- Email: [email protected]
A PSHaver for Centromeric Histones
Molecular Cell
Previews A PSHaver for Centromeric Histones H. Diego Folco1 and Arshad Desai1,* 1Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92037, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.10.031
In this issue of Molecular Cell, Hewawasam et al. (2010) and Ranjitkar et al. (2010) identify and characterize Psh1, an E3 ubiquitin ligase that specifically targets the centromeric histone Cse4 in budding yeast and limits its misincorporation at noncentromeric regions. In eukaryotes, chromosome segregation is directed by centromeres, the DNA regions upon which the kinetochore, the molecular machine that connects to spindle microtubules, is assembled during cell division. While centromeric DNA varies from a specific 125 base pair sequence in budding yeast to megabases of repetitive alpha satellite sequences in humans, active centromeres invariably correlate with the presence of the histone H3 variant CENP-A/CenH3 (Cse4 in budding yeast). An important unanswered question in centromere biology is how CENP-A is restricted to centromeres and excluded from ectopic locations in the genome. Previous work has implicated ubiquitin-mediated proteolysis as a critical component in controlling levels of CENPA and minimizing its deposition outside of centromeres (Collins et al., 2004; Moreno-Moreno et al., 2006). However, the specific components involved in this surveillance pathway were unknown. In this issue, Hewawasam et al. (2010) and Ranjitkar et al. (2010) report the discovery that, in the budding yeast Saccharomyces cerevisiae, Psh1 is an E3 ubiquitin ligase that specifically recognizes Cse4 and reduces its misincorporation at noncentromeric regions. Mass spectrometry of Cse4 purifications led both groups to discover Psh1 (named for Pob3/Spt16 histone associated). Psh1 interacts with Cse4 but not with histone H3, is able to ubiquitinate Cse4 in vitro, and contributes to Cse4 ubiquitination and stability in vivo. Previous work has shown that the centromere-targeting domain (known as the CATD) of CENP-A, an 40 amino acid region in the histone fold, is essential and sufficient for centromere localization; a recent crystal structure of human
CENP-A/H4 tetramers provides insight into the precise differences from H3/H4 (Sekulic et al., 2010 and references therein). Ranjitkar et al. (2010) show that the CATD domain is the recognition module for Psh1, explaining how Psh1 discriminates Cse4 from histone H3 (Figure 1A). Psh1 is able to bind a H3CATD chimera (histone H3 with insertion of the CATD from Cse4) but is unable to bind a chimera of the opposite structure (i.e., Cse4 in which the CATD was replaced with the corresponding histone H3 region). In agreement with these findings, and similar to Cse4, the stability of the H3CATD chimera is increased in vivo upon deletion of Psh1. The action of Psh1 as an E3 ubiquitin ligase for Cse4 is likely to be counterbalanced by the presence of CENP-A specific chaperones. Such chaperones have been discovered in humans (known as HJURP), as well as budding and fission yeasts (known as Scm3)—while divergent in primary sequence, informatic analysis and functional studies indicate that these chaperones are related (Sanchez-Pulido et al., 2009). HJURP interacts with the CATD region of CENP-A (Foltz et al., 2009), and it is likely that Scm3 does the same. The similar recognition mechanism employed to discriminate Cse4 by HJURP/Scm3 and by Psh1 suggests a potential antagonistic relationship in controlling Cse4 stability. Consistent with this, Hewawasam et al. (2010) show that Scm3 binding protects Cse4 from Psh1mediated degradation. First, Psh1 cannot ubiquitinate Cse4 if Scm3 is added to the in vitro ubiquitination assay. Second, by exploiting their prior observation that overexpression of Cse4 rescues the inviability of an SCM3 deletion, Hewawasam et al. (2010) provide in vivo evidence for
