Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Major target audiences include, but are not limited to, fellow scientists, undergraduate, graduate and postdoctoral trainees, members of the biotechnology and pharmaceutical industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Three graduate and four undergraduate students have received training in basic research in the areas of genetics, molecular biology, biochemistry and biophysics while conducting this research. How have the results been disseminated to communities of interest? Publications, presentations, talks and posters at scientific meetings, teaching. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We focus on 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 and gene expression. We study how an epigenetic process called silencing is inherited in the budding yeast Saccharomyces cerevisiae and similar processes in mammalian cells. Silencing analogous to epigenetic gene regulation in organisms including humans, Arabidopsis, and Drosophila. Epigenetic processes regulate gene expression, ribosomal RNA expression, telomere structure, centromere function and, in multicellular organisms, control cell differentiation. We have been exploring how epigenetic gene regulation is influenced by external (environmental and dietary) and genetic factors. We have been characterizing the relationship between histone modifications, DNA replication, DNA damage and the formation and stability of epigenetic processes. We have discovered that several essential, evolutionarily conserved, DNA replication proteins affect epigenetic processes by influencing chemical modification of several histone residues and are currently characterizing how the activities of proteins involved in epigenetic processes are regulated as a function of the cell cycle and DNA damage responses using biochemical, genetic and single molecule approaches. We have discovered protein-protein interactions important for chromatin assembly during DNA replication and repair as well as for the regulation of silencing. We have conducted synthetic interaction analyses to identify genetically the relationship between genes that regulate epigenetic processes and DNA damage responses. We also are collaboratively developing and applying single molecule technologies to detect and quantify changes in DNA modifications and histone modifications as well as changes in gene expression in response to environmental factors or genetic backgrounds in cell lysates and individual cells.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Miller Andrew and Kirchmaier Ann L. (2014) Analysis of Silencing in Saccharomyces cerevisiae. in Yeast Genetics: Methods and Protocols, Methods in Molecular Biology Vol. 1205, Jeffery S. Smith, Daniel Burke, Eds., Ch. 17, pp. 275-302. Humana Press, Springer Science+Business Media, NY.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Characterizing the Interactions between CAF-1 and Cdc7 in Silencing and Responses to DNA Damage. Tiffany J. Young, Yi Cui, Joseph Irudayaraj, Ann L. Kirchmaier. 2014 Midwest Yeast Meeting, Northwestern University, Evanston, IL; 9/27-28/14.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Characterization of Interactions between the Kinase Cdc7 and the Nucleosome Assembly Factor CAF-1. Tiffany J. Young, Yi Cui, Joseph Irudayaraj, Ann L. Kirchmaier. 2014 Midwest Chromatin and Epigenetics Meeting, University of Wisconsin, Madison, WI; 5/18-20/14.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Differentiation-dependent changes in the epigenomic landscape of human myeloid leukemia cells. Basudev Chowdhury, Zhiping Wang, Yunlong Liu, Arun Seetharam, Jyothi Thummapuram, Joseph Irudayaraj, Ann L. Kirchmaier. 2014 Midwest Chromatin and Epigenetics Meeting, University of Wisconsin, Madison, WI; 5/18-20/14.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Major target audiences include, but are not limited to, fellow scientists, undergraduate, graduate and postdoctoral trainees, members of the biotechnology and pharmaceutical industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate and three undergraduate students have received training in basic research in the areas of genetics, molecular biology, biochemistry and biophysics while conducting this research. How have the results been disseminated to communities of interest? Publications. Presentations, talks and posters at scientific meetings, teaching. What do you plan to do during the next reporting period to accomplish the goals? Continuation of current lines of inquiry; application of single molecule technologies, as well as genetic, molecular biology and biochemistry strategies to probe epigenetic processes.
Impacts
What was accomplished under these goals?
