ACETYLATION OF P45/NF E2 BY PCAF AND ITS CONSEQUENCES

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Hematopoiesis is the process in which pluripotential progenitors acquire lineage-specific phenotype and eventually give rise to mature circulating blood cells. Environmental signals that control hematopoiesis are ultimately interpreted by sets of transcription factors which regulate the expression of genes associated with lineage-specific functions. The long-term objectives of this proposal are to define the signaling events leading to the modulation and activation of hematopoietic transcription factors. This proposal aims at characterizing the regulation of the erythroid/megakaryocytic transcription factor NF-E2 by the acetyltransferase PCAF. The hematopoietic subunit of NF-E2, p45, was found to be acetylated by PCAF at a highly conserved lysine residue. This observation forms the basis of our hypothesis that the activity of p45 may be modulated by acetylation. The specific aims are to: 1) Determine the mechanistic consequences of p45 acetylation. p45 a is acetylated by PCAF in the CNC domain which participates in DNA binding. The effects of p45 acetylation on DNA binding will be determined. Acetylation could also change the conformation of p45 thus could modulate its interaction with other proteins. Finally, the transcriptional activity of mutant p45 lacking the acetylation site will be examined by gene complementation in p45-null erythroid cells. 2) Monitor acetylation of p45 during megakaryocytic and erythroid differentiation. To establish the correlation between p45 acetylation/deacetylation and the activation of p45 target genes in differentiating cells, antibodies specifically recognizing acetylated p45 will be generated. A pepetide encompassing the in vivo acetylation site will be used to generate antibodies specific for acetylated p45. The acetylation status of p45 will be followed in both leukemic cell lines and primary cells undergoing differentiation. The effects of both differentiation-inducing chemicals and cytokines on p45 acetylation will be examined. Together, these studies may yield important insights into the regulation of hematopoietic transcription factors by acetyltransferases. Since these enzymes present excellent targets for pharmacological intervention, this knowledge could be useful for the design of drugs used to influence gene expression and cellular differentiation in patients with hematological disorders.