Elevated plasma cholesterol levels are considered responsible for excess cardiovascular morbidity and mortality. Cholesterol in plasma is tightly controlled by cholesterol within cells. Here, we developed and applied an integrative functional genomics strategy that allows systematic identification of regulators of cellular cholesterol levels. Candidate genes were identified by genome-wide gene-expression profiling of sterol-depleted cells and systematic literature queries. The role of these genes in cholesterol regulation was then tested by targeted siRNA knockdown experiments quantifying cellular cholesterol levels and the efficiency of low-density lipoprotein (LDL) uptake. With this strategy, 20 genes were identified as functional regulators of cellular cholesterol homeostasis. Of these, we describe TMEM97 as SREBP target gene that under sterol-depleted conditions localizes to endo-/lysosomal compartments and binds to LDL cholesterol transport-regulating protein Niemann-Pick C1 (NPC1). Taken together, TMEM97 and other factors described here are promising to yield further insights into how cells control cholesterol levels.

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A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.

Several oncogenes isolated by the NIH/3T3 transformation assay, i.e., dbl, dbs, lbc, lfc, lsc, net, ost and tim, contain a Dbl homology (DH) and a pleckstrin-homology (PH) domain and act as GEFs (guanine nucleotide exchange factors) for Rho-like GTPases. In a search for genes with oncogenic potential in DNA from the monocytic leukaemia cell line U937, we identified an amino-terminal truncated form of gef-h1, a gene encoding a GEF for RhoA. These data support the idea that a systematic search for mutations and/or deletions of GEFs in human cancer is promising.