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«by Swathi Krishnan A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biological Chemistry) ...»

-- [ Page 1 ] --

STRUCTURAL AND BIOCHEMICAL INSIGHTS INTO METHYLATION SITE AND

STATE SPECIFICITY OF JMJD2 LYSINE DEMETHYLASES

by

Swathi Krishnan

A dissertation submitted in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

(Biological Chemistry)

in the University of Michigan

Doctoral Committee:

Associate Professor Raymond C. Trievel, Chair

Professor Carol A. Fierke Assistant Professor Georgios Skiniotis Professor John J. G. Tesmer Associate Professor Xiaochun Yu © Swathi Krishnan 2012 To my family ii

ACKNOWLEDGEMENTS

It has been a valuable learning experience for me at the University of Michigan, Ann Arbor. Firstly, I thank Dr. Ray Trievel for giving me an opportunity to work in his lab. Ray has been helpful in many ways and been very generous with many expensive reagents and peptides that have been instrumental in my thesis research. I also thank him for encouraging me to apply for various grants and awards, writing review articles and reviewing manuscripts and helping me become a well-rounded researcher. I thank my thesis committee members for providing valuable suggestions and discussions and for making it all the way to the MSRB buildings for my 9am meetings.

I thank Paul, for helping me with almost everything in the lab and for all the great conversations and Stacie, who taught me crystallography when I knew nothing about it, and helped me get my driver’s license! Thanks to Kim for being a vibrant presence in the lab and always encouraging me to smile through tough times. I also thank the Biochem students and staff, particularly, Beth for all the help and support.

I would not be the person I am today without my mom and dad and I am immensely grateful to them for being my source of strength even when they were 9000 miles away. Thanks Preethi for being my moral support, friend and confidante for the past 25 years! You are the best sister anyone can ask for. Lastly, but by no means the least, I thank Rozario who, made me fall in love with New York City and him, showed me how baseball can be fun and changed my life in the most wonderful ways possible. As long as I am with you, I will consider myself the luckiest person in the world.

iii

TABLE OF CONTENTS

DEDICATION

ACKNOWLEDGEMENTS

LIST OF FIGURES

LIST OF TABLES

LIST OF ABBREVIATIONS

Abstract

CHAPTER 1: HISTONE LYSINE DEMETHYLASES

Histone Modifications and Regulation of Gene Expression

Biological Functions of Histone Lysine Methylation

Mechanisms of Histone Lysine Demethylation

JMJD2 KDMs in Biology and Disease

JMJD2 KDMs as Drug Targets

Methylation Site and State Specificities in the JMJD2 KDMs

Objectives of This work

REFERENCES

CHAPTER 2: OPTIMIZATION OF THE FORMALDEHYDE DEHYDROGENASECOUPLED LYSINE DEMETHYLASE ASSAY FOR JUMONJI ENZYMES.................21 The Formaldehyde Dehydrogenase (FDH) Coupled Demethylase Assay...............21 Inhibition of JmjC KDMs by Transition State Metals

MATERIALS AND METHODS

Expression and Purification of Strep(II)-Tagged JMJD2A and JMJD2D................24 Expression and Purification of Recombinant Formaldehyde Dehydrogenase.........25 Substrate Histone Peptides

Determination of Metal Content of JMJD2A and JMJD2D

Reagents for the FDH- Coupled Demethylase Assay

Setting up the FDH- Coupled Demethylase Assay

NADH Calibration Assay

Determination of Kinetic Parameters

RESULTS

Purity of Recombinant Formaldehyde Dehydrogenase

Purity of Strep(II)-Tagged JMJD2A and JMJD2D

Metal Content Analysis of Strep(II)-tagged JMJD2A and JMJD2D

Kinetic Analysis

Use of Methyllysine Analog Bearing Substrates

DISCUSSION

ivACKNOWLEDGEMENTS

CHAPTER 3: METHYLATION SITE SPECIFICITY STUDIES-CRYSTAL

STRUCTURE OF HUMAN JMJD2D

MATERIALS AND METHODS

Cloning, Expression and Purification of His-JMJD2D

Crystallization of JMJD2D•2-OG•H3K9me3 Ternary Complex

Optimization of Crystals by Surface Entropy Reduction

X-Ray Diffraction Data Collection, Processing and Structure Determination.........62 RESULTS

