Analysis of DNase I-Hypersensitive Sites in the Chromatin of the Chicken C/EBPbeta Gene Reveals Multiplecis-Regulatory Elements

Abstract

CCAAT box/enhancer binding protein beta (C/EBPbeta), a member of the basic region-leucine zipper (bzip) class of transcription factors, plays important roles during differentiation of certain cell types, such as liver cells, fat cells and myelomonocytic cells. C/EBPbeta is highly expressed in these cell types, and activates specific genes during their differentiation. In the hematopoietic system, C/EBPbeta expression is restricted to the myelomonocytic lineage. To investigate the molecular basis of the cell-type specific expression of the C/EBPbeta gene in hematopoietic cells we have cloned the chicken C/EBPbeta gene and mapped DNase I hypersensitive sites (DHS) in the vicinity of the gene, using myelomonocytic as well as other cell types. We show that there are multiple nuclease-sensitive sites, most of which are cell-type specific, suggesting that they might act as cell-type specific cis-regulatory DNA elements. To study the possible function of these elements we have constructed reporter genes containing these sequences and analyzed their activity in different cell types. Our results show that several of the nuclease-sensitive regions act as cis-acting stimulatory elements in myelomonocytic but not in other cells. Taken together, our data suggest that the expression of the C/EBPbeta gene in myelomonocytic cells is controlled by multiple cell-type specific cis-acting sequences located both upstream and downstream of the gene.

Regulation of C/EBPβ mRNA expression and C/EBPβ promoter activity by protein kinases A and C in a myelomonocytic cell line (HD11)

“…For this purpose, a PCR-amplifi ed DNA fragment containing the 5´ fl anking region from +24 to -704 of the C/EBPb gene was linked to the CAT reporter gene. This region contains no further hypersensitive site except that at the promoter [27]. For control, the CAT gene driven by the human Corticotropin-releasing-hormone (CRH) promoter containing a perfect cAMP-responsive element (CRE) was used.…”

Identification of a Myb-responsive enhancer of the chicken C/EBPβ gene

“…Previously, we have mapped a number of DHSs in the chicken C/EBPb gene region (Kintscher et al, 2003), most of which were specific for myelomonocytic cells, which express the gene at a high level. One of these sites, located downstream of the gene, appeared to be more prominent in the v-mybtransformed myeloblast cell line BM2 than in a myelomonocytic cell line (HD11) not expressing v-myb or c-myb (Kintscher et al, 2003). Since the C/EBPb gene is a Myb target gene, we became interested to study this site in more detail.…”

“…The luciferase reporter gene pGL3-tk-Luc containing tk promoter sequences from À81 to þ 53 bp has been described (Kintscher et al, 2003). Plasmid pGL3-tk S6004 was obtained by cloning a 0.6 kb SacI fragment from downstream of the C/EBPb gene and containing the þ 3 kb DHS region into the SacI site of pGL3-tk-Luc.…”

“…The b-galactosidase reporter gene pCMVb was obtained from Clontech. DNA transfection was performed by calcium phosphate co-precipitation or by electroporation, as described previously (Kintscher et al, 2003). Cells were harvested 24 h after transfection and analysed for luciferase and b-galactosidase activity as previously described (Burk et al, 1993).…”

“…In an attempt to identify additional cis-acting sequences that control the expression of the gene in myelomonocytic cells, we have recently mapped DNase I hypersensitive sites (DHSs) in the chromatin surrounding the C/EBPb gene (Kintscher et al, 2003). We have identified a number of such sites, most of which were specific for myelomonocytic cells.…”