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Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes

The FACT (FAcilitates Chromatin Transactions) complex influences transcription initiation and enables passage of RNA polymerase (Pol) II through gene body nucleosomes during elongation. In the budding yeast, ∼280 noncoding RNA genes highly transcribed in vivo by Pol III are found in the nucleosome-free regions bordered by positioned nucleosomes. The downstream nucleosome dynamics was found to regulate transcription via controlling the gene terminator accessibility and hence, terminator-dependent Pol III recycling. As opposed to the enrichment at the 5′-ends of Pol II-transcribed genes, our genome-wide mapping found transcription-dependent enrichment of the FACT subunit Spt16 near the 3′-end of all Pol III-transcribed genes. Spt16 physically associates with the Pol III transcription complex and shows gene-specific occupancy levels on the individual genes. On the non-tRNA Pol III-transcribed genes, Spt16 facilitates transcription by reducing the nucleosome occupany on the gene body. On the tRNA genes, it maintains the position of the nucleosome at the 3′ gene end and affects transcription in a gene-specific manner. Under nutritional stress, Spt16 enrichment is abolished in the gene downstream region of all Pol III-transcribed genes and reciprocally changed on the induced or repressed Pol II-transcribed ESR genes. Under the heat and replicative stress, its occupancy on the Pol III-transcribed genes increases significantly. Our results show that Spt16 elicits a differential, gene-specific and stress-responsive dynamics, which provides a novel stress-sensor mechanism of regulating transcription against external stress. By primarily influencing the nucleosomal organization, FACT links the downstream nucleosome dynamics to transcription and environmental stress on the Pol III-transcribed genes.


IA. Spt16 ChIP-seq data analysis
The quality control results showed high reproducibility among the biological replicates of the acquired ChIP-seq data (Supplemental Figs. S1A,B,C). The plotPCA function of deeptools (2.3.2) was used to generate the panels S1B and S1C. In the PCA plot (Fig. S1B), there is a strong effect of "genomic positions" on the principal component 1 (PC1). However stacking of majority of samples on the same vertical line suggest this effect is similar across samples.
The treatment conditions have greater influence on PC2 in this case. The result (Fig. S1C) shows the first 2 or 3 principal components give maximum amount of variability in the data (>90%). The rest of principal components account for a small proportion of the variability and are trivial. Overall, panels S1B and S1C show robust clustering of the data (experimental conditions) and least variability between the replicates. Moreover, despite resolution differences, the Spt16-TAP occupancy profile on the pol II-transcribed ORFs normalized to Input/mock IP (ChIP-seq, this study) is similar and comparable (Supplemental Fig. S1D) to the earlier reported Input-normalized profile obtained by using untagged cells as controls (ChIP-chip, Mayer et al. 2010). Normalization with Input instead of the mock returned the same occupancy profile showing highest Spt16 levels at the 5' end of ORFs ( Fig. 2A and Supplemental Fig. S2D) and in the DS region on the tRNA genes (Supplemental Fig. S1E).
The differences in peak heights on the tRNA genes after normalizations (Supplemental Fig.   S1E) could be related to the differences in the Input and mock values at the positions around the tRNA genes (Supplemental Fig. S1F). These comparisons also show that the US peak is not due to the Spt16 enrichment there, as it could not be validated by the ChIP and Real Time PCR measurements as well (Fig. 2C). However, as mock is a better control (Flensburg et al. 2014) and we could get good quality mock data, it was used for all tRNA ChIP normalizations.
Similar to the tRNA genes, input-or mock-normalized occupancy profiles on the non-tRNA SNR6 were found largely similar to each other (Supplemental Fig. S2F). In contrast, we found that normalization with either mock (Supplemental Figs. S2G) or input ( Fig. 2D) gave significantly different Spt16 occupancy profiles for the pol III-transcribed non-tRNA genes RPR1 and SCR1. This could be due to the abrupt decrease in the mock signal causing large differences in the profiles of the two normalizers for these genes (Supplemental Fig. S2H, I) at the marked positions. As ChIP-Real Time PCR measurements matched the input-normalized Spt16 profiles better, all the data for the three non-tRNA genes were normalized with input for further use.

