Key Publications

The Grunstein lab discovered that nucleosomes and histone acetylation sites regulate gene activity in vivo.

The Grunstein laboratory now studies the function of histones and histone interacting proteins in heterochromatin and euchromatin. Topics of special interest are the roles of histone modification in mammalian DNA replication and in pluripotency.

Key publications:

1981. Histone H2B variants are redundant in vivo

It was known that complex eukaryotes contained variants of histone proteins. Wallis et al (1980) showed that yeast contains histone variants. Rykowski et al (1981) showed that histone H2B variant genes can be disrupted and can substitute functionally for each other in vivo. This was the first genetic analysis directed towards histone proteins.

1988. Nucleosomes repress transcription initiation in vivo.

Nucleosomes were previously known to prevent gene activity in vitro but were widely assumed to be transparent to the transcription apparatus in vivo. Han et al (1988) showed that histones could be depleted genetically in vivo. When they were, nucleosomes were lost, and every repressed gene analyzed was activated. This study showed that nucleosomes are represssors of TATA elements in vivo and led to the realization that nucleosomes regulate transcription (Grunstein, 1990).

1991. Histone acetylation sites regulate gene activity

While nucleosomes are repressors Durrin et al (1991) showed that the acetylation sites of histone H4 are required for gene activity in vivo. This was the first demonstration that histone acetylation sites regulate gene activity and led to the first demonstration that histone acetylation is not merely a result of transcription but a cause of gene activity (Durrin et al., 1991; Grunstein, 1990; 1992).

1996. Purification and function of histone deacetylases

Carmen et al (1996) and Rundlett et al (1996) demonstrated the identification and purification of Hda1 and Rpd3 histone deacetylase complexes from yeast independently of the Schreiber laboratory. They also first identified the family of five related histone deacetylases (Rpd3, Hda1, Hos1, Hos2, Hos3) in yeast and the related acetylpolyamine amidohydrolase (APH) in bacteria (Rundlett et al., 1996) and showed that deacetylases Rpd3 and Hda1 modify a sharply defined domain next to the promoter (Rundlett et al., 1998; Wu et al., 2001). Hda1 and Hos3 deacetylase complexes were also the first histone deacetylase complexes purified to homogeneity (Carmen et al., 1999; Wu et al., 2002)

2000. Global histone acetylation is functionally distinct from that targeted to promoters. Global regulation of transcription and DNA replication.

This study found that two acetyltranferases and two deacetylases function not only at promoters, but globally, affecting most nucleosomes. This `background’ acetylation is important for gene regulation, for the reversibility of activation or repression, and for the regulation of DNA replication (Vogelauer et al., 2000; 2002). The work of Siavash Kurdistani and colleagues has also shown that global acetylation state is indicative of clinical prognosis in low grade prostate tumors (Seligson et al., 2005).

2002. Identifying where deacetylases affect histone acetylation genome wide

Genome wide analysis was developed to analyze histone acetylation genome wide. This study determined where the five yeast histone deacetylases (and Sir2) affected histone acetylation throughout the yeast genome. This study (Robyr et al., 2002) also identified subtelomeric chromosomal domains that were uniquely affected by Hda1 histone deacetylase. A related study showed where Rpd3 binds chromatin genome wide (Kurdistani et al., 2002) and that it is restricted from subtelomeric domains..

2002. Hos2 histone deacetylase is a general activator of gene activity

While deacetylases are generally believed to repress gene activity, Hos2 was shown to bind preferentially and deacetylate chromatin of genes during activity and to be required for gene activity (Wang et al., 2002). Thus histone deacetylases are not only repressors but also activators of transcription.

2004. Unique patterns of histone acetylation are important for gene activity

Using microarray approaches and antibodies to most sites of N terminal histone acetylation this paper showed that different gene classes utilize complex patterns of histone acetylation. In particular, some sites are relatively acetylated while others are relatively deacetylated when genes are active (Kurdistani et al., 2004). Acetylation and deacetylation of histone N terminal lysines are believed to form interaction surfaces for distinct regulatory proteins (Kurdistani et al., 2004). This helps explain why deacetylation by Hos2 of unique lysines can activate transcription (Wang et al., 2002).

