BACKGROUND
transcription by rna polymerase  ii is subject to complex regulation at the level of chromatin modification and assembly.
several classes of chromatin modifying proteins have been identified; these contribute to gene activation by promoting chromatin decondensation, thereby allowing access of rna pol ii to regulatory sequences and facilitating movement of rna pol ii and associated factors through transcription units.
atp-dependent remodeling factors, including swi/snf and nurf, disrupt nucleosome structure and increase dna accessibility  <cit> .
histone acetyltransferases  promote chromatin opening, nucleosome fluidity and protein interactions by acetylating histones at specific lysine residues  <cit> .
histone methyltransferases  modify histones at lysine and arginine; methylation of particular residues, for example, on histone h <dig> at k <dig> and k <dig>  is linked to active transcription and may mark recently transcribed regions  <cit> .
efficient transcriptional elongation through chromatin requires additional factors, including fact, spt <dig> and asf <dig>  which are required for histone eviction and histone redeposition  <cit> .
mutation of each of these factors is associated with inappropriate nucleosomal positioning and activation of cryptic promoters within coding regions  <cit> .
fact and asf <dig> may facilitate elongation by stimulating the eviction of h2a/h2b or h3/h <dig> dimers, respectively, from chromatin.
the paf <dig> complex, which was first identified in yeast as a functional and physical cluster of proteins including paf <dig>  ctr <dig>  cdc <dig>  rtf <dig> and leo <dig>  <cit> , is also required for efficient transcriptional elongation through chromatin  <cit> .
the paf <dig> complex is associated with transcriptionally active rna pol ii and interacts with the transcription elongation factors spt <dig>  spt <dig>  fact and tfiis  <cit> .
paf <dig> and rtf <dig> are required for monoubiquitylation of histone h2b by rad6-bre <dig> at k <dig>  <cit> .
this modification, in turn, is essential for the di- and tri-methylation of histone h <dig> by the set <dig> and dot <dig> methyltransferases at k <dig> and k <dig>  respectively  <cit> .
moreover, paf <dig> and rtf <dig> promote the recruitment of set <dig> and set <dig>  a histone h <dig> k <dig> methyltransferase, to rna pol ii at sites of active transcription  <cit> .
in this communication we show that depletion of the ctr <dig> or paf <dig> component of the paf <dig> complex is associated with delayed loss of histones from the gal <dig> gene upon induction, decreased association of rna pol ii with gal <dig> and altered distribution of the polymerase along the gal <dig> locus.
these observations are consistent with the interpretation that the paf <dig> complex enhances transcriptional elongation by promoting nucleosomal destabilization and histone removal.
RESULTS
correlation of transciption initiation frequency with occupancy by ctr <dig> and paf1
we began by examining association of ctr <dig> and paf <dig> with genes whose expression increased, decreased or remained unchanged upon depletion of ctr <dig> or paf <dig>  as determined by microarray analysis .
endogenous ctr <dig> and paf <dig> were each tagged at the carboxyl terminus with multiple c-myc epitopes; expression of tagged proteins was confirmed by immunoblotting .
association of tagged paf <dig> and ctr <dig> with specific open reading frames   was assayed by chromatin immunoprecipitation .
contrary to expectation, occupancy of an orf by either protein was not correlated with its change in expression upon depletion of ctr <dig> .
for example, association of ctr <dig> and paf <dig> was greatest with act <dig> and adh <dig>  two genes whose expression was unchanged in the ctr9Δ strain, while association of these proteins was lowest with est <dig>  whose expression decreased in response to ctr <dig> depletion.
rather, the occupancy of a specific orf by ctr <dig> or paf <dig> was positively correlated with the frequency of transcriptional initiation at that locus, as estimated by young and coworkers .
these findings suggest that the occupancy of a locus by the paf <dig> complex is positively correlated with its occupancy by rna pol ii.
we next sought a common feature of transcriptional regulation among the genes in the downregulated set.
a subset of bna genes, which function in nad+ biosynthesis, is down regulated in ctr9Δ cells .
these genes are coordinately repressed by the sir <dig> homologue hst <dig>  a sensor of intracellular nad+  <cit> .
this observation suggested that ctr <dig> might function, directly or indirectly, in relief of repression by hst <dig>  although the down regulation of bna genes observed in ctr9Δ cells might have resulted from an increase in intracellular nad+, no significant change in the amount of intracellular nad+ was found .
thus, the effect of ctr <dig> depletion on expression of nad+ biosynthetic genes was not likely to be mediated by nad+ itself, but rather through a more direct effect on transcription.