We focus on 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 and gene expression. We study how a group of proteins, called Sir proteins, participate in a type of epigenetic gene regulation called silencing in the budding yeast Saccharomyces cerevisiae. Silencing in S. cerevisiae is akin to epigenetic gene regulation in organisms including maize, flies, and mammals. S. cerevisiae epigenetically regulates several cellular activities including controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. We have been exploring how epigenetic gene regulation is influenced by external (environmental and dietary) and genetic factors. We have been characterizing the relationship between histone modifications, DNA replication, DNA damage and the formation and stability of epigenetic processes. We have discovered that several essential, evolutionarily conserved, DNA replication proteins affect epigenetic processes by influencing chemical modification of several histone residues and are currently characterizing their activities. We have been applying biochemical, genetic and single molecule approaches to understand how these proteins interact in the cell. We have defined protein-protein interactions important for chromatin assembly during DNA replication and repair as well as for the regulation of transcription. We have conducted synthetic interaction analyses to identify genes that function in regulating epigenetic processes, DNA damage responses, and genome integrity. We also are collaboratively developing and applying single molecule technologies to detect and quantify changes in chemical modifications to chromatin (DNA modifications and histone modifications) as well as changes in chromatin-associated factors in response to changes in cellular environments or genetic backgrounds in cell lysates, nuclear preps and individual cells. We are also developing strategies to assess transcripts and DNA replication–related processes in these systems.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Kirchmaier AL (2013) Creating Memories of Transcription. Proc. Natl. Acad. Sci. USA 110:13701-13702.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Saatchi F and Kirchmaier AL. (2013) HATs, HDACs and the Regulation of Cellular Processes. in Acetate: Versatile Building Block of Biology and Chemistry, D. A. Sanders, Ed., Nova Science Publishers, Inc. Hauppauge, NY. ISBN:978-1-62808-565-5. Ch. 3, pp. 29-64.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: We focus on 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 and gene expression. We study how a group of proteins, called Sir proteins, participate in a type of epigenetic gene regulation called silencing in the budding yeast Saccharomyces cerevisiae. Silencing in S. cerevisiae is akin to epigenetic gene regulation in organisms including maize, flies, and mammals. S. cerevisiae epigenetically regulates several cellular activities including controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. We have been exploring how epigenetic gene regulation is influenced by external (environmental and dietary) and genetic factors. We have been characterizing the relationship between histone modifications, DNA replication, DNA damage and the formation and stability of epigenetic processes. We have discovered that several essential, evolutionarily conserved, DNA replication proteins affect epigenetic processes by influencing chemical modification of several histone residues. This past year we have been taking biochemical, genetic and single molecule approaches to define proteins that function in a network of overlapping chromatin assembly and modification pathways involved in packaging newly synthesized DNA into nucleosomes and higher order chromatin structures as well as in responses to DNA damage and in epigenetic processes. We also are collaboratively developing and applying single molecule technologies to detect and quantify changes in chemical modifications to chromatin in response to changes in cellular environments or genetic backgrounds in yeast and human cells. We have defined the composition of nucleosomes containing multiple histone variants and chemical modifications associated with these nucleosomes in cell extracts and fixed cells. This area of research is being expanded to include assessing DNA modifications in addition to histone modifications in regulating gene expression and epigenetic processes. PARTICIPANTS: Andrew Miller (Research Associate), Ann Kirchmaier (Principal Investigator), Faeze Saatchi (Graduate Student), Tiffany Young (Graduate Student), Elizabeth Bell (Undergraduate Student), Jessica Grabbard (Undergraduate Student), Alex Kosiak (Undergraduate Student) and Fakhry Daowd (Undergraduate Student). Both undergraduate and graduate students participated in the discussed research. TARGET AUDIENCES: Major target audiences include, but are not limited to, fellow scientists, undergraduate, graduate and postdoctoral trainees, members of the biotechnology and pharmaceutical industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We study the epigenetic processes that regulate gene expression and their relationship to DNA replication. Epigenetic modifications are chemical modifications that are added on top of the DNA without changing its sequence. These modifications are affected by environmental and dietary factors, are crucial during normal development of plants, animals and humans and are often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate epigenetic processes requires an understanding of how epigenetic modifications are created and what regulates their heritability.
