Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone
Abstract 
Funding : PH is a senior fellow of the Canadian Institute For Advanced Research ( CIFAR ) and acknowledges support from the National Institutes of Health ( operating grant R01 : 4R01CA158162 ) and Canadian Institutes of Health Research ( CIHR operating grant MOP-38096 ) . 
MSK is a Senior Fellow of CIFAR and is funded by CIHR operating grant MOP-119383 and MOP-119372 . 
YAC acknowledges scholarship support from the Natural Sciences and Engineering Research Council of Canada , as well as the Roman M. Babicki Fellowship in Medical Research . 
MJA and PYTL were supported by Frederick Banting and Charles Best Canada graduate scholarships from CIHR . 
PCS was a fellow of the Terry Fox Foundation ( # 700044 ) , the Michael Smith Foundation for Health Research and is currently supported by the Cancer Research Society . 
The funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript . 
Introduction
Elevated DNA : RNA hybrid formation due to defects in RNA processing pathways leads to genome instability and replication stress across species [ 1 -- 7 ] . 
R loops threaten genome stability and often form under abnormal conditions where nascent mRNA is improperly processed or RNA half-life is increased , resulting in RNA that can hybridize with template DNA , displacing the nontranscribed DNA strand [ 8 ] . 
A recent study also found that hybrid formation can occur in trans via Rad51-mediated DNA-RNA strand exchange [ 9 ] . 
Persistent R loops pose a major threat to genome stability through two mechanisms . 
First , the exposed nontranscribed strand is susceptible to endogenous DNA damage due to the increased exposure of chemically reactive groups . 
The second , more widespread mechanism , identified in Escherichia coli , Saccharomyces cerevisiae , Caenorhabditis elegans and human cells , involves the R loops and associated stalled transcription complexes , which block DNA replication fork progression [ 3,4,8,10,11 ] . 
R loop-mediated instability is an area of great interest primarily because genome instability is considered an enabling characteristic of tumor formation [ 12 ] . 
Moreover , mutations in RNA splicing / processing factors are frequently found in human cancer , heritable diseases like Aicardi-Goutieres syndrome , and a degenerative ataxia associated with Senataxin mutations [ 13 -- 17 ] . 
To avoid the deleterious effects of R loops , cells express enzymes for the removal of abnormally formed DNA : RNA hybrids . 
In S. cerevisiae , RNH1 and RNH201 , each encoding RNase H are responsible for one of the best characterized mechanisms for reducing R loop formation by enzymatically degrading the RNA in DNA : RNA hybrids [ 8 ] . 
Another extensively studied anti-hybrid factor is the THO/TREX complex which functions to suppress hybrid formation at the level of transcription termination and mRNA packaging [ 4,11,18,19 ] . 
In addition , the Senataxin helicase , yeast Sen1 , plays an important role in facilitating replication fork progress through transcribed regions and unwinding RNA in hybrids to mitigate R loop formation and RNA polymerase II transcription-associated genome instability [ 5,20 ] . 
Several additional anti-hybrid mechanisms have also been identified including topoisomerases and other RNA processing factors [ 2,6,7,9,21 -- 23 ] . 
To add to the complexity of DNA : RNA hybrid management in the cell , hybrids also occur naturally and have important biological functions [ 24 ] . 
In human cells , R loop formation facilitates immunoglobulin class switching , protects against DNA methylation at CpG island promoters and plays a key role in pause site-dependent transcription termination [ 25 -- 28 ] . 
Transcription of telomeres by RNA polymerase II also produces telomeric repeatcontaining RNAs ( TERRA ) , which associate with telomeres and inhibit telomere elongation in a DNA : RNA hybrid-dependent fashion [ 29 -- 31 ] . 
Noncoding ( nc ) RNA such as antisense transcripts , perform a regulatory role in the expression of sense transcripts that may involve R loops [ 32 ] . 
The proposed mechanisms of antisense transcription regulation are not clearly understood and involve different modes of action specific to each locus . 
Current models include chromatin modification resulting from antisense-associated transcription , antisense transcription modulation of transcription regulators , collision of sense and antisense transcription machineries and antisense transcripts expressed in trans interacting with the promoter for sense transcription [ 32 -- 40 ] . 
