the packaging of eukaryotic genomes into chromatin has fundamental effects on gene expression but how this is brought about still remains poorly understood .
histone proteins are highly conserved and are subject to many different posttranslational modifications ( ptms ) including acetylation and methylation ( rando and winston , 2012 ) .
these modifications can influence nucleosome occupancy and position as well as the recruitment of a wide range of effector proteins implicated in a variety of cellular processes .
histone methylation can be influenced by the conformation of the n - terminal region of histone h3 , particularly cis - trans isomerization of bonds around proline residues ( lu et  al .
, 2007 ; nelson et  al . , 2006 ; youdell et  al . ,
the peptidyl - prolyl isomerase ( ppiase ) fpr4 increases the rate of cis - trans isomerization at all three prolines ( 16 , 30 , and 38 ) on h3 in  vitro ( monneau et  al . , 2013 ) and
genome - wide mapping studies show that modification patterns are correlated with both gene structure and gene activity , often showing characteristic distributions on active or repressed genes ( liu et  al . , 2005 ; pokholok et  al . ,
one such modification is set1-dependent methylation of lysine 4 on histone h3 ( k4 ) , present in most eukaryotes at active or potentially active genes ( santos - rosa et  al . , 2002 ) .
this has led some to assume that k4 methylation by set1 is an activating modification yet , in yeast , most evidence points to k4 methylation having a repressive or adaptive role , silencing some rdna repeats ( briggs et  al . , 2001 ; bryk et  al . , 2002 ) , repressing genes in midsporulation and during exponential growth ( carvin and kladde , 2004 ; fingerman et  al . , 2005 ; guillemette et  al . , 2011 ;
, 2011 ; wang et  al . , 2011 ) , and facilitating the transcriptional response to diamide stress ( weiner et  al . , 2012 )
. paradoxically , but consistent with a modulatory role , k4 methylation facilitates the recruitment of both lysine acetyl transferases ( kats ) and histone deacetylases ( hdacs ) ( vermeulen and timmers , 2010 ) .
set1 is found in a complex ( set1c ) with seven other subunits ( swd1 , swd3 , bre2 , sdc1 , spp1 , swd2 , and shg1 ) ( briggs et  al . , 2001 ;
2003 ) that contribute to complex integrity and/or degree of methylation ( k4me1 , k4me2 or k4me3 ) .
spp1 is required for k4me3 ( morillon et  al . , 2005 ) and contains a phd motif that interacts with k4me2 and k4me3 ( murton et  al . , 2010 ;
shi et  al . , 2007 ) thus maintaining high local levels of k4me3 .
loss of k4me3 from chromatin involves either histone dilution during dna replication ( radman - livaja et  al . , 2010 ) or active demethylation by jhd2 ( ingvarsdottir et  al .
, 2007 ; liang et  al . , 2007 ; seward et  al . , 2007 ; tu et  al . , 2007 ) .
jhd2 is particularly important in sporulation to keep certain genes expressed ( xu et  al . ,
methylated k4 is influenced by the modification state at distant residues on nucleosomal histones including h2bub1 and k14ac promoting , and h3r2me and k4ac antagonizing k4me2 and k4me3 to varying extents ( briggs et  al .
, 2002 ; guillemette et  al . , 2011 ; kirmizis et  al .
, 2007 ; maltby et  al . , 2012 ; nakanishi et  al . , 2008 ) .
cells alternate between phases of growth and quiescence , known as the yeast metabolic cycle ( ymc ) ( tu et  al . , 2005 ) .
global levels of k4me3 remain fairly constant but levels of h3 acetylation vary , for example on k14 ( cai et  al . , 2011 ; tu et  al . , 2005 ) .
this may reflect fluctuations in the levels of acetyl coa , a cofactor for kats , which are lower in quiescent cells .
