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Role of the Polyadenylation Factor CstF-50 in regulating the BRCA1/BARD1 E3 Ubiquitin (Ub) Ligase Activity
Danae Fonseca Graduate Center, City University of New York
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Role of the Polyadenylation Factor CstF-50 in regulating the
BRCA1/BARD1 E3 Ubiquitin (Ub) Ligase Activity
by
Danae Fonseca
A dissertation submitted to the Graduate Faculty in Biochemistry in partial fulfillment of the
requirements for the degree of Doctor of Philosophy, The City University of New York.
2014
This manuscript has been read and accepted for the Graduate Faculty in Biochemistry in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy.
Dr. Frida E. Kleiman
Date Chair of Examining Committee
Dr. Edward J. Kennelly
Date Executive Officer
Dr. Richard Baer
Dr. Karen Hubbard
Dr. Maria Figueiredo-Pereira
Dr. Hualin Zhong
Supervisory Committee
THE CITY UNIVERSITY OF NEW YORK
ii
ABSTRACT
Role of the Polyadenylation Factor CstF-50 in regulating the BRCA1/BARD1 E3
Ubiquitin (Ub) Ligase Activity
By
Danae Fonseca
The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. The studies presented in this dissertation focus on the determination and characterization of the role of mRNA processing factor CstF-50 and escort protein p97 in the regulation of the BRCA1/BARD1 E3 ubiquitin (Ub) ligase activity during the
DNA damage response (DDR).
As part of the studies presented in Chapter II, I determined that the polyadenylation factor CstF plays a direct role in DDR, specifically in transcription-coupled repair (TCR), and that it localizes with RNA polymerase II (RNAP II) and BARD1 to sites of repaired DNA. My results also indicated that CstF plays a role in the UV-induced ubiquitination and degradation of
RNAP II. In Chapter III, I determined that the carboxy-terminal domain of p53 associates with factors that are required for the ultraviolet (UV)-induced inhibition of the mRNA 3’ cleavage step of the polyadenylation reaction, such as the tumor suppressor BARD1 and the polyadenylation factor CstF-50. These results were part of a study that identified a novel 3’ RNA processing inhibitory function of p53, adding a new level of complexity to the DDR by linking
RNA processing to the p53 network. In addition, in Chapter IV I showed that CstF-50 can interact not only with BRCA1/BARD1 E3 Ub ligase but also with ubiquitin (Ub), the escort-
iii factor p97 and some of BRCA1/BARD1 substrates, such as RNAP II, H2A and H2B. I also demonstrate that CstF-50-associated p97 activates the BRCA1/BARD1-dependent RNAP II poly-ubiquitination, H2A and H2B monoubiquitination as well as BRCA1/BARD1 autoubiquitination. Together my results provide evidence that CstF-50-associated p97 regulates
BRCA1/BARD1 Ub ligase activity during DDR, helping in the assembly and/or stabilization of the ubiquitination complex. Extending these studies, in Chapter V, I showed that UV-treatment induces changes in the localization of BRCA1, BARD1, CstF-50, p97 and some of
BRCA1/BARD1 substrates in different nuclear fractions, and that these changes depend on
BRCA1/BARD1 and CstF-50 expression. Further, my results demonstrate that the content of monoubiquitinated H2B in the chromatin of genes with different levels of expression changes during DDR and this is mediated by BRCA1/BARD1 and CstF-50. The data presented in this chapter show new insights into the role of mRNA 3’ processing factor CstF-50 in regulating the
Ub pathway, resulting in epigenetic control during DDR. Finally, in Chapter VI, I identified the
RNA binding protein HuR as a new substrate for BRCA1/BARD1/CstF-50/p97 Ub ligase activity in different cellular conditions.
All together, the studies presented in this dissertation revealed unexpected insights into the role of the RNA processing factor CstF-50, tumor suppressors BRCA1/BARD1 and p53, the
Ub pathway and chromatin structure during DDR.
iv
SIGNIFICANCE
The integrity of the genome is constantly challenged by numerous genotoxic agents and environmental stress. It has been estimated that ten to hundred thousand DNA lesions are introduced each day in a mammalian cell with diverse and adverse consequences. That is why is not surprising that organisms have acquired a sophisticated system, the DNA damage response
(DDR), to neutralize the genetic erosion caused by these DNA damaging agents. Importantly,
DDR includes not only a set of DNA repair mechanisms and cell-cycle checkpoints but also transcriptional and mRNA processing regulation and programmed cell death. Therefore, the effectiveness of DDR and its proper regulation is a vital cellular event since genome instability is a hallmark of cancer and a major contributor to tumorigenesis.
In the past few years, it has becoming more evident that the interplay among different pathways is crucial in the regulation of the intricate DDR mechanism. For instance, the dynamic nature of the mRNA 3’ end processing machinery allows the regulation of mRNAs levels and has the potential to contribute to the cells rapid response to stress. Tumors suppressors, such as
BRCA1/BARD1 and p53, transiently regulate mRNA processing after UV damage through its interaction with the mRNA processing factor CstF-50. Besides, compelling evidence has unveiled that the ubiquitin (Ub)-proteasome pathway plays a pivotal role in the regulation of
DNA repair. It has been shown that several pathways relevant to DDR are modulated by the Ub- dependent modification of certain proteins in a programmed manner which is essential to ensure appropriate DNA repair. Post-translational modifications of histones that result in chromatin remodeling during DDR has become a central subject of study. Chromatin structure influences access to the damaged DNA, and often serves as a docking or signaling site for the recruitment
v of repair factors and signaling proteins. Therefore, regulation of nucleosome dynamics during
DNA repair is another important element in the response to DNA damage.
The findings reported in this dissertation are highly significant because they show that the cleavage stimulation factor CstF-50 is not only part of the mRNA 3’ end processing machinery but is also a bridge connecting several pathways, establishing a novel interplay among mRNA 3’ end processing, tumor suppression, the Ub pathway, chromatin remodeling and DNA repair.
These studies specifically focus on CstF-50 functional interconnection with
BRCA1/BARD1, RNA polymerase II (RNAP II), histones, the escort factor p97 and Ub and their role in the complex cellular response to DNA damage. Importantly, results presented in this dissertation support a regulatory mechanism involving CstF-50 and p97 control of the
BRCA1/BARD1 E3 Ub ligase activity, resulting in epigenetic control during DDR through the ubiquitination of RNAP II and histones. The presented evidence provides a broader mechanistic understanding of transcription-coupled RNA processing/DNA repair.
mRNA binding protein antigen R (HuR) is an AU-rich elements (ARE)-binding protein that increases mRNA stability and translation of its target mRNAs after stress. The implication of HuR in carcinogenesis is not surprising since dozens of HuR target mRNAs encodes tumor suppressors and DDR proteins. Understanding the complexity of the signaling in the 3’ untranslated region of genes and the functional interaction between the 3’ processing machinery and factors involved in DDR/tumor suppression might help us to understand cell-specific profiles, improving the developing of new therapies and the identification of cancer subtypes.
Furthermore, despite our understanding of the biochemistry of the BRCA1/BARD1 E3
Ub ligase, little is known about its cellular targets, how these targets are chosen and recruited to
DNA damage sites, and how this ligase is regulated during DDR. The findings presented in this
vi dissertation have answered some of these questions. A better understanding of how
BRCA1/BARD1 E3 ligase is involved in DNA repair, mRNA 3’ processing, the Ub pathway and chromatin remodeling will provide valuable opportunities for modulation of DNA repair and the development of new drugs to treat various human pathologies, including immunodeficiencies, neurodegenerative diseases, and cancer. As germline mutations of BRCA1 and BARD1 are present in most of inherited breast and ovarian cancer cases, it is easy to imagine that all the processes studied in this dissertation might be affected by deficiencies in BRCA1/BARD1 functions contributing to the development of those cancers. Finally, the studies presented in this dissertation will serve as a valuable framework both for understanding these critical biological processes and for developing appropriate therapeutic approaches to the varied disorders mentioned above.
vii
ACKNOWLEDGMENTS
It has been a long journey… since I first came to Hunter 10 years ago. I still remember the almost empty room I started working in, the same room that is now filled with so many memories, good experiences, lots of friendships, some difficulties and, of course, great research.
My gratitude to Frida, my mentor, is beyond words. Since day one she has supported me at so many levels: as her employee and student but most importantly as a friend. Her willingness to always find a way to overcome adversities no matter how many options we needed to try, took me to the point I am right now. Her warm, optimistic and hardworking character is unparalleled; it gave me and everybody around the best environment to develop and mature as scientists, as well as individuals.
To all my fellow students, co-workers and friends I have been so blessed to have such a great company along the way. Many thanks to Fathima, Murat, Nurit, Jorge, Xiaokan, and Emral we have shared so many things, from our rich scientific discussions to our personal happiness and also some sad moments and struggles. We have care and support each other all the way, that is why we are such a great team!
I have had the privilege to have known and trained so many talented high school and undergraduate students that I am afraid that if I try to list them all I am going to forget many names. From my very first undergrad Andrea Arata, who helped me unpack many of the equipment and reagents that we still have in the lab today, to my last ones Shadaqur Rahman and
Esraa Soliman, that have helped me to complete my dissertation project… thank you all!
I have been fortunate enough to have taught more than 300 students during these years, I also would like to thank them because when research was not very productive or rewarding, I truly found new inspiration and enthusiasm in the lab room with you all!
viii
Many thanks to all the members of the Chemistry Department, especially to the ladies in the chemistry office and stockroom that always made my life much easier. I am also indebted to
Biochemistry Assistant Program officer Judy Lee, former executive officer Dr. Lesley Davenport and current executive officer Dr. Edward Kennelly.
I am very appreciative with the members of my committee: Dr. Maria Pereira-Figueiredo,
Dr. Karen Hubbard, Dr. Hualin Zhong and Dr. Richard Baer for the guidance and support throughout my graduate years. Their helpful comments and insightful questions helped me to be able to complete this dissertation.
Last but certainly not the least, I am very grateful to my family, for all the love and encouragement from the distance, and to my beloved husband Richard, I thank him for his patience, support and love and specially for always remind me what is the most important thing of all……
ix
TABLE OF CONTENTS
Chapter I. Background. 1
DNA damage response (DDR) 2
Nucleotide excision repair pathway 3
Ubiquitin pathway 5
BRCA1/BARD1 ubiquitin ligase 14 mRNA processing and the cleavage stimulation factor CstF-50 19
The Ubiquitin pathway and mRNA processing machinery 23
Chapter II. The 3’ processing factor CstF functions in the DNA repair response under different cellular conditions. 26
Introduction 27
Results 30
Discussion 36
Chapter III. p53 inhibits mRNA 3’ processing through its interaction with the CstF/BARD1 complex. 42
Introduction 43
Results 45
Discussion 46
Chapter IV. Role of the polyadenylation factor CstF1 in regulating the BRCA1/BARD1 E3
Ubiquitin Ligase Activity. 51
Introduction 52
x
Results 54
Discussion 76
Chapter V. CstF-50 regulates the BRCA1/BARD1 Ub Ligase activity resulting in chromatin remodeling during DDR. 81
Introduction 82
Results 85
Discussion 93
Chapter VI. BRCA1/BARD1 associated CstF-50/p97 ubiquitinates the mRNA binding protein
Human Antigen R (HuR). 98
Introduction 99
Results 103
Discussion 109
Chapter VII. Future directions 113
Chapter VIII. Experimental procedures 123
Chapter IX. References 131
xi
LIST OF FIGURES
Figure 1: Mammalian nucleotide excision repair pathway. 5
Figure 2: The Ubiquitin pathway. 7
Figure 3: Consequences of different linkage types (signal structure) in the final fate of ubiquitinated substrates. 9
Figure 4: Structure of the proteasome. 11
Figure 5: Structure of the BRCA1/BARD1 heterodimer. 15
Figure 6: Schematic representation of the mammalian mRNA 3’ end formation. 20
Figure 7: CstF and CstF-50 structure. 22
Figure 8: Cells with reduced levels of CstF show enhanced sensitivity to UV treatment. 32
Figure 9: Cells with reduced levels of CstF show reduced ability to ubiquitinate RNAP II.
34
Figure 10: RNAP II, CstF and BARD1 localize to sites of repaired DNA. 36
Figure 11: Model for the role of CstF in the DNA damage response. 41
Figure 12: CstF-50 and BARD1 interact with the C-terminal domain of p53. 47
Figure 13: GST-CstF-50 can form a complex with Ub and BRCA1/BARD1 E3 Ub ligase .
56
Figure 14: Ub, CstF-50, BRCA1, BARD1 and RNAP II co-immunoprecipate from the NEs of
HeLa cells. 58
Figure 15: CstF-50 increases the autoubiquitination of ΔBRCA1/BARD1 heterodimer. 59
Figure 16: p97 and CstF-50 can form a complex. 62
Figure 17: p97 relocates from the cytoplasm to the chromatin after UV damage. 63
xii
Figure 18: p97 cellular localization in chromatin-bound fractions depends on BRCA1/BARD1 and CstF-50 expression under non damaging conditions. 64
Figure 19: The functional interaction between p97 and CstF-50 regulates BRCA1/BARD1 Ub ligase activity. 65
Figure 20: CstF-50 can activate the ubiquitination of RNAP IIO by BRCA1/BARD1 in vitro.
66
Figure 21: siRNA-mediated knockdown of CstF-50 expression decreases the UV-induced degradation of RNAP II. 67
Figure 22: CstF-50 activates the polyubiquitination of RNAP II in vivo 69
Figure 23: CstF-50 can activate the monoubiquitination of H2A and H2B by BRCA1/BARD1.
71
Figure 24: CstF-50 activates H2B and H2A ubiquitination in vivo. 73
Figure 25: H2B ubiquitination in chromatin-bound fractions depend on CstF-50 expression.
74
Figure 26: CstF-50 interacts directly with H2A and H2B in vitro. 75
Figure 27: p97 play a role in the ubiquitination of RNAPII and histones in different compartments. 77
Figure 28: Cellular fractionation in HeLa cells samples. 86
Figure 29: UV-treatment induces changes in the localization of BRCA1, BARD1, CstF-50 and some of its substrates in nuclear fractions. 87
Figure 30: UV-treatment induces changes in the localization of BRCA1, BARD1, CstF-50 and some of its substrates in nuclear fractions. 89
xiii
Figure 31: BRCA1/BARD1 and CstF-50 play a role in the ubiquitination of RNAP II and histones in different nuclear fractions. 90
Figure 32: BRCA1/BARD1/CstF-50 has a distinctive effect on chromatin structure of genes differentially expressed. 92
Figure 33: Model for the role of polyadenylation factor CstF-50, the Ub-escort factor p97 and the BRCA1/BARD1 Ub ligase during the progression of DDR. 96
Figure 34: Schematic representation of HuR structure. 100
Figure 35: BRCA1/BARD1 E3 Ub ligase can ubiquitinate HuR. 104
Figure 36: HuR is concomitantly ubiquitinated and phosphorylated. 105
Figure 37: HuR binds directly to the mRNA processing factor CstF-50. 106
Figure 38: HuR ubiquitination by BRCA1/BARD1 E3 Ub ligase is inhibited by GST-CstF-50.
107
Figure 39: Ubiquitination of HuR by BRCA1/BARD1 E3 Ub ligase is inhibited by His-p97.
108
Figure 40: HuR is concurrently monoubiquitinated and phosphorylated in nuclear extracts.
109
Figure 41: Model of regulation of p53 mRNA steady-states levels by CstF-50-regulated
BRCA1/BARD1 ubiquitination of HuR. 112
Figure 42: Analysis of quality and quantity of the recombinant proteins used in this study.
125
xiv
CHAPTER I
BACKGROUND
1
DNA DAMAGE RESPONSE (DDR)
Physical and chemical environmental agents, such as ultraviolet (UV) and ionizing radiation (IR), as well as chemicals present in food and from air pollution induce a wide a variety of DNA lesions. DNA damage has several diverse and adverse consequences from impeded gene transcription and DNA replication to cell cycle arrest and /or cell death. DNA lesions might interfere with cell division avoiding proper chromosome segregation and some aberrations might lead to carcinogenesis (Dinant et al., 2008; Nag and Smerdon, 2009). Besides, DNA damage results in transient alterations in the expression of affected genes due to transcription ((Khobta and Epe, 2012) and mRNA 3’ processing inhibition (Cevher and Kleiman, 2010).
To keep the integrity of the genome and counteract the severe biological effects of DNA damage the cell has evolved an intricate and sophisticated network of genome surveillance mechanisms or DNA damage response (DDR) processes. The center of this defense system is formed by several DNA repair mechanisms that correct different types of DNA insults. So far six major DNA damage repair pathways have been described in mammals: base excision repair
(BER) that removes damaged bases, homologous recombination (HR) and non-homologous end joining (NHEJ) eliminate breaks in the DNA backbone, mismatch repair (MR) that recognizes base incorporation errors and base damage, cross-link repair that removes interstrand cross-links, and nucleotide excision repair (NER) that removes bulky DNA adducts (Li et al., 2011; Sirbu and Cortez, 2013). As NER has been investigated in this dissertation, I will describe this repair mechanism in more detail.
2
NUCLEOTIDE EXCISION REPAIR PATHWAY
NER repairs a wide variety of single-strand helix distorting DNA lesions generated by
UV irradiation including cyclobutane pyrimidine dimers, pyrimidone photoproducts and bulky chemical adducts. NER is the most versatile of all repair mechanisms and it is also considered the main repair mechanism in the cell. In eukaryotes, around 30 proteins are involved in this pathway. NER is deficient in patients with Xeroderma pigmentosum syndrome, which includes mutations in XPA and XPG genes, and Cockayne syndrome, which includes mutations in CSA and CSB genes (Li et al., 2011; Nag and Smerdon, 2009). NER pathway consists of five basic steps including: damage recognition, DNA unwinding and incision of the damaged strand, excision of the damage-containing oligonucleotide, filling the gap by DNA polymerase, and final ligation. NER is further categorized into global genome repair (GGR) and transcription-coupled repair (TCR) depending on the factors involved in lesion recognition. GGR acts on the entire genome comprising unexpressed regions and non-transcribed strands of active genes. In the other hand, TCR preferentially removes DNA damage in transcriptionally active regions, particularly on the transcribed strand of actively expressed genes.
In GGR, XPC and UV-damaged DNA-binding protein (UV-DDB) complex recognize the
UV-induced lesions to later recruit XPA to the damage site (Lans et al., 2012; Scharer, 2013)
(Figure 1). On the other hand, the TCR subpathway is initiated by RNA polymerase II (RNAP II) physical blockage and triggers the subsequent recruitment of CSA and CSB that will help the loading of XPA to the site of damage. Although GGR and TCR defer in this first step of lesion recognition, the further steps occurs via a common pathway. The DNA at the damaged site is then unwind by XPB and XPD, which carry out 5’ to 3’ and 3’ to 5’ helicase activities, respectively. This step is followed by incisions on the 3’ and 5’ sides of the damage executed by
3 the endonucleases XPG and XPF-ERCC1, respectively, which lead to the removal of 25-30 nucleotides of single-stranded DNA. Then, the resulting gap is filled by the replication machinery, including DNA polymerases δ, Ƙ, and ε and proliferating cell nuclear antigen
(PCNA), the DNA synthesis is done using the complementary strand as a template, enabling error-free repair. Finally, the gap is sealed by DNA ligase III completing the NER process
(Figure 1).
Proper regulation of NER is crucial to ensure the fidelity of the genomic information. It has been demonstrated that post-translational modifications play important roles in regulating
NER activity, specifically ubiquitination has proven to be critical in this regulation either in a proteolysis dependent or independent manner. Ubiquitination directly regulates NER at different levels, XPC and DDB are ubiquitinated in the recognition step, ubiquitinated PCNA is required for the recruitment of polymerases involved in the gap filling, and in late phase of TCR the ubiquitination and degradation of CSB is needed for efficient transcription recovery ((Li et al.,
2011). As shown in this dissertation, RNAP II is ubiquitinated and degraded after UV irradiation
(Figures 9, 14, 21, 22) (Ratner et al., 1998). Although the UV-induced ubiquitination and degradation of RNAP II has been traditionally considered part of TCR pathway, in the last few years the consensus among scientist in the field is that these changes in RNAP II are so drastic that are considered an entirely separate pathway, which acts as an alternative to TCR (Wilson et al., 2013). As part of this pathway, a molecule of RNAP II that is permanently stalled at a UV- induced lesion and cannot reengage in transcription becomes ubiquitinated and degraded by the proteasome. This process is extremely regulated to ensure that the degradation will only affect those RNAP II molecules that cannot be salvaged. Even though TCR and degradation can be viewed as two independent pathways, they are still functionally interconnected.
4
GGR TCR
Damage
recognition
and incision and
DNA unwind unwind DNA
Excision Gap filling Gap ligation and
Figure 1: Mammalian nucleotide excision repair pathway. NER consist of 5 steps:
damage recognition, DNA unwinding and incision, excision of the damage patch, gap
filling by DNA polymerase δ and ε and ligation by DNA ligase III. Different initial
recognition complexes are established in the 2 subpathways of NER: Global genome
repair (GGR) and transcription-coupled repair (TCR) (Modified from (Lans et al., 2012).
THE Ub PATHWAY:
Protein modification by covalent conjugation of the ubiquitin (Ub) molecule is one of the most highly versatile and dynamic posttranslational modifications that control virtually all types
5 of cellular events. This process called ubiquitination was originally described as a mechanism by which cells disposed short-lived, damaged or abnormal proteins. This pathway plays a central regulatory role in a number of eukaryotic cellular processes such as transcription, cell-cycle control, growth factor signaling, gene silencing, receptor endocytosis, stress response, and DNA repair (Jadhav and Wooten, 2009).
Degradation of a protein via the Ub-proteasome pathway involves two successive steps: tagging of the substrate by covalent attachment of multiple Ub molecules followed by degradation of the tagged protein by the 26S proteasome complex with release of free and reusable Ub. This process is mediated by a multienzyme cascade and involves the formation of thiol esters with at least two, and, in most instances, three distinct types of enzymes: E1 (Ub‐ activating enzyme), E2 (Ub conjugating enzyme), and E3 (Ub ligase). A “middle part” of this pathway has been described, where escort protein p97 and other ubiquitin elongation factors named E4 guide the Ub-substrate to the proteasome (Halawani and Latterich, 2006; Koegl et al.,
1999). Protein ubiquitination occurs in a sequential manner via a three steps mechanism (Figure
2). The reaction starts with the formation of a thiol-ester linkage between the glycine residue at the C-terminus of Ub and the active Cys residue of the first enzyme of the system, E1, which activates Ub in an ATP-dependent manner. The Ub molecule is subsequently transferred to the cysteinyl group of the enzyme E2. Lastly, through the action of an Ub ligase E3, Ub and the putative substrate are linked together via an amide (isopeptide) bond; this conjugation involves attachment of C-terminal Gly of Ub to the ε-amino group in Lys residues of the targeted protein.
This ability of an E3 to recognize and bind both the target substrate and the Ub-E2 enzyme suggests that this enzyme provides specificity to the Ub reaction (Glickman and Ciechanover,
2002).
6
Conjugation
E4 HECT RING
or
p97 Cofactors “Middle” + E4
Degradation
Figure 2: The Ub pathway. Schematic showing the main steps of the Ub pathway:
conjugation of the substrate with Ub through the successive action of different enzymes
(E1, E2, E3, and E4), “middle” or escorting substrates through p97/VCP and other
associated cofactors and degradation through the 26S proteasome. The scheme also
includes a description of two different types of E3s: Homologous to E6-AP C Terminus
HECT) and Really Interesting New Gene (RING). Modified from (Ciechanover, 1998).
The ubiquitination reaction may result in the addition of a single Ub molecule to a single target site, which is known as monoubiquitination. However, as Ub is a 76 amino acid polypeptide (8.5 kDa) that harbors 7 Lys residues, the ubiquitination step can also add single molecules of Ub to several Lys in the target protein giving rise to multiubiquitination.
Alternatively, after the initial Ub is conjugated to a substrate, it can also be conjugated to another
7 molecule of Ub and an isopeptide bond can be formed between Gly76 of one Ub to the ε-NH2 group of one of those seven potential Lys (K6, K11, K27, K29, K33, K48 or K63) of the preceding Ub, giving rise to many different types of polyubiquitinated proteins (Adhikari and
Chen, 2009). These polyubiquitin chains can vary in length with respect to the number of Ub molecules, resulting in different topologies and, ultimately different functional consequences.
Both monoubiquitination and polyubiquitination possess non-proteasomal regulatory functions such as control of protein localization, enzyme activity and protein-protein interactions (Hershko and Ciechanover, 1998; Pickart, 2001) (Figure 3).
The formation of polyubiquitin chains by linkage of the carboxyl terminus of a new Ub molecule to Lys‐48 of the last Ub moiety of the chain has been shown to mark a protein for proteolysis by the 26S proteasome (Chau et al., 1989). However, it has been shown that polyubiquitin chains are also assembled through conjugation to Lys residues of Ub other than
Lys‐48, and the resulting chains seem to function in distinct biological processes. For example, a polyubiquitin chain composed of Lys‐63–linked Ub moieties has been implicated in different cellular processes such as ribosomal function (Spence et al., 2000), signal transduction
(Mukhopadhyay and Riezman, 2007; Sun and Chen, 2004), the stress response (Arnason and
Ellison, 1994), and DNA repair (Hofmann and Pickart, 1999; Spence et al., 1995). Interestingly,
K63-chains have also been shown to serve as a targeting signal for the 26S proteasome (Saeki et al., 2009; Seibenhener et al., 2004). Another mode of conjugation involves linking Ub molecules via Lys-29 (Mastrandrea et al., 1999; You and Pickart, 2001). Moreover, the formation of multiubiquitin chains linked via Lys-6, or Lys-11 have also been described. Although these types of chains can bind to the proteasomal subunit Rpn10/S5a, it is still not clear if they can actually target substrates for degradation (Baboshina and Haas, 1996). To complicate things even more, it
8 has been reported that non Lys moieties can serve as Ub acceptor sites as well, the fusion of the first Ub moiety occurs at their NH2-terminal residue rather than in an internal Lys. Examples of ubiquitination occurring at this noncanonical site have been described for the latent membrane protein-1 of Epstein-Barr virus (Aviel et al., 2000), transcription factor MyoD (Breitschopf et al.,
1998), and p21 (Bloom et al., 2003).
Figure 3: Consequences of different linkage types (signal structure) in the final fate
of ubiquitinated substrates. The linkages through different Ub lysine residues of the
corresponding polyUb chains are denoted by distinctive color squares, as defined in the
arrows on the left. Taken from Pickart and Fushman (2004).