More recently , studies in Arabidopsis thaliana found an antisense transcript that forms R loops , which can be differentially stabilized to modulate gene regulation [ 41 ] . 
Similarly , in mouse cells the stabilization of an R loop was shown to inhibit antisense transcription [ 42 ] . 
Here we describe , for the first time , a genome-wide profile of DNA : RNA hybrid prone loci in S. cerevisiae by DNA : RNA immunoprecipitation followed by hybridization on tiling micro-arrays ( DRIP-chip ) . 
We found that DNA : RNA hybrids occurred at highly transcribed regions in wild type cells , including some identified in previous studies . 
Remarkably , we observed that DNA : RNA hybrids were significantly associated with genes that have corresponding antisense transcripts , suggesting a role for hybrid formation at these loci in gene regulation . 
Consistently , we found that genes whose expression was altered by overexpression of RNase H were also significantly associated with antisense transcripts . 
A small-scale cytological screen found that diverse 
RNA processing mutants had increased hybrid formation and additional DRIP-chip studies revealed specific hybrid-site biases in the RNase H , Sen1 and THO complex subunit Hpr1 mutants . 
These genome-wide analyses enhance our understanding of DNA : RNA hybrid-forming regions in vivo , highlight the role of cellular RNA processing activities in suppressing hybrid formation , and implicate DNA : RNA hybrids in control of a subset of antisense regulated loci . 
Results
The genomic distribution of DNA:RNA hybrids
DNA : RNA hybrids have been previously immunoprecipitated at specific genomic sites such as rDNA , selected endogenous loci , and reporter constructs [ 2,5 ] . 
Subsequently , DRIP coupled with deep sequencing in human cells has demonstrated the prevalence of R loops at CpG island promoters with high GC skew [ 26 ] . 
To investigate the global profile of DNA : RNA hybrid prone loci in a tractable model , we performed genome-wide DRIP-chip analysis of wild type S. cerevisiae ( ArrayExpress E-MTAB-2388 ) using the S9 .6 monoclonal antibody which specifically binds DNA : RNA hybrids , as characterized previously [ 43,44 ] . 
DRIP-chip profiles were generated in duplicate ( spearman 's r = 0.78 when comparing each of over 2 million probes after normalization and data smoothing , Supplementary Figure S1 ) and normalized to a no antibody control . 
Overall , our DRIP-chip profiles identified several previously reported DNA : RNA hybrid prone sites including the rDNA locus and telomeric repeat regions ( Figure 1 , Supplementary Tables S1 , S2 ) [ 2,29 -- 31 ] . 
DNA : RNA hybrids were also observed at 1217 open reading frames ( ORFs ) ( containing greater than 50 % of probes above the threshold of 1.5 and found in both wild type replicates ) ( Supplementary Table S3 ) . 
These were generally shorter in length than average ( p = 4.29 e ) , highly transcribed 258 26 ( Wilcoxon rank sum test p = 2.21 e ) , and had higher GC content 250 ( p = 2.52 e ) ( Figure 2A , 2B and 2C , Supplementary Figure S2 ) . 
Importantly , despite the correlation between DNA : RNA hybrid association and transcriptional frequency , the wild type DRIP-chip profiles compared to the localization profile of the RNA polymerase II subunit Rpb3 revealed very low correlation ( r = 0.0097 ; [ 45 ] ) . 
This suggests that the DRIP-chip method was not unduly biased towards the short DNA : RNA hybrids that could theoretically have been captured within active transcription bubbles . 
Importantly , because genes with high GC content also have high transcriptional frequencies ( Supplementary Figure S3 ) , it is not clear from our findings whether GC content or transcriptional frequency contributed more to DNA : RNA hybrid forming potential . 
Furthermore , we observe that DNA : RNA hybrid prone loci do not encode for mRNA transcripts with particularly long half-lives ( Supplementary Figure S2D ) , suggesting that the act of transcription is vital to DNA : RNA hybrid formation and supporting the notion of co-transcriptional hybrid formation as the major source of endogenous DNA : RNA hybrids . 
