THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE
DEPARTMENT OF BIOLOGY
THE IDENTIFICATION AND CHARACTERIZATION OF SUFU INTERACTING PROTEINS
EMILY VALERIO SPRING 2013
A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biology with honors in Biology
Reviewed and approved* by the following:
Aimin Liu Associate Professor of Biology Thesis Supervisor
Gong Chen Associate Professor of Biology Honors Adviser
* Signatures are on file in the Schreyer Honors College. i
ABSTRACT
The Hedgehog (Hh) pathway, activated by a special family of proteins, is a prominent pathway in mammalian development and also in the formation of various cancers. When interacting with cells, Hh ligands are responsible for enhancing target Hh gene expression through the activation of Gli-transcriptional activators. Suppressor of Fused (Sufu) is a specific
Gli-interacting protein that functions in negatively regulating Gli activity and by doing so, suppressing Gli-activated tumor formation. The extent of how Sufu functions is not yet understood in mammals. In an effort to identify proteins that may interact with Sufu in this pathway, over 50 candidate proteins were identified through a yeast-two hybrid screen. Through much background research, four particular proteins were selected due to their known function and location in the cell: ran-binding protein 9 (RanBP9), transmembrane 131-like precursor (T131L),
COP9 signalosome complex subunit 1 isoform (Gps1), and hypothetical protein LOC67513.
After performing co-immunoprecipitation assays, we confirmed interactions with T131L, Gps1 and LOC67513. These proteins have also been recognized to interact with both structural domains of Sufu, the N-terminal domain and C-terminal domain. Luciferase reporter assays have indicated that these three proteins have a role in mammalian Hh signaling through promotion of
Gli activity. These experiments will hopefully help us gain better understanding of the function of Sufu and the Hedgehog pathway in the human body as well as possibly giving insight in the future as to how it may affect disease development.
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TABLE OF CONTENTS
List of Figures...... iii
List of Tables ...... iv
Acknowledgements ...... v
1. Introduction ...... 1
1.1 Hh signaling in development and disease ...... 1 1.2 Our current understanding of Hh signaling in mammals ...... 5 1.3 The roles and remaining questions of Sufu ...... 8
2. Materials & Methods...... 11
2.1 Construction of Plasmids ...... 11 2.2 Co-Immunoprecipitation Assays ...... 17 2.3 Dual-Luciferase Reporter Assays ...... 19
3. Results ...... 21
3.1 Identification of Sufu-interacting proteins through a Yeast Two-Hyrbid Screen ...... 21 3.2 The interaction between Sufu and T131L, Gps1, Loc67513 fragments are confirmed in mammalian cells ...... 22 3.3 Full-length T131L, Gps1 and Loc67513 interact with Sufu in mammalian cells ..... 23 3.4 The three proteins interact with both the N- and C-terminal domains of Sufu ...... 25 3.4 The three proteins significantly activate Gli-dependent reporters in Sufu -/- Mef cells ...... 27 3.5 T131L significantly activates Gli-dependent reporter activation in wild-type Mef cells ...... 29 3.6 T131L activates Gli-dependent reporter in dose-dependent manner ...... 32
4. Discussion ...... 35
Appendix ...... 39 REFERENCES...... 41 iii
LIST OF FIGURES
Figure 1-1. The expression patterns of mammalian Hedgehog homologues...... 1
Figure 1-2. The Drosophila Hh signaling pathway mechanism ...... 5
Figure 1-3 The inactive and active states of mammalian Hh sigaling in cilia...... 7
Figure 2-2. Basic mechanism for co-immunoprecipitation assay ...... 17
Figure 3-1. Co-Immunoprecipitation assay results with protein fragments………………….22
Figure 3-2. Co-Immunoprecipitation assay results with full-length proteins……………...…24
Figure 3-3. Co-Immunoprecipitation assay results with Sufu truncations…………………...26
Figure 3-4. Relative activity of proteins in Sufu -/- Mef cells………………………………...27
Figure 3-5. Relative activity of proteins in Sufu -/- Mef cells with over-expressed Sufu….…28
Figure 3-6. Relative activity of proteins in wild-type Mef cells………………………………30
Figure 3-7. Relative activity of proteins in wild-type Mef cells with over-expressed Sufu….31
Figure 3-8. Relative activity of doses of T131L in wild-type Mef cells………………………33
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LIST OF TABLES
Table 2-1. Phusion PCR environments ...... 12
Table 2-2. Vector and insert digestion information...... 14
Table 5-1. Fusion PCR reaction setups...... 39
Table 5-2. Recipe for 6x loading buffer………………………………………………………39
Table 5-3. Transfection setup…………………………………………………………………39
Table 5-4. Recipe for 1% triton lysis buffer…………………………………………………..40
Table 5-5. Recipe for 5ml SDS Separating Gel……………………………………………….40
Table 5-6. Recipe for 2.5ml SDS Stacking Gel………………………………………………..40
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ACKNOWLEDGEMENTS
I would like to sincerely thank Aimin Liu for allowing me the opportunity to work and learn in his lab for the past two years and for guiding me in the thesis writing process. I would also like to thank all of the members of the Liu lab including Huiqing Zeng, Xuan Ye, Hongchen
Cai, Rachel Chang and Keren Kohath for instructing me during my time here, and especially
Jinling Liu for mentoring me along the way. I would also like to thank my honors advisor, Gong
Chen, for reading my thesis and providing me with helpful recommendations. Finally, I would like to thank Penn State and the Eberly College of Science for providing funding to carry out our experiments.