quiescent cells are more resistant to stress ( lu et  al . , 2009 ; slavov et  al . ,
2012 ) and express genes that negatively correlate with growth rate ( brauer et  al . , 2008 ) and genes whose expression increases during the common environmental stress response ( esr ) ( gasch et  al . ,
2014 ) . feeding quiescent cells metabolic intermediates , such as acetate , induces cycling and growth ( cai et  al .
moreover , the lysine acetyltransferase , gcn5 ( kat2 ) a component of saga ( baker and grant , 2007 ) , is required for metabolic cycling ( cai  et  al . , 2011 ) .
this suggests that acetylation on histone h3 will  reflect the metabolic state of the cell and environmental growth - related signals . as k14 acetylation
is reported to be required for k4me3 ( maltby et  al . , 2012 ;
2008 ) and k14 is subject to gcn5/saga - dependent acetylation ( jiang et  al . , 2007 ; zhang et  al . ,
1998 ) , we wanted to explore in more detail the relationship between k14 and k4me3 . here
, we show that k14 modulates the conformation of the h3 tail at the alanine 15-proline 16 peptide bond ( cis - trans isomerization ) to  control k4me3 .
substitutions at k14 ( a ,  r , and q ) and p16 ( v and a ) to mimic neutral , positively charged , acetylated or trans conformation of the h3 tail respectively reveal the different contributions of these residues in promoting spp1 association with chromatin and antagonizing jhd2-mediated demethylation of k4me3 .
strains lacking spp1 show a similar reduction ( 5-fold decrease ) in global k4me3 levels to the k14a strain .
this residual level of k4me3 is due to a spp1- , k14- , and p16-independent mechanism for deposition of k4me3 , most evident at the ribosomal protein genes ( rpgs ) .
by contrast , environmental stress response ( esr)-induced genes are most dependent on spp1 , k14 , and p16 for k4me3 . we propose that changes in growth conditions and the availability of metabolic intermediates and regulators allow variable control over levels of k4me3 at different genic loci .
proline is unique in its ability to form cis or trans peptide bonds with the preceding residue . we were interested in whether , as at p38 ( nelson et  al . , 2006 ) ,
cis - trans isomerization of p16 is important for h3 tail modifications . for evidence that p16 adopts cis or trans conformations in  vivo
, we raised antibodies against short h3 peptides in which the a15-p16 peptidyl - prolyl bond was fixed predominantly in the cis or trans configuration by hydroxylation of the proline ring ( taylor et  al . , 2005 ) .
after extensive selection for specificity using the peptides ( figure  1a ) , we obtained antibodies that specifically recognized the peptide with hydroxylated p16 in the desired conformation ( the unmodified peptide will have the majority of p16 in trans ) .
we further characterized these antibodies by western blot using a wt strain ( both cis and trans conformations ) and a strain with p16 substituted for valine , which fixes the a15-v16 bond in trans ( lu et  al .
as expected , we observed a higher p16cis signal in the wt over the p16v strain but a higher p16trans signal in the p16v strain relative to the wt .
the antibodies were used to perform a chromatin immunoprecipitation ( chip ) experiment to detect a15-p16 in cis and in trans in the chromatin over the long gene fmp27 ( figure  1c and figure  s1a available online ; adh1 ) .
p16trans is low at the 5 region of fmp27 and increases toward the 3 end of the gene .
conversely , we observed p16cis at the 5 region of the genes , with a similar distribution to that we had previously observed for k4me3 .
both valine and alanine substitutions at p16 significantly reduced global levels of k4me3 ( figures 1d and 1e ) .
we tested other modifications on h3 but none , apart from k4me3 , k14ac , and k18ac , were influenced by the p16 substitution ( figure  s1b ) .
dot blot analysis demonstrated that antibodies raised against k14ac and k18ac were unable to bind peptides containing their epitope and a p16v substitution ( figure  s1c ) , but mass spectrometry revealed the level of k14ac in a p16v strain was roughly equivalent to wt ( figure  s1d ) .
