SUPPLEMENTARY INFORMATION

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The coding regions are divided into six equally sized bins.

1 doi: /nature11326 Supplementary Figure 1: Histone exchange increases over the ORF in a set2 mutant. (a) Gene average analysis. Schematic representation of the bin distribution over the coding and intergenic regions. The coding regions are divided into six equally sized bins. Promoters and terminators (within 500bp upstream or downstream of the gene) are divided into two bins each. (b-c) ChIP-on-chip assays were performed on 4x44K yeast genomic arrays from Agilent Technologies. The log2 ratios of IP over input 1

RESEARCH SUPPLEMENTARY INFORMATION obtained from the microarrays were subjected to a modified average gene analysis. The beige box indicates the coding region.

2 RESEARCH SUPPLEMENTARY INFORMATION obtained from the microarrays were subjected to a modified average gene analysis. The beige box indicates the coding region. TSS and TE are the Transcription Start Site and the TErmination site, respectively. The key for the distribution is provided within the plot area. The grey shading extending from the traces denote the 95% confidence interval (C.I). (b) The genome-average plot for the distribution of Myc-H3 in the wildtype and set2 exchange strains. (c) The genome-average plot for the distribution of Flag-H3 (normalized to the Myc-H3 distribution) in the wild-type and set2 mutant. (d) A majority of long genes show increased histone exchange upon loss of Set2. Genes in each category as shown in Fig. 1b were analyzed for the levels of histone exchange over the ORF. Genes with mean histone exchange greater than 0 over the ORF were identified and tabulated as above. The last column shows the percentage of genes in each category that have increased histone exchange over the ORF. (e) The change in histone exchange distribution in Figure 1a was divided into five subclasses based on transcription frequency. The average of each subclass was plotted. This data shows the inverse relation between histone exchange and transcription frequency, confirming previously published data that the Set2 pathway affects less transcribed genes. Supplementary Figure 2: H3 K56 acetylation increases over the ORF in a set2 mutant (BY4741 background). ChIP-on-chip data were plotted as in Supplementary Figure 1. (a) The genome-average plot for the distribution of H3 in the wildtype and set2 mutant. (b) The genome-average plot for the distribution of H3K56 2

RESEARCH acetylation (normalized to the H3 distribution) in the wildtype and set2 mutant. The grey shading extending from the traces denote the 95% C.I.

3 RESEARCH acetylation (normalized to the H3 distribution) in the wildtype and set2 mutant. The grey shading extending from the traces denote the 95% C.I. Supplementary Figure 3: Histone H4 acetylation increases over the ORF in a set2 mutant. ChIP-on-chip data were plotted as in Supplementary Figure 1. (a) The genome-average plot for the distribution of H4 acetylation (normalized to the Myc-H3 distribution) in the wild-type and set2 exchange strains. (b) The genome-average plot for the change in acetylated H4 distribution in a set2 exchange strain over the wildtype. (c) The data in (b) was divided into four subclasses based on the ORF length. The average of each subclass was plotted. The data presented here is a ratio of set2 over wildtype. The grey shading extending from the traces denote the 95% C.I. 3

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 4: Genes in cluster 1 longer than 1000 bps show strong correlation between histone exchange and H4ac.

4 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 4: Genes in cluster 1 longer than 1000 bps show strong correlation between histone exchange and H4ac. ChIP-on-chip data were plotted as in Supplementary Figure 1. (a) The genes in cluster 1 obtained by the k-means clustering of the acetylated H4 increase profile (Fig. 2a, red trace) were separated into four subclasses according to their gene lengths. The average of each subclass was plotted. (b) The histone exchange increase profile of genes in cluster 1 (Fig. 2b, red trace) was separated into four subclasses according to their gene lengths. The average of each subclass was plotted. (c) Correlation between the acetylated H4 data and the histone exchange data for cluster1 in the exchange strain. The Pearson s coefficient and the p-value were calculated using R for the pair of data in each gene-length group. (d) The distribution of H3 K36 tri- 4

RESEARCH methylation (normalized to the H3 distribution) in the wild-type strain for the Cluster 1 genes from Figure 2a was divided into four subclasses based on the ORF length.

