Regulation of gene expression by the
Mediator kinase CDK19
by
Katherine Audrey Audetat
B.S., University of Arizona, 2009
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirement for the degree of
Doctor of Philosophy
Department of Molecular, Cellular, and Developmental Biology
2016
This thesis entitled:
Regulation of gene expression by the Mediator kinase CDK19
Written by Katherine Audrey Audetat
has been approved for the
Department of Molecular, Cellular, and Developmental Biology by:
______
Dylan J. Taatjes, Ph.D.
______
Corrella S. Detweiler, Ph.D.
Date______
The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards
of scholarly work in the above-mentioned discipline. Audetat, Katherine Audrey (Ph.D., Molecular, Cellular, Developmental Biology)
Regulation of gene expression by the Mediator kinase CDK19
Thesis directed by Dylan J. Taatjes, Ph.D.
Abstract
The Mediator complex is a required co-activator of RNA Polymerase II (Pol II), the enzyme responsible for the transcription of all protein coding genes in eukaryotes.
Mediator facilitates transcription factor-dependent gene expression by directly interacting with gene specific transcription factors and Pol II and the general transcription machinery. Mediator consists of up to 29 subunits; a four-subunit “CDK module” reversibly associates with Core-Mediator and alters its structure and activity.
Most of the work on the CDK module has been done in yeast, and has focused on the
CDK module subunits CDK8, MED12, and MED13. These three subunits have paralog proteins CDK19, MED12L, and MED13L that are present only in vertebrates. Information about these CDK module paralog subunits is limited. CDK19, the paralog of CDK8, associates with all other CDK module subunits and with Core-
Mediator. As one of the only two enzymatic subunits of Mediator, the CDK19 kinase likely has important roles in transcription, but these roles remain uncharacterized.
Here I initiated experiments to define a role for CDK19 in human cancer cells.
Using osteosarcoma cells (SJSA) that naturally lack endogenous CDK8 protein, I have identified a role for CDK19 in cellular proliferation under standard growth conditions,
iii and I have demonstrated that this function is not dependent upon its kinase activity.
Furthermore, using a combination of RNA-Seq and qRT-PCR in SJSA cells with stable
CDK19 knockdown, I have established evidence of a role for CDK19 in the transcriptional response to 5-fluorouracil (5-FU), an inducer of genotoxic and metabolic stress. These experiments also revealed a specific requirement for the CDK19 protein in the activation of p53 target genes during 5-FU treatment. To further probe a potential role for CDK19 in the p53 response, I completed studies in control vs. CDK19 knockdown cells treated with Nutlin, a highly specific, well-tested activator of p53. Remarkably, I observed that the presence of CDK19 enhanced survival in Nutlin- treated cells and was required for SJSA cells to return to a proliferative state following
Nutlin treatment. As with the general proliferative defect under normal growth conditions, this effect was observed to be kinase-independent. Taken together, the results summarized herein implicate CDK19 as a key regulator of the p53 stress response in SJSA cells. These data also suggest that in cancers that are sensitive to
Nutlin, concomitant targeting of CDK19 might represent an effective therapeutic strategy.
iv Acknowledgements
My heart is full of gratitude. This has been quite a trip, and I am only here because of the efforts of many people.
Danny, my life changed the day I met you. Thank you for the love, help, and encouragement you have freely and constantly given. Thank you for my son. You are the best person I know.
Dylan, you saved my life. I would not be here without you. Thank you for taking me in and giving me a place to think and work, and finish my degree. Your patience, guidance, and reassurance changed my perspective on science and research. Thank you to the rest of the Taatjes lab for accepting me.
Thank you to my ladies: Amber, Liz, Hestia, Roni, Stefanie, Charli, and Cecie.
Thank you to my guys: Ben, Zach, Ryan, Tom, Joel, and Lavan. Your support in and out of lab has been the best part of grad school.
Thank you to my committee members, who have been my critics and my advocates, supported me and kept me on a path to this day.
Thank you to the department for seeing to my education from start to finish, including providing considerable financial support, especially during the transition.
Thank you to my dogs, Toby and Gixxer, for staying with me for so long.
Thank you to the Marines, for getting me through a decade of school and life debt free.
Thank you to my wonderful family, immediate and extended, across the country and world. You have loved me and cheered me on, and been everything I needed.