Our data also revealed DNA : RNA hybrids highly associated with Ty1 and Ty2 subclasses of retrotransposons ( Figure 2E , Supplementary Table S4 ) . 
Consistent with our findings at ORFs , the levels of DNA : RNA hybrids correspond well with the known levels of expression of these elements . 
In general , Ty1 which constitutes one of the most abundant transcripts in the cell has the highest levels of DNA : RNA hybrids . 
Ty3 and Ty4 that are only slightly expressed have much lower levels of hybrids , and the lone Ty5 retrotransposon which is transcriptionally silent is not enriched for DNA : RNA hybrids ( Figure 2E ) ( [ 46 -- 48 ] ) . 
In contrast to the trends observed with ORFs , GC content in retrotransposons is not highly correlated with the levels of expression , suggesting that expression is the main contributor to 
DNA : RNA hybrid formation . 
Specifically , Ty3 retrotransposons have the highest GC content but have only modest levels of expression and DNA : RNA hybrids . 
DNA : RNA hybrids are significantly correlated with genes associated with antisense transcripts 
Certain DNA : RNA hybrid enriched regions identified by our DRIP-chip analysis such as rDNA and retrotransposons are associated with antisense transcripts [ 49,50 ] . 
Therefore , we checked if this was a common feature of DNA : RNA prone sites by comparing our list of DNA : RNA prone loci to a list of antisense-associated genes ( [ 51 ] ) . 
Because the expression of antisense-associated transcripts may be highly dependent on environmental conditions , we based our analysis on a list of transcripts identified in S288c yeast grown to mid-log phase in rich media which most closely mirrors the growth conditions of our cultures analyzed by DRIP-chip ( [ 51 ] ) . 
DNA : RNA hybrid enriched genes significantly overlapped with antisense-associ-ated genes , suggesting that DNA : RNA hybrids may play a role in antisense transcript-mediated regulation of gene expression ( Fisher 's exact test p = 1.03 e ) ( Figure 3A , 3B and 3C , 212 Supplementary Table S5 ) . 
RNase H overexpression reduces detectable levels of DNA : RNA hybrids in cytological screens and suppresses genomic instability associated with R loop formation presumably through the degradation of DNA : RNA hybrids [ 7,52,53 ] . 
To test for a potential role of DNA : RNA hybrids in antisense-mediated gene regulation , we performed gene expression microarray analysis of an RNase H overexpression strain compared to an empty vector control ( GEO GSE46652 ) . 
This identified genes that had increased mRNA levels ( upregulated n = 212 ) or decreased mRNA levels ( downregulated n = 88 ) as a result of RNase H overexpression . 
A significant portion of the genes with increased mRNA levels were antisense-associated ( Fisher exact test p = 2.9 e ) 27 ( Figure 3D , Supplementary Table S5 ) and tended to have high GC content , similar to DNA : RNA hybrid enriched genes in wild type ( Supplementary Figure S4 ) . 
However , the genes with increased mRNA levels under RNase H overexpression and the antisense-associated genes enriched for DNA : RNA hybrids in our DRIP experiment both tended towards lower transcriptional frequencies ( Figure 3E ) . 
These findings suggest that antisense-associated DNA : RNA hybrids moderate the levels of gene expression . 
Indeed , genes that were both modulated by RNase H overexpression and enriched for DNA : RNA hybrids were all found to be antisense-associated ( Figure 3F ) . 
The mechanism underlying altered gene expression in cells overexpressing RNase H remains unclear . 
While the association with antisense transcription is compelling , alternative models exist . 
One possibility is that the stress of RNase H overexpression triggers gene expression programs that coincidentally are antisense regulated . 
We analyzed gene ontology ( GO ) terms enriched among genes whose expression was changed by RNase H overexpression . 
Consistent with previous work , genes for iron uptake and incorporation were strongly activated by RNase H overexpression ( p = 2.21 e ) ( Figure 4A , Supplementary 212 Table S6 ) and several of these iron transport genes ( i.e. FRE4 , FRE2 , FRE3 , FET3 , FET4 ) are antisense-associated ( [ 51,54 ] ) suggesting that overexpression of RNase H activates transcription of these genes by perturbing antisense-mediated regulation . 