this suggests that the antibodies specific for k14ac and k18ac require p16 as part of their epitope .
thus p16 adopts distinct conformations in  vivo and is required for optimal levels of k4me3 .
it has been reported that substitutions at k14 also result in reduced k4me3 but not k4me1/2 ( nakanishi et  al . , 2008 ) .
given the effect of p16 substitution on k4me3 , we wanted to reexplore the relationship between k14 and k4me3 .
as described previously , substitution of k14 with alanine ( a ) or glutamine ( q ) causes reductions in the global level of k4me3 .
in contrast , substitution of k14 with arginine ( r ) resulted in less severe reductions in global k4me3 ( figures 2a and 2b ) , confirmed in a range of strain backgrounds ( figure  2c ) .
lysines for which k to r and k to q substitutions have opposite effects are known acetylation substrates , such as k14 ( weiner et  al . , 2012 ) . as there are robust levels of k4me3 in the k14r strain ,
a lysine at position 14 is not absolutely required for k4me3 and this also rules out a strict dependency on acetylation of k14 for k4me3 .
moreover , as the arginine substitution was used to mimic the positively charged , unmodified lysine residue whereas glutamine is a mimic of the neutral , acetylated lysine ( wang and hayes , 2008 ) , this suggests that unmodified k14 is associated with k4me3 .
we suggest that k14 in its unmodified state is associated with k4me3 , whereas three different groups have concluded , using strains lacking acetyltransferases such as gcn5 or sas3 , that acetylation is required for k4me3 ( govind et  al .
2012 ) . as these strains have a significantly reduced growth rate ( zhang et  al . , 1998 ) , it is possible that the reduction in k4me3 is not a direct consequence of reduced k14ac . to test this , gcn5 or sas3 with ada2 ( ada2 is required for the kat activity of gcn5 ) , were deleted in the k14r strain and a strain with wt histone h3 .
deletion of gcn5 , or sas3 with ada2 , reduced the global level of k4me3 in the presence of a wt copy of histone h3 , as reported previously .
however , deletion of gcn5 or sas3 with ada2 in the k14r strain further reduced the level of k4me3 when compared to the control k14r strain ( figure  2d ) .
because there is already no k14ac in the k14r strain , the additional reduction in k4me3 can not be due to loss of this modification .
therefore , gcn5 and sas3/ada2 must be able to modulate k4me3 independently of k14ac , possibly via acetylation of other lysines , global changes in transcription or indirectly through the reduced growth rate of the deletion strains .
many gene deletions cause slow growth and result in a transcriptional signature that correlates strongly with the common environmental stress response ( esr ) ( oduibhir et  al . , 2014 ) .
however , there was no obvious difference in growth rate or stress resistance of the k14 and p16 substitution strains compared to wt that might explain reduced k4me3 ( figure  2e ) . together , the varying levels of k4me3 in the p16 and the three k14 substitution strains point toward a more modulatory role for these residues rather than a strict requirement for acetylation at k14 . to investigate if k14 and p16 were influencing k4me3 through a common pathway , k14 p16 double substitution strains were created ( no growth defect observed , figure  s2a ) and levels of k4me3 were assessed globally or by chip at the 5 ends of rps15 , sen1 , fmp27 , and pgk1 ( figure  3 ) .
the global levels ( figures 3a and 3b ) and the chip signals ( figure  3c ) for k4me3 in the k14a p16v and k14q p16v strains were similar to the k14a and k14q single substitutions , respectively .
by contrast , the signals in the k14r p16v strain were most reduced compared to the single k14r substitution .
together , these findings imply that there may not be a simple linear pathway between k14 , p16 , and k4me3 , perhaps consistent with a structural role  for p16 and an additional function at k14 .
we note that the relative k4me3 levels by chip varied at the four genes ( figure  3c ) , suggesting that genes respond nonuniformly to these substitutions .
next , we wanted to uncover more about the mechanism by which the k14 and p16 substitutions modulate levels of k4me3 .