Thank you to my particularly splendid sisters; you are the best.
v Table of Contents
Abstract...... iii
Acknowledgements...... v
Table of Contents...... vi
List of Tables...... x
List of Figures...... xi
Chapter 1- Introduction...... 1
1.1- Transcription Initiation...... 2
1.1.1- RNA Polymerase II...... 3
1.1.2- PIC Assembly...... 4
1.1.3- Mediator Complex...... 5
1.1.4- General Transcription Factors...... 5
1.2- Transcription Elongation...... 6
1.2.1- Pausing...... 6
1.2.2- Pause Release...... 6
1.2.3- Productive Elongation...... 7
1.3- Transcription Termination...... 7
1.4- Pol II CTD...... 8
1.5- Co-transcriptional processing...... 10
1.5.1- Capping...... 10
1.5.2- Splicing...... 11
1.5.3- Polyadenylation...... 11
1.6- Post-transcriptional processing...... 12
vi 1.6- Export, Translation, Degradation...... 12
1.7- The Mediator Complex...... 13
1.7.1- Mediator is a required co-activator of RNA Polymerase II...... 13
1.7.2- Conservation of Mediator subunits...... 15
1.7.3- Structural shifts due to cofactor binding...... 17
1.8- The CDK module...... 18
1.8.1- Subunit composition...... 18
1.8.2- Paralog proteins...... 21
1.8.3- Interaction with Core Mediator...... 23
1.8.4- Mutations and disease association...... 24
1.9- Mediator Kinases CDK8 and CDK19...... 25
1.9.1- Similarities between CDK8 and CDK19...... 26
1.9.2- Differences between CDK8 and CDK19...... 27
1.10- The tumor suppressor protein p53...... 29
1.10.1- p53 as a transcription factor...... 29
1.10.2- Cytoplasmic roles of p53...... 31
1.10.3- Role in cancer...... 32
1.10.4- Interaction with Mediator...... 33
1.10.5- Regulation of p53 activity...... 33
Chapter 2- CDK19 as an effector of p53 response in SJSA cells...... 36
2.1- the SJSA osteosarcoma cell line...... 36
2.2- Establishment and characterization of CDK19 knockdown line...... 38
2.2.1- CDK19 knockdown cells proliferate slower but remain viable...... 38
vii 2.2.2- Analysis of CDK module subunits in control and CDK19 knockdowns.....40
2.2.3- Gene expression changes due to CDK19 knockdown...... 42
2.3- CDK19 and the response to 5-Fluorouracil treatment...... 45
2.3.1- 5-Fluorouracil mechanisms of action...... 45
2.3.2- CDK19 knockdown does not increase sensitivity to 5-FU...... 46
2.3.3- Gene expression changes in response to 5-FU are diminished in CDK19 knockdown cells...... 47
2.3.4- CDK19 knockdown does not affect transcription machinery recruitment..50
2.3.5- CDK19 knockdown does not affect transcript stability...... 53
2.4- CDK19 and the response to Nutlin treatment...... 55
2.4.1- Nutlin-3a mechanism of action...... 55
2.4.2- CDK19 knockdown sensitizes SJSA cells to Nutlin-3 treatment...... 56
2.4.3- CDK19 kinase activity is not required to rescue Nutlin sensitivity...... 57
2.4.4- CDK19 knockdown results in decreased p53 target gene expression...... 60
2.5- Discussion...... 62
2.6- Methods...... 68
Bibliography...... 73
Appendices
Appendix 1- CDK19 as an effector of p53 response in HCT116 cells...... 85
A1.1- HCT116 cells...... 85
A1.1.1- Colorectal cancer review...... 85
A1.1.2- Characterization of CDK19 knockdown...... 85
A1.2- p53 target gene induction is diminished in CDK19 knockdown cells...... 88
viii A1.2.1- Ingenuity Pathway Analysis of published results...... 88
A1.2.2- qRT-PCR analysis of p53 target genes...... 88
A1.3- Methods...... 89
Appendix 2- HIF1A utilizes multiple co-activators in the response to hypoxia...... 91
A2.1- The TIP60 complex is a co-activator of HIF1A...... 91
A2.2- CDK19 knockdown results in decreased HIF stabilization in SJSA cells...... 94
A2.3- Methods...... 95
Appendix 3- Cortistatin A inhibition of CDK19 kinase activity...... 96
A3.1- Isolation and characterization of CA activity...... 96
A3.2- CA inhibits CDK19 phosphorylation of SIRT1 ...... 98
A3.3- Methods...... 99
ix List of Tables
Table 1.1- List of Mediator Subunits...... 16
Table 2.1- shRNA sequences ...... 68
Table 2.2- qRT-PCR primer sequences ...... 69
Table 2.3- Antibodies used in this study ...... 69
Table 2.4- ChIP qPCR primer sequences ...... 70
Table 2.5- Site-Directed Mutagenesis primer sequences...... 72
x List of Figures
Chapter 1
Figure 1.1- DNA is wrapped around histones and packaged into chromatin...... 2
Figure 1.2- Histone proteins are modified to control access to genes...... 3
Figure 1.3- The Pre-Initiation Complex...... ………………………………...... 5
Figure 1.4- The CTD of Pol II is phosphorylated during transcription……………………..8
Figure 1.5- Co-transcriptional processing of mRNA……………………………………….10
Figure 1.6- Structure and organization of Mediator……………………………………...... 17