Publications
- Young, TJ and Kirchmaier, AL. (2012) Cell Cycle Regulation of Silent Chromatin Formation. Biochem. Biophys. Acta. 1819:303-312.
- Chen J, Miller A, Kirchmaier AL, and Irudayaraj JMK. (2012) Single Molecule Tools Elucidate H2A.Z Nucleosome Composition. J. Cell Sci. 125:2954-2964.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: We focus on 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 and gene expression. We study how a group of proteins, called Sir proteins, participate in a type of epigenetic gene regulation called silencing in the budding yeast Saccharomyces cerevisiae. Silencing in S. cerevisiae is akin to epigenetic gene regulation in organisms including maize, flies, and mammals. S. cerevisiae epigenetically regulates several cellular activities including controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. We have been exploring how epigenetic gene regulation is influenced by external (environmental and dietary) and genetic factors. We have been characterizing the relationship between histone modifications, DNA replication, DNA damage and the formation and stability of epigenetic processes. We have discovered that several essential, evolutionarily conserved, DNA replication proteins affect epigenetic processes by influencing chemical modification of several histone residues. Our initial findings provide evidence for replication-coupled histone modifications occurring through interactions between PCNA and two histone acetyltransferases via the chromatin assembly factors CAF-1 or Asf1p. This past year we have been taking biochemical, genetic and single molecule approaches to define protein:protein interactions that occur in a network of overlapping chromatin assembly and modification pathways involved in packaging newly synthesized DNA into nucleosomes and higher order chromatin structures. PCNA, CAF-1, Asf1p and the histone acetyltransferases are members of this network. We have been defining how this network participates in ensuring silent chromatin is inherited epigenetically each cell generation and how branches of this network participate in cellular responses to DNA damage. We are also collaboratively developing and applying single molecule technologies to detect and quantify changes in chemical modifications to chromatin in response to changes in cellular environments or genetic backgrounds in yeast and human cells. We have defined the composition of nucleosomes containing rare histone variants and the chemical modifications associated with these nucleosomes in cell extracts and fixed cells and our initial findings from this work is under review. Ongoing work in this area includes deciphering the role of histone modifications in regulating gene expression and epigenetic processes. PARTICIPANTS: Andrew Miller (Research Associate) Ann Kirchmaier (Principal Investigator) Jennifer Jacobi (Graduate Student) Fan Hu (Graduate Student) Tiffany Young (Graduate Student) Zach McBride (Undergraduate Student) Amanda Campbell (Undergraduate Student) Alex Kosiak (Undergraduate Student) Both undergraduate and graduate students participated in the discussed research. TARGET AUDIENCES: Major target audiences include, but are not limited to, fellow scientists, undergraduate, graduate and postdoctoral trainees, members of the biotechnology and pharmaceutical industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We study the epigenetic processes that regulate gene expression and their relationship to DNA replication. Epigenetic modifications are chemical modifications that are added on top of the DNA without changing its sequence. These modifications are affected by environmental and dietary factors, are crucial during normal development of plants, animals and humans and are often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate epigenetic processes requires an understanding of how epigenetic modifications are created and what regulates their heritability.
Publications
- Kirchmaier, A.L. (2011) Ub-Family Modifications at the Replication Fork: Regulating PCNA-Interacting Components. FEBS Letters 585:2920-2928.