together, these results raised the possibility that deleterious effects of paf <dig> or ctr <dig> depletion on transcription might be most readily observed at loci that are subject to fluctuation between active and silent states or that are capable of rapid induction.
impaired transcription of telomeric genes upon loss of ctr9
to test whether the paf <dig> complex contributes to the establishment of transcriptionally permissive chromatin, we examined the effect of ctr <dig> or paf <dig> depletion on transcription of ura <dig> in its natural setting and at three other sites.
expression of ura <dig> from its natural locus was unaffected by loss of ctr <dig> .
additionally, expression of ura <dig> incorporated into sites not subject to telomeric silencing, either  <dig>  or  <dig> kb downstream of the natural telomere on chromosome xv  <cit>  was unperturbed .
wild-type and ctr9Δ cells, as well as the control strains npt1Δ and bna1Δ, which carry deletions in genes implicated in maintenance of silencing, grew similarly on plates lacking uracil, while growth was completely inhibited by 5-foa.
in contrast, when ura <dig> was integrated adjacent to an artificial telomere on chromosome vii  <cit> , growth of ctr9- or paf1-deficient cells was inhibited on medium lacking uracil , indicating a context-dependent effect on ura <dig> expression.
the growth of these cells was also impaired in the presence of 5-foa, consistent with impairment of telomeric silencing, as previously noted  <cit> , although the effects of ctr <dig> or paf <dig> deletion were less severe than observed for a set1Δ strain .
one interpretation of these results is that deletion of ctr <dig> or paf <dig> impairs gene expression in the setting of an artificial telomere, while simultaneously reducing silencing.
this interpretation was supported by examining expression of ade <dig>  integrated adjacent to an artificial telomere on chromosome v  <cit> , in the presence or absence of ctr <dig> .
in this assay, cells expressing ade <dig> appear white, while cells in which ade <dig> is silenced appear red.
on a wild-type background, as expected, sporadic loss of telomeric silencing at the ade <dig> locus was associated with the appearance of red and white sectored colonies .
loss of npt <dig>  which encodes a component of the nad+ salvage pathway, completely abolished silencing; as a result all colonies were white.
on a ctr9Δ background, in contrast, colonies exhibited a uniform pink colour .
this phenotype is consistent with expression of ade <dig> at an intermediate level in most or all cells, as would be expected if loss of ctr <dig> were to relieve telomeric silencing and at the same time to reduce transcriptional efficiency.
these observations demonstrate position-dependent impairment of gene expression in the absence of ctr <dig> and paf <dig>  and once again suggest that the paf <dig> complex facilitates transcription through regions of chromatin that are undergoing a transition from inactive to active states.
we proceeded to test this directly by examining the effects of ctr <dig> and paf <dig> depletion on induction of repressed loci.
impaired induction of gal genes in the absence of ctr <dig> or paf1
ctr <dig> and paf <dig> are required for monoubiquitylation of h2b at k <dig>  <cit> , a process which in turn is necessary for efficient expression of gal genes  <cit> .
we examined the effects of ctr <dig> or paf <dig> depletion on induction of transcription at gal loci.
strains were grown with raffinose as the sole carbon source before induction with galactose; this allowed us to bypass the process of derepression and focus solely on transcriptional activation.
induction of gal <dig> rna was impaired in both the ctr9Δ and paf1Δ strains, relative to wild-type .
deletion of ctr <dig> or paf <dig> was associated with a 2- to 3-fold reduction in gal <dig> rna at  <dig> min after induction by galactose.
we asked whether this decrease in rna accumulation was accompanied by a change in transcriptional kinetics by assaying induction of gal <dig> by galactose at 24°c.
by inducing expression at a reduced temperature we hoped to unmask differences in the rate of transcript accumulation that might have been less pronounced at 30°c.
we also used additional isolates of the wild-type, ctr9Δ and paf1Δ strains to militate against potential effects of secondary mutations cause by loss of ctr <dig> or paf <dig>  deletion of ctr <dig> or paf <dig> had substantial effects on the initial accumulation of gal <dig> transcripts, reducing rna levels by about  <dig> fold at  <dig> min; by  <dig> min the difference in transcript levels between wild-type and the mutants was  <dig> fold and by  <dig> min this was reduced to only a 2-fold difference .
we obtained similar results for gal <dig> .
these results indicated that loss of the paf <dig> complex is associated with delayed induction of gal loci, consistent with preferential impairment of early transcription in the absence of the paf <dig> complex, as has been described  <cit> .
loss of ctr <dig> or paf <dig> impairs displacement of histones upon gal <dig> induction
induction of transcription at gal loci is accompanied by loss of histones at promoter and coding regions  <cit> .
we assessed the effects of ctr <dig> or paf <dig> deletion on eviction of histones from the gal <dig> locus upon induction.
