Vishwanath R. Iyer

Systematic variation in gene expression patterns in human cancer cell lines

We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institutes screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.

Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure.

Many yeast promoters contain homopolymeric dA:dT sequences that affect nucleosome formation in vitro and are required for wild‐type levels of transcription in vivo. Here, we show that poly(dA:dT) is a novel promoter element whose function depends on its intrinsic structure, not its interaction with sequence‐specific, DNA‐binding proteins. First, poly(dA:dT) stimulates Gcn4‐activated transcription in a manner that is length dependent and inversely related to intracellular Gcn4 levels. Second, Datin, the only known poly(dA:dT)‐binding protein, behaves as a repressor through poly(dA:dT) sequences. Third, poly(dG:dC), a structurally dissimilar homopolymer that also affects nucleosomes, has transcriptional properties virtually identical to those of poly(dA:dT). Three probes of chromatin structure including HinfI endonuclease cleavage in vivo indicate that poly(dA:dT) increases accessibility of the Gcn4 binding site and adjacent sequences in physiological chromatin. These observations suggest that, by virtue of its intrinsic structure, poly(dA:dT) locally affects nucleosomes and increases the accessibility of transcription factors bound to nearby sequences.

Coordinate Regulation of Yeast Ribosomal Protein Genes Is Associated with Targeted Recruitment of Esa1 Histone Acetylase

The Esa1-containing NuA4 histone acetylase complex can interact with activation domains in vitro and stimulate transcription on reconstituted chromatin templates. In yeast cells, Esa1 is targeted to a small subset of promoters in an activator-specific manner. Esa1 is specifically recruited to ribosomal protein (RP) promoters, and this recruitment appears to require binding by Rap1 or Abf1. Esa1 is important for RP transcription, and Esa1 recruitment to RP promoters correlates with coordinate regulation of RP genes in response to growth stimuli. However, following Esa1 depletion, H4 acetylation decreases dramatically at many loci, but transcription is not generally affected. Therefore, the transcription-associated targeted recruitment of Esa1 to RP promoters occurs in a background of more global nontargeted acetylation that is itself not required for transcription.

Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays

In Saccharomyces cerevisiae, copper ions regulate gene expression through the two transcriptional activators, Ace1 and Mac1. Ace1 mediates copper-induced gene expression in cells exposed to stressful levels of copper salts, whereas Mac1 activates a subset of genes under copper-deficient conditions. DNA microarray hybridization experiments revealed a limited set of yeast genes differentially expressed under growth conditions of excess copper or copper deficiency. Mac1 activates the expression of six S. cerevisiae genes, including CTR1,CTR3, FRE1, FRE7, YFR055w, and YJL217w. Two of the last three newly identified Mac1 target genes have no known function; the third, YFR055w, is homologous to cystathionine γ-lyase encoded by CYS3. Several genes that are differentially expressed in cells containing a constitutively active Mac1, designated Mac1up1, are not direct targets of Mac1. Induction or repression of these genes is likely a secondary effect of cells because of constitutive Mac1 activity. Elevated copper levels induced the expression of the metallothioneins CUP1 andCRS5 and two genes, FET3 and FTR1, in the iron uptake system. Copper-induced FET3 andFTR1 expression arises from an indirect copper effect on cellular iron pools.

The chromo domain protein Chd1p from budding yeast is an ATP‐dependent chromatin‐modifying factor

CHD proteins are members of the chromo domain family, a class of proteins involved in transcription, DNA degradation and chromatin structure. In higher eukaryotes, there are two distinct subfamilies of CHD proteins: CHD1 and CHD3/4. Analyses carried out in vitro indicate that the CHD3/4 proteins may regulate transcription via alteration of chromatin structure. However, little is known about the role of CHD proteins in vivo, particularly the CHD1 subfamily. To understand better the cellular function of CHD proteins, we initiated a study on the Chd1p protein from budding yeast. Using genomic DNA arrays, we identified genes whose expression is affected by the absence of Chd1p. A synthetic‐lethal screen uncovered genetic interactions between SWI/SNF genes and CHD1. Biochemical experiments using Chd1p purified from yeast showed that it reconfigures the structure of nucleosome core particles in a manner distinct from the SWI–SNF complex. Taken together, these results suggest that Chd1p functions as a nucleosome remodeling factor, and that Chd1p may share overlapping roles with the SWI–SNF complex to regulate transcription.

Mechanism of differential utilization of the his3 TR and TC TATA elements.

The yeast his3 promoter region contains two TATA elements, TC and TR, that are differentially utilized in constitutive his3 transcription and Gcn4-activated his3 transcription. TR contains the canonical TATAAA sequence, whereas TC is an extended region that lacks a conventional TATA sequence and does not support transcription in vitro. Surprisingly, differential his3 TATA-element utilization does not depend on specific properties of activator proteins but, rather, is determined by the overall level of his3 transcription. At low levels of transcription, the upstream TC is preferentially utilized, even though it is inherently a much weaker TATA element than TR. The TATA elements are utilized equally at intermediate levels, whereas TR is strongly preferred at high levels of transcription. This characteristic behavior can be recreated by replacing TC with moderately functional derivatives of a conventional TATA element, suggesting that TC is a collection of weak TATA elements. Analysis of promoters containing two biochemically defined TATA elements indicates that differential utilization occurs when the upstream TATA element is weaker than the downstream element. In other situations, the upstream TATA element is preferentially utilized in a manner that is independent of the overall level of transcription. Thus, in promoters containing multiple TATA elements, relative utilization not only depends on the quality and arrangement of the TATA elements but can vary with the overall level of transcriptional stimulation. We suggest that differential TATA utilization results from the combination of an intrinsic preference for the upstream element and functional saturation of weak TATA elements at low levels of transcriptional stimulation.

Genome-wide maps of DNA-protein interactions using a yeast ORF and intergenic microarray

We have made a DNA microarray that includes not only all the open reading frames (ORFs) and other features in the yeast genome, but also all the intergenic regions. We are using this as a tool to construct genome-wide maps of DNA-protein interactions for proteins that interact directly or indirectly with DNA or chromatin in vivo. Proteins are crosslinked to DNA in vivo using formaldehyde and the crosslinked DNA is extracted and sheared. DNA specifically associated with any protein of interest is immunoprecipitated using a specific antibody against the protein or an epitope tag that may be fused to the protein. The selected DNA, representing loci that the protein interacts with in vivo, can be identified by fluorescently labelling it and hybridizing it to the microarray along with an appropriate reference probe. This approach is being used to map the genome-wide interactions of sequence-specific DNA binding proteins, components of the transcription machinery and chromatin components, under a variety of conditions.