i Lack of Aryl Hydrocarbon Receptor Alters Gene Expression and Functional Capacity of Murine ...

152 Pages·2015·4.32 MB·English

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Lack of Aryl Hydrocarbon Receptor Alters Gene Expression and Functional
Capacity of Murine Hematopoietic Stem Cells
by
John A. Bennett

Submitted in Partial Fulfillment of the
Requirements for the Degree
Doctor of Philosophy

Supervised by
Professor Thomas A. Gasiewicz

Department of Environmental Medicine
School of Medicine and Dentistry

University of Rochester
Rochester, New York

2015
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Biographical Sketch
John Allen Bennett was born in Sylva, North Carolina on March 4th, 1986. He grew up in
Cullowhee, North Carolina and graduated cum laude from Western Carolina University
with a Bachelor of Science in Biology (concentration in Molecular Biology). He began
doctoral studies at the University of Rochester in 2009. His research involved the
characterization of a tissue-restricted knockout mouse model to better understand the role
of the aryl hydrocarbon receptor in the regulation of murine hematopoietic stem cell
signaling and function, under the direction of Dr. Thomas A. Gasiewicz. His work has
been presented at the International Society for Stem Cell Research on multiple occasions,
and utilized as part of competitive grant renewals.

The following publications were a result of work conducted during doctoral study:

Gasiewicz TA, Singh KP, Bennett JA (2014) The role of the Ah receptor in stem cell
cycling, regulation and quiescence. Ann N Y Acad Sci. 2014 Mar;1310:44-50

Singh KP, Bennett JA, Casado FL, Walrath JL, Welle SL, Gasiewicz TA (2014) Loss of
aryl hydrocarbon receptor promotes gene changes associated with premature
hematopoietic stem cell exhaustion and development of a myeloproliferative disorder in
aging mice. Stem Cells Dev. 2014 Jan 15;23(2):95-106
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Bennett JA, Singh KP, Welle SL, Gasiewicz TA (2015) Deficiency in aryl hydrocarbon
receptor (AhR) expression throughout aging alters gene expression profiles in murine long-
term hematopoietic stem cells (submitted to PLOS One, 4/8/2015)
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Acknowledgements

I would like to thank my advisor, Tom Gasiewicz, for his constant scientific and
personal support. He is always driving his students to consider the experiments that will
best answer the questions being addressed, and to consider all the possible explanations
for every finding. He drives us to be better scientists, and without his support I would not
be the scientist I am today. He is always available for discussions, professional and
personal, and I truly feel that he is a paragon of a mentor who truly cares about his
students and their personal and professional development.
I also wish to thank Drs. Ellen Henry and Kameshwar Singh for being my closest
colleagues, as well as friends. Ellen is always willing to listen to my questions and help
formulate new ideas and directions, and without her assistance as a mentor and
collaborator my project and publications would not be of the quality they are. Dr. Singh
was always at my side, helping me with the day to day planning of experiments as well as
the execution of experimental plans that would have been extraordinarily difficult with
only one set of hands. Dr. Singh has a wealth of scientific knowledge, and my project
greatly benefitted from his suggestions and guidance. On a more personal note, Drs.
Henry and Singh both made laboratory time very entertaining, because we share a lot of
the same passions in life for music, food, gardening and outdoor activities. I will always
treasure our discussions and time together.
Thank you to the members of my research advisory committee (Drs. Paige
Lawrence, Steve Georas and James Palis) for their personal and scientific support. They
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have been invaluable with formulating my project, and navigating the process of
interpreting experimental data and modulating my plans in accordance. They are
incredibly friendly and supportive, and they always made meeting with them a
pleasurable experience. My scientific work has greatly benefitted from their expert
advice, and I truly appreciate how much effort and care they place into training,
supporting and guiding their students.
Last but certainly not least, I would like to thank my family and friends for their
constant support throughout my doctoral studies. My grandmother (Ruby Bennett) is
always pushing me to hurry up and finish, and our daily conversations helped me to never
feel homesick or alone. I would like to thank my dog (Kirby) and my cat (Ella) for their
unadulterated love and affection, as they make coming home a joy and always help keep
me happy and prop me up when I am feeling uncertain or worried. Thanks to Jason
Walrath and Michael Moses for our constant jokes and discussion. The ability to discuss
complex science in a humorous fashion helped me to both formulate and refine ideas in a
way that always felt fun and entertaining.
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Abstract