we first used micrococcal nuclease digestion of chromatin to examine nucleosomal occupancy and spacing before and after induction by galactose.
examination of bulk chromatin revealed no effect of ctr <dig> or paf <dig> deletion on the general deposition or spacing of histones .
specific interrogation of the gal <dig> locus, however, indicated that loss of either ctr <dig> or paf <dig> was associated with delayed displacement of histones from the gene.
at  <dig> min, a discrete distribution of nucleosomal digestion products was observed in all samples.
in the wild-type sample, this discrete distribution was largely lost by  <dig> min after galactose induction, and this pattern persisted at  <dig> min after induction .
this suggested that in the wild-type strain, nucleosomes had been lost or their phasing disrupted by  <dig> min after induction.
in contrast, discrete nucleosomal laddering was still observed at  <dig> min after galactose induction in the ctr9Δ and paf1Δ strains .
by  <dig> min, nucleosome occupancy or phasing had been lost in all strains.
the difference between wild-type and ctr9Δ or paf1Δ strains with respect to the kinetics of phased nucleosome loss was consistent with the interpretation that the paf <dig> complex is required for the timely removal of histones from the gal <dig> gene, or their displacement over the locus, upon induction of transcription.
to distinguish between these possibilities, we used chip to determine whether depletion of ctr <dig> or paf <dig> affects the association of histones and rna pol ii with the gal <dig> locus upon activation.
association of histone h <dig> and rna pol ii with 5', middle and 3' regions of gal <dig>  were examined in wild-type, ctr9Δ and paf1Δ strains maintained at 24°c before and at various times after induction by galactose.
in wild-type cells, occupancy of gal <dig> by histone h <dig> decreased sharply from  <dig> to  <dig> min after induction and remained low for the remainder of the time course .
deletion of either ctr <dig> or paf <dig> impaired removal of histone h <dig> from the gal <dig> locus .
before the  <dig> min time point, no statisically significant differences in histone h <dig> levels were detected among wild-type, ctr9Δ and paf1Δ samples.
by  <dig> min, histone h <dig> occupancy was about 30% lower in wild-type cells than in the ctr9Δ and paf1Δ mutants and this difference increased to 40% by  <dig> min.
these differences were determined to be highly significant .
by  <dig> min after induction, depletion of histone h <dig> over the gal <dig> locus in the ctr9Δ and paf1Δ cells began to approach that of wild-type, but nonetheless remained statistically higher .
these results suggest that the paf <dig> complex promotes eviction of histones from the gal <dig> locus during early stages of transcriptional induction.
moreover, these findings indicate that in the absence of the paf <dig> complex, nucleosomes can be removed from the gal <dig> locus upon activation, but with delayed kinetics relative to wild-type.
in the nuclease digestion experiments we had observed more unphased dna fragments of intermediate size in the wild-type sample than in the ctr9Δ and paf1Δ strains at  <dig> min .
while this in principal could have reflected greater occupancy of the gal <dig> locus by unphased nucleosomes in wild-type cells, our observation that removal of histones from gal <dig> is more rapid in the wild-type than in ctr9Δ or paf1Δ strains argues against this point.
we observed an inverse relationship between histone displacement and the association of rna pol ii with the gal <dig> locus.
in wild-type cells, association of rna pol ii with gal <dig> increased rapidly after addition of galactose .
this association was apparent at  <dig> min after induction and continued to increase throughout the time course.
the amount of rna pol ii at the 5' region was consistently greater than at the middle or 3' region of the gene, although the kinetics of polymerase association with these regions paralleled the kinetics observed for the 5' region .
these findings are consistent with a report describing kinetics of association of rna pol ii with a galactose-inducible, synthetic locus  <cit> .
loss of ctr <dig> or paf <dig> was associated with changes in the association of rna pol ii with gal <dig> and its distribution over the locus following induction.
while the confidence limits determined for these differences were not below the significance threshold, similar trends were observed for the ctr9Δ and paf1Δ mutants.
as with wild-type cells, association of rna pol ii with the 5' region of gal <dig> was detectable in ctr9Δ and paf1Δ cells at  <dig> min after addition of galactose, but the amount of association was reduced .
throughout the time course, the amount of rna pol ii over the 5' region was consistently lower in the ctr9Δ and paf1Δ strains than in wild-type.
interestingly, loss of either ctr <dig> or paf <dig> was associated with a relative increase in the amount of rna pol ii associated with the middle region of the gal <dig> locus , despite the reduction in the amount of polymerase associated with the 5' region.
only small differences in polymerase occupancy of the 3' region of gal <dig> were observed among the strains .