5 RESEARCH methylation (normalized to the H3 distribution) in the wild-type strain for the Cluster 1 genes from Figure 2a was divided into four subclasses based on the ORF length. The average of each subclass was plotted. The grey shading extending from the traces denote the 95% C.I. Supplementary Figure 5: H3 K9 acetylation and H4 K12 acetylation increases over the ORF in a set2 mutant (BY4741 background). ChIP-on-chip data were plotted as in Supplementary Figure 1. (a) The genome-average plot for the distribution of H3 K9 acetylation (normalized to the H3 distribution) in the wildtype and set2 mutant. (b) The genome-average plot for the change in distribution of H3K9 acetylation (normalized to the H3 distribution) as a ratio of set2 over wild-type. (c) The data in (b) was divided into four subclasses based on the ORF length. The average of each subclass was plotted. The data presented here is a ratio of set2 over wildtype. (d) The genome-average plot for the distribution of H4 K12 acetylation (normalized to the H3 distribution) in the wildtype and set2 mutant. (e) The genome-average plot for the change in distribution of H4K12 acetylation (normalized to the H3 distribution) as a ratio of set2 over wildtype. 5

RESEARCH SUPPLEMENTARY INFORMATION (f) The data in (e) was divided into four subclasses based on the ORF length. The average of each subclass was plotted.

6 RESEARCH SUPPLEMENTARY INFORMATION (f) The data in (e) was divided into four subclasses based on the ORF length. The average of each subclass was plotted. The data presented here is a ratio of set2 over wildtype. The grey shading extending from the traces denote the 95% C.I. Supplementary Figure 6: The exchange-specific H3K9ac, H3K56ac, H4 K12ac increase profiles over the ORF correlates with H4ac and histone exchange. ChIP-onchip data were analyzed and plotted as in Supplementary Figure 1. (a) The ratio of H3 K9 acetylation in the set2 strain over wild-type strains for the same three clusters of genes 6

7 RESEARCH as in Figure 2a were averaged and plotted. (b) The ratio of H3 K56 acetylation in the set2 strain over wild-type strains for the same three clusters of genes as in Figure 2a were averaged and plotted. (c) The ratio of H4 K12 acetylation in the set2 strain over wild-type strains for the same three clusters of genes as in Figure 2a were averaged and plotted. (d) The cluster 1 averaged increase profiles of histone exchange, acetylated H4 (both normalized to Myc-H3), H3K56ac, H3K9ac and H4K12ac (all normalized to H3) are plotted together. (e) Correlation between the data sets tested in pairs is tabulated and represented as correlation coefficient (top) / p-value(bottom). The Pearson s coefficient and the p-value were calculated using R for the pair of data for cluster 1. The grey shading extending from the traces denote the 95% C.I. Supplementary Figure 7: Loss of Asf1 or Rtt109-mediated exchange in set2 decreases the enrichment of acetylated histones over the coding regions. ChIP-onchip data were plotted as in Supplementary Figure 1. The averaged data is presented separately for the different strains. (a, d) The whole genome average plot of the enrichment of acetylated H4 for the indicated strains over the gene. (b, e) The whole 7