IB. High enrichment of Spt16 on tRNA genes
This study has found an unexpected enrichment of the yeast FACT subunit Spt16 at the region downstream (DS) of the 3'-ends of the pol III-transcribed genes. An earlier genomewide study had reported "hyper-ChIPability" of 238 sites including 145 tRNA genes and genes highly transcribed by Pol II as listed in the Table S1 of Teytelman et al. (2013).
According to the authors, this may not be due to a particular tag or protein and may have more to do with a step in ChIP sample preparation and processing. Though the real cause is not clear, the clear association of hyper-ChIPability with highly transcribed regions suggests involvement of open chromatin structure found on such regions. Since our mappings have been made on pol III-transcribed genes, we made some checks on our data in the context of hyper-ChIPability, high transcription and open chromatin structure of these genes and found the following.
(1) We had earlier reported the nucleosomal arrangement on the pol III-transcribed loci (Kumar and Bhargava, 2013), which showed that all tRNA genes reside in nucleosome-free regions (NFRs) of different lengths. We did not find any correlation of hyper-ChIPable tRNA genes with either the longest or any other specific NFR length. Grouping of all genes according to their NFR lengths found all hyper-ChIPable genes distributed among all the groups. This means open chromatin structure may not be the cause of hyper-ChIPability of the yeast tRNA genes.
(2) We had earlier mapped genome-wide yeast pol III occupancy (Kumar and Bhargava 2013) and found pol III on >90 pol II-transcribed genes (unpublished data). As pol III is a protein known to occupy the tRNA genes, we checked whether these pol II genes were listed as hyper-ChIPable. Interestingly, we could find only 7 of these pol III-occupied genes in the list of hyper-ChIPable pol II-transcribed genes. Moreover, all of the seven genes were not the highly transcribed ones, suggesting the pol III occupancy on pol II-transcribed genes may not be related to their highly transcribed nature.
(3) We did not find any difference in Spt16 distribution over the tRNA genes classified as hyper-ChIPable or otherwise (Supplemental Fig. S2E). Spt16 occupancy also did not show a correlation with transcription from individual tRNA genes as measured by the nascent RNA levels (Fig. 3A, Supplemental Fig. S3A, B, C).
(4) In our genome-wide data on Spt16 occupancy, average Spt16 levels on the tRNA gene bodies were found similar to the highest average levels of Spt16 at the 5' end of the pol IItranscribed genes (cf. Figs. 2A and 2B). If Spt16 was associated with tRNA genes due to their hyper-ChIPability, we should not have seen it on the tRNA gene bodies at levels similar to levels on all those tRNA genes, which do not show hyperChIPability (Supplemental Fig.   S2E) or to average levels on pol II-transcribed genes.
(5) All tRNA genes are similarly occupied by pol III and highly transcribed. Therefore, not finding all of them in the list of hyperChIPable regions may have some other reasons, not specific for tRNA genes.
(6) Out of seven genes used for validations by ChIP and Real Time qPCR in this study, three are found in the list of hyperChIPable tRNA genes. One of the seven, tE(UUC)E1 is not in the list of hyperChIPable genes but shows highest Spt16 occupancy levels; higher than tY(GUA)J2, tV(AAC)M1 and tQ(CUG)M which are found in the list.
All these observations make us believe that despite the earlier report on hyper-ChIPability of the tRNA genes, our Spt16 occupancy data represent a specific association profile of Spt16 on the pol III-transcribed genes.

IC. The tRNA genes are not the Phantom binding sites for Spt16
The "phantom" binding sites have been reported in other systems, including IgG pulldowns of TAP-tagged proteins, which we used for Spt16 here (Park et al. 2013;Jain et al. 2015).
We did not use "No tag" control but included other control (mock) like Protein A/G sepharose in place of IgG sepharose beads for the TAP tag. We have used Input as background control and used mock for normalization of the data. ORFs as reported by us (Supplemental Fig. S1D). We have also used a polyclonal Spt16specific antibody, in our ChIP Real Time PCR quantifications, along with mock immunoprecipitation involving protein A/G sepharose and non-specific antibody IgG. These measurements did not find Spt16 enrichment in the mock, again suggesting that Spt16 associations found in our data represent specific enrichments. Moreover, both the methods showed a similar trend of variations among different genes and showed highest Spt16 occupancy on the tE(UUC)E1 gene which is not in the list of hyperChIPable tRNA genes.
We have noticed that in most of the measurements, tRNA genes behave in a gene-specific manner, giving a non-uniform level of quantitative changes. Thus, the measured effects may appear small on some of the tested gens but there are other genes in the same estimation, which show larger changes. These observations are reflected in the trend seen in the wild type Spt16 DS occupancy values in the Figs 2C (using TAP tag) and the Fig. 6B (using Spt16-specific antibody) for the corresponding genes. High enrichment in the DS region as well as gene-specific trend of levels, regardless of using TAP-tag or Spt16-specific antibody for the ChIP was consistently seen.

ID. The myc tag at the Rpc160 C-terminus is masked and Rpc160 protein is not degraded in the spt16-197 cells
Rpc160-myc ChIP was comparatively very low at 30 0 C or 37 0 C in the mutant cells (Supplemental Fig. S3E). We noticed, despite confirmation of the myc tag insertion at the right genomic location by sequencing, the Rpc160-myc protein shows highly reduced myc signal in the mutant cells at both the temperatures (Supplemental Fig. S3F). As a pronounced myc signal is seen in the wild type cells, the negligible signal in the mutant cells appears to be spt16 mutation-specific and not temperature-or tag-related. This implied either degradation of Rpc160 or a masking of the myc tag, making it non-available for immunodetection in western or immuno-capture in ChIP from the mutant cells.  S3G). We ruled out the loss of Myc tag coding sequence from the genomic DNA by repeated genomic locus sequencings after several times sub-culturing of the positive clones having tagged Rpc160 gene. Considering all the above-mentioned checks, it appears that Myc tag is not detected in our probings/ ChIP because of its masking by some cellular component and not because of the degradation of the Rpc160 protein. Although our further preliminary checks on this were encouraging, further probings are beyond the scope of this manuscript.
Finally, since the normal growth of the mutant (Malone et al. 1991) may not be possible if pol III levels are severely compromised, we inferred that the Rpc160 levels are similar in both cell types, which is consistent with the similar cellular levels of total tRNAs in the wild type and spt16-197 cells (Fig. 3B, Supplemental Fig. S3D).