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Area of Expertise: Epigenetic processes that mediate heritable modifications to chromatin
The research in my lab focuses on understanding how cells
regulate epigenetic processes. Our research examines the role of
the cell cycle and DNA replication in assembly or maintenance of
chromatin structures and the effects of these structures on DNA
replication. We are interested in how the cell restricts
heterochromatin to specific genomic loci, why heterochromatin
formation is regulated by the cell cycle, how transcription of
genes is prevented in silenced regions, and whether epigenetic
processes are influenced by or influence events including DNA
damage and the initiation of DNA replication. We are intrigued
by how epigenetic states are maintained throughout the cell
cycle and are duplicated and inherited each time the chromosome
itself is replicated and the cell divides. We investigate how
environmental factors perturb epigenetic processes and can
contribute to inappropriate gene expression, developmental
defects, tumorogenesis and other catastrophic disorders.
Our research explores the interface between
epigenetic processes, histone modifications, chromatin assembly,
DNA replication and the cell cycle, Our laboratory combines
molecular biology, biochemical and quantitative microscopy-based
approaches with mammalian cell culture and the power of yeast
genetics to understand the impact of genetic and external
factors on epigenetic gene regulation.
Silencing inSaccharomyces cerevisiae
We use silent chromatin in S.
cerevisiae as a
model for understanding how epigenetically regulated chromatin
structures are established, maintained and inherited. Silenced
chromatin in budding yeast, is akin to heterochromatin in
organisms such as maize, flies, and mammals. S.
epigenetically inherited chromosomal structures to regulate a
variety of cellular activities including controlling cell-type
specific gene expression, modulating ribosomal RNA levels, and
preserving telomere structure and stability. Silenced
regions in S.
are regulated, in part, by the Silent Information Regulator, or
Sir, proteins include the silent mating-type loci, HML and HMR,
the rDNA locus and the telomeres.
To mediate silencing at a given site on a
chromosome, an organism must first have a way to recruit the
proteins that compose silenced chromatin to that locus. In
yeast, regulatory sites known as silencers flank the silent
mating-type loci. Silencers contain binding sites for the Origin
Recognition Complex (ORC), and the transcriptional regulators
Rap1p and Abf1p. In addition, the Sir proteins, Sir1p, Sir2p,
Sir3p and Sir4p, are structural components of silenced chromatin
in yeast. Unlike ORC, Rap1p and Abf1p, however, the Sir proteins
do not bind to DNA site-specifically. Instead, Sir proteins
associate with silencers through protein-protein interactions
between each other, proteins bound at silencers, and histones H3
and H4. Once initiated, silencing spreads along the chromosome
over several kilobases of DNA and inactivates gene expression.
Once established, the transcriptionally inactive state of this
region of the chromosome is maintained throughout the cell cycle
and can be stably inherited in subsequent generations. Many
proteins involved in silencing in yeast have homologs in a wide
variety of organisms, including humans where several are
involved in development and differentiation or have been
implicated in cancer.
Epigenetic Processes and Replication-Coupled
We are investigating how a network of chromatin
assembly factors, histone-modifying enzymes and replication
factors interact to assemble appropriately modified nucleosomes
during DNA replication and thereby promote epigenetic processes
and genome integrity. We have been characterizing how silencing
is affected by the DNA polymerase processivity factor PCNA
through pathways involving several factors including the
chromatin assembly factors Asf1p and CAF-1, the PCNA loading
complex RF-C and the histone acetyltransferase Rtt109p. As
nucleosomal DNA serves as the foundation upon which silent
chromatin is built, perturbations in replication-coupled
chromatin assembly can alter the efficiency and location of
silent chromatin formation as well as lead to defects in
maintaining and inheriting epigenetic states. Repair of DNA
damage is also often compromised in mutants with defects linked
to histone modifications and replication-coupled chromatin
Defining the Composition of Chromatin
The simplest structural unit of chromatin, the
nucleosome, can exist in a variety of configurations depending
on the histone variants and post-translational modifications
present. The composition of individual nucleosomes influences
chromatin structure and function and provides signals to the
cellular machinery to promote gene activation or repression.
