Valor nutricional

The occupancy profile of C-terminally TAP-tagged Spt6 on the yeast genome showed a high correlation to the profiles of Pol II subunits Rpb3 (Pearson correlation coefficient 0.86) and Rpb7 (Pearson correlation coefficient 0.87) (Jasiak et al, 2008). Thus, the pearson coefficient is only slightly lower than the correlation coefficient of Rpb3 and Rpb7 (0.91), which are both part of the same multisubunit complex. This means that Spt6 localizes together with Pol II on

Figure 18: Genome-wide occupancy profiling of transcription elongation factor Spt6

A representative 230 kilobase pair sample on chromosome one (genomic positions 0-230.000) of the profiles for Rpb3 and Spt6 are depicted. Each green dot represents the signal for a single oligonucleotide probe on the tiling array, which has one probe every 32 bp. An example for a region in the genome with different Pol II/Spt6 occupancy is marked red.

(A) Difference signal between averaged Rpb3 (B) and Spt6 (C) occupancy profiles.

(B) Average over three biological replicate traces for Rpb3, one of which with interchanged fluorescent dyes (Jasiak et al., 2008)

(C) Average over two biological replicate traces for Spt6, both with interchanged fluorescent dyes (D) Genomic features based on the Saccharomyces genome database annotations

3 Structure and requirement of the Spt6 SH2 domain 65

a genome-wide scale. The significance of these profiles is described in Jasiak et al (2008): housekeeping genes as well snoRNA genes show a high occupancy, which is expected expected, because these genes are highly expressed. tRNA-genes, that are expressed by RNA polymerase III show no increased occupancy. Fig. 18 shows a representative 230 kilobase sample of the profiles for Spt6 and Rpb3. The profiles are virtually similar, but show slight differences in some positions (Fig. 18, marked red).

We asked, if our data shows recruitment of Spt6 relative to Pol II that parallels Ser2-

phosphorylation of the CTD. We would expect to observe an increase of Spt6 occu-pancy towards the 3’ end of the transcribed regions, since it was shown that the SH2 domain binds exclusively to the CTD when phosphorylated at Ser2 residues (Yoh et al, 2007), which

predominate only in 3’ regions of a gene (Komarnitsky et al, 2000). We could not observe this general behavior of Spt6 in the occupancy profile.

To further analyze this question, we asked if the effect of an interaction between the CTD and the SH2 domain can be seen at genes of similar length, when the ChIP signals of these genes are averaged. For this, based on the Rpb3-data, we filtered out genes with an average ChIP signal < 0.2 to eliminate genes that are not transcribed. In addition, overlapping genes (both on the same strand and on the opposite strand) were eliminated to get clean signals from single genes. For the same reason we excluded neighboring genes that were too close to assign clean signals because of a "smearing" of ChIP signals in 5’ and 3’ regions. This resulted in 638 genes that showed a distribution in length as shown in Fig. 19 A. We used those classes for our analysis, that contained a sufficiently high number of genes (green in Fig. 19 A).

The results of our analysis are shown in Fig. 19 B. The averaged Rpb3 ChIP-signal is high around the Start-codon (green, dashed line) and has its maximum shortly after that. This behavior can be seen in all classes of gene length and can be explained by polymerases paused at promoter proximal sites. In contrast to that, the averaged Spt6 signal shows a delay in comparison to the Pol II occupancy, with increasing density toward the 3’ end of transcribed regions. This effect decreases with increasing gene length. Thus, Spt6 shows an occupancy profile that roughly parallels Ser2-phosphorylation of the CTD, but only when the

signals of many genes of the same length are averaged. Since it is crosslinking efficiency that is directly measured in a ChIP-experiment this effect could be explained by a change in crosslinking efficiency of Spt6 relative to Rpb3. Since it can only be seen in the averaged data, the effect seems to be very subtle and can rather be explained by a rearrangement between Spt6 and Rpb3 (or Pol II) than by a recruitment of Spt6 to the transcription machinery by Ser2-phosphorylated CTD.

Unexpectedly, the Spt6 occupancy has a peak around the Stop codon and shows a prolonged signal beyond the end of the coding region in comparison to Pol II (Fig. 19 B). To test if this is significant or an artifact of our data processing, we averaged the signals of all genes, independent of their length, around the Start codon (+/- 500 bp) and the Stop codon

3 Structure and requirement of the Spt6 SH2 domain 67

Figure 19 (preceding page): Differences in averaged occupancy profiles of Pol II and Spt6

(A) Distribution of genes into classes of different length after filtering out untranscribed, overlapping and closely spaced genes (see text for details). Length classes that were used for further analysis are marked green.

(B) Plots of the averaged ChIP-on-chip signals of the different length classes from (A). Classes are indicated above the plots. Averaged Rpb3- and Spt6-ChIP signals are shown in red and blue, respectively. The absolute values of different datasets cannot be related to each other.

(C) Plots of averaged ChIP-on-chip signals of the Rpb3-, Rpb7- and Spt6-datasets around regions of the Start and the Stop codon. The ChIP-on-chip signal is averaged from all genes. Rpb3-, Rpb7- and Spt6-signals are shown in red, green and blue, respectively. Rpb3- and Rpb7-ChIP-on-chip data is from Jasiak et al (2008). The plots were kindly provided by Matthias Siebert.

(+/- 500 bp). For this we used the Rpb3 and the Spt6 datasets and in addition the data of the Rpb7 ChIP-on-chip experiment (Jasiak et al, 2008). The ChIP-data from Rpb7 is independent from the Rpb3-data, but both proteins are Pol II subunits. Indeed, Rpb3 and Rpb7 show similar profiles proximal to the Start and the Stop codon, whereas Spt6 again shows a maximum around the Stop codon and a longer retention in the 3’ regions of the genes relative to Pol II, which is strengthening the significance of our data analysis.