Alternatively , changes in RNase H levels may increase the cellular iron requirements since sensitivity to low iron concentration is associated with DNA damage and repair [ 55 ] . 
To test this alternative hypothesis , we tested the RNase H deletion and sen1-1 mutants for sensitivity to low iron conditions compared to a fet3D positive control ( Figure 4B ) . 
The sen1-1 mutant , RNase H depletion or overexpression did not induce sensitivity to low iron ruling out the possibility that the transcriptional response in cells overexpressing RNase H was a result of cellular iron requirement . 
Collectively , our DRIP-chip and microarray analysis suggest that DNA : RNA hybrids may be an important player in antisensemediated gene regulation . 
Cytological profiling of RNA processing mutants for R loop formation
Transcription-coupled DNA : RNA hybrids have been shown to accumulate in a diverse set of transcription and RNA processing mutants involved in a wide range of transcription related processes ( Table 1 ) . 
To gain a broader understanding of factors involved in R loop formation , we performed a cytological screen of RNA processing , transcription and chromatin modification mutants for 
DNA : RNA hybrids using the S9 .6 antibody . 
Importantly , previous work in our lab has shown that all of the mutants screened exhibit chromosome instability ( CIN ) , which would be consistent with increased hybrid formation [ 53 ] . 
Significantly elevated hybrid levels were found in 22 of the 40 mutants tested compared to wild type , including a SUB2 mutant which has been previously linked to R loop formation ( Figure 5 , [ 4 ] ) . 
We also assayed some of the well-characterized R-loop forming mutants , RNase H , Sen1 and Hpr1 , as positive controls for elevated DNA : RNA hybrid levels ( Figure 5 ) . 
In our screen , we detected hybrids in mutants affecting several pathways linked to DNA : RNA hybrid formation such as transcription , nuclear export and the exosome ( Figure 5 , Table 1 ) . 
Consistent with findings in metazoan cells , we also observed hybrid formation in some splicing mutants ( Figure 5 , Table 1 ; [ 56 ] ) . 
Several rRNA processing mutants were enriched for DNA : RNA hybrids ( 7 out of the 22 positive hits ) , likely due to DNA : RNA hybrid accumulation at rDNA genes , a sensitized hybrid formation site ( Figure 1 ; [ 2 ] ) . 
It is possible that , as seen in mRNA cleavage and polyadenylation mutants , DNA : RNA hybrid formation may contribute to their CIN phenotypes [ 6 ] . 
Currently , there are 52 yeast genes whose disruptions have been found to lead to DNA : RNA hybrid accumulation , 21 of which were newly identified by our screen ( Table 1 ) . 
The success of this small-scale screen suggests that most RNA processing pathways suppress hybrid formation to some degree and that many DNA : RNA hybrid forming mutants remain undiscovered . 
DRIP-chip profiling of R loop forming mutants
To better understand the mechanism by which cells regulate DNA : RNA hybrids , we performed DRIP-chip analysis of rnh1Drnh201D , hpr1D , and sen1-1 mutants in order to determine if these contribute differentially to the DNA : RNA hybrid genomic profile . 
The rnh1Drnh201D , hpr1D , and sen1-1 mutants are particularly interesting because they have well established roles in the regulation of transcription dependent DNA : RNA hybrid formation . 
Our DRIP-chip profiles revealed that , similar to wild type profiles , the mutant profiles were enriched for DNA : RNA hybrids at rDNA , telomeres , and retrotransposons ( Figure 6 , Supplementary Tables S1 , S2 , S3 ) . 
The rnh1Drnh201D , hpr1D , and sen1-1 mutants also exhibited DNA : RNA hybrid enrichment in 1206 , 1490 and 1424 ORFs respectively compared to the 1217 DNA : RNA hybrid enriched ORFs identified in wild type ( Supplementary Table S4 ) . 
Interestingly , in addition to the similarities described above , our profiles also identified differential effects of the mutants on the levels of DNA : RNA hybrids . 