deletion of ctr <dig> or paf <dig> had similar effects on rna pol ii occupancy as well as histone displacement at the gal <dig> locus .
these findings suggest that upon induction of transcription at gal <dig> and gal <dig> loci, the paf <dig> complex promotes histone displacement and more efficient transcriptional elongation.
we considered the possiblity that inappropriate internal initiations in the ctr9Δ and paf1Δ strains might contribute to alterations in the distribution of rna pol ii, but this seems unlikely as we observed no truncated gal <dig> or gal <dig> transcripts in hybridization assays of mutant cells using double-stranded probes, which would have detected transcripts from either strand .
discussion
it has been reported that the paf <dig> complex associates with rna pol ii and that loss of the ctr <dig> or paf <dig> components of this complex impairs transcription of a subset of genes.
we find that the amount of paf <dig> associated with a particular gene is positively correlated with its rate of transcription initiation.
the paf <dig> complex supports specific histone modifications that are essential for maintenance of telomeric silencing, including monoubiquitylation of histone h2b and methylation of histone h <dig> at lysine  <dig>  in agreement with previous reports, we detect a loss of telomeric silencing in ctr <dig> and paf <dig> deletion mutants, but demonstrate in addition that the loss of these genes also impairs transcription of telomeric markers.
the paf <dig> complex is likely required for efficient transcriptional elongation  <cit> , but the underlying mechanism has remained unclear.
a recent report indicates that paf <dig> complex-dependent ubiquitylation of h2b is required for efficient transcription elongation in vitro, and further suggests that ubiquitylation of h2b permits fact to efficiently displace h2a/h2b histone dimers  <cit> .
here we have shown that the loss of ctr <dig> or paf <dig> in vivo is associated with delayed removal of histones from the gal <dig> locus upon induction of transcription.
this delay in histone displacement may contribute to the decrease in association and altered distribution of rna pol ii that we observed in ctr9- or paf1-deficient cells.
our findings may also explain the relative delay in induction of gal transcripts in ctr9Δ and paf1Δ strains, relative to wild-type.
we have considered the possibility that a reduction in recruitment of rna pol ii, rather than an impairment of histone eviction, might be responsible for the delay in histone displacement that we observed.
in support of our interpretation are two reports documenting recruitment of the paf <dig> complex to promoter regions after, but not before, initiation of transcription.
in mammalian cells the paf <dig> complex is recruited to the rarβ <dig> promoter after recruitment of rna pol ii and fact  <cit> .
moreover, rna pol ii binds to the yeast arg <dig> promoter in the absence of the paf <dig> complex; subsequent recruitment of the paf <dig> complex is dependent on prior binding of the spt <dig> transcription elongation factor and phosphorylation of rna pol ii at ser <dig>  <cit> .
lastly, association of rad <dig> and the saga complex with the gal <dig> promoter occurs independently of h2b monoubiquitylation  <cit> .
taken together, these observations suggest that recruitment of rna pol ii to promoter regions occurs prior to and independent of the actions of ctr <dig> or paf <dig>  our results are consistent with a direct role for the paf <dig> complex in promoting histone eviction but do not exclude the possibility that paf <dig> and ctr <dig> exert their effects through one or more additional components of the elongating rna pol ii complex.
it has been proposed that newly transcribed genes undergo a so-called "pioneer round" of transcription in which rna pol ii functions in concert with specialized machinery for chromatin modification  <cit> .
based on our work and earlier findings we suggest that upon initial induction of gal <dig>  gal <dig>  rad6/bre <dig>  saga and rna pol ii, among other factors, are recruited and initiate transcription  <cit> .
subsequent association of the paf <dig> complex with rna pol ii would promote monoubiquitylation of h2b at k <dig> by rad6/bre <dig> as the polymerase complex traverses the coding sequence  <cit> .
we imagine that ubiquitylation of k <dig> could confer upon fact and asf <dig> the ability to evict histones from the dna in addition to the established roles of fact and asf <dig> in histone displacement and redeposition.
our results suggest that in the absence of the paf <dig> complex histones are inefficiently removed and transcriptional elongation is impaired.
while it has been suggested that the paf <dig> complex does not affect transcriptional elongation from a gal <dig> promoter  <cit> , this conclusion was based on observations made at  <dig>  hr after galactose induction.
because our data indicate that the effects of ctr <dig> and paf <dig> depletion on histone eviction are most evident early after induction, it is not surprising that differences in elongation rates were not observed at a relatively late time point.
our results suggest that the paf <dig> complex exerts its effects on histone displacement during the initial stages of transcriptional activation, and that at later times after induction, histones can be removed even in the absence of the paf <dig> complex.
the paf <dig> complex and the h2b monoubiquitylation that it promotes are clearly not universally required for gene expression  <cit> .
unlike fact, the genes encoding components of the paf <dig> complex and rad6/bre <dig> are inessential  <cit> .
the available data suggest that the paf <dig> complex enhances transcriptional efficiency in those settings in which environmental changes demand rapid transcriptional responses, such as induction of metabolic enzymes or stress response genes.
in this regard, it is particularly interesting that one salient phenotype of paf1Δ and ctr9Δ strains is sensitivity to a broad range of environmental stressors  <cit> .
