Table Of ContentAMINO TERMINAL CLEAVAGE AND MULTISITE PHOSPHORYLATION
REGULATE THE FUNCTION OF THE COHESIN DEPOSITION FACTOR, SCC2
by
JULIE ANN WOODMAN
B.S., Colorado State University, 2009
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Molecular Biology Program
2015
This thesis for the Doctor of Philosophy degree by
Julie Ann Woodman
has been approved for the
Molecular Biology Program
by
Mingxia Huang, Chair
Kirk Hansen
Chad Pearson
Robert Sclafani
Andrew Thorburn
Paul Megee, Advisor
Date: 5/06/2015
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Woodman, Julie Ann (Ph.D., Molecular Biology)
Amino Terminal Cleavage and Multisite Phosphorylation Regulate the Function of
the Cohesin Deposition Factor, Scc2
Thesis directed by Associate Professor Paul C. Megee.
ABSTRACT
Sister chromatid cohesion plays vital roles in chromosome biology, including
the regulation of gene expression, the facilitation of DNA repair, the mediation of
chromosome condensation, and the tethering, or cohesion, of replicated sister
chromatids to promote chromosome biorientation and proper segregation. The
four-subunit cohesin complex mediates cohesion and its deposition onto
chromosomes requires a separate conserved loading complex, composed of
Scc2 and Scc4 (Scc2/Scc4) in budding yeast. How the Scc2/Scc4 deposition
complex regulates the spatiotemporal association of cohesin with chromosomes
is not understood. The work presented here advances the understanding of
cohesin deposition by exploring how Scc2 is regulated through specific
proteolysis and phosphorylation. I demonstrate that dephosphorylation of whole
cell extract promotes Scc2 amino terminal cleavage, thereby disrupting Scc2’s
interaction with Scc4 and its cohesin deposition activity. I have also identified
phosphorylation sites on Scc2 residues and altered its phosphorylation state by
genetic manipulation to explore the impact of Scc2 phosphoregulation on cohesin
function. I provide evidence using scc2 mutants with phosphomimetic substitution
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of phosphorylated residues that suggests that constitutive phosphorylation at
several Scc2 residues results in decreased stability of the cohesin complex
subunit, Mcd1. While cohesin association on chromosome arms is significantly
reduced in the phosphomimetic mutants, it apparently remains above a key
threshold, as sister chromatid cohesion is only modestly perturbed. However, the
mutants exhibit dramatic defects in chromosome condensation, perhaps
explaining previous observations that the cohesin deposition complex has a
critical post-S phase role required for maintaining cell viability. While Mcd1 that is
prevented from being deposited onto chromosomes remains stable in wild type
cells, the Mcd1 instability that is observed in scc2 phosphomutants potentially
reveals a previously unappreciated requirement for the deposition complex in
maintaining cohesin complex integrity during a critical step in cohesin activity.
The form and content of this abstract are approved. I recommend its
publication.
Approved: Paul C. Megee
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Great are the works of the Lord, studied by all who delight in them.
Psalm 111:2
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ACKNOWLEDGMENTS
The work accomplished in this thesis is a result of so many people going
above and beyond what is expected of them. Thank you to the Molecular Biology
Program for providing funds through the T32 Training Grant and the Bolie
Scholarship and to the Cornelia de Lange Foundation for additional funds and for
allowing me to see how my work impacts patient lives. Also, thank you to
program administrators, Jean Sibley and Sabrena Heilman, for all of the
encouragement and support that you have both provided. Special thanks to my
committee members for offering insightful suggestions to my project, taking the
time to really think about my results, and for always being available for questions
and scientific discussion.
I’d also like to thank the people that directly contributed to the work presented
in this thesis. Thank you to Drs. Kirk Hansen and Monika Dzieciatkowska for their
countless hours spent answering questions as well as processing and analyzing
my MS data as it was absolutely critical for the advancement of my research. And
a very special thank you to current and former lab members for providing moral
support throughout my time in lab and lending a helping hand with technical
aspects. Thank you to Matt, in particular, for trudging through all of the
phosphomutant cloning so that I could sweep in and do the fun experiments!
I especially thank Dr. Paul Megee for excellent mentorship. As an advisor,
you taught me to think like a scientist and as a friend, you offered the
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encouragement and support to guide my success. Thank you for the interesting
discussions and for challenging me in ways that I can only appreciate in
hindsight. J I value my years spent in your lab and am so grateful for the
opportunity to have learned from the best!