8 RESEARCH SUPPLEMENTARY INFORMATION genome average plot of the enrichment of H3 for the indicated strains over the gene. (c, f) The whole genome average plot of the enrichment of acetylated H4 (normalized to H3) for the indicated strains over the gene. The grey shading extending from the traces denote the 95% C.I. Supplementary Figure 8: KAT activity of most known histone acetyltransferases is not required for initiation of cryptic transcription upon loss of Set2. Total RNA from the indicated strains were prepared and subjected to Northern blot analysis. The probe used in these assays was from the 3 end of the STE11 gene. An SCR1 probe was used as a loading control. The full-length transcript (FL) and the short cryptic transcripts (*) are indicated. (a) Northern blot analysis of KAT catalytic subunit deletion mutants either singly or in combination with Set2. RNA from the indicated strains was used in these blots. (b) Quantitation of cryptic transcripts produced in the strains assayed in Figure 4b and Supplementary Figure 8a. The cryptic transcript levels are expressed as a ratio of mutant over set2 Error bars represent +/- s.e.m. 8

9 RESEARCH Supplementary Figure 9: H3 K36 methylation increases over the ORF in an asf1 mutant over the wildtype. ChIP-qPCR assays were performed with primers spanning the entire length of the PYK1 gene. The IP over Input ratios from the H3K36 tri-methyl and H3 ChIPs were normalized to the values obtained from the inactive STE3 coding region. (a) Distribution of H3 K36 tri-methylation for the PYK1 gene in an asf1 mutant and wildtype. (b) Distribution of H3 for the PYK1 gene in an asf1 mutant and wild-type. Error bars represent +/- s.e.m. 9

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 10: H4 acetylation or H3 K9 acetylation is not required for initiation of cryptic transcription upon loss of Set2.

10 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 10: H4 acetylation or H3 K9 acetylation is not required for initiation of cryptic transcription upon loss of Set2. Total RNA from the indicated strains was prepared and subjected to Northern blot analysis as detailed in Supplementary Figure 8. (a) Northern blot analysis of histone H4 acetylation site mutants, H4 K5, 8, 12R (H4 3KR) or H3 K9A in combination with SET2 deletion. Total RNA from the indicated strains was used in this blot. (b) Quantitation of the cryptic transcripts produced in the strains assayed in Supplementary Fig.10a. Cryptic transcription in the set2 strain was used a reference (=1) to calculate the fold change in the level of transcription in the histone acetylation site, set2 -double mutants tested. All values were normalized to the SCR1 loading control. The values presented are the average of three independent experiments. Error bars represent +/- s.e.m. 10

11 RESEARCH Supplementary Figure 11: H3K56R mutation along with SET2 is synthetic sick. (a) Wild-type and set2 strains were transformed with either the wild-type pdm18 plasmid or the mutant plasmid containing H3K56R and 4-fold dilutions were spotted either on trp ura plates (left) or trp +5-FOA plates (right). (b) ChIP-on-chip data were plotted as in Supplementary Figure 1. The distribution of H3 K36 tri-methylation (normalized to the H3 distribution) in the wild-type strain was plotted along with the Flag-H3 (normalized to Myc-H3) histone exchange distribution in the wild-type exchange strain to emphasize the anti-correlation (Pearson s correlation coefficient= , P=0.003) between the two distributions. The grey shading extending from the traces denote the 95% C.I. 11

12 RESEARCH SUPPLEMENTARY INFORMATION 12

13 RESEARCH Supplementary Figure 12: H3 K56R suppresses sense cryptic transcript initiation in combination with H3K36A. mrna was prepared from the K36A, K56R, K36AK56R and wild-type histone shuffle strains and subjected to gene expression analysis on an Agilent array that was designed to detect sense transcripts alone. (a) Whole genome gene expression ratios of mutant to wildtype was subjected to a modified gene averaging analysis. The data over the ORF of each gene was averaged and plotted as in Supplementary Figure 1. The increase towards the 3 ends of the genes indicates an accumulation of cryptic transcription. Both the K56R and the K36AK56R mutants show a significant reduction in the levels of cryptic transcription. All three mutants show a decrease in full length transcription with respect to the wildtype as evidenced by a dip at the 5 end below the reference line. The grey shading extending from the traces denote the 95% C.I. (b-m) Gene expression ratios of each mutant over wildtype for different genes were normalized by loess and plotted. Figures 12b-j are examples of genes known to demonstrate cryptic initiation. Figures 12k-m are examples of genes that have high transcription rates. 13

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 13: H3 K56R suppresses antisense cryptic transcript initiation in combination with H3K36A.

