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Identification of an ancient Bmp4 mesoderm enhancer located 46 kb from the promoter.

ABSTRACT: Bone morphogenetic protein 4 (Bmp4) is a multi-functional, developmentally regulated gene that is essential for mouse development, as most Bmp4-null mouse embryos die at the onset of gastrulation and fail to develop mesoderm. Little is known about the transcriptional regulation of Bmp4. To identify potential long-range cis-regulatory elements that direct its complex spatiotemporal expression patterns, we surveyed the mouse Bmp4 locus using two overlapping bacterial artificial chromosome (BAC) reporter transgenes. Our findings indicate that tissue-specific cis-regulatory elements reside greater than 28 kb 5' or 3' to the mouse Bmp4 transcription unit. In addition, comparative analyses identified three noncoding evolutionarily conserved regions (ECRs), spaced around the gene and conserved from mammals to fish, that are maintained in a syntenic group across vertebrates. Deletion of one of these conserved sequences (ECR2) from a BAC transgene revealed a tissue-specific requirement for ECR2 in driving Bmp4 expression in extraembryonic and embryonic mesoderm. Furthermore, a 467 bp mouse sequence containing ECR2 reproducibly directed lacZ minigene expression in mesoderm. Taken together, this shows that an ancient, mesoderm-specific cis-regulatory element resides nearly 50 kb 5' to mouse Bmp4.

SUBMITTER: Chandler KJ

PROVIDER: S-EPMC2846791 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Identification of an ancient Bmp4 mesoderm enhancer located 46 kb from the promoter.

Chandler Kelly J KJ Chandler Ronald L RL Mortlock Douglas P DP

Developmental biology 20090103 2

Bone morphogenetic protein 4 (Bmp4) is a multi-functional, developmentally regulated gene that is essential for mouse development, as most Bmp4-null mouse embryos die at the onset of gastrulation and fail to develop mesoderm. Little is known about the transcriptional regulation of Bmp4. To identify potential long-range cis-regulatory elements that direct its complex spatiotemporal expression patterns, we surveyed the mouse Bmp4 locus using two overlapping bacterial artificial chromosome (BAC) re ...[more]

PMID: 19159624

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Project description:C/EBPα plays a key role in specifying myeloid lineage development. HoxA9 is expressed in myeloid progenitors, with its level diminishing during myeloid maturation, and HOXA9 is over-expressed in a majority of acute myeloid leukemia cases, including those expressing NUP98-HOXD13. The objective of this study was to determine whether HoxA9 directly represses Cebpa gene expression. We find 4-fold increased HoxA9 and 5-fold reduced Cebpa in marrow common myeloid and LSK progenitors from Vav-NUP98-HOXD13 transgenic mice. Conversely, HoxA9 decreases 5-fold while Cebpa increases during granulocytic differentiation of 32Dcl3 myeloid cells. Activation of exogenous HoxA9-ER in 32Dcl3 cells reduces Cebpa mRNA even in the presence of cycloheximide, suggesting direct repression. Cebpa transcription in murine myeloid cells is regulated by a hematopoietic-specific +37 kb enhancer and by a more widely active +8 kb enhancer. ChIP-Seq analysis of primary myeloid progenitor cells expressing exogenous HoxA9 or HoxA9-ER demonstrates that HoxA9 localizes to both the +8 kb and +37 kb Cebpa enhancers. Gel shift analysis demonstrates HoxA9 binding to three consensus sites in the +8 kb enhancer, but no affinity for the single near-consensus site present in the +37 kb enhancer. Activity of a Cebpa +8 kb enhancer/promoter-luciferase reporter in 32Dcl3 or MOLM14 myeloid cells is increased ~2-fold by mutation of its three HOXA9-binding sites, suggesting that endogenous HoxA9 represses +8 kb Cebpa enhancer activity. In contrast, mutation of five C/EBPα-binding sites in the +8 kb enhancer reduces activity 3-fold. Finally, expression of a +37 kb enhancer/promoter-hCD4 transgene reporter is reduced ~2-fold in marrow common myeloid progenitors when the Vav-NUP98-HOXD13 transgene is introduced. Overall, these data support the conclusion that HoxA9 represses Cebpa expression, at least in part via inhibition of its +8 kb enhancer, potentially allowing normal myeloid progenitors to maintain immaturity and contributing to the pathogenesis of acute myeloid leukemia associated with increased HOXA9.

Dataset Information

Identification of an ancient Bmp4 mesoderm enhancer located 46 kb from the promoter.

Publications

Identification of an ancient Bmp4 mesoderm enhancer located 46 kb from the promoter.

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