Once marked by polyubiquitin chains, proteins are rapidly degraded by the 26S proteasome
(Figure 4). The proteasome holoenzyme is a large 2.5 MDa ATP-dependent multi-catalytic
9 protease complex made up of two copies each of at least 32 different subunits that are highly conserved among all eukaryotes that degrades poly-ubiquitinated proteins to small peptides. This large structure contains the central 20S proteasome, in which proteins are cleaved, and two 19S complexes, which provide substrate specificity and regulates the function of the former.
Specifically, it is composed of two subcomplexes: a 20S core particle (CP) that carries the catalytic activity and a regulatory 19S regulatory particle (RP). The 20S CP is a barrel-shaped structure composed of four stacked rings of seven subunits each, two identical outer α-rings and two identical inner β-rings. Certain β subunits contain the proteases active sites that are oriented facing inward into the proteolytic chamber. Each extremity of the 20S barrel can be capped by a
19S RP. On the other hand, the 19S RP itself can be further dissected into two multisubunit substructures, a lid and a base. The base containing the proteasomal ATPases attaches to the α- ring of the CP. The 19S RP recognizes the polyubiquitinated proteins and opens a channel to deliver the target protein to the 20S core particle (Glickman and Ciechanover, 2002).
Ubiquitination is a reversible and highly regulated process which is controlled by the opposing activities of the E3 Ub ligases that attach Ub molecules covalently to target proteins and de-ubiquitinating enzymes (DUBs) that remove the Ub from target proteins (Wilkinson,
1997). Reversible covalent modification allows cells to rapidly and efficiently convey signals across different sub-cellular locations and different functional pathways. A simplified view of the hierarchical structure of the Ub conjugation machinery is that a single E1 activates Ub for all conjugation reactions and transfers it to all known E2s. Typically, each E2 interacts with several
E3s and each E3 targets several substrates. The interactions of the ligases among themselves and with many of the target substrates may differ from this “classical” cascade. For instance, a single
10
Figure 4: Structure of the proteasome. The proteasome is a large catalytic protease
complex composed by a 20S subunit that carries the catalytic activity and 19S subunits
that functions in substrate recognition (Modified from Glickman and Ciechanover, 2002).
E1 can interact with 2 distinct E2s and a single E3 can have several distinct recognition sites targeting different classes of substrates. Additionally, some substrates can be targeted by different E3s, recognizing different motifs. Therefore, this hierarchy is more complicated and cannot be viewed simply as a pyramid structure, but rather as a very complex network of overlapping interactions between multiple components. Finally, a single enzyme, the proteasome, degrades all substrates targeted for proteolysis by ubiquitination (Glickman and
Ciechanover, 2002). It has been predicted that the human genome encodes three Ub-protein E1 enzymes, about fifty Ub-protein E2 conjugating complexes, over 600 Ub ligases and about 100
DUBs (Kaiser and Huang, 2005).
Among the distinct enzymes implicated in the Ub pathway, the Ub-protein ligases E3s are the last but not the least in this enzymatic cascade and most likely they are the most important components in the Ub conjugation system because they play an important role in controlling target specificity. The E3s recruit target proteins, position them for optimal transfer
11 of the Ub moiety from the E2 to a Lys residue in the target protein, and initiate the conjugation; therefore, they are able to bind both the E2 and the substrate. Besides, the interaction with the substrate can be direct or via ancillary proteins (Glickman and Ciechanover, 2002; Jadhav and
Wooten, 2009). Ub E3 ligases can be either monomeric proteins or multimeric complexes with the most common type of Ub ligases grouped into two classes depending on their modular architecture and catalytic mechanism. First, typically the E3s that contain a HECT domain
(Homologous to E6-AP C Terminus) form a direct thioester bond with Ub. Their approximately
350 amino acid HECT domains contain a conserved Cys residue that participates in the direct transfer of activated Ub from the E2 to a target protein. On the other hand, RING (Really
Interesting New Gene) finger domain ligases consist of conserved Cys and His residues that form a cross-brace structure that coordinate two Zn++ ions. The globular architecture of this domain primarily functions as a scaffold or bridge that position the substrate close to the E2s and their target proteins allowing optimal Ub transfer. Hence, these ligases require a structural and/or catalytic motif that facilitates ubiquitination without directly forming a bond with Ub. RING finger domain containing E3s comprise the largest ligase family, and contain both monomeric and multimeric Ub ligases. (Hershko and Ciechanover, 1998; Pickart, 2001).
Many E3 ligases are known to interact directly with their specific substrates or the specificity could be provided by scaffold proteins. These types of proteins facilitate the interaction between the E3 enzymes and their substrates through their multi-domain architecture.
One clear example of such scaffold proteins is p62. This protein acts as a scaffold by interacting with the RING E3 TRAF6 as well as other proteins (Wooten et al., 2001). Some of the substrates that have been shown to be K63- polyubiquitinated by the TRAF6/p62 complex are the tyrosine kinase receptor A (Geetha et al., 2005a) and the neurotrophin receptor interacting factor (Geetha
12 et al., 2005b). The carboxy-terminal domain of p62 has been shown to non-covalently bind Ub
(Mueller and Feigon, 2002). Additionally, p62 can function as an escort factor for polyubiquitinated substrates by binding the ubiquitinated proteins and the 26S proteasome
(Wooten et al., 2005).
It has been shown that some proteins function as “Ub chain elongation factors” catalyzing polyubiquitin chain formation (Imai et al., 2002; Koegl et al., 1999; Matsumoto et al.,
2004; Wu and Leng, 2011b; Wu et al., 2011). This group of enzymes named E4s seems to be an additional subset of E3s that serves as scaffold/adaptor to help in the transfer of Ub from the E2 to a previously conjugated substrate with one Ub moiety. They can also elongate short polyubiquitin chains by mediating transfer of Ub to a previously conjugated Ub molecule rather than to the substrate itself, resulting in a fully elongated polyubiquitin chain (Koegl et al., 1999).
An E3 Ub ligase would still be needed in this scenario to attach the first Ub to the substrate.
Additionally, some of them can also function as Ub ligases independent of the action of an E3 in vitro (Glickman and Ciechanover, 2002). However, the lack of evidence of direct interaction between E4s and E2 enzymes and the inability of E4 enzymes to replace an E3 ligase in vivo strongly suggests that E4 represents a novel class of enzymes with an activity distinct from E3 ligases (Hoppe, 2005). Furthermore, it has been shown that the E4 enzyme UFD2 cooperates with a set of Ub-binding factors in an escort pathway to transfer and deliver polyubiquitinated substrates to the 26S proteasome (Kuhlbrodt et al., 2005). E4-dependent polyubiquitination seems to be tightly controlled by a group of different Ub-binding proteins. This regulation causes not only the restriction in the number of Ub molecules added by the E4 enzyme to the growing polyubiquitin chain, but also in the subsequent targeting of the substrate to the proteasome
(Richly et al., 2005). Such an escort mechanism can provide another layer of regulation and
13 specificity in the Ub system (Kuhlbrodt et al., 2005). The escort pathway has helped to understand an unclear part of the Ub pathway that has been defined as the “middle part” of the
Ub-proteasome pathway (Bays and Hampton, 2002; Jentsch and Rumpf, 2007; Richly et al.,
2005; Rumpf and Jentsch, 2006). The main protein involved in this pathway is p97/VCP that can directly bind both the proteasome and the polyubiquitinated substrates (Dai and Li, 2001).
p97 is a conserved chaperone-like ATPase in eukaryotic cells. Its primary function is to bind ubiquitinated substrates and segregate them from their binding partners or protein complexes, and escort them to the proteasome. p97 is also able to control the degree of ubiquitination of the bound substrates by binding to substrate-processing cofactors that either regulate polyubiquitination or deubiquitination of the bound polyubiquitinated protein (Jentsch and Rumpf, 2006). Besides, p97 interacts physically with BRCA1 in the nucleus and participates in DDR as an ATP transporter, possibly facilitating TCR (Zhang et al., 2000). Importantly, it has been recently described that p97 play an important role in ubiquitin-dependent eviction from chromatin during DDR ((Dantuma and Hoppe, 2012).
BRCA1/BARD1 E3 Ub LIGASE:
One interesting example of the E3 family is provided by the heterodimer formed by tumor suppressors BRCA1 and BARD1, which are the first RING-dependent Ub ligases described. BRCA1 and BARD1 together exhibit strong E3 Ub ligase activity, and the complex catalyze its autoubiquitination as well as the transubiquitination of other substrates (Chen et al.,
2002). The BRCA1 gene encodes a protein of 1,863 amino acids with a molecular weight of 220 kDa (Miki et al., 1994). This protein consists of an amino-terminal RING finger domain, an E2 binding motif, and two tandem BRCT domains in its C-terminal region (Figure 5). Its RING
14 finger constitutes the interaction platform with E2 UbcH5 and BARD1. BRCA1 plays an important role in multiple biological pathways that regulate cell-cycle progression, centrosome duplication, cell growth, apoptosis, transcriptional regulation, and DNA damage repair. (Deng and Brodie, 2000). Germline mutations in BRCA1 are present in nearly 50% of inherited breast cancer cases indicating that BRCA1 is an important tumor-suppressor gene (King et al., 2003).
BRCA1 associates with a myriad of different proteins whose interactions are not limited to the
RING and BRCT domains. These proteins include transcription factors, chromatin-modifying proteins, oncogenes and proteins involved in DDR (Scully and Livingston, 2000).
One of the most important BRCA1-interacting proteins is BARD1 (BRCA1-associated
RING domain 1). BARD1 is a 97 kDa protein that has a similar conformation as BRCA1: a
RING finger in the N-terminal domain and C-terminal tandem BRCT domains (Figure 5) (Wu et al., 1996). The association of BRCA1 and BARD1 is required for the E3 Ub ligase activity that is the only biochemical activity ascribed to BRCA1 so far (Baer and Ludwig, 2002). BARD1 markedly increases the intrinsically low ligase activity of BRCA1 and is required for the function
CstF-50
Figure 5: Structure of the BRCA1/BARD1 heterodimer. Schematic of BRCA1 and
BARD1 composition showing the RING and BRCT domains for both proteins, and
ankyrin repeats for BARD1. The BARD1 binding site for CstF-50 is also depicted
(Modified from (Baer and Ludwig, 2002).
15
of this tumor suppressor protein in protecting genomic integrity (Chen et al., 2002; Hashizume et al., 2001).The increase in BARD1-dependent BRCA1 E3 activity is attributed to the fact that
BARD1 interaction is required to stabilize the proper conformation of the BRCA1 RING domain for E3 activity (Brzovic et al., 2003). Another hypothesis for the mechanism of BARD1- mediated activation is that the RING domain of BARD1 helps to stabilize the interaction between the RING domain of BRCA1 and UbcH5 (E2) (Chen et al., 2002). Interestingly, mutations related to ovarian and breast cancer have been described for BARD1 (Thai et al.,
1998).
The fact that BRCA1/BARD1 heterodimer possesses Ub ligase activity has helped us to understand how BRCA1 functions, and how it elicits its tumor suppressor activity. The ubiquitination activity is dependent on critical residues within the RING finger of BRCA1 that are found to be mutated in breast and ovarian tumors (Hashizume et al., 2001; Ruffner et al.,
2001). Those mutations in BRCA1 abolish the Ub ligase activity of the BRCA1/BARD1 heterodimer affecting the interaction between the two proteins (Hashizume et al., 2001). As many mutations are also found on the RING domain of BARD1 (Thai et al., 1998), it is probable that their Ub ligase activity is important for their DNA-repair and tumor suppressor activities
(Huang and D'Andrea, 2006). Unexpectedly, it has been reported that BRCA1 tumor suppression depends on its BRCT domain and not its RING domain, indicating that the E3 ligase activity of
BRCA1 is dispensable for tumor suppression (Shakya et al., 2011).
Several putative substrates of BRCA1/BARD1 have been identified: histones (Mallery et al., 2002), γ-tubulin (Starita et al., 2004), estrogen receptor α (ERα) (Eakin et al., 2007), nucleophosmin/B23 (Sato et al., 2004), RNAP II (Kleiman et al., 2005; Starita et al., 2005), CtIP
16
(Yu et al., 2006), progesterone receptor-A (PR-A) (Poole et al., 2006), transcription factor TFIIE
(Horwitz et al., 2007), Topoisomerase IIα and Npm1 (Starita and Parvin, 2006), SAFB2 and Tel2
(Song et al., 2011), CSB (Wei et al., 2011), Claspin (Sato et al., 2012), and cyclin B and Cdc25C
(Shabbeer et al., 2013). Despite our understanding of the biochemistry of the BRCA1/BARD1
E3 ligase, we know very little of its cellular targets, how these targets are chosen and the mechanism by which they are recruited to DNA damage sites. In fact, the biological consequences for the ubiquitination of the above substrates are not completely understood.
It has been shown that BRCA1/BARD1 complex interacts in vitro and in intact cells with cleavage stimulation factor 50 (CstF-50) and this interaction inhibits polyadenylation following
UV damage (Kleiman and Manley, 1999, 2001). Supporting the physiological significance of these results, a previously identified tumor-associated germline mutation in BARD1 (Gln564His) reduced binding to CstF and abrogated inhibition of polyadenylation. The interaction between the CstF-50 WD-40 domain and BARD1 involves the ankyrin-BRCT linker but do not require ankyrin or BRCT domains (Edwards et al., 2008; Kleiman and Manley, 1999) (Figure 5).
Interestingly, the UV-induced inhibition of polyadenylation results not simply from the
CstF/BARD1/BRCA1 interaction but also from proteasome-mediated degradation of RNAP II, which is an activator of polyadenylation (Hirose and Manley, 1998; McCracken et al., 1997) and a BRCA1/BARD1 substrate (Kleiman et al., 2005; Starita et al., 2005). It has been suggested that
BRCA1/BARD1, as part of the RNAP II holoenzyme (Chiba and Parvin, 2002), senses sites of
DNA damage and repair, and the inhibitory interaction with CstF ensures that the nascent RNAs are not erroneously polyadenylated at such sites, avoiding the expression of deleterious proteins.
These functional interactions provide a link between transcription-coupled RNA processing and
DDR (Kleiman and Manley, 1999; Mirkin et al., 2008).
17
In living cells, most of BRCA1 exists in association with BARD1, suggesting that these proteins function together as a heterodimeric complex possessing RING-dependent Ub ligase activity (Chen et al., 2002). Previous experiments indicated that the BRCA1/BARD1 heterodimer catalyses its own ubiquitination by synthesizing a novel type of polyubiquitin chains through
Lys6 (Nishikawa et al., 2004; Wu-Baer et al., 2003), which stabilizes BRCA1 and potentiates its
E3 Ub ligase activity by more than 20-fold (Mallery et al., 2002) and represents a regulatory positive feedback mechanism for the heterodimer activity. It has been proposed that the activation of its E3 Ub ligase activity might occur either by allosteric changes in the heterodimer due to autoubiquitination or changes in the polyubiquitin chains themselves that are able to increase the affinity of BRCA1/BARD1 for its substrates or for its corresponding E2s, enhancing in this way the Ub transfer rate (Mallery et al., 2002). Another hypothesis proposed for this activation is that the RING domain of BARD1 helps to stabilize the interaction between the
BRCA1 RING domain and the E2 (Chen et al., 2002). Those studies indicate that autoubiquitination does not target the heterodimer to proteasomal degradation (Chen et al., 2002).
Instead, it might serve as a signaling event such as in DNA repair or in regulating the BARD1- mediated inhibition of mRNA polyadenylation after DNA damage (Kleiman and Manley, 2001).
In fact, it was recently found that HERC2 is the E3 Ub ligase that targets BRCA1 for degradation by the proteasome, and this degradation occurs in BARD1-uncoupled BRCA1 (Wu et al., 2010).
Interestingly, autoubiquitinated BRCA1/BARD1 complex has also an increased affinity for binding to DNA as well as for DNA repair intermediates (Simons et al., 2006). However,
BRCA1 and BARD1 stabilize each other not only by the interaction of their RING domains but also through the BRCT domain of BRCA1, indicating that ubiquitination might not be the only aspect involved in the stability of the complex. For example, BRCA1/BARD1 interaction blocks
18 a nuclear export signal situated near the BRCA1 RING domain causing the retention of the heterodimer in the nucleus (Rodriguez et al., 2004).
The heterodimer E3 Ub ligase activity can be down-regulated by cyclin-dependent kinase
2 (CDK2), during the cell cycle causing its degradation and nuclear export (Hayami et al., 2005).
It has also been shown that UBX domain protein 1 (UBXN1), negatively regulates the
BRCA1/BARD1 enzymatic function in an ubiquitination status-dependent manner (Wu-Baer et al., 2010). Curiously, UBXN1 recognizes autopolyubiquitinated BRCA1/BARD1 complex through a bipartite interaction in which its C-terminal domain binds the heterodimer in an Ub- dependent manner and the UBA domain interacts with the K6-poly Ub chains. Even though
BRCA1/BARD1 heterodimer has been extensively studied for many years, so far not a single activator has been described for its E3 Ub ligase activity.
mRNA PROCESSING AND THE CLEAVAGE STIMULATION FACTOR CstF-50
Almost all eukaryotic mRNA precursors undergo a co-transcriptional modification at the
3' end. The mRNA 3’ end processing includes a two-step reaction: an initial cleavage step followed by the synthesis of a 200-adenosine residue tail to the 3’ end of the cleaved product
(Shatkin and Manley, 2000; Zhao et al., 1999). Polyadenylation plays a fundamental role in regulating mRNA stability, translation and nuclear export, and thus is essential for the proper control of mRNA levels and of gene expression in eukaryotes. One of the first steps of the reaction is the recognition of the highly conserved hexamer AAUAAA located at 10 to 30 nucleotides upstream of the cleavage site by the cleavage and polyadenylation specific factor
(CPSF) and of the G/U- and U-rich region located further downstream by the cleavage stimulation factor (CstF) (Takagaki and Manley, 1997). While a relatively simple signal
19 sequence in the precursor mRNA is required for the reaction, many diverse and specific interactions between a large number of protein factors are involved in the formation of polyadenylation complex and regulation of 3’ end processing in different tissues and in different cellular conditions. While CPSF, CstF, cleavage factors 1 and 2 (CF I and CF II), RNAP II and poly(A) polymerase (PAP) play a role in the cleavage reaction; CPSF, PAP, symplekin and poly(A) binding protein (PABP) are involved in the polyadenylation step.
Figure 6: Schematic representation of the mammalian mRNA 3’ end formation. A)
The cleavage step of mRNA 3’ end processing is initiated by the assembly of cleavage
complex through a cooperative binding of CstF at the G/U- and U-rich region and CPSF
at the AAUAAA signal. CPSF directly interacts with CstF and PAP (not shown). CF I,
CF II and RNAP II also play a role in the cleavage reaction. B) After the cleavage step,
CPSF and PAP remain bound to the cleaved RNA and elongate a 200-adenosine residue
poly(A) tail to the 3’ end of the cleaved product in the presence of PABP. Taken from
(Russell, 2009).
20
CstF is one of the essential 3’ processing factors required for the endonucleolytic cleavage step and helps to specify the site of RNA processing. CstF is a heterotrimeric protein consisting of subunits CstF-50 (50 kDa), CstF-64 (64 kDa) and CstF-77 (77 kDa). CstF-64 is largely responsible for RNA binding (MacDonald et al., 1994; Takagaki and Manley, 1997), while CstF-77 is needed for interactions with other polyadenylation factors (Takagaki and
Manley, 1994).The third subunit, CstF-50, plays important roles in regulation of mRNA processing by interacting with other factors. It contains seven WD-40 repeats, which are characteristic of regulatory proteins (Neer et al., 1994) and are involved in protein-protein interactions (Figure 6) (Takagaki et al., 1992).
CstF-50 has been shown to interact with the C-terminal domain of RNAP II largest subunit (RNAP II LS), likely facilitating the RNAP II-mediated activation of mRNA 3’ processing (Hirose and Manley, 1998; McCracken et al., 1997). It has also been shown that CstF-
50 interacts with BARD1 (Kleiman and Manley, 1999) (Figure 14), Ub (Figure 13-14) and with the DNA replication/repair factor PCNA (Kleiman and Manley, 1999). BARD1-mediated inhibition of mRNA polyadenylation and degradation of RNAP II are abrogated in BARD1 phosphorylation-deficient mutants, suggesting that phosphorylation of BARD1 is critical for the
DNA damage functions of the BRCA1/BARD1 complex (Kim et al., 2006). CstF-50 also interacts with C-terminal domain of the deadenylation factor PARN following UV-induced DNA damage, and this is accompanied with the activation of PARN-mediated deadenylation and the inhibition of 3’ cleavage as a result of repressed CstF activity (Cevher et al., 2010). Recently, it has been shown that CstF-50 associates with the tumor suppressor p53 (Nazeer et al., 2011).
These two proteins can coexist in complexes with BARD1 in extracts of UV-treated cells. As part of those studies, it has been described that p53 can inhibit mRNA 3’ cleavage and that there is a
21 reverse correlation between the levels of p53 expression and the levels of mRNA 3’ cleavage under different cellular conditions.
Previous reports have also shown that the DNA-damage induced inhibition of polyadenylation correlates with increasing amounts of a CstF/BARD1/BRCA1 complex formation (Kleiman and Manley, 2001). As mentioned before, the UV-induced inhibition of 3’ cleavage results not simply from the CstF/BARD1 interaction but also from the proteasome- mediated degradation of RNAP II (Kleiman and Manley, 2001; Kleiman et al., 2005), which is an activator of polyadenylation (Hirose and Manley, 1998; McCracken et al., 1997). These
CstF
CstF-64 2 3 ← CstF-50 1 CstF-77
CstF-50
Figure 7: CstF and CstF-50 structure. CstF is a heterotrimeric protein with three
subunits: CstF-50, CstF-64 and CstF-77. In the cleavage reaction, CstF-64 is responsible
for G/U- and U-rich region recognition and RNA binding, while CstF-77 directly
interacts with CPSF-160. The other subunit, CstF-50, contains 7 WD-40 repeats that are
characteristics of regulatory proteins. CstF-50 mediates various protein-protein
interactions to regulate the cleavage step in the polyadenylation reaction (Mandel et al.,
2008).
22 studies suggest the existence of several, possibly redundant, mechanisms to explain the inhibitory effect of UV irradiation on mRNA 3’ processing. Supporting this data, the siRNA- mediated depletion of both BRCA1 and BARD1 prevented degradation of RNAP II and rescued the inhibition of 3’ cleavage after DNA damage (Kleiman et al., 2005). It has been shown that the large subunit of RNAP IIO is a specific target of the BRCA1/BARD1 heterodimer (Kleiman et al., 2005). It has also been shown that CstF plays a direct role in the DDR (Mirkin et al.,
2008). Cells with reduced levels of CstF display decreased viability following UV treatment, reduced ability to ubiquitinate RNAP II, and defects in repair of DNA damage. These studies indicate that CstF plays an active role in DDR, providing a link between transcription-coupled
RNA processing and DNA repair.
As also mentioned before, CstF-50 can bind not only to BRCA1/BARD1 but also to Ub and RNAP II (Figures 13-14), which is a BRCA1/BARD1 substrate. Interestingly, the inhibition of mRNA 3’ processing and the ubiquitination/degradation of RNAP II occur as part of the
DDR. Based on these studies, it is easy to propose that CstF-50 might play a role in the assembly or stabilization of the ubiquitination complex, linking transcription-coupled RNA processing and
DNA repair to the Ub-proteasome pathway.
THE UB PATHWAY AND mRNA PROCESING MACHINERY
The Ub pathway is of special relevance in the epigenetic control of gene expression by regulating either transcription or mRNA processing. The interplay between histone modifications and mRNA processing, particularly alternative mRNA splicing, is now well documented. For example, U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 (U2AF65), an essential pre-mRNA splicing factor, inhibits the ubiquitin-dependent proteolysis of the human
23 telomeric protein TRF1 (Kim and Chung, 2014). U2AF65 stabilizes TRF1 protein by inhibiting the interaction between TRF1 and Fbx4 E3 Ub ligase. These findings suggest that U2AF65 is a positive regulator of TRF1 protein level and represents a new pathway for modulating TRF1 function at telomeres. Another example is the homeostatic regulation of Serine/Arginine rich
Splicing Factor 1 (SRSF1) protein levels (Moulton et al, 2014). Recently, it has been shown that
T cell activation in normal and systemic lupus erythematosus cells induces a rapid and significant increase in SRSF1 mRNA levels, but this increase is not reflected at the protein level.
Control of SRSF1 protein levels during the early activation phase is regulated through ubiquitination and proteasome-mediated degradation upon stimulation. This study also suggested that very likely ubiquitination of SRSF1 also regulates other aspects of SRSF1 function such as cellular localization and activity (Moulton et al, 2014).
Many studies have shown that histone ubiquitination is functionally linked to mRNA processing. For example, Swd2, a component of both the methyltransferase complex and the cleavage and polyadenylation factor CPF, has been shown to be the link between H2B ubiquitination and H3 methylation in yeast (Mellor, 2008; Vitaliano-Prunier et al., 2008). It has been suggested that different levels of H2B ubiquitination could serve as a switch controlling the amount of Swd2 in either complex, favoring either the balance towards methylation at the end of genes or mRNA processing and termination (Mellor, 2008; Vitaliano-Prunier et al., 2008).
Another report showed that the cap binding complex facilitates pre-mRNA splicing of one component of the H2B Ub protease machinery by modifying the ubiquitination state of H2B, indicating that the ubiquitination and the mRNA processing pathways can be mutually regulated
(Hossain et al., 2009). Other studies have shown that CDK9-mediated monoubiquitination of
H2B regulates 3’ end processing of histones mRNAs (Pirngruber et al, 2009). The CDK9-
24 dependent histone modification might influence the accessibility of the newly synthesized mRNA or the recruitment of 3’ end processing machinery by modifying the chromatin structure.
Interestingly, Ub ligases that bind RNA have been described and predicted to bind and regulate RNA stability, linking ubiquitination with mRNA turnover (Cano et al., 2010). E3 Ub ligase hRUL138/2A-HUB possesses a RING domain-E3 activity and a RNA-binding region, suggesting that both are likely mechanistically linked. It has been shown that this E3 Ub ligase has a cytoplasmic function regulating mRNA stability and a nuclear one acting as a transcriptional repressor (Kreft and Nassal, 2003; Zhou et al., 2008). Another RNA-binding
RING-dependent Ub ligase is CCR4-NOT, it has also cytoplasmic and nuclear functions, the first one being regulation of mRNA turnover through its deadenylase activity and the second one controlling mRNA synthesis (Morita et al., 2009). An additional example is MDM2, a RING E3
Ub ligase that targets p53 to proteasomal degradation as well as stimulates p53 translation by binding to p53 mRNA (Elenbaas et al., 1996; Naski et al., 2009). Although MDM2 does not possess a defined RNA-binding motif; it binds RNA through its RING domain. It has also been demonstrated that this interaction suppresses its E3 Ub ligase activity. However, how these
RNA-binding E3 Ub ligases play a role in different mRNA-associated functions is still not known.