2005. Acetylation of a site (K56) within the globular domain of histone H3 regulates gene activity.

Most acetylation sites studied are found at the histone N termini. This paper has identified a site (K56) in H3 that is present at the entry-exit points of the nucleosomal DNA superhelix. The data here argue that acetylation of K56 through the acetyltransferase SPT10 is an initial nucleosome remodeling event at the entry-exit gate that enables recruitment of the SWI/SNF remodeling complex to specific genes (Xu et al., 2005).

Heterochromatin.

1988. Histone H4 N terminus is uniquely required for silencing heterochromatin.

Histones are ubiquitous and condense chromatin almost everywhere in the genome, yet this study finds that unlike the H2B N terminus (Wallis et al., 1983) the H4 N terminus has a specific role in silencing the HM silent mating loci (now called HM heterochromatin to distinguish it from telomeric heterochromatin) (Kayne et al., 1988). Other genes were not affected strongly by mutations that affected the HM loci. This studied showed for the first time that a histone (in particular the H4 N terminal domain) is required for heterochromatin function.

1990. A genetic interaction between histone H4 and Sir3 helps explain why histone H4 mutations have unique effects on heterochromatin.

The study (Kayne et al., 1988) using nested deletions points to H4 lysine 16 as the only acetylation site that may be important for silencing. This is confirmed by single substitutions (Johnson et al., 1990). Here it was also shown that two mutations in Sir3, a protein that affects heterochromatin, can each suppress the strong H4 mutation that disrupts silencing. Thus H4 and Sir3 interact genetically.

1995. Histones and Sir proteins interact in vitro. A new paradigm for gene regulation in which regulators function through their interaction with histone tails suggests a model for the spreading of heterochromatin.

The regions in histones H3 and H4 that affect silencing by silent mating locus (HM) and telomeric heterochromatin also interact with both Sir3 and Sir4 in vitro (Hecht et al., 1995). This not only helps explain how heterochromatin proteins spread, by interacting with the platform formed by the histone tails, but also describes the first example of a new paradigm in gene regulation. Regulators interact reversibly with histone tails. Moreover charge changes (Johnson et al., 1990; Hecht et al., 1996) and acetylation (Carmen et al., 2002) disrupt these interactions in vivo and in vitro. There are now many such examples in the binding of regulators to unacetylated or acetylated lysines and in binding of other proteins to methylated lysines.

1996. Sir proteins interact physically with heterochromatin in vivo. A new modification to chromatin immunoprecipitation utilizing PCR.

It was previously known from the work of Herskowitz and Gottschling and their colleagues that Sir proteins are required for silencing of HM and telomeric heterochromatin. But it was not known whether Sir proteins were chromosomal proteins. Varshavsky’s laboratory invented the procedure of chromatin immunoprecipitation. This study (Hecht et al., 1996) and a related study (Strahl-Bolsinger et al., 1997) described an altered chromatin immunoprecipitation protocol that employed PCR for the first time allowing rapid and convenient ChIP analysis. This study also described for the first time the finding that Sir proteins bind chromatin and spread along heterochromatin. This interaction requires one particular acetylatable lysine K16 in histone H4 in the charged (`deacetylated’) state (Johnson et al., 1990; Hecht et al., 1996).

2001. All lysines analyzed in telomeric and HM heterochromatin are deacetylated.

New highly specific antibodies were generated to 12 individual sites of acetylation at all four core histones. This study found using ChIP that heterochromatin in yeast whether at the telomere or the HM silent mating loci forms a fully hypoacetylated domain. The antibodies generated in these studies made future work on acetylation state and genome wide patterns of acetylation (above) feasible.