mutual antagonism between Scm3 and Psh1 in regulation of Cse4 levels. These results suggest that Psh1 acts as a proteolytic buffer that eliminates Cse4 not bound to Scm3 and, in turn, this reduces ectopic incorporation of Cse4 (Figures 1A and 1B). While the discovery of a ubiquitin ligase specifically controlling the level of a centromere-specific histone variant is a significant advance, it is surprising that both groups report that the deletion of PSH1 does not exhibit a phenotype unless Cse4 is massively overexpressed. This observation suggests that additional mechanisms must be working in parallel. Consistent with this, prior work showed that a lysine-free Cse4 that is unable to be ubiquitinated is still degraded, albeit more slowly than wild-type Cse4 (Collins et al., 2004); the lysine-free Cse4 mutant also does not exhibit a significant growth defect. The alternative pathway(s) that regulates Cse4 stability remains to be elucidated in future work. The two papers convincingly link Psh1 to Cse4, but the location of this proteolytic regulation remains unclear. Psh1 may limit Cse4 misincorporation by controlling the level of soluble Cse4 and/or by targeting Cse4 that is misincorporated into noncentromeric regions for degradation (Figure 1B). Both groups show that Psh1 interacts with and ubiquitinates Cse4 in solution, favoring the first possibility. Neither group tested whether Psh1 ubiquitinates Cse4 reconstituted into nucleosomes, so whether DNA-associated Cse4 is a Psh1 substrate remains an open question. With respect to Psh1 potentially targeting misincorporated Cse4, it is important to note that Psh1 was initially discovered based on its binding to the FACT (facilitates chromatin transcription)
Molecular Cell 40, November 12, 2010 ª2010 Elsevier Inc. 351
Molecular Cell
Previews A
Psh1
Ub
Cse4/H4
Ub Ub
CATD
CATD
Proteasomal degradation
Scm3
B
Scm3-Bound Cse4 Pool Scm3-Free Cse4 Pool
Ub Ub Ub
i) Psh1
Kinetochore ii)
expression or other genome functions. Psh1 apparently lacks homologs in metazoans, albeit this is not conclusive given that the conservation of the catalytic domain harbored by many E3 ubiquitin ligases impairs homology assessments. CENP-A proteolysis has been detected in human cells undergoing senescence or infection with herpes virus (Lomonte et al., 2001; Maehara et al., 2010) and components of the regulated proteolysis machinery, including RING finger domain ligases, have been copurified with CENP-A chromatin in human cells (Obuse et al., 2004). Thus, specific proteolysis machinery targeting centromeric histones is likely to be conserved. Future work, stimulated by the findings reported in this issue, should reveal whether this is indeed the case as well as elucidate the mechanism and physiological function of this proteolytic regulation. REFERENCES Collins, K.A., Furuyama, S., and Biggins, S. (2004). Curr. Biol. 14, 1968–1972.
Misincorporated in Chromatin
CEN
Figure 1. Psh1 Is a Ubiquitin Ligase Specific for the Centromeric Histone Variant Cse4 in Budding Yeast (A) Psh1 recognizes the CATD region of Cse4 and targets it for ubiquitination and subsequent proteasomal degradation. The chaperone Scm3 probably also binds the CATD and antagonizes the action of Psh1. (B) Two scenarios for Psh1 mechanism of action that are not mutually exclusive: (i) Psh1 targets the Scm3free soluble Cse4 pool thereby limiting availability of Cse4 for misincorporation or (ii) Psh1 targets misincorporated Cse4 nucleosomes, potentially with the aid of FACT. Centromeric Cse4 is depicted as being protected from Psh1-mediated degradation by Scm3 and/or kinetochore assembly.
complex (Krogan et al., 2002). FACT is well studied for its role in transcriptional elongation on chromatin templates and has also been copurified with CENP-A chromatin in higher eukaryotes (Obuse et al., 2004). Thus, FACT might guide Psh1 to Cse4 misincorporated in actively transcribed regions. In this scenario, the FACT-associated Psh1 may recognize the CATD domain of Cse4 misincorporated into transcribed regions and target it for degradation. Consistent with this view, Ranjitkar et al. show that the interaction between Cse4 and the FACT complex is mediated through Psh1. However, the inability to independently assay turnover of chromatin-bound and soluble Cse4 pools and the essential nature of FACT
precluded further analysis. As Psh1 is also detected at centromeres (Hewawasam et al., 2010), centromeric Cse4 must be protected from Psh1. Either the presence of Scm3 and/or assembly of kinetochore components may limit the susceptibility of centromeric Cse4 to Psh1 (Figure 1B). In higher eukaryotes, overexpression of CENP-A can lead to ectopic formation of kinetochores and significant chromosome missegregation (Heun et al., 2006). Such an outcome is not likely in budding yeast given that Cse4 nucleosomes cannot elicit kinetochore formation without the specific centromeric DNA sequence in cis. However, noncentromeric Cse4 may have detrimental effects on gene