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1. Introduction
1.1 Hh signaling in development and disease
There is an amalgamation of developmental pathways that are at work in organisms as they grow. One pathway in particular, the Hedgehog (Hh) signaling pathway, is prominent in organisms ranging from Drosophila, or fruit flies, to mammals. It not only is significant in developmental patterning, but also has major implications in human disease (Varjosalo and
Taipale, 2008). Primarily, this pathway is active during early embryogenesis, but it is also circumstantially active in adults (Fig. 1-1).
Figure 1-1. The expression patterns of the mammalian Hedgehog signaling homologues in Mus Musculus (Varjosalo and Taipale, 2008). 2 There are three Hh signaling molecules in mammals. As shown in figure 1-1, these include Sonic Hedgehog (Shh), Desert Hedgehog (Dhh) and Indian Hedgehog (Shh) (Varjosalo and Taipale, 2008). Development of many body areas are dependent upon these morphogenic ligands, and in their absence, defects occur.
Of the three ligands, Shh has the most broad expression pattern. It is involved in the determination of the dorso-ventral axis and the left-right axis due to its presence in midline tissues; these tissues include the notochord, floor plate, prechordal plate of the axial mesoderm and ventral forebrain, among others (Varjosalo and Taipale, 2008). It is also present in the zone of polarizing activity, making it a major regulator of limb development and distal limb element patterning, such as in the digits. The development of less critical features, such as teeth and hair growth are also dependent on Shh (Varjasalo and Taipale, 2008). Due to some of its vital functions, the absence of Shh leads to major malformations. Cyclopia, limb defects and even lethal malformations involving the ventral neural tube, ribs, somites, brain and vertebrae occur in
Shh null mutants (Beachy et al, 2010).
The expression of Dhh, unlike Shh, is confined to a small area. As seen in Figure 1-1, it is mainly involved in testis and ovarian development and functions in concordance with Ihh expression (Varjosalo and Taipale, 2008). Dhh is expressed in female granulosa cells and male sertoli calls between 11.5 and 13.5 dpc (Yao, Whoriskey and Capel, 2002). In males, Dhh receptors are located within Leydig cells and peritubular cells, which are responsible for the development of the seminiferous tubules. The expression of Dhh ligands results in the differentiation and normal development of these adult cells; it therefore plays a big part in spermatogenesis (Yao, Whoriskey and Capel, 2002). Male Dhh null mutants express various phenotypes, including incomplete spermatogenesis, feminized genitalia, malformed seminiferous tubules, and sterility (Clark, Garland and Russell, 2000). Because these defects are not life- threatening, the embryos are still viable, unlike Shh null mutants (Varjosalo and Taipale, 2008). 3 A second function of Dhh not shown in figure 1-1 is perineural formation (Sharghi-
Namini et al, 2006). Dhh is expressed in Schwann cells in the peripheral nervous system.
Absence of this ligand results in axon and brain vasculature abnormalities, an ill-developed immune system and defects in the blood-brain barrier (Sharghi-Namