The amino acid composition following H2BK123 is not important for H2B ubiquitination and H3K4 methylation.
Previously, to test whether the SUMO moiety can substitute for ubiquitin at the H2B C terminus to mediate the trans
-histone cross talk (4
), we created a chimeric H2B by introducing two consensus sumoylation sites to replace H2B residues T122 and K123 [H2B(2SU)] (Fig. ). We confirmed that H2B(2SU) is modified by sumoylation and ubiquitination (4
) (Fig. , lanes 2 and 3). Replacement of the lysine residues (K1 and K2) (Fig. ) in H2B(2SU) with leucine (L) showed that only sumoylation and only ubiquitination occurred in H2B(2SU)-K1L
, respectively (Fig. , lanes 4 and 5). However, sumoylation at the H2B C-terminal region cannot support H3K4 methylation (Fig. ). Although H2B(2SU)-K2L
contains ubiquitination levels similar to those of the wild type, H3K4me3 is reduced in this mutant (Fig. , lane 5). This is likely due to the differences in the amino acid composition of H2B and H2B(2SU)-K2L (Fig. ); the latter contains a substitution (threonine to isoleucine) and a seven amino acid insertion adjacent to K123. Therefore, the reduced H3K4me3 levels in this mutant suggested a novel possibility that residues surrounding K123 might also play a role in controlling H3K4 methylation in addition to H2Bub1.
We changed all the residues after K123 to alanines [H2B(124-129A)] (Fig. ) to determine their role in controlling H2Bub1 and H3K4 methylation. H2B(124
had no apparent effect on H2Bub1 and H3K4 methylation (Fig. ). However, eliminating all the residues after K123 (H2B-Y124stop
; Fig. ) resulted in a severe decrease in both of the histone modifications (Fig. ). Therefore, even though most of the residues following K123 form an unstructured C-terminal “tail” domain (54
), they are still essential for maintaining normal levels of H2Bub1 and H3K4 methylation, but their exact amino acid sequences are not important for these processes.
Yeast strains used in this study contain N-terminally Flag epitope-tagged H2B or its derivatives and were derived from parental histone shuffle strain Y131 (39
). Importantly, this strain does not contain any extra mutation that suppresses the effect of the H2B-K123R
mutant on H3K4 and -K79 methylation (11
). Moreover, previously reported H2A and H2B mutations with or without a Flag epitope exerted the same effect on H3K4 methylation and H2Bub1 in Y131 and in other independent yeast strains (32
Mutations in H2B T122 differentially affect H2Bub1 and H3K4, but not H3K79, methylation.
Since the amino acid composition following H2B residue K123 is not important for H2Bub1 and H3K4 methylation, the decrease in H3K4me3 in H2B(2SU)-K2L (Fig. ) is likely due to the threonine-to-isoleucine substitution at position 122. Therefore, we tested whether the residues preceding K123 play any role in establishing H2Bub1 and H3K4 methylation. We replaced T122 in H2B with the following differently charged residues, an alanine (T122A), arginine (T122R), or aspartate (T122D), and assessed their effects on H2Bub1 and H3K4 methylation. Compared to wild-type levels, H2Bub1 levels were reduced in H2B-T122R but remained unaffected in H2B-T122A (Fig. ). In contrast, the H2Bub1 level was enhanced in H2B-T122D. A ~4.6-fold increase in H2Bub1 levels relative to unmodified H2B was observed in this mutant compared to that in the wild type (Fig. ).
FIG. 2. H2B C-terminal residues R119 and T122 control H2Bub1 levels, H3K4 methylation, and telomeric gene silencing. (A and D) Western blots for H2Bub1, as described in the legend to Fig. . (B and E) Western blots for H3K4 and -K79 methylation. (more ...)
