Cloning of the Novel Human Myeloid-Cell-Speciﬁc C/EBP- e Transcription Factor

Chicken NF-M transcription factor, in cooperation with either c-Myb or v-Myb, is active in the combinatorial activation of myeloid-cell-speciﬁc genes in heterologous cell types, such as embryonic ﬁbroblasts. In humans, similar effects were observed with homologous members of the CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators, especially the human homolog of chicken NF-M, C/EBP- b (NF-IL6). However, the NF-IL6 gene is expressed in a variety of nonmyeloid cell types and is strongly inducible in response to inﬂammatory stimuli, making it an unlikely candidate to have an exclusive role as a combinatorial differentiation switch during myelopoiesis in human cells. By using a reverse transcription-PCR-based ap- proach and a set of primers speciﬁc for the DNA-binding domains of highly homologous members of the C/EBP family of transcriptional regulators, we have cloned a novel human gene encoding a member of the C/EBP gene family, identiﬁed as the human homolog of CRP1, C/EBP- « . A 1.2-kb cDNA encoding full-length human C/EBP- « was cloned from a promyelocyte-late myeloblast-derived l gt11 library. Molecular analysis of the cDNA and genomic clones indicated the presence of two exons encoding a protein with an apparent molecular mass of 32 kDa and a pI of 9.5. Primer extension analysis of C/EBP- « mRNA detected a single major transcription start site approximately 200 bp upstream of the start codon. The putative promoter area is similar to those of several other myeloid-cell-speciﬁc genes in that it contains no TATAAA box but has a number of purine-rich stretches with multiple sites for the factors of the Ets family of transcriptional regulators. Northern blot analyses indicated a highly restricted mRNA expression pattern, with the strongest expression occurring in promyelocyte and late-myeloblast-like cell lines. Western blot and immunoprecipita- tion studies using rabbit anti-C/EBP- « antibodies raised against the N-terminal portion of C/EBP- « (amino acids 1 to 115) showed that C/EBP- « is a 32-kDa nuclear phosphoprotein. The human C/EBP- « protein exhibited strong and speciﬁc binding to double-stranded DNA containing consensus C/EBP sites. Cotransfec- tion of the C/EBP- « sense and antisense expression constructs together with chloramphenicol acetyltransferase reporter vectors containing myeloid-cell-speciﬁc c- mim and human myeloperoxidase promoters suggested a role for C/EBP- « transcription factor in the regulation of a subset of myeloid-cell-speciﬁc genes. Transient tranfection of a promyelocyte cell line (NB4) with a C/EBP- « expression plasmid increased cell growth by sevenfold, while antisense C/EBP- « caused a ﬁvefold decrease in clonal growth of these cells. 72 8 C for 1.5 min. The PCR ampliﬁcation products were analyzed by gel electrophoresis; candidate bands were excised and used as probes in Northern blot hybridizations with total RNA prepared from numerous hematopoietic and nonhematopoietic cells. Fragments showing a tissue-speciﬁc expression pattern were cloned into an Sma I site of the pTZ19R vector (Pharmacia) and sequenced by the dideoxy chain termination method (Sequenase II kit; U.S. Biochemicals). cDNA and genomic DNA library screening and isolation of C/EBP- « clones. A partial cDNA fragment of C/EBP- e obtained by RT-PCR was used to probe an HL60 cell line-derived l gt11 cDNA library (Clontech) under high-stringency conditions as previously described (53) except that a Hybond N-Plus membrane (Amersham) was used. Eleven positive clones were plaque puriﬁed and sequenced, with 10 of them representing C/EBP- e cDNA fragments of variable length and 1 representing human C/EBP- a , which is also expressed in HL60 cells. Similarly, human and murine genes for C/EBP- e were cloned using genomic l DASH (Stratagene) libraries according to the protocol of the provider and mapped by partial digestion with restriction enzymes and hybridization with arm-speciﬁc probes (derived against the T7 and T3 promoters, respectively).