Identification of Target Genes of an Erythroid Transcription Factor Complex Containing SCL (TAL1).

Haematopoiesis is the process whereby haematopoietic stem cells give rise to mature blood cell lineages. The SCL (TAL1) gene, originally identified by chromosomal translocations associated with T-cell acute lymphocytic leukaemia, encodes a key transcription factor (TF) which is expressed in various blood lineages and is essential for haematopoietic development. It has been shown that the SCL protein forms a multi-protein complex during erythroid development with other TFs (GATA1, E2A, LDB1, and LMO2) which binds to a sequence-specific motif to regulate the expression of glycophorin A and c-kit. We have used two complementary approaches to identify novel target genes regulated by this TF complex during erythroid development. In the first approach, we have transfected short interfering RNAs (siRNAs) into the K562 cell line to knockdown transiently the TFs of the erythroid complex. For all members of the complex, a knockdown efficiency of at least 70% was confirmed at the mRNA and protein level within 48 hours after transfection. The consequences of the knockdown at the level of gene expression were observed using Affymetrix GeneChips in order to identify downstream events associated with the erythroid complex in transcriptional programmes. In the second approach, chromatin immunoprecipitation (ChIP) was performed for each member of the complex in the K562 cell line and the ChIP material hybridised to a human transcription factor promoter microarray. A number of novel target genes for the SCL erythroid complex have been identified and verified independently using both approaches. Our data shows that members of the erythroid complex are involved in auto-regulation and regulate genes which control chromatin structure and function. These findings demonstrate that the expression of this TF complex is tightly controlled and point to an important role for it in orchestrating fundamental biological processes which have profound effects on gene expression in erythroid development.