Identification of β-catenin binding regions in colon cancer cells using ChIP-Seq

Deregulation of the Wnt/β-catenin signaling pathway is a hallmark of colon cancer. Mutations in the adenomatous polyposis coli (APC) gene occur in the vast majority of colorectal cancers and are an initiating event in cellular transformation. Cells harboring mutant APC contain elevated levels of the β-catenin transcription coactivator in the nucleus which leads to abnormal expression of genes controlled by β-catenin/T-cell factor 4 (TCF4) complexes. Here, we use chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) to identify β-catenin binding regions in HCT116 human colon cancer cells. We localized 2168 β-catenin enriched regions using a concordance approach for integrating the output from multiple peak alignment algorithms. Motif discovery algorithms found a core TCF4 motif (T/A–T/A–C–A–A–A–G), an extended TCF4 motif (A/T/G–C/G–T/A–T/A–C–A–A–A–G) and an AP-1 motif (T–G–A–C/T–T–C–A) to be significantly represented in β-catenin enriched regions. Furthermore, 417 regions contained both TCF4 and AP-1 motifs. Genes associated with TCF4 and AP-1 motifs bound β-catenin, TCF4 and c-Jun in vivo and were activated by Wnt signaling and serum growth factors. Our work provides evidence that Wnt/β-catenin and mitogen signaling pathways intersect directly to regulate a defined set of target genes

Characterization of an extracellular polyhydroxyalkanoate depolymerase from Streptomyces sp. SFB5A

A poly(-3-hydroxybutyrate) (PHB) degrading bacterium, Streptomyces sp. SFB5A, was isolated from hardwood mulch. It synthesized an extracellular poly(3-hydroxyalkanoate) (PHA) depolymerase during growth on PHB, poly(-3- hydroxyvalerate) (PHV), PHB-PHV copolymer (PHBV), or 3-hydroxybutyrate. The purified enzyme had a subunit relative molecular weight of 47,000 and a broad pH optimum of 7.0 to 8.5; was stimulated by Ca+2 and Mg+2; and was inhibited by ethylenediaminetetraacetic acid, dithiothreitol, and non-ionic detergents. The enzyme degraded PHB, PHV, and PHBV to a mixture of monomers, dimers, and trimers, with monomers predominating. The level of trimers peaked and then decreased over time, suggesting that they were produced early and were subsequently degraded to monomers and dimers. The enzyme did not degrade poly(3-hydroxyoctanoate), indicating that it was a short-chain-length PHA depolymerase. The PHA depolymerase gene was cloned and sequenced. The deduced amino acid sequence included three features typical of extracellular PHA depolymerases: a catalytic domain type 1, a fibronectin type III linker domain, and a substrate-binding domain. A mechanism accounting for the transient production of PHA trimers was proposed, based on modeling of the enzyme’s tertiary structure and amino acid sequence homology to related PHA depolymerases