Structure of JMJD2D Apoenzyme

Structure of the JMJD2D•2-OG•H3K9me3 Complex

Interactions of JMJD2D with Substrate H3K9me3

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

CHAPTER 4: METHYLATION SITE SPECIFICITY STUDIES-DIFFERENTIAL

SPECIFICITY OF JMJD2D AND JMJD2A

MATERIALS AND METHODS

Protein Expression and Purification

Histone Substrate Peptides

FDH-Coupled Demethylase Assay

Molecular Docking of JMJD2D and H3K36me3

RESULTS

Comparison of H3K9me3 Recognition by JMJD2D and JMJD2A

Role of H3T11ph in the Recognition of H3K9me3 by JMJD2 KDMs

Mode of H3K36me3 Occlusion by JMJD2D

Activity of JMJD2D and JMJD2A toward hybrid peptides

DISCUSSION

Mode of H3K9me3 recognition and H3K36me3 occlusion by JMJD2 KDMs......115 Comparing the JMJD2 and UTX Enzyme Families





ACKNOWLEDGEMENTS

REFERENCES

CHAPTER 5: SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS................123 Methods for Purifying and Assaying Fe(II)-dependent Dioxygenases

Structural Basis for Methylation Site Specificity in JMJD2 KDMs

Separation of H3K9 and H3K36 Site Specificities of JMJD2 KDMs

Implications of this Study in Drug Design and Therapy

REFERENCES

APPENDIX A: METHYLATION STATE SPECIFICITY STUDIES USING JMJD2A

AND JMJD2D

MATERIALS AND METHODS

v Cloning, Expression and Purification of JMJD2A and JMJD2D Mutants.............137 Incorporation of para-Aminophenylalanine (pAF) in JMJD2A

Crystallization and Structure Determination of JMJD2A _Y177pAF

Histone Substrate Peptides and FDH-Coupled Demethylase Assay

RESULTS

Active Site Residues Determine Methylation State Specificity

JMJD2A_Y177pAF is Catalytically Inactive

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

–  –  –

Figure 1.1 Crystal Structure of a Nucleosome Core Particle

Figure 1.2 Catalytic Mechanism of JmjC Lysine Demethylases

Figure 1.3 Differential Methylation Site and State Specificity in JmjC KDMs

Figure 1.4 Domain architecture of the human JMJD2 enzymes

Figure 2.1 Schematic of the FDH-coupled Demethylase Assay

Figure 2.2 Strep-Tactin purification of Strep(II)-tagged JMJD2A

Figure 2.3 Gel Filtration Purification of JMJD2A

Figure 2.4 Purification of recombinant FDH on S200 column

Figure 2.5 FDH samples prepared with excess Dithiothreitol (DTT)

Figure 2.6 Optimization of 2-OG concentration in the coupled demethylase assay.

.................32 Figure 2.7 Optimization of the FDH concentration in the coupled demethylase assay..............33 Figure 2.8 NADH calibration curve

Figure 2.9 Linearity between JMJD2 enzyme concentration and initial velocity

Figure 2.10 Kinetic analysis of JMJD2A and H3K9me3

Figure 2.11 Kinetic analysis of JMJD2D and H3K9me3

Figure 2.12 Schematic of site-specific installation of methyllysine analogs in histones.

...........48 Figure 2.13 Kinetic analysis of JMJD2A and H3K9Cme3

Figure 3.1 Sequence alignment of the JMJD2 family of KDMs

Figure 3.2 Ni(II) column purification of JMJD2D

Figure 3.3 S200 purification of JMJD2D

vii Figure 3.4 Crystals of the JMJD2D•2-OG•H3K9me3 ternary complex

Figure 3.5 Crystals of JMJD2D apoenzyme (K93A/K94A)

Figure 3.6 Structure of JMJD2D apoenzyme at 2.

5 Å resolution

Figure 3.7 Structural conservation among JMJD2 KDMs

Figure 3.8 Structure of the JMJD2D•2-OG•H3K9me3 ternary complex at 1.

8 Å resolution....69 Figure 3.9 Simulated annealing omit map of 2-OG and Ni(II)

Figure 3.10 Simulated annealing omit map of the H3K9me3 peptide

Figure 3.11 Comparison of the JMJD2D•2-OG•H3K9me3 structure and 3DXT

Figure 3.12 Crystal contacts in 3DXT

Figure 3.13 Steric clashes in the 3DXT structure

Figure 3.14 Recognition of the H3K9me3 peptide by JMJD2D

Figure 4.1 Recognition of R8 in the H3K9me3 substrate

Figure 4.2 Conservation of R8-recognizing residues

Figure 4.3 Recognition of S10 and T11 in the H3K9me3 substrate

Figure 4.4 Activity of JMJD2A and JMJD2D toward an H3K9me3_T11S peptide.

.................94 Figure 4.5 Activity of JMJD2 enzymes for an H3K9me3T11ph peptide

Figure 4.6 T11ph modeled in the JMJD2D peptide binding cleft

Figure 4.7 JMJD2D docked with the H3K36me3 peptide

Figure 4.8 Occlusion of H39 and R40 by JMJD2D

Figure 4.9 Favorable recognition of H39 and R40 by JMJD2A

Figure 4.10 Activity of JMJD2A for an H3K36me3_R40A peptide

Figure 4.11 Kinetic Analysis of JMJD2A_I71L

Figure 4.12 Electrostatic surface representations of JMJD2D and JMJD2A

–  –  –

Figure 4.14 Hybrid peptides between H3K9me3 and H3K36me3

Figure 4.15 Activity of JMJD2D toward hybrid peptides

Figure 4.16 Rationale for the H3K36K9_V35R substrate

Figure 4.17 Activity of JMJD2A toward hybrid peptides

Figure 4.18 Similarities in the H3K9me3 and H1.

4K26me3 sites

Figure 4.19 Divergence of residues involved in H3K36me3 discrimination

Figure 4.20 Structural alignment of the ternary complexes of JMJD2D and UTX.

.................120 Figure A.1 Expression analysis of JMJD2A_Y177pAF

Figure A.2 Crystals of JMJD2A_Y177pAF obtained by streak seeding

Figure A.3 Comparison of active sites of JMJD2D and JMJD2A

Figure A.4 Activity of JMJD2A, JMJD2D and their state specificity mutants

Figure A.5 Alignment of the JMJD2A_WT and JMJD2A_Y177pAF structures

–  –  –

Table 1.1 Functions of Histone H3 Lysine Methylation

Table 1.2 Biological functions, disease implications and specificity of JMJD2 KDMs.

...........12 Table 2.1 Step-wise protocol for performing the FDH-coupled demethylase assay..................35 Table 2.2 Step-wise protocol for performing the NADH calibration assay

Table 2.3 Transition state metal content analysis of JMJD2A and JMJD2D

Table 2.4 Kinetic constants of replicate purifications of Strep-Tactin column purified JMJD2A and JMJD2D

Table 3.1 Crystallographic Data and Refinement Statistics of JMJD2D

Table 4.1 List of the H3 peptides used in the kinetic analysis of JMJD2D and JMJD2A.

.........86 Table 4.2 Kinetic Characterization of JMJD2D and JMJD2A with mutant peptides.................89 Table 4.3 Kinetic Analysis of JMJD2 KDMs

Table 4.4 Comparison of the kinetic parameters of JMJD2A_WT and JMJD2A_I71L.

........105 Table 4.5 List of mutations in JMJD2D that were tested for H3K36me3 catalytic activity.....109 Table 5.1 Mutations in JMJD2A that can potentially enable separation of specificity............129 Table A.1 Primer sequences for Site-Directed Mutagenesis of JMJD2A and JMJD2D..........138 Table A.2 Crystallographic Data and Refinement Statistics of JMJD2A_Y177pAF...............142 Table A.3 Kinetic analysis of JMJD2A, JMJD2D, JMJD2A_S288A and JMJD2D_A292S with H3K9me2 and H3K9me3

–  –  –

2-OG, 2-oxoglutarate AR, androgen receptor BSA, bovine serum albumin ChIP, chromatin immunoprecipitation DTT, dithiothreitol EDTA, ethylenediaminetetraacetic acid ERα, estrogen receptor α FAD, flavin adenine dinucleotide FDH, formaldehyde dehydrogenase FPLC, fast performance liquid chromatography HP1, heterochromatin protein 1 HPLC, high-performance liquid chromatography IC50, apparent inhibitor constant ICP-HRMS, inductively coupled plasma-high resolution mass spectroscopy Il12b, interleukin 12b IMAC, immobilized metal affinity chromatography ING, inhibitor of growth IPTG, isopropyl β-D-1-thiogalactopyranoside JmjC, jumonji C

–  –  –

KMT, lysine methyltransferase LSD1, lysine specific demethylase Mdc, macrophage-derived chemokine MLL, mixed lineage leukemia NAD+, nicotinamide adenine dinucleotide NADH, nicotinamide adenine dinucleotide-reduced NOG, N-oxalylyglycine Oct4, octamer-binding transcription factor 4 pAF, para-aminophenylalanine PEG, polyethylene glycol PHD, plant homeodomain PSA, prostate specific antigen RFU, relative fluorescence units RMSD, root mean square deviation SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis SER, surface entropy reduction TEV, tobacco etch virus TFF1, trefoil factor 1 TSS, transcription start site WT, wildtype

–  –  –



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