because spp1 is required for k4me3 , we asked whether k14 and p16 substitutions influence the association of spp1 with chromatin .
chip of spp1-ha at six genes in the four histone substitution strains revealed levels at or above wt in the k14r strain , reduced levels in the p16v strain and low levels in the k14a and k14q substitution strains , generally reflecting global levels of k4me3 in these strains ( figure  4a ) .
no change in global spp1-ha protein levels , or swd1-ha in the set1c , is observed in the h3 substitutions ( figure  s2b ) .
this supports a role for k14 and p16 in spp1-dependent k4me3 . once again , as the k14r substitution has near wt levels of both spp1 chromatin association and global k4me3 , acetylation of k14 can not be required for spp1 binding and k4me3 .
we asked whether spp1 and substitutions at k14/p16 are  epistatic by deleting spp1 from the k14/p16 substitution strains .
levels of k4me3 were further reduced in the k14/p16-substituted spp1 strains but remained in proportion to the amount of k4me3 in the individual k14/p16 substitutions when spp1 is present ( figures 4b and 4c ) .
interestingly , levels of k4me2 are also substantially lower in the k14a spp1 and k14q spp1 strains than in the k14r spp1 strain , suggesting that these k14 substitutions render the residual k4 methylation in the spp1 strain more sensitive to demethylation by jhd2 ( figure  4b ) . to test this , jhd2 was deleted in a variety of strains .
first , we examined k4me3 in jhd2 strains with a wt copy of histone h3 .
an increase in k4me3 was observed upon jhd2 deletion at both the global level ( figures 4c and 4d ) and at the 5 end of fmp27 by chip ( figure  4e ) in the presence or absence of spp1 .
we note that levels of jhd2 protein do not change in these strains ( figure  s2c ) . upon jhd2 deletion , global levels of k4me3
whether any of the substitutions resulted in a greater increase in k4me3 in the absence of jhd2 ( jhd2 ) compared to the increase in the wt histone h3 strain , we assessed the fold change in k4me3 ( figure  4f ) .
the k14a , k14q , and p16v strains all showed a larger fold change than observed in the wt or k14r strains , suggesting that these substitutions result in increased action of jhd2 toward k4me3 , accounting in part for the reduced levels compared to wild - type ( wt ) .
finally , we examined the effects of the double spp1jhd2 deletions in the k14/p16-substituted strains ( figures 4b and 4c ) .
although levels are low , in all spp1jhd2 strains we observed an increase in k4me3 compared to the spp1 k14/p16 substitutions .
interestingly , deletion of jhd2 did not even partially rescue levels of k4me2 in the k14a spp1 or k14q spp1 strains ( figure  4b ) .
therefore , reduced k4me2 in these strains can not be explained by increased demethylation , but could instead result from increased histone turnover or from the k14a / q substitutions compromising the capacity of the set1c to both di- and trimethylate k4 in the absence of spp1 .
thus the h3 substitutions are altering the balance of k4 methylation by set1/spp1 and demethylation by jhd2 to result in reduced levels of k4me3 .
so far we have demonstrated how k14 and p16 are both required for optimal k4me3 .
these residues influence k4me3 in an overlapping but distinct manner with similar altering of the balance of spp1 and jhd2 action .
thus , we addressed how changes to the h3 tail around k14 and p16 might influence k4me3 genome - wide . to examine the effect of substitutions at k14 and p16 on levels of  k4me3 genome - wide , chip - sequencing ( chip - seq ) experiments for k4me3 were performed with the wt , k14a , and p16v substitution strains , normalized to h3 levels in each strain .
the k4me3 levels in the wt strain correlated well with the previously published data ( kirmizis et  al .
all nondubious genes ( n  = 5,771 ) were ranked according to the ratio ( from largest to smallest ) of the average k4me3 over the transcription unit in the substitution strains relative to the wt strain ( table s1 ) .
