Analysis of previously published sets of DNA microarray gene expression data by singular value decomposition has uncovered underlying patterns or ''characteristic modes'' in their temporal profiles. These patterns contribute unequally to the structure of the expression profiles. Moreover, the essential features of a given set of expression profiles are captured using just a small number of characteristic modes. This leads to the striking conclusion that the transcriptional response of a genome is orchestrated in a few fundamental patterns of gene expression change. These patterns are both simple and robust, dominating the alterations in expression of genes throughout the genome. Moreover, the characteristic modes of gene expression change in response to environmental perturbations are similar in such distant organisms as yeast and human cells. This analysis reveals simple regularities in the seemingly complex transcriptional transitions of diverse cells to new states, and these provide insights into the operation of the underlying genetic networks.T he recent development of DNA microarray technology has enabled the genome-wide measurement of temporal changes in gene expression levels (1, 2). Analysis of the expression patterns obtained with large gene arrays has revealed the existence of groups or ''clusters'' of genes with similar expression patterns (3-6). Not surprisingly, gene clusters often contain genes that encode proteins required for a common function, and, hence, co-clustering has been helpful in identifying the functions of unknown gene products. However, such cluster analyses provide little insight into the relationships among groups of co-regulated genes or the behavior of biological networks as a whole.In this paper, we report the results of subjecting several large published gene expression data sets to singular value decomposition (SVD), a standard and straight-forward analytic procedure. We show that highly complex sets of gene expression profiles can be represented by a small number of ''characteristic modes'' that capture the temporal patterns of gene expression change. These modes are somewhat analogous to the characteristic vibration modes of a tuned violin string. The tone produced by the vibrating string can be entirely specified by the contributions of its characteristic vibration modes. We show here that a gene expression profile, similarly, can be precisely represented by specifying the magnitude and sign of the contribution of each of its characteristic modes. This type of ''spectral'' analysis yields a hierarchical interpretation of the expression data and provides insights into the nature and behavior of genetic networks. MethodsThe mathematical analysis is carried out straightforwardly by using SVD (7). The gene expression data of n genes, each measured at m discrete time points, may be written as an n ϫ m matrix, A. Following the procedures outlined in ref. 6, we have polished the data by requiring that the rows and columns have a zero mean by subtracting the mean values of the raw...
The recruitment of the TATA box-binding protein (TBP) to promoters in vivo is often rate limiting in gene expression. We present evidence that TBP negatively autoregulates its accessibility to promoter DNA in yeast through dimerization. The crystal structure of TBP dimers was used to design point mutations in the dimer interface. These mutants are impaired for dimerization in vitro, and in vivo they generate large increases in activator-independent gene expression. Overexpression of wild-type TBP suppresses these mutants, possibly by heterodimerizing with them. In addition to loss of autorepression, dimerization-defective TBPs are rapidly degraded in vivo. Direct detection of TBP dimers in vivo was achieved through chemical cross-linking. Taken together, the data suggest that TBP dimerization prevents unregulated gene expression and its own degradation.