25
CHAPTER II
THE 3’ PROCESSING FACTOR CstF-50 FUNCTIONS IN THE DNA DAMAGE
RESPONSE (DDR)
26
INTRODUCTION
The cellular response to DNA damage involves changes in the properties of a number of nuclear proteins, resulting in coordinated control of gene expression and DNA repair. One example is provided by the transient decrease in mRNA levels following UV irradiation
(Hanawalt, 1994; Ljungman et al., 1999). Although the mechanism underlying this response is still unresolved, it has been suggested that the UV-induced inhibition of transcription, reflecting turnover of the RNAP II largest subunit (RNAP II LS), is responsible for the decrease (Donahue et al., 1994). This indeed is likely a significant part of the mechanism. However, those studies have not considered the important effect of RNA processing on mRNA levels. Indeed, it has been shown that processing of mRNA precursors, and specifically 3’ end formation, is also affected by DNA damage. Previous data indicated that mRNA polyadenylation in cell extracts is strongly but transiently inhibited following treatment of cells with DNA damage inducing agents
(Kleiman and Manley, 2001). These results suggested a functional interaction between RNA processing and DNA repair.
The poly(A) tail found on almost all eukaryotic mRNAs plays important roles in regulation of mRNA stability, translation and RNA transport from the nucleus (Anderson, 2005;
Mangus et al., 2003; Neugebauer, 2002). The polyadenylation reaction consists of an endonucleolytic cleavage followed by synthesis of the poly(A) tail (reviewed in (Colgan and
Manley, 1997; Shatkin and Manley, 2000; Zhao et al., 1999). While a relatively simple signal sequence in the mRNA precursor is required for the reaction, a surprisingly large number of protein factors are necessary for 3’ processing. Cleavage stimulation factor (CstF) is one of the essential 3’ processing factors. Genetically modified chicken B cells deficient in CstF-64, a CstF subunit, undergo cell cycle arrest and apoptotic death (Takagaki and Manley, 1998). Another
27 subunit, CstF-50, has been shown to interact with the C-terminal domain of the RNAP II LS
(CTD), likely facilitating the RNAP II-mediated activation of 3’ processing (Hirose and Manley,
1998; McCracken et al., 1997). The stimulatory role of RNAP II in polyadenylation highlights the link between RNA processing and transcription. This link is supported by a variety of chromatin immunoprecipiation experiments documenting the association of polyadenylation factors with transcribed genes (e.g. (Calvo and Manley, 2005; Kim et al., 2004; Venkataraman et al., 2005).
Links between mRNA 3’ processing and other nuclear events have also been described.
For example, the association between CstF and the BRCA1/BARD1 tumor suppressor complex was uncovered and characterized. The direct interaction between CstF-50 and BARD1 inhibits 3’ processing in vitro (Kleiman and Manley, 1999). The complex formation increases transiently following DNA damage-inducing treatments and results in inhibition of 3’ processing in extracts from the treated cells (Kleiman and Manley, 2001). It has also been shown that DNA damage- induced BARD1 phosphorylation is critical for inhibition of polyadenylation and RNAP II LS degradation (Kim et al., 2006). After UV treatment, a fraction of RNAP II LS is phosphorylated, ubiquitinated and degraded by the proteasome (reviewed by (Muratani and Tansey, 2003; van den Boom et al., 2002). While UV-induced turnover of RNAP IIA, the form engaged at promoters, occurs by phosphorylation and conversion to RNAP IIO, turnover of RNAP IIO, which functions in elongation, occurs by ubiquitination and degradation (Lee et al., 2002; Luo et al., 2001; McKay et al., 2001; Mitsui and Sharp, 1999; Ratner et al., 1998; Woudstra et al.,
2002). Dr. Kleiman and colleagues showed that degradation of RNAP IIO in fact contributes to inhibition of 3’ processing in response to DNA damage (Kleiman et al., 2005), suggesting the existence of another, possibly redundant, mechanism to explain the inhibitory effect of UV
28 irradiation. As part of those studies, I determined that both BRCA1 and BARD1 are necessary for ubiquitination of RNAP II LS and its turnover in response to UV treatment (Kleiman et al.,
2005; Starita et al., 2005). UV-induced turnover of RNAP II is part of the transcription-coupled repair (TCR) response (reviewed by (Muratani and Tansey, 2003; van den Boom et al., 2002).
TCR is a pathway that operates on certain types of DNA damage found in the transcribed strand of expressed genes. Accumulated evidence suggests that the blockage of elongating RNAP II at sites of DNA damage is an early event that initiates TCR. Levels of mRNA are transiently decreased, and normal recovery depends on TCR (Derheimer et al., 2005; Hanawalt, 1994;
Ljungman et al., 1999; Mullenders, 1998). One of the earliest indications of the existence of
TCR was the key observation that when mammalian cells are exposed to UV light, RNA synthesis resumes before any significant amount of UV-induced damage is removed from the bulk of the genome by global genome repair (Mellon et al., 1987). One reason for this may be that TCR serves to repair transcription-blocking lesions and, therefore, to facilitate a rapid recovery of transcription. Transcription complexes can be extremely stable when they are stalled at endogenous pause sites or at sites of damage (Svejstrup, 2002). It has been suggested that
RNAP II stalled at sites of DNA damage could respond in either of two ways. If the lesion is repaired rapidly, RNAP II re-engages and continues transcription, but if the lesion persists,
RNAP II is ubiquitinated and degraded (Brueckner et al., 2007; Tornaletti et al., 2003; Woudstra et al., 2002). Stalling and/or degradation of RNAP II have another potential function: to prevent transcription across sites of DNA repair and thereby prevent formation of potentially deleterious proteins. However, this could result in release of prematurely terminated transcripts, and inhibition of the 3’ processing machinery would then function to prevent polyadenylation and stabilization of such RNAs.
29
Extending the studies on the functional links between mRNA 3’ processing and DNA repair pathways, Dr. Mirkin and colleagues (2008) found that UV treatment affects both transcription and polyadenylation of nascent mRNAs, resulting in the transient formation of prematurely terminated polyadenylated RNAs in the absence of the BRCA1/BARD1/CstF-50 complex. They also showed that CstF depletion causes defects in DNA damage repair and significant delays in TCR. As part of those studies, I determined that depletion of CstF in DT40 cells enhances sensitivity to UV treatment and reduces UV-induced ubiquitination of RNAP II.
My studies also provided evidence that following UV treatment BRCA1/BARD1, RNAP II and
CstF associate at sites of repaired DNA. Taken together, our results indicate that CstF plays active roles not only in mRNA 3’ processing but also in DNA repair, providing a link between transcription-coupled RNA processing and DNA repair.
RESULTS
Previous studies provided evidence that mRNA 3’ processing is affected by DNA damage and that the mRNA cleavage factor CstF functionally interacts with factors involved in
DNA repair after UV-treatment. Extending those studies, I investigated whether CstF itself might function in DNA repair. To this end, I used genetically modified chicken DT40 cells in which the only source of CstF-64 is from a tet-repressible transgene (DT40–64, Takagaki and Manley,
1998). These cells allow tet-dependent depletion of CstF-64, which destabilizes the entire CstF complex. CstF-64 became undetectable in DT40–64 cells treated with 10 µg/ml of tet for 48 h as measured by Western blot (Figure 8). First, I determined whether the presence of CstF affects the ability of the cells to recover following UV treatment. While the cells were still viable at 48 h treatment, they stopped growing after 3–4 days in tet-containing medium and started to die
30 shortly thereafter (data not shown; (Takagaki and Manley, 1998). DT40–64 cells were treated with tet for 48 h, exposed to UV light (20 Jm-2), and cell viability determined after 5 h, first measured simply by the appearance of cell death, which appeared significantly enhanced in the tet-treated cells compared to untreated controls (Figure 8A). Cell viability was quantified by trypan blue staining, which showed that the cells with reduced levels of CstF indeed displayed enhanced sensitivity to UV treatment.
Then I tested whether the heightened UV sensitivity was specific to CstF depletion, or might be a characteristic of DT40 cells poised to undergo cell death. To address this, DT40-ASF cells, which express the essential splicing factor ASF/SF2 under tet control (Wang et al., 1996), were analyzed as before (Figure 8B). Significantly, DT40-ASF cells did not show enhanced sensitivity to UV, supporting the idea that CstF has a specific role in recovery from exposure to
UV. As CstF is a general polyadenylation factor that functions in the 3’ processing of many if not most mRNA precursors, it is conceivable in principle that the effect of CstF on DNA repair might be indirect. However, Takagaki and Manley (Takagaki and Manley, 1998) showed that depletion of CstF in DT40–64 cells did not detectably affect the steady-state levels of actin mRNA and several other less abundant transcripts, at least over time courses such as employed in our experiments.
To determine whether this might also apply to mRNAs encoding proteins involved in
DNA repair, I examined levels of CSA and CSB proteins, which are involved in TCR (Groisman et al., 2006; Laine and Egly, 2006), following depletion of CstF and UV treatment. Tet- dependent depletion of CstF for 24 h and UV treatment did not significantly affect the expression levels of CSA and CSB (Figure 8C), supporting the idea that any effect of the depletion of CstF on DNA repair (see subsequently) is in fact due to a direct role of this RNA processing factor in
31 this response. As the samples analyzed were obtained 2 h after UV treatment, my Western blot analysis did not show the CSA-dependent degradation of CSB by the ubiquitin-proteasome pathway that occurs 3–4 h after UV irradiation (Groisman et al., 2006).
A) 1 No UV 5 hrs UV tet/ no UV tet/ 5 hrs UV 0.8 no UV
blue) 0.6 UV
0.4 10 µg tet trypan ( 10 µg tet/UV % viability % cell 0.2 0 0 hr 0.5 hr 1 hr 2 hr 5 hr recovering time after UV
B) 1 DT40-64 DT40-ASF 0.8 DT40-ASF no tet tet tet
blue) 0.6 UV: 0 0.5 1 2 5 0 0.5 1 2 5 0 0.5 1 2 5 hr 0.4 DT40-64
-CstF trypan
( 0.2 55- % viability % cell 0 0 hr 0.5 hr 1 hr 5 hr -actin 2 hr 36- recovering time after UV
C) no tet tet UV: 0 2 0 2 hr -CSB 130- 55- -CSA
-actin 36-
Figure 8: Cells with reduced levels of CstF show enhanced sensitivity to UV
treatment. A) DT40–64 cells were grown in presence or absence of tet (10 mg/ml) for 48
h. Then cells were exposed to UV irradiation (20 Jm-2) and allowed to recover for the
times indicated. Viable cells are seen as bright dots by contrast microscopy, while non-
viable cells are seen as condensed dark dots. The percentage of viable cells after UV
treatment determined by trypan blue staining is also shown. B) DT40–64 and DT40-ASF
cells were treated with tet and UV as in (A). C) One hundred microgram of each cell
extract was analyzed by immunoblotting with antibodies against CstF-64, CSA, CSB and
actin.
32
UV-induced degradation of RNAP IIO LS, initiated by BRCA1/BARD1 ubiquitination, contributes to inhibition of mRNA 3’ processing (Kleiman et al., 2005). Next, I determined whether CstF functions in DNA damage-induced ubiquitination of RNAP II, and again used
DT40–64 cells. Ubiquitination and degradation of RNAP IIO LS was examined by Western blot using antibodies directed against the Ser 2-phosphorylated CTD epitope of RNAP II (H5), which reflects elongating RNAP IIO. The proteasome inhibitor MG132 was added to the cells immediately after UV exposure to prevent degradation of RNAP II, and cell extracts were prepared at different times after UV/MG132 treatment. With degradation blocked, I was able to observe apparent ubiquitinated forms of RNAP IIO in cells expressing normal levels of CstF
(Figure 9A, lanes 2 and 3). Importantly, cells with reduced expression levels of CstF showed lower accumulation of ubiquitinated RNAP IIO. This was apparent after 24 h tet treatment and essentially complete after 48 h (Figure 9A, lanes 12 and 13). In the absence of MG132, UV- induced degradation of RNAP IIO was observed in the presence of CstF (Figure 8B, lanes 3 and
4), but strikingly, turnover was reduced (24 h) or completely blocked (48 h) when CstF was depleted (Figure 9B, lanes 13 and 14). Taken together these results indicate that CstF is required for UV-induced proteasomal degradation of RNAP II.
The data presented before provides evidence that CstF participates in ubiquitination of
RNAP II in response to DNA damage, and in the TCR response itself. Based on this and on our previous data establishing an interaction between CstF and BRCA1/BARD1, I next determined whether RNAP II, CstF and BRCA1/BARD1 all associate at sites of repaired DNA damage. To this end, a variation of the chromatin immunoprecipitation (ChIP) assay was employed (Orlando,
2000; Takahashi et al., 2000). This method has been used largely to study chromatin associated factors, but has also been valuable in analysis of proteins apparently associated with
33
A) MG132 treatment after UV no tet 24 hrs tet 48 hrs tet UV: 0 0.5 1 2 5 0 0.5 1 2 5 0 0.5 1 2 5 250- -RNAP II-Ub(n) -RNAP II
-CstF 55- -actin 36- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
B) no tet 24 hrs tet 48 hrs tet UV: 0 0.5 1 2 5 0 0.5 1 2 5 0 0.5 1 2 5 250- -RNAP II
-CstF 55- -actin 36- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Figure 9: Cells with reduced levels of CstF show reduced ability to ubiquitinate
RNAP II. A) DT40–64 cells were grown in presence or absence of tet (10 mg/ml) for 48
h. Then cells were exposed to UV irradiation (20 Jm-2) and allowed to recover for the
times indicated. CstF-64, RNAP IIO and actin protein levels in cell extracts were
monitored by Western blot of DT40–64 cells treated or not treated with UV. As indicated
in the figure, cells were incubated (A) or not (B) with the proteasome inhibitor MG132
immediately after UV treatment.
elongating RNAP II (e.g. (Komarnitsky et al., 2000; Schroeder et al., 2000). In this experiment,
BrdU was added to HeLa cells immediately after exposure to UV light to label repaired DNA.
Cells were crosslinked with formaldehyde at different times after UV exposure. As ubiquitination of RNAP II occurs within 15 min of exposing cells to UV and persists for about
8–12 h (Bregman et al., 1996), I performed this analysis in a period between 0–5 h after UV treatment. Extracts of these cells were prepared and following sonication DNA–protein
34 complexes were IPed by incubation with an anti-BrdU monoclonal antibody. Following reversal of crosslinks, rather than analyzing DNA by PCR, I determined whether specific proteins were associated with the BrdU-containing DNA by Western blot. Samples from cells not treated with
UV were used as a control.
The data showed that RNAP II, CstF and BARD1 all associated with repaired/BrdU- containing DNA (Figure 10). These findings corroborate earlier observations that part of RNAP
IIO does not dissociate from the damaged DNA during the assembly of the TCR complex
(Fousteri et al., 2006). The presence of RNAP II supports the hypothesis proposed in yeast that
RNAP II is not always degraded at sites of DNA damage and might re-engage and continue transcription (Brueckner et al., 2007; Fousteri et al., 2006; Woudstra et al., 2002). RNAP II associated with the repaired DNA was detected at the earliest time after UV irradiation (0.4 h), suggesting that the arrest of the RNAP II is an early event in TCR. Consistent with previous results, the Western blot analysis also revealed that UV treatment decreased accumulation of
RNAP II at later times (Figure 10, 2 h after UV treatment; (Fousteri et al., 2006; Kleiman et al.,
2005), likely reflecting the turnover of stalled RNAP II shown in Figure 9 A-B. Significantly, we also detected CstF-64 and BARD1 associated with the BrdU-containing DNA, with a time course very similar to that displayed by RNAP II. Together, this data supports the idea that
RNAP II, CstF and BARD1 associate at sites of DNA repair and play a direct role in the DNA repair response.
35
Supernatant IP
UV: 0 0 .4 1 2 5 0 0 .4 1 2 5 hrs
250- -RNAP II
-BARD1 95-
-CstF 55-
-actin 36-
Figure 10: RNAP II, CstF and BARD1 localize to sites of repaired DNA. Analysis of
protein complexes associated with BrdU-labeled DNA after UV treatment and the
indicated recovery times. BrdU was added to HeLa cells immediately after exposure to
UV light. Cells were cross-linked with formaldehyde after UV exposure at the times
indicated. Sonicated cell extracts were IPed with anti-BrdU antibody. Equivalent amounts
of the pellets (IP) and normalized amounts of the supernatants, which represent 7% of the
input, were analyzed by immunoblotting with anti-RNAP II (H5), anti-CstF-64, anti-
BARD1 and anti-actin antibodies.
DISCUSSION
Previous work showed that polyadenylation is inhibited after DNA damage as a result of both BRCA1/BARD1/CstF complex formation (Kleiman and Manley, 2001) and proteasome- mediated degradation of RNAP II (Kleiman et al., 2005). As CstF-50 can interact with BARD1 to inhibit polyadenylation (Kleiman and Manley, 1999) and with the CTD of RNAP II to activate polyadenylation (McCracken et al., 1997), it was proposed that CstF plays an important role in the response to DNA damage. In this study, Mirkin and colleagues (2008) provided evidence that prematurely terminated polyadenylated transcripts can be detected in vivo following DNA
36 damage, especially under conditions when the BRCA1/BARD1/CstF complex is not formed. I contributed to this work as first joint author. As part of these studies, I determined that cells with reduced levels of CstF displayed enhanced sensitivity to UV treatment and that the depletion of
CstF correlated with decreases in both ubiquitination and turnover of RNAP IIO. Besides,
Mirkin and colleagues (2008) also found that the decreased in CstF expression also correlated with diminished levels of repair of the transcribed DNA strand, which are events in the TCR response (Bregman et al., 1996; Fousteri et al., 2006; Luo et al., 2001; McKay et al., 2001;
Ratner et al., 1998). Consistent with the model for CstF function, I also found that RNAP IIO,
BARD1 and CstF were all transiently associated with sites of repaired DNA. This finding also suggests that a fraction of RNAP II elongation complexes arrested at sites of DNA damage are stable and remain associated with the DNA. Taken together, our results suggest that the polyadenylation machinery, specifically CstF, plays an important role in the response to DNA damage.
The data presented before provides evidence that DNA damage can induce premature transcription termination and polyadenylation, likely at sites of DNA damage and that accumulation of such species is blocked by the activation of the BRCA1/BRAD1/CstF complex.
How could this complex prevent such RNAs from accumulating? Milligan et al. (Milligan et al.,
2005) observed not only reduction in the levels of different mRNA species but also of truncated
RNAs in yeast strains with a defective poly(A) polymerase. Defective polyadenylation of prematurely terminated transcripts is known to activate a nuclear surveillance pathway, eliminating those mRNAs by deadenylation and exosome-mediated degradation (Milligan et al.,
2005; Thiebaut et al., 2006; Wyers et al., 2005). Extending this idea, work at Dr. Kleiman’s lab indicates that another CstF-50-interacting protein is the poly(A) specific ribonuclease (PARN;
37
(Mitchell and Tollervey, 2000; Wilusz et al., 2001; Wu et al., 2005). The CstF-50/PARN complex formation is induced under DNA damaging conditions (Cevher et al., 2010). PARN has been shown to co-purify with essential nonsense-mediated decay factors (Maquat, 2004) and
PARN down-regulation abrogates nonsense-mediated decay (Lejeune et al., 2003). The CstF-
50/PARN complex formation has a role in the inhibition of 3’ cleavage and activation of deadenylation upon DNA damage (Cevher et al., 2010). Extending these results, it was found that the tumor suppressor BARD1, which is involved in the UV-induced inhibition of 3’ cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-
50/BARD1 can revert the cap-binding protein-80 (CBP80)-mediated inhibition of PARN activity. PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions, suggesting that the interplay between polyadenylation, deadenylation and tumor-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.
My results suggest that CstF is involved in DNA damage-induced ubiquitination of
RNAP II LS, which is an important event in the TCR response (Fousteri et al., 2006; Luo et al.,
2001; McKay et al., 2001; Ratner et al., 1998). As CstF-50 can bind BARD1 (17), the CTD of
RNAP II (McCracken et al., 1997; Hirose and Manley, 1998) and ubiquitin (Ub, Figure 13), it is possible that it functions to help in the assembly or stabilization of the ubiquitination complex. In this scenario, CstF-50 might function as a cofactor for ubiquitination of RNAP II by
BRCA1/BARD1 (Kleiman et al., 2005; Starita et al., 2005). Recent studies suggest that Ub- binding proteins may be critical in determining substrate specificity and substrate fate (Elsasser and Finley, 2005; Hicke et al., 2005; Kim and Rao, 2006). In this respect CstF-50 could function
38 to help the BRCA1/BARD1 heterodimer recognize some of its substrates, such as RNAP IIO. By this model, loss of CstF would have a negative effect on clearance of the stalled RNAP II from sites of damage, by preventing ubiquitination and degradation of RNAP IIO. This could in turn block access of repair enzymes to the DNA, thereby interfering with the repair process and enhancing cell death. My results with DT40 cells depleted of CstF showing deficiencies not only in recovery from UV treatment but also in repair of UV-induced DNA damage support this idea.
Mirkin and colleagues (2008) data indicate that CstF plays a role in the DNA repair response. As just discussed, CstF could affect DNA repair by inhibiting the erroneous processing of nascent, truncated RNAs, by inducing RNAP II ubiquitination, and/or by re-engaging and continuing transcription with stalled RNAP II complexes. First, CstF interacts with the DNA replication and repair factor PCNA (Kleiman and Manley, 1999). It has been shown that PCNA co-localizes with BRCA1/BARD1 at sites of DNA repair (Scully et al., 1997; Wang et al., 2000) and associates with DNA repair proteins as part of the TCR response (Balajee et al., 1998). It is possible that PCNA is the repair factor that links the stalled RNAP II complex to the repair machinery during TCR. Second, several polyadenylation factors have been shown to interact with DNA repair factors. For example, cleavage factor CFIIm co-purifies with the BRCA1- associated protein hMre11 (de Vries et al., 2000), which has been implicated in DNA repair and cancer predisposition (reviewed by (Petrini, 2000)). Additionally, the transcriptional co-activator
PC4 interacts not only with CstF-64 (Calvo and Manley, 2005) but also with the DNA repair protein XPG (Wang et al., 2004a). XPG is known to function in multiple DNA repair pathways.
XPG recruits PC4 to the bubble-containing DNA substrate, PC4 displaces XPG and forms a
DNA-PC4 complex (Wang et al., 2004a). PC4 can also interact with the elongating RNAP IIO through CstF-64, preventing premature termination during the elongating phase (Calvo and
39
Manley, 2005). It is thus possible that the interaction of PC4 with CstF-64 mediates the damage- induced association of the stalled RNAP II and the DNA repair machinery. In any case, these data have provided evidence that CstF plays a role in TCR, reinforcing the functional interaction between components of the transcription, 3’ processing and DNA repair machineries.
Based on the results presented in the work of Mirkin and colleagues (2008) and this dissertation, the following model was proposed (Figure 11; (Fousteri et al., 2006; Kleiman and
Manley, 1999, 2001). After exposure to DNA damage-inducing agents, the elongating RNAP II-
CstF holoenzyme complex stalls at sites of damage. A BRCA1/BARD1-containing complex is activated and recruited to sites of repair, inhibiting RNAP II and the associated polyadenylation machinery by ubiquitination followed by degradation of the RNAP IIO. This process facilitates repair by allowing access to the repair machinery, while simultaneously preventing polyadenylation of aborted nascent mRNAs, which are eliminated by exosome-mediated degradation in a nuclear surveillance pathway. Alternatively, the RNAP II complexes arrested at certain DNA lesions are not degraded, CSA and CSB proteins regulate the recruitment of chromatin remodeling and TCR factors (Fousteri et al., 2006), and then RNAP II re-engages and continues transcription once repair is completed. Given that CstF-50 can functionally interact with all the elements of this model, we propose an important role for this protein in the transcription-coupled DDR.
40
ub ub ub ub ub
Figure 11: Model for the role of CstF in the DNA damage response. Coupling polyadenylation, transcription and DNA repair.
41
CHAPTER III
p53 INHIBITS mRNA 3’ PROCESSING THROUGH ITS INTERACTION WITH THE
CstF/BARD1 COMPLEX
42
INTRODUCTION
The p53 gene is the most commonly mutated target in human tumors. Activation of p53 affects the expression level of a large set of genes and mediates several cellular responses such as
DNA repair, cell cycle arrest and/or apoptosis (Levine et al., 2006; Vogelstein et al., 2000).
Although it is well established that p53 is a transcriptional regulator, transactivation-independent functions of p53 have been described (reviewed by (He et al., 2007; Takwi and Li, 2009). For example, certain microRNAs are transactivated by p53, and these microRNAs cause dramatic changes in gene expression, offering an indirect p53-mediated control of gene expression at the posttranscriptional level (Chang et al., 2007). It has been described that the induction of p53 expression upon ultraviolet (UV) treatment is associated with changes in the levels of total poly(A)+ mRNA (Ljungman et al., 1999; McKay and Ljungman, 1999). Interestingly, this p53- associated changes in poly(A)+ RNA levels might also be functionally related to the UV-induced inhibition of mRNA polyadenylation (Kleiman and Manley, 2001). As mRNA poly(A) tails are important for the regulation of mRNA stability, it is possible that these changes of poly(A)+ mRNA levels might represent another mechanism of p53-mediated control of gene expression.
The 3’ end of the mRNA is processed by the cleavage of the mRNA followed by the addition of a non-templated polyadenylated tail, which in mammalian cells is of approximately
200–300 adenosines. The assembly of the cleavage/polyadenylation machinery requires specific signal sequences in the mRNA precursor as well as interactions of a large number of protein factors (reviewed by (Mandel et al., 2008). It has been shown that the regulation of mRNA 3’ end formation can have significant roles in cancer (Kleiman and Manley, 2001; Rozenblatt-
Rosen et al., 2009; Scorilas, 2002; Topalian et al., 2001). Most importantly, alternative mRNA cleavage and polyadenylation changes the length of the 3’ untranslated region and regulates gene
43 expression of different mRNAs in cancer cells (Mayr and Bartel, 2009; Singh et al., 2009) and during cell differentiation (Ji et al., 2009; Sandberg et al., 2008; Zlotorynski and Agami, 2008).