genome - wide , we observed a continuum from least to  most affected genes ( figure  s3b ) .
there was a positive correlation between the ratio of k4me3 ( k14a / wt and p16v / wt ) and  nascent sense transcription ( rs  = 0.326 and 0.425 , respectively ) , but no strong correlation with gene length ( rs  = 0.063 and 0.003 ) .
the 200 genes that maintained the highest k4me3 in the k14a or p16v stains relative to wt were classed as largely k14/p16-independent whereas the 200 genes that lost the most k4me3 in the h3 substitution strains were classed as most k14/p16-dependent ( table s1 ; figures 5a and s3b ) .
these classes were used in gene ontology ( go ) ( huang et  al . , 2009a , 2009b ) and overlap analyses , which revealed that the k14/p16-independent and k14/p16-dependent genes function in very different biological processes and show different transcriptional dependencies on saga ( table s2 ; figures 5b and s3c ) .
genes with the highest ratios of k4me3 , in both the k14a and p16v strains relative to wt , are predominantly involved in translation , particularly ribosomal protein genes ( rpgs ) , and are enriched in genes expressed during the oxidative phase of the ymc and repressed during the esr .
in contrast , k14/p16-dependent genes , with k4me3 most reduced by the k14a or p16v substitutions , are predominantly regulated by saga , are involved in the response to environmental stresses and metabolic processes , and tend to be induced during the esr and reductive phase of the ymc .
interestingly , in the p16v substitution strain , the genes with k4me3 most reduced encode proteins involved in sporulation and meiosis , in addition to some of the stress response pathways common to the k14a - affected genes ( table s2 ) .
despite these differences , the  spearman correlation between the ranked  genes from the k14a and p16v strains is 0.772 .
this suggests that although the general trends are similar , k14 and p16 may  contribute to different extents to k4me3 levels at individual genes , consistent with both overlapping and distinct functions .
further analysis of the 200 k14/p16-independent and k14/p16-dependent genes revealed distinct differences ( figures 5c and 5d ) . in the wt strain ,
the k14/p16-independent genes are particularly enriched in nascent sense transcription ( churchman and weissman , 2011 ) and nrd1 ( mayer et  al . , 2012 ) , one of the few genome - wide transcription termination factors shown to be dependent on k4me3 ( schulz et  al . , 2013
; terzi et  al . , 2011 ) , in a pattern distinct from the majority of genes . by contrast , the k14/p16-dependent genes are depleted for nrd1 and nascent sense transcription and enriched for antisense transcription ( as expected due to low nrd1 levels ) , suggesting distinct regulation of these genes .
we used chip at six genes to validate our genome - wide data and confirm that at two rpgs ( rps15 and rpl10 ) a strain with a k14a substitution retains high levels of k4me3 ( figure  5e ; see also figure  3c ) . at four other genes , occupying various positions in the ranking ,
given the link between k14 and spp1 , we asked how loss of spp1 affects k4me3 , using chip - qpcr at the same six representative genes ( figure  5e ) .
the profile of k4me3 in the spp1 strain is very similar to the k14a strain .
spp1 is not required for k4me3 at rpgs but is required at the remaining four genes .
we observed no significant change in k14ac at these genes in a spp1 strain that might explain changes in k4me3 ( figure  5f ) .
this suggests that spp1 , like k14 , is not essential for k4me3 and further links spp1 function to the integrity of k14 and p16 .
we conclude ( 1 ) that the integrity of k14 and p16 play a major role in determining k4me3 levels at spp1-dependent genes , and ( 2 ) there may be spp1-dependent and spp1-independent forms of set1c in  vivo .
given the similar effects of k14 and p16 substitution on k4me3 on different genes and the results from the k14  p16 double substitution experiments , we wanted to learn more  about the relationship between these two residues , particularly how the k14r substitution might differ from other k14 substitutions .