CONCLUSIONS
in this communication we have shown that loss of either paf <dig> or ctr <dig> is associated with impaired induction of transcription at the gal <dig> and gal <dig> loci and altered recruitment and distribution of rna pol ii across the gal <dig> locus.
these effects are accompanied by delayed removal of histones from the gal <dig> locus upon induction of transcription in paf1- or ctr9-deficient strains.
our findings suggest that the paf <dig> complex and the histone modifications that it mediates increase the efficiency of transcriptional elongation by promoting destabilization of nucleosomes and histone removal.

 transcription by rna polymerase ii is subject to complex regulation at the level of chromatin modification and assembly. several classes of chromatin modifying proteins have been identified; these contribute to gene activation by promoting chromatin decondensation, thereby allowing access of rna pol ii to regulatory sequences and facilitating movement of rna pol ii and associated factors through transcription units. atp-dependent remodeling factors, including swi/snf and nurf, disrupt nucleosome structure and increase dna accessibility. histone acetyltransferases promote chromatin opening, nucleosome fluidity and protein interactions by acetylating histones at specific lysine residues. histone methyltransferases modify histones at lysine and arginine; methylation of particular residues, for example, on histone h at k and k is linked to active transcription and may mark recently transcribed regions. efficient transcriptional elongation through chromatin requires additional factors, including fact, spt and asf which are required for histone eviction and histone redeposition. mutation of each of these factors is associated with inappropriate nucleosomal positioning and activation of cryptic promoters within coding regions. fact and asf may facilitate elongation by stimulating the eviction of h2a/h2b or h3/h dimers, respectively, from chromatin. the paf complex, which was first identified in yeast as a functional and physical cluster of proteins including paf ctr cdc rtf and leo, is also required for efficient transcriptional elongation through chromatin. the paf complex is associated with transcriptionally active rna pol ii and interacts with the transcription elongation factors spt spt fact and tfiis. paf and rtf are required for monoubiquitylation of histone h2b by rad6-bre at k. this modification, in turn, is essential for the di- and tri-methylation of histone h by the set and dot methyltransferases at k and k respectively. moreover, paf and rtf promote the recruitment of set and set a histone h k methyltransferase, to rna pol ii at sites of active transcription. in this communication we show that depletion of the ctr or paf component of the paf complex is associated with delayed loss of histones from the gal gene upon induction, decreased association of rna pol ii with gal and altered distribution of the polymerase along the gal locus. these observations are consistent with the interpretation that the paf complex enhances transcriptional elongation by promoting nucleosomal destabilization and histone removal.  correlation of transciption initiation frequency with occupancy by ctr and paf1 we began by examining association of ctr and paf with genes whose expression increased, decreased or remained unchanged upon depletion of ctr or paf as determined by microarray analysis. endogenous ctr and paf were each tagged at the carboxyl terminus with multiple c-myc epitopes; expression of tagged proteins was confirmed by immunoblotting. association of tagged paf and ctr with specific open reading frames was assayed by chromatin immunoprecipitation. contrary to expectation, occupancy of an orf by either protein was not correlated with its change in expression upon depletion of ctr. for example, association of ctr and paf was greatest with act and adh two genes whose expression was unchanged in the ctr9Δ strain, while association of these proteins was lowest with est whose expression decreased in response to ctr depletion. rather, the occupancy of a specific orf by ctr or paf was positively correlated with the frequency of transcriptional initiation at that locus, as estimated by young and coworkers. these findings suggest that the occupancy of a locus by the paf complex is positively correlated with its occupancy by rna pol ii. we next sought a common feature of transcriptional regulation among the genes in the downregulated set. a subset of bna genes, which function in nad+ biosynthesis, is down regulated in ctr9Δ cells. these genes are coordinately repressed by the sir homologue hst a sensor of intracellular nad+. this observation suggested that ctr might function, directly or indirectly, in relief of repression by hst although the down regulation of bna genes observed in ctr9Δ cells might have resulted from an increase in intracellular nad+, no significant change in the amount of intracellular nad+ was found. thus, the effect of ctr depletion on expression of nad+ biosynthetic genes was not likely to be mediated by nad+ itself, but rather through a more direct effect on transcription. together, these results raised the possibility that deleterious effects of paf or ctr depletion on transcription might be most readily observed at loci that are subject to fluctuation between active and silent states or that are capable of rapid induction. impaired transcription of telomeric genes upon loss of ctr9 to test whether the paf complex contributes to the establishment of transcriptionally permissive chromatin, we examined the effect of ctr or paf depletion on transcription