- Jacobi, JL and Kirchmaier, AL. (2011) Propagation of Epigenetic States during DNA Replication. in Fundamental Aspects of DNA Replication, Jelena Kusic-Tisma Ed. InTech Publishing, Vienna, Austria. ISBN: 978-953-307-259-3, pp. 245-270.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: We focus on 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 and gene expression. We study how a group of proteins, called Sir proteins, participate in a type of epigenetic gene regulation called silencing in the budding yeast Saccharomyces cerevisiae. Silencing in S. cerevisiae is akin to epigenetic gene regulation in organisms including maize, flies, and mammals. S. cerevisiae epigenetically regulates several cellular activities including controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. We have been exploring how epigenetic gene regulation is influenced by external (environmental and dietary) and genetic factors. We have been characterizing the relationship between histone modifications, DNA replication, DNA damage and the formation and stability of epigenetic processes. We have discovered that several essential, evolutionarily conserved, DNA replication proteins affect epigenetic processes by influencing chemical modification of several histone residues. We have defined replication-coupled genetic pathways regulating acetylation of K9 and K56 on histone H3 via the histone acetyltransferase Rtt109p and acetylation of K16 on histone H4 via the histone acetyltransferase Sas2p in the SAS-I complex. Our findings provide evidence for replication-coupled histone modifications occurring through interactions between PCNA and Rtt109p (related to human transcription regulator CBP/p300) or the SAS-I complex (related to the human MYST family of genes involved in tumor suppression, e.g. MOF/TIP60) via the chromatin assembly factor intermediates CAF-1 or Asf1p, thereby linking cell cycle-regulated histone modification to epigenetic gene expression. We also participate in collaborative studies to develop and apply single molecule technologies to detect and quantify changes in chemical modifications to chromatin in response to changes in cellular environments or genetic backgrounds in yeast and human cells. In our proof of concept work, we have defined the composition of nucleosomes containing rare histone variants and the chemical modifications called acetylation and methylation associated with these nucleosomes. Ongoing work includes deciphering the role of histone variants in forming specialized histone modification patterns and the biological functions of these patterns with respect to gene expression. PARTICIPANTS: Andrew Miller (Research Associate) Ann Kirchmaier (Principal Investigator) Jennifer Jacobi (Graduate Student) Fan Hu (Graduate Student) Katherine Turpen (Undergraduate Student) Zach McBride (Undergraduate Student) Amanda Campbell (Undergraduate Student) Caleb Peck (Undergraduate Student) Both undergraduate and graduate students participated in the discussed research. TARGET AUDIENCES: Major target audiences include, but are not limited to, fellow scientists, undergraduate, graduate and postdoctoral trainees, members of the biotechnology and pharmaceutical industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We study the epigenetic processes that regulate gene expression and their relationship to DNA replication. Epigenetic modifications are chemical modifications that are added on top of the DNA without changing its sequence. These modifications are affected by environmental factors, are crucial during normal development of plants, animals and humans and are often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate epigenetic processes requires an understanding of how epigenetic modifications are created and what regulates their heritability.
Publications
- Miller A, Chen J, Takasuka TE, Jacobi JL, Kaufman PD, Irudayaraj JMK, and Kirchmaier AL. (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.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: We focus on 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 and gene expression. We study how a group of proteins, called Sir proteins, participate in a type of epigenetic gene regulation called silencing in the budding yeast Saccharomyces cerevisiae. Silencing in S. cerevisiae, is akin to epigenetic gene regulation in organisms including maize, flies, and mammals. S. cerevisiae epigenetically regulates several cellular activities including controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. We have been exploring how epigenetic gene regulation is influenced by external and genetic factors. We have been characterizing the relationship between histone modifications, DNA replication, DNA damage and the formation and stability of silent chromatin. We have discovered that misregulation of several DNA replication and chromatin assembly factors affect epigenetic processes by influencing the modification status of several histone residues. These modifications include acetylation of K9 and K56 on histone H3 via the histone acetyltransferase Rtt109p and acetylation of K16 on histone H4 via the histone acetytransferase Sas2p in the SAS-I complex. Chromatin-associated histones in replication factor mutants are hypoacetylated at these residues, resulting in epigenetic defects. Together, our findings provide evidence for the existence of replication-coupled