Cleavage stimulation factor (CstF) is one of the essential 3’ processing factors and is most likely active as a dimer of an heterotrimer, consisting of three protein factors called CstF-77, CstF-64 and CstF-50. CstF-64 interacts directly with the mRNA, and cells deficient in CstF-64 undergo cell cycle arrest and apoptotic death (Takagaki and Manley, 1998). Both the CstF-50 and CstF-
77 subunits interact specifically with the C-terminal domain of RNA polymerase II, likely facilitating the RNA polymerase II-mediated activation of 3’ end processing (Hirose and
Manley, 1998; McCracken et al., 1997). After DNA damage, mRNA 3’ processing is inhibited as a result of CstF/BARD1/BRCA1 complex formation (Kleiman and Manley, 1999) and of the proteasome-mediated degradation of RNA polymerase II (Kleiman et al., 2005), suggesting the existence of possibly redundant mechanisms to explain the inhibitory effect of UV irradiation.
Since the mechanisms involved in the p53-dependent control of gene expression following DNA damage have not been completely elucidated, Nazeer and colleagues (2011) examined the functional interaction between p53 with BARD1 and CstF-50 and its effect on mRNA 3’ processing as well as on the polyadenylation of cellular RNAs. As part of these studies, I determined that the carboxy-terminal domain of p53 associates with factors that are required for the UV-induced inhibition of the mRNA 3’ cleavage step of the polyadenylation reaction, such as the tumor suppressor BARD1 and the 3’ processing factor CstF-50. Nazeer and colleagues (2011) showed using competition assays that p53 is also part of the previously described BARD1/CstF-50 complex and plays a role stabilizing the complex. As part of those studies, it was shown that UV treatment induced the interaction between those three factors, supporting the idea that p53 and CstF might be simultaneous binding partners of BARD1. The
44 co-precipitation of p53 with CstF was irrespective of DNA damage, indicating that the formation of the CstF/p53 complex might be independent of BARD1. Extending these studies, it was described that p53 inhibits 3’ cleavage in vitro and that the same region of p53 required for binding CstF-50 and BARD1 is necessary for inhibiting mRNA 3’ cleavage and that p53 expression levels inversely correlate with levels of mRNA 3’ cleavage in different cell types under different cellular conditions. Supporting these results, a tumor-associated mutation in p53 not only decreases the interaction with BARD1 and CstF, but also decreases the UV-induced inhibition of 3’ processing, all of which is restored by wild-type-p53 expression. Finally, Nazeer and collaborators (2011) also found that p53 expression levels affect the polyadenylation levels of housekeeping genes (β-actin and GAPDH), but not of p21 and c-fos genes, which are involved in the DNA damage response (DDR). These studies have identified a novel 3’ RNA processing inhibitory function of p53, adding a new level of complexity to the DDR by linking RNA processing to the p53 network.
RESULTS
It has been shown that p53 interacts with BARD1 independently of BRCA1 to induce an apoptotic response to genotoxic stress (Feki et al., 2005). A germline mutation in BARD1
(Gln564His) reduces the binding to p53 and induction of apoptosis (Irminger-Finger et al.,
2001). BARD1 can also interact with the 3’ processing factor CstF-50, inhibiting mRNA 3’ processing and linking it to the DDR (Kleiman and Manley, 1999). Interestingly, the Gln564His mutation of BARD1 also reduces the binding of CstF-50 to BARD1, interfering with the role of
BARD1 in mRNA 3’ processing (Kleiman and Manley, 2001). Taken together, these studies
45 suggest a functional interaction between BARD1, CstF-50 and p53 under DNA-damaging conditions.
My contribution to this paper was to further investigate this possibility by examining the physical association of p53 with BARD1 and CstF-50 using the recombinant proteins shown in
Figure 12A. The three derivatives that I expressed and purified included the amino-terminal domain, which included transactivation and DNA binding domains (aa 1-293), carboxy-terminal domain (aa 94-393), which included DNA binding and regulatory domains, and a derivative encompassing just the DNA binding domain (aa 94-293) (Figure 12A). Then, I performed pull- down assays by incubating GST, GST-BARD1 and GST-CstF-50 bound to glutathione sepharose beads to either full-length-His-p53 or His-p53 derivatives followed by gel electrophoresis and immunodetection using anti-p53 antibodies.
The results showed that both full-length His-p53 and the C-terminal fragment of p53 interacted directly in vitro with not only GST-BARD1 (Figure 12B, lane 6), but also GST-CstF-
50 (Figure 12B, lane 3). Neither CstF-50 nor BARD1 bound to the two derivatives that lacked the C-terminal domain of p53 or to GST alone (Figure 12B, lane 9). Taken together, these results indicate that the C-terminal domain of p53, which has been described to have regulatory functions in the DDR (Sauer et al., 2008), constitutes the CstF-50 and BARD1 interaction domain.
DISCUSSION
This is the first study showing a 3’ mRNA processing inhibitory function of p53, adding a new level of complexity to the DDR by linking RNA processing to the p53 network. Several
46 lines of evidence support the hypothesis that p53 is an inhibitor of mRNA 3’ processing. First, the direct interaction of the C-terminal domain of p53 with CstF1 and BARD1 (Figure 12),
A
BARD1 (1-777)
CstF-50 (1-397)
p53 (FL) p53 (1-293) p53 (94-393) p53 (94-293)
B
GST-CstF-50 GST-BARD1 GST SN PD SN PD SN PD 55- -FL-p53
-p53 (1-293) 36-
-p53 (94-293) 36-
-p53 (94-393) 36- 1 2 3 4 5 6 7 8 9
Figure 12: CstF-50 and BARD1 interact with the C-terminal domain of p53. A)
Diagram of BARD1, CstF-50 and p53 derivatives. B) Requirement of p53 C-terminal
domain for both CstF-50 and BARD1 interaction. Recombinant His-p53 or the indicated
His-p53 derivatives were incubated with purified GST, GST-CstF-50 or GST-BARD1.
Protein samples were treated with RNase A. Bound proteins were eluted and analyzed by
western blot with anti-p53 antibodies. Five percent of His-p53 or His-p53 derivatives
used in the reaction are shown as input.
47
both of which are involved in the UV-induced inhibition of mRNA 3’ processing, and the existence of protein complexes of these factors in extracts of different cell lines (Nazeer et al.,
2011). Second, p53 can inhibit the 3’ cleavage reaction in vitro and p53 expression levels inversely correlate with levels of mRNA 3’ cleavage (Nazeer et al., 2011). Third, the tumor- associated S241F mutation in p53 reduces binding to BARD1 and CstF and disrupts the
CstF/BARD1/p53 complex association, and this complex formation is restored by the induced expression of WT-p53 (Nazeer et al., 2011). Furthermore, cells expressing this mutant p53 show reduced inhibition of mRNA 3’ cleavage, which is restored by the induced expression of WT- p53. Fourth, it was determined that the expression of WT-p53 has different effects on the polyadenylation state of the housekeeping genes (Nazeer et al., 2011). Taken together, our results suggest a novel function of p53 as an inhibitor of the mRNA 3 ’processing machinery.
Previous studies from Dr. Kleiman’s lab proposed that there is a general effect of DNA- damaging conditions on mRNA levels, and that the UV-induced inhibition of 3’ end processing plays an important role in decreasing the total cellular mRNA levels as part of this response
(Cevher and Kleiman, 2010). Consistent with this, the levels of poly(A)+ mRNA of genes not involved in DDR decrease after DNA damage (Dheda et al., 2004; Ljungman et al., 1999;
Maccoux et al., 2007; Mirkin et al., 2008). It has been shown that full recovery of total mRNA levels within 6 h after the DNA-damaging exposure correlates with cellular protection against apoptosis in a p53-dependent manner (McKay et al., 2001). On the other hand, there is also a gene-specific effect of DNA-damaging conditions on the levels of poly(A)+ mRNAs of genes involved in the DDR, those genes are either down- or upregulated at different time points after
DNA damage (reviewed by (Cevher and Kleiman, 2010). Nazeer and collaborators (2011)
48 discovered that under DNA-damaging conditions, p53 in association with the 3’ processing factor CstF and the tumor suppressor BARD1 can control mRNA 3’ processing of housekeeping genes, but not of p21 and c-fos genes, which are involved in DDR. On the basis of these studies, it has been proposed that p53, a protein with compromised expression in most cancers, plays a role in the general response to DNA damage by stabilizing the CstF/BARD1 complex and inhibiting 3’ processing of aborted nascent RNA products, allowing the elimination of prematurely terminated transcripts to avoid the expression of deleterious proteins and facilitating
DNA repair by clearing the area around the lesion. Defective polyadenylation of prematurely terminated transcripts is known to activate a nuclear surveillance pathway, allowing the elimination of those mRNAs by exosome-mediated degradation (reviewed by (Cevher and
Kleiman, 2010). As DNA repair proceeds, the levels of p53 expression decrease allowing the recovery of total mRNA levels. The results obtained by Nazeer and colleagues (2011) indicate that different cell lines exhibit different 3’ processing profiles depending on p53 expression levels, consistent with the idea proposed by Singh et al. (2009) that the interaction of the 3’ processing machinery and factors involved in the DDR/tumor suppression might result in cell- specific 3’ processing profiles.
Supporting the idea that the 3’ processing machinery is interconnected with the p53 pathway, it has been shown that Rbbp6, a p53-binding protein, and Ku-70 subunit of DNA-PK, which is involved in the BARD1-mediated phosphorylation of p53 Ser15 upon DNA damage
(Fabbro et al., 2004), are part of the pre-mRNA 3’ processing complex (Shi et al., 2009). Other tumor suppressors, such as CSR1 and Cdc73, have also been shown to functionally associate with 3’ processing factors, such as CPSF3 (Zhu et al., 2009b) and CPSF/ CstF (Rozenblatt-
Rosen et al., 2009). Recently, it has been shown that the use of alternative mRNA 3’ cleavage
49 and polyadenylation sites can control the expression of certain genes by eliminating or including several cis-acting elements, such as microRNA target sites and AU-rich elements, in cancer cells and during development (Ji et al., 2009; Mayr and Bartel, 2009; Sandberg et al., 2008; Singh et al., 2009; Zlotorynski and Agami, 2008). Although more work is necessary to determine the functional relevance of the CstF/BARD1/p53 interaction in the regulation of expression of specific genes involved in DDR; it is possible that the p53-mediated inhibition of mRNA 3’ processing might also play a role in the selection of different alternative mRNA 3’ cleavage sites and, consequently, in the regulation of the mRNA levels of genes involved in DDR. Considering that the p53 pathway is tightly controlled in cells following DNA damage (reviewed by
(Vousden, 2006), the p53-associated control of mRNA 3’ processing could be an effective mechanism employed to control gene expression in cells upon DNA-damaging conditions.
Taken together, these studies identify a novel 3’ RNA processing inhibitory function of p53 and suggest that the CstF/BARD1/p53 interaction contributes to UV-induced inhibition of pre-mRNA 3’ processing, providing evidence of another link between mRNA 3’ processing and tumor suppression.
50
CHAPTER IV
ROLE OF THE POLYADENYLATION FACTOR CstF-50 IN REGULATING THE
BRCA1/BARD1 E3 UBIQUITIN LIGASE ACTIVITY
51
INTRODUCTION
All eukaryotes have evolved an elaborate surveillance mechanism to maintain genomic integrity against variety of cellular insults. The DNA damage response (DDR) not only includes repair mechanisms but also regulation of cell cycle, transcription ((Khobta and Epe, 2012;
Warmerdam and Kanaar, 2010) as well as mRNA processing (Cevher and Kleiman, 2010). The tumor suppressors BRCA1 and BRCA1-associated RING domain protein (BARD1) play an important role in the regulation of different nuclear events during DDR, including mRNA 3’ end processing.
BRCA1/BARD1 complex plays a role in the UV-induced inhibition of mRNA 3’ processing (Kleiman and Manley, 2001), resulting in the transient decrease of the cellular levels of polyadenylated transcripts. Most eukaryotic mRNA precursors undergo a cleavage followed by the synthesis of a 200-adenosine residue tail to the 3’ end of the cleaved product (Shatkin and
Manley, 2000; Zhao et al., 1999). CstF-50 is one of the essential polyadenylation factors that interacts with other mRNA 3’ processing factors and specifically with RNA polymerase II
(RNAP II), facilitating the RNAP II-mediated activation of 3’ end cleavage (Hirose and Manley,
1998; McCracken et al., 1997). Furthermore, 3’ end cleavage can be repressed after DNA damage as a result of the interaction of CstF-50 and BRCA1/BARD1 complex (Kleiman and
Manley, 2001; Kleiman et al., 2005; Nazeer et al., 2011) and the proteasome-mediated degradation of RNAP II, which is a BRCA1/BARD1 substrate (Kleiman et al., 2005; Starita et al., 2005). Importantly, DNA damage functions of BARD1, such as interaction with CstF-50, inhibition of mRNA 3’ processing and RNAP II degradation, are abrogated in phosphorylation mutants of BARD1 (Kim et al., 2006). Interestingly, the CstF-50/BARD1 complex also plays a
52 role regulating the mRNA levels of different genes during DDR by its association with p53
(Nazeer et al., 2011) and PARN deadenylase (Cevher et al., 2010; Devany et al., 2013).
Based on these studies, I am proposing a model whereby BRCA1/BARD1, as part of the
RNAP II holoenzyme (Chiba and Parvin, 2002), senses sites of DNA damage and repair, and the inhibitory interaction with CstF ensures that the nascent RNAs are not erroneously polyadenylated at such sites, avoiding the expression of deleterious proteins (Kleiman et al.,
2005; Mirkin et al., 2008). In that scenario, CstF-50 is involved in DNA damage-induced ubiquitination of RNAP II, which is an important event in DDR (Fousteri et al., 2006; Luo et al.,
2001). By this model, loss of CstF-50 would have a negative effect on clearance of the stalled
RNAP II from sites of damage, by preventing RNAP IIO from ubiquitination and degradation.
This could in turn block access of repair enzymes to the DNA, thereby interfering with the repair process and enhancing cell death. Supporting this model, the depletion of CstF in DT40 in UV treated cells results in a decreased viability, reduced ability to ubiquitinate RNAP II and defects in repair of DNA damage (Mirkin et al., 2008). Consistent with this, the siRNA-mediated knockdown of BRCA1/BARD1 in Hela cells reduces the UV-induced ubiquitination and degradation of RNAP II (Kleiman et al., 2005).
Ubiquitination regulates many cellular processes but its potential functional connection with mRNA 3’ processing and DNA damage has not been well characterized. Accumulated evidence shows that BRCA1 and BARD1 form a heterodimer that exhibits E3 Ub ligase activity and is quickly recruited to DNA lesions during DDR. In that scenario, BRCA1/BARD1 regulates chromatin structure by histones ubiquitination (Zhu et al., 2011). Interestingly, it has been shown that UV treatment induces monoubiquitination of H2A (Bergink et al., 2006), resulting in the eviction of Ub-H2A from UV-damaged chromatin (Lan et al., 2012). Another BRCA1/BARD1
53
Ub ligase target is the LS of RNAP IIO, which functions in elongation, but not of RNAP IIA, the form engaged in promoters (Kleiman et al., 2005). Despite our understanding of the biochemistry of the BRCA1/BARD1 E3 ligase, we know very little of its cellular targets, how these targets are chosen and the mechanism by which they are recruited to DNA damage sites.
Here I provide evidence of the role of polyadenylation factor CstF-50 and the Ub-escort factor p97 in regulating BRCA1/BARD1 E3 Ub ligase, resulting in the control of transcription and chromatin remodeling during DDR. I am showing that CstF-50 can interact directly not only with BRCA1/BARD1 but also with Ub, the escort-factor p97 and some of BRCA1/BARD1 substrates, such as RNAP II, H2A and H2B. Besides, CstF-50 activates BRCA1/BARD1- dependent RNAP II polyubiquitination, H2A and H2B monoubiquitination as well as
BRCA1/BARD1 autoubiquitination. Taken together, my results provide evidence that CstF-50- associated p97 regulates BRCA1/BARD1 Ub ligase activity during DDR, helping in the assembly and/or stabilization of the ubiquitination complex.
RESULTS
The discovery of BRCA1/BARD1 E3 Ub ligase activity triggered a search for substrates that could explain how BRCA1 functions in different cellular processes. Although many
BRCA1/BARD1 substrates have been described in the last few years, many of them remain unknown. Previous studies have identified RNAP II as an enzymatic substrate for
BRCA1/BARD1 E3 Ub ligase (Kleiman et al., 2005; Starita et al., 2005). As part of those studies, my results showed that the large subunit of RNAP IIO, but not RNAP IIA, is a specific target of the BRCA1/BARD1 Ub ligase activity. CstF-50 can bind not only BARD1 (Kleiman and Manley, 1999) but also the C-terminal domain of RNAP II (Hirose and Manley, 1998;
54
Kleiman et al., 2005; McCracken et al., 1997). Besides, Ub was one of the CstF-50 interactors detected when using a yeast two-hybrid screening (Kleiman and Manley, 1999; Mirkin et al.,
2008). Together these studies suggest that CstF-50 might play a role in the ubiquitination of
BRCA1/BARD1 substrates, either helping in the assembly or stabilization of the ubiquitination complex.
To determine the proposed role of CstF-50 in the BRCA1/BARD1-dependent ubiquitination I further analyzed the physical association of Ub and CstF-50 by performing pull- down assays. I used recombinant GST-CstF-50, His-Ub and Ub-agarose in those assays. These three pull-down assays indicate that CstF-50 interacts directly with Ub (Figure 13A). The interaction was detected only when GST-CstF-50 but not GST was used in these assays. To test whether Ub might also be part of the previously described CstF-50/BARD1 complex, I carried out His-Ub pull-down assays using nuclear extracts (NEs) of HeLa cells treated or non-treated with UV irradiation. HeLa cells were treated with UV (20 Jm-2), and allowed to recover for 2 h.
His-Ub was able to pull-down CstF-50, RNAP II and BRCA1 from NEs independently of UV treatment (Figure 13B). Interestingly, an increase in the amount of BARD1 pulled-down by His-
Ub was observed after UV treatment. Topoisomerase II (Topo II) did not interact with His-Ub.
Together these experiments indicate that CstF-50 can interact directly and non-covalently with
Ub. Although these results do not show how many complexes Ub can form with CstF-50,
BARD1, BRCA1 and RNAP II, they clearly show that Ub can interact with these factors.
55
A) B) Input PD UV: - + - + -Topo II 10- -His-Ub 130-
1 2 3 250- -RNAP II
His-Ub 250- -BRCA1
95- -BARD1
-GST-CstF-50 72- 1 2 3 55- -CstF-50 1 2 3 1 2 3 4 Ub-Agarose
-GST-CstF-50 72- 1 2 3
Figure 13: CstF-50 can form a complex with Ub and BRCA1/BARD1 E3 Ub ligase.
A) CstF-50 interacts directly with Ub. Immobilized GST-CstF-50 (upper panel) on glutathione-agarose beads was incubated with His-Ub. Immobilized His-Ub on nickel beads (middle panel) or Ub-agarose (lower panel) were incubated with GST-CstF-50.
Equal protein concentration of GST was used as control. Equivalent amounts of the pull- downs (PD) were analyzed and proteins were detected by immunoblotting with either anti-Ub or anti-CstF-50 antibodies. 5% of His-Ub or GST-CstF-50 were loaded as input.
B) Ub, CstF-50, BARD1, BRCA1 and RNAP II can form protein complex(es).
Immobilized His-Ub on nickel beads was incubated with NEs from Hela cells exposed or not to UV treatment. Cells were exposed or not to UV (20 Jm-2) and allowed to recover for 2 h. Samples were analyzed by immunobloting with the antibodies indicated in the figure. 5% of the NEs used in the pull-down assays were loaded as input.
56
To examine the CstF-50/Ub complex formation in cellular extracts I performed co- immunoprecipitation assays using NEs from HeLa cells. The co-immunoprecipitation (co-IP) assays indicated that CstF-50 can form (a) protein complex(es) with Ub in NEs from HeLa cells under non-stress conditions and after UV treatment (Figure 14A), reflecting the strong interaction between CstF-50 and Ub observed in the pull-down assays (Figure 13A). CstF-50 antibody was also able to coimmunoprecipitate RNAP II and BARD1. These results confirm previously published data that showed that an antibody against CstF-64, which is another subunit of the CstF heterodimer, can coimmunoprecipitate BARD1 (Nazeer et al., 2011) and RNAP II
(Kleiman et al., 2005), confirming that the whole CstF complex is involved in these interactions.
As previously described, my results showed the UV-induced degradation of RNAP II (Kleiman et al., 2005) and the UV-induced increase in the CstF-50/BARD1 complex formation (Kleiman and Manley, 2001). Similar results were obtained in the reciprocal co-IP analysis using antibodies against Ub (Figure 14B). Together these results indicate that CstF-50 interact non- covalently with Ub, BRCA1/BARD1, and RNAP II. As CstF-50 interacts with Ub, the Ub E3 ligase and one of its substrate, RNAP II (Kleiman et al., 2005; Starita et al., 2005), CstF-50 might participate in the BRCA1/BARD1-dependent ubiquitination of RNAP II during DDR by acting as a scaffold/adaptor to help in transfering of Ub from the E3 or in the elongation of short polyubiquitin chains.
To determine whether CstF-50 plays a role in regulating the BRCA1/BARD1 Ub ligase activity I performed in vitro ubiquitination assays using recombinant proteins. Plasmids encoding
His-wt-BARD1/∆BRCA1, His-Ub and His-UbcH5c were kindly provided by Dr. Baer,
(Columbia University). First, I analyzed the effect of increasing amounts of CstF-50 on the autoubiquitination of BRCA1/BARD1. A heterodimeric complex (ΔBRCA1/BARD1-wt)
57
A) B)
Input CstF-50 IP Input Ub IP UV: - + - + UV: - + - + -Topo II -Topo II 130- 130- 250- 250- -RNAP II -RNAP II
250- -BRCA1 250- -BRCA1
95- -BARD1 95- -BARD1
55- -CstF-50 55- -CstF-50 1 2 3 4
10- -Ub
1 2 3 4
Figure 14: Ub, CstF-50, BRCA1, BARD1 and RNAP II co-immunoprecipate from
the NEs of HeLa cells. CstF-50 and Ub co-immunoprecipitation is irrespective of UV
irradiation. Co-immunoprecipitation assays were performed using NEs of cells exposed
or not to UV irradiation and allowed to recover for 2 h as described before. NEs were
immunoprecipitated with either (A) anti-CstF-50 (polyclonal A301-250A, Bethyl) or (B)
anti-Ub (monoclonal P4D1, Santa Cruz) antibodies. Equivalent amounts of the pellets
(IP) and 5% of NEs as input were resolved by SDS-PAGE and proteins were detected by
immunoblotting with antibodies against anti-BARD1, RNAP II, BRCA1, Ub and CstF-
50. Antibodies against Topo II were used as a loading and specificity control.
comprised of truncated BRCA1 (residues 1–304, encompassing the RING domain) and full- length BARD1 was incubated with E1, its cognate E2 (UbcH5c), His-Ub and increasing amounts of GST-CstF-50. The reaction products were analyzed by Western blotting with an anti-BRCA1 antibody as well as an anti-Ub antibody to measure total, nonspecific ubiquitination. As indicated by the low mobility species in Figure 15A-B, the heterodimer autoubiquitination
58 augmented with increasing amounts of GST-CstF-50 (25-100 ng). As expected, a decrease in unmodified ∆BRCA1 (Figure 15A lower panel) was observed. Non-specific ubiquitination was detected by blotting with antibodies against Ub (Figure 15B). Together, these results indicate that CstF-50 can enhance the autoubiquitination of BRCA1/BARD1 in vitro. We will extend these studies using an heterodimer comprised of full length BRCA1.
A) B)
CstF-50 (ng): 0 25 50 100 0 CstF-50 (ng): 0 25 50 100 0
BRCA1/BARD1 polyUb chains 250- 250-
130- 130-
95- 95-
55- 55- -∆BRCA1
55-
55- 1 2 3 4 5 -∆BRCA1
1 2 3 4 5
Figure 15: CstF-50 increases the autoubiquitination of ΔBRCA1/BARD1
heterodimer. In vitro ubiquitination reactions were conducted in the presence of
ΔBRCA1/BARD1, E1, His-E2, His-Ub, ATP and increasing amounts of recombinant
GST-CstF-50. Samples were fractionated by SDS-PAGE and immunoblotted with anti-
BRCA1 (A) and anti-Ub (B) specific antibodies. Small blot shows a longer exposure of
the anti-BRCA1 immunoblot of the lower part of A).
59
In an effort to understand the mechanisms behind the CstF-50-mediated activation of
BRCA1/BARD1 activity, we have considered different alternative possibilities that are not mutually exclusive by which CstF-50 might exert its function. First, CstF-50 might interact with the BRCA1/BARD1 complex and activate its enzymatic activity. Second, CstF-50 might work as a scaffold protein helping in the assembly of the ubiquitination complex by binding BARD1, Ub and the substrate. Lastly, CstF-50 can help in the sorting of ubiquitinated substrates to the proteasome by binding the substrate and factors of the escort pathway.
p97/VCP is the central escort protein involved in this pathway, ferrying ubiquitinated substrates to its final destination, the proteasome, for subsequent degradation. p97/VCP is a chaperon-like ATPase having a segregase activity that binds to and separates Ub-proteins from their binding partners or extract them from protein complexes (E2-E3-E4-Ub-protein complex), singling and preparing them before passing through the 26S proteasome (Meyer et al., 2002;
Rape et al., 2001). p97 binds monoUb (Rape et al., 2001) but has higher affinity for multiUb chains (Dai and Li, 2001) as well as associates with substrates directly by its amino-terminal domain or indirectly through Ub-binding cofactors (Meyer et al., 2002; Richly et al., 2005).
Remarkably, p97 has another important function controlling the degree of ubiquitination of the bound substrates through its Ub binding cofactors or substrate-processing cofactors that either activate or inhibit polyubiquitination or even deubiquitinate the Ub-substrate (Jentsch and
Rumpf, 2007; Koegl et al., 1999; Richly et al., 2005; Wang et al., 2004b). Moreover, downstream stages in the escort pathway involve the recognition of Ub-substrates by the proteasome. This is mediated directly by proteasome subunits RPN10 and RPN13, or indirectly by Ub receptors like PLICI and hHR23b (Fatimababy et al., 2010). Over a decade ago, it was shown that p97 binds to E4 enzymes, implying that this type of enzymes serve as Ub-binding
60 cofactors (Koegl et al., 1999). Additionally, it has also been shown that E4s can also interact with Ub receptors RAD23 and DSK2, the yeast homologs of PLICI and hHR23b, respectively
(Hanzelmann et al., 2010; Richly et al., 2005).
