Home

Swathi Krishnan, Ph.D.

Mentor: Ray Trievel

swathik@umich.edu

Thesis Defense: November 2, 2012
Structural and Biochemical Insights into Methylation Site and State Specificity of JMJD2 Lysine Demethylases

Thesis Abstract:

The human JMJD2/KDM4 family of histone lysine demethylases comprises four homologs: JMJD2A, JMJD2B, JMJD2C and JMJD2D. These enzymes have been implicated in a number of biological processes such as transcriptional activation, development and cell cycle control. The biological functions of these enzymes are defined by their distinct methylation site and state specificities. JMJD2A, JMJD2B and JMJD2C display dual specificity for trimethylated histone H3 Lys9 and Lys36 (H3K9me3 and H3K36me3), whereas JMJD2D is specific for H3K9me3. Furthermore, while most JMJD2 homologs are predominantly trimethyllysine-specific, JMJD2D can demethylate both tri- and dimethyllysines. To enable quantitative kinetic studies of JMJD2 demethylases, we developed and applied a new affinity purification protocol that minimizes contamination by transition state metals. In order to delineate the molecular basis of site and state specific demethylation by the JMJD2 enzymes, we determined the first crystal structure of JMJD2D in the apoenzyme form and in a ternary complex with 2-OG and an H3K9me3 peptide. Our site specificity studies with JMJD2A and JMJD2D revealed surprising differences in H3K9me3 recognition by these enzymes despite the overall similarity in the substrate binding conformation. In addition, docking studies with H3K36me3 and biochemical analysis with histone H3 hybrid peptides underscored the role of steric clashes, electrostatic clashes and loss of productive hydrogen bonds in occluding the recognition of the H3K36me3 site by JMJD2D. Our structural and biochemical analysis of the active site also revealed the basis for differential state specificity in the JMJD2 enzymes and highlighted the role of CH•••O hydrogen bonds in di- and trimethyllysine substrate recognition. Together, these structural and biochemical studies elucidate the molecular basis of different substrate specificities within the JMJD2 family, which is not only key to understanding their distinct biological functions but will also aid in the structure-based design of selective inhibitors of JMJD2 enzymes implicated in disease.

Most recent position: Postdoctoral Fellow, New York University School of Medicine

Undergraduate Institution: Anna University



Research Profile

My research focuses on the human histone lysine demethylases of the JMJD2 family. I am currently working on resolving the crystal structure of JMJD2C and JMJD2D. The other part of my research is to develop physiologically relevant substrates to use them in demethylase assays and understand the kinetic mechanisms of these enzymes. In order to develop methylated histone substrates, I have been installing methyl lysine analogs in the desired position using an established protocol [1]. To this end, I have prepared Xenopus Histone H3K9 and H3K36 trimethylated substrates which, show at least 20 times more activity in the demethylase assays than their peptide counterparts. I am currently working on developing H3-H4 tetramers bearing these methyl-lysine analogs.

References:

1. Simon, M.D. et.al., The Site-Specific Installation of Methyl-Lysine Analogs into Recombinant Histones , Cell 2007

Presentations

30th Summer Symposium in Molecular Biology: Chromatin and Epigenetic Regulation of Transcription. (June 21-24, 2011) Pennsylvania State University, State College, PA. Poster presentation, "Purification and assay of Jumonji C lysine demethylases"

Awards

2013 The Dziewiatkowski Award (Most Outstanding Ph.D. Thesis)
2012 Lee Murphy Memorial Prize (Most Significant Paper or Series of Papers)
2012 Rackham Predoctoral Fellowship
2011 Rackham Travel Grant
2011 Anthony and Lillian Lu Award, (Academic Achievement and Potential as a Scientist)
2010 Rackham Graduate Student Research Grant (Candidate)
2009 Rackham Graduate Student Research Grant (Pre-Candidate)