14 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 13: H3 K56R suppresses antisense cryptic transcript initiation in combination with H3K36A. mrna was prepared from the K36A, K56R, K36AK56R and wild-type histone shuffle strains and subjected to gene expression analysis on an Agilent array that was designed to detect antisense transcripts alone. (a) Whole genome gene expression ratios of mutant to wildtype were plotted as detailed in Supplementary Figure 1. The grey shading extending from the traces denote the 95% C.I. The increase towards the 5 ends of the genes indicates an accumulation of antisense cryptic transcription. Both the K56R and the K36AK56R mutants show a significant reduction in the levels of cryptic transcription. (b-j) Gene expression ratios of each mutant over wildtype for different genes were normalized by loess and plotted. Figures 13b-j are genes known to demonstrate antisense cryptic initiation. 14

RESEARCH Supplementary Figure 14: H3 K56R mutant produces sense cryptic transcripts in 130 genes, 56% of which also show cryptic transcription in an H3 K36A mutant.

alone. (a) Whole genome gene expression ratios of H3 K56R mutant to wildtype were plotted as detailed in Supplementary Figure 1 The grey shading extending from the traces denote the 95% C.I.

15 RESEARCH Supplementary Figure 14: H3 K56R mutant produces sense cryptic transcripts in 130 genes, 56% of which also show cryptic transcription in an H3 K36A mutant. mrna prepared from the K56R and wild-type histone shuffle strains was subjected to gene expression analysis on separate Agilent arrays that were designed to detect sense or antisense transcripts alone. (a) Whole genome gene expression ratios of H3 K56R mutant to wildtype were plotted as detailed in Supplementary Figure 1 The grey shading extending from the traces denote the 95% C.I. The increase towards the 3 ends of the genes indicates an accumulation of sense cryptic transcripts. The absence of an increase towards the 5 ends of the genes for antisense cryptic transcripts indicates that H3 K56R does not produce antisense cryptic transcripts. (b) To determine which genes demonstrate cryptic transcription, the sense cryptic transcript mean probe signals were determined for the 5 and 3 ends of open reading frames and genes selected with a 3 /5 ratio of log2 > 0.5 (1.4 fold), for the H3K36A and H3K56R strains. We identified 1043 and 130 genes 15

RESEARCH SUPPLEMENTARY INFORMATION respectively, and found that 56% of cryptic transcript genes overlap between the two mutants. The P-value was calculated using the hypergeometric test.

16 RESEARCH SUPPLEMENTARY INFORMATION respectively, and found that 56% of cryptic transcript genes overlap between the two mutants. The P-value was calculated using the hypergeometric test. Supplementary Figure 15: H3K56R suppresses antisense cryptic transcript initiation in combination with H3K36A while an H3K56Q mutant does not. Total RNA from the indicated strains were prepared and subjected to Northern blot analysis. The probe used in these assays was from the 3 end of the STE11 and PCA1 genes. An SCR1 probe was used as a loading control. (a) Northern blot analysis of H3K56R and H3K56Q point mutants either singly or in combination with H3K36A mutant. RNA from the indicated strains was used in these blots. (b) Quantitation of cryptic transcripts produced in the strains assayed in (a). Cryptic transcription in the H3 K36A strain was used a reference (=1) to calculate the fold change in the level of transcription in the double mutants tested. All values were normalized to the SCR1 loading control. The values presented are the average of three independent experiments. Error bars represent +/- s.e.m. 16