352 Molecular Cell 40, November 12, 2010 ª2010 Elsevier Inc.
Foltz, D.R., Jansen, L.E., Bailey, A.O., Yates, J.R., 3rd, Bassett, E.A., Wood, S., Black, B.E., and Cleveland, D.W. (2009). Cell 137, 472–484. Heun, P., Erhardt, S., Blower, M.D., Weiss, S., Skora, A.D., and Karpen, G.H. (2006). Dev. Cell 10, 303–315. Hewawasam, G., Shivaraju, M., Mattingly, M., Venkatesh, S., Martin-Brown, S., Florens, L., Workman, J.L., and Gerton, J.L. (2010). Mol. Cell 40, this issue, 444–454. Krogan, N.J., Kim, M., Ahn, S.H., Zhong, G., Kobor, M.S., Cagney, G., Emili, A., Shilatifard, A., Buratowski, S., and Greenblatt, J.F. (2002). Mol. Cell. Biol. 22, 6979–6992. Lomonte, P., Sullivan, K.F., and Everett, R.D. (2001). J. Biol. Chem. 276, 5829–5835. Maehara, K., Takahashi, K., and Saitoh, S. (2010). Mol. Cell. Biol. 30, 2090–2104. Moreno-Moreno, O., Torras-Llort, M., and Azorı´n, F. (2006). Nucleic Acids Res. 34, 6247–6255. Obuse, C., Yang, H., Nozaki, N., Goto, S., Okazaki, T., and Yoda, K. (2004). Genes Cells 9, 105–120. Ranjitkar, P., Press, M.O., Yi, X., Baker, R., MacCoss, M.J., and Biggins, S. (2010). Mol. Cell 40, this issue, 455–464. Sanchez-Pulido, L., Pidoux, A.L., Ponting, C.P., and Allshire, R.C. (2009). Cell 137, 1173–1174. Sekulic, N., Bassett, E.A., Rogers, D.J., and Black, B.E. (2010). Nature 467, 347–351.
Previews A PSHaver for Centromeric Histones H. Diego Folco1 and Arshad Desai1,* 1Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92037, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.10.031
In this issue of Molecular Cell, Hewawasam et al. (2010) and Ranjitkar et al. (2010) identify and characterize Psh1, an E3 ubiquitin ligase that specifically targets the centromeric histone Cse4 in budding yeast and limits its misincorporation at noncentromeric regions. In eukaryotes, chromosome segregation is directed by centromeres, the DNA regions upon which the kinetochore, the molecular machine that connects to spindle microtubules, is assembled during cell division. While centromeric DNA varies from a specific 125 base pair sequence in budding yeast to megabases of repetitive alpha satellite sequences in humans, active centromeres invariably correlate with the presence of the histone H3 variant CENP-A/CenH3 (Cse4 in budding yeast). An important unanswered question in centromere biology is how CENP-A is restricted to centromeres and excluded from ectopic locations in the genome. Previous work has implicated ubiquitin-mediated proteolysis as a critical component in controlling levels of CENPA and minimizing its deposition outside of centromeres (Collins et al., 2004; Moreno-Moreno et al., 2006). However, the specific components involved in this surveillance pathway were unknown. In this issue, Hewawasam et al. (2010) and Ranjitkar et al. (2010) report the discovery that, in the budding yeast Saccharomyces cerevisiae, Psh1 is an E3 ubiquitin ligase that specifically recognizes Cse4 and reduces its misincorporation at noncentromeric regions. Mass spectrometry of Cse4 purifications led both groups to discover Psh1 (named for Pob3/Spt16 histone associated). Psh1 interacts with Cse4 but not with histone H3, is able to ubiquitinate Cse4 in vitro, and contributes to Cse4 ubiquitination and stability in vivo. Previous work has shown that the centromere-targeting domain (known as the CATD) of CENP-A, an 40 amino acid region in the histone fold, is essential and sufficient for centromere localization; a recent crystal structure of human