These signals tend to be dynamic and can vary as a function of
the cell cycle or growth conditions. Yet, our understanding of
the patterns found within individual nucleosomes and presented
to the cellular machinery is limited. Population-based
approaches widely used to characterize chromatin composition
have been valuable in identifying and mapping individual
modifications within histones, but have been limited in
describing which modifications are combined within the same
nucleosomes. As a powerful complement to standard approaches, we
are utilizing single molecule strategies in
vitro and in
single mammalian and yeast cells to probe epigenetic processes
that regulate transcriptional states, responses to DNA damage,
centromere function and differentiation in several collaborative
Awards & Honors
(1988) Robert B. and Sophia Whiteside Scholarship.
(1987) LB (In-College) Scholarship.
(1988) Odden Scholarship.
(1988) Alliss Educational Foundation Scholarship. Alliss Educational Foundation.
(1991) National Science Foundation Fellow. Summer Institute in Japan for U.S. Graduate Students in Science and Engineering.
(1995) Predoctoral Fellowship. National Institutes of Health.
(2001) National Research Service Award. National Institutes of Health.
(2002) Senior Postdoctoral Fellowship. American Cancer Society.
(2009) Ellison Foundation Fellowship, Molecular Biology of Aging Workshop. Ellison Foundation.
(2014) University Faculty Scholar. Purdue University.
Miller, A., & Kirchmaier, A. (2014). Analysis of silencing in Saccharomyces cerevisiae. In Yeast Genetics: Methods and Protocols, Methods Mol. Biol. (1205, 275-302). NY: Humana Press, Springer Science+Business Media.
Kirchmaier, A. (2013). Creating memories of transcription. Proc. Natl. Acad. Sci. USA, 110, 13701-13702. Retrieved from http://www.pnas.org/content/110/34/13701.full.pdf+html
Saatchi, F., & Kirchmaier, A. (2013). HATs, HDACs and the Regulation of Cellular Processes. In Acetate: Versatile Building Block of Biology and Chemistry (1, 29-64). Hauppauge, NY: Nova Science Publishers, Inc.
Young, T. (2012). Cell Cycle Regulation of Silent Chromatin Formation. Biochim. Biophys. Acta, 1819, 303-312. Retrieved from http://ac.els-cdn.com/S1874939911001829/1-s2.0-S1874939911001829-main.pdf?_tid=5b0d693e311053e0f99a75f7f8cdb662&acdnat=1339704549_a37db7c27e84da68aaefbb24174efb0b
Chen, J., Miller, A., Kirchmaier, A., & Irudayaraj, J. K. (2012). Single-molecule tools elucidate H2A.Z nucleosome composition. J. Cell Sci, 125, 2954-2964. Retrieved from http://jcs.biologists.org/content/125/12/2954.full.pdf+html
Jacobi, J. (2011). Propagation of Epigenetic States during DNA Replication. In Fundamental Aspects of DNA Replication (1st, 245-270). Vienna, Austria: InTech Publishing. Retrieved from http://www.intechopen.com/articles/show/title/propagating-epigenetic-states-during-dna-replication
Ub-Family Modifications at the Replication Fork: Regulating PCNA-Interacting Components. (2011). FEBS Lett, 585, 2920-2928. Retrieved from http://www.febsletters.org/article/S0014-5793(11)00598-9/
Miller, A., Chen, J., Takasuka, T., Jacobi, J., Kaufman, P., & Irudayaraj, J. K. (2010). Proliferating Cell Nuclear Antigen (PCNA) is Required for Cell Cycle-Regulated Silent Chromatin on Replicated and Nonreplicated Genes. J. Biol. Chem, 285, 35142-35154. Retrieved from http://www.jbc.org/content/early/2010/09/02/jbc.M110.166918.abstract
Yang, B., & Miller, A. (2008). HST3/HST4 - dependent Deacetylation of Lysine 56 of Histone H3 in Silent Chromatin. Mol. Biol. Cell, 19(11), 4993-5005. Retrieved from http://www.molbiolcell.org/cgi/content/full/19/11/4993
Yang, B., & Britton, J. (2008). Insights into the Impact of Histone Acetylation and Methylation on Sir Protein Recruitment, Spreading. J. Mol. Biol, 381(4), 826-844. Retrieved from http://www.sciencedirect.com/science/article/pii/S0022283608007754