Figure 1.7- Mediator adopts different conformations upon transcription factor or Pol II binding ……………………………………………………………………………...…….……18
Figure 1.8- The CDK module links to Core Mediator via MED13 subunit………….……20
Figure 1.9- Paralogs are mutually exclusive as CDK module subunits………………….22
Figure 1.10- Alignments of CDK8 and CDK19…………………………….……………….27
Figure 1.11- p53 functions in the nucleus and cytoplasm……………….………………..31
Figure 1.12- p53 induces apoptosis in the cytoplasm.………...………….…………….…32
Figure 1.13- Post-translational modifications regulate p53..…………....……….………..34
Chapter 2
Figure 2.1- SJSA cells express CDK19 but no detectable CDK8 protein....…...... ……37
Figure 2.2- Stable CDK19 knockdown in SJSA cells………………………………………39
Figure 2.3- Slowed proliferation can be rescued with wild-type or kinase-dead CDK19 expression………………………………………………………………………………....…...40
Figure 2.4- CKM subunits in CDK19 knockdown cells……………………………....…….42
Figure 2.5- Gene expression changes due to CDK19 knockdown……………………….44
xi Figure 2.6- CDK19 knockdown does not increase sensitivity to 5-FU…………………...46
Figure 2.7- Gene expression changes in response to 5-FU are diminished in CDK19 knockdown cells…………………………………………………………………………….….48
Figure 2.8- CDK19 knockdown does not affect transcription machinery recruitment.....51
Figure 2.9- CDK19 knockdown does not affect transcript stability……………....……….54
Figure 2.10- SJSA cells respond to Nutlin-3a………………………………....……………55
Figure 2.11- CDK19 knockdown sensitizes cells to Nutlin-3a…………………………….58
Figure 2.12- Figure 4.12 Nutlin treatment causes decreased gene expression and increased levels of apoptosis in the CDK19 knockdown cells…………………….....…...61
Appendix 1
Figure A1.1- HCT116 cells express CDK8 and CDK19 and tolerate knockdown of both kinases...... 86
Figure A1.2- CDK19 is required for activation of p53 targets during 5-FU treatment in
HCT116 cells ...... 87
Appendix 2
Figure A2.1- TIP60 Depletion Impairs Expression of Specific HIF1A Target Genes in
HCT116 cells...... 93
Figure A2.2- HIF stabilization during hypoxia is decreased in CDK19 knockdown...... 94
Appendix 3
Figure A3.1- Cortistatin A is a selective inhibitor of CDK8 and CDK19 kinase activity...97
Figure A3.2- CA inhibits phosphorylation of SIRT1 by CDK19...... 98
xii CHAPTER 1- Introduction
Gene expression is a multi-step process that underlies the central dogma in biology; template DNA is transcribed into an RNA molecule, which is then translated into protein-the functional unit of the cell. From the simplest bacterial cell to humans, gene expression is the first step in life, survival, and the response to our environment.
Gene regulation becomes increasingly complex as an organism increases in cell number and developmental stages. In higher order eukaryotes, transcription is an exceptionally intricate and regulated process requiring the concerted effort of multiple proteins. Transcription occurs in well-defined stages, with many processing events occurring simultaneously. The stages of transcription are: initiation, elongation, and termination. Many mRNA processing events occur co-transcriptionally, and are important for transcript stability, removal of noncoding regions, and export to the cytoplasm. Once in the cytoplasm, the mRNA is translated into protein, and degraded thereby completing the process.
1.1 Transcription Initiation
Transcription Initiation is the process by which the necessary proteins assemble onto DNA and begin the synthesis of RNA. This stage is a critical regulatory step, as gaining access to the DNA is limited by chromatin structure. Genomic DNA is packaged into nucleosomes, which consist of ~146 base pairs of DNA wrapped around a histone octamer (Figure 1.1). Chromatin remodeling complexes change the architecture of packaged genomic DNA to allow access by other proteins (Clapier and Cairns, 2009).
The histone proteins play a key role in DNA access; the individual histones can be heavily modified with post-translational marks such as phosphorylation, methylation, and acetylation, and these marks can change gene expression patterns (Bannister and
Kouzarides, 2011) (Figure 1.2).
Figure 1.1 DNA is wrapped around histones and packaged into chromatin. DNA is wrapped around histone octamers to form nucleosomes, and then further condensed into chromatin and chromosomes. (Felsenfeld and Grouding, Nature 2003)
2
Figure 1.2 Histone modifications dictate specific biological readouts. Histones can be modified in numerous ways, which affects the recruitment of chromatin remodeling enzymes. This control over access to genes is called the histone code, and represents one of the first levels of transcription regulation (Munsh