17 RESEARCH Supplementary Figure 16: H3 K56 acetylation increases over the ORF in a set2 mutant over the wildtype. ChIP-qPCR assays were performed with primers spanning the entire length of the STE11 genes. The IP over Input ratios from the H3K56 acetyl and H3 ChIPs were normalized to the values obtained from the inactive STE3 coding region. The data is represented as the H3 K56 distribution in a set2 mutant over the wildtype after being normalized for histone occupancy. Error bars represent +/- s.e.m. 17

18 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 17: H3 K56 acetylation increases over the ORF in a H3 K36A expressing point mutant. ChIP-on-chip data were analyzed and plotted as in Supplementary Figure 1. (a) The genome-average plot for the distribution of H3K56 acetylation in the wildtype and H3K36A point mutant. (b) The genome-average plot for the distribution of H3K56 acetylation (normalized to the H3 distribution) in the wild-type and H3K36A point mutant. (c) The genome-average plot for the change in distribution of H3K56 acetylation (normalized to the H3 distribution) as a ratio of H3K36A point mutant over wildtype. (d) The data in (c) was divided into four subclasses based on the ORF length. The average of each subclass was plotted. The data presented here is a ratio of H3K36A point mutant over wildtype. The grey shading extending from the traces denote the 95% C.I. 18

The purified proteins were separated on SDS-PAGE gels and visualized by silver stain or coomassie staining. (b) Rco1-TAP complex binds H3K36me3 peptides.

19 RESEARCH Supplementary Figure 18: Purification of proteins used in the peptide pulldown assays. (a) The Asf1, Spt16 and Rco1 TAP tagged protein complexes were purified from yeast strains. Spt6-Flag was purified from Sf21 cells infected with recombinant baculovirus. The purified proteins were separated on SDS-PAGE gels and visualized by silver stain or coomassie staining. (b) Rco1-TAP complex binds H3K36me3 peptides. Interaction between unmodified or modified H3 peptide and the Rco1-Tap complex was analyzed by Western blotting the peptide pulldown with the anti-cbp antibody. This confirms previously published data that the H3K36 trimethylated peptide binds the Rpd3S complex. 19