In particular , we observed that deletion of HPR1 resulted in higher levels of DNA : RNA hybrids along the length of most ORFs with a preference for longer genes compared to wild type ( Figure 7A , 7B and 7C ) . 
This observation is consistent with Hpr1 's role in bridging transcription elongation to mRNA export and its localization at actively transcribed genes ( [ 4,57 -- 59 ] ) . 
In contrast , mutating SEN1 resulted in higher levels of DNA : RNA hybrids at shorter genes ( Figure 7A and 7B ) , which is consistent with Sen1 's role in transcription termination particularly for short proteincoding genes ( [ 5,60,61 ] ) . 
The rnh1Drnh201D mutant revealed higher levels of DNA : RNA hybrids at highly transcribed and longer genes ( Figure 7A and 7B ) which is supported by a wealth of evidence of RNase H 's role in suppressing R loops in long genes to prevent collisions between transcription and replication 
Further inspection of our profiles also revealed that rnh1Drnh201D and sen1-1 mutants but not the hpr1D mutant had increased DNA : RNA hybrids at tRNA genes ( two tailed unpaired Wilcox test p = 1.56 e in the rnh1Drnh201D mutant and 219 1.68 e in the sen1-1 mutant ) ( Figure 8A , 8B and 8C , 215 Supplementary Table S7 ) and this was confirmed by DRIP-quantitative PCR ( qPCR ) of two tRNA genes in wild type and rnh1Drnh201D ( Supplementary Figure S5 ) . 
Because tRNAs are transcribed by RNA polymerase III , this observation indicates that Hpr1 is primarily involved in the regulation of RNA polymerase II specific DNA : RNA hybrids while RNase H and Sen1 have roles in a wider range of transcripts . 
Mutation of SEN1 also led to increased levels DNA : RNA hybrids at snoRNA ( two tailed unpaired Wilcox test p = 1.81 e ) ( Figure 8D , 8E and 8F , 26 Supplementary Table S8 ) consistent with its role in 39 end processing of snoRNAs ( [ 63 ] ) . 
Discussion
The genomic profile of DNA:RNA hybrids
Identifying the landscape of genomic loci predisposed to DNA : RNA hybrids is of fundamental importance to delineating mechanisms of hybrid formation and the contributions of various cellular pathways . 
Although our profiles depend on the specificity of the anti-DNA : RNA hybrid S9 .6 monoclonal antibody , this aspect has been well characterized [ 44 ] and several of our observations are consistent with what has been reported in the literature . 
Locus specific tests showed that DNA : RNA hybrids occur more frequently at genes with high transcriptional frequency and GC content [ 4,5,18 ] . 
Moreover , in rnh201D cells , there is an inverse relationship between GC content and gene expression levels , suggesting that DNA : RNA hybrids accumulate at regions of high GC content and block transcription in the absence of RNase H [ 64 ] . 
Our work extends the knowledge of DNA : RNA hybrids from a few locus-specific observations to show that , in wild type , there are potentially hundreds of hybrid prone genes that tend to be shorter in length , frequently transcribed and high in GC content [ 2,4,56 ] . 
The latter is consistent with recent studies in human cells that demonstrated that genomic regions with high GC skew are prone to R loop formation , which plays a regulatory role in DNA methylation [ 26,27 ] . 
However , while we determined the relationship between GC content and DNA : RNA hybrid formation , we were unable to do the same analysis for GC skew , likely due to the low level of GC skew and lack of DNA methylation in Saccharomyces . 
This is unsurprising since the best characterized functional element associated with GC skew , CpG island promoters [ 26,27 ] , are not found in yeast . 
Importantly , our findings at retrotransposons support the notion that expression levels and not GC content contribute more to DNA : RNA hybrid forming potential . 
Additionally , DRIP-chip analysis of wild type cells identified hybrid enrichment at rDNA , retrotransposons , and telomeric regions . 
Along with previous studies , our DRIP-chip analysis confirms that rDNA is a hybrid prone genomic site and suggests that many factors of rRNA processing and ribosome assembly suppress potentially damaging rDNA : rRNA hybrid formation [ 2,7 ] . 