All mature cell lineages of the peripheral blood and adaptive immune system are
generated from bone marrow stem cells known as hematopoietic stem cells (HSCs). Lack
of aryl hydrocarbon receptor (AhR) signaling, or treatment with AhR agonists such as
TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin) , has been shown to alter the phenotype and
functional ability of HSCs, suggesting a role for the AhR in regulating HSC function and
output. Most classical studies on AhR have been undertaken using TCDD or other
xenobiotic ligands to modulate AhR activity. While these studies have revealed a wealth
of information regarding AhR target genes and signaling networks in a variety of cell
types (including HSCs), it has remained difficult to determine which endpoints /
regulatory target genes are adaptive or toxic responses due to exposure to AhR ligands
versus normal targets and functions under homeostatic conditions.
Studies were performed using both global AhR-null allele (AhR-KO) mice as
well as hematopoietic system restricted AhRfx/fxVav1-Cre (CKO) mice in order to better
understand the HSC-intrinsic physiological role of AhR in regulating HSC function and
output. These studies revealed a surprising lack of phenocopy between the two strains.
Aged KO animals display an expansion of peripheral white blood cells which is not
found in aged CKO animals. KO animals have enhanced levels of oxidative stress in
bone marrow progenitors, as well as evidence of DNA damage and decreased expression
of p16. CKO animals only display enhanced oxidative capacity at the time point
examined. Microarray studies on long term HSCs were undertaken in order to examine
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the differences in cell signaling and gene expression that could be driving these
unexpected differences in phenotype. The two strains display very little overlap in genes
with differential expression relative to control at young ages, but become more similar
with aging. Gene Set Enrichment Analysis revealed significant enrichment in a variety of
gene sets that are related to HSC function, or involved in the etiology of certain diseases
such as leukemia or myeloproliferation. A subset of differentially expressed genes that
changed expression due to both aging and lack of AhR expression (Pdgf-D, Smo, Zbtb37,
and Zfp382), and that contain AhR binding sites in their upstream regulatory regions,
change expression when AhR expression is silenced in wild-type hematopoietic cells.
Taken together, these data reveal potential genes that AhR may intrinsically regulate in
HSCs throughout aging of the hematopoietic system. Given the lack of phenocopy and
the small degree of overlap differentially expressed genes in HSCs between KO and
CKO mice, these data also implicate AhR in non-hematopoietic stromal cells of the bone
marrow as having a regulatory effect on HSC function and output.
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Contributors and Funding Sources

This work was supervised by a committee consisting of Dr. Thomas Gasiewicz
(primary advisor) and Dr. Paige Lawrence of the Department of Environmental
Medicine, Dr. James Palis of the Department of Pediatrics, Hematology and Oncology
and Dr. Steve Georas of the Department of Medicine, Pulmonary Diseases and Critical
Care. Staff in the URMC Functional Genomics Center collected microarray data. Dr.
Stephen Welle generated the normalized probe signals used for microarray analyses,
uploaded raw data to the Gene Expression Omnibus for public accession, ran ANOVA on
array data and prepared data files for GSEA analysis. Drs. Ellen Henry and Kameshwar
Singh assisted with experimental planning and execution. Dr. Singh performed tail vein
injections for transplant experiments. Jason Walrath maintained mouse colonies and
performed genotyping. All other work conducted for the dissertation was completed
independently by the student.
The work in this thesis was supported by NIH grants T32ES07026, ES004862 and
ES023068.
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Table of Contents

Chapter 1 : Introduction .......................................................................................................1
1.1 Hematopoietic Stem Cells........................................................................................1
1.2 The Aryl Hydrocarbon Receptor ..............................................................................7
1.3 Evidence for a Physiological Role of AhR ............................................................12
1.4 Evidence Supporting a Role for AhR in HSC Function ........................................15
1.5 Animal Models for Global and Conditional Deletion of AhR ...............................19
1.6 Rationale, Overall Hypothesis and Specific Aims .................................................22
Chapter 2 : Materials and Methods ....................................................................................24
2.1 Mice ......................................................................................................................24
2.2 Complete Blood Counts and Organ / Bone Marrow Cell Isolation ......................25
2.3 Bone Marrow Lineage Depletion...........................................................................27
2.4 Flow Cytometry and Cell Sorting .........................................................................29
2.5 Spleen Colony Forming Unit Assay .....................................................................33
2.6 Serial Repopulation Assay ....................................................................................35
2.7 Microarray Analyses .............................................................................................36
2.8 Gene Set Enrichment Analysis .............................................................................38
2.9 Ingenuity Pathway Analysis .................................................................................38
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2.10 qPCR Validation of Array Data ............................................................................39
2.11 Viral Knockdown of AhR .....................................................................................41
Chapter 3 : Conditional lack of AhR produces a limited hematopoietic phenotype and
does not entirely phenocopy global AhR deletion .............................................................44
3.1 CKO animals display excision of the Ahr gene in hematopoietic cells, reduced
levels of AhR protein, and are non-responsive to TCDD .............................................44
3.2 CKO animals are healthy and do not display gross phenotypic alterations ..........48
3.3 AhR-CKO mice do not display significant alterations in bone marrow
subpopulations, peripheral blood, or cell cycling .........................................................50
3.4 18-month-old AhR-CKO mice do not entirely phenocopy 18-month-old AhR KO
mice ...............................................................................................................................54
3.5 Significance ...........................................................................................................62
Chapter 4 : Conditional lack of AhR produces a limited hematopoietic phenotype and
does not entirely phenocopy global AhR deletion .............................................................63
4.1 Conditional deletion of AhR in hematopoietic cells is sufficient to alter gene
expression in LT-HSCs from 8-week old mice ..............................................................63
4.2 Gene sets enriched in LT-HSCs of 8-week-old AhR-CKO mice are consistent
with the hypothesized regulatory role of AhR ...............................................................65