Identification of novel binding elements and gene targets for the homeodomain protein BARX2.

BARX2 is a homeobox transcription factor that influences cellular differentiation in various developmental contexts. To begin to identify the gene targets that mediate its effects, chromatin immunoprecipitation (ChIP) was used to isolate BARX2 binding sites from the human MCF7 breast cancer cell line. Cloning and sequencing of BARX2-ChIP-derived DNA fragments identified 60 potential BARX2 target loci that were proximal to or within introns of genes involved in cytoskeletal organization, cell adhesion, growth factor signaling, transcriptional regulation, and RNA metabolism. The sequences of over half of the fragments showed homology with the mouse genome, and several sequences could be mapped to orthologous human and mouse genes. Binding of BARX2 to 21 genomic loci examined was confirmed quantitatively by replicate ChIP assays. A combination of sequence analysis and electrophoretic mobility shift assays revealed homeodomain binding sites within several fragments that bind to BARX2 in vitro. The majority of BARX2 binding fragments tested (14/19), also affected transcription in luciferase reporter gene assays. Mutation analyses of three fragments showed that their transcriptional activities required the HBS, and suggested that BARX2 regulates gene expression by binding to DNA elements containing paired TAAT motifs that are separated by a poly(T) sequence. Inhibition of BARX2 expression in MCF7 cells led to reduced expression of eight genes associated with BARX2 binding sites, indicating that BARX2 directly regulates their expression. The data suggest that BARX2 can coordinate the expression of a network of genes that influence the growth of MCF7 cells.

Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins.

BACKGROUND: The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs. RESULTS: The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis. CONCLUSIONS: The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.