Next, we tested whether mutations in T122 also affected H3K4 methylation. Similar to its effect on H2Bub1 levels, H2B-T122A
did not alter H3K4 methylation (Fig. ). H2B-T122R
led to a slight reduction in H3K4me3 (Fig. ), although it caused a significant decrease in H2Bub1 levels. This observation is similar to the finding that bur1
and certain rad6
mutants cause a severe reduction in H2Bub1 levels but maintain near normal levels of H3K4 methylation (4
). Given the increase in H2Bub1 levels in H2B-T122D
, we anticipated an increase in H3K4 methylation. Surprisingly, H3K4me1 and -me2 were not affected, and the levels of H3K4me3 were reduced in this mutant (Fig. ). This decrease in H3K4me3 even in the presence of large amounts of H2Bub1 reveals a novel and distinct role for the H2B C-terminal helix in modulating the function of Set1-COMPASS.
H2Bub1 also regulates Dot1-mediated H3K79 methylation (3
). However, both H2B-T122D
had no effect on H3K79me2 or -me3 (Fig. ). Therefore, these mutations specifically affect H3K4me3. To the best of our knowledge, this is the first demonstration of an uncoupling of the H2Bub1-mediated coregulation of H3K4 and -K79 methylation by specific H2B mutations.
Mutations in H2B R119 affect only H2Bub1 and H3K4 methylation without affecting H3K79 methylation.
The increased H2Bub1 level observed in H2B-T122D
is reminiscent of the effect observed in yeast strains lacking H2Bub1-specific deubiquitinases (UBP8
). Since H3K4 methylation was increased in ubp8
Δ and/or ubp10
), the reduced H3K4me3 in H2B-T122D
is not due to the elevated levels of H2Bub1. Instead, it suggests a role for the H2B C-terminal helix in controlling the function of Set1-COMPASS. Next, we postulated that in addition to T122, the other residue(s) preceding K123 might also be important for this trans
-histone pathway. To test this hypothesis, we mutated the arginine (R) residue at position 119. The H2B R119 residue was targeted because it is spatially adjacent to, and resides on the same phase of the H2B C-terminal helix as, T122 and K123 (Fig. ). Western analyses revealed that the levels of H2Bub1 and H3K4 methylation were severely reduced in the H2B-R119D
mutant (Fig. ). In H2B-R119K
, the levels of H2Bub1, H3K4, and -K79 methylation were similar to those in the wild type (data not shown). Importantly, similar to H2B-T122D
enhances H2Bub1 levels (~2.4 fold change) (Fig. ) and severely reduces H3K4me3 (Fig. ). A slight reduction in H3K4me2 was also observed in H2B-R119A
(Fig. ). Moreover, H3K79 methylation was not reduced in H2B-R119D
(Fig. ), further demonstrating an uncoupling of the H2Bub1-mediated coregulation of H3K4 and -K79 methylation. The effect of H2B-R119A
appears to be highly selective toward H3K4 methylation, as they do not affect H3K56 or H4K16 acetylation (data not shown). Additionally, the reduced H3K4 methylation in these H2B mutants is not due to enhanced demethylation, since global Jhd2 levels are not altered by the H2B-R119A
mutant (data not shown).
The increase in H2Bub1 levels observed in H2B-R119A
is not due to any increase in overall H2B transcript levels, and these H2B mutations do not induce transcription of the extraneous GAL1/GAL10
promoter-driven copy of HTA2-HTB2
(data not shown), which is present in the yeast strain used in this study (Y131) (33
). Additionally, the effects of both mutations on H2Bub1 and H3K4 methylation were reproducibly observed in an Y131-derived strain (FGY8), which lacks the GAL1/GAL10
, and in a completely independent strain background (FY406) (data not shown). Collectively, we conclude that the R119 and T122 residues preceding H2BK123 play an important role in modulating both H2Bub1 and H3K4 methylation levels.
H2B-R119A and H2B-T122D confer a telomeric silencing defect.