The presence of TERRA-DNA hybrids at telomeres is supported by our observation of significant hybrid signal at telomeric repeat regions across all DRIP-chip experiments . 
Antisense association of DNA:RNA hybrids
The DRIP-chip dataset is a resource for future studies seeking to elucidate the localization of DNA : RNA hybrids across antisense-associated regions and the impact of DNA : RNA hybrid removal on genome-wide transcription . 
We observed that genes associated with antisense transcripts were significantly enriched for 
DNA : RNA hybrids and modulated at the transcript level by RNase H overexpression . 
Antisense regulation has been reported at mammalian rDNA and yeast Ty1 retrotransposons , loci that were also enriched for DNA : RNA hybrids in our DRIP-chip [ 49,50 ] . 
The role of DNA : RNA hybrids and RNase H in antisense regulation is currently unclear . 
However , there are several non-exclusive models of antisense gene regulation . 
One model proposes that the physical presence of the antisense transcripts is crucial to antisense gene regulation . 
For instance , trans-acting antisense transcripts have been shown to control Ty1 retrotransposon transcription , reverse transcription and retrotransposition [ 65 ] . 
Another study has further shown that trans-acting antisense transcripts that only overlap with the sense strand promoter can block sense transcription , potentially by hybridizing with the nontemplate DNA strand [ 33 ] . 
These suggest that antisense transcription in cis is not necessary as long as the antisense transcript is present . 
It is possible that DNA : RNA hybrids may be formed by the antisense or the sense transcript with genomic DNA . 
Moreover , DNA : RNA hybrids may play a functional role in antisense transcription regulation as shown by antisense-associated genes both enriched for DNA : RNA hybrids and affected transcriptionally by RNase H overexpression . 
Experiments comparing the ratio of antisense versus sense transcripts and determining the amount of DNA : RNA hybrid formation by either transcript under conditions known to regulate the particular gene will further elucidate the role of RNase H and DNA : RNA hybrids in antisense regulation . 
DRIP-chip analysis of hybrid-resolving mutants
Our investigation of mutant-specific DNA : RNA hybrid formation sites is consistent with the existing literature on Hpr1 , Sen1 and RNase H. Significantly , the hpr1D and rnh1Drnh201D mutants exhibited increased DNA : RNA hybrid levels along the length of long genes , while the sen1-1 mutant exhibited increased 
DNA : RNA hybrid levels along the length of short genes ( Figure 7A ) . 
This coheres with Hpr1 's function in transcription elongation and mRNA export , and RNase H 's role in preventing transcription apparatus and replication fork collisions , which carry greater consequence for long genes ( [ 4,57 -- 59,62 ] ) . 
In contrast , Sen1 is particularly important for transcription termination at short genes ( [ 61 ] ) . 
In addition , the RNase H deletion and sen1-1 mutants had increased hybrids at tRNA genes , suggesting that they are both required to prevent tRNA : DNA hybrid accumulation . 
Interestingly , a recent study found that the mRNA levels of genes encoding RNA polymerase III and proteins that modify tRNA are increased in an rnh1Drnh201D mutant [ 64 ] , which may be in response to a lack of properly processed tRNA transcripts . 
The finding that both tRNA and snoRNA genes were enriched for hybrids in sen1-1 highlights the role of Sen1 in RNA polymerase I , II and III transcription termination and transcript maturation [ 60,63,66 ] . 
More broadly , our data and the literature support the notion that transcripts from RNA polymerases I , II and III can be subject to DNA : RNA hybrid formation especially in RNA processing mutant backgrounds . 
Perspective
Factors regulating ectopic , genome destabilizing DNA : RNA hybrids are best characterized in yeast , although less is known about the functions of native R loop structures . 
The genome-wide maps of DNA : RNA hybrids presented here recapitulate the known sites of hybrid formation but also add important new insights to potential functions of R loops . 
Most importantly , we demonstrate the usefulness of DRIP profiling for detecting biologically meaningful differences in mutant strains . 
