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The +37 kb Cebpa Enhancer Is Critical for Cebpa Myeloid Gene Expression and Contains Functional Sites that Bind SCL, GATA2, C/EBPα, PU.1, and Additional Ets Factors.

Cooper S, Guo H, Friedman AD - PLoS ONE (2015)

Bottom Line: Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells.These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression.In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
The murine Cebpa gene contains an evolutionarily conserved 453 bp enhancer located at +37 kb that, together with its promoter, directs expression to myeloid progenitors and to long-term hematopoietic stem cells in transgenic mice. In human acute myeloid leukemia cases, the enhancer lacks point mutations but binds the RUNX1-ETO oncoprotein. The enhancer contains the H3K4me1 and H3K27Ac histone modifications, denoting an active enhancer, at progressively increasing levels as long-term hematopoietic stem cells transition to granulocyte-monocyte progenitors. We previously identified four enhancer sites that bind RUNX1 and demonstrated that their integrity is required for maximal enhancer activity in 32Dcl3 myeloid cells. The +37 kb Cebpa enhancer also contains C/EBP, Ets factor, Myb, GATA, and E-box consensus sites conserved in the human +42 kb CEBPA enhancer. Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells. In 293T gel shift assays, exogenous C/EBPα binds both C/EBP sites, c-Myb binds the Myb site, PU.1 binds the second Ets site, PU.1, Fli-1, ERG, and Ets1 bind the sixth Ets site, GATA2 binds both GATA sites, and SCL binds the second E-box. Endogenous hematopoietic RUNX1, PU.1, Fli-1, ERG, C/EBPα, GATA2, and SCL were previously shown to bind the enhancer, and we find that endogenous PU.1 binds the second Ets site in 32Dcl3 cells. Using CRISPR/Cas9, we developed 32Dcl3 lines in which the wild-type enhancer alleles are replaced with a variant mutant in the seven Ets sites. These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression. In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation.

No MeSH data available.


Related in: MedlinePlus

PU.1, Fli-1, ERG, and Ets1 bind cognate cis elements in the +37 kb Cebpa enhancer.A) 293T cell nuclear extracts expressing PU.1, Fli-1, ERG, or Ets1, or a control extract, were assessed for binding to radio-labeled ETSa, ETSb, ETSc, ETSd, or ETSe probes, containing the first to fifth Ets sites. B) Binding of these factors to the ETSf or ETSg probes, containing the sixth and seventh Ets sites, was assessed similarly. C) 293T nuclear extract expressing PU.1 was assessed for binding to the ETSb probe in the presence of no competitor or 5- or 25-fold excess WT or mutant competitor (left panel), and nuclear extract expressing Fli-1 was assessed similarly for binding to the ETSf probe (right panel).
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pone.0126385.g004: PU.1, Fli-1, ERG, and Ets1 bind cognate cis elements in the +37 kb Cebpa enhancer.A) 293T cell nuclear extracts expressing PU.1, Fli-1, ERG, or Ets1, or a control extract, were assessed for binding to radio-labeled ETSa, ETSb, ETSc, ETSd, or ETSe probes, containing the first to fifth Ets sites. B) Binding of these factors to the ETSf or ETSg probes, containing the sixth and seventh Ets sites, was assessed similarly. C) 293T nuclear extract expressing PU.1 was assessed for binding to the ETSb probe in the presence of no competitor or 5- or 25-fold excess WT or mutant competitor (left panel), and nuclear extract expressing Fli-1 was assessed similarly for binding to the ETSf probe (right panel).

Mentions: The Ets factors PU.1, Fli-1, and ERG were previously shown to bind the Cebpa +37 kb enhancer in ChIP-Seq assays [13], and RUNX1, which also binds the enhancer, cooperates with Ets1 in gene activation [22]. We therefore generated 293T extracts expressing PU.1, Fli-1, ERG, or Ets1 and compared their ability to bind radio-labeled oligonucleotides corresponding to the seven Ets sites. Strong binding of PU.1 to the ETSb probe was detected, no specific binding was seen to the ETSa, ETSc, ETSd, ETSe, or ETSg probes, and PU.1, Fli-1, ERG, and Ets1 each bound the ETSf probe (Fig 4A and 4B). Interaction of PU.1 with ETSb or Fli-1 with ETSf was effectively competed by excess unlabeled WT ETSb or ETSf DNAs but not by variants carrying a 2 bp mutation in their Ets consensus sites, indicating specific binding at these sites (Fig 4C).