H3K4 methylation is important for maintaining telomeric gene silencing in yeast (49
). Therefore, we tested whether the H2B C-terminal helix mutations induce expression from the silent URA3
gene integrated at the left-end telomere of chromosome VII (49
). Since H2B-R119D
caused a severe reduction in H3K4 methylation (Fig. ), it failed to grow on 5-fluoroorotic acid (5FOA)-containing medium, similar to H2B-K123R
(Fig. ). Moreover, H2B-R119A
, which reduced only H3K4me3, also conferred a severe telomere-silencing defect similar to that of H2B-K123R
(Fig. ). This finding confirms a role for H3K4me3 in maintaining telomeric silencing, as reported previously (8
). However, it conflicts with the findings from another study (43
), which showed that H3K4me3 is not required for telomeric silencing. Indeed, H2B-T122R
also caused a reduction in H3K4me3, albeit to a lesser degree than H2B-R119A
(Fig. ), but it did not confer 5FOA sensitivity (Fig. ). Therefore, our findings suggest that a certain overall threshold level of H3K4me3 is needed for maintaining telomeric silencing.
H2B-R119A and H2B-T122D increase H2Bub1 levels by hindering deubiquitination.
H2B-R119A and H2B-T122D yield ~2.4- and ~4.6-fold increases in H2Bub1 levels, respectively (Fig. and A, left). These differences in H2Bub1 levels in the mutants may be due to various degrees of stimulation of Rad6-mediated ubiquitin conjugation. This possibility was tested by examining the steady-state H2Bub1 levels of wild-type H2B, H2B-R119A, and H2B-T122D in the absence of deubiquitinases (ubp8Δ ubp10Δ) to circumvent any contributions from an altered deubiquitination. Western analysis showed that the overall H2Bub1 levels in the H2B mutants were similar to those in wild-type H2B in ubp8Δ ubp10Δ (Fig. , right). Notably, the ~2-fold difference in H2Bub1 levels between H2B-R119A and H2B-T122D, which was evident in the wild-type strain (Fig. and Fig. , left), was not observed in ubp8Δ ubp10Δ (Fig. , right). This result suggests that the increased H2Bub1 levels in the H2B-R119A or H2B-T122D mutant (in the UBP8 UBP10 background) is likely not due to a differential stimulation of Rad6-mediated ubiquitination, but instead, these levels might be due to defects in the function of deubiquitinases.
FIG. 3. H2B-R119A and H2B-T122D increase H2Bub1 levels by affecting deubiquitination. (A) Western blot for H2Bub1, as described in the legend to Fig. . (B) Western blot analysis of Ubp8-3Flag, Rad6-9Myc, and Ubp10-3HA in the whole-cell extracts (more ...)
Next, we examined the global chromatin-bound levels of Rad6, Ubp8, and Ubp10 in strains harboring wild-type or mutant H2B using standard chromatin fractionation analysis (1
). Briefly, nuclei isolated from the wild type or mutants were lysed using a hypotonic solution to obtain chromatin. An equal amount of chromatin from each strain was subjected to Western analyses. To ensure equal loading of chromatin, we examined the levels of H3. Compared to the levels in the wild type, a similar fold reduction in the chromatin-bound levels of Rad6 and Ubp8 was observed in H2B-R119A
, and Ubp10 levels remained unchanged (Fig. ). This finding suggests that the augmentation of H2Bub1 levels in both of the mutants is not due to the increased chromatin-bound Rad6 level; instead, it is due to the reduced chromatin association of Ubp8 and defects in deubiquitination.
