Sequential chromatin immunoprecipitation protocol: ChIP-reChIP.

Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene transcription. Therefore, it has become relevant to determine the cohabitation of several proteins in a particular developmental stage and cell type. Furthermore, multiple post-translational histone modifications can be analyzed on the same genomic location with the aim of deciphering the combinatorial pattern of histone modifications associated to specific transcriptional stages during cell commitment. Here we describe the ChIP-reChIP assay that represents a direct strategy to determine the in vivo colocalization of proteins interacting or in close contact in a chromatinized template on the basis of double and independent rounds of immunoprecipitations with high-quality ChIP grade antibodies.

Globin genes transcriptional switching, chromatin structure and linked lessons to epigenetics in cancer: a comparative overview.

At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.

R-Ras promotes tumor growth of cervical epithelial cells.

BACKGROUND: R-Ras is 55% identical to H-Ras. However, these two oncogenes seem to have different tumor-transforming potential. R-Ras induced cell transformation in fibroblasts but not in other cell types. R-Ras also reportedly induces a more invasive phenotype in breast epithelial cells through integrin activation. The authors studied the mechanisms whereby R-Ras induces a malignant phenotype. METHODS: Dominant negative (R-Ras43N) and constitutively active (R-Ras87L) mutants of R-Ras were stably transfected into human cervical epithelium C33A cells. Transfected cells were analyzed for adhesion, cell spreading, migration, and growth in culture and in nude mice. The activity of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K) also was determined by Western blot analysis and by in vitro kinase assays. RESULTS: R-Ras87L-transfected cells, but not R-Ras43 N-transfected cells, had a higher growth rate in nude mice and in culture compared with control cells. None of the transfected C33A cells showed an increase in cell adhesion to fibronectin or collagen I, nor did they show an increment of beta1 integrin affinity. However, cells that expressed R-Ras87L, but not cells that expressed R-Ras 43N, presented a marked increase in cell spreading and migration through collagen-coated membranes. Increases in cell proliferation, spreading, and migration induced by R-Ras87L were inhibited by the PI 3-K inhibitor LY294002. In addition, PI 3-K activity, but not ERK activity, was increased only in cells that expressed R-Ras87L. CONCLUSIONS: These data suggest that the oncogene R-Ras promotes tumor growth of cervical epithelial cells and increases their migration potential over collagen through a pathway that involves PI 3-K.