Systematic analysis of transcription factors (TFs) and associated gene regulation networks is of central relevance to medicine. The combination of RNAi and genome-wide expression profiling provides a powerful strategy for elucidating gene function, and gaining insights into the nature and complexity of regulatory networks controlling eukaryotic gene expression at the level of transcription.

We have knocked down the expression of ca. 150 human transcription factors potentially involved in key developmental processes and human pathologies in a panel of human cell lines via RNAi technique. The downstream effects for each knocked-down transcription factor is monitored on the global cell transcriptome. In order to evaluate off-target effects affecting the expression of unintended gene targets, we performed independent experiments using different silencing molecules, chemically synthesized siRNAs and endoribonuclease-prepared siRNAs (esiRNAs-cooperation with Dr. F. Buchholz MPI-CBG Dresden).

Establishing transcriptional regulatory networks requires knowledge of TFs bound to promoter, enhancer and repressor elements. To address in vivo targets across the human genome for selected TFs, we are using chromatin immunoprecipitation coupled with second-generation sequencing technology (ChIP-seq). Genome-wide location analysis is a tool for further dissecting gene regulatory networks by identifying binding sites at which proteins reside through the genome in vivo.

We have developed a highly specific ChIP-seq protocol and are applying it to those HSA21 TFs that are endogenously expressed in HEK293 cells. Sequencing of the ChIP DNAs delivers >10 million reads for each TF. Data analysis identifies peak regions enriched in reads that serve as basis for extracting weight matrices for bound DNA motifs. Peak locations are determined relative to nearby genes, and potential target genes are annotated. Data is also being integrated with data from RNAi experiments coupled to expression profiling, giving a glimpse of consequences on the expression of the target genes resulting from knock-down of the corresponding TFs, allowing to chart associated regulatory networks in HEK293 cells.

This work is funded by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN-2) and by the European Union (AnEUploidy FP6 Integrated project).