Genes Identified in a Hemopoietic Lineage Switch Influence Transcription.

The J2E erythroblastoid cell line responds to erythropoietin (Epo) by morphological maturation and hemoglobin synthesis. However, on rare occasions, these cells have undergone a spontaneous lineage switch and display features of monoblastoid cells which do not respond to Epo. Amongst the genes up-regulated in the monoblastoid variants were Hemopoietic lineage switch (Hls) 5 and 7. Hls5 is a recently identified member of the RING finger, B Box, Coiled coil (RBCC) or tripartite motif (TRIM) family, which includes PML, a gene involved in acute promyelocytic leukaemia. Hls7 is the murine orthologue of Myeloid leukaemia factor 1 (Mlf1), a gene involved in a t(3;5), associated with acute myeloid leukaemia. We have shown previously that Hls7/Mlf1 imposes a dramatic phenotypic change upon the erythroid cells, rendering them monoblastoid (Williams, J. et al EMBO 1999). We have studied the role of Hls5 and Mlf1 in erythroid commitment and differentiation. Ectopic expression of Hls5 inhibits globin production in erythroid cells and suppresses development of B-FUE and C-FUE. A yeast-two-hybrid screen identified FOG-1 as a binding partner of Hls5. Significantly, FOG-1 is a transcriptional co-regulator of GATA-1, which controls globin gene expression. While Hls5 is able to enhance the repression of GATA-1 activity imposed by FOG-1, it is also able to repress GATA-1 transcriptional activity in the absence of FOG-1. Using electrophoretic mobility shift assay we have shown that Hls5 is able to reduce GATA-1 binding to DNA in a dose dependant manner. This observation that Hls5 reduces GATA-1 binding to promoter elements is mirrored by chromatin immunoprecipitation assays. Expression of MLF1 is highest in CD34+ cells, but is markedly down regulated during erythroid differentiation. Microarray analysis identified a number of known transcriptional regulators differentially expressed in the presence of Mlf1 including ets1, Myc intron binding protein and Tbr2. Mlf1 is able to bind DNA and luciferase reporter assays demonstrated that Mlf1 is able to affect transcription. In addition, Mlf1 interacts with a novel member of the hnRNP family viz Mlf1 associated nuclear protein (Manp). Manp binds to DNA, is able to influence the subcellular localisation of Mlf1 by translocating Mlf1 from the cytoplasm to the nucleus. Importantly, Manp also has an affect on transcription. These data demonstrate that both Hls5 and Mlf1 affect transcription of genes associated with erythroid differentiation.