The MDS-Associated Gene DLK Modulates Gene Expression in Human Myeloid Cells by Altering the Activity of Multiple Transcription Factors.

Delta-Like 1 (DLK) is an EGF-like transmembrane protein, which is overexpressed in myelodysplastic syndrome (MDS) CD34+ cells. We have previously shown that ectopic DLK expression inhibits HL-60 cell differentiation and proliferation through intracellular domain interactions. To further investigate mechanisms underlying DLK effects on myeloid cell differentiation and proliferation, we compared gene expression profiles of DLK expressing and control HL-60 cells, with or without differentiating induction with ATRA, using Affymetrix HG-U133A arrays. Gene expression data was analyzed using affy and limma (linear model of microarray analysis) packages in the open-source BioConductor project (v 1.6). Raw data were processed using robust multi-chip average (RMA) algorithm, a linear model fit to each gene, and the following comparisons were made: (a) effects of DLK expression in unstimulated cells, (b) effects of DLK expression in ATRA exposed cells, (c) effects of ATRA induction on R1 cells, (d) effects of ATRA induction on DLK+ cells, and (e) differences in the response of DLK+ vs. control cells to ATRA. Adjusted P values and log odds of differential expression (B statistic, 50% probability when B=0) were calculated. B values > 0 were considered statistically significant. 523 genes were differentially expressed between unstimulated control and DLK+ cells, 343 genes were differentially expressed between control and DLK+ cells after ATRA stimulation, and 204 genes were common to the two sets. 802 genes were differentially expressed after ATRA stimulation in control cells, 742 genes in DLK+ cells, with 550 genes common to the two sets. 13 genes were differentially expressed when ATRA responses of control and DLK+ cells were compared. Gene ontology (GO) analyses indicated that "Biological processes" significantly affected by DLK overexpression included signal transduction, cell cycle, proliferation, cell death, protein metabolism and enzyme cascades, and "Molecular functions" most affected included nucleotide/DNA binding and protein kinase activity. These observations are consistent with observed cellular effects of DLK. Using MotifRegressor software, we performed promoter analysis correlating common transcription factor-binding motifs with expression profiles of genes differentially expressed between DLK+ and control cells. We identified the transcription factors (TF) PBX, GATA-1, c-Myc: Max, HIF-1, DEC1, Hand1, Lmo2, NKX25, GKLF and AP-1 as being potentially involved in DLK-mediated changes in gene expression. The observed patterns of differential gene expression were consistent with altered activities of these TF. Electrophoresis mobility shift assays (EMSA) indicated increased PBX and reduced HIF-1 and GATA-1 activities in DLK+ cells. Interestingly, Hand1, c-Myc: Max and Dec1 are basic Helix-loop-Helix (b-HLH) factors with E box binding sites, which are known to associate and form regulatory complexes with other TF. TF such as GATA-1, GLKF and Lmo2, also identified in our analysis, are known to be associated with such complexes. In conclusion, gene expression profiles of DLK expressing human myeloid cells are consistent with observed alterations in cell proliferation and differentiation. We have identified TF that may act individually and/or in concert to induce the observed changes in gene expression in DLK+ cells. Further evaluation of their role of these TF in mediating DLK effects and in abnormal hematopoietic cell growth in MDS is warranted.