Since the identification of Dot1 as a HMT, chromatin modification by this enzyme has been implicated in many nuclear processes, namely establishment of telomeric chromatin boundaries, transcription elongation, and DNA damage response (Shilatifard, 2006
; Wysocki et al., 2005
). Methylation of H3 lysine 79 is prevalent in budding yeast chromatin and is found in coding regions of transcribed genes and near telomeres (Millar and Grunstein, 2006
). It is therefore seen as a euchromatin-specific modification. A functional role of methylated K79 in DNA damage response is to allow binding of ScRad9 to chromatin near DSBs through the protein tudor domain (Wysocki et al., 2005
). However, the precise mechanistic roles of Dot1/K79 methylation during transcription elongation and establishment telomeric heterochromatin boundaries are not understood. Our findings showing that Dot1 interacts with a short region of histone H4 tail in chromatin and that this interaction is essential for methylation of lysine 79 on H3 provide critical new information that shed light on its function in blocking the spreading of heterochromatin.
We showed that Sir3, the chromatin-binding component of the SIR complex, which spreads from chromosome ends to form heterochromatin and silence gene expression, is a potent inhibitor of Dot1-dependent methylation. This is explained by the fact that Sir3 and Dot1 compete for the same binding site on H4 tail, a short basic patch between residues 16 and 20. It was known that Sir3 binding to H4 tail is disrupted by acetylation lysine 16, but this is not the case for Dot1. This supports the concept that the regulation of H4 K16 acetylation by Sas2 acetyltransferase and Sir2 deacetylase is a key primary step controlling the establishment of heterochromatin boundaries () (Millar et al., 2004
). Our results indicate that a pivotal function of H4 K16 acetylation is to displace Sir3, which in turn allows Dot1 to bind and methylate H3 K79. The role of AcK16 in Dot1-dependent methylation is restricted to the region near Sir3-containing chromatin since K16 mutation has no effect on bulk levels of MeK79 or in vitro HMT activity ( and ).
Model of Succession of Events during the Establishment of Telomeric Heterochromatin Boundaries in Yeast
Our results also reconcile contradictory data present in the literature. While it was known that deletion of DOT1
, its overexpression, or mutation of H3 lysine 79 to an alanine had similar effect on telomeric silencing (van Leeuwen et al., 2002
), it was puzzling to see that a K79R mutation, which cannot be methylated, had no phenotype or even increased silencing (Park et al., 2002
). These data suggest that Dot1 binding to the H4 tail, even without K79 methylation, is sufficient to block SIR complex spreading into euchromatin regions. It is clearly not that simple, since expression of catalytically dead mutants of Dot1 only slightly suppresses the telomeric silencing defects of DOT1
deletion/overexpression (van Leeuwen et al., 2002
). Since Dot1 does not appear to methylate other histone residues in vitro (van Leeuwen et al., 2002
), these results could reflect a lower affinity of the Dot1 mutants for chromatin. Maybe AdoMet binding by Dot1 induces a conformational change allowing coordinated binding to both H4 tail and H3 K79 region at the surface of the nucleosome. As for the H3 K79A mutation, it suggests that losing the positive charge has a similar effect as methylation (which does not remove the charge). Since mutation of other basic residues neighboring H3 K79 in the nucleosome also creates defect in silencing (Fry et al., 2006
; Park et al., 2002
; Thompson et al., 2003
), we tested if in fact Sir3 was also interacting with a second basic patch in the nucleosomes, centered on H3 K79. We showed that it was the case and that the interaction is extremely sensitive to K79 methylation (). Altogether, our results indicate that Sir3 and Dot1 target the same two positively charged patches on nucleosomes. Binding of Sir3 to both targets is apparently necessary for its function, while Dot1 binding to the H4 tail/ nucleosome without methylation could be sufficient to block Sir3 spreading (based on K79A/R phenotypes) ().
The data and concepts presented in this report could also impact our understanding of DNA DSB repair. While Dot1-dependent methylation has been implicated for binding of ScRad9 and checkpoint activation (Wysocki et al., 2005
), the SIR complex is also recruited near DSBs but at later time points (Martin et al., 1999
). In this situation, one would expect that H3 K79 would have to be demethylated in order to allow local SIR (Sir3) binding.
It will be interesting to investigate the role of Dot1 in the establishment of chromatin boundaries in higher eukaryotes, since the mechanisms may significantly differ. The H4 K16 acetyltransferase and deacetylase have been identified in hMOF and Sirtuins (Michan and Sinclair, 2007
; Smith et al., 2005
), but no homolog of Sir3 or SIR complex has been characterized. Artificial targeting of human H4 K16 deacetylase Sirt1 to a gene in cultured cells results in local loss of H3 K79 methylation, heterochromatinization, and gene silencing (Vaquero et al., 2004
). Furthermore, in higher eukaryotes, H4 is methylated on K20 in heterochromatin (Kouzarides, 2007
). The impact of this modification on mammalian Dot1-binding activity needs to be studied. It is tempting to speculate that H4 MeK20 could regulate Dot1 binding to the basic patch as Sir3 does in yeast. Accordingly, single H4 molecules do not carry simultaneous AcK16 and MeK20 residues, and H4 K16 acetylation inhibits K20 methylation in vitro (and vice versa) (Garcia et al., 2007
; Nishioka et al., 2002
). Finally, a recent report identified two homologs of Dot1 in trypanosoma, each one being selective for di- or trimethylation of H3. Such selectivity in the level of methylation argues for distinct functional roles (Janzen et al., 2006
The data presented here underline the critical role played by a short basic patch of histone H4 tail in chromatin function. This small region is now known to physically interact with Sir3, Sas2/MOF HAT, Sir2 HDAC, Dot1 HMT, and the ISWI family of ATP-dependent chromatin remodelers. Coordinated interaction of Sir3 or Dot1 with both the H4 tail and H3 K79 region is compatible with the previously established close proximity of these two regions in the nucleosome structure (Luger et al., 1997
; Thompson et al., 2003
). An independent study also identified Dot1 interaction with H4 tail and mapped the region necessary for binding to a short acidic patch in Dot1 (Fingerman et al., 2007
Altogether, our results explain through a histone H4-H3 crosstalk why Dot1 methyltransferase activity can only modify chromatin substrates, not free histones/peptides. Even more importantly, they suggest a precise succession of molecular events leading to the establishment of telomeric heterochromatin boundaries, local control of histone H4 K16 acetylation specifying the direct binding of Sir3 or Dot1. Both proteins compete for the same molecular targets at the surface of the nucleosome. If Dot1 is allowed to bind, then methylation of H3 K79 directly blocks further Sir3 association/spreading from telomeres. The reported role of H2A variant Htz1 (H2AZ) in blocking heterochromatin spreading is also dependent on the H4 tail, since Htz1 incorporation near telomeres requires K16 acetylation () (Shia et al., 2006
). On the other hand, the impact of Htz1 on telomeric silencing seems less dramatic than Dot1, suggesting that the incorporation and the role of Htz1 could be subsequent and accessory to the H4 acetylation/H3 methylation steps (Dhillon and Kamakaka, 2000
). It will be very interesting to investigate the role of Dot1 in chromatin boundaries in higher eukaryotes. Distinct histone modifications could functionally interact with Dot1/H3 K79 methylation, but the primary role of H4 basic patch as regulatory target certainly remains. These findings also impact research efforts at dissecting Dot1 function in DNA damage response, transcription elongation, and leukemogenesis.