histone modifications in yeast through interactions between PCNA and Rtt109p or the SAS-I complex, thereby linking cell cycle-regulated histone modifications to epigenetic gene expression. We have also initiated collaborative studies to develop single molecule tools to detect and quantify changes in chromatin modifications in response to changes in cellular environments or genetic backgrounds. PARTICIPANTS: Andrew Miller (Research Associate) Ann Kirchmaier (Principal Investigator) Rebecca Funk (Undergraduate Student/Technician) Michelle Weyreter (Undergraduate Student) Caleb Peck (Undergraduate Student) Amanda Campbell (Undergraduate Student) Gabriella Mavaro Velez (Undergraduate Student) Jennifer Jacobi (Graduate Student) Bo Yang (Graduate Student) Both undergraduate and graduate students participated in the discussed research. TARGET AUDIENCES: Major target audiences include, but are not limited to, fellow scientists, undergraduate, graduate and postdoctoral trainees, members of the biotechnology and pharmaceutical industries PROJECT MODIFICATIONS: None
Impacts
We study the formation, maintenance and epigenetic inheritance of chromosome structures that affect gene expression and DNA replication. These processes are affected by environmental factors, are crucial during normal development of an organism and are often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate these effects requires an understanding of what events trigger epigenetic processes and what regulates their heritability.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: We focus on 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 and gene expression. We study how a group of proteins, called Sir proteins, participate in a type of epigenetic gene regulation called silencing in the budding yeast Saccharomyces cerevisiae. Silencing in S. cerevisiae, is akin to epigenetic gene regulation in organisms including maize, flies, and mammals. S. cerevisiae epigenetically regulates several cellular activities including controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. We have been exploring how epigenetic gene regulation is influenced by external and genetic factors. We have been characterizing the relationship between histone modifications, DNA replication, DNA damage and the formation and stability of silent chromatin. We have discovered that the histone H3 K56-specific deacetylases Hst3p and Hst4p that are involved cellular responses to DNA damage are also required for silencing. We have also discovered that misregulation of several DNA replication and chromatin assembly factors affect epigenetic processes by influencing the modification status of histone H3 on K56 through a pathway involving the histone acetyltransferase Rtt109p. Mutating histone residues that are targets of these enzymes also modulates silencing. Together our findings couple newly discovered, cell cycle-regulated histone modifications to epigenetic gene expression. PARTICIPANTS: Andrew Miller (Research Associate) Ann Kirchmaier (Principal Investigator) Jeanette Britton (Undergraduate Student) Tiaun Foster (Undergraduate Student) Jennifer Jacobi (Graduate Student) Bo Yang (Graduate Student) Both undergraduate and graduate students participated in the discussed research. TARGET AUDIENCES: Major target audiences include, but are not limited to, fellow scientists, undergraduate, graduate and postdoctoral trainees, members of the biotechnology and pharmaceutical industries. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We study the formation, maintenance and epigenetic inheritance of chromosome structures that affect gene expression and DNA replication. These processes are affected by environmental factors, are crucial during normal development of an organism and are often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate these effects requires an understanding of what events trigger epigenetic processes and what regulates their heritability.
Publications
- Yang B, Miller A and Kirchmaier AL. (2008) HST3/HST4-Dependent Deacetylation of Lysine 56 of Histone H3 in Silent Chromatin. Mol. Biol. Cell. 19:5005-5017.
- Yang B, Britton J and Kirchmaier AL. (2008) Insights into the Impact of Histone Acetylation and Methylation on Sir Protein Spreading and Silencing in Saccharomyces cerevisiae. J. Mol. Biol. 381:826-844.
- Miller A, Yang B, Foster T and Kirchmaier AL. (2008) Proliferating Cell Nuclear Antigen and ASF1 Modulate Silent Chromatin in Saccharomyces cerevisiae via Lysine 56 on Histone H3. Genetics 179:793-809.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Research at Purdue University focuses on how chromosomes structures are built and how these structures can affect gene expression. The model organism used is budding yeast because many of the proteins that "turn on and off" genes have been maintained over the course of evolution and so are used also by plants and animals, including humans. A series of experiments demonstrated that when a protein makes chemical changes to the chromosome structure, that chemical reaction creates places for other proteins to bind and spread across the chromosome. These experiments also demonstrated that these steps were necessary but not sufficient to "turn off" genes and the chemical reaction is needed for an additional unknown event. The research defined a new step in a multi-step process involved in "turning off" genes.