To examine the possible role of CstF-50 in the escort pathway, I analyzed the complex formation of CstF-50 with p97 in extracts from HeLa cells by co-immunoprecipitation assays.
For these studies, I used antibodies directed against p97. As shown in Figure 16A, an important fraction of CstF-50 and BARD1 co-precipitated with p97 independently of UV treatment. I extended these studies by further confirming the direct interaction between p97 and CstF-50 by performing GST- and His-pull-down assays using recombinant GST-CstF-50 and His-p97, kindly provided by Dr. Monteiro (University of Maryland). As shown in Figure 16B, the interaction was detected only when GST-CstF-50 but not GST was used to pull-down His-p97, confirming the direct interaction between these two proteins. The interaction of p97 with
BARD1 and CstF-50 is consistent with the fact that p97 can interact with several E3 Ub ligases
(Halawani and Latterich, 2006) and E4 enzymes (Koegl et al., 1999). The immunoprecipitation assay showed that CstF-50 can form a complex with p97 in different cellular conditions, suggesting a possible functional interaction between CstF-50 and the p97-mediated escort pathway.
Supporting a role of p97 in DDR, p97 underwent a change in cellular localization after
UV damage, being very abundant in the cytoplasmic fraction before UV (Figure 17C) and relocating to the chromatin fraction after UV treatment (Figure 17B). Soluble nuclear levels were also increased after UV (Figure 17A). The levels of p97 were unaffected after UV treatment in whole cell extracts (WCE) (Figure 17D), indicating that the observed change in p97 protein levels are not due to changes in the expression but due to changes in its cellular localization
61
A) B) Input p97 IP UV: - + - +
-Topo II 95- -His-p97 130- 1 2 3 250- -BRCA1
95- -p97
95- -BARD1
55- -CstF-50
1 2 3 4
Figure 16: p97 and CstF-50 can form a complex. A) p97, CstF-50 and BARD1 co-
immunoprecipitate on NEs of HeLa cells irrespective of UV-treatment. Co-
immunoprecipitation assays were performed using NEs of cells exposed or not to UV
irradiation and allowed to recover for 2 h. NEs were immunoprecipitated with anti-p97
antibodies (polyclonal A300-589A, Bethyl). Equivalent amounts of the pellets (IP) and
5% of NEs as input were resolved by SDS–PAGE and proteins were detected by
immunoblotting with antibodies against anti-BARD1, RNAP II, BRCA1 and CstF-50.
Antibodies against Topo II were used as a loading and specificity control. B) CstF-50
interacts directly with p97 in vitro. Either immobilized GST or GST-CstF-50 was
incubated with His-p97. Bound proteins were eluted, resolved in SDS-PAGE and
detected with p97 antibodies. 5% of the His-p97 used in the reaction was loaded as
input.
upon DNA damage. As shown in Figure 17, our fractionation assays indicated that p97 colocalized with BRCA1/BARD1/CstF-50 in soluble nuclear and chromatin-bound fractions.
62
A) B) C) D) sol nuclear chrom-bound cytoplasmic whole cell extracts UV: - + UV: - + UV: - + UV: - +
-Topo II 15- -H2B -GAPDH 130- 35- 35- -GAPDH
250- -BRCA1 250- -BRCA1 -p97 -Topo II 95- 130- 95- 95- -BARD1 -BARD1 15- -H2B
55- 55- -CstF-50 -CstF-50 95- -p97
-p97 95- -p97 95-
Figure 17: p97 relocates from the cytoplasm to the chromatin after UV damage.
Cytoplasmic, soluble nuclear and chromatin-bound fractions were prepared as described
in Experimental procedures. A) Whole cell extracts (WCE), B) cytoplasmic, C) soluble
nuclear and D) chromatin bound fractions were separated by SDS-PAGE and analyzed
with the indicated antibodies.
Then I determined whether the p97 relocalization depended on the expression of
BRCA1/BARD1 and CstF-50. As seen in Figure 18, depletion of CstF-50 and BRCA1/BARD1 expression increased the amount of p97 in the chromatin under non-stress conditions (compare lane 1 to 3 and 5), suggesting that BRCA1/BARD1 and CstF-50 might regulate p97 cellular localization in normal conditions.
To further analyze the functional interaction of BRCA1/BARD1, CstF-50 and p97 I performed in vitro ubiquitination assays using recombinant proteins as in Figure 15. Limiting amounts of ∆BRCA1/BARD1 and increasing amounts of His-p97 and GST-CstF-50 were used.
63
siRNA: Control CstF-50 BRCA1/ BARD1 UV: - + - + - +
-Topo II 130-
250- -BRCA1
95- -BARD1
55- -CstF-50
-p97 95-
1 2 3 4 5 6
Figure 18: p97 localization in chromatin-bound fractions depends on
BRCA1/BARD1 and CstF-50 expression under non-damaging conditions.
Chromatin-bound fractions were prepared, as described in Experimental procedures, from
cells treated with control, CstF-50 or BRCA1/BARD1 siRNAs and immublotted with the
indicated antibodies. Anti-Topo II antibodies were used as loading control.
Surprisingly, increasing amounts of His-p97 further activated BRCA1/BARD1 autoubiquitination in the presence of optimal amounts of GST-CstF-50 (Figure 19A-B), suggesting a synergism between p97 and CstF-50 in activating the BRCA1/BARD1 Ub ligase activity. The addition of p97 by itself did not have any effect on BRCA1/BARD1 autoubiquitination. While further experiments are necessary to determine the details of this pathway, these data indicated the existence of an unexpected connection between mRNA 3’ processing, DNA damage and the Ub-escort pathway.
BRCA1/BARD1 E3 Ub ligase can transubiquitinate numerous protein substrates, but how this complex selects those substrates and the biological consequences of these substrates ubiquitination are not completely understood.
64
A) B)
CstF-50 (ng): 0 25 50 50 50 0 0 CstF-50 (ng): 0 25 50 50 50 0 0 p97 (ng): 0 0 0 25 50 50 0 p97 (ng): 0 0 0 25 50 50 0 BRCA1/BARD1 250- polyUb chains 250-
130- BRCA1/BARD1 130- polyUb chains
95- 95-
55- 55-
-∆BRCA1
1 2 3 4 5 6 7 1 2 3 4 5 6 7
Figure 19: The functional interaction between p97 and CstF-50 regulates
BRCA1/BARD1 Ub ligase activity. In vitro ubiquitination reactions were carried out as
described in Figure 15 but using limiting amounts of ΔBRCA1/BARD1 (5 ng) and
increasing amounts of GST-CstF-50 and of His-p97. Samples were fractionated by SDS-
PAGE and immunoblotted with anti-BRCA1 (A) and anti-Ub (B) specific antibodies.
Since there are a large number of BRCA1/BARD1 substrates I decided to study those ones involved in DDR. RNAP IIO is an enzymatic substrate for BRCA1/BARD1 E3 Ub ligase.
(Kleiman et al., 2005). To test whether the mRNA 3’ processing factor CstF-50 plays a direct role in the ubiquitination of RNAP IIO by BRCA1/BARD1 I performed in vitro ubiquitination assays in the presence of increasing amounts of CstF-50 (Figure 20). The heterodimeric complex
BRCA1/BARD1-wt was incubated with increasing amounts of recombinant GST-CstF-50,
RNAP IIO and the reaction mix described in Experimental Procedures. The reaction products were analyzed by Western blotting with an anti-RNAP II antibody that only recognizes the elongating form of RNAP II. As indicated by the low mobility species in Figure 20, RNAP IIO
65 ubiquitination increased when CstF-50 was added to the complete reaction. The very high molecular weight of these species indicated the addition of multiple Ub monomers, presumably in the form of polyubiquitin chains. Together, these experiments indicated that CstF-50 has a strong stimulatory effect on the ubiquitination of RNAP IIO by BRCA1/BARD1 in vitro.
CstF-50 (ng): polyUb-RNAP IIO 250- -RNAP IIO
1 2 3 4 5 6
Figure 20: CstF-50 can activate the ubiquitination of RNAP IIO by BRCA1/BARD1
in vitro. In vitro ubiquitination reactions were conducted in the presence of E1, His-E2,
∆BRCA1/BARD1-WT, His-Ub, ATP, RNAP IIO (100 ng) and increasing amounts of
GST-CstF-50. Samples were fractionated by SDS-PAGE and immunoblotted with RNAP
IIO (H5, Covance) specific antibodies. Purification and separation of the two RNAP II
isoforms (IIA and IIO) from NE pellets was done as described (Hirose and Manley
1998).
To confirm that CstF-50 indeed activates the ubiquitination of RNAP IIO by
BRCA1/BARD1, I knocked-down CstF-50 expression in HeLa cells by taking advantage of the siRNA technique. As the ubiquitination of RNAP II is induced by UV treatment, cells were exposed to UV irradiation (20 Jm-2) and allowed to recover for 2 h. Two different CstF-50 siRNAs were used giving same knockdown efficiencies. As shown in Figure 21A, the siRNA– mediated depletion of CstF-50 (75-80%) resulted in the stabilization of RNAP IIO after UV damage. CstF-50 depletion resulted in a 30% decrease in RNAP IIO degradation (Figure 21B).
66
B) A) 120 100 siRNA: Control CstF-50 80 UV: - + - + 60 40
-Topo II protein protein levels % of % RNAP II 20 130- 0 siRNA: Control Control CstF-50 CstF-50 250- -RNAP II UV UV
250- -BRCA1 C)
100 95- 90 -BARD1 80 70 60 55- 50 -CstF-50 40
30 protein protein levels % of % RNAP II 20 1 2 3 4 10 0 siRNA:
Figure 21: siRNA-mediated knockdown of CstF-50 expression decreases the UV-
induced degradation of RNAP II. A) NEs from HeLa cells treated with either control or
CstF-50 siRNAs and UV irradiation were analyzed by immunoblotting using antibodies
against RNAP II (8WG16, Covance), BRCA1, BARD1 and CstF-50. Antibodies against
Topo II were used as a loading control. B) Quantification of RNAP II protein levels of
the Western blots analyzed in (A) are shown. C) Quantification of RNAP II protein levels
in samples from BRCA1/BARD1 knockdown cells as described in Kleiman et al. (2005).
Interestingly, the magnitude of the decrease in RNAP IIO degradation in CstF-50 depleted cells was similar to the one previously observed in BRCA1/BARD1 depleted cells (Figure 21C)
(Kleiman et al., 2005), suggesting that CstF-50 and BRCA1/BARD1 are part of the same UV- induced ubiquitination pathway.
To further analyze the role of CstF-50 in UV-induced RNAP II ubiquitination, HeLa cells were transfected with HA-tagged Ub expression vector concomitantly with either control or
67
CstF-50 siRNAs (Figure 22A). Then, samples were IPed with HA-antibodies and analyzed by
Western blot. The effect of the proteasomal inhibitor MG132 on UV-treated cells was also tested.
As previously described (Mirkin et al., 2008), CstF-50 depletion increased the levels of RNAP II after UV treatment (Figure 22B). Interestingly, analysis of the HA-IPed samples confirmed that
CstF-50 depletion decreased the levels of RNAP II ubiquitination after UV treatment (compare lanes 7-9 to 10-12). Supporting these results, the increase in RNAP II ubiquitination in
UV/MG132 treated control cells was not observed in CstF-50 depleted cells (compare lanes 9 and
12), suggesting that CstF-50 plays a role in the UV-induced ubiquitination of RNAP II in vivo. It is important to highlight that RNAP II ubiquitination was observed in non UV-treated cells and this was independent of CstF-50 expression (Figure 22B). These results are consistent with previously published data showing that monoubiquitinated RNAP II is present at low levels in unstressed cells (Wilson et al., 2013) and with the possibility that RNAP II is targeted for ubiquitination in vivo by more than one E3 ligase (Kleiman et al., 2005; Mitsui and Sharp, 1999).
Our results confirm that CstF-50 is necessary for UV-induced proteasomal degradation of RNAP
II. The data presented here is the first description of an activator for BRCA1/BARD1 E3 Ub ligase activity. So far, only inhibitors of BRCA1/BARD1 activity have been described. For example, UBXN1 dramatically reduces the E3 ligase activity of BRCA1/BARD1 by recognizing autoubiquitinated BRCA1 (Wu-Baer et al., 2010). It has also been shown that BRCA1/BARD1- mediated ubiquitination of NPM and autoubiquitination of BRCA1 are dramatically inhibited by
CDK2 (Hayami et al., 2005).
68
A)
siRNA: Control CstF-50 MG132: - - + - - + UV: - + + - + +
-Topo II 130-
55- -CstF-50
1 2 3 4 5 6
B) Input HA-IP
siRNA: Control CstF-50 Control CstF-50 MG132: - - + - - + - - + - - + UV: - + + - + + - + + - + +
-Topo II 130-
250- -RNAP II
1 2 3 4 5 6 7 8 9 10 11 12
Figure 22: CstF-50 activates the polyubiquitination of RNAP II in vivo. A) Extracts
from Hela cells transfected with control or CstF-50 siRNA, HA-Ub and treated or not
with UV and MG-132 were analyzed by immunoblot with anti-CstF-50 antibody to
confirm the knockdown efficiency. Antibodies against Topo II were used as a loading
control. B) Cells extracts from samples described in (A) were prepared and IPed with
anti-HA agarose beads. Equivalent amounts of the pellets (IP) were resolved by SDS-
PAGE and proteins were detected by immunoblotting using the indicated antibodies. 5%
of the extracts used in the IP reaction are shown as input.
Previous reports have demonstrated that different histones are also enzymatic substrates for BRCA1/BARD1 E3 Ub ligase (Mallery et al., 2002). In normal cellular conditions, approximately 10 % of total H2A and 1-2 % of total H2B are monoubiquitinated in mammals
(Spencer and Davie, 1999), and this modification has been associated with normal active
69 transcription (Davie and Murphy, 1990). It has also been postulated that the ubiquitination of histones may possibly alter nucleosome or chromatin structures, thereby opening up the DNA to allow the access of the transcriptional machinery (Spencer and Davie, 1999). Based on this information, it is possible that histone ubiquitination might also have a profound influence in other DNA-template processes like DNA repair (Zhu and Wani, 2010) and mRNA processing. In fact, it has been recently shown that H2B ubiquitination does interfere with chromatin compaction leading to an open and biochemically more accessible chromatin conformation due to the bigger size of this modification and its unique position in the nucleosomal surface (Fierz et al., 2011).
Consistent with this, H2A ubiquitination, a modification located in the opposite side of the nucleosomal surface, does not appear to hinder chromatin compaction (Jason et al., 2001).
An “access-repair-restore” model has been postulated that conceptually describes how
DNA repair might occur in chromatin environment. This model states that after DNA damage, chromatin is locally destabilized to allow the access of the repair machinery to the lesions. After the repair is completed, the original chromatin structure has to be restored to its original state.
The histone modifications describe above are easily integrated to this model since H2B ubiquitination opens up the chromatin and may allow the recruitment of the repair factors into the damaged sites, and the DDR-dependent H2A ubiquitination may be the post-repair process related to chromatin restoration (Zhu et al., 2009a).
To better understand the role of CstF-50 in the BRCA1/BARD1-mediated ubiquitination of histones the effect of increasing amounts of CstF-50 on the ubiquitination of H2A and H2B by
BRCA1/BARD1 was tested in in vitro assays. The heterodimeric complex ∆BRCA1/BARD1 was incubated with increasing amounts of recombinant GST-CstF-50 and either H2A or H2B.
The reaction products were then analyzed by Western blotting with an anti-histones antibody, as
70 well as an anti-Ub antibody to measure total, nonspecific ubiquitination. Interestingly, CstF-50 increased the levels of monoubiquitination of H2A and H2B (Figure 23A-B). Together, these experiments indicated that CstF-50 has a strong stimulatory effect on the monoubiquitination of histones by BRCA1/BARD1.
A) B)
CstF-50(ng): 0 25 50 100 150 - - CstF-50 (ng): 0 25 50 100 150 - -
25- -Ub-H2A
-H2A 15- 55-
-Ub-H2B 36- 25-
25- -Ub-H2A 15- -H2B 1 2 3 4 5 6 7 1 2 3 4 5 6 7
Figure 23: CstF-50 can activate the monoubiquitination of H2A and H2B by
BRCA1/BARD1. A) In vitro ubiquitination reactions were conducted in the presence
of E1, His-E2, His-Ub, ∆BRCA1/BARD1-WT, ATP and increasing amounts of GST-
CstF-50. One microgram of commercially available H2A and H2B (New England
Biolabs) were used as substrates. Samples were fractionated by SDS-PAGE and
immunoblotted with anti-histones antibodies (monoclonal MAB052, Millipore). B)
Blot of the H2A in vitro ubiquitination reactions shown in A) immunoblotted with anti-
Ub antibodies (P4D1, Santa Cruz Biotechnology).
It is important to highlight that the recombinant proteins that I have expressed in E. coli to perform the in vitro assays are devoid of Ub modifications since prokaryotes lack the enzymatic machinery for Ub conjugation (Wu-Baer et al., 2010). Therefore, these reactions are a good
71 system to analyze ubiquitination and the factors that could modify the activity of the E3 ligases involved in the reaction. As these reactions might be regulated by other proteins or in certain cellular conditions, it is important to analyze these reactions in intact cells. To investigate the effect of BRCA1/BARD1/CstF-50 on the ubiquitination of histones in vivo, HeLa cells were transfected with HA-tagged Ub expression vector, and FLAG-histones expression vectors (H2A or H2B), concomitantly with either control or CstF-50 siRNAs (Figure 24A-B). Whole cell extracts were prepared and IPed with anti-FLAG M2 magnetic beads and analyzed by Western blot (Figure 24C-D). The FLAG-tagged histones (H2A, H2B) were constructed by inserting each histone coding sequence into the FLAG-pCMV10 vector. The cloned genes were obtained by
PCR from HeLa cells cDNAs. The HA-Ub constructs and FLAG-pCMV10 vector were kindly provided by Dr. Zhong (Hunter College, CUNY). As shown in Figure 24C and D, knockdown of
CstF-50 expression abolished the monoubiquitination of H2A and H2B independently of UV treatment. Western blot analysis with HA antibodies of Flag-IPed samples confirmed the role of
CstF-50 in histones monoubiquitination. No changes in the levels of total H2A and H2B were detected after either UV treatment or CstF-50 depletion. Together, the in vitro and in vivo ubiquitination assays indicated that the mRNA 3’ processing factor CstF-50 is an activator of the ubiquitination of some BRCA1/BARD1 substrates.
Chromatin-bound fractions from Hela cells transfected as described in Figure 24 were analyzed for H2B ubiquitination by immunoblot. Monoubiquitination of H2B decreased in chromatin fractions of both CstF-50 depleted and UV treated cells (Figure 25). Interestingly, concomitant UV/MG132 treatments caused a dramatic decrease of Ub-H2B in the chromatin and the accumulation of higher molecular ubiquitinated forms. A similar lack of signal in our
72
A) B)
siRNA: Control CstF-50 siRNA: Control CstF-50 UV: - + - + UV: - + - +
-Topo II -Topo II 130- 130- 55- -CstF-50 55- -CstF-50
-Ub-Flag-H2A -Ub-Flag-H2B
15- -Flag-H2A 15- -Flag-H2B 1 2 3 4 1 2 3 4
C) D) Input Flag- IP Input Flag- IP
siRNA: Control CstF-50 Control CstF-50 siRNA: Control CstF-50 Control CstF-50 UV: - + - + - + - + UV: - + - + - + - + -Topo II -Topo II 130- 130- -Ub-Flag-H B -Ub-Flag-H2A 2 -Flag-H B 15- -Flag-H2A 15- 2
-HA-Ub-Flag-H A 25- 2 25- -HA-Ub-Flag-H2B 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Figure 24: CstF-50 activates H2B and H2A ubiquitination in vivo. A) Cell extracts
from Hela cells transfected with control or CstF-50 siRNA concomitantly with HA-Ub
and with either FLAG-H2A or H2B were analyzed by immunoblot with the indicated
antibodies. The effect of UV treatment was also analyzed. Antibodies against Topo II
were used as a loading control. B) Samples analyzed in A) were IPed with anti-FLAG
M2 magnetic beads (Sigma) followed by Western blot analysis with the indicated
antibodies. 5% of the cell extracts used in the IP reaction is shown as input.
Western blot analysis was observed by others (Mimnaugh et al., 1997). This phenomenon occurs not only by a decrease in H2A and H2B ubiquitination due to depletion of unconjugated Ub but also by an increase in H2A and H2B deubiquitination, explaining the lack of signal in our
Western blot analysis. The unconjugated Ub is exhausted by the accumulation of non-degraded
73
siRNA: Control CstF-50 MG132: - - + - - + UV: - + + - + +
-Topo II 130- 55- -CstF-50
-Flag-H2B 15- Chromatin- bound HA-poly-Ub
-HA-Ub-Flag-H2B 15-
-HA-Ub-Flag-H2B 15- 1 2 3 4 5 6
Figure 25: H2B ubiquitination in chromatin-bound fractions depend on CstF-50
expression. HeLa cells were treated with either control or CstF-50 siRNAs
concomitantly with HA-Ub and FLAG-H2B. UV and MG132 treatment was also
performed. Chromatin-bound fractions were prepared as described in Experimental
Procedures followed by Western blot analysis with the indicated antibodies. 5% of the
cell extracts used in the IP reaction is shown as input. To better observe the UV-induced
decrease in FLAG-H2B a lower exposure of the Western blot with anti-HA antibodies is
shown.
ubiquitinated proteins (Figure 25, lane 3). Extending these studies, I analyzed the direct interaction of CstF-50 with some other BRCA1/BARD1 substrates, such as H2A and H2B. The direct interaction between these proteins was confirmed by performing GST pull-down assays using recombinant GST-CstF-50 and commercially available H2A and H2B for the pull-down.
The interaction was evident only when GST-CstF-50 but not GST was used to pull-down both
74
H2A and H2B (Figure 26). Together these experiments indicate that CstF-50 can interact directly with several BRCA1/BARD1 substrates, such as RNAP II, H2A and H2B.
-H A -H B 15- 2 15- 2 1 2 3 4 5 6
Figure 26: CstF-50 interacts directly with H2A and H2B in vitro. Immobilized GST-
CstF-50 or GST on glutathione-agarose beads was incubated with H2A or H2B (1 μg,
New England Biolabs). Bound proteins were eluted, resolved in SDS-PAGE and detected
with anti-histones antibodies. 5% of the histones used in the reaction were loaded as
input.
I have shown so far that ubiquitination RNAP II and histones by BRCA1/BARD1 is activated by CstF-50 (Figures 20-25). I have also uncovered a role for p97 in regulating the
BRCA1/BARD1 E3 Ub ligase activity (Figure 19). To further analyze the CstF-50-associated
BRCA1/BARD1 ubiquitination pathway I extended my studies to the p97 factor. I tested the effect of p97 on the ubiquitination of some of the BRCA1/BARD1 substrates. First, I determined whether p97 is involved in the RNAP II ubiquitination in mammalian cells by treating HeLa cells with increasing amounts of the p97 inhibitor DeBQ (LifeSensors) in non-damaging as well as under UV conditions. DeBQ is an inhibitor that reduces the ATPase activity of p97 as described by Chou and colleagues (Chou et al., 2011). Cellular fractionations were prepared as described in Experimental Procedures and analyzed by Western blot (Figure 27). When cells were treated with both UV and DeBQ, RNAP II levels were stabilized in both soluble nuclear 75 and chromatin-bound fractions (Figure 27A-B). Consistent with this, it has been shown that p97 facilitates the UV-dependent turnover of RNAP II in yeast and that an adaptor downstream of the
E3 Ub ligase is involved in this reaction (Verma et al., 2011). These results suggest that p97 might be the protein responsible for escorting polyUb-RNAP II to the proteasome where it is finally degraded. Besides, our data suggest that CstF-50 might be the adaptor needed downstream of BRCA1/BARD1 E3 Ub ligase in mammalian cells. The inhibition of p97 had a similar effect on RNAP II levels to that observed by depletion of BRCA1/BARD1 (Kleiman et al., 2005) and CstF (Mirkin et al., 2008); Figures 21-22), supporting the idea that all these factors participate in the same ubiquitination pathway.
Then I determined whether DeBQ inhibitor has any effect on the BRCA1/BARD1/CstF-
50-mediated ubiquitination and consequent eviction of histones from chromatin. The experiment was performed as described above for RNAP II (Figure 27B). Interestingly, the treatment with
UV and low concentration of DeBQ (10 µM) increased the levels of Ub-H2A and Ub-H2B in the chromatin, supporting the idea that p97 plays a role in the extraction from chromatin of factors involved in DDR (Vaz et al., 2013). The treatment with higher concentrations of p97-inhibitor
(20 µM) and UV irradiation had a similar effect to that observed with UV/MG132 treatment
(Figures 25 and 31B). This data indicate that both ATPase activity of p97 and CstF-50 are required for activation of the UV-induced BRCA1/BARD1 E3 Ub ligase.
DISCUSSION
BRCA1, in concert with BARD1, possesses significant E3 Ub ligase activity which might account for many of BRCA1/BARD1 roles in DNA repair, genome stability, and gene expression during DDR. Although a lot of work has been done to understand the biochemistry of the
76
A) B) chromatin-bound Sol nuclear DeBQ (µM): 0 20 0 10 20 DeBQ (µM): 0 20 0 10 20 UV: - - + + + UV: - - + + + -Topo II -Topo II 130- 130- 250- -RNAP II 250- -RNAP II
1 2 3 4 5 25- -Ub-H2A
-Ub-H B 25- 2
15- -H2B 1 2 3 4 5
Figure 27: p97 plays a role in the ubiquitination of RNAPII and histones in different
compartments. Soluble nuclear and chromatin-bound fractions were prepared as
described in Experimental Procedures. Soluble nuclear A) and chromatin-bound fractions
B) from cells treated with UV irradiation and increasing amount of p97 inhibitor DBeQ
(Life Sensors) were analyzed by immunoblot with the indicated antibodies.
BRCA1/BARD1 E3 ligase, very little is known of its cellular targets, how these targets are chosen and the mechanism by which they are recruited to DNA damage sites. Previous studies showed that the formation of a complex between BRCA1/BARD1 and the mRNA 3’ processing factor CstF-50 regulates mRNA 3’ processing after DNA damage (Cevher et al., 2010; Kleiman and Manley, 2001; Mirkin et al., 2008; Nazeer et al., 2011); and proteasome-mediated degradation of RNAP II (Kleiman et al., 2005). In addition, it has been shown that the large subunit of RNAP IIO is a specific target of the BRCA1/BARD1 E3 Ub ligase activity (Kleiman et al., 2005) and that CstF plays a role in the ubiquitination and turnover of RNAP IIO as well as in the repair of the transcribed DNA strand, which are known events in the DDR (Figures 9;
77
(Mirkin et al., 2008). Based on these results I proposed in this dissertation that CstF could play an important role in the response to DNA damage.
