Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

Abstract

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromnatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pul and C-EBPa as enhancer-binding activities. Pul is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBPoi exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBPfI is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP6 expression and functional recruitment of C-EBPJ and C-EBPj8 to the nucleus.Our results implicate Pul and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.While the molecular mechanisms of lineage choice remain largely unknown, the functional balance of transcription factors and the accessibility of cis-acting DNA regulatory elements are likely to be important components in lineage determination (1, 2). The 13-globin and immunoglobulin loci have long served as models for erythroid and lymphoid specific transcriptional regulation, respectively, but considerably less is known about the molecular control of myeloid-specific locus activation.To gain further insight into the early steps involved in commitment and gene activation in myeloid cell differentiation, we have been analyzing the transcriptional regulation of the myeloperoxidase (MPO) gene, whose expression is restricted to the promyelocyte stages of granulocytic differentiation (3, 4). We have recently identified a myeloid lineagespecific enhancer -3.1 kb upstream of the murine MPO gene that resides in a DNase I hypersensitive region (5'HS4) in myeloid cells but lies within inaccessible chromatin in nonmyeloid cell types (5). An important question is how the accessibility and functional status of this upstream enhancer are developmentally linked to both granulocyte lineage commitment and subsequent stable expression of MPO mRNA and protein specifically in promyelocytes.We have now addressed this issue by using multipotential hemopoietic stem cells that retain granulocyte differentiation potential (6, 7) to analyze the chromatin structure and transcription factor status of the MPO gene before commitment to the granulocyte lineage. To ...

CCAAT/enhancer-binding proteins are required for granulopoiesis independent of their induction of the granulocyte colony-stimulating factor receptor

“…Similarly, MPO RNA is rapidly induced by C/EBP␣WT-ER but not in the presence of cycloheximide, a finding that may reflect regulation of the MPO distal enhancer by both C/EBPs and PU.1 and the ability of C/EBP␣-ER to induce PU.1 expression. 25,56 Unexpectedly, K␣ER did not prevent induction of the PU.1 RNA by G-CSF. We previously found that C/EBP␣WT-ER rapidly induces PU.1 RNA in both 32D cl3 and Ba/F3 cells, within 4 hours, in the presence of cycloheximide but not actinomycin D, suggesting that C/EBP␣ directly regulates the PU.1 gene.…”

CCAAT/enhancer binding protein ɛ is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing

“…The myeloperoxidase gene initiates transcription from two promoters separated by approximately 400 bp (35), and is a target of C͞EBP proteins and PU.1 during granulocytic differentiation (36). C͞EBP␣, C͞EBP␤, and C͞EBP␦ are expressed in an overlapping manner during in vitro granulocytic differentiation of A4 multipotential progenitor cells (36). Based on its expression pattern C͞EBP may also interact with the myeloperoxidase promoter regions.…”

“…The transactivation potential of the long form of C͞EBP on G-CSF promoter reporter construct, although low under our experimental conditions, indicates that C͞EBP may also be involved in the regulation of this gene. The myeloperoxidase gene initiates transcription from two promoters separated by approximately 400 bp (35), and is a target of C͞EBP proteins and PU.1 during granulocytic differentiation (36). C͞EBP␣, C͞EBP␤, and C͞EBP␦ are expressed in an overlapping manner during in vitro granulocytic differentiation of A4 multipotential progenitor cells (36).…”

RARα-PLZF oncogene inhibits C/EBPα function in myeloid cells

“…C/EBPα is important for optimal Csf3r expression in vivo (22), although the induction of granulocytic differentiation by C/EBPα could be Csf3r-independent (23). Therefore, we analyzed the expression levels of additional C/EBPα target genes that are involved in myeloid differentiation, i.e., Ltf, Mpo, Ela2 as well as miR-223 (24)(25)(26)(27), all produced at the promyelocyte stage of differentiation. Interestingly, these C/EBPα target genes were strongly reduced in RP7, RP11, and RP-TAP19 clones compared with parental cells or control cells expressing the empty TAP vector ( Fig.…”