CENP-A/H4 tetramers provides insight into the precise differences from H3/H4 (Sekulic et al., 2010 and references therein). Ranjitkar et al. (2010) show that the CATD domain is the recognition module for Psh1, explaining how Psh1 discriminates Cse4 from histone H3 (Figure 1A). Psh1 is able to bind a H3CATD chimera (histone H3 with insertion of the CATD from Cse4) but is unable to bind a chimera of the opposite structure (i.e., Cse4 in which the CATD was replaced with the corresponding histone H3 region). In agreement with these findings, and similar to Cse4, the stability of the H3CATD chimera is increased in vivo upon deletion of Psh1. The action of Psh1 as an E3 ubiquitin ligase for Cse4 is likely to be counterbalanced by the presence of CENP-A specific chaperones. Such chaperones have been discovered in humans (known as HJURP), as well as budding and fission yeasts (known as Scm3)—while divergent in primary sequence, informatic analysis and functional studies indicate that these chaperones are related (Sanchez-Pulido et al., 2009). HJURP interacts with the CATD region of CENP-A (Foltz et al., 2009), and it is likely that Scm3 does the same. The similar recognition mechanism employed to discriminate Cse4 by HJURP/Scm3 and by Psh1 suggests a potential antagonistic relationship in controlling Cse4 stability. Consistent with this, Hewawasam et al. (2010) show that Scm3 binding protects Cse4 from Psh1mediated degradation. First, Psh1 cannot ubiquitinate Cse4 if Scm3 is added to the in vitro ubiquitination assay. Second, by exploiting their prior observation that overexpression of Cse4 rescues the inviability of an SCM3 deletion, Hewawasam et al. (2010) provide in vivo evidence for
mutual antagonism between Scm3 and Psh1 in regulation of Cse4 levels. These results suggest that Psh1 acts as a proteolytic buffer that eliminates Cse4 not bound to Scm3 and, in turn, this reduces ectopic incorporation of Cse4 (Figures 1A and 1B). While the discovery of a ubiquitin ligase specifically controlling the level of a centromere-specific histone variant is a significant advance, it is surprising that both groups report that the deletion of PSH1 does not exhibit a phenotype unless Cse4 is massively overexpressed. This observation suggests that additional mechanisms must be working in parallel. Consistent with this, prior work showed that a lysine-free Cse4 that is unable to be ubiquitinated is still degraded, albeit more slowly than wild-type Cse4 (Collins et al., 2004); the lysine-free Cse4 mutant also does not exhibit a significant growth defect. The alternative pathway(s) that regulates Cse4 stability remains to be elucidated in future work. The two papers convincingly link Psh1 to Cse4, but the location of this proteolytic regulation remains unclear. Psh1 may limit Cse4 misincorporation by controlling the level of soluble Cse4 and/or by targeting Cse4 that is misincorporated into noncentromeric regions for degradation (Figure 1B). Both groups show that Psh1 interacts with and ubiquitinates Cse4 in solution, favoring the first possibility. Neither group tested whether Psh1 ubiquitinates Cse4 reconstituted into nucleosomes, so whether DNA-associated Cse4 is a Psh1 substrate remains an open question. With respect to Psh1 potentially targeting misincorporated Cse4, it is important to note that Psh1 was initially discovered based on its binding to the FACT (facilitates chromatin transcription)
Molecular Cell 40, November 12, 2010 ª2010 Elsevier Inc. 351
Molecular Cell
Previews A
Psh1
Ub
Cse4/H4
Ub Ub
CATD
CATD
Proteasomal degradation
Scm3
B
Scm3-Bound Cse4 Pool Scm3-Free Cse4 Pool
Ub Ub Ub
i) Psh1
Kinetochore ii)
expression or other genome functions. Psh1 apparently lacks homologs in metazoans, albeit this is not conclusive given that the conservation of the catalytic domain harbored by many E3 ubiquitin ligases impairs homology assessments. CENP-A proteolysis has been detected in human cells undergoing senescence or infection with herpes virus (Lomonte et al., 2001; Maehara et al., 2010) and components of the regulated proteolysis machinery, including RING finger domain ligases, have been copurified with CENP-A chromatin in human cells (Obuse et al., 2004). Thus, specific proteolysis machinery targeting centromeric histones is likely to be conserved. Future work, stimulated by the findings reported in this issue, should reveal whether this is indeed the case as well as elucidate the mechanism and physiological function of this proteolytic regulation. REFERENCES Collins, K.A., Furuyama, S., and Biggins, S. (2004). Curr. Biol. 14, 1968–1972.
