THE ANALYSIS OF THE MCF7 CANCER MODEL SYSTEM AND THE EFFECTS OF 5-AZA-2’-DEOXYCYTIDINE TREATMENT ON THE CHROMATIN STATE USING A NOVEL MICROARRAY-BASED TECHNOLOGY FOR HIGH RESOLUTION GLOBAL CHROMATIN STATE MEASUREMENT APPROVED BY SUPERVISORY COMMITTEE Harold Garner, Ph.D. Elliott Ross, Ph.D. Thomas Kodadek, Ph.D. John Minna, M.D. Keith Wharton, M.D., Ph.D. DEDICATION Omnibus qui adiuverunt THE ANALYSIS OF THE MCF7 CANCER MODEL SYSTEM AND THE EFFECTS OF 5-AZA-2’-DEOXYCYTIDINE TREATMENT ON THE CHROMATIN STATE USING A NOVEL MICROARRAY-BASED TECHNOLOGY FOR HIGH RESOLUTION GLOBAL CHROMATIN STATE MEASUREMENT by MICHAEL RYAN WEIL DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas March, 2006 Copyright by Michael Ryan Weil All Rights Reserved THE ANALYSIS OF THE MCF7 CANCER MODEL SYSTEM AND THE EFFECTS OF 5-AZA-2’-DEOXYCYTIDINE TREATMENT ON THE CHROMATIN STATE USING A NOVEL MICROARRAY-BASED TECHNOLOGY FOR HIGH RESOLUTION GLOBAL CHROMATIN STATE MEASUREMENT Publication No. MICHAEL RYAN WEIL, B.S. The University of Texas Southwestern Medical Center at Dallas, 2006 Supervising Professor: Harold Ray (Skip) Garner, Ph.D. A microarray method to measure the global chromatin state of the human genome was developed in order to provide a novel view of gene regulation. The 'chromatin array' employs traditional methods of chromatin isolation, microarray technology, and advanced data analysis, and was applied to a cancer model system. Chromatin is first separated by its condensation state using chromatin fractionation. By probing with a comparative genomic hybridization-style microarray, the chromatin condensation state of thousands of individual loci in an MCF7 tumor model cell line was determined and correlated with transcriptional activity. The chromatin array showed a significant portion (>3,000) of the genes were in a condensation state that was neither condensed or relaxed as a result of heterogeneity in the v condensation states in the population. The utility of the chromatin array in deciphering gene regulation was demonstrated in a MCF7 cell line treated with 5 Aza dC, which disrupts genome methylation, and as a result causes global relaxation of chromatin structure. 5 Aza dC treatment results in strong changes in expression, and a normalized global chromatin relaxation of two-fold. A significant subset of 378 genes was condensed by 5 Aza dC treatment, indicating that a mechanism of chromatin regulation exists that can resist the effects of 5 Aza dC treatment. The genes with the largest changes in response to 5 Aza dC treatment showed a strong correlation with CpG island-based regulation (p < 0.0001), and a restoration of transcription patterns associated with normal mammary tissue. Analysis using splice-form specific microarray probes demonstrated that the chromatin state was not uniform across a gene. These findings indicate that certain gene regions exhibit differential sensitivity to 5 Aza dC treatment, and therefore may be regulated independently. Using functional annotation, expression microarray, and comparative genomic hybridization data, this work should provide a framework through which the biological implications of the relationship between chromatin accessibility and expression may be deciphered. vi TABLE OF CONTENTS Chapter One: Background 1-1 Introduction .............................................................................................................. 20 1-2 The basics of chromatin............................................................................................ 22 1-2-1 The history of chromatin research: structural versus ...................................... 24 1-2-2 Modern chromatin research: The unification of structure and function.......... 26 1-2-3 Chromatin ImmunoPrecipitation..................................................................... 29 1-3 Microarray technology ............................................................................................. 30 1-3-1 A history of transcriptomics............................................................................ 30 1-3-2 A history of microarray technology ................................................................ 32 1-3-3 Spotted versus light-directed synthesis ............................................................35 1-3-4 Expression microarray methodology ...............................................................38 1-3-5 Microarray-based comparative genomic hybridization ..................................42 1-3-6 Chromatin immunoprecipitation on microarrays .............................................44 1-4 Thesis and organization.............................................................................................47 1-5 References ................................................................................................................48 Chapter Two: Development of techniques for robust hybridization of human genomic DNA to cDNA microarrays 2-1 Specificity and sensitivity of cDNA versus BAC microarrays .................................54 2-2 Measurement of human genomic DNA coverage on cDNA microarrays.................56 2-3 Refinements to the high-resolution aCGH method ...................................................61 2-4 Materials and methods...............................................................................................64 vii 2-4-1 Genomic DNA Labeling ..................................................................................64 2-4-2 RNA Labeling ..................................................................................................65 2-4-3 Microarray Production .....................................................................................65 2-4-4 Microarray Clean Up and Hybridization..........................................................66 2-4-5 Washing and Scanning.....................................................................................66 2-4-6 Analysis............................................................................................................67 2-5 References ................................................................................................................68 Chapter Three: Development of a microarray-based assay to measure chromatin condensation state at a subgene resolution 3-1 Development of methods to recover chromatin by its condensation state in preparation for hybridization ...........................................................................................70 3-1-1 Fragment Length Selection .............................................................................71 3-1-2 Chromatin Fractionation .................................................................................74 3-2 Validation of the microarray-based method ..............................................................77 3-2-1 Validation by traditional methods of chromatin state measurement................77 3-2-2 Validation of the chromatin array method .......................................................78 3-2-3 Cross validation of the two chromatin state fractionation techniques .............82 3-3 Insight into transcriptional regulation revealed by global high resolution chromatin studies ..............................................................................................................................83 3-3-1 Combining chromatin array and expression data increases the robustness of the functional classification .............................................................................................84 viii 3-3-2 Novel information provided by high-throughput global chromatin state measurements of synchronized cell culture to resolve heterogeneity issues .............87 3-3-3 Novel information resulting from chromatin state measurements of genes with expression state too low for microarray-based measurement....................................89 3-3-4 The chromatin state data provides significant insight into the relationship between chromosomal alteration and transcription ...................................................91 3-4 Materials and methods...............................................................................................94 3-4-1 Cell culture .......................................................................................................94 3-4-2 Cell cycle synchronization by serum starvation and population analysis........95 3-4-3 DNase I digestion and fragment length selection.............................................95 3-4-4 Micrococcal nuclease digestion and the chromatin fractionation assay ..........96 3-4-5 Microarray characteristics................................................................................96 3-4-6 DNA labeling ...................................................................................................97 3-4-7 Chromatin hybridization ..................................................................................97 3-4-8 RNA extraction and hybridization ...................................................................98 3-4-9 Data extraction, normalization and analysis ....................................................98 3-4-10 Data interpretation..........................................................................................99 3-5 References ...............................................................................................................100