of ura in its natural setting and at three other sites. expression of ura from its natural locus was unaffected by loss of ctr. additionally, expression of ura incorporated into sites not subject to telomeric silencing, either or kb downstream of the natural telomere on chromosome xv was unperturbed. wild-type and ctr9Δ cells, as well as the control strains npt1Δ and bna1Δ, which carry deletions in genes implicated in maintenance of silencing, grew similarly on plates lacking uracil, while growth was completely inhibited by 5-foa. in contrast, when ura was integrated adjacent to an artificial telomere on chromosome vii, growth of ctr9- or paf1-deficient cells was inhibited on medium lacking uracil, indicating a context-dependent effect on ura expression. the growth of these cells was also impaired in the presence of 5-foa, consistent with impairment of telomeric silencing, as previously noted, although the effects of ctr or paf deletion were less severe than observed for a set1Δ strain. one interpretation of these results is that deletion of ctr or paf impairs gene expression in the setting of an artificial telomere, while simultaneously reducing silencing. this interpretation was supported by examining expression of ade integrated adjacent to an artificial telomere on chromosome v, in the presence or absence of ctr. in this assay, cells expressing ade appear white, while cells in which ade is silenced appear red. on a wild-type background, as expected, sporadic loss of telomeric silencing at the ade locus was associated with the appearance of red and white sectored colonies. loss of npt which encodes a component of the nad+ salvage pathway, completely abolished silencing; as a result all colonies were white. on a ctr9Δ background, in contrast, colonies exhibited a uniform pink colour. this phenotype is consistent with expression of ade at an intermediate level in most or all cells, as would be expected if loss of ctr were to relieve telomeric silencing and at the same time to reduce transcriptional efficiency. these observations demonstrate position-dependent impairment of gene expression in the absence of ctr and paf and once again suggest that the paf complex facilitates transcription through regions of chromatin that are undergoing a transition from inactive to active states. we proceeded to test this directly by examining the effects of ctr and paf depletion on induction of repressed loci. impaired induction of gal genes in the absence of ctr or paf1 ctr and paf are required for monoubiquitylation of h2b at k, a process which in turn is necessary for efficient expression of gal genes. we examined the effects of ctr or paf depletion on induction of transcription at gal loci. strains were grown with raffinose as the sole carbon source before induction with galactose; this allowed us to bypass the process of derepression and focus solely on transcriptional activation. induction of gal rna was impaired in both the ctr9Δ and paf1Δ strains, relative to wild-type. deletion of ctr or paf was associated with a 2- to 3-fold reduction in gal rna at min after induction by galactose. we asked whether this decrease in rna accumulation was accompanied by a change in transcriptional kinetics by assaying induction of gal by galactose at 24°c. by inducing expression at a reduced temperature we hoped to unmask differences in the rate of transcript accumulation that might have been less pronounced at 30°c. we also used additional isolates of the wild-type, ctr9Δ and paf1Δ strains to militate against potential effects of secondary mutations cause by loss of ctr or paf deletion of ctr or paf had substantial effects on the initial accumulation of gal transcripts, reducing rna levels by about fold at min; by min the difference in transcript levels between wild-type and the mutants was fold and by min this was reduced to only a 2-fold difference. we obtained similar results for gal. these results indicated that loss of the paf complex is associated with delayed induction of gal loci, consistent with preferential impairment of early transcription in the absence of the paf complex, as has been described. loss of ctr or paf impairs displacement of histones upon gal induction induction of transcription at gal loci is accompanied by loss of histones at promoter and coding regions. we assessed the effects of ctr or paf deletion on eviction of histones from the gal locus upon induction. we first used micrococcal nuclease digestion of chromatin to examine nucleosomal occupancy and spacing before and after induction by galactose. examination of bulk chromatin revealed no effect of ctr or paf deletion on the general deposition or spacing of histones. specific interrogation of the gal locus, however, indicated that loss of either ctr or paf was associated with delayed displacement of histones from the gene. at min, a discrete distribution of nucleosomal digestion products was observed in all samples. in the wild-type sample, this discrete distribution was largely lost by min after galactose induction, and this pattern persisted at min after induction. this suggested that in the wild-type strain, nucleosomes had been lost or their phasing disrupted by min after induction. in contrast, discrete nucleosomal laddering was still observed at min after galactose induction in the ctr9Δ and paf1Δ strains. by min, nucleosome occupancy or phasing had been lost in all strains. the difference between wild-type and ctr9Δ or paf1Δ strains with respect to the kinetics of phased nucleosome loss was consistent with the interpretation that the paf complex is required for the timely removal of histones from the gal gene, or