Misincorporated in Chromatin
CEN
Figure 1. Psh1 Is a Ubiquitin Ligase Specific for the Centromeric Histone Variant Cse4 in Budding Yeast (A) Psh1 recognizes the CATD region of Cse4 and targets it for ubiquitination and subsequent proteasomal degradation. The chaperone Scm3 probably also binds the CATD and antagonizes the action of Psh1. (B) Two scenarios for Psh1 mechanism of action that are not mutually exclusive: (i) Psh1 targets the Scm3free soluble Cse4 pool thereby limiting availability of Cse4 for misincorporation or (ii) Psh1 targets misincorporated Cse4 nucleosomes, potentially with the aid of FACT. Centromeric Cse4 is depicted as being protected from Psh1-mediated degradation by Scm3 and/or kinetochore assembly.
complex (Krogan et al., 2002). FACT is well studied for its role in transcriptional elongation on chromatin templates and has also been copurified with CENP-A chromatin in higher eukaryotes (Obuse et al., 2004). Thus, FACT might guide Psh1 to Cse4 misincorporated in actively transcribed regions. In this scenario, the FACT-associated Psh1 may recognize the CATD domain of Cse4 misincorporated into transcribed regions and target it for degradation. Consistent with this view, Ranjitkar et al. show that the interaction between Cse4 and the FACT complex is mediated through Psh1. However, the inability to independently assay turnover of chromatin-bound and soluble Cse4 pools and the essential nature of FACT
precluded further analysis. As Psh1 is also detected at centromeres (Hewawasam et al., 2010), centromeric Cse4 must be protected from Psh1. Either the presence of Scm3 and/or assembly of kinetochore components may limit the susceptibility of centromeric Cse4 to Psh1 (Figure 1B). In higher eukaryotes, overexpression of CENP-A can lead to ectopic formation of kinetochores and significant chromosome missegregation (Heun et al., 2006). Such an outcome is not likely in budding yeast given that Cse4 nucleosomes cannot elicit kinetochore formation without the specific centromeric DNA sequence in cis. However, noncentromeric Cse4 may have detrimental effects on gene
352 Molecular Cell 40, November 12, 2010 ª2010 Elsevier Inc.
Foltz, D.R., Jansen, L.E., Bailey, A.O., Yates, J.R., 3rd, Bassett, E.A., Wood, S., Black, B.E., and Cleveland, D.W. (2009). Cell 137, 472–484. Heun, P., Erhardt, S., Blower, M.D., Weiss, S., Skora, A.D., and Karpen, G.H. (2006). Dev. Cell 10, 303–315. Hewawasam, G., Shivaraju, M., Mattingly, M., Venkatesh, S., Martin-Brown, S., Florens, L., Workman, J.L., and Gerton, J.L. (2010). Mol. Cell 40, this issue, 444–454. Krogan, N.J., Kim, M., Ahn, S.H., Zhong, G., Kobor, M.S., Cagney, G., Emili, A., Shilatifard, A., Buratowski, S., and Greenblatt, J.F. (2002). Mol. Cell. Biol. 22, 6979–6992. Lomonte, P., Sullivan, K.F., and Everett, R.D. (2001). J. Biol. Chem. 276, 5829–5835. Maehara, K., Takahashi, K., and Saitoh, S. (2010). Mol. Cell. Biol. 30, 2090–2104. Moreno-Moreno, O., Torras-Llort, M., and Azorı´n, F. (2006). Nucleic Acids Res. 34, 6247–6255. Obuse, C., Yang, H., Nozaki, N., Goto, S., Okazaki, T., and Yoda, K. (2004). Genes Cells 9, 105–120. Ranjitkar, P., Press, M.O., Yi, X., Baker, R., MacCoss, M.J., and Biggins, S. (2010). Mol. Cell 40, this issue, 455–464. Sanchez-Pulido, L., Pidoux, A.L., Ponting, C.P., and Allshire, R.C. (2009). Cell 137, 1173–1174. Sekulic, N., Bassett, E.A., Rogers, D.J., and Black, B.E. (2010). Nature 467, 347–351.