Proteomic analysis of chromatin-modifying complexes in Saccharomyces cerevisiae identifies novel subunits.

Epigenetics is the alteration of phenotype without affecting the genotype. An underlying molecular mechanism of epigenetics is the changes of chromatin structure by covalent histone modifications and nucleosome reorganization. In the yeast, Saccharomyces cerevisiae, two of the most well-studied macromolecular complexes that perform these epigenetic changes are the ATP-dependent Swi/Snf chromatin-remodelling complex and the SAGA histone acetyltransferase complex. To understand fully the mechanism by which these large protein complexes perform their functions in the cell, it is crucial that all the subunits of these complexes are identified. In an attempt to identify new subunits associated with SAGA and Swi/Snf, we used tandem affinity purification, followed by a multidimensional protein identification technology to analyse the subunit composition. Our analysis identified two novel proteins, one associated with SAGA, YPL047W (Sgf11), and another associated with Swi/Snf, Rtt102.

Transcriptional mechanisms responsible for the overexpression of the keratin 18 gene in cells of a human colon carcinoma cell line.

The keratin 18 (K18) gene is overexpressed in cells of tumorigenic clones isolated from the SW613-S human colon carcinoma cell line, compared to cells of nontumorigenic clones. The isolated minimal promoter (TATA box and initiation site) of the K18 gene has by itself a differential activity in tumorigenic and nontumorigenic cells. An Sp1 binding site located upstream of the TATA box contributes to the high level of expression of the gene in tumorigenic cells. We report here that the Sp1 gene is not differentially expressed between the two cell types and that this is also the case for genes coding for factors of the preinitiation complex known to directly interact with the Sp1 protein. Further, DNase I footprinting experiments and mutagenesis analysis indicated that the mechanism responsible for the differential activity of the minimal K18 promoter apparently does not involve the binding of a factor to a specific sequence. During the course of these experiments, it was found that the initiation site of the K18 promoter is actually located 11 bp upstream of the +1 position previously reported and that the TATA box is the only essential element of the minimal promoter. Treatment of the cells with histone deacetylase inhibitors was more efficient at stimulating the activity of the K18 promoter in nontumorigenic cells than in tumorigenic cells. We propose that overexpression of the K18 gene in tumorigenic cells could result from of a high level of acetylation of histones and/or of factors controlling the activity of the transcription complex.

Deregulated expression of the keratin 18 gene in human colon carcinoma cells.

The keratin 18 (K18) gene is expressed at a normal level in cells of nontumorigenic clones derived from the SW613-S human colon carcinoma cell line, but is overexpressed in cells of tumorigenic clones. A high level of expression was also found in the cells from 10 of 15 other human colon carcinoma cell lines. The expression of the gene is downregulated in differentiating Caco-2 cells, resulting in a normal expression level. Determination of K18 mRNA half-life in growing and confluent Caco-2 cells indicated that this downregulation does not take place at a posttranscriptional level. The density of RNA polymerase molecules on the K18 gene, as measured in nuclear run-on experiments, is the same in growing and confluent Caco-2 cells, but the rate of synthesis of K18 transcripts in confluent Caco-2 cells, as determined by in vivo pulse-labeling, is 35% of that in growing cells. Nuclear run-on experiments carried out with nuclei prepared from growing or confluent Caco-2 cells treated with 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) indicated that a reduction in both the initiation and elongation rates of RNA polymerase molecules occurs on the K18 gene in confluent Caco-2 cells. This leads to a decreased rate of K18 transcript production with no reduction in the polymerase density on the gene. Evidence is provided that the mechanisms responsible for the differential expression of the K18 gene between tumorigenic and nontumorigenic SW613-S cells and between growing and differentiating Caco-2 cells share some similarities.