Aporte de nutrientes

3.3.12 The Spt6 SH2 domain has a widespread function in vivo

Results in this section are the work of Andreas Mayer and are presented here for discussion. To investigate the importance of the Spt6 SH2 domain in vivo, we carried out Affymetrix gene expression profiling (Affymetrix GeneChip Yeast Genome 2.0) with a yeast strain lacking the C-terminal region of Spt6 that includes the SH2 domain (Youdell et al, 2008) (strain spt6C, 2.1.1, Table 3). Compared to a wild type strain, 790 out of 5665 genes that were present on the array showed significantly altered mRNA levels using a fold-change cut-off value of greater +2.0 or smaller than -2.0. Thus the Spt6 SH2 domain is necessary for the regulation of a subset of genes (14%) in Saccharomyces cerevisiae. The extent of deregulation of gene expression is comparable to strains carrying deletions of other Pol II elongation factor genes, including genes encoding subunits of the Paf1 complex (13% for paf1 and 15% for ctr9) (Penheiter et al, 2005). Of the mRNAs with significantly altered levels, 465 were up-regulated and 325 were down-regulated (Fig. 17 A), suggesting a repressive function of the SH2 domain at a majority of genes. Western blotting revealed that yeast cells adapt to the deletion by increasing the Spt6 protein levels (Fig. 16), which could compensate a failure of the mutant protein to localize to the transcription machinery. However, this does not restore the wild type phenotype in terms of growth, since the mutant strain shows a slow growth phenotype. Elevated Spt6 levels in the mutant can also root in a less efficient degradation of the TAP-tagged mutant protein. We were not able to resolve this problem because no antibody against the yeast Spt6 protein is available.

Figure 16: The cellular concentration of Spt6 increases when SH2 containing C-terminus is deleted

Western Blot of Spt6 proteins with a C-terminal TAP-tag, resolved in a 8% SDS-PAG. The mutant variant of Spt6 shows elevated expression levels relative to wild-type Spt6 and tubulin.

We next analyzed biological processes that were significantly affected by deletion of the SH2 domain with the Gene Ontology Enrichment Analysis Software Toolkit (GOEAST) and the web-based Gene Ontology (GO) tools (Ashburner et al, 2000; Zheng & Wang, 2008). This analysis showed that very diverse biological processes were over-represented, including genes involved in the response to toxins, in copper ion transport, and in thiamin metabolic processes.

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A deconvolution of gene expression microarray data is generally difficult as it represents the result of primary and secondary effects during gene expression. We nevertheless aimed at detecing a possible global, chromatin-related function of the SH2 domain in the transcriptome data, with use of correlation analysis. We first investigated whether the deregulated genes correlate to genes that were previously described to show cryptic transcription initiation in a spt6 mutant that carried an internal deletion of amino acids 931- 949 (corresponding to 930-993 in C. glabrata and comprising the HhH-domain marked purple in Fig. 15). From the 960 ORFs that showed cryptic transcription (Cheung et al, 2008), only 147 were included in our spt6C differential gene expression profile (Appendix Fig. A3). We also investigated whether our set of differentially expressed genes shows any correlation with gene length or an unusual number of associated nucleosomes (Lee et al, 2007). However, we could not find any significant correlations. These results are consistent with the view that Spt6 has multiple functions and is not only required for nucleosome assembly but also for mRNA splicing and

export (Yoh et al, 2007), and that it contains different functional surfaces (Fig. 15) that are perturbed in the different mutants.

To address the problem of secondary effects that influence the microarray data, we compared the differentially expressed genes in the spt6C mutant to a list of transcription factors (Hu et al, 2007). 33 genes of transcription factors are contained in our list of genes, which is more than 4% of all affected genes in the spt6C mutant (Appendix Fig. A4). This suggests that a number of alternatively expressed genes can probably be related to secondary effects induced by the Spt6 mutation. With the tools available, it is currently not possible to reveal details of this defective regulation networks.

To analyze whether the expression of similar genes is affected by deletion or mutation of different Pol II elongation factor genes, we compared our gene expression data to available data for the yeast strains dst1 (DST1 is the gene encoding TFIIS, (Koschubs et al, 2009),

spt4, and rtf1,(Hu et al, 2007). We expected similarity between these data sets since Spt6 interacts genetically with TFIIS (Hartzog et al, 1998) and with the Rtf1-containing Paf1 complex (Costa & Arndt, 2000; Mueller & Jaehning, 2002), and since Spt6 binds the Spt4- Spt5 complex (Krogan et al, 2002). An unsupervised hierarchical cluster analysis showed that the differential expression data from the spt4 and rtf1 strains form a distinct cluster within a dendrogram, indicating similarity of their gene expression profiles (Fig. 17 B, lanes 2 and 3, Methods). However, the spt6C mutant exhibits a very different expression profile (Fig. 17 B, lane 1), suggesting that the function of the Spt6 SH2 domain is clearly distinct from the functions of Spt4-Spt5 and the Paf1 complex in vivo. This analysis additionally revealed that dst1 showed the most distinct expression profile (Fig. 17 B, lane 4), maybe because TFIIS is not only required during elongation, but also during initiation (Guglielmi et al, 2007; Kim et al, 2007). Additional correlation studies confirmed these results.

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Figure 17: The Spt6 SH2 domain is required for normal gene expression in vivo

(A) Differentially expressed genes as detected by microarray analysis of the spt6C strain. The fraction of up- and down-regulated genes is shown in red and green, respectively. In total, 790 genes showed significantly altered mRNA levels between Spt6 wild-type and the spt6C strain. All gene expression analysis was performed with biological duplicates.

(B) Cluster analysis of differential gene expression profiles of different Pol II elongation factor deletion strains. The cluster diagram was calculated for 1350 yeast genes of spt6C, spt4, rtf1 and dst1 mutant strains, depicted in lanes 1, 2, 3, and 4, respectively. Each row corresponds to a particular gene and each column corresponds to a particular elongation factor mutant. Changes in mRNA levels compared with the isogenic wild-type strain are depicted in red (increase), green (decrease) or black (no change; see intensity bar). Both rows and columns were clustered using a hierarchical cluster algorithm (Saeed et al, 2003). The dendrogram for column clustering is shown.

(C) Pearson’s correlation matrix for gene expression profiles of yeast strains spt6C, spt4, rtf1 and dst1. The corresponding correlation coefficients are given.

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A weak correlation was detected between expression profiles of spt4 and rtf1 strains, but no significant correlations were seen between the profiles of the remaining mutant strains (Fig. 17 C). Taken together, deletion or mutation of various elongation factor genes results in different changes in the transcriptome, despite the observed genetic and physical interations between these factors.

Taken together, this data shows that the deletion of the Spt6 SH2 domain from the genome has a dramatic effect on gene expression. It is currently not resolved if this observation is mainly due to the absence of the domain and a resulting defect of Spt6 to recognize the Ser2-

phosphorylated CTD of Pol II, or to raised Spt6-levels in the mutant strain. However, this data shows the general importance of Spt6 and especially of its SH2 domain for correct transcription of the yeast genome.

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