20 RESEARCH SUPPLEMENTARY INFORMATION Table S1: List of strains used in the study Name Genotype Parental Strain BY4741 MATa his3 1 leu2 0 met15 0 ura3 0 BY4741 YSV100 MATa his3 1 leu2 0 met15 0 ura3 0 set2 ::URA3 BY4741 This Study YSV101 MATa his3 1 leu2 0 met15 0 ura3 0 asf1 ::KAN BY4741 Open Biosytems YSV102 MATa his3 1 leu2 0 met15 0 ura3 0 rtt109 ::KAN BY4741 Open Biosytems YSV103 MATa his3 1 leu2 0 met15 0 ura3 0 gcn5 ::KAN BY4741 Open Biosytems YSV104 MATa his3 1 leu2 0 met15 0 ura3 0 hat1 ::KAN BY4741 Open Biosytems YSV105 MATa his3 1 leu2 0 met15 0 ura3 0 sas2 ::KAN BY4741 Open Biosytems YSV106 MATa his3 1 leu2 0 met15 0 ura3 0 sas3 ::KAN BY4741 Open Biosytems YSV107 MATa his3 1 leu2 0 met15 0 ura3 0 elp3 ::KAN BY4741 Open Biosytems YSV108 MATa his3 1 leu2 0 met15 0 ura3 0 asf1 ::KAN set2 ::URA3 BY4741 This Study YSV109 MATa his3 1 leu2 0 met15 0 ura3 0 rtt109 ::KAN set2 ::URA3 BY4741 This Study YSV110 MATa his3 1 leu2 0 met15 0 ura3 0 gcn5 ::KAN set2 ::URA3 BY4741 This Study YSV111 MATa his3 1 leu2 0 met15 0 ura3 0 hat1 ::KAN set2 ::URA3 BY4741 This Study YSV112 MATa his3 1 leu2 0 met15 0 ura3 0 sas2 ::KAN set2 ::URA3 BY4741 This Study YSV113 MATa his3 1 leu2 0 met15 0 ura3 0 sas3 ::KAN set2 ::URA3 BY4741 This Study YSV114 MATa his3 1 leu2 0 met15 0 ura3 0 elp3 ::KAN set2 ::URA3 BY4741 This Study YBL574 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2-hhf2(trp1) S288C Kind Gift Dr. Fred Winsto YSV115 hhf2) ::HIS3 Ty lacZ::his4 set2 ::KAN pdm18-hht2-hhf2(trp1) S288C This Study YSV116 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2(k9a)-hhf2(trp1) S288C This Study YSV117 hhf2) ::HIS3 Ty lacZ::his4 set2 ::KAN pdm18-hht2(k9a)-hhf2(trp1) S288C This Study YSV118 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2-hhf2(k5,8,12r)(trp1) S288C This Study YSV119 hhf2) ::HIS3 Ty lacZ::his4 set2 ::KAN pdm18-hht2- HHF2(K5,8,12R)(TRP1) S288C This Study YSV120 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2(k36a)-hhf2(trp1) S288C This Study YSV121 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2(k56r)-hhf2(trp1) S288C This Study YSV122 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2(k36a K56R)-HHF2(TRP1) S288C This Study YSV123 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2(k56q)-hhf2(trp1) S288C This Study YSV124 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2(k36a K56Q)-HHF2(TRP1) S288C This Study YSV125 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2-hhf2(trp1) bar1 ::CloNAT S288C This Study YSV126 hhf2) ::HIS3 Ty lacZ::his4 pdm18-hht2 (K36A)-HHF2(TRP1) bar1 ::CloNAT S288C This Study MDY510 MATa lys2-801 ade2-101 trp1d63 his3d200 leu2d1 hht1-hhf1::pwz405-f2f9- LEU2 hht2-hhf2::pwz403-f4f10-his3 [pnoy439(cen6 ARS4 TRP1 MYC- HHT2-HHf2] URA3::YIplac211pGAL1/10-FLAG-HHT1-HHF1 bar1 ::KAN MDY510 Kind Gift Dr. Oliver Rando YSV127 MATa lys2-801 ade2-101 trp1d63 his3d200 leu2d1 hht1-hhf1::pwz405-f2f9- LEU2 hht2-hhf2::pwz403-f4f10-his3 [pnoy439(cen6 ARS4 TRP1 MYC- HHT2-HHf2] URA3::YIplac211pGAL1/10-FLAG-HHT1-HHF1 bar1 ::KAN set2 ::HphB MDY510 This Study YSV128 MATa his3 1 leu2 0 met15 0 ura3 0 bar1 ::LEU2 BY4741 This Study YSV129 MATa his3 1 leu2 0 met15 0 ura3 0 set2 ::URA3 bar1 ::LEU2 BY4741 This Study YSV130 MATa his3 1 leu2 0 met15 0 ura3 0 asf1 ::KAN bar1 ::LEU2 BY4741 This Study YSV131 MATa his3 1 leu2 0 met15 0 ura3 0 asf1 ::KAN set2 ::URA3 bar1 ::LEU2 BY4741 This Study YSV132 MATa his3 1 leu2 0 met15 0 ura3 0 rtt109 ::KAN bar1 ::LEU2 BY4741 This Study YSV133 MATa his3 1 leu2 0 met15 0 ura3 0 rtt109 ::KAN set2 ::URA3 bar1 ::LEU2 BY4741 This Study 20

21 RESEARCH Table S2: List of antibodies used in the study Antibody Supplier Catalog # Flag Sigma F1804 Myc Sigma M4439 Histone H3 Abcam ab1791 Pan-acetylated H4 Millipore H3K56 acetyl Millipore H3K9 acetyl Millipore H4K12 acetyl Abcam ab1761 H3K36 trimethyl Abcam ab9050 Anti-CBP (TAP) Prepared by Workman lab 21