To directly test the effects of H2B-R119A and H2B-T122D in maintaining H2Bub1 levels, we examined the ability of these mutations to affect deubiquitination in vitro. When isolated under denaturing conditions, wild-type and mutant H2B showed similar H2Bub1 levels in ubp8Δ ubp10Δ (Fig. ). Therefore, we used these strains for the experiment, as they contained equal amounts of H2Bub1 but differed only in their H2B sequences. Since H2Bub1 is a very labile modification and is rapidly removed by deubiquitinases, initially we prepared extracts from the ubp8Δ ubp10Δ strain expressing either wild-type or mutant H2B under native conditions and monitored the differences in the loss of H2Bub1 due to nonspecific removal by other yeast deubiquitinases. As shown in Fig. , wild-type and mutant H2B retained similar H2Bub1 levels, even when isolated under native conditions (mock, 0 min) and following prolonged incubation (mock, 10 min and 40 min). These data confirm that Ubp8 and Ubp10 are the major H2Bub1-specific deubiquitinases in yeast. Therefore, we incubated extracts from the ubp8Δ ubp10Δ strain harboring wild-type or mutant H2B with an extract from a wild-type yeast strain, which contains intact Ubp8 and Ubp10. All extracts were prepared under native conditions to preserve active deubiquitinases. H2Bub1 levels remained unchanged in extracts containing H2B-R119A or H2B-T122D, even after 40 min of incubation with extracts containing Ubp8 and Ubp10 (Fig. ). This is in striking contrast to near-complete loss of H2Bub1 in extracts containing wild-type H2B under the same condition. This finding demonstrates that H2B-R119A and H2B-T122D impede the ability of Ubp8 and Ubp10 to remove H2Bub1. Collectively, we conclude that the increased H2Bub1 levels in H2B-R119A and H2B-T122D are the result of decreased chromatin association and reduced enzymatic action of deubiquitinases, especially Ubp8.
H2B-R119A and H2B-T122D alter the occupancy of H2Bub1 and H3K4 methylation on transcribed genes.
Next, we used chromatin immunoprecipitation (ChIP) assays to assess overall occupancy and distribution of H2Bub1 and H3K4 methylation on chromatin in H2B-R119A
. We looked at the relative enrichment of these histone modifications at the 5′, middle, and 3′ ORF regions of constitutively expressed genes PMA1
(Fig. A). To evaluate the occupancy of H2Bub1, we carried out a chromatin double-immunoprecipitation assay (4
). In parallel ChIP assays, we used methylation-specific antibodies to directly isolate chromatin containing different methylated forms of H3K4.
FIG. 4. H2B-R119A and H2B-T122D increase H2Bub1 levels and alter H3K4 methylation levels on transcribed genes. (A) Schematic diagrams of the gene organization for PMA1 and DMA2 are shown, drawn to scale. Solid black lines represent the 5′, middle (M), (more ...)
A uniform distribution of H2Bub1 across the ORFs of PMA1 and DMA2 was observed in the wild type, and this distribution pattern was not disrupted in H2B-R119A and H2B-T122D (Fig. ). However, the overall occupancy of H2Bub1 in both genes was considerably increased in the mutants. Also, the level of H2Bub1 occupancy was generally higher in H2B-T122D than in H2B-R119A. These findings correlate well with the increase in steady-state H2Bub1 levels in these mutants (Fig. ). Taken together, these findings demonstrate that the H2B C-terminal helix residues R119 and T122 play an important role in modulating H2Bub1 levels during transcription.
Next, assessment of occupancy and distribution of H3K4 methylation on PMA1 and DMA2 showed that the general distribution of H3K4me3 across both ORFs in both mutants was similar to that of the wild type, but its occupancy levels were reduced in all regions, and the reduction in H3K4me3 was more severe in H2B-R119A (Fig. ). This result is consistent with the Western analyses (Fig. ). Importantly, this finding reveals that H2B residues R119 and T122 play an important role in the establishment of H3K4me3 during transcription.
