the other two ETS family members tested, GABPα and ETS1, was enriched at the MEIS1 promoter region (35). This strong association of ELF1 to the MEIS1 promoter region was also confirmed in human primary samples enriched with the hematopoietic stem/progenitor cells where MEIS1 was highly expressed.…”

Section: Discussionsupporting

confidence: 91%

Identification of E74-like factor 1 (ELF1) as a transcriptional regulator of the Hox cofactor MEIS1

Xiang

Argiropoulos

et al. 2010

Experimental Hematology

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Objective-MEIS1 is a Hox cofactor known for its role in development and strongly linked to normal and leukemic hematopoiesis. Although previous studies have focused on identifying protein partners of MEIS1 and its transcriptionaly regulated targets, little is known about the upstream transcriptional regulators of this tightly regulated gene. Understanding the regulation of MEIS1 is important toward the understanding of normal hematopoiesis and leukemogenesis.Materials and Methods-Here we describe our studies focusing on the evolutionary conserved putative MEIS1 promoter region. Phylogenetic sequence analysis followed and reporter assays in MEIS1 expressing (K562) and non-expressing (HL60) leukemic cell line models were used to identify key regulatory regions and potential transcription factor binding sites within the candidate promoter region followed by functional and expression studies of one identified regulator in both cell lines and primary human cord blood and leukemia samples. Results-The chromatin status of MEIS1 promoter region is associated with MEIS1 expression.Truncation and mutation studies coupled with reporter assays revealed that a conserved ETS family member binding site located 289bp upstream of the annotated human MEIS1 transcription start site (AHTSS) is required for promoter activity. Of the three ETS family members tested, only ELF1 was enriched on the MEIS1 promoter as assessed by both electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments in K562. This finding was confirmed in MEIS1 expressing primary human samples. Moreover, siRNA -mediated knockdown of ELF1 in K562 cells was associated with a decreased MEIS1 expression.Conclusions-We conclude that the ETS transcription factor ELF1 is an important positive regulator of MEIS1 expression.

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Section: Discussionsupporting

confidence: 91%

Identification of E74-like factor 1 (ELF1) as a transcriptional regulator of the Hox cofactor MEIS1

Xiang

Argiropoulos

et al. 2010

Experimental Hematology

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“…24 For example, whole genome studies with chromatin immunoprecipitation have allowed a better definition of consensus binding sites that have a much stronger predictive value. 25,26 Often these consensus sequences are much larger than the previously described or include influence from neighbouring nucleotides. Unfortunately, there are only a few of these studies and they do not include RUNX1.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the functional relevance of a putative regulatory SNP of PDCD1, PD1.3, associated with systemic lupus erythematosus

et al. 2008

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This study aimed to test the functional effects of the PD1.3 single nucleotide polymorphism (SNP) (rs11568821), which were proposed based on its association to systemic lupus erythematosus (SLE) susceptibility and in electrophoretic mobility shift assays (EMSA) results. We analysed transcriptional effects of the PD1.3 locus by enhancer reporter assays. Results were against the hypothesis that the PD1.3 locus acts as enhancer in transcriptional regulation of PDCD1. In addition, they excluded a differential effect of the PD1.3 alleles. EMSA results confirmed that oligonucleotides with the PD1.3 G allele bind RUNX1 but not those with the A allele. However, binding to PD1.3 G oligonucleotides was much lower than binding to positive control oligonucleotides. Criss-cross experiments showed that this was due to flanking nucleotides in the PD1.3 sequence that negatively affect RUNX1 binding. These results cast doubts on the functional relevance of the PD1.3 SNP and, together with the lack of association in several studies, put into question its role as an SLE susceptibility factor. Investigation of other PDCD1 polymorphisms is needed to uncover the possible effect of this gene on SLE susceptibility.

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“…(1) The ChIP assay often yields low signals in comparison to negative controls, which can lead to inconclusive results; (2) it is difficult to determine the precise binding site for a factor because of the limited resolution of the assay; and (3) ChIP is not a functional assay and cannot by itself demonstrate the functional significance of a protein or modified histone found to be located at a genomic region of interest. This final limitation is especially problematic because many studies have now provided strong evidence that proteins associate with genomic sites at which they have no obvious function (Walter et al 1994;Li and Johnston 2001;Martone et al 2003;Phuc Le et al 2005;Beima et al 2006;Hollenhorst et al 2007;Dong et al 2008;Li et al 2008). It has been difficult to design experiments to rigorously test the possibility that specific histone modifications, as opposed to transcription factor-DNA interactions, are not functionally important at specific genomic locations at which they are found.…”

Section: Limitations Of the Chip Methodsmentioning

confidence: 99%

Chromatin Immunoprecipitation (ChIP)

Carey¹,

Peterson²,

Smale³

2009

Cold Spring Harb Protoc

231

174

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INTRODUCTIONChromatin immunoprecipitation (ChIP) is an invaluable method for studying interactions between specific proteins or modified forms of proteins and a genomic DNA region. ChIP can be used to determine whether a transcription factor interacts with a candidate target gene and is used with equal frequency to monitor the presence of histones with post-translational modifications at specific genomic locations. In early ChIP studies, UV light from a transilluminator was used to cross-link proteins to DNA irreversibly. The cross-linked chromatin was then either sonicated or cleaved with restriction enzymes to generate smaller DNA fragments, followed by immunoprecipitation with the desired antibodies. The precipitated protein-DNA adducts were then purified, treated with a protease, and analyzed by dot blot or Southern blot using a radiolabeled probe derived from the cloned DNA fragment of interest. The use of formaldehyde as a reversible protein-DNA and protein-protein cross-linking agent for ChIP and the use of polymerase chain reaction (PCR) to detect precipitated DNA fragments were later added as components of the modern ChIP procedure. The protocol below represents a standard ChIP procedure for use in mammalian cells. Cross-linking is performed by adding formaldehyde to growing cells, and chromatin is prepared, sheared by sonication, and precleared to reduce nonspecific immunoprecipitation. Immunoprecipitation is performed with a specific antibody. After elution of the protein-DNA complexes from protein A- or protein G-agarose resin, the samples are heated to reverse the covalent cross-links. The DNA fragments are purified and analyzed by PCR or real-time PCR.

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Genome-wide analyses reveal properties of redundant and specific promoter occupancy within theETSgene family

Cited by 267 publications

References 56 publications

Identification of E74-like factor 1 (ELF1) as a transcriptional regulator of the Hox cofactor MEIS1

Identification of E74-like factor 1 (ELF1) as a transcriptional regulator of the Hox cofactor MEIS1

Analysis of the functional relevance of a putative regulatory SNP of PDCD1, PD1.3, associated with systemic lupus erythematosus

Chromatin Immunoprecipitation (ChIP)

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