their displacement over the locus, upon induction of transcription. to distinguish between these possibilities, we used chip to determine whether depletion of ctr or paf affects the association of histones and rna pol ii with the gal locus upon activation. association of histone h and rna pol ii with 5', middle and 3' regions of gal were examined in wild-type, ctr9Δ and paf1Δ strains maintained at 24°c before and at various times after induction by galactose. in wild-type cells, occupancy of gal by histone h decreased sharply from to min after induction and remained low for the remainder of the time course. deletion of either ctr or paf impaired removal of histone h from the gal locus. before the min time point, no statisically significant differences in histone h levels were detected among wild-type, ctr9Δ and paf1Δ samples. by min, histone h occupancy was about 30% lower in wild-type cells than in the ctr9Δ and paf1Δ mutants and this difference increased to 40% by min. these differences were determined to be highly significant. by min after induction, depletion of histone h over the gal locus in the ctr9Δ and paf1Δ cells began to approach that of wild-type, but nonetheless remained statistically higher. these results suggest that the paf complex promotes eviction of histones from the gal locus during early stages of transcriptional induction. moreover, these findings indicate that in the absence of the paf complex, nucleosomes can be removed from the gal locus upon activation, but with delayed kinetics relative to wild-type. in the nuclease digestion experiments we had observed more unphased dna fragments of intermediate size in the wild-type sample than in the ctr9Δ and paf1Δ strains at min. while this in principal could have reflected greater occupancy of the gal locus by unphased nucleosomes in wild-type cells, our observation that removal of histones from gal is more rapid in the wild-type than in ctr9Δ or paf1Δ strains argues against this point. we observed an inverse relationship between histone displacement and the association of rna pol ii with the gal locus. in wild-type cells, association of rna pol ii with gal increased rapidly after addition of galactose. this association was apparent at min after induction and continued to increase throughout the time course. the amount of rna pol ii at the 5' region was consistently greater than at the middle or 3' region of the gene, although the kinetics of polymerase association with these regions paralleled the kinetics observed for the 5' region. these findings are consistent with a report describing kinetics of association of rna pol ii with a galactose-inducible, synthetic locus. loss of ctr or paf was associated with changes in the association of rna pol ii with gal and its distribution over the locus following induction. while the confidence limits determined for these differences were not below the significance threshold, similar trends were observed for the ctr9Δ and paf1Δ mutants. as with wild-type cells, association of rna pol ii with the 5' region of gal was detectable in ctr9Δ and paf1Δ cells at min after addition of galactose, but the amount of association was reduced. throughout the time course, the amount of rna pol ii over the 5' region was consistently lower in the ctr9Δ and paf1Δ strains than in wild-type. interestingly, loss of either ctr or paf was associated with a relative increase in the amount of rna pol ii associated with the middle region of the gal locus, despite the reduction in the amount of polymerase associated with the 5' region. only small differences in polymerase occupancy of the 3' region of gal were observed among the strains. deletion of ctr or paf had similar effects on rna pol ii occupancy as well as histone displacement at the gal locus. these findings suggest that upon induction of transcription at gal and gal loci, the paf complex promotes histone displacement and more efficient transcriptional elongation. we considered the possiblity that inappropriate internal initiations in the ctr9Δ and paf1Δ strains might contribute to alterations in the distribution of rna pol ii, but this seems unlikely as we observed no truncated gal or gal transcripts in hybridization assays of mutant cells using double-stranded probes, which would have detected transcripts from either strand. discussion it has been reported that the paf complex associates with rna pol ii and that loss of the ctr or paf components of this complex impairs transcription of a subset of genes. we find that the amount of paf associated with a particular gene is positively correlated with its rate of transcription initiation. the paf complex supports specific histone modifications that are essential for maintenance of telomeric silencing, including monoubiquitylation of histone h2b and methylation of histone h at lysine in agreement with previous reports, we detect a loss of telomeric silencing in ctr and paf deletion mutants, but demonstrate in addition that the loss of these genes also impairs transcription of telomeric markers. the paf complex is likely required for efficient transcriptional elongation, but the underlying mechanism has remained unclear. a recent report indicates that paf complex-dependent ubiquitylation of h2b is required for efficient transcription elongation in vitro, and further suggests that ubiquitylation of h2b permits fact to efficiently displace h2a/h2b histone dimers. here we have shown that the loss of ctr or paf in vivo is associated with delayed removal of histones from the gal locus upon induction of transcription. this delay in histone displacement may contribute to the decrease in association