PARTICIPANTS: Bo Yang, Andrew Miller, and Jennifer Henkly did research on this project in the lab.
TARGET AUDIENCES: Biochemists
Impacts
Purdue researchers discovered that the "on-off switch" for certain genes requires more steps than previously recognized. This basic research stands as the precursor to deciphering the "rules" a cell follows when choosing to regulate gene expression by changing the structure of its chromosomes. As a result, new studies will have the starting point needed to consider the most applicable methods for gene therapy and therapeutic drugs to treat various diseases and forms of cancer where normal chromosome structure is altered.
Publications
- Yang, B and Kirchmaier, A.L. (2006). Bypassing the Catalytic Activity of Sir2 for SIR Protein Spreading in S. cerevisiae. Mol. Biol. Cell. 17: 5287-5297.
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Progress 10/01/05 to 09/30/06
Outputs
We focus on 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 and gene expression. We study how a group of proteins, called Sir proteins, participate in a type of epigenetic gene regulation called silencing in the budding yeast Saccharomyces cerevisiae. Silencing in S. cerevisiae, is akin to epigenetic gene regulation in organisms including maize, flies, and mammals. S. cerevisiae epigenetically regulates several cellular activities including controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. We have been exploring how epigenetic gene regulation is influenced by external and genetic factors. We have discovered that hydroxyurea (a drug used to treat cancer that inhibits a protein called ribonucleotide reductase which makes the building blocks of DNA for DNA replication), triggers a change
to the chromosome that prevents a late step in the initial formation of silent chromatin during S phase of the cell cycle (Kirchmaier & Rine, MCB, 2006). We have demonstrated that hydroxyurea inhibits a late step in silent chromatin formation that occurs after the Sir proteins form a blanket across the chromosome. We have also determined that this change to the chromosome that acts as an inhibitory signal is somehow linked to DNA replication; this inhibitory signal does not form on DNA that cannot undergo DNA replication. Also, this inhibitory signal only prevents the formation of new silent chromatin; it does not disrupt preexisting silent chromatin (Kirchmaier & Rine, MCB, 2006). We have also created a unique experimental system that has allowed us to analyze the biological role of the chemical reaction that is facilitated by one of the Sir proteins, the evolutionarily conserved enzyme Sir2p. We have identified a way to form the blanket of Sir proteins along the chromosome without
this chemical reaction occurring. During the course of this analysis, we also have provided evidence that the removal of additional chemical modifications from unknown proteins by Sir2p is critical for turning off gene expression after the blanket of Sir proteins forms. We also identified a way to both build the blanket of Sir proteins on the chromosome and shut off gene expression in the absence of the chemical reaction facilitated by Sir2p (Yang & Kirchmaier, In Press).
Impacts
We study the formation, maintenance and epigenetic inheritance of chromosome structures that affect gene expression and DNA replication. These processes are affected by environmental factors, are crucial during normal development of an organism and are often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate these effects requires an understanding of what events trigger epigenetic processes and what regulates their heritability.
Publications
- Kirchmaier AL* and Rine J. (2006). Cell Cycle Requirements in Assembling Silent Chromatin in Saccharomyces cerevisiae. Mol. Cell. Biol. 26:852-862. (*Corresponding author.)