The +37 kb Cebpa Enhancer Is Critical for Cebpa Myeloid Gene Expression and Contains Functional Sites that Bind SCL, GATA2, C/EBPα, PU.1, and Additional Ets Factors.

Cooper S, Guo H, Friedman AD - PLoS ONE (2015)

PU.1, Fli-1, ERG, and Ets1 bind cognate cis elements in the +37 kb Cebpa enhancer.A) 293T cell nuclear extracts expressing PU.1, Fli-1, ERG, or Ets1, or a control extract, were assessed for binding to radio-labeled ETSa, ETSb, ETSc, ETSd, or ETSe probes, containing the first to fifth Ets sites. B) Binding of these factors to the ETSf or ETSg probes, containing the sixth and seventh Ets sites, was assessed similarly. C) 293T nuclear extract expressing PU.1 was assessed for binding to the ETSb probe in the presence of no competitor or 5- or 25-fold excess WT or mutant competitor (left panel), and nuclear extract expressing Fli-1 was assessed similarly for binding to the ETSf probe (right panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4418761&req=5

pone.0126385.g004: PU.1, Fli-1, ERG, and Ets1 bind cognate cis elements in the +37 kb Cebpa enhancer.A) 293T cell nuclear extracts expressing PU.1, Fli-1, ERG, or Ets1, or a control extract, were assessed for binding to radio-labeled ETSa, ETSb, ETSc, ETSd, or ETSe probes, containing the first to fifth Ets sites. B) Binding of these factors to the ETSf or ETSg probes, containing the sixth and seventh Ets sites, was assessed similarly. C) 293T nuclear extract expressing PU.1 was assessed for binding to the ETSb probe in the presence of no competitor or 5- or 25-fold excess WT or mutant competitor (left panel), and nuclear extract expressing Fli-1 was assessed similarly for binding to the ETSf probe (right panel).
Mentions: The Ets factors PU.1, Fli-1, and ERG were previously shown to bind the Cebpa +37 kb enhancer in ChIP-Seq assays [13], and RUNX1, which also binds the enhancer, cooperates with Ets1 in gene activation [22]. We therefore generated 293T extracts expressing PU.1, Fli-1, ERG, or Ets1 and compared their ability to bind radio-labeled oligonucleotides corresponding to the seven Ets sites. Strong binding of PU.1 to the ETSb probe was detected, no specific binding was seen to the ETSa, ETSc, ETSd, ETSe, or ETSg probes, and PU.1, Fli-1, ERG, and Ets1 each bound the ETSf probe (Fig 4A and 4B). Interaction of PU.1 with ETSb or Fli-1 with ETSf was effectively competed by excess unlabeled WT ETSb or ETSf DNAs but not by variants carrying a 2 bp mutation in their Ets consensus sites, indicating specific binding at these sites (Fig 4C).

Bottom Line: Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells.These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression.In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
The murine Cebpa gene contains an evolutionarily conserved 453 bp enhancer located at +37 kb that, together with its promoter, directs expression to myeloid progenitors and to long-term hematopoietic stem cells in transgenic mice. In human acute myeloid leukemia cases, the enhancer lacks point mutations but binds the RUNX1-ETO oncoprotein. The enhancer contains the H3K4me1 and H3K27Ac histone modifications, denoting an active enhancer, at progressively increasing levels as long-term hematopoietic stem cells transition to granulocyte-monocyte progenitors. We previously identified four enhancer sites that bind RUNX1 and demonstrated that their integrity is required for maximal enhancer activity in 32Dcl3 myeloid cells. The +37 kb Cebpa enhancer also contains C/EBP, Ets factor, Myb, GATA, and E-box consensus sites conserved in the human +42 kb CEBPA enhancer. Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells. In 293T gel shift assays, exogenous C/EBPα binds both C/EBP sites, c-Myb binds the Myb site, PU.1 binds the second Ets site, PU.1, Fli-1, ERG, and Ets1 bind the sixth Ets site, GATA2 binds both GATA sites, and SCL binds the second E-box. Endogenous hematopoietic RUNX1, PU.1, Fli-1, ERG, C/EBPα, GATA2, and SCL were previously shown to bind the enhancer, and we find that endogenous PU.1 binds the second Ets site in 32Dcl3 cells. Using CRISPR/Cas9, we developed 32Dcl3 lines in which the wild-type enhancer alleles are replaced with a variant mutant in the seven Ets sites. These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression. In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation.

No MeSH data available.


Related in: MedlinePlus