MLL fusion genes arise as the consequence of chromosome translocations associated with leukemia. Among the known targets of regulation by MLL are the type I homeobox (HOX) genes that have important roles incell-lineage commitment and differentiation. Cyp33 is a cyclophilin that binds the third PHD finger of MLL and inhibits the MLL transactivating activity at silent genetic loci. The MLL fusion proteins cannot bind Cyp33 because they lack the PHD fingers. Thus, they function as constitutive activators that promote ectopic expression of HOX genes and other MLL target genes, and prevent commitment of hematopoietic progenitor cells, leading to their immortalization,and eventually leukemogenesis. Cyp33 can bind either RNA or MLL through its RRM domain. Therefore, nascent RNA at promoters and enhancers can titer Cyp33 releasing MLL from its control. This would provide a mechanism for the MLL complex to recognize active genes in early embryogenesis and maintain their expression through subsequent development.
Specific Aim 1. a) Using modified MLZ.-fusion genes expressed in human cell lines, to test the role of RD2 and the;3rdPHD finger of MLL in HDAC recruitment and in regulation of HOX gene expression,b) Using modified A/LZ,-fusion genes in retroviral expression vectors transduced into mouse bone marrow cells, to test the role of RD2 and the 3rdPHD finger of MLL in immortalizationof hematopoietic progenitor cells, c) Using RNAi,to test the role of Cyp33 in regulation of HOX gene expression in human cell lines and in immortalization of mouse bone marrow cells, d) Using DNA-arrays, to compare the gene expression profile of cells over-expressing Cyp33, having normal levels of Cyp33 or knocked-down Cyp33 expression.
Specific aim 2 : a) To test the inductionof HOX geneexpression by AU-rich RNA produced from an expression vector transfected into human cells or Drosophila SL2cells (transinduction). b) Using an expression vector for AU-rich RNA in Cyp33-expressing cells, to test the Cyp33- dependence of trans-induction, c) Using an expression vector for AU-rich non-coding RNA (nc-RNA) in MLL-/- MEFs, to test the MLL-dependence of trans-induction, d) Using FISH, to determine the sub-nuclearlocalizationof nc- RNA, the nc-RNA plasmid, and cellular HOX gene loci. To test the need for Cyp33 in early embryogenesis in Drosophila, by knockingdown Cyp33 in embryos, e) Using EMSA, to test the relative affinity of Cyp33 for different RNA sequence motifs and for the MLL/trx PHD fingers, f) To characterize the intergenic noncoding transcriptsIn the HOX gene cluster. These studies will contribute to our understanding of leukemogenesis and hematopoietic stem cell biology.
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