As part of this dissertation, I have extended these investigations and provided unexpected insights into the role of CstF-50 and BRCA1/BARD1 in the Ub pathway and chromatin structure during DDR. First, I showed the direct interaction between CstF-50 not only with Ub but also with some of BRCA1/BARD1 substrates, such as RNAP II, H2A and H2B (Figures 13-26). As an important fraction of nuclear Ub associates with CstF-50 independently of UV treatment (Figure
14) and the BRCA1/BARD1/CstF complex formation is induced only after UV treatment
(Kleiman and Manley, 2001; Kleiman et al., 2005), it is possible that UV treatment might induce the interaction between CstF-50/Ub and BARD1 (Figure 14). Second, I determined that CstF-50 is able to activate the Ub ligase activity of BRCA1/BARD1 by enhancing its autoubiquitination
(Figure 15), suggesting a potential role for CstF-50 as an E4 enzyme that helps BRCA1/BARD1 to recognize the substrate. Supporting this idea, CstF-50 can interact with p97 (Figure 16), which is one of the factors involved in the escort pathway of ubiquitinated substrates to the proteasome, and that p97 is able to further activate the BRCA1/BARD1 Ub ligase activity having a synergistic effect with CstF-50 (Figures 19, 27). Third, I found that CstF-50 is also an activator of transubiquitination reactions of known BRCA1/BARD1 substrates involved in the DDR, such as RNAP IIO and histones H2A and H2B (Figures 20-25). Together these results reveal an unexpected functional connection between the mRNA 3’ processing machinery and the Ub pathway.
The results presented in this Chapter provide evidence that CstF-50 plays a role in
BRCA1/BARD1-mediated ubiquitination, which is part of DDR. Therefore, as CstF-50 can directly bind BARD1, different BRCA1/BARD1 substrates and Ub, it is possible that it functions
78 to help in the assembly or stabilization of the ubiquitination complex, affecting the chromatin structure and, hence, gene expression after DNA damage. These results also support the idea that
CstF-50 might have E4 enzymatic activity, binding Ub and helping in determining substrate specificity for ubiquitination by BRCA1/BARD1 and/or substrate fate by escorting ubiquitinated substrates to the proteasome. Ub chain elongation factors E4s have been shown to participate in the elongation of short poly(Ub) chains by mediating transfer of Ub to a previously conjugated
Ub molecule rather than to the substrate itself, resulting in a fully elongated poly(Ub) chain
(Koegl et al., 1999; Wu and Leng, 2011a). Consistent with this, BRCA1/BARD1/CstF-50 complex functionally interacts with p97, which coordinates the elongation of the Ub chain of ubiquitinated substrates and their delivery to the proteasome through the binding to Ub chain receptors (Meyer et al., 2002). Interestingly, p97-mediated regulation of substrates ubiquitination occurs in the presence of Ub-binding or substrate-processing cofactors that either activate or inhibit polyubiquitination or deubiquitination of the substrate (Koegl et al., 1999; Richly et al.,
2005; Rumpf and Jentsch, 2006). The results shown in this dissertation indicate that
BRCA1/BARD1/CstF-50 complex associated with p97 participate in the UV-induced degradation of RNAP II (Figure 27). Consistent with this, it was shown in yeast that p97 facilitates the UV-dependent turnover of RNAP II at sites of stalled transcription and that an adaptor to the CUL3 Ub ligase, UbX5, facilitates the degradation of chromatin-bound RNAP II
(Verma et al., 2011). Besides, it has been shown that p97 binds Ub-binding factors E4s (Koegl et al., 1999).
The data presented in this dissertation is the first description of an activator of
BRCA1/BARD1 E3 Ub ligase activity. Several inhibitors of BRCA1/BARD1 activity have been described: UBXN1 that reduces the E3 ligase activity by recognizing autoubiquitinated BRCA1
79
(Wu-Baer et al., 2010); CDK2 that reduces both BRCA1/BARD1-mediated ubiquitination of
Nucleophosmin and autoubiquitination of BRCA1 (Hayami et al., 2005). Further studies on substrates and factors involved in this CstF-50/p97-mediated regulation of BRCA1/BARD1 Ub ligase activity may allow us to better understand the function of this tumor suppressor complex in
DDR and disease. This study provided evidence of a link between transcription, mRNA 3’ processing, DNA repair and the Ub-pathway.
80
CHAPTER V
CstF-50 REGULATES THE BRCA1/BARD1 Ub LIGASE ACTIVITY RESULTING IN
CHROMATIN REMODELING DURING DDR
81
INTRODUCTION
In eukaryotic cells, genomic DNA is packed in a complex and dynamic structure called chromatin, which basic building blocks are the nucleosomes. Nucleosomes consist of a segment of 147 bp segment of DNA wrapped around a histone octamer, including a tetramer of histone
H3 and H4, flanked by two H2A-H2B dimers. These core histones are predominantly globular with the exception of their unstructured amino terminal “tails”, which can undergo a large number of modifications. Nucleosomes are usually packed together with the aid of a histone H1 to form a 30 nm large fiber allowing the formation of a higher level of organization (Kouzarides,
2007; Zhu and Wani, 2010). Since highly condense chromatin is refractory to DNA repair enzymes and blocks access of these factors to the damage DNA, packaging of DNA into chromatin represents a strong barrier for efficient DNA repair (Nag and Smerdon, 2009).
Mechanisms must exist to assure that chromatin is flexible and modifiable both at the nucleosome and at a higher level of packaging to allow accessibility of repair factors to damaged
DNA. Chromatin dynamic, which oscillates between highly compact (heterochromatin) and more loosely packed (euchromatin) states, is regulated by a combined effect of rearrangements.
These rearrangements are accomplished by different mechanisms: ATP-dependent chromatin remodeling enzymes, histone variants, histone chaperones, and histone prost-translational modifications (Nag and Smerdon, 2009).
All core histones are subjected to a vast array of post-translational modifications that include phosphorylation, acetylation, methylation, SUMOylation, proline isomerization, citrullination, ribosylation, and ubiquitination. These modifications occur within the amino or carboxy-terminal tail region of each histone but a growing number of modifications at the nucleosome core level have been also described (Peterson and Almouzni, 2013). It is very likely
82 that a “crosstalk” between modifications occurs due to abundance and overlapping of the modified sites. For instance, acetylation, methylation and ubiquitination all happen at Lys residues, causing some form of antagonism since these modifications are functionally different and mutually exclusive (Kouzarides, 2007). Other possibilities are that the binding of one protein can be disrupted by an adjacent modification as well as the enzymatic activity of a protein can be disturbed or enhanced by modification of its substrate recognition site. Besides, a trans-tail regulation has also been established, where the modification on one histone influences the modification on a different histone (Mellor, 2008).
There are two broad mechanisms by which histone modifications control chromatin plasticity. First, post-translational modifications can indirectly influence chromatin organization by creating or eliminating binding sites for non-histone proteins that influence its structure (eg.
ATP-dependent chromatin remodelers). Second, post-translational modifications can directly impact either the stability of individual nucleosomes or the ability of chromatin to fold into higher order compaction (Peterson and Almouzni, 2013).
Ubiquitination is the bulkiest post-translational modification of histones because it conjugates an 8.5 kDa peptide (Ub) to lysine residues. In particular, the modification of both histones and repair factors by ubiquitin has recently been demonstrated to play a crucial role in the detection and repair of DNA damage. Ubiquitination of H2A has been generally associated with gene silencing during transcription, while H2B ubiquitination has been related to both silencing and gene activation (Dinant et al., 2008). In addition, histones H3 and H4 are less abundantly ubiquitinated and there are still no functional consequences assigned to this modification (Dinant et al., 2008).
83
It was recently showed that BRCA1/BARD1 regulates chromatin structure by histones ubiquitination (Zhu et al., 2011). BRCA1 binds to satellite DNA regions and ubiquitinates histone H2A in vivo. Cells deficient in BRCA1 show an impaired heterochromatin integrity that causes the disruption of gene silencing at tandem repeated DNA regions due to the loss of Ub-
H2A (Zhu et al., 2011). Interestingly, it has been shown that UV treatment induces monoubiquitination of H2A (Bergink et al., 2006), resulting in the eviction of Ub-H2A from UV- damaged chromatin (Lan et al., 2012).
These rearrangements in chromatin structure have been described as the “Access-Repair-
Restore” model that involves the disorganization and subsequent re-organization of chromatin
(Adam and Polo, 2012). The mechanisms by which these rearrangements occur are still poorly understood. Regulation of chromatin structure has also an effect on DNA-based processes like transcription, RNA processing, replication and DDR, which occur in euchromatin (Kouzarides,
2007; Marteijn et al., 2009)
The data presented in Chapter IV indicate that CstF-50-associated BRCA1/BARD1 can activate histone monoubiquitination (Figures 23-25) as well as RNAP II polyubiquitination
(Figures 20-22). Interestingly, some of these ubiquitination events have been described as part of
DDR (Bergink et al., 2006; Kleiman et al., 2005; Mirkin et al., 2008). At this point, it is critical to understand the role of these factors in chromatin remodeling and their effects on the regulation of gene expression in different cellular conditions.
In this Chapter, I show that UV-treatment induces changes in the localization of BRCA1,
BARD1, CstF-50, p97 and some of BRCA1/BARD1 substrates in different nuclear fractions, and that these changes depend on BRCA1/BARD1 and CstF-50 expression. The content of monoubiquitinated H2B in the chromatin of genes with different levels of expression changes
84 during DDR and this is mediated by BRCA1/BARD1 and CstF-50. Taken together, my results provide evidence that CstF-50-associated p97 regulates BRCA1/BARD1 Ub ligase activity facilitating chromatin remodeling during the progression of DDR.
RESULTS
As some of the substrates of the CstF-50-associated BRCA1/BARD1 E3 Ub ligase are located in the chromatin, I decided to evaluate the effect of UV treatment and expression of
CstF-50 and BRCA1/BARD1 on their cellular localization by carrying out cellular fractionation assays. Samples from HeLa cells were separated in cytoplasmic, soluble nuclear and chromatin- bound fractions, and analyzed by Western blot to confirm the proper separation of the different cellular compartments and to make sure there was no leakage from one compartment to the other
(Figure 28).
Western blot analysis of soluble nuclear (Figure 29A) and chromatin-bound fractions
(Figure 29B) indicated that BRCA1, BARD1, CstF-50, CstF-64 as well as RNAP II co-localized in those fractions. As expected, H2A, H2B and their modified isoforms were detected only in the chromatin-bound fractions. I extended these studies to p97, the escort protein in the Ub pathway
(Meyer et al., 2002). As BRCA1/BARD1/CstF-50 complex, p97 was also detected in both soluble and chromatin fractions. Importantly, UV treatment had little effect on the composition of soluble nuclear fractions, except for an increase in p97 and a decrease in RNAP II due to its
UV-induced proteasomal degradation (Figures 29A). However, several changes were observed in the chromatin-bound fraction. First, like in the soluble nuclear fraction, RNAP II levels decreased after UV treatment. Surprisingly, there was a reduction in the levels of CstF-50,
BRCA1, BARD1, monoubiquitinated H2A (Ub-H2A) and monoubiquitinated H2B (Ub-H2B)
85 after UV damage (Figure 29B). No evident changes were observed for the levels of unmodified histones in these conditions. The reduction of Ub- histones in the chromatin after UV irradiation was also observed in acidic nuclear extracts prepared as described in Experimental procedures
(Figure 29C).
Cellular compartments
250- -RNAP II
250- -BRCA1
-Topo II 130-
-p97 95-
95- -BARD1
72- -Lamin
-CstF-64 55-
55- -CstF-50
55- -actin
35- -GAPDH
17- -H2A
17- -H2B
1 2 3
Figure 28: Cellular fractionation of HeLa cell samples. Cytoplasmic, soluble nuclear
and chromatin-bound fractions were prepared as described in Experimental Procedures.
Fractions were subsequently analyzed by Western blot with the antibodies indicated in
the figure to ensure proper fractionation. 86
A) B) sol nuclear chrom-bound UV: - + UV: - + -Topo II -Topo II 130- 130- 250- 250- -RNAP II -RNAP II
250- 250- -BRCA1 -BRCA1
-p97 95- -BARD1 95-
95- -BARD1 95- -p97 72- 72- -CstF-64 -CstF-64
55- -CstF-50 55- -CstF-50
1 2 25- -Ub-H2B C) Acidic
Extracts 25- -Ub-H2A UV: - +
17- -H2A 25- -Ub-H2A
17- -H2B 25- -Ub-H2B 1 2 1 2
Figure 29: UV-treatment induces changes in the localization of BRCA1, BARD1,
CstF-50 and some of its substrates in nuclear fractions. Soluble nuclear and
chromatin-bound fractions were prepared as described in Experimental Procedures. A)
Soluble nuclear fractions from cells exposed to non-damaging and UV treatment were
analyzed by Western blot with the indicated antibodies. B) Chromatin-bound fractions
were analyzed as in (A). C) Acidic extracts were prepared as described in Experimental
Procedures and analyzed by immunoblot using antibodies specific for Ub-H2A and Ub-
H2B.
These results are consistent with previous studies that show the eviction of Ub-H2A with the concomitant release of the corresponding E3 Ub ligase from the UV-damaged chromatin
87
(Lan et al., 2012). Moreover, p97 was recruited to the chromatin after UV treatment (Figure
29B), supporting the proposed role for p97 in protein extraction from chromatin during DDR
(Vaz et al., 2013). The co-fractionation of these factors with chromatin-associated monoubiquitinated histones suggests that these factors might be functionally associated during
DDR. Furthermore, the treatment with both the proteasomal inhibitor MG132 and UV irradiation resulted in the stabilization of RNAP II, BRCA1 and BARD1 (Figure 30). Supporting the UV- induced ubiquitination of BRCA1, it has been shown that HERC2, a protein implicated in DDR, is an E3 ligase that target BRCA1 for degradation (Wu et al., 2010). However, ubiquitinated histones were not detected in the chromatin after MG132/UV treatment (Figure 30). This unexpected event was previously described by Mimnaugh and colleagues (1997). They presented evidence showing that the inhibition of proteasomal activity not only decreases H2A and H2B ubiquitination due to depletion of unconjugated Ub but also increases H2A and H2B deubiquitination, explaining the lack of signal in the Western blot analysis. These results suggest that the UV-induced change in the localization of these factors might play a role in DDR by modifying the local content of factors in the chromatin and allowing the access of the repair machinery to the damaged site.
Then I determined whether the expression of BRCA1/BARD1 and CstF-50 has an effect on the localization and ubiquitination of these factors. The siRNA-mediated depletion of
BRCA1/BARD1 and CstF-50 diminished the UV-induced degradation of RNAP II in both soluble nuclear (Figure 31A) and chromatin bound-fractions (Figure 31B). This is consistent with our previous studies that showed that BRCA1/BARD1 (Kleiman et al., 2005) and CstF (Mirkin et al., 2008; Figure 9) are involved in the UV-induced ubiquitination and degradation of RNAP II in nuclear samples. Importantly, both CstF-50 and BRCA1/BARD1 expression were necessary for
88 the UV-induced decrease in Ub-H2A and Ub-H2B in the chromatin fractions (Figure 31B).
Together these results indicate that the UV-induced formation of the BRCA1/BARD1/CstF-50 complex does not only have an effect on the ubiquitination of some chromatin components but also in chromatin remodeling during DDR.
chrom-bound MG132: - - + UV: - + + -Topo II 130-
250- -RNAP II
250- -BRCA1
-p97 95-
95- -BARD1
72- -CstF-64
55- -CstF50
25- -Ub-H2B
25- -Ub-H2A
17- -H2A
17- -H2B
1 2 3
Figure 30: UV-treatment induces changes in the localization of BRCA1, BARD1,
CstF-50 and some of its substrates in nuclear fractions. Soluble nuclear and
chromatin-bound fractions were prepared as described in Experimental Procedures.
Chromatin-bound fractions treated with proteasome inhibitor MG132 were analyzed by
Western blot with the indicated antibodies.
89
A) B)
soluble nuclear chromatin-bound siRNA: Control CstF-50 BRCA1/ siRNA: Control CstF-50 BRCA1/ BARD1 BARD1 UV: - + - + - + UV: - + - + - +
-Topo II -Topo II 130- 130- 250- -RNAP IIo 250- -RNAP II -RNAP IIa
250- 250- -BRCA1 -BRCA1
95- -BARD1 95- -p97
-p97 95- -BARD1 95-
55- 55- -CstF-50 -CstF-50
1 2 3 4 5 6 25- -Ub-H2B
25- -Ub-H2A
17- -H2B
17- -H2A 1 2 3 4 5 6
Figure 31: BRCA1/BARD1 and CstF-50 play a role in the ubiquitination of RNAP
II and histones in different nuclear fractions. Soluble nuclear and chromatin-bound
fractions were prepared as described in Experimental Procedures. Soluble nuclear (A)
and chromatin-bound (B) fractions from cells exposed to UV treatment and to control,
CstF-50 or BRCA1/BARD1 siRNAs were examined by Western blot with the indicated
antibodies.
Finally, I determined the implications of the BRCA1/BARD1/CstF-50-mediated changes in chromatin structure at gene specific level after UV damage using chromatin IP assays (ChIP) followed by qPCR. Briefly, chromatin extracts from crosslinked HeLa cells treated with UV and either control, CstF-50 or BRCA1/BARD1 siRNAs were prepared as described in Experimental
Procedures (Figure 32A). Samples were then IPed with a specific antibody against Ub-H2B, and
90 the IPed DNA fragments were analyzed by qPCR using primers for differentially expressed genes. The primers were designed on the transcribed region of each gene since it was previously shown that Ub-H2B modifications are preferentially targeted within this area (Minsky et al.,
2008). I tested highly expressed genes (DHFR and GAPDH), silent genes (CD4 and insulin) and
DDR-related genes (p53 and CHEK2). Non-IPed chromatin extracts were used as normalization control. To facilitate the comparison among different conditions the ratio of the amount of DNA co-IPed by Ub-H2B from UV-treated to no-UV-treated samples was analyzed. The DNA quatification by qPCR in UV-treated samples were lower than the ones in non-treated samples in all the conditions analyzed (not shown), reflecting the UV-induced decrease in Ub-H2B in the chromatin observed by Western blots (Figures 29B and 30B). The depletion of BRCA1/BARD1 and CstF-50 expression increased the UV/no UV ratio in highly expressed genes (Figure 32B), supporting the idea that BRCA1/BARD1/CstF-50 complex ubiquitinated H2B after UV treatment resulting in a decrease of its content in the chromatin in this group of genes (Figure 31B). This is consistent with our previous studies showing the role of BRCA1/BARD1 and CstF-50 in transcription-coupled RNA processing/DNA repair (Mirkin et al., 2008). Interestingly, there was no change in the UV/no UV ratio in silent genes (Figure 32C), reflecting that the content of Ub-
H2B in the chromatin was not affected by either BRCA1/BARD1 and CstF-50 expression or UV treatment in this group of genes. Strikingly, the expression of BRCA1/BARD1 and CstF-50 had an opposite effect on genes involved in DDR compared to that observed in highly expressed genes (Figure 32D). The knockdown of BRCA1/BARD1 and CstF-50 expression decreased the
UV/no UV ratio in genes involved in DDR, indicating that the level of Ub-H2B in the chromatin increased. Although more studies are necessary to better understand these results, it is possible that we are addressing a post repair or chromatin restoration event. As these studies were
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A) B) chromatin extracts siRNA: Control CstF50 BRCA1/ BARD1 Highly transcribed genes UV: - + - + - + 70 -Topo II 60 130- 50 250- 40 GAPDH -BRCA1 30 DHFR
20 Ratio UV/no(%) UV 95- -BARD1 10 0 siRNA Control CstF BRCA1/BARD1 55- -CstF-50
1 2 3 4 5 6 C) D)
Silent genes DNA damage response genes 25 60
20 50 40 15 30 10 CD4 p53 INSULIN 20 CHEK2
Ratio UV/no(%) UV 5
Ratio Ratio UV/no UV (%) 10 0 0 Control CstF BRCA1/BARD1 siRNA siRNA Control CstF BRCA1/BARD1
Figure 32: BRCA1/BARD1/CstF-50 has a distinctive effect on chromatin structure of genes differentially expressed. Chromatin from cells treated with either control,
CstF-50 or BRCA1/BARD1 siRNAs as well as with UV damage were prepared as described in Experimental Procedures. A) Chromatin extracts were analyzed by immunoblotting using antibodies against the indicated proteins. B-D) Chromatin extracts were IPed with Ub-H2B specific antibody (Millipore) followed by qPCR analysis of the
IPed DNA fragments using primers for differentially expressed genes described in
Experimental Procedures. 1% of non-IPed chromatin extracts were used as normalization control. The ratio of qPCR values from UV/no UV treated samples was plotted.
92 performed 2 hrs after UV-treatment, genes involved in the progression of DDR are probably completely repaired. Together these ChIP assays indicate that differentially expressed genes show different content of ubiquitinated H2B in the chromatin in a BRCA1/BARD1- and CstF-50- dependent manner, suggesting a specific mechanism of regulation of chromatin structure for each group of genes.
DISCUSSION:
BRCA1, in concert with BARD1, possesses significant E3 Ub ligase activity. Previous studies showed that the formation of a complex between BRCA1/BARD1 and the mRNA 3’ processing factor CstF-50 regulates mRNA 3’ processing reaction after DNA damage (Cevher et al., 2010; Kleiman and Manley, 2001; Kleiman et al., 2005; Mirkin et al., 2008; Nazeer et al.,
2011). Extending those studies, I am providing in this dissertation insights into the role of CstF-
50 and BRCA1/BARD1 in the Ub pathway and chromatin structure during DDR. As shown in
Figure 31A-B, the CstF-50-mediated activation of BRCA1/BARD1 Ub ligase resulted in the polyubiquitination and degradation of RNAP II in soluble nuclear- as well as in chromatin- fractions. The monoubiquitination of H2A and H2B and their eviction from chromatin after UV irradiation were also affected by the CstF-50-mediated activation of BRCA1/BARD1 in chromatin-fractions (Figure 31B). My results also indicated that BRCA1/BARD1 E3 Ub ligase, its activator CstF-50, Ub-H2A and Ub-H2B change their nuclear localization after UV treatment, resulting in a BRCA1/BARD1/CstF-50 dependent-chromatin remodeling (Figure 31).
Importantly, BRCA1/BARD1/CstF-50 has a distinctive effect on chromatin structure of genes differentially expressed during DDR (Figure 32), suggesting a specific mechanism of regulation of chromatin structure for each group of genes. Consistent with this, it was described that actively
93 transcribed genes and silenced or repressed areas of the genome might require different chromatin remodeling mechanisms for efficient repair (Nag and Smerdon, 2009).
It has been shown that histone ubiquitination destabilizes the organization of nucleosomes during DDR, suggesting that this modification facilitates access to damaged chromatin by promoting histone eviction from damaged nucleosomes (Lan et al., 2012). Besides, H2A ubiquitination has been proposed to be involved in post-repair chromatin restoration by serving as a signal for the recruitment of chromatin remodelers that allow the reposition of newly formed nucleosomes (Adam and Polo, 2012). A less robust chromatin remodeling is necessary in nucleotide excision repair (NER) compared to other repair pathways because the damages restored by this repair mechanism are single-helix distorting lesions that produce small repair patches of 25-30 nucleotides within a single nucleosome. In contrast, repair of double-strand breaks involves the restoration of hundreds to thousands base pairs and need higher levels of chromatin remodeling (Lans et al., 2012). Furthermore, there are certain chromatin landscapes, such as actively transcribed genes, that might require less remodeling compare to those in silenced or transcriptionally repressed genes that could need more intense chromatin rearrangements for efficient repair (Nag and Smerdon, 2009). Less remodeling is required for transcrition-coupled repair (TCR) because chromatin has already been made more accessible by the transcription machinery (Lans et al., 2012).
Based on the results presented in this dissertation and previous work from Dr. Kleiman’s lab, I propose a model whereby BRCA1/BARD1 coordinates a ubiquitous response to DNA damage by a mechanism that includes interactions with RNAP II and components of the mRNA
3’ processing machinery, specifically CstF-50 (Figure 33). In normal conditions, where chromatin structure is such that allows transcription and mRNA processing, the elongating
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RNAP IIO is interacting with CstF-50 as part of the RNAP II holoenzyme. After DNA damage, the elongating RNAP IIO is stalled at DNA lesions, causing premature termination or arrest of transcription. At this point, chromatin must be locally destabilized to allow the access of the repair machinery to the lesions. In this scenario, BRCA1/BARD1 complex interacts with RNAP
IIO and CstF-50 at sites of DNA damage, preferentially on active genes (Chiba and Parvin, 2001,
2002; Kleiman et al., 2005; Krum et al., 2003; Mirkin et al., 2008). Then, the formation of the
BRCA1/BARD1/CstF-50 complex allows not only the UV-induced ubiquitination of RNAP IIO but also the inhibition of the mRNA 3’ processing machinery (Kleiman and Manley, 2001). The
BRCA1/BARD1/CstF-50 complex at the DNA damaged site might also ubiquitinate H2B, resulting in the remodeling of the nucleosome structure to open up the chromatin to allow the access of the repair machinery to the damaged DNA (Zhu and Wani, 2010). After the damage is repaired, BRCA1/BARD1/CstF-50 complex might also ubiquitinate H2A allowing the restoration of the chromatin structure (Zhu et al., 2009a). This mechanism is in agreement with the described “access-repair-restore model” that conceptually describes how DNA repair occurs in the chromatin environment (Zhu and Wani, 2010).
In fact, it has been recently shown that H2B ubiquitination leads to a biochemically more accessible chromatin conformation due to the size of this modification and its position in the nucleosomal surface (Fierz et al., 2011). Ubiquitination of H2A occurs in the opposite side of the nucleosomal surface and it does not appear to hinder chromatin compaction (Jason et al., 2001).
Moreover, H2A ubiquitination requires active NER (Bergink et al., 2006), suggesting that this posttranslational modification could be a post repair event and serves as a signal for the recruitment of factors to help in the reposition of nucleosomes (Zhu et al., 2009a).