The TATA binding protein (TBP) is a central component of the eukaryotic transcription machinery and is subjected to both positive and negative regulation. As is evident from structural and functional studies, TBP's concave DNA binding surface is inhibited by a number of potential mechanisms, including homodimerization and binding to the TAND domain of the TFIID subunit TAF1 (yTAF II 145/130). Here we further characterized these interactions by creating mutations at 24 amino acids within the Saccharomyces cerevisiae TBP crystallographic dimer interface. These mutants are impaired for dimerization, TAF1 TAND binding, and TATA binding to an extent that is consistent with the crystal or nuclear magnetic resonance structure of these or related interactions. In vivo, these mutants displayed a variety of phenotypes, the severity of which correlated with relative dimer instability in vitro. The phenotypes included a low steady-state level of the mutant TBP, transcriptional derepression, dominant slow growth (partial toxicity), and synthetic toxicity in combination with a deletion of the TAF1 TAND domain. These phenotypes cannot be accounted for by defective interactions with other known TBP inhibitors and likely reflect defects in TBP dimerization.Activation of eukaryotic genes is a multistep process, involving the coalescence of promoter-specific activators, chromatinremodeling complexes, and components of the general transcription machinery at promoters. An important part of the activation process is the removal of inhibitors associated with latent activators, promoters, and the general transcription machinery. One component of the general transcription machinery that is subjected to substantial inhibition is the TATA binding protein (TBP) (reviewed in reference 75). Virtually all genes require TBP for function, and its association with promoters is generally linked to transcriptional activity (57, 63). Preventing unregulated promoter binding by TBP may be critical for preventing unregulated gene expression. TBP access might be prevented in part by nucleosome formation over the TATA box (43, 80). However, many quiescent genes are not derepressed upon histone depletion (83), indicating that other inhibitory mechanisms might prevent TBP from binding to promoters.A number of proteins inhibit TBP function. These include the TAF1 (yTAF II 145/130) subunit of TFIID, NC2, Mot1, the Spt3/Spt8 subunits of SAGA, the Ccr4-Not complex, and a second molecule of TBP in the form of homodimers. Here we focus on two inhibitory interactions which are directed at TBP's concave surface: TBP dimerization and the TAF1 TAND domain.TFIID is a multisubunit complex consisting of TBP and TAFs (19,77,78). TFIID is required for activated transcription but is intrinsically inhibitory toward TBP-TATA interactions (78). At least part of this inhibitory activity might reside within the amino-terminal TAND domain of the TFIID subunit, TAF1 (11,52,70). Mutagenesis studies have delineated Drosophila and yeast TANDs as two subdomains, I and II (52, 54). ...
A kinetic analysis of dimer dissociation, TATA DNA binding, and thermal inactivation of the yeast Saccharomyces cerevisiae and human TATA binding proteins (TBP) was conducted. We find that yeast TBP dimers, like human TBP dimers, are slow to dissociate in vitro (t(1/2) approximately 20 min). Mild mutations in the crystallographic dimer interface accelerate the rate of dimer dissociation, whereas severe mutations prevent dimerization. In the presence of excess TATA DNA, which measures the entire active TBP population, dimer dissociation represents the rate-limiting step in DNA binding. These findings provide a biochemical extension to genetic studies demonstrating that TBP dimerization prevents unregulated gene expression in yeast [Jackson-Fisher, A. J., Chitikila, C., Mitra, M., and Pugh, B. F. (1999) Mol. Cell 3, 717-727]. In the presence of vast excesses of TBP over TATA DNA, which measures only a very small fraction of the total TBP, the monomer population in a monomer/dimer equilibrium binds DNA rapidly, which is consistent with a simultaneous binding and bending of the DNA. Under conditions where other studies failed to detect dimers, yeast TBP's DNA binding activity was extremely labile in the absence of TATA DNA, even at temperatures as low as 0 degrees C. Kinetic analyses of TBP instability in the absence of DNA at 30 degrees C revealed that even under fairly stabilizing solution conditions, TBP's DNA binding activity rapidly dissipated with t(1/2) values ranging from 6 to 26 min. TBP's stability appeared to vary with the square root of the TBP concentration, suggesting that TBP dimerization helps prevent TBP inactivation.
We have built a microarray database, StressDB, for management of microarray data from our studies on stress-modulated genes in Arabidopsis. StressDB provides small user groups with a locally installable web-based relational microarray database. It has a simple and intuitive architecture and has been designed for cDNA microarray technology users. StressDB uses Windows 2 2000 as the centralized database server with Oracle 2 8i as the relational database management system. It allows users to manage microarray data and data-related biological information over the Internet using a web browser. The source-code is currently available on request from the authors and will soon be made freely available for downloading from our website athttp://arastressdb.cac.psu.edu.
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