Source: PURDUE UNIVERSITY submitted to
BIOCHEMICAL ANALYSIS OF HISTONE METHYLTRANSFERASES AND DEMETHYLASES
 
PROJECT DIRECTOR: Briggs, S. D.
 
PERFORMING ORGANIZATION
Biochemistry
PURDUE UNIVERSITY
WEST LAFAYETTE,IN 47907
 
NON TECHNICAL SUMMARY: Our research on histone methyltransferases and demethylases are important because these factors help control how genes are turned on and off. The study of how genes are turned on and off is what we call regulation of gene expression. Because the histone methyltransferases/demethylases and histone modifications that we study are found in yeast, humans, animals, insects and plants, our genetic and biochemical studies in yeast will allow us to understand how human, animal, insects and plant cells turn on and off genes. How gene expression is regulated is vital to understanding human health and agriculture production. In regards to human health, inappropriate gene expression can result in human developmental defects and cancers such as acute myeloid leukemia, breast cancer and multiple myeloma. For agriculture purposes, our understanding of how genes are regulated will likely impact crop and livestock production as well as controlling pathogenic yeast or other insects that can either infect animals or damage plants.
 
OBJECTIVES: The major objective of this research is to further our understanding of the biochemical function of the histone H3 Lysine 4 (H3K4) methyltransferase Set1 and the histone H3K4 demethylase Jhd2. Because histone methyltransferases and demethylases and site of histone methylation is conserved in animals and plants, our studies will provide us a better understanding of how eukaryotic genomes function as well as key insights into human health and agricultural production. To understand how Set1 and Jhd2 mediate histone H3K4 methylation, we will: A. Identify and characterize novel protein motifs and domains within Set1-complex members that are required for proper histone H3K4 methylation and gene expression. B. Define how Set1 associates with RNA Polymerase II and determine if Jhd2 binds to RNA Polymerase II. C. Determine if binding of Jhd2 to the H3 N-terminal tail facilitates histone H3K4 demethylation. Overall, these studies will likely provide insight that will impact human health and agriculture production (crops and livestock).
 
APPROACH: To experimentally address our objectives, we will use a combination of genetic, biochemical, and molecular approaches using budding yeast, Saccharomyces cerevisiae, as the main model system. These experiments will help further our biochemical understanding of how histone methyltransferases and demethylases function. To address objective A, we will use yeast genetics, Western blot analysis, chromatin immunoprecipitation analysis and quantitative real-time PCR analysis to characterize novel protein motifs and domains that are required for proper histone H3K4 methylation and gene expression. To address objective B, we will use coimmunoprecipitation and Western blot analysis and in vitro binding studies to determine if Set1 and/or Jhd2 associate with RNA Polymerase II. To address objective C, we will use in vitro GST-pulldown assays to determine an association between Jhd2 and histone H3.
 
CRIS NUMBER: 0198869 SUBFILE: CRIS
PROJECT NUMBER: IND011075 SPONSOR AGENCY: NIFA
PROJECT TYPE: HATCH PROJECT STATUS: REVISED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2010 TERMINATION DATE: Sep 30, 2015

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
201402010002.234%
201402010402.233%
201402010802.233%

CLASSIFICATION HEADINGS
KA201 - Plant Genome, Genetics, and Genetic Mechanisms
S4020 - Fungi
F1080 - Genetics
F1000 - Biochemistry and biophysics
F1040 - Molecular biology
G2.2 - Increase Efficiency of Production and Marketing Systems


RESEARCH EFFORT CATEGORIES
BASIC 100%
APPLIED (N/A)%
DEVELOPMENTAL (N/A)%

KEYWORDS: chromatin~gene expression~cancer~histone~methylation~demethylases~methyltransferases~human disease~agriculture production~livestock production~plant development~pathogenic yeast

PROGRESS: Oct 1, 2011 TO Sep 30, 2012
OUTPUTS: The main focus of my laboratory is to understand how histone methylation and demethylation occurs and regulates gene expression. Histone methyltransferases and demethylases are highly conserved in yeast, plants, animals and humans. Several human histone methyltransferases and demethylases been mutated or overexpressed in human cancers suggesting they play a key role in the proliferation of cancers cells. In S. cerevisiae, the Set1 histone H3K4 methyltransferase forms a high molecular weight protein complex and this complex is conserved in plants and animals. The key subunits that associate with Set1 are Bre2, Sdc1, Spp1, Swd1, Swd3, Swd2, and Shg1. In a recently published study, we defined for the first time how two subunits of the Set1 histone methyltransferase complex interact. We determined that the C-terminus of Bre2 contains a critical protein-protein interaction domain that binds to the Dpy-30 domain of Sdc1. We also determined that the human homologs of Bre2 and Sdc1, ASH2L and DPY-30, respectively, interact in a similar manner, suggesting this interaction is maintained from yeast to humans. Therefore, this novel protein-protein interaction domain in Bre2 and ASH2L was defined as the Sdc1 Dpy-30 Interaction (SDI). More importantly, we determined that this interaction is necessary for proper histone methylation and gene expression. We have recently identified a new protein-protein interaction involving a charge-based interaction between Set1 and Swd1. This charge-based interaction is critical for histone methylation, protein stability of Set1 and gene expression. Future work is to determine how the various Set1 complex subunits interact with one another, to define the precise determinants for protein-protein interactions, and to understand how these interactions impact gene expression. PARTICIPANTS: The people who work on this project are: Scott D. Briggs - Principle Investigator Hai-Ning Du - Postdoctoral Fellow Paul South - Graduate student Past members Ian Fingerman - Postdoctoral Fellow, obtained another position Douglas Mersman - Graduate student, Graduated TARGET AUDIENCES: Biochemists working on histone methylation PROJECT MODIFICATIONS: Not relevant to this project.

IMPACT: 2011-10-01 TO 2012-09-30 Although much attention has been given to understanding DNA as our genetic material, an emerging area of research is epigenetic gene regulation. Epigenetics is the process by which gene expression can stably change without altering the DNA sequence. Furthermore, any disruption of the normal epigenetic process can result in aberrant gene expression and human disease. One of the key mechanisms used to properly maintain and control proper epigenetic gene expression (activation or repression) in a cell is histone lysine methylation. Histone methylation is mediated by lysine methyltransferases and is removed by histone demethylases. Histone methyltransferases and demethylases are conserved in eukaryotic species ranging from yeast to humans and are important because many have been found to be mutated or over produced in human cancers such as leukemia, multiple myeloma, prostate, and breast. Furthermore, it is believed that altered histone methylation is one of the key contributing factors in these cancers leading to aberrant changes in gene expression. Therefore, understanding how histone methyltransferases and demethylases biochemically and biologically function will provide us with a better understanding of how eukaryotic genomes are epigenetically regulated and how mis-regulation of these modifying activities can lead to human, animal, and plant diseases.

PUBLICATION INFORMATION: 2011-10-01 TO 2012-09-30
South and Briggs, S.D. Understanding the Structure and Function of ASH2L. Atlas of Genetics and Cytogenetics in Oncology and Haematology: URL: http://atlasgeneticsoncology.org//Deep/ASH2LFunctionID20097.html, June 2011.

PROJECT CONTACT INFORMATION
NAME: Briggs, S. D.
PHONE: 765-494-0112
FAX: 765-497-7897