Intratail regulation of histone modifications has been described. The existing paradigms, however, involve the control of one modification by the modification state of another residue located within close proximity and within the active site of the enzyme (19
). Here we describe a novel mechanism where H3 K36me3 is regulated by residues of H3 predicted to be located outside of the catalytic site of Set2. The crystal structure of most SET domains with histone peptides reveal no more than 9 amino acids total within the active site of the protein, and contacts with residues on each side of the modification site are observed (12
). More relevant to our data, Set2 can methylate a histone peptide containing only amino acids 29 to 41 (42
). This suggests that the reduction in Set2 activity observed in the H3 mutants examined here is not merely due to an alteration of the interaction of residues within the active site of Set2. Thus, to our knowledge, the stimulatory effect of the H3 tail on Set2 activity is unique to these other forms of intratail regulation. It is certain that the H3 tail plays multiple roles in transcription activation and they are not restricted to regulating K36me. Deleting SET2
had a small effect on the activation of RNR3
, less than that of the tail mutants (not shown). Another function suggested by data presented here is regulation of the recruitment or activity of SAGA. Future studies will be required to understand all of the roles of the H3 tail in the transcription of RNR3
Our data are fully consistent with the tail affecting Set2 catalytic activity. Deleting the tail had no effect on the interaction between Set2 and nucleosomes, once the differences in the turnover of nucleosomes were corrected for by using the catalytic mutant of Set2 (Fig. ). The binding of the tail to Set2 could affect its activity through an allosteric mechanism, or it could position K36 within the active site of Set2. In the latter case, the H3 N terminus interacts with Set2 to position lysine 36 next to the catalytic residues or restricts movement of the tail within the active site of Set2 (Fig. , left panel). Deleting the H3 tail, or mutating the lysines to neutral glutamines, could result in the misalignment of K36 within the active site, reducing the modification of the residue (Fig. , middle and right panels, respectively). We favor this scenario, versus the allosteric mechanism, based on the previously described regulation of K36me by the isomerization of an adjacent proline residue, P38, by the prolyl-isomerase Frp4 (42
). According to this model, Set2 can methylate lysine 36 only when P38 is in the trans
conformation, which orients K36 in the active site. An unresolved question about the proposed prolyl isomerization switch is what favors the active isomer versus the inactive form of P38. Prolyl isomerization is highly reversible and subject to equilibrium conditions. Interactions between the H3 tail and Set2, which could be regulated by modification or cleavage by cellular proteases, could favor the active conformation.
FIG. 8. Model for the regulation of Set2 activity by the H3 tail. The H3 tail interacts with Set2 to properly position lysine 36 in the Set2 active site. Deletion of the H3 tail or mutation of the charged lysine residues to neutral glutamines results in a reduction (more ...)
The region within Set2 that interacts with the H3 tail is not yet fully characterized. We observed that the SET domain of Set2 (amino acids 1 to 261) has greater activity on wild-type nucleosomes than those lacking the H3 tail (Fig. ). From this, we conclude that the intratail regulatory interaction occurs within the first 261 amino acids of Set2. Since deleting the tail or mutating the lysines to glutamine results in similar phenotypes, this suggests that the charge of the H3 tail is important for mediating the interaction with Set2. Accordingly, we show that lysine to arginine mutations do not negatively affect K36me3 in vivo. The pI of the SET domain of Set2 (amino acids 1 to 261) is 4.94, and it is possible that an acidic patch on Set2 could act as the interaction partner for the basic H3 tail. Determining the structure of the SET domain of Set2 could provide further evidence for this, and allow for the direct testing of this model, but unfortunately this has not been achieved yet.
An intriguing possibility is that modification of residues within the tail of H3, such as acetylation, can regulate the levels of K36me3. Histone H3 acetylation, found predominantly at promoter regions where H3 K36 methylation is low (29
), also neutralizes the charge of H3 lysine residues similar to the glutamine mutations. It is possible that acetylation of H3 helps create a barrier between the promoter and ORF regions by limiting H3 K36 methylation. This might be especially important when the end of one gene is in close proximity to the promoter of another gene. Alternatively, cotranscriptional acetylation within genes may serve to dampen Set2 activity ahead of the elongating polymerase. Histone H3 acetylation and Set2 methylation may negatively regulate the activity of each other to strike the proper balance of histone modifications. We attempted to directly test this model in vivo by deleting GCN5
, the histone acetyltransferase responsible for the DNA damage-induced H3 acetylation at RNR3
), and examining the change in H3K36me3 patterns and levels at RNR3
. While we did observe an increase in H3 K36me3 relative to RNAPII density in the gcn5Δ
mutant at RNR3
, we also observed a loss of H3 K36Ac as well (data not shown). Thus, we could not exclude the possibility that the increase in H3 K36me3 is the result of more “methylatable” K36 residues due to the loss of K36Ac.
The histone tail deletion mutants used here mimic a naturally occurring phenomenon in eukaryotic cells. The programmed “clipping” of the H3 tail at residue 21 occurs in vivo in S. cerevisiae
within nucleosomes at the promoters of activated sporulation control genes (51
). While it has not been described yet, tail cleavage could occur within other regions of the genome. Our data suggest a consequence of the removal of the H3 tail, reduced Set2 methylation. Clipping the H3 tail will inhibit Set2-dependent K36me, a mark whose function is to reset and repress chromatin by the recruitment of HDACs. Reducing K36me by tail cleavage could increase the expression of certain genes. This is not occurring at RNR3
, as expression of this gene is impaired in the tail mutant. As mentioned above, the H3 tail may be playing multiple roles in transcription at RNR3
. Furthermore, cleavage of the H3 tail has been identified in mouse embryonic stem cells during differentiation (15
). While the purpose of this cleavage is not well understood, the authors found that histone acetylation inhibited H3 proteolysis. Given that histones are found to be mostly deacetylated in differentiating cells, cleavage of the H3 tail may provide another mechanism to regulate lysine 36 methylation patterns during differentiation or under circumstances when changes in acetylation alone are insufficient. The novel intratail regulation described here provides insight into the depth of the regulatory mechanisms controlling histone modifications and may reveal a consequence of the recently described programmed proteolysis of H3.