Therefore , DRIP profiling of yeast genomes in various mutant backgrounds will be key to understanding the causes and consequences of inappropriate R loop formation and how these are modulated by other cellular pathways . 
Methods
Strains and plasmids
All strains are listed in Supplementary Table S9 . 
For RNase H overexpression experiments , recombinant human RNase H1 was expressed from plasmid p425-GPD-RNase H1 ( 2m , LEU2 , GPDpr-RNase H1 ) and compared to an empty control plasmid p425-GPD ( 2m , LEU2 , GPDpr ) [ 7 ] . 
DRIP-chip and qPCR
Briefly , cells were grown overnight , diluted to 0.15 OD600 and grown to 0.7 OD600 . 
Crosslinking was done with 1 % formaldehyde for 20 minutes . 
Chromatin was purified as described previously [ 67 ] and sonicated to yield approximately 500 bp fragments . 
40 mg of the anti-DNA : RNA hybrid monoclonal mouse antibody S9 .6 ( gift from Stephen Leppla ) was coupled to 60 mL of protein A magnetic beads ( Invitrogen ) . 
For ChIP-qPCR , crosslinking reversal and DNA purification were followed by qPCR analysis of the immunoprecipitated and input DNA . 
DNA was analyzed using a Rotor-Gene 600 ( Corbett Research ) and PerfeCTa SYBR green FastMix ( Quanta Biosciences ) . 
Samples were analyzed in triplicate on three independent DRIP samples for wild type and rnh1Drnh201D . 
Primers are listed in Supplementary Table S11 . 
For DRIP-chip , precipitated DNA was amplified via two rounds of T7 RNA polymerase amplification ( [ 68 ] ) , biotin labeled and hybridized to Affymetrix 1.0 R S. cerevisiae microarrays . 
Samples were normalized to a no antibody control sample ( mock ) using the rMAT software and relative occupancy scores were calculated for all probes using a 300 bp sliding window . 
All profiles were generated in duplicate and replicates were quantile normalized and averaged . 
Spearman correlation scores between replicates are listed in Supplementary Table S10 . 
Coordinates of enriched regions are available in Dataset S1/S2/S3 / S4/S5/S6 / S7 / S8 . 
DRIP-chip data is available at ArrayExpress E-MTAB-2388 . 
DRIP-chip analysis
Enriched features had at least 50 % of the probes contained in the feature above the threshold of 1.5 . 
Only features enriched in both replicates were reported . 
Transcriptional frequency [ 69 ] , GC content ( [ 70 ] ) and gene length were compared using the Wilcoxon rank sum test . 
Antisense association was analyzed by the Fisher 's exact test using R. Statistical analysis of genomic feature enrichment was performed using a Monte Carlo simulation , which randomly generates start positions for the particular set of features and calculates the proportion of that feature that would be enriched in a given DRIP-chip profile if the feature were distributed at random [ 67 ] . 
500 simulations were run per feature for each DRIP-chip replicate to obtain mean and standard deviation values . 
These values were used to calculate the cumulative probability ( P ) on a normal distribution of seeing a score lower than the observed value by chance . 
DRIP-chip visualization
CHROMATRA plots were generated as described previously ( [ 71 ] ) . 
Relative occupancy scores for each transcript were binned into segments of 150 bp . 
Transcripts were sorted by their length , transcriptional frequency or GC content and aligned by their Transcription Start Sites ( TSS ) . 
For transcriptional frequency transcripts were grouped into five classes according to their transcriptional frequency described by Holstege et al 1998 . 
For GC content transcripts were grouped into four classes according to their GC content obtained from BioMart ( [ 70 ] ) . 
Average gene , tRNA or snoRNA profiles were generated by averaging all the probes that were encompassed by the features of interest . 
For averaging ORFs , corresponding probes were split into 40 bins while 1500 bp of UTRs and their probes were split into 20 bins . 
For smaller features like tRNAs and snoRNAs corresponding probes were split into only 3 bins . 
Average enrichment scores were calculated using in house scripts that average the score of all the 
Gene expression microarray
Gene expression microarray data is available at GEO GSE46652 . 
Strains harboring the RNase H1 over-expression plasmid or empty vector were grown in SC-Leucine at 30uC . 