Unlike H3K4me3, the occupancy and distribution of H3K4me1 and -me2 on PMA1 and DMA2 in the H2B-R119A and H2B-T122D mutants showed a few surprising differences. In general, changes in the levels of H3K4me1 and -me2 on PMA1 in the two mutants closely resemble the changes in the global levels of these modifications (Fig. ), albeit with minor region-specific differences. The occupancy of H3K4me2 on PMA1 was severely reduced in H2B-R119A compared to H2B-T122D and the wild type (Fig. , left). Also, H3K4me1 occupancy on PMA1 remained unchanged in the mutants, except for a reduction in the 3′ region (Fig. , left). However, both the distribution and occupancy of H3K4me1 and -me2 on DMA2 were dramatically altered in the H2B mutants relative to the wild type (Fig. , right), and they differ from PMA1 and the results of Western analyses (Fig. ). Occupancy of H3K4me2 was reduced in the middle and 3′ regions of DMA2 in H2B-R119A and H2B-T122D. In contrast, an increase in H3K4me2 occupancy at the 5′ region of DMA2 was observed in both mutants (Fig. , right). Compared to the wild type, the occupancy of H3K4me1 on DMA2 in the mutants showed a decrease in the 3′ region, but an increase in 5′ region, with a dramatic 6-fold increase in H2B-R119A (Fig. , right). Therefore, these findings suggest that H2B R119 and T122 play an important role in modulating all forms of H3K4 methylation during transcription. Importantly, since H2B-R119A and H2B-T122D affect both global and local levels of H3K4 methylation even in the presence of high levels of H2Bub1 (Fig. and ), our findings further suggest that these H2B mutations might modulate the functions of Set1-COMPASS independent of H2Bub1.
H2B-R119A and H2B-T122D affect Set1-COMPASS-mediated H3K4 methylation independent of H2Bub1.
To test whether H2B-R119A and H2B-T122D affect Set1-COMPASS functions independent of H2Bub1, we examined H3K4 methylation levels in yeast strains harboring the wild-type or mutant H2B but lacking the deubiquitinases Ubp8 and Ubp10. The ubp8Δ ubp10Δ strain harboring either wild-type or mutant H2B showed very high but similar levels of H2Bub1 (Fig. ). However, H3K4me3 levels were still reduced in strains harboring mutant H2B (Fig. A). This finding supports the hypothesis that H2B residues R119 and T122 can affect Set1-COMPASS function independent of H2Bub1.
FIG. 5. H2B-R119A and H2B-T122D adversely affect the functions of normal and hyperactive Set1 in an H2Bub1-independent manner. (A to D) Western blots for H3K4 methylation in the indicated strains. Wild-type Set1 or its dominant, hyperactive form (Set1D-G990E) (more ...)
To gain insight into the mechanism by which H2B-R119A
affect H3K4 methylation, we tested whether enhancing the Set1 activity could restore H3K4me3 in these mutants. To this end, we used a dominant Set1 allele (Set1D
-G990E), which increases all forms of H3K4 methylation in the wild type and has the ability to partially restore them even in the absence of H2Bub1 (42
). Expression of Set1D
-G990E did not restore H3K4me3 levels in H2B-R119A
to those present in the wild type (Fig. , compare lane 1 to lanes 6 and 9). Additionally, in both mutants, the ability of Set1D
-G990E to produce H3K4me3 is reduced (Fig. , compare lane 3 to lanes 6 and 9).
cause an increase in H2Bub1 levels (Fig. ), the reduced H3K4me3 by Set1 and Set1D
-G990E in these mutants might be due to the following two reasons: H2B C-terminal helix mutations might debilitate the stimulation of methyltransferase activities by H2Bub1, or H2B mutations might directly affect the function of Set1 or Set1D
-G990E independent of H2Bub1. To test these possibilities, Set1 or Set1D
-G990E was expressed in strains lacking H2Bub1 (rad6
Δ) and containing either wild-type or mutant H2B. Consistent with a previous finding (42
-G990E was able to partially restore H3K4me1 and -me2 in rad6
Δ (Fig. , lane 4). However, its ability to catalyze H3K4me1 was reduced in the rad6
strains (Fig. , lanes 7 and 10). Importantly, Set1D
-G990E-mediated H3K4me2 and -me3 were completely abolished by the H2B mutations (Fig. , lanes 7 and 10). These results further confirm our novel finding that the H2B C-terminal helix residues R119 and T122 can modulate the function of methyltransferases (Set1 and Set1D
-G990E) independent of H2Bub1.
In the absence of H2Bub1, Set1 produces low levels of H3K4me1, but it cannot catalyze H3K4me2 and -me3 (5
) (Fig. , lane 2). Interestingly, the low level of H3K4me1 seen in rad6
Δ is reduced in the rad6
strains (Fig. ; compare lane 2 to lanes 5 and 8). This result shows that H2B-R119A
affect Set1 functions even in the absence of H2Bub1. Collectively, our findings clearly establish that the H2B C-terminal residues R119 and T122 can modulate Set1-COMPASS independent of H2Bub1.