2002. How telomeric heterochromatin initiates, spreads and stops spreading.

Luo et al (2002) demonstrate that Rap1 recruitment of Sir4 in the absence of other Sir proteins initiates the formation of heterochromatin. This followed earlier work from the David Shore laboratory showing that Rap1which binds to repeated sequences at telomeres interacts with Sir proteins. From there, Sir proteins along the histone tails (Hecht et al., 1995; 1996). How does spreading stop? One important factor is the acetylation state of histone H4 lysine 16. Not only does its deacetylation by Sir2 allow spreading but its global acetylation by Sas2 (a K16 specific MYST acetyltransferase) in adjacent euchromatin prevents the spreading of heterochromatin. The barrier to telomeric heterochromatin spreading is not a sequence element as for the HM loci (Kamakaka laboratory) but the presence of global acetylation of K16 in euchromatin.

References:

1. Wallis, J.W., Hereford, L., and Grunstein, M. (1980). Histone H2B Genes of Yeast Encode Two Different Proteins. Cell. 22: 799-805.

2. Rykowski, M.C., Wallis, J., Choe, J., and Grunstein, M. (1981). Histone H2B Subtypes are Dispensable During the Yeast Cell Cycle. Cell. 25: 477-487.

3. Wallis, J., Rykowski, M., and Grunstein, M. (1983). Yeast Histone H2B Containing Large Amino Terminus Deletions Can Function in vivo. Cell. 35: 711-719.

4. Han, M., Chang, M., Kim, U.-J., and Grunstein, M. (1987). Histone H2B Repression Causes Cell-Cycle-Specific Arrest in Yeast: Effects on Chromosomal Segregation, Replication and Transcription. Cell. 48: 589-597.

5. Kayne, P.S., Kim, U.-J., Han, M., Mullen, J.R., Yoshizaki, F., and Grunstein, M. (1988). Extremely Conserved Histone H4 N-Terminus is Dispensable for Growth but Essential for Repressing the Silent Mating Loci in Yeast. Cell. 55: 27-39.

6. Han, M., and Grunstein, M. (1988). Nucleosome Loss Activates Yeast Downstream Promoters In Vivo. Cell. 55: 1137-1145.

7. Johnson, L.M., Kayne, P.S., Kahn, E.S., and Grunstein, M. (1990). Genetic Evidence for an Interaction Between SIR3 and Histone H4 in the Repression of the Silent Mating Loci in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA. 87: 6286-6290.

8. Grunstein, M. (1990). Histone Function in Transcription. In: Annual Review of Cell Biology, Vol. 6 (G. E. Palade, ed.), Annual Reviews, Inc. pp. 643-678.

9. Durrin, L., Mann, R., Kayne, P., and Grunstein M. (1991). Yeast Histone H4 N-Terminal Sequence is Required for Promoter Activation In Vivo. Cell. 65: 1023-1031.

10. Grunstein, M. (1992). Histones as Regulators of Genes. Scientific American. 267: 68-74.

11. Thompson, J., Ling, X. and Grunstein, M. (1994). Histone H3 Amino Terminus is Required for Telomeric and Silent Mating Locus Repression in Yeast. Nature. 369: 245-247.

12. Hecht, A., Laroche, T., Strahl-Bolsinger, S., Gasser, S., and Grunstein, M. (1995). Histone H3 and H4 N-Termini Interact with SIR3 and SIR4 Proteins: A Molecular Model for the Formation of Heterochromatin in Yeast. Cell. 80: 583-592.

13. Carmen, A., Rundlett, S.E., and Grunstein, M. (1996). HDA1 and HDA3 are Components of a Yeast Histone Deacetylase (HDA) Complex. J. Biol. Chem. 271:15837-15844.

14. Hecht, A., Strahl-Bolsinger, S., and Grunstein, M. (1996). Spreading of Transcriptional Repression by SIR3 from Telomeric Heterochromatin. Nature. 383: 92-96.