we have already demonstrated that the p16v substitution prevents antibodies raised against k14ac or k18ac from recognizing their epitopes on peptides ( see figure  s1c ) .
we developed a second assay that involves monitoring the effect of the p16v substitution on the binding of the spt7 bromodomain to an h3 peptide modified by acetylation at k14 and/or k18 using surface plasmon resonance ( spr ) ( figures 6a , s4a , and s4b ) or by pull downs ( figures 6b and s4c ) .
bromodomains are acetyl - lysine binding domains , found in a number of chromatin - associated proteins ( taverna et  al . , 2007 ) including spt7 .
the function and binding specificity of the spt7 bromodomain are currently unknown , but it preferentially interacts with hyperacetylated histones in  vitro ( hassan et  al . , 2007 ) .
the spt7 bromodomain ( residues 363619 ) specifically recognized acetylated k18 , as evidenced by the lack of binding to the unmodified h3 peptide , or a peptide acetylated at k14 ( figures 6a and 6b ) , or of the bromodomain carrying alanine substitutions in residues known to influence acetyl - lysine binding ( y500 , y520 , and n521 ) ( figure  s4c ) .
the binding of the spt7 bromodomain to the p16v - substituted k18ac peptide improved binding by 2-fold .
we reasoned that this could reflect an effect of the side chain on binding or an influence on the conformation of the a15-p16 bond . to test this
, we introduced a valine substitution at p16 in the k14r - k18ac peptide , thereby fixing the a15-v16 peptide bond in the trans configuration and restored binding at levels between the p16v - k18ac and k18ac peptides .
this suggests that in the k14r - k18ac peptide , the a15-p16 peptidyl - prolyl bond adopts the cis configuration , reducing binding of the bromodomain to k18ac , which can be restored by fixing the bond in trans .
the corollary of this is that acetylation of k14 would correlate with the a15-p16 bond in the trans configuration , supporting ideas that the peptidyl - prolyl bond conformation is mainly determined by local effects in proteins ( reimer et  al . , 1998 ) .
we tested this using a peptide acetylated at both k14 and k18 and show binding to the spt7 bromodomain similar to the p16v - k18ac peptide , consistent with our hypothesis that acetylation of k14 influences the conformation of the a15-p16 peptide bond to favor the trans configuration .
to test more directly the influence of substitutions or modifications at k14 on cis versus trans conformations of the a15-p16 peptide bond , we used an established protease - coupled proline  isomerization assay ( fischer et  al . , 1984 )
we  designed four peptides , in which k14 was unmodified or acetylated , or substituted with r or q and where p16 is followed by a phenylalanine and paranitroaniline ( f - pna ) group .
chymotrypsin cleaves these peptides to release pna only if the a15-p16 peptide bond is in the trans conformation and with kinetics that are much faster than the spontaneous rate of cis - trans isomerization of the proline .
thus , the initial rate of the chymotrypsin - dependent release of pna , measured spectrophotometrically at 395  nm , is a function of the amount of peptide with a trans proline conformation and for the unmodified peptide is comparable to that reported previously in a similar assay ( nelson et  al . , 2006 ) .
acetylation of k14 increases the initial rate of cleavage of the peptide 3-fold , indicating that a higher proportion of the a15-p16 peptide bond is in the trans conformation ( figure  6d ) .
the k14q substitution resulted in an increase in  the initial rate of cleavage of the peptide , consistent with an increase in the amount of peptide in the trans conformation .
crucially , a k14r substitution reduced the initial rate of cleavage of the peptide below the unmodified peptide consistent with an increase in the cis conformation . taken together these data suggest that ( 1 ) the conformation of the a15-p16 peptidyl - prolyl bond influences the binding of protein effectors to histone h3 , ( 2 ) the conformation of the a15-p16 peptidyl - prolyl bond is likely to be influenced by the nature of the residue at n-2 and that acetylation of k14 promotes the trans conformation ( figure  6e ) , and ( 3 ) k4me3 is promoted by unmodified k14 increasing a15-p16cis ( figure  6f ) .