H2B-R119A and H2B-T122D reduce the chromatin-bound levels of Set1-COMPASS components Sdc1 and Spp1.
Next, we investigated how the H2B C-terminal region might directly modulate the function of Set1-COMPASS. Both Set1 and Set1D
-G990E are present as holoenzymes, copurifying with all COMPASS components, and their activities are dependent on Bre2 (42
). Both enzymes are also dependent on other COMPASS components Swd3, Sdc1, and Spp1 for their activities (Fig. ). Since COMPASS subunits modulate Set1 activity, this puts forth a possibility that H2B-R119A
might affect the association of COMPASS subunits with chromatin and cause changes in H3K4 methylation by Set1, especially a decrease in H3K4me3.
It has been suggested that the biochemical activity of the Set1 complex purified from the total cell population does not reflect the events on chromatin in vivo
). Therefore, we used a combinatorial approach of fractionation and ChIP assays to examine the global and gene-specific changes in Set1-COMPASS subunits on chromatin, respectively. The global chromatin associations of Set1 (Fig. G, top), Swd1, Swd2, Swd3, and Bre2 (Fig. , respectively) in H2B-R119A
were similar to that of the wild type. Also, steady-state levels of Set1 and all other COMPASS components in whole-cell extracts were not affected by these mutations (Fig. ). However, the chromatin-bound Spp1 and Sdc1 levels were reduced in both mutants (Fig. ). The levels of chromatin-bound Sdc1 are lower in H2B-R119A
than in H2B-T122D
(Fig. ), correlating well with the observed decrease in the global H3K4me2 and -me3 levels in H2B-R119A
H2B-R119A and H2B-T122D affect the integrity of Set1-COMPASS on chromatin during transcription.
Next, we examined the Set1-COMPASS integrity on chromatin by evaluating the distribution and occupancy of some of the regulatory subunits on constitutively expressed genes PMA1 and DMA2 using ChIP assays. In general, the overall distribution for most factors remained unchanged in both H2B-R119A and H2B-T122D compared to that of the wild type (Fig. ). Nevertheless, a gene- and region-specific decrease in factor occupancy was observed, further confirming that both mutations affect the integrity of Set1-COMPASS on chromatin. Set1 occupancy was affected by H2B-T122D in the 3′ regions of both genes (Fig. ). Swd2 occupancy was reduced in all regions of PMA1 in H2B-R119A but was affected only in the 5′ region of DMA2 (Fig. ). Bre2 occupancy remained mostly unchanged in both mutants (Fig. ). Although overall chromatin-bound Sdc1 levels were reduced in H2B mutants (Fig. ), the occupancy of Sdc1 was reduced only in the middle and 3′ regions of DMA2 in H2B-T122D but remained unaffected in most regions of both genes (Fig. ). The following two possibilities can be envisaged for the discrepancy in the chromatin-bound Sdc1 levels observed in fractionation and ChIP assays. First, Sdc1 occupancy is reduced on other genes, but not on PMA1 and DMA2. Second, the interaction of Sdc1 with Set1-COMPASS and/or chromatin may be weakened by H2B-R119A and H2B-T122D. This weakened intermolecular interaction causes dissociation of Sdc1 during fractionation, culminating in the apparent reduced levels on chromatin (Fig. ). However, formaldehyde cross-linking stabilizes these weakened interactions and prevents the detection of changes in chromatin interaction of Sdc1 in ChIP assays. Consistent with the reduction in overall chromatin-bound Spp1 levels in fractionation (Fig. ), Spp1 occupancy was reduced in almost all regions of PMA1 and in some regions of DMA2 in both H2B mutants (Fig. ). Collectively, our findings from chromatin fractionation and ChIP assays suggest that the H2B-R119A and H2B-T122D reduce H3K4 methylation levels by affecting the Set1-COMPASS integrity on chromatin, probably by disrupting the direct chromatin association or the binding of some of the regulatory subunits.
