professor of biology
Ph.D., University of Massachusetts at Amherst
Fields of Interest
Molecular biology; chromatin assembly and histone modifications in mammalian cells and fission yeast.
The nuclear DNA of eukaryotes is organized with structural and regulatory proteins to form the nucleoprotein complex termed chromatin. The primary functional unit of chromatin is the nucleosome, a particle containing histone proteins and approximately 200 base pairs of DNA. Research in my laboratory is directed toward understanding the processes involved in nucleosome assembly during DNA replication. Just as the DNA in dividing cells must be replicated once each cell cycle, so too must sufficient histones (and other chromatin proteins) be synthesized to assemble nucleosomes on the newly replicated DNA. The proper assembly of chromatin during cell division is of vital importance, because the presence or absence of nucleosomes (and the precise positioning of nucleosomes with respect to DNA sequences) can determine which genes are transcribed, and when. To make our results as relevant as possible to human cell biology, our experiments are performed using HeLa cells, a transformed human cell line maintained in spinner culture.
The faithful transmission and assembly of chromatin requires that many independent cellular processes be coordinated. As DNA is being replicated, histones are synthesized, then modified by enzymatic acetylation, transported to the nucleus, and assembled into nucleosomes. Moreover, supercoiled chromatin higher-order structures must first "unwind" to allow access to the DNA, and then condense again after replication is completed. In my laboratory the specific questions currently being investigated include: the modification status of parental histones that are segregated to progeny chromosomes, and the mechanisms of histone deposition onto newly replicated DNA; the involvement of histone acetylation in nucleosome assembly, and the properties of enzymes (acetyltransferases) involved; the role of histone phosphorylation in regulating chromatin folding; and the isolation and characterization of somatic nucleosome "assembly factors" to define the in vivo assembly pathway.
In order to address these questions we use a number of approaches, including DNA replication systems (in vivo and in vitro), histone acetylation assays, in vitro assembly reactions using purified components, and DNA supercoiling studies. We also take advantage of antibodies directed against specific histone and non-histone chromatin proteins, to purify and analyze newly replicated nucleosomes and their assembly intermediates. Our aims are to identify major cellular components needed to generate nucleosomes in vivo, and to characterize the stages of chromatin biosynthesis. Ultimately, these studies should provide a better understanding of the regulation of chromatin organization during DNA replication, and of the processes involved in the faithful assembly of transcriptionally active and inactive chromatin structures.
Benson, L.J., Phillips, J.A., Gu, Y., Parthun, M.R., Hoffman, C.S., Annunziato, A.T. 2007. Properties of the type B histone acetyltransferase HAT1: H4 tail interaction, site preference, and involvement in DNA repair. Journal of Biological Chemistry 282(2): 836–42 (link to PubMed abstract).
Benson, L.J., Gu, Y., Yakovleva, T., Tong, K., Barrows, C., Strack, C.L., Cook, R.G., Mizzen, C.A., and Annunziato, A.T. 2006. Modifications of H3 and H4 during chromatin replication, nucleosome assembly, and histone exchange. Journal of Biological Chemistry 281: 9287–9296 (link to PubMed abstract).
Annunziato, A.T. 2005. Split decision: What happens to nucleosomes during DNA replication? Journal of Biological Chemistry 280: 12065–12068 (link to PubMed abstract).
Benson, L.J., and Annunziato, A.T. 2004. In vitro analysis of histone acetyltransferase activity. Methods 33: 45–52 (link to PubMed abstract).
Makowski, A.M., Dutnall, R.N., and Annunziato, A.T. 2001. Effects of acetylation of histone H4 at lysines 8 and 16 on activity of the Hat1 histone acetyltransferase. Journal of Biological Chemistry 276: 43499–502 (link to PubMed abstract).
Annunziato, A.T., and Hansen, J.C. 2000. Role of histone acetylation in the assembly and modulation of chromatin structures. Gene Expression 9: 37–61 (link to PubMed abstract).
Chang, L., Ryan, C.A., Schneider, C.A., and Annunziato, A.T. 1999. Preparation/analysis of nascent chromatin replicated in vivo and in isolated nuclei. In: Methods in Enzymology: Chromatin, Vol. 304 (P. Wassarman & A. Wolffe, Eds.) Academic Press, San Diego, pp. 76–99 (link to PubMed abstract).
Ryan, C.A. and Annunziato, A.T. 1999. Separation of histones in a Triton-acid-urea gel system. In: Current Protocols in Molecular Biology (Chanda, V., Ed.) John Wiley and Sons. Inc., New York, Chapter 21; Unit 2.2, pp. 2.3–2.10.
Chang, L., Loranger, S.S., Mizzen, C., Ernst, S.G., Allis, C.D., and Annunziato, A.T. 1997. Histones in transit: cytosolic histone complexes and H4 diacetylation during nucleosome assembly in human cells. Biochemistry 36: 469–480 (link to PubMed abstract).
Annunziato, A.T., Eason, M.B., and Perry, C.A. 1995. Interaction between methylation and acetylation of arginine-rich histones in cycling and arrested HeLa cells. Biochemistry 34: 2916–2924 (link to PubMed abstract).
Sobel, R.E., Cook, R.G., Perry, C.A., Annunziato, A.T., and Allis, C.D. 1995. Conservation of deposition-related acetylation sites in newly synthesized H3 and H4. Proceedings of the National Academy of Sciences of the USA 92: 1237–1241 (link to PubMed abstract).