we have shown that acetylation at k14 influences the local conformation of histone h3 at the a15-p16 peptide bond in  vitro , promoting a trans conformation , and we describe how these changes reduce levels of k4me3 in  vivo .
we propose that the h3 tail alternates between two dynamic forms in  vivo : one with k14ac and p16trans and the other with unmodified k14 and p16cis ( figure  6f ) .
moreover , substitutions at k14 and p16 affect the balance of methylation by set1c / spp1 and demethylation by jhd2 to modulate k4me3 levels .
we find that the amount of k4me3 lost on genes in strains lacking spp1 or with substitutions at k14 or p16 is not uniform .
this is particularly evident on genes that are differentially regulated during the yeast metabolic cycle and environmental stress response ( figure  6 g ) .
we infer at least two distinct mechanisms for deposition of k4me3 , one of which is sensitive to loss of spp1 from set1c and the conformation of the n - terminal region of histone h3 .
spp1 has been proposed to be critical for stimulating the trimethylase activity of set1 in set1c ( takahashi et  al . , 2009 ) .
a remarkable finding from this study is that k4 trimethylation ( k4me3 ) at the rpgs is largely independent of spp1 and mostly insensitive to the acetylation status of k14 or cis - trans isomerization of p16 , which may regulate the association of spp1 with chromatin .
this suggests an additional mechanism for stimulating k4me3 that predominates at rpgs but also functions at other genes to varying extents .
set1 contains rrm rna binding motifs and the binding of rna by set1 is proposed to stimulate its k4me3 activity ( schlichter and cairns , 2005 ; trsaugues et  al . , 2006 ) .
high levels of sense transcription , intronic rna ( weiner et  al . , 2012 ) , antisense transcripts ( margaritis et  al . , 2012 ; murray et  al . ,
2012 ) , and nrd1-attenuated noncoding transcripts ( schulz et  al . , 2013 ) at rpgs may contribute to spp1-independent alternative anchoring of set1 to this gene class . at the majority of yeast genes , including esr - induced genes ,  maintenance of k4me3 depends on the integrity of k14 , a15-p16cis ( bent ) and spp1 .
these genes have lower levels of sense transcription that may not be sufficient for the deposition of k4me3 by a k14/p16-independent mechanism .
although often thought of as unstructured , 50% of the h3 n - terminal tail adopts an -helical conformation in the nucleosome ( banres et  al . , 1997 ) .
proline is known to disrupt  helices and may  act as hinge points in h3 , dramatically affecting the relative trajectories of the flanking helices ( lu et  al . , 2007 ) and
thus the ability of modifying enzymes to find their substrates . rather than direct cause and effect , our data support a highly dynamic scenario in which levels of k4me3 are influenced by the action of the set1c methyltransferase with spp1 and the jhd2 demethylase , which in turn are influenced by the modification status of k14 and the conformation of the a15-p16 bond ( figure  6 g ) .
more generally , acetylation in proximity to proline residues may act as a metabolically controlled switch within intrinsically disordered regions of proteins to facilitate the alternative conformations required for interactions with a range of different proteins .
although ppiases promote protein folding by increasing the rate of proline isomerization , in disordered regions the cis - trans switch may rely on the natural slower isomerization rate with local ptms , such as acetylation , influencing one state over the other , perhaps by steric hindrance . in  vivo
, these states could be captured by binding proteins to induce local folding . in summary , we describe a function for lysine acetylation in determining local protein conformation , in this case histone h3 , by directly influencing proline 16 cis - trans isomerization .
the potential for antibody occlusion in yeast expressing substituted histones or off - target effects resulting from hdac inhibition preclude further reliable in  vivo validation of this relationship , although tools to explore this are being developed .