15. Rundlett, S., Carmen, A., Turner, B., Kobayashi, R., Bavykin, S., and Grunstein, M. (1996). HDA1 and RPD3 are Members of Functionally Distinct Yeast Histone Deacetylase Complexes. Proc. Nat. Acad. Sci. USA. 93: 14503-14508.

16. Strahl-Bolsinger, S. Hecht, A. Luo, K., and Grunstein, M. (1997). SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin of yeast. Genes and Development. 11: 83-93.

17. Rundlett, S.E., Carmen, A.A., Suka, N., Turner, B.M., and Grunstein, M. (1998). Transcriptional repression by UME6 involves deacetylation of lysine 5 of histone H4 by RPD3. Nature 392:831-835.

18. Grunstein, M. (1998). Yeast Heterochromatin: regulation of its assembly and inheritance by histones. Cell. 93:325-328.

19. Hecht, A., and Grunstein, M. (1999). Mapping DNA interaction sites of chromosomal proteins using immunoprecipitation and PCR. Methods in Enzymology 304:399-414.

20. Carmen, A.A., Griffin, P.R., Calaycay, J.R., Rundlett, S.E., Suka, Y., and Grunstein, M. (1999). Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity. Proc. Nat. Acad. Sci. USA 96:12356-12361.

21. Vogelauer, M., Wu, J., Suka, N., and Grunstein, M. (2000). Global histone acetylation and deacetylation in yeast. Nature 408:495-498.

22. Wu, J., Carmen, A. A., Kobayashi, R., Suka, N. and Grunstein, M. (2001). HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. Proc. Nat. Acad. Sci. USA 98:4391-4396.

23. Suka, N., Suka, Y., Carmen, A.A., Wu, J., and Grunstein, M. (2001). Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin. Molecular Cell. 8:473-479.

24. Carmen, A. A., Milne, L. and Grunstein, M. (2002). Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3. J. Biol. Chem. 277:4778-4781.

25. Robyr, D., Suka, Y., Xenarios, I., Wang, A., Suka, N., and Grunstein, M. (2002). Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases Cell 109:437-446.

26. Kurdistani, S.K., Robyr, D., Tavazoie, S. and Grunstein, M. (2002). Genome-wide binding map of the RPD3 histone deacetylase in yeast. Nature Genetics 31:248-254.

27. Luo, K., Vega-Palas, M. A. and Grunstein, M. (2001). Rap1-Sir4 binding independent of other Sir, yKu, or histone interactions initiates the assembly of telomeric heterochromatin in yeast. Genes and Dev. 16:1528-1539.

28. Vogelauer M., Rubbi, L., Lucas, I., Brewer, B and Grunstein, M. (2002). Rpd3 histone deacetylase regulates the timing of DNA replication origins in yeast. Molecular Cell 10:1223-1233.

29. Wang, A., Kurdistani, S. K. and Grunstein, M. (2002). Yeast HOS2 histone deacetylase promotes gene activity. Science 298:1412-1414.

30. Suka, N., Luo, K. and Grunstein, M. (2002). SIR2 and SAS2 opposingly regulate acetylation of yeast histone H4 lysine 16 and spreading of heterochromatin. Nature Genetics 32:378-383.

31. Kurdistani, S. K. and Grunstein, M. (2003). In vivo protein-protein and protein-DNA crosslinking for genomewide binding microarray. Methods 31:90-95

32. Robyr, D. and Grunstein, M. (2003). Histone acetylation arrays. Methods 31:83-89.

33. Kurdistani, S. K., Tavazoie, S, and Grunstein, M. (2004). Mapping global histone acetyation patterns to gene expression. Cell 117: 721-733.

34. Xu, F., Zhang, K. and Grunstein, M. (2005). Acetylation in the histone H3 globular domain regulates gene expression in yeast. Cell 121:375-385

35. Seligson , D.B., Horvath, S., Shi, T., Yu, H., Tze, S., Grunstein, M. and Kurdistani, S. K. (2005). Global histone modification patterns predict risk of prostate cancer recurrence . Nature. In Press: