IDENTIFICATION OF A NOVEL, RAPID MECHANISM TO ALLEVIATE THE NUCLEOSOME ...

244 Pages·2012·14.67 MB·English

IDENTIFICATION OF A NOVEL, RAPID MECHANISM TO ALLEVIATE
THE NUCLEOSOME BARRIER TO TRANSCRIPTION

A Dissertation
Presented to the Faculty of the Graduate School
of Cornell University
In Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy

by
Steven Joseph Petesch
May 2012
© 2012 Steven Joseph Petesch
IDENTIFICATION OF A NOVEL, RAPID MECHANISM TO ALLEVIATE THE
NUCLEOSOME BARRIER TO TRANSCRIPTION

Steven Joseph Petesch, Ph. D.
Cornell University 2012

To efficiently transcribe genes, RNA Polymerase II (Pol II) must overcome
the barrier imposed by nucleosomes and higher order chromatin structure.
Many genes, including Drosophila melanogaster Hsp70, undergo changes in
chromatin structure upon activation. It has long been thought that changes to
chromatin structure occur co-transcriptionally as a result of Pol II movement
through the gene and recruitment of Pol II associated factors that disrupt
chromatin structure. In this dissertation, I demonstrate that, upon activation,
the changes in chromatin structure of Drosophila melanogaster’s Hsp70 gene
occur in an extremely rapid manner that is independent of active transcription
of the gene. In addition, these changes extend beyond the gene encoding unit
to natural chromatin insulating elements. From a series of targeted RNAi
screens I indentified four proteins necessary for the rapid, transcription-
independent loss of nucleosomes at the Hsp70 locus following heat shock and
an ordered mechanism through which they function. The first factor identified,
heat shock factor (HSF), is the master transcriptional activator of heat shock
genes and is rapidly recruited to the gene within seconds of heat shock, and
binds cooperatively with the second factor, GAGA Factor, already bound to the
gene. HSF is necessary for the recruitment of the third identified factor,
dTip60, a histone acetyltransferase that acetylates histone H2A lysine 5. This
acetylation is necessary for the enzymatic activation of the fourth and final
factor, Poly(ADP)-Ribose Polymerase (PARP), which catalyzes the formation
Poly(ADP-ribose). PARP is associated with the 5’ end of Hsp70 before heat
shock, and its enzymatic activity is rapidly induced by heat shock. This
activation causes PARP to redistribute throughout the Hsp70 loci and
Poly(ADP-ribose) to concurrently accumulate in the wake of PARP’s
redistribution. Both the protein PARP and its catalytic activity are necessary
for the rapid loss in nucleosome structure of Hsp70 upon heat shock and full
transcriptional activation of Hsp70. In this dissertation I propose a novel
mechanism to overcome the nucleosome barrier to achieve full transcriptional
activation through the enzymatic activation of PARP which results in a rapid,
transcription-independent, locus-wide disruption of chromatin structure.
BIOGRAPHICAL SKETCH
I was born on August 24, 1983 in Seattle, WA to two accountants, Doug and
Mary Anne Petesch. I grew up in Seattle and infamously did not speak much
of my early life, as my older sister Anne did most of the talking for me. I was
said to be content sitting and doing puzzles quietly. As I grew older, and my
younger sister Julie appeared I found myself most happy doing math
homework, both teaching it to my classmates and sometimes to my sisters as
well. I graduated from St. Mark’s Catholic Grade School as the class
valedictorian where I convinced my 8th grade math teacher, without the
support of my classmates, to teach the entirety of our math textbook for the
first time in twenty years. I followed my sister to Seattle Preparatory High
School where at the end of my freshman year I met my wife-to-be, Krissy
Hope, as I helped manage the school’s girl’s volleyball team. It was here that I
was the editor-in-chief of our school’s newspaper, was the first student to take
the BC AP Calculus test and helped convince the school to implement a BC
AP Calculus class, and received the math award and class valedictorian at
graduation. I decided to leave Washington for Harvey Mudd College in
southern California mainly because I wanted to attend a challenging math and
science college as I thought that I wanted to be an engineer and also because
I still had far-fetched dreams of becoming a volleyball player. By my second
year of college I realized that engineering was no longer rewarding enough for
me and there was no chance of me ever becoming a professional volleyball
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player. It was also at this time that I took an inspiring organic chemistry class
that convinced me I would be most happy transferring into the chemistry
department. Harvey Mudd College hired a new Biochemistry professor, Dr.
Haushalter, the following year that had just finished a post doc in Jim
Kadonaga’s lab, working with single-molecule techniques, chromatin, and
DNA repair proteins. It was in Dr. Haushalter’s lab that I first gained laboratory
experience in biochemistry and molecular biology research, was introduced
into chromatin biology, and I fell in love with it all. After spending four years
apart in college, I decided that I would follow Krissy to Cornell to do my
graduate school work, where she was already in her first year of graduate
school. It was here at Cornell that I was first able to experience all things that I
am most passionate about at once: science, teaching, Krissy, and volleyball.
As I leave Cornell and head back to Seattle I hope to continue pursuing
science in creative ways, challenging others when it comes to what they are
capable of achieving, and maybe, if there is still time, become a better
volleyball player.

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I would like to dedicate this work to my family, friends, and particularly my wife,
who, through perseverance, is now appropriately named Dr. Hope.
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ACKNOWLEDGMENTS
I would first, and foremost, like to acknowledge my advisor, John Lis,
for accepting me into his lab and being incredibly supportive of allowing me to
investigate the things that most interested me. John is an advisor whose
enthusiasm and passion for science is nothing short of infective. John is an
incredibly creative thinker that always is looking towards developing unique
techniques that allow us to visually observe and quantitatively measure the
many aspects of transcription. I have learned a lot from him throughout the
years as to how to go about picking interesting questions to answer and to
push the limits of a technology’s current capabilities to provide the most
comprehensive and accurate answer to those questions. In my years of being
in the lab with John I can honestly say that after every time I spoke with him
about my project I came away from those discussions more inspired and
always looking at some aspect of my project from a different perspective than
before. I attribute much of the success of my project to his critical thinking and
providing a creative soundboard through which this project was cultivated and
grew into the story it is today.
I would also like to acknowledge my two committee members, Eric
Alani and Michelle Wang. I had the privilege of rotating through Eric’s lab as a
first year graduate student and learned a lot from him, and his class, about
how to think about a problem from a geneticist’s point of view. His discussions
of how to identify the target of PARP’s actions at Hsp70 were always very
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helpful. I also would like to thank him for his cheery disposition as I looked
forward to updating him about my recent progress and developments in my
project and future career goals. I would also like to thank Michelle for her
added expertise about how to think about this project through the prospective
of a physicist. Her continued suggestions of how to improve the temporal and
spatial resolution of my assays to better describe the potential models for how
nucleosomes could be lost or how PARP redistributes were very beneficial. I
could not have asked for a better committee as their background and input
complimented each other greatly.
In addition to thanking all the members of the John’s lab that I have
overlapped with in my time, I would like to mention a few in particular. Upon
rotating through the lab, I was guided by both Behfar Ardehali and Nicholas
Fuda. Throughout the years they have provided me with exceptional technical
guidance and some of the most useful experimental suggestions throughout
our many subgroup and group meetings. Their selfless, thoughtful, and critical
perspectives provided me with role models as to how to think and approach
science in a laboratory setting. I would also like to thank Abbie Saunders for
her willingness to help edit and revise my first publication and to help me
achieve a writing style that was more “sexy” for publication purposes. I lastly
have to thank Janis Werner, John’s lab manager, who has provided an easy,
organized, and efficient lab work environment while I was here.
I finally have to thank my family, friends, and wife. I first have to thank
my parents for their willingness to send me to a great college and pay for my
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education, even after telling them that I no longer wanted to be an engineer
but would rather pursue chemistry. I would also like to acknowledge my
friends, both scientists and nonscientists, for being able to share in my
excitement and celebrate with me my accomplishments. Finally, I would like
to thank my wife, Krissy Hope, who, as a scientist, has provided me with just
as much feedback, insights, and criticisms regarding my project than the
members of my lab.
I would like to end my acknowledgements with an explanation of how
John’s patience and creative input helped mold this project from the beginning.
After joining John’s lab in June of my first year, he and I talked almost every
day for multiple months about potential ideas or questions that we had
surrounding the interface of chromatin and transcription. Besides an ongoing
RNAi screen in the lab, I did not pick up a pipette for my own project for
virtually months. Instead, my many ideas that were presented were met with
either fair criticisms or tepid responses. Although frustrated, and slightly
concerned that John might soon regret his decision to let me join the lab, I
always felt inspired from my discussions with John to think of a new idea and
grateful for his patience with me. It was ultimately after one group meeting
that a discussion with him resulted in us both excited about the prospect
probing the chromatin landscape near the paused polymerase of Hsp70 both
before and after heat shock to understand the fate of transcribed nucleosomes
at high resolutions. I look back and acknowledge that these first few months in
the lab of critically thinking and talking with John as what truly helped me
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