FIG. 7. H2B-R119A and H2B-T122D affect the occupancy of Set1-COMPASS subunits on transcribed genes. (A to E) Distribution and occupancy of the indicated Set1-COMPASS subunits in 5′, middle, and 3′ ORF regions of PMA1 and DMA2 in the indicated (more ...) Spp1 interacts with H2B, and this interaction can be disrupted by H2B-R119AT122D.
We used GST pulldown assays to test whether components of Set1-COMPASS bind to the H2B C-terminal helix. Since the chromatin binding of Spp1 and Sdc1 was reduced by H2B-R119A and H2B-T122D (Fig. ), we purified recombinant Spp1 and Sdc1 expressed in bacteria containing N-terminal hexahistidine and GST tags (His6GST) and incubated them with a bacterial lysate containing yeast H2B before binding to glutathione-Sepharose beads. Only His6GST-Spp1, but not His6GST-Sdc1, was able to bind H2B (Fig. A). Next, we tested whether residue R119 or T122 play a role in this interaction between Spp1 and H2B. To this end, we incubated His6GST-Spp1 with bacterial lysates containing either wild-type or mutant H2B. Compared to wild-type H2B, only H2B-T122D caused a slight reduction in Spp1 binding (Fig. ). Therefore, we tested whether a double-site mutant H2B containing both R119A and T122D might compromise Spp1 binding. Indeed, H2B-R119AT122D showed severely reduced binding to Spp1 (Fig. ). Collectively, our findings show that Spp1 interacts with H2B via the R119 and T122 residues and suggest that disruption of this interaction results in changes in H3K4 methylation and reduced chromatin binding of Set1-COMPASS in H2B-R119A and H2B-T122D. Importantly, our findings provide mechanistic insight into the role of the H2B C-terminal helix in the trans-histone cross talk independent of H2Bub1 by modulating the Set1-COMPASS association with the nucleosome.
FIG. 8. H2B C-terminal helix residues R119 and T122 are necessary for the interaction of Spp1 with H2B. (A) A bacterial lysate expressing H2B was incubated with purified GST, His6GST, His6GST-Spp1, or His6GST-Sdc1 before binding to glutathione-Sepharose beads. (more ...) H2B-R119AT122D affects H2Bub1, H3K4 methylation, cell growth, and transcription.
Since H2B-R119AT122D binds poorly to Spp1 (Fig. ), we tested the effect of double mutation on overall H2Bub1, H3K4, and H3K79 methylation levels. The steady-state H2Bub1 levels were dramatically increased in H2B-R119AT122D relative to the wild type (Fig. A) and the single-site H2B mutants (Fig. , bottom). Furthermore, H2B-R119AT122D showed a very severe reduction in all forms of H3K4 methylation compared to the wild type (Fig. ) and single mutants (Fig. ). However, no change in H3K79 methylation was observed (Fig. ). These findings show that H2B-R119AT122D affects only Set1-COMPASS, but not Dot1, function independent of H2Bub1. The effect of H2B-R119AT122D on Set1-COMPASS function is also evident from the severe reduction in the low levels of H3K4me1 present in the rad6Δ strain (Fig. ; long exposure blot), further demonstrating the H2Bub1-independent direct modulation of Set1-COMPASS by the H2B C-terminal helix residues.
FIG. 9. H2B-R119AT122D causes drastic changes in H2Bub1, H3K4 methylation, and gene expression levels and affects cell growth. (A to B) Western blots for H2Bub1, H3K4, and -K79 methylation in the indicated strains. Short exp., short exposure; long exp., long (more ...)