Last but certainly not least, I am humbled by the love and support offered
from my friends and family. Thank you to the amazing friends that I found here,
especially Nancy and Laura. And to my family, thank you for the sacrifices that
you made and the support that you offered. To Trish and Steve, who always ask
about the progress of my research and genuinely act interested when I respond
with too much detail, thank you for all of your support and encouragement. Thank
you to my parents who have always been my biggest cheerleaders, my strongest
support system, and incredible role models of hard work and determination. I
pursued science because you showed me that I could do anything and I am so
appreciative for all of your encouragement over the years. Thank you to my
husband who has had to deal with me through all of the highs and lows of
graduate school, you are an incredible man and you have made this experience
a true adventure. To my sweet Annie, you inspire me to give my all… thank you.
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TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION…………………………………………………………..........1
Introduction ……………………………………………………………………1
Roles of cohesin ………………….…………………………………………..1
The cohesin complex………………………………………………………. 2
Models of cohesin’s chromatin association ………………………………4
Cohesin’s deposition factor, Scc2/Scc4 …………………………………..6
The spatiotemporal regulation of
cohesin’s chromatin association ……………………..……………………9
Cohesin’s spatial distribution on chromosomes ….………………..9
Cohesin’s temporal regulation ……………………………………..10
Scc2/Scc4’s contribution to
cohesin’s spatiotemporal regulation ………………………………13
Phosphoregulation ………………………………………………………….15
Mcd1 instability ……………………………………………………………...16
Cohesinopathies: Roberts Syndrome and
Cornelia de Lange Syndrome ……………………………………………...17
Summary of background and relation to thesis work ……..……………..18
II. METHODS ……………………………………………………………………..20
Strain construction…………………..……………………………………..20
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Yeast cell culture and cell cycle staging…………………………………25
Chromatin fractionation …………………………………………………..26
In vitro cleavage assay ………………….………………………………..27
Immunoblot quantitation ………………………………………………….27
Antibody production ………………………….……………………………28
Mass spectrometry ………………………………………………………..28
Fe-NTA phosphopeptide enrichment and identification ………………31
Phosphomutant construction …………………………………………….32
Chromosome condensation assay ………………………………………32
III. SCC2 DEPHOSPHORYLATION REULTS IN AMINO TERMINAL
CLEAVAGE AND DISRUPTS COHESIN DEPOSITION ………………….34
Introduction ………………………………………………………………...34
Results ……………………………………………………………………..35
Scc2’s biphasic chromatin association correlates
with the presence of two distinct Scc2 species …………………….35
Scc2 inactivation in
post-S phase cells reduces cellular viability ………………………..40
The appearance of Scc2 species is cell cycle regulated ………….40
Dephosphorylation promotes Scc2 instability ……………………...42
Phosphorylation protects Scc2 from cleavage ……………………..47
Scc2 cleavage eliminates its interactions with Scc4 ………………48
Discussion ………………………………………………………………….51
IV. SCC2 PHOSPHORYLATION REGULATES
COHESIN COMPLEX INTEGRITY ………………………………………….56
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Introduction ………………………………………………………………...56
Results ……………………………………………………………………..57
Scc2 is phosphorylated at multiple residues ……………………….57
Scc2 phosphomimetic mutants delay
cell cycle progression and decrease viability ………………………58
scc2-8A mutant acquires novel phosphorylation sites …………….66
Scc2 phosphomutants interact
with Scc4 and bind chromatin ………………………………………..67
Mcd1 stability is reduced
in scc2 phosphomimetic mutants ……………………………………70
Mcd1 instability is independent of Esp1 and Eco1 ………………..72
Cohesin binding to chromosome arms
is reduced in scc2 phosphomutants ………………………………...73
scc2 phosphomutants have
defects in chromosome condensation ………………………………76
Discussion ………………………………………………………………….81
V. DISCUSSION ………………………………………………………………….89
Summary and conclusion ………………………………………………...89
Model of Scc2 function ……………………………………………….91
Cell cycle regulation of Scc2 phosphorylation ……………………..95
A post-replicative role for Scc2 ………………………………………96
Scc2/Scc4 interactions ……………………………………………….97
Mcd1 instability ………………………………………………………..99
Protein structure………………………………………………………100
Future directions …………………………………………………………102
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Description:cell extract promotes Scc2 amino terminal cleavage, thereby disrupting Scc2's Thank you to Matt, in particular, for trudging through all of the also prepared for phosphopeptide enrichment using immobilized metal affinity.