and altered distribution of rna pol ii that we observed in ctr9- or paf1-deficient cells. our findings may also explain the relative delay in induction of gal transcripts in ctr9Δ and paf1Δ strains, relative to wild-type. we have considered the possibility that a reduction in recruitment of rna pol ii, rather than an impairment of histone eviction, might be responsible for the delay in histone displacement that we observed. in support of our interpretation are two reports documenting recruitment of the paf complex to promoter regions after, but not before, initiation of transcription. in mammalian cells the paf complex is recruited to the rarβ promoter after recruitment of rna pol ii and fact. moreover, rna pol ii binds to the yeast arg promoter in the absence of the paf complex; subsequent recruitment of the paf complex is dependent on prior binding of the spt transcription elongation factor and phosphorylation of rna pol ii at ser. lastly, association of rad and the saga complex with the gal promoter occurs independently of h2b monoubiquitylation. taken together, these observations suggest that recruitment of rna pol ii to promoter regions occurs prior to and independent of the actions of ctr or paf our results are consistent with a direct role for the paf complex in promoting histone eviction but do not exclude the possibility that paf and ctr exert their effects through one or more additional components of the elongating rna pol ii complex. it has been proposed that newly transcribed genes undergo a so-called "pioneer round" of transcription in which rna pol ii functions in concert with specialized machinery for chromatin modification. based on our work and earlier findings we suggest that upon initial induction of gal gal rad6/bre saga and rna pol ii, among other factors, are recruited and initiate transcription. subsequent association of the paf complex with rna pol ii would promote monoubiquitylation of h2b at k by rad6/bre as the polymerase complex traverses the coding sequence. we imagine that ubiquitylation of k could confer upon fact and asf the ability to evict histones from the dna in addition to the established roles of fact and asf in histone displacement and redeposition. our results suggest that in the absence of the paf complex histones are inefficiently removed and transcriptional elongation is impaired. while it has been suggested that the paf complex does not affect transcriptional elongation from a gal promoter, this conclusion was based on observations made at hr after galactose induction. because our data indicate that the effects of ctr and paf depletion on histone eviction are most evident early after induction, it is not surprising that differences in elongation rates were not observed at a relatively late time point. our results suggest that the paf complex exerts its effects on histone displacement during the initial stages of transcriptional activation, and that at later times after induction, histones can be removed even in the absence of the paf complex. the paf complex and the h2b monoubiquitylation that it promotes are clearly not universally required for gene expression. unlike fact, the genes encoding components of the paf complex and rad6/bre are inessential. the available data suggest that the paf complex enhances transcriptional efficiency in those settings in which environmental changes demand rapid transcriptional responses, such as induction of metabolic enzymes or stress response genes. in this regard, it is particularly interesting that one salient phenotype of paf1Δ and ctr9Δ strains is sensitivity to a broad range of environmental stressors.  in this communication we have shown that loss of either paf or ctr is associated with impaired induction of transcription at the gal and gal loci and altered recruitment and distribution of rna pol ii across the gal locus. these effects are accompanied by delayed removal of histones from the gal locus upon induction of transcription in paf1- or ctr9-deficient strains. our findings suggest that the paf complex and the histone modifications that it mediates increase the efficiency of transcriptional elongation by promoting destabilization of nucleosomes and histone removal. 
this delay in histone removal is accompanied by a decrease in association of rna pol ii with gal and altered distribution of the polymerase along the locus. these observations may explain why initial induction of gal transcripts is reduced in ctr9- or paf1-deficient cells, and is consistent with a model suggesting that the paf complex and the histone modifications that it mediates increase efficiency of transcriptional elongation by promoting nucleosomal destabilization and histone removal. the yeast paf protein complex is required for efficient transcription elongation by rna polymerase ii, but the precise role of the complex has been unclear.
 the yeast paf protein complex is required for efficient transcription elongation by rna polymerase ii, but the precise role of the complex has been unclear.  here we show that depletion of the ctr or paf component of the paf complex delays the loss of histones from the gal gene upon induction. this delay in histone removal is accompanied by a decrease in association of rna pol ii with gal and altered distribution of the polymerase along the locus.  these observations may explain why initial induction of gal transcripts is reduced in ctr9- or paf1-deficient cells, and is consistent with a model suggesting that the paf complex and the histone modifications that it mediates increase efficiency of transcriptional elongation by promoting nucleosomal destabilization and histone removal. 