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Progress 10/01/04 to 09/30/05
Outputs
We focus on 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 investigate the regulation of such epigenetic processes in two systems: in the budding yeast Saccharomyces cerevisiae and in human B cells infected with the human tumor virus, Epstein-Barr virus. Silenced chromatin in S. cerevisiae, is akin to heterochromatin in organisms including maize, flies, and mammals. S. cerevisiae epigenetically regulates several cellular activities including controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. We have been analyzing the role of the cell cycle in forming silent chromatin and have determined that all known protein components of silent chromatin are localized to discrete sites on the chromosome in G1 of the cell cycle prior to silencing gene expression at those sites. Similarly,
chromatin modifications that are known to occur in silent chromatin are present in G1 of the cell cycle. However, silencing of those sites will not occur until after cells pass through S phase of the cell cycle. Surprisingly, we have found that entry into S phase is sufficient to enable silencing of nonreplicated templates, whereas passage through S phase is required to silence replicated templates. This study is currently submitted for publication. We are now analyzing the role of DNA replication factors in establishing epigenetically inherited chromatin. Further analysis is ongoing. We study mechanisms that regulate the stably heritable repressed state of lytic viral genes during latency and the roles of host cellular proteins in this process. We have monitored viral and host gene expression and chromatin modifications by chromatin immunoprecipitation and real time PCR to learn how viral gene expression is regulated. Further analysis is ongoing.
Impacts
We study the formation, maintenance and epigenetic inheritance of chromosome structures that affect gene expression and DNA replication. These processes are affected by environmental factors, are crucial during normal development of an organism and often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate these effects requires an understanding of what events trigger epigenetic processes and what regulates their heritability.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/29/04
Outputs
The research in my lab focuses on 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 investigate the regulation of such epigenetic processes in two systems: the silent mating-type loci of budding yeast Saccharomyces cerevisiae and the life cycle of the human tumor virus, Epstein-Barr virus, EBV. Silencing in Saccharomyces cerevisiae Silenced chromatin in S. cerevisiae, is akin to heterochromatin in organisms such as maize, flies, and mammals. S. cerevisiae uses epigenetically inherited chromosomal structures to regulate a variety of cellular activities such as controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. We are developing a system to isolate biochemically a single chromosomal locus and all proteins associated with silent chromatin at this locus. To date, we now have completed
strain construction, have all steps in the purification process working separately. We have initiated combining the separate purification steps and scale-up procedures. We have also successfully optimized sample preparation for mass spec analysis. Further analysis is ongoing. Heritable Repression of Epstein-Barr Viral Gene Expression: We study mechanisms that regulate the stably heritable repressed state of lytic viral genes during latency and the roles of host cellular proteins in this process. To date, the renovation of laboratory space designated for our BL-2 tissue culture facilities within the Biochemistry Building was completed and we set up our tissue culture facilities. Cell lines and strains, bacterial strains, plasmid stocks, and other reagents were acquired and transferred to our own collection. We have subsequently designed, tested and optimized primers for real-time PCR for both gene expression and chromatin immunoprecipitation studies for open reading frames of every
latent gene, every latent viral promoter, immediate early viral gene promoters and open reading frames, the latent and lytic origins of replication and control loci in the host cell. We are currently analyzing by real-time PCR viral gene expression during latency and upon induction of the lytic cycle in EBV-infected cell lines and subclones that we have recently generated. We have also optimized conditions for chromatin immunoprecipitation experiments in mammalian cell lines infected with EBV. We are currently performing chromatin immunoprecipitation experiments to determine histone modifications present at these sites on the viral genome during latency and upon induction of the lytic cycle in multiple EBV-positive tumor cell lines. Further analysis is ongoing.
