Genome-wide Analysis of the DNA Topoisomerase II and its Relationship to the CP190 Insulator Protein Público

Torre, Eduardo Andres (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/0g354f302?locale=pt-BR
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Abstract

Abstract


Topoisomerase II (Topo II) is an enzyme known to reduce DNA super-coiling by
introducing negative coils in the double helix. It is involved in processes such as
chromosome segregation, condensation, homologous recombination, DNA replication,
and DNA transcription,. Chromatin immunoprecipitation (ChIP) followed by microarray
analysis (ChIP-chip) experiments were done to determine the genome-wide binding
pattern of Topo II. This study shows that Topo II binds to genomic intervals suggesting
that it may bind DNA in a structure- specific rather than in a sequence-specific manner.
In addition, I show that Centrosomal Protein 190 (CP190), a protein essential for the
proper function of most known Drosophila insulators, delimits the distribution of this
enzyme and that RNA interference- (RNAi) mediated knock down of CP190 leads to a
change in the distribution pattern of Topo II. These data suggest that insulators may play
an essential role in fine-tuning the distribution of Topo II and cells may use the controlled
recruitment of CP190 as a means of regulating Topo II.







Genome-wide Analysis of the DNA Topoisomerase II and its Relationship to the
CP190 Insulator Protein

By

Eduardo A. Torre
Advisor: Victor G. Corces, PhD
A thesis submitted to the Faculty of the James T. Laney School of Graduate Studies of
Emory University in partial fulfillment of the requirements for the degree of Master of
Science in Biology
2011




Table of Contents


TABLE OF CONTENTS

INTRODUCTION
1
MATERIALS AND METHODS
9
ANTIBODIES
10
ChIP-chip ANALYSIS
10
REAL-TIME PCR ANALYSIS
11
RNA INTERFERENCE
11
PEAK ANALYSIS
12
RESULTS
13
TOPO II OPERATES ON GENOMIC INTERVALS
14
TOPO II DISTRIBUTION IS DELIMITED BY CP190
19
LOSS OF CP190 LEADS TO A CHANGE IN TOPO II DISTRIBUTION
24
THE BEHAVIOR OF TOPO II AT CP190 SITES IS NOT THE SAME IN ALL INSULATOR SUBGROUPS
28
DISCUSSION
33
REFERENCES
41

TABLE OF IMAGES


FIGURE 1 - GENOME-WIDE LOCALIZATION OF TOPO II..........................................................................16
FIGURE 2 - STATISTICAL ANALYSIS OF TOPO II RAW DATA USING CMARRT ......................................18
FIGURE 3 - TOPOII IS FLANKED AND INTERRUPTED BY CP190 ............................................................21
FIGURE 4 - COHESIN LOCALIZATION AROUND TOPO II AND CP190 .....................................................23
FIGURE 5 - CP190 KNOCK DOWN............................................................................................................25
FIGURE 6 - TOPO II ChIP-chip ON CP190 KNOCK DOWN ....................................................................26
FIGURE 7 - DISTRIBUTION OF TOPO II AROUND CP190 AFTER CP190 RNAI ...................................27
FIGURE 8 - VENN DIAGRAM OF THE DIFFERENT CP190 SUBGROUPS....................................................30
FIGURE 9 - TOPO II DISTRIBUTION AROUND DIFFERENT CP190 SUBGROUPS.....................................31
TABLE 1 - qPCR VERIFICATION OF ChIP-chip ......................................................................................17

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