We have recently cloned a novel human gene (C/EBPepsilon) which is a member of large family of transcriptional factors known as the CCAAT enhancer-binding proteins (C/EBP). C/EBPepsilon is uniquely expressed in granulocytic precursor cells, and this expression is markedly enhanced by retinoids. C/EBP protein exhibits strong and specific binding to double-stranded DNA containing consensus C/EBP sites. Cotransfection of either the sense or anti-sense C/EBPepsilon expression constructs together with CAT-reporter vectors containing myeloid- specific c-mim or human myeloperoxidase promoters suggest a role for C/EBPepsilon in the regulation of a subset of myeloid-specific genes. Likewise, over- and under-expression of C/EBPepsilon in a myeloid line (NB4) increased and decreased the clonal growth of the cells, respectively.
Specific Aim 1 will study the modulation of expression of C/EBPepsilon in myeloid cells including: A.) Which myeloid cells express C/EBPepsilon; B.) What is the mechanism by which retinoids induce expression of C/EBPepsilon; C.) How is expression of C/EBPepsilon regulated.
In Specific Aim 2, we will determine partners of C/EBPepsilon and how these partners influence the biology of C/EBPepsilon. Using the yeast two hybrid selection system, we have identified CREB2 as one of the frequent partners of C/EBPepsilon. Binding of CREB2 to C/EBPepsilon will be confirmed by several additional in vitro and in vivo techniques and its influence on C/EBPepsilon function will be explored. Other partners of C/EBPepsilon will also be identified and studied.
Specific Aim 3 will elucidate biological function of C/EBPepsilon in vitro and in mice. Using both transient and stable sense (S)-and antisense (AS)- expression vectors and oliogonucleotides of C/EBPepsilon, the role that C/EBPepsilon plays in the combinatorial process of myeloid differentiation will be explored using human myeloid cell lines and the murine 32D myeloid cells. In vivo experiments will include: 1.) Study of CMV-driven C/EBPepsilon S- and AS-expressing transgenic mice. 2.) Analysis of C/EBPepsilon germline knock-out mice and C/EBPepsilon deletional chimeras.
Specific Aim 4 will dissect the transcriptional activity of the C/EBPepsilon molecule and identify its target genes. The first set of experiments, will define regions of transcriptional activation and repression of the C/EBPepsilon molecule. In further experiments, we will define the consensus binding sequences of C/EBPepsilon using CASTing and whole genome PCR in order to begin to identify novel C/EBPepsilon target sequences and genes. In addition, we will study in detail the ability of C/EBPepsilon to transactivate the myeloperoxidase and neutrophil elastase genes. Taken together, these studies will provide an understanding of C/EBPepsilon, a retinoid inducible, myeloid specific transcriptional activator.
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