All profiles were generated in duplicate . 
Total RNA was isolated from 1 OD600 of yeast cells using a RiboPure Yeast kit ( A&B Applied Biosystems ) , amplified , labeled , fragmented using a Message-Amp III RNA Amplification Kit ( A&B Applied Biosystems ) and hybridized to a GeneChIP Yeast Genome 2.0 microarray using the GeneChip Hybridization , Wash , and Stain Kit ( Affymetrix ) . 
Arrays were scanned by the Gene Chip Scanner 3000 7G and expression data was extracted using Expression Console Software ( Affymetrix ) with the MAS5 .0 statistical algorithm . 
All arrays were scaled to a median target intensity of 500 . 
A minimum cut off of pvalue of 0.05 and signal strength of 100 across all samples were implemented and only transcripts that had over a 2-fold change in the RNase H over-expression strain compared to wild type were considered significant . 
The correlation between duplicate biological samples was : control ( r = 0.9955 ) , RNase H over-expression ( r = 0.9719 ) . 
For statistical analysis , GC content , transcription frequencies and antisense association were analyzed as for DRIP ¬ 
Yeast chromosome spreads
Cells were grown to mid-log phase in YEPD rich media at 30uC and washed in spheroplasting solution ( 1.2 M sorbitol , 0.1 M potassium phosphate , 0.5 M MgCl2 , pH 7 ) and digested in spheroplasting solution with 10 mM DTT and 150 mg/mL Zymolase 20T at 37uC for 20 minutes similar to previously described ( [ 72 ] ) . 
The digestion was halted by addition of ice-cold stop solution ( 0.1 M MES , 1 M sorbital , 1 mM EDTA , 0.5 mM MgCl2 , pH 6.4 ) and spheroplasts were lysed with 1 % vol/vol Lipsol and fixed on slides using 4 % wt/vol paraformal-dehyde/3 .4 % wt/vol sucrose ( [ 73 ] ) . 
Chromosome spread slides were incubated with the mouse monoclonal antibody S9 .6 ( 1 mg/mL in blocking buffer of 5 % BSA , 0.2 % milk and 16 PBS ) . 
The slides were further incubated with a secondary Cy3-conjugated goat anti-mouse antibody ( Jackson Laboratories , # 115-165-003 , diluted 1:1000 in blocking buffer ) . 
For each replicate , at least 100 nuclei were visualized and manually counted to obtain the fraction with detectable DNA : RNA hybrids . 
Each mutant was assayed in triplicate . 
Mutants were compared to wild type by the Fisher 's exact test . 
To correct for multiple hypothesis testing , we implemented a cut off of p ,0.01 divided by the total number of mutants compared to wild type , meaning mutants with p ,0.00024 were considered significantly different from wild type . 
BPS sensitivity assay
10-fold serial dilutions of each strain was spotted on 90 mM BPS plates with FeSO4 concentrations of 0 , 2.5 , 20 or 100 mM and grown at 30uC for 3 days [ 55 ] . 
A summary of this paper was presented at the 26 International th Conference on Yeast Genetics and Molecular Biology , August 2013 [ 74 ] . 
Supporting Information
Dataset S1 Wild type replicate 1 enriched region coordinates . 
( XLSX ) 
Dataset S2 Wild type replicate 2 enriched region coordinates . 
( XLSX ) 
Acknowledgments
The RNase H1 plasmid and anti-DNA : RNA hybrid antibody S9 .6 were kind gifts from Doug Koshland and Stephen Leppla respectively . 
We thank Alice Wang and Grace Leung for their assistance with the DRIP-chip protocol and Nigel O'Neil for helpful discussions . 
We thank Gian Luca Negri for helpful discussions of the chip-on-chip data analysis and for providing scripts . 
Author Contributions
Conceived and designed the experiments : YAC MJA AH PCS . 
Performed the experiments : YAC MJA ZL AH . 
Analyzed the data : YAC MJA PCS . 
Contributed reagents/materials/analysis tools : MJA PYTL ZL MSK PH. Wrote the paper : YAC MJA PCS PH.