k4me3 interacts with a variety of protein effectors in yeast ( vermeulen and timmers , 2010 ) , such as spp1 , the kat components yng1 and sgf29 , the chromatin remodeling atpase isw1 , and the hdac complex rpd3l , potentially leading to acetylation or deacetylation of residues , including k14 , in the vicinity .
the resulting positive and negative feedback loops , coupled to variable concentrations of intracellular nadph and acetyl coa during the metabolic cycle and growth , could facilitate dynamic regulation of k14 acetylation / deacetylation , cis - trans isomerization at p16 , and k4 demethylation / trimethylation at different gene classes , allowing the rapid switching between transcriptional states and adaptation to changing conditions .
differential  control of k4me3 could modulate many other processes , including initiation of dna replication ( kan et  al . , 2008 ) ,
2013 ) , and epigenetic memory ( muramoto et  al . , 2010 ) . given the many different enzymes and proteins capable of methylating , demethylating , and interacting with k4me3 in multicellular organisms and the variety of processes associated with k4me3 ( vermeulen and timmers , 2010 ) , fine tuning mechanisms such as those described here would extend the versatility of this near universal but still poorly understood histone modification .
yeast cells were grown at 30c , shaking at 200  rpm in ypd , to exponential phase ( 1.25   10 cells / ml ) for whole cell extracts , subject to western blots , and visualized using chemiluminescence ( pierce ) and exposure to x - ray film .
peptide synthesis and antibody production to raise polyclonal antibodies against p16ohcis and p16ohtrans peptides were performed by pacific immunology and antibody affinity purified with 90  g p16ohtrans or p16ohcis peptides , respectively and 90  g unmodified h3 peptides dotted onto nitrocellulose for 48  hr at 4c .
the p16 proline isomerase assay was performed as described ( shan et  al . ,
1994 ) using 10  l of 7.8  mm pna peptide ( proteogenix ) solution per 1  ml assay reaction and pna release monitored at 395  nm .
chip - qpcr was performed as described ( morillon et  al . , 2005 ) .
dna was multiplexed during library preparation and subjected to 50 nt paired - end single lane sequencing .
the peptides used in these experiments had a minimum purity , determined by maldi - tof mass spectrometry and hplc , of 84% .
spt7 bromodomain ( amino acids 363619 ) was expressed and purified as described ( boubriak et  al . , 2009 ) .
resin ( 200  l ) was incubated with 350  l of bromodomain ( final concentration of 30  g / ml ) for 2  hr at 4c .
after multiple washes with binding buffer , high salt ( 350  mm nacl ) , and te buffer , bound material was eluted by heating at 80c with 120  l lds ( invitrogen ) .
spr data for peptides binding to immobilized spt7 bromodomain was generated on biacore t100 and t200 instruments at 25c .
sensorgrams , binding curves , and kd values were analyzed with bia t100 and t200 evaluation software ( ge healthcare ) using a 1:1 binding model .
 summarygene transcription responds to stress and metabolic signals to optimize growth and survival . 
 histone h3 ( h3 ) lysine 4 trimethylation ( k4me3 ) facilitates state changes , but how levels are coordinated with the environment is unclear . here 
 , we show that isomerization of h3 at the alanine 15-proline 16 ( a15-p16 ) peptide bond is influenced by lysine 14 ( k14 ) and controls gene - specific k4me3 by balancing the actions of jhd2 , the k4me3 demethylase , and spp1 , a subunit of the set1 k4 methyltransferase complex . 
 acetylation at k14 favors the a15-p16trans conformation and reduces k4me3 . 
 environmental stress - induced genes are most sensitive to the changes at  k14 influencing h3 tail conformation and k4me3 . by contrast 
 , ribosomal protein genes maintain k4me3 , required for their repression during stress , independently of spp1 , k14 , and p16 . thus , the plasticity in control of k4me3 , via signaling to k14 and isomerization at p16 , informs distinct gene regulatory mechanisms and processes involving k4me3 . 
Introduction
Results
Discussion
Experimental Procedures