Next, we examined the changes in distribution and overall occupancy of H3K4 methylation on PMA1 and DMA2 in H2B-R119AT122D by ChIP assay. Consistent with Western analyses (Fig. ), both H3K4me2 and H3K4me3 were severely reduced in all regions of both genes in H2B-R119AT122D compared to the wild type (Fig. ) and single-site mutants (Fig. ). Distribution and occupancy of H3K4me1 showed no change in PMA1 but was altered in DMA2 in H2B-R119AT122D (Fig. ), similar to those in the single mutants (Fig. ). Collectively, these findings suggest that the drastic reduction in H3K4 methylation is likely to due to a severe debilitation of chromatin-association of Set1-COMPASS in H2B-R119AT122D.
Given the dramatic change in H3K4 methylation on chromatin (Fig. ), we examined the effect of H2B-R119AT122D on the transcription of PMA1 and DMA2. Measurement of the steady-state mRNA levels showed that PMA1 transcripts were reduced in H2B-R119A and H2B-R119AT122D, whereas the DMA2 transcript levels were not reduced in any mutant (Fig. ). These findings reveal the intrinsic differences in the regulation of PMA1 and DMA2. Importantly, they suggest that the control of Set1-COMPASS-mediated H3K4 methylation by the H2B C-terminal helix may not be tightly coupled to ongoing transcription.
Consistent with reduced H3K4 methylation (Fig. ), H2B-R119AT122D conferred the silencing defect (data not shown), similar to the single mutants (Fig. ). Importantly, H2B-R119AT122D conferred a severe slow-growth defect compared to the wild type or single mutants (Fig. ). Altogether, our results demonstrate that the H2B C-terminal helix plays an important role in maintaining cell growth, in controlling the active transcription of certain genes, and in the establishment of H3K4 methylation by Set1-COMPASS.
Set1-COMPASS integrity is compromised by H2B-R119AT122D.
To test how H2B-R119AT122D affects the chromatin association of Set1-COMPASS, we examined the distribution and occupancy of individual subunits on PMA1 and DMA2 using ChIP assays. In general, a drastic reduction in the occupancy of Swd2, Bre2, Sdc1, and Spp1 was observed in almost all regions of both genes (Fig. B to E). Set1 occupancy remained unchanged in PMA1 but was reduced in DMA2 (Fig. ). Unlike the single mutations (Fig. ), the reduced occupancy of many subunits of COMPASS in almost all regions of PMA1 and DMA2 suggests that the chromatin binding of the entire Set1-COMPASS is disrupted by H2B-R119AT122D.
FIG. 10. H2B-R119AT122D affects chromatin association and overall stability of Set1-COMPASS. (A to E) ChIP assay for the distribution and occupancy of Set1-COMPASS subunits in PMA1 and DMA2 in the indicated strains was done as described in the legends to Fig. (more ...)
Next, we performed fractionation to determine the effect of H2B-R119AT122D
on the overall chromatin association of Set1-COMPASS. Chromatin-bound levels of Set1, Swd1, and Swd2 were reduced in H2B-R119AT122D
(Fig. ). Since Set1 and Swd1 are essential for the complex integrity (40
), our finding suggests that the H2B-R119AT122D
reduces not only the binding but also the Set1-COMPASS integrity on chromatin. The chromatin-bound Sdc1 levels are nearly absent in the H2B-R119AT122D
mutant (Fig. ). This result further confirms the finding that H2B residues R119 and T122 are important for the association of Sdc1 with chromatin, as seen from the partial loss of chromatin-bound Sdc1 in the single mutants (Fig. ). Importantly, the chromatin-bound levels of Spp1 were decreased (Fig. ), consistent with the reduced binding of H2B-R119AT122D with Spp1 in GST pulldown assays (Fig. ). Surprisingly, in control experiments, we found a reduction in the global, steady-state levels of Set1-COMPASS subunits even in whole-cell extracts, especially at a near total loss of Sdc1 (Fig. ). However, the transcript levels of all Set1-COMPASS subunits in H2B-R119AT122D
were similar to those in the wild type (Fig. ). Therefore, the reduced chromatin and global levels of Set1-COMPASS subunits in H2B-R119AT122D
demonstrate, for the first time, that the H2B C-terminal helix residues R119 and T122 are important for chromatin binding, integrity, and stability of Set1-COMPASS.