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P P P 2 3 1 H2B H2A H2B H2A H2B H2A H2B H2A
H4 H3 H4 H3 H4 H3 H4 H3
P 3 P P 2 1 UV proteasome
p97 Ub-histone eviction
P
P 2 P 3 1 H2B H2A H2B H2A H2B H2A H2B H2A
H4 H3 H4 H3 H4 H3 H4 H3
Reassembly Disassembly
H2B H2A DNA Repair H2B H2A
H4 H3 H4 H3
Figure 33: Model for the role of polyadenylation factor CstF-50, the Ub-escort factor p97 and the BRCA1/BARD1 Ub ligase during the progression of DDR. After exposure to UV treatment, CstF-50-associated to the elongating RNAP IIO recruits a
BRCA1/BARD1-containing complex, inducing RNAP II ubiquitination and its CstF-
50/p97-mediated escorting to the proteasome for degradation. In that scenario, CstF-50 contribute to the BRCA1/BARD1-mediated ubiquitination of histones H2B, allowing the opening of the chromatin at the damage site and facilitating the DNA repair. The reconstitution of the chromatin structure occurs by the BRCA1/BARD1/CstF-50- mediated ubiquitination of histones H2A. As CstF-50 can functionally interact with elements from the transcription, mRNA processing, DNA repair and the Ub- pathways, we propose a key role for CstF-50 in the progression of DDR.
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BRCA1/BARD1 plays a role in the ubiquitination of histones and cells deficient in BRCA1 show abnormalities in chromatin structure, most likely due to the loss of Ub-H2A (Zhu et al.,
2011). Interestingly, H2A has been shown to be monoubiquitinated after UV treatment (Bergink et al., 2006). As part of this dissertation, I described a BRCA1/BARD1/CstF-50 dependent- chromatin remodeling event during DDR (Figure 31) that includes the eviction of the Ub ligase complex, Ub-H2B and Ub-H2A. Consistent with this, earlier studies showed that the E3 Ub ligase
DDB1-2-CUL4B, which monoubiquitinates H2A in non-transcribed genomic regions,is evicted from the chromatin altogether with Ub-H2A during DDR, allowing the assembly of repair factors at the lesion by the destabilization of the nucleosome (Lan et al., 2012). Interestingly, previous studies from Dr. Kleiman’s lab showed that RNAP II, CstF and BARD1 all associated with repaired/BrdU-containing DNA and play a direct role in the DDR (Mirkin et al., 2008).
Finally, the fate of evicted Ub-histones is still under investigation. It is possible that Ub- histones might be targeted for degradation to ensure elimination of damaged histones, an event described to occur during transcription (Chen and Qiu, 2012). Polyubiquitination of histones has not been reported in eukaryotes but there is a possibility that histones are degraded in a ubiquitination-independent manner by other mechanisms like lysosomal degradation, autophagy or by some non-proteasomal proteases (Chen and Qiu, 2012). Alternatively, histones that are removed from damaged chromatin might be recycled by histone chaperones contributing to chromatin reorganization after DNA repair (Adam and Polo, 2012). Taken together, my results describe a novel link between transcription, mRNA processing, DNA repair, the Ub-proteasome and escort pathway, and chromatin remodeling during DDR.
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CHAPTER VI
BRCA1/BARD1-ASSOCIATED CstF-50/p97 UBIQUITINATES the mRNA BINDING
PROTEIN HUMAN ANTIGEN R (HuR)
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INTRODUCTION
Gene expression in mammalian cells is regulated at the transcriptional and post- transcriptional level. Among the post-transcriptional events, control of mRNA stability and translation is particularly important because it can provide a dynamic cellular response to internal and external cues. mRNAs half-lives and translation rates are controlled by a set of
RNA-binding proteins (RBPs). One of the most well characterized RBPs involved in the control of gene expression during the progression of DNA damage response (DDR) is the Human
Antigen R (HuR) mRNA binding protein (Kim et al., 2010).
HuR is an ubiquitously expressed RBP that belongs to the Hu (ELAV) family of RNA-
BPs, which also includes the neuro-specific proteins HuB, HuC, and HuD. HuR interaction with its target mRNAs is mediated by its three RNA recognition motifs (RRMs). A hinge region was also identified between RRM2 and RRM3 that contains the HuR nucleocytoplasmic shuttling domain (HNS, Figure 34). Phosphorylation at this region is implicated in both HuR nuclear retention in G2/M transition during the cell cycle in unstressed cells and HuR translocation to the cytoplasm upon stress (Kim and Gorospe, 2008). HuR binds the 3’ untranslated region (3’UTR) of target mRNAs, specifically the AU-rich element (ARE) binding sites, helping in the stabilization of those mRNA targets by increasing their half-life (Fan and Steitz, 1998; Zou et al.,
2006). Besides, HuR binding can block the interaction of destabilizing factors, such as deadenylases-associated RBPs, that also bind the same ARE sequence leading to the destabilization and degradation of target mRNAs (Srikantan and Gorospe, 2012). Although HuR is localized mainly in the nucleus, most of the functional studies have focused on its effect on the expression of target mRNAs in the cytoplasm. HuR nuclear function is largely unknown except for a poorly defined role in mRNA splicing (Izquierdo, 2008; Wang et al., 2010) and, more
99 recently, in the coupling of different mRNA processing events (Mukherjee et al., 2011). HuR has been suggested to bind its substrates in the nucleus, as early as co-transcriptionally, and escort them to the cytoplasm and polysomes to stabilize and enhance translation (Fan and Steitz, 1998;
Mukherjee et al., 2011).
HuR HNS
Figure 34: Schematic representation of HuR structure: HuR has three RNA
recognition motifs (RRM, grey), and a hinge region (white) containing a unique nuclear-
cytoplasmic shuttle sequence (HNS, green). Modified from (Venigalla and Turner, 2012).
Many of HuR target mRNAs encode factors implicated in cellular stress-responses. For example, HuR promotes the translation (Mazan-Mamczarz et al., 2003) and mRNA stability
(Zou et al., 2006) of one of the most important DDR regulated genes, the tumor suppressor p53.
This tumor suppressor is the most mutated gene in cancer. It regulates gene expression by activation of transcription of target genes (Tokino and Nakamura, 2000) and of certain miRNAs
(Chang et al., 2007). p53 can also regulate mRNA 3’ processing (Devany et al., 2013; Nazeer et al., 2011). Therefore, the regulation of p53 levels under different cellular conditions is extremely critical. It is well established that p53 protein accumulates in the cell upon DNA damage via the degradation of Mdm2, which ubiquitinates and targets p53 protein for degradation under normal
100 conditions (Honda et al., 1997). As part of the DDR, p53 expression should be rapidly relieved from repression, contributing to rapid response to stress.
Growing evidence suggests that regulation of p53 at the mRNA level is also important for
DDR. A number of proteins have been shown to regulate p53 mRNA through 5’UTR, coding region and 3’UTR (reviewed in (Candeias, 2011; Vilborg et al., 2010). Studies from Dr.
Kleiman’s lab have shown that poly(A) specific ribonuclease (PARN) regulates p53 mRNA steady-state levels under normal conditions through ARE-mediated deadenylation (Devany et al.,
2013) and microRNA-mediated deadenylation (Zhang et al., unpublished data). PARN is the major nuclear deadenylase in mammalian cells and is activated upon UV by CstF-50/BARD1
(Cevher et al., 2010) and p53 (Devany et al., 2013). Interestingly, after UV treatment, the changes in the levels of p53 mRNA and p53 protein were PARN-independent, indicating there is(are) other mechanism(s) involved in the regulation of p53 expression during DDR.
Preliminary data from Dr. Kleiman’s lab indicate that the nuclear localization of elements that control p53 mRNA stability, such as Ago2, miR125b and miR504, changes after UV treatment
(Zhang et al., unpublished data). After DNA damage, p53 mRNA has to be relieved from miRNA- and PARN-mediated repression rapidly, so p53 can contribute to cells rapid response to stress. In a recent study, it has been reported that over 75% of mRNAs with Ago-2 binding sites also have HuR binding sites (Mukherjee et al., 2011). Most of these Ago-2 and HuR binding site pairs are in less than 10 nt distance, suggesting a competitive or cooperative regulation of target mRNAs (Mukherjee et al., 2011). Besides, HuR sites in 3'UTRs of mRNAs overlap extensively with predicted miRNA target sites (Uren et al., 2011), suggesting interplay between the functions of HuR and miRNAs.
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Dr. Devany and collaborators have recently shown that after UV treatment HuR can regulate p53 expression in a UV-dependent manner not only in the cytoplasm but also in the nucleus by reverting the PARN-mediated destabilization of p53 mRNA (Dr. Devany, personal communication). PARN/Ago2 and HuR can compete for binding to p53 3’UTR, providing a mechanism to regulate p53 expression during the progression of DDR (Devany et al, unpublished data). However, the molecular mechanism(s) by which HuR is kept away from the
ARE binding site of the target mRNA allowing PARN/Ago2 to bind is completely unknown.
It has been shown that HuR is post-transcriptionally modified by phosphorylation and ubiquitination. It was reported that HuR phosphorylation by Cdk1 at Ser202 contributes to its nuclear retention (Kim and Gorospe, 2008), and that Chk2 phosphorylates HuR RNA recognition motifs (RRM1 and RRM2), modulating HuR binding to target mRNAs (Kim et al., 2010).
Besides, transient ubiquitin (Ub)-mediated proteolysis of HuR in response to moderate heat shock has been also described (Abdelmohsen et al., 2009). Zhou and colleagues (2013) have shown that p97 destabilizes p21 mRNA, which is an mRNA target of HuR, in the cytoplasm by promoting the extraction of ubiquitinated HuR from the target mRNA-bound to ribonucleoprotein complex under non-stress conditions. Moreover, p97 needs the help of its cofactor, UBXD8, to accomplish this task. These studies suggest a new mechanism through which HuR might control mRNA stability in different conditions that involves the Ub pathway
(Zhou et al., 2013). Zhou and colleagues (2013) did not identify the E3 Ub ligase involved in the ubiquitination of HuR.
The studies presented in this Chapter are aimed to elucidate the mechanism(s) behind
HuR-mediated stabilization of p53 mRNA upon UV damage in the nucleus. The results in
Chapter IV indicate that p97 is involved in the CstF-50-mediated activation of BRCA1/BARD1
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Ub ligase. In this Chapter, I explore the possibility that BRCA1/BARD1 is the E3 Ub ligase that modifies HuR. In fact, my results indicate that BRCA1/BARD1 can promote monoubiquitination
(monoUb) of HuR in vitro. Strikingly, p97-associated CstF-50 inhibits the monoUb of HuR in in vitro assays. Consistent with the idea that CstF-50 is a co-factor or scaffold protein that binds to different BRCA1/BARD1 substrates (Figure 26, Chapter IV), CstF-50 is also able to bind directly to HuR. My results also show that HuR ubiquitination is decreased after UV irradiation.
Based on these results we propose a model where under non-stress conditions BRCA1/BARD1 ubiquitinates HuR, inducing HuR release from the mRNA and allowing the PARN/Ago2- mediated destabilization of mRNAs involved in DDR. After UV treatment, HuR ubiquitination by BRCA1/BARD1 activity is inhibited by CstF-50/p97, resulting in HuR binding to target mRNAs and the stabilization and expression of mRNAs involved in DDR.
RESULTS
Data from Dr. Kleiman’s lab has shown that under UV damaging conditions HuR is the mRNA binding protein that competes with PARN deadenylase/Ago2 complex for binding to the
ARE site located in 3’UTR of p53 (Dr. Devany personal communication). The binding of HuR to the p53 mRNA ARE site results in the stabilization of p53 mRNA and, consequently, the increase in p53 protein levels upon DNA damage. Under non-damaging conditions, HuR binding to p53 mRNA ARE element decreases by an unknown mechanism, favoring the binding of
PARN/Ago2 complex to the same site resulting in the destabilization of p53 mRNA. Recently, it has also been shown that ubiquitination of HuR by an unknown E3 Ub ligase promotes its disassembly from RNPs in a p97-dependent manner (Zhou et al., 2013). My results in Chapter
IV indicate that p97 plays a role in the CstF-50-mediated activation of the BRCA1/BARD1 E3
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Ub ligase activity. To test whether BRCA1/BARD1 is the E3 Ub ligase that modifies HuR I performed in vitro ubiquitination reactions as described (Figure 15) but using His-HuR as a substrate. Significantly, BRCA1/BARD1 was not only able to monoubiquitinate HuR but to weakly diubiquitinate HuR in vitro (Figure 35).
E1 + E2 + ∆BRCA1/BARD1 Ub: + - 55- -diUb-His-HuR -? -Ub-His-HuR
35- -His-HuR
1 2
Figure 35: BRCA1/BARD1 E3 Ub ligase can ubiquitinate HuR. In vitro
ubiquitination reactions were conducted as described in Figure 15 (Chapter IV) in the
presence of 1 μg of His-HuR. Samples were fractionated by SDS-PAGE and
immunoblotted with anti-HuR antibodies.
Interestingly, another band was also present right above the monoubiquitinated form of
HuR. The change in mobility did not correspond to the addition of an extra Ub moiety, so I suspected that phosporylation of the monoubiquitinated form might be a possibility. To determine whether this extra band comes from concomitant ubiquitination and phosphorylation of HuR I performed the in vitro ubiqutination reactions followed by calf intestine phosphatase
(CIP) treatment. As shown in Figure 36, these preliminary studies showed that treatment with
CIP not only decreased the upper band but also the lower band. Unfortunatelly, these results cannot address if phosphorylation occured after ubiquitination or if phosphorylation was required for the ubiquitination of HuR. Further studies will be designed to confirm whether HuR is both
104 phosphorylated and ubiquitinated. I propose to use anti-phospho specific antibodies for HuR, which are available upon request.
E1 + E2 + ∆BRCA1/BARD1
Ub: + + + + - CIP (U): 0 30 20 10 0 55- -diUb-His-HuR -p-Ub-His-HuR -Ub-His-HuR
-His-HuR 35-
1 2 3 4 5
Figure 36: HuR is concomitantly phosphorylated and ubiquitinatinated. In vitro
ubiquitination reactions were conducted as in Figure 15 in the presence of increasing
amounts of calf intestine phosphatse (CIP). Samples were fractionated by SDS-PAGE
and immunoblotted with anti-HuR antibodies.
To determine whether CstF-50 and p97 play a role in the ubiquitination of HuR by
BRCA1/BARD1, first, I checked the interaction of CstF-50 with this newly identified
BRCA1/BARD1 substrate. In vitro pull-down assays were performed as described (Figure 13,
Experimental Procedures). The GST pull-down assays showed that GST-CstF-50 and His-HuR interacted directly. The interaction was observed only when GST-CstF-50 but not GST alone was used in the pull-down assay (Figure 37A). Then, I examined the complex formation of CstF-
50 with HuR in nuclear extracts (NEs) from HCT16 cells by co-immunoprecipitation assays.
Polyclonal antibodies against p97 were used. As shown in Figure 37B, an important fraction of
CstF-50, BRCA1, BARD1 and HuR co-precipitated with p97 independently of UV treatment,
105 indicating that HuR can form a complex with all these proteins in different cellular conditions, suggesting a possible functional interaction between them.
A) B) Input p97 IP UV: - + - +
-Topo II 35- -His-HuR 130-
1 2 3 250- -BRCA1
95- -BARD1
95- -p97
55- -CstF-50
35- -HuR
1 2 3 4
Figure 37: HuR binds directly to the mRNA processing factor CstF-50. A)
Immobilized GST or GST-CstF-50 on glutathione-agarose beads was incubated with His-
HuR. Equivalent amounts of the pull-downs (PD) were analyzed and proteins were
detected by immunoblotting with anti-HuR antibodies. 5% of His-HuR was loaded as
input. B) HuR forms a complex with CstF-50, BRCA1/BARD1 and p97 in NEs from
HCT116 cells exposed or not to UV irradiation. NEs were prepared as described in
Experimental Procedures and IPed with anti-p97-antibodies (polyclonal A300-589A,
Bethyl). Equivalent amounts of the pellets (IP) were resolved by SDS-PAGE and proteins
were detected by immunoblotting using the indicated antibodies. 5% of the NE used in
the IP reaction is shown as input.
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Evidence showed that CstF-50 and p97 play a role in the UV-induced ubiquitination of
RNA polymerase IIO (RNAP IIO) and histones by regulating the BRCA1/BARD1 E3 Ub ligase activity (Chapter IV) and that p97 plays an important part in the release of HuR from the ARE binding site of p21 (Zhou et al., 2013). Extending those studies, I determine the role of CstF-50 and p97 in the ubiquitination of HuR by BRCA1/BARD1. In vitro ubiquitination reactions were carried out as described in Figure 35, adding increasing amounts of GST-CstF-50 or His-p97 to the reactions. Strikingly, both GST-CstF-50 and His-p97 were able to inhibit the monoUb of
HuR by BRCA1/BARD1 (Figures 38A and 39). There was no inhibition of monoUb-HuR when using equal amounts of GST in the reactions (Figure 38B). Surprisingly, these results indicate that CstF-50 and p97 could be either an activator or an inhibitor of the BRCA1/BARD1 E3 Ub ligase activity depending on the cellular mechanism or pathway and cellular conditions. Studies will be designed to further understand this phenomenon.
A) B) E1 + E2 + ∆BRCA1/BARD1 E1 + E2 + GST (ng): 0 0 0 20 50 0 ∆BRCA1/BARD1 GST-CstF-50 (ng): 0 5 10 25 50 100 0 GST-CstF-50 (ng): 0 20 50 0 0 0 Ub: + + + + + + - Ub: + + + + + - 55- 55- -Ub-His-HuR -Ub-His-HuR
35- -His-HuR 35- -His-HuR
1 2 3 4 5 6 7 1 2 3 4 5 6
Figure 38: HuR ubiquitination by BRCA1/BARD1 E3 Ub ligase is inhibited by
GST-CstF-50. In vitro ubiquitination reactions were conducted as in Figure 36 but in the
presence of increasing amounts of GST-CstF-50 (A) and GST alone (B). Samples were
fractionated by SDS-PAGE and immunoblotted with anti-HuR antibodies.
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E1 + E2 + ∆BRCA1/BARD1
His-p97 (ng): 0 5 10 25 50 100 0 Ub: + + + + + + - 55- -Ub-His-HuR
35- -His-HuR
1 2 3 4 5 6 7
Figure 39: Ubiquitination of HuR by BRCA1/BARD1 E3 Ub ligase is inhibited by
His-p97. In vitro ubiquitination reactions were conducted as in Figure 36 but adding
increasing amounts of His-p97. Samples were fractionated by SDS-PAGE and
immunoblotted with anti-HuR antibodies.
To test whether HuR ubiquitination occurs in the cell nucleus I analyzed HuR ubiquitination in NEs from HCT116 cells treated or not with UV and the proteasomal inhibitor
MG132. As shown in Figure 40, HuR monoUb was enriched in samples from non-treated cells.
Interestingly, the phosphorylated and ubiquitinated forms of HuR were also detected in these nuclear samples. The lower mobility band decreased in samples from UV-treated cells but increased after MG132 treatment in non-stress conditions. Inhibition of proteasomal activity did not change the pattern of bands after UV treatment, suggesting that these ubiquitination events did not target HuR for degradation.
Additional studies need to be done to further elucidate the regulatory role of CstF-50 and p97 on the BRCA1/BARD1-mediated ubiquitination of HuR in vivo using siRNA-mediated depletion of these factors. These assays will reveal new insight on the identity of the E3 Ub ligase involved in this pathway and cofactors involved in this pathway.
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HCT116 cells MG-132: - + - + UV: - - + +
-Topo II
55-
-Ub-HuR
35- -HuR
1 2 3 4
Figure 40: HuR is concurrently monoubiquitinated and phosphorylated in nuclear
extracts. NEs from HCT116 cells exposed or not to UV treatment were analyzed by
Western blot with the indicated antibodies. Samples from cells treated with proteasome
inhibitor MG132 were also analyzed.
DISCUSSION
In Chapters II, IV and V of this dissertation I have shown a new role for mRNA processing factor CstF-50 has a regulator of the BRCA1/BARD1 E3 Ub ligase activity, activating the polyubiquitination of RNAP II and the monoUb of histones H2A and H2B during the progression of DDR. I also presented evidence of the involvement of escort protein p97 in this process. In this Chapter, I have unveiled a new link between mRNA processing, the Ub pathway and the regulation of mRNA turnover/stability and, hence, gene expression through the interaction of polyadenylation factor CstF-50 with the RNA-binding protein HuR. I have also identified that HuR is a new substrate for BRCA1/BARD1 complex (Figure 35). HuR is monoUb in in vitro assays and this modification is inhibited by CstF-50 and p97 (Figures 38-39).
The monoUb-HuR is also detected in NEs from cells under non-stress conditions, and this modification is decreased after UV damage (Figure 40).
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The preliminary data presented in this Chapter suggest that HuR is concurrently phosphorylated and ubiquitinated (Figure 35-36). In this respect, connections between phosphorylation and ubiquitination of different factors have been already established. It has been shown that HuR is more efficiently degraded in cells with reduced levels of HuR phosphorylation after heat shock (Abdelmohsen et al., 2009), suggesting that HuR polyubiquitination might be increased when phosphorylation is inhibited. Besides, phosphorylation of RNAP IIO at Ser2 is a requisite for the ubiquitination and degradation of the elongating form of RNAP II (Kleiman et al., 2005). Furthermore, phosphorylation of H2A.X is pre-requisite for its ubiquitination (Huen et al., 2007). Our preliminary data (Figures 35-39) shows the presence of a doublet in the in vitro ubiquitination assays suggesting that either HuR monoUb might occur concomitantly with phosphorylation or one modification might be indispensable for the occurrence of the other.
Based on Dr. Devany’s findings (unpublished data) and the results described in this
Chapter, a mechanism for the association/dissociation of HuR from the target mRNA during the progression of DDR can be proposed (Figure 41). Our model is that under non-stress conditions
HuR is monoUb by BRCA1/BARD1, serving as a signal for its release from the ARE binding site in p53 mRNA. Consistent with this, HuR ubiquitination has been shown to be signal for its released from mRNA (Zhou et al., 2013). Consequently, the unoccupied ARE binding site at the target mRNA is available for the binding of the destabilizing factors, PARN/Ago2, causing p53 mRNA destabilization through activation of deadenylation followed by degradation. Under DNA damaging conditions, the BRCA1/BARD1/CstF-50/p97 complex is formed as previously shown
(Kleiman and Manley, 2001)Chapter IV) inhibiting the monoUb of HuR and promoting the binding of unmodified HuR to the ARE binding site of the p53 mRNA. HuR binding to p53
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3’UTR outcompetes PARN/Ago2 resulting in the stabilization of the p53 mRNA, promoting the export of HuR and its cargo mRNA to the cytoplasm and, consequently, increasing p53 translation and p53 protein levels after UV damage. Supporting this model, HuR has been shown to bind p53 mRNA in a UV-dependent manner and translocates it to the polysomes, where translation of p53 mRNA is achieved (Mazan-Mamczarz et al., 2003). It is worth to mention that since ubiquitination is the largest among the post-translational modifications known, the monoUb of HuR could likely interfere with its association to the target mRNA by steric hindrance allowing the dissociation of HuR.
Surprisingly, my studies indicate that CstF-50 is able to either activate (Figures 20-23) or inhibit (Figures 38-39) the BRCA1/BARD1-mediated ubiquitination of different substrates. This might be explained by the nature and the cellular function of those substrates, depending on the architecture of the modification and/or the scenario where CstF-50 functions are implicated
(nucleoplasm or chromatin). It is possible that CstF-50 could orchestrate the regulation of the
BRCA1/BARD1 E3 Ub ligase activity in a spatiotemporal manner. In that scenario, p97 could also contribute to BRCA1/BARD1 differential regulation by its interaction which several other factors, such as deubiquitinases, under different cellular conditions.
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nucleus HuR Ub Ub
AGO AREBP PAS p53 ORF ARE ARE AAAAA mRNA A A
Activation of deadenylation
Low p53 levels
UV
nucleus CstF
AGO AREBP
p97 p97 HuR PAS AAAAAAAAAAAAA mRNA p53 ORF ARE ARE
mRNA export
Increase in mRNA levels and translation/High p53 levels
Figure 41: Model of regulation of p53 mRNA steady-state levels by BRCA1/BARD1- mediated ubiquitination of HuR. Under non-stressed conditions, HuR is monoUb by
BRCA1/BARD1, resulting in the release of Ub-HuR from p53 mRNA and causing its destabilization and degradation through the binding of PARN/Ago2 to the ARE binding site. After UV damage, BRCA1/BARD1/CstF-50/p97 complex is formed, inhibiting the ubiquitination of HuR and triggering the binding of unmodified HuR to the ARE site of the p53 mRNA. These results in the stabilization of p53 mRNA and increase in its translation, resulting in elevated levels of p53 protein during DDR.
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CHAPTER VII
FUTURE DIRECTIONS
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Although BRCA1/BARD1 E3 Ub ligase has been extensively studied, little is known about its cellular targets, how these targets are chosen, the mechanism by which they are recruited to DNA damage sites, which are the cofactors implicated in the BRCA1/BARD1 mediated ubiquitination during DNA damage response (DDR), and how all these events contribute to cancer development.
The findings presented in this dissertation provide evidence of the role of the polyadenylation factor CstF-50 and the Ub-escort factor p97 in regulating BRCA1/BARD1 E3
Ub ligase, resulting in the control of transcription and chromatin remodeling during DDR. I also show that CstF-50 can interact directly not only with BRCA1/BARD1 but also with ubiquitin
(Ub), the escort-factor p97 and some of BRCA1/BARD1 substrates, such as RNA polymerase II
(RNAP II), and histones H2A and H2B. Besides, CstF-50 activates BRCA1/BARD1-dependent
RNAP II polyubiquitination, H2A and H2B monoubiquitination as well as BRCA1/BARD1 autoubiquitination. Moreover, as part of this dissertation I describe that UV-treatment induces changes in the localization of BRCA1, BARD1, CstF-50, p97 and some of BRCA1/BARD1 substrates in different nuclear fractions, and these changes depend on BRCA1/BARD1 and CstF-
50 expression. Importantly, the content of monoubiquitinated H2B in the chromatin of genes with different levels of expression changes during DDR and this is mediated by BRCA1/BARD1 and
CstF-50. Extending these studies, I also determine that the escort factor p97 is involved in this
BRCA1/BARD1/CstF-50-dependent response. Taken together, my results provide evidence that
CstF-50-associated p97 regulates BRCA1/BARD1 Ub ligase activity during DDR, helping in the assembly and/or stabilization of the ubiquitination complex, and affecting the chromatin structure, DNA repair and, hence, gene expression.
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The studies presented in this dissertation are innovative because they are the first showing a regulator (CstF-50/p97) that can have a dual pro and anti-regulatory effect on the ubiquitination of BRCA1/BARD1 substrates depending on the cellular context and under different cellular conditions. Besides, this study has revealed a new role for CstF-50/p97 mediated
BRCA1/BARD1 E3 Ub ligase in regulating the Ub pathway resulting in epigenetic control during DDR. I also identified Human Antigen R (HuR) as a new target for BRCA1/BARD1 E3 ligase. BRCA1/BARD1 monoubiquitintes HuR and this modification is inhibited by CstF-50 and p97.