Impacts
We study the formation, maintenance and epigenetic inheritance of chromosome structures that affect gene expression and DNA replication. These processes are affected by environmental factors, are crucial during normal development of an organism and often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate these effects requires an understanding of what events trigger epigenetic processes and what regulates their heritability.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Since I arrived at Purdue University in August, 2002, the lab is now mostly set up and experiments are underway. My research program involves the genetic and biochemical analysis of epigenetic processes. In Saccharomyces cerevisiae, the silent mating-type loci, HML and HMR, are repressed by their assembly into heterochromatin. The formation of this heterochromatin requires a cell cycle event that occurs in S phase (Miller & Nasmyth, Nature 312:247, 1984). We found that this cell cycle-restricted event is neither the initiation of replication at the silencers nor the passage of the DNA replication fork through HMR (Kirchmaier & Rine, Science 291:646, 2001). We have identified discrete, separable stages in heterochromatin formation. These are the loading of Sir1 protein, the other Sir proteins on the silencer and the spreading of the Sir proteins throughout the silencer regions. We have demonstrated that spreading requires the deacetylase activity of Sir2p, an enzyme
which is conserved from Archae to humans (Rusche, Kirchmaier & Rine, Mol. Biol. Cell. 13:2207-2222, 2002). Remarkably, we have observed that the S phase requirement for silencing appears to act post-recruitment of Sir proteins. We are beginning both genetic and biochemical approaches to characterize the components of silent chromatin to better define this cell cycle regulated event. Since arriving at Purdue, I also co-wrote and published a comprehensive review of the progress in the silencing field over the past seven years designed to provide a thought-provoking critique of the current models for heterochromatin formation and function in Saccharomyces cerevisiae. (See below: Rusche, L.N., A.L. Kirchmaier and J. Rine, Annu. Rev. Biochem., 72:481-516, 2003.)
Impacts
We study the formation, maintenance and epigenetic inheritance of chromosome structures that affect gene expression and DNA replication. These processes are affected by environmental factors, are crucial during normal development of an organism and often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate these effects requires an understanding of what events trigger epigenetic processes and what regulates their heritability.
Publications
- Laura N. Rusche, Ann L. Kirchmaier and Jasper Rine. The Establishment, Inheritance, and Function of Silenced Chromatin in Saccharomyces cerevisiae. Annu. Rev. Biochem., 72:481-516, 2003.
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Progress 08/01/02 to 10/31/02
Outputs
I recently arrived at Purdue University (August, 2002). I am in the process of setting up a research laboratory and establishing my research program that involves both the genetic and biochemical analysis of heterochromatin formation using the model organism Saccharomyces cerevisiae. In Saccharomyces cerevisiae, the silent mating-type loci, HML and HMR, are repressed by their assembly into heterochromatin. The formation of this heterochromatin requires a cell cycle event that occurs in S phase (Miller & Nasmyth, Nature 312:247, 1984). We found that this cell cycle-restricted event is neither the initiation of replication at the silencers nor the passage of the DNA replication fork through HMR (Kirchmaier & Rine, Science 291:646, 2001). We have identified discrete, separable stages in heterochromatin formation. These are the loading of Sir1 protein, the other Sir proteins on the silencer and the spreading of the Sir proteins throughout the silencer regions. We have
demonstrated that spreading requires the deacetylase activity of Sir2p, an enzyme which is conserved from Archae to humans (Rusche, Kirchmaier & Rine, Mol. Biol. Cell. 13:2207-2222, 2002). Remarkably, we have observed that the S phase requirement for silencing appears to act post-recruitment of Sir proteins. Analysis of the S phase requirement for silencing is ongoing in my laboratory. Since arriving at Purdue, I also co-wrote and submitted the manuscript "The Establishment, Inheritance and Function of Silenced Chromatin in Saccharomyces cerevisiae." Rusche, L.N., A.L. Kirchmaier and J. Rine to the Annual Review of Biochemistry. This is a comprehensive review of the progress in the silencing field over the past seven years designed to provide a thought-provoking critique of the current models for heterochromatin formation and function in Saccharomyces cerevisiae.
Impacts
We study the formation, maintenance and epigenetic inheritance of chromosome structures that affect gene expression and DNA replication. These processes are affected by environmental factors, are crucial during normal development of an organism and often perturbed in cancer. Understanding how these processes occur is critical when considering their effects on human and animal health or on the value of agricultural products. Future development of therapeutic or industrial strategies designed to modulate these effects requires an understanding of what events trigger epigenetic processes and what regulates their heritability.
Publications
- No publications reported this period
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