Now, it is important to further elucidate the total consequences of the CstF-50/p97 mediated regulation of BRCA1/BARD1 E3 Ub ligase activity. The following proposed studies might help to understand some other aspects of the working model shown in this dissertation.
Further characterization of BRCA1/BARD1/CstF-50-mediated ubiquitination and its consequences at genomic scale.
To understand the functional consequences of the BRCA1/BARD1/CstF-50-mediated ubiquitination in different cellular pathways, it is also important to determine the architecture of the Ub chain of each identified substrates. This will help us to understand deeper the mechanism of action of BRCA1/BARD1 as an E3 Ub ligase and will also help to understand the final fate of the ubiquitinated substrates. It has been shown that various BRCA1/BARD1 substrates are not degraded by the proteosome, such as NPM1, CtIP, histones, TFIIE and BRCA1 itself. (Wu et al.,
2008). It has been determined that BRCA1/BARD1 complex catalyzes unconventional polyubiquitin chains that include Lys6-type of linkage when undergoing autoubiquitination
(Nishikawa et al., 2004; Wu-Baer et al., 2003). Besides it has been described that E4 enzymes
115 can catalyze a linkage switch in the substrates that are been ubiquitinated. It has been suggested that E4 enzymes might regulate the selection of Lys residues used for Ub-Ub linkages during polyubiquitin-chain assembly. For example, yeast UFD2 (E4) catalyzes a linkage switch from
Lys29 used for mono-ubiquitination to Lys48 to further elongate the Ub chain (Saeki et al.,
2004). Moreover, short Lys-29 chains have been shown to be involved in recruitment of the chain-elongating factor E4 (Koegl et al., 1999). As the studies presented in this dissertation have indicated that CstF-50 might have E4 activity, it is now important to perform assays to find out the linkage-type of the BRCA1/BARD1 substrates studied in this dissertation. It is also important to test whether CstF-50 determines the type of linkage used or causes a linkage switch affecting the final fate of the ubiquitinated proteins.
My studies indicate that the ubiquitination of RNAP II and histones by
BRCA1/BARD1/CstF-50 during DDR results in a dynamic change in their nuclear localization and in the content of Ub-H2B in the chromatin of genes with different levels of expression. It is now important to determine the role of BRCA1/BARD1 and CstF-50 in the regulation of chromatin remodeling during DDR at the genomic scale. This can be accomplished by comparing the effect of histone H2B ubiquitination on the chromatin structure by ChIP-seq analysis in normal conditions as well as during DDR in cells depleted in BRCA1/BARD1 and CstF-50. This type of analysis can be accomplished by using the traditional ChIP analysis followed by next generation sequencing (ChIP-seq). This technique allows a finer insight into the target genomic regions both at the specific-gene and the genome-wide level harboring this type of modification during specific cellular conditions. The ChIP-seq technique involve a massively parallel sequencing of the DNA-bound to Ub-histone H2B in conjunction with whole-genome sequence databases to analyze the interaction pattern of Ub-histone H2B with DNA. Using ChIP-seq, is
116 possible to map genomic locations harboring Ub-H2B and study nucleosome positioning as well as analyze the distribution of Ub-H2B in expressed (active) and non-expressed (non-active) genes at the genome-wide level.
Identification of new BRCA1/BARD1/CstF-50 substrates involved in DDR.
PCNA: Although many BRCA1/BARD1 substrates have been already identified, only some of them have been implicated in DDR. Taking into account that BRCA1/BARD1 heterodimer play important roles in the response to DNA damage and that many factors implicated in nucleotide excision repair (NER) are subject to ubiquitination, it is now crucial to address if some of them could be substrates for BRCA1/BARD1.
The proliferating cell nuclear antigen (PCNA) is a protein that encircles and slides along double-stranded DNA, acting as a “sliding clamp” that localizes proteins, such as polymerases to
DNA. PCNA has been implicated in DNA replication as well as in the synthesis step of NER. A similar molecular mechanism has been assumed for both pathways: the replication factor C) clamp participates in the loading of PCNA at the double-strand/single-strand DNA template- primer terminus allowing the access of the DNA polymerase to the replication site through its interaction with PCNA (Ogi et al., 2010). Interestingly, PCNA is monoubiquitinated upon UV irradiation and that monoubiquitinated PCNA specifically interacts with DNA polymerases
(Kannouche et al., 2004). Besides, PCNA monoubiquitination functions as a modulator of the residence time of PCNA at sites of DNA damage by increasing its accumulation at the damaged area and, consequently, the time that the polymerases are associated at those sites (Bienko et al.,
2005; Essers et al., 2005; Plosky et al., 2006). It was determined that Ub-PCNA associates with protein factors that are involved in the late step of NER (Guo et al., 2008). Although PCNA
117 ubiquitination was Rad18 (E3)-dependent, the subsequent recruitment of DNA polymerase was not (Ogi et al., 2010). These results suggest that PCNA ubiquitination is not required for the loading of the polymerase into damaged DNA and that the loading actually takes place through unmodified PCNA or other factors (Ogi et al., 2010). However, another very likely possibility that was not considered in that study is the requirement of a different E3 Ub ligase to exert the ubiquitination of PCNA and the subsequent loading of the polymerase. This idea is supported by the fact that polymerases bind stronger with Ub-PCNA compared to unmodified PCNA (Guo et al., 2008) due to the presence of the highly conserved Ub-binding domains within the polymerases (Bienko et al., 2005).
Interestingly, previous studies identified PCNA as one of the interactors of CstF-50 in a two-hybrid screening (Kleiman and Manley, 1999). I hypothesize that the CstF-50-associated E3
Ub ligase, BRCA1/BARD1, could be involved in the ubiquitination of PCNA helping in the recruitment of the corresponding polymerase during NER. In fact, my in vitro preliminary studies showed that PCNA is ubiquitinated by BRCA1/BARD1 (not shown). The
BRCA1/BARD1/CstF-50-mediated ubiquitination of PCNA fit the model proposed in this dissertation: when transcription stalls at sites of DNA damage, CstF-50-associated
BRCA1/BARD1 complex ubiquitinates RNAP II and changes chromatin structure by histone ubiquitinaion to allow repair factors to find and repair the damaged DNA. At the same time,
BRCA1/BARD1/CstF-50 complex might also ubiquitinate PCNA, enhancing its affinity for the
DNA repair machinery, specifically for the polymerase in charge of the synthesis of the repaired patch (Bienko et al., 2005; Plosky et al., 2006). Future work should address the role of
BRCA1/BARD1 and CstF-50 in PCNA ubiquitination.
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Histone variant H2A.X: Histone variant H2A.X has been widely implicated in the maintenance of genomic stability in response to DNA double strand breaks (DSBs) after ionizing radiation
(IR). Both IR and UV irradiation induces ATR-dependent phosphorylation and foci formation of
H2A.X. Phosphorylated H2A.X, has been implicated in remodeling of higher order chromatin structures (Downs et al., 2000). H2A.X foci colocalize with PCNA and BRCA1 at sites of DNA lesions (Ward and Chen, 2001). Mice lacking H2A.X exhibit sensitivity to DNA damage and profound chromosomal instability (Celeste et al., 2002). In addition, H2A.X is monoubiquitinated in vitro by BRCA1/BARD1 (Chen et al., 2002; Mallery et al., 2002). BRCA1/BARD1 heterodimer mediates the monoubiquitination of both phosphorylated and un-phosphorylated
H2A.X. The biological relevance of this modification is poorly understood. Interestingly, H2A.X is ubiquitinated after UV irradiation (Sharma et al., 2014). My preliminary data indicate that
H2A.X is able to interact directly with CstF-50 (data not included in the dissertation). Future studies will test whether BRCA1/BARD1/CstF-50 ubiquitinates this histone variant in response to UV damage.
Identification of deubiquitinases associated with BRCA1/BARD1/CstF-50 complex.
Ubiquitination is a reversible process which role in DDR has been very well established.
In contrast, the reverse pathway, deubiquitination, remains poorly defined. There is a balance between the opposing actions of specific E3 ligases and deubiquitinases (DUBs) which makes protein ubiquitination a versatile and dynamic posttranslational modification. The activity of E3 ligases can be antagonized by DUBs rescuing ubiquitinated proteins from proteasomal degradation, or altering its fate by editing the length and topology of its Ub chain (Cao and Yan,
2012). Some DUBs can be very specific for certain Ub linkages, while others show a notable
119 promiscuity with respect to the type of Ub linkage they hydrolyze. Several DUBs not only have a regulatory function but also play a housekeeping function in the maintenance of the cellular pool of free Ub chain (Cao and Yan, 2012).
It appears increasingly clear that the DDR is critically regulated by ubiquitination and deubiquitination. Future studies should focus on the identification of DUBs that balance the activity of BRCA1/BARD1 and CstF-50 during DDR. Interestingly, Liu and collaborators
(2012) showed that p97 interacts with DUBs from the endoplasmic reticulum-associated degradation (ERAD) pathway. As p97 can associate with both Ub ligases and DUBs, enzymes with opposing biochemical activities, they proposed that DUBs may act as Ub chain trimming or editing enzymes that modulate the length or topology of Ub chains rather than a chain eliminator
(Cao and Yan, 2012)). In consequence, future works will address whether p97-associated DUBs regulates the BRCA1/BARD1 enzymatic activity with different substrates in different conditions.
Ub-specific protease 1 (USP1) is one of the best-characterized DUBs that plays an important role in the regulation of DNA repair processes. USP1 regulates ubiquitination of
PCNA, FANCD2 and FANC1 during DDR (Garcia-Santisteban et al., 2013). Importantly, USP1 is able to revert the monoubiquitination of PCNA after UV damage (Niimi et al., 2008). As
PCNA is a potential new target for BRCA1/BARD1 E3 ligase, USP1 will be included in our future studies. USP3 is one of DUBs involved in histone deubiquitination, it can deubiquitinate
Ub-H2A and Ub-H2A.X in response to UV damage (Sharma et al., 2014). As described in this dissertation, histone modifications play a crucial role in DDR by either facilitating DDR signaling or by influencing chromatin folding/organization. Like other histone modifications, monoubiquitination of histones H2A, H2B and H2A.X is reversible. Deubiquitination of histones
120 may eliminate repair factors or chromatin remodelers binding sites, thereby regulating their recruitment (Vissers et al., 2008). Another DUBs is the putative tumor suppressor BAP1, which is a Ub hydrolase enzyme that associate with BRCA1 (Jensen et al., 1998) and BARD1
(Nishikawa et al., 2009). Even though it was initially reported that BAP1 does not affect the
BRCA1/BARD1 Ub ligase activity (Mallery et al., 2002), it was later reported that in fact BAP1 is able to perturb the formation of the BRCA1/BARD1 heterodimer resulting in the inactivation of its E3 Ub ligase activity (Nishikawa et al., 2009).
Another DUBs, USP7, removes Ub moieties from autoubiquitinated BRCA1/BARD1 and from some of its substrates. Several lines of evidence indicate that USP7 could be the deubiquitinase that associates and regulates BRCA1/BARD1/CstF-50 complex. First, USP7 can rescue the hypophosphorylated form of RNAP II from transcription-coupled repair (TCR) complexs avoiding RNAP II degradation after UV irradiation (Zhang et al., 2012). Second, elongating RNAP IIO stalled at DNA damage sites can be deubiquitinated by USP7, allowing transcription resumption after damaged is repaired. Third, USP7 resides in chromatin- immunoprecipitated TCR complexes in a UV-dependent manner (Nakazawa et al., 2012;
Schwertman et al., 2012; Zhang et al., 2012). Considering previous studies from Dr. Kleiman’s lab showing the involvement of CstF-50 in TCR (Mirkin et al., 2008) and my own results demonstrating a role for CstF-50 regulating BRCA1/BARD1 activity during DDR and facilitating chromatin remodeling (Chapter IV and V), it is feasible that USP7 could be involved in the BRCA1/BARD1/CstF-50 pathway.
It is important to emphasize that a subset of human DUBs with function in DDR, have been increasingly recognized as potential targets in cancer treatment. For this reason, targeting
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DUBs enzymes might be an approach to overcome resistance to conventional therapy (Garcia-
Santisteban et al., 2013)(García-Santisteban et al., 2013).
Further characterization of the mechanism of HuR binding/exclusion from mRNA-bound ribonucleoproteins (mRNPs) during the progression of DDR.
I previously showed in Chapter VI that BRCA1/BARD1 can monoubiquitinate HuR in vitro and this modification is inhibited by CstF-50 and p97. The monoUb-HuR is also detected in
NEs from cells under non-stress conditions, and this modification is decreased after UV damage.
Interestingly, I also detected a lower mobility band in those assays that likely is a double modified HuR isoform (phosphorylated and ubiquitinated). Further studies on the effect of post- translational modifications of HuR protein on the mechanism of regulation of p53 mRNA levels, in non-stress conditions and after DNA damage are crucial for understanding this important mechanism. I will extend those studies to other genes involved in DDR that are HuR targets.
Studies will be designed to confirm whether HuR is both phosphorylated and ubiquitinated under different cellular conditions by using anti-phospho specific antibodies for HuR, which are available upon request. More experiments are needed to confirm whether BRCA1/BARD1 is able to ubiquitinate HuR in vivo. Besides, additional studies need to be done to further elucidate the regulatory role of CstF-50 and p97 on the BRCA1/BARD1-mediated ubiquitination of HuR in vivo using siRNA-mediated depletion assays.
As mentioned before, understanding how p53 mRNA and other genes involved in DDR are regulated under different cellular conditions may help us to improve therapeutic approaches to diseases such as cancer.
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CHAPTER VIII
EXPERIMENTAL PROCEDURES
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Tissue culture and treatments: HeLa and HCT116 cells were cultured in Dubelcco’s modified
Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) as described (Nazeer et al.,
2011, Cevher et al., 2010). Cells were treated with 2 µM of proteasome inhibitor MG132
(SIGMA) for 4 h or 10-20 µM of p97 inhibitor DBeQ (Life Sensors) for 4 h. Calf intestine phosphatase (CIP) treatments were performed with 10-30 U of the enzyme at 37 °C for 1 h.
DNA-damaging agents: 90% confluent cultures were exposed to UV and harvested at different times. UV doses (20 or 40 Jm-2) were delivered in two pulses using a Stratalinker (Stratagene).
Medium was removed prior to pulsing, and replaced immediately after treatment.
Purification of recombinant proteins: Plasmids encoding His-Ub and His-UbcH5c (kindly provided by Dr. Baer, Columbia University), His-HuR (kindly provided by Dr Bhattacharyya,
Friedrich Miescher Institute Basel, Switzerland), His-p97 (kindly provided by Dr. Monteiro,
University of Maryland), GST, and GST-CstF-50 were transformed into BL21 cells. Plasmid encoding His-BARD1/∆BRCA1 (kindly provided by Dr. Baer, Columbia University) were transformed into Rosetta (DE3)plysS. His-fusion proteins were expressed and purified by binding to and elution from Ni-Agarose beads (QIAGEN) as described (Wu-Baer et al, 2003; Kleiman et al, 2005; Kleiman and Manley, 1999). GST-fusion proteins were purified by binding to and elution from glutathione–agarose beads using NETN Buffer and eluted with reduced glutathione as described (Kleiman and Manley, 2001). Purified proteins were separated by SDS-PAGE and analyzed by Coomassie Blue or Silver staining.
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A) B) C)
mg BSA mg BSA M 0.1 0.25 0.5 1 2 5 M 0.10.25 0.5 1 2 5 72 - 250 - 55 - -BARD1 36 - 130 - 28 - 95 -
17 - 72-
55 - 11- -BRCA1 36- 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 1 2
Figure 42: Analysis of quality and quantity of the recombinant proteins used in this
study. A) and B) Purified recombinant proteins, His-Ub, His-UbcH5c, His-p97, GST,
and GST-CstF-50 were separated in SDS-PAGE and quantified with bovine serum
albumin (BSA) by staining with Coomassie Blue. C) Purified His-BARD1/∆BRCA1 was
separated by PAGE and analyzed by Silver staining and Western blot with antibodies
against BRCA1 and BARD1.
Nuclear extracts (NEs) preparation: After UV treatment, NEs were prepared from harvested cells essentially as described (Cehver et al., 2010; Nazeer et al., 2011; Lee et al., 1988). Cells were lysed by douncing in 4 ml of 10 mM Tris pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were centrifuged for 10 min at 2,000 rpm, and pellets were resuspended in 20 mM Tris pH 7.9, 1.5 mM MgCl2, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 0.3 M NaCl.
Preparations were incubated for 30 min at 4 °C and centrifuged for 15 min at 13,000 rpm.
Supernatants were quick frozen and stored at −80°C.
Immunoblotting: Equivalent amounts of proteins were subjected to SDS-PAGE and proteins were detected by immunoblotting using antibodies against BRCA1 (C-20 and K-18, Santa Cruz
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Biotechnology), BARD1 (H-300, Santa Cruz Biotechnology), RNAP II (N-20, Santa Cruz
Biotechnology), Topoisomerase II (H-8, Santa Cruz), GAPDH (G-9, Santa Cruz Biotechnology),
Lamin A (H-102, Santa Cruz Biotechnology), Ub (P4D1, Santa Cruz Biotechnology), Histone
H2A (2578, Cell Signaling), Histone H2B (8135, Cell Signaling), CstF-50 (A301-250A, Bethyl),
CstF-64 (A301-092A, Bethyl), p97 (A300-589A, Bethyl), Ub-H2A (05-678, Millipore), Ub-H2B
(05-1312, Millipore), histones (MABE71, Millipore), HA.11 (MMS-101P, Covance), RNAP II
(8WG16, MMS-126R, Covance), RNAP IIO (H5, MMS-129R, Covance), actin (A2066,
SIGMA), and FLAG M2 (F3165, SIGMA). Approximately 60 g of each NE was analyzed by immunoblotting.
Acidic Extracts preparation: Acidic extracts were prepared after UV treatment by lysing cells on TBE Buffer (0.5% Triton X-100, 2 mM PMSF and 0.02% NaN3 on ice for 15 min followed by extraction of histones in 0.2 N HCl overnight at 4 °C. Extracts were centrifuged at 4 °C for 10 min at 2000 rpm. Supernatants were quick frozen and stored at −80°C.
Protein–protein interaction assays: The GST and His protein interaction assays with GST-
CstF-50 and His-Ub were performed as described (Kleiman and Manley, 2001; Cevher et al,
2010; Nazeer et al, 2011). Briefly, 2 µg of His-tagged proteins were incubated with Ni-Magnetic beads for 2 h at 4 °C in 300 μl final volume of binding buffer (20 mM HEPES pH 7.9, 0.5 M
KCl, 0.5% NP-40, 10% glycerol, 2 mM mercaptoethanol and 2.5 mM imidazole). Beads were treated extensively with washing buffer (binding buffer plus different concentrations of NaCl).
Then 2 µg of the GST-tagged proteins were added and incubated for 3 h at 4 ºC. Samples were washed as described above. Alternatively, 2 µg of GST-tagged proteins were incubated
Glutathione Sepharose beads in binding Buffer (1× phosphate-buffered saline (PBS): 137 mM
NaCl, 3 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.04% Albumin, 0.5mM PMSF, 0.001%
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NP-40) for 2 h, washed six times with buffer and incubated with 2 µg of His-tagged proteins for another 2 h. Washing conditions were as described before. Additionally, 200 μg of HeLa cell
NEs were incubated with 2 μg of the indicated His-fusion proteins, the binding and washing conditions were as described before. Protein samples were treated at 4 °C with 50 µg of RNase
A/ml for 10 min. Equivalent amounts of pellets and 5% of the original samples were analyzed by immunoblotting as described before.
Knockdown expression of CstF-50 and BRCA1/BARD1 in Hela cells: siRNA specific for human CstF-50, BRCA1, BARD1 and a control siRNA were obtained from Dharmacon RNA technologies. 80% confluent cells were transfected with 30 l of Lipofectamine RNAiMAX according to the manufacturer’s protocol (Invitrogen) and 100 nM of each siRNA for a total of
48 h. After treating the cells with the siRNAs in reduced serum Opti-MEM medium for 8 h, medium was changed to complete medium. After additional 16 h, cells were transfected again and harvested for analysis 48 h after the initial transfection. To determine the effectiveness and specificity of each siRNAs used, protein levels were monitored by Western blot.
In vitro ubiquitination reactions: Autoubiquitination and RNAP II ubiquitination reactions were conducted at 37 °C for 1 h in a 30 μl volume containing 50 mM Tris-HCl, pH 7.4, 5 mM
MgCl2, 2 mM NaF, 10 nM okadaic acid, 2 mM ATP, 0.6 mM dithiothreitol, and 20 ng rabbit E1
(LifeSensors), 0.25 μg of (E2) UbcH5c, 10 ng of ΔBRCA1/BARD1 heterodimer and 1 μg of Ub monomer. Others transubiquitination reactions were performed using ubiquitination Buffer containing 50 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 15 mM KCl, 0.01% Triton X-100, 1% glycerol, 0.7 mM DTT and 2 mM ATP with 0.3 μg E1(LifeSensors), 0.3 μg of (E2) UbcH5c, 50 ng of ΔBRCA1/BARD1 heterodimer, 1.5 μg of Ub monomer, and either 1 μg of recombinant histones, 1 μg of His-HuR or 300 ng RNAP IIO. Increasing amounts of GST-CstF-50 (5-300 ng)
127 and/or His-p97 (5- 100 ng) were used as indicated in the corresponding figures. The reactions were stopped by adding 4X SDS-Loading Buffer. The reaction products were fractionated by
SDS-PAGE and detected by immunoblotting using antibodies indicated in the figures.
Immunoprecipitation Assays: 100 μg of proteins from each NE were pre-cleared with 50 µl of protein-A–Sepharose and immunoprecipitated with antibodies against CstF-50, p97, and Ub.
Antibodies were bound to PureProteome protein A magnetic beads (Millipore). The antibodies were coupled to the protein A-magnetic beads for 30 min at RT in PBS-T buffer.
Immunoprecipitations were carried out for 3 h at 4 °C in 200 μl of buffer A (1× phosphate- buffered saline (PBS): 137 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.1%
Tween 20, 0.5mM PMSF). The beads were recovered and treated at 4 °C with 50 μg of RNase
A/ml for 10 min. Washing was performed with PBS-T buffer. Samples were incubated at 100˚C with loading buffer for 10 min. 5% of the original sample was loaded as input along with the immunoprecipitated samples and analyzed by SDS-PAGE and immunoblotting.
Chromatin Immunoprecipitation Assays: Chromatin was prepared from control, CstF-50 and
BRCA1/BARD1 siRNA and/or UV treated cells using the MAGNA-EZ ChIP kit (Millipore) following manufacturer instructions. Immunoprecipitations were carried out using a ChIP grade
Ub-H2B antibody (Millipore). DNA samples were analyzed by quantitative PCR (qPCR) as described below. qPCR Assays: Equal amount of DNA from ChIP samples described above were quantitatively amplified using TaqMan Gene Expression Assay Kit with primers for differentially expressed genes. 1% of the original chromatin samples were amplified in parallel as loading control. The primers were designed on the transcribed region of each gene based on Minsky et al. (2008).
Primers were designed in the transcribed region of each gene and purchase from Integrated DNA
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Technologies (IDT). GAPDH: Forward primer 5’-CTAGGCAGCAGCAAGCATTC-3, Reverse primer 5’-CCCAACACCCCCAGTCATAC-3; DHFR: Forward primer 5’-AATTCCATCG-
GCAGTCCTCA-3, Reverse primer 5’-GGTGGGGCTTAGATTAGGCA-3; Insulin: Forward primer 5’-CCTGTAGGTCCACACCCAGT-3, Reverse primer 5’-GGAGGACACAGT-
CAGGGAGA-3’; CD4: Forward primer 5’-TAGGCGACAGAGCAAGACTC-3’, Reverse primer 5’-GTGTGTGTGTGTGTGTGCTT-3’; p53: Forward primer 5’-AGTGTCCGAAGA-
GAATGGGC-3’, Reverse primer 5’-CACCCTTCCCCACCTGATAC-3’; and CHECK2:
Forward primer 5’-GCTCACTGTGGCCTTGATCT-3’, Reverse primer 5’-TAAGGCAGGAG-
GATGGGTTG-3’. Relative levels were calculated using ΔCτ method.
Cellular Fractionation: Cytoplasmic, soluble nuclear and chromatin-bound fractions were prepared from HeLa cells using a Cell Fractionation Kit (Pierce) following the manufacturer instructions.
Construction of Flag-tagged Histones: Flag-tagged recombinant WT histones H2A and H2B were generated by subcloning the full-length coding sequence of each histone by PCR amplification from a Hela cDNA library into the pCMV10 expression vector containing 3X flag tags (Sigma). The primers sequences are as follows: H2A: Forward primer 5’-GCGGCGGCC-
GCTATGTCGGGACGCG-3’; Reverse primer 5’-GCGGGATCCTTATTTGCCTTTGGCCT-
TG-3’; H2B: Forward primer 5’-GCGGCGGCCGCTATGCCTGAACCGGC-3’; Reverse primer
5’-GCGGGATCCTCACTTGGAGCTGGTGTA-3’. The PCRs were performed using high- fidelity pfu enzyme (Stratagene) following the manufacturer instructions and products were confirmed by sequencing. The PCR products were digested with NotI and BamHI and the resulting DNA fragment was inserted into the p3xFLAG-CMV-10 vector.
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In vivo ubiquitination assays: RNAP IIO: HeLa cells were transfected with either control or
CstF-50 siRNAs concomitantly with HA-Ub and treated or not with UV and MG-132. Cells extracts were prepared using RIPA Buffer (50 mM Tris-HCl pH 8.0, 150mM NaCl. 1% NP-40,
0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM PMSF, 1X protease inhibitor cocktail) and IPed with EZview Red anti-HA Affinity Gel beads (E6779, SIGMA). Equivalent amounts of the pellets (IP) were resolved by SDS-PAGE and proteins were detected by immunoblotting. 5% of the extracts used in the IP reaction were separated along as input. Histones: HeLa cells were transfected and treated as described above for RNAP II with an additional transfection with either 3XFLAG-H2A or 3xFLAG-H2B. Cell extracts were prepared using Lysis Buffer (50 mM
Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5 mM PMSF, 1X protease inhibitor cocktail), IPed with anti-FLAG M2 Magnetic Beads (M8823, SIGMA) and eluted using
3x FLAG peptide (F4799, SIGMA) followed by Western blot analysis. 5% of the cell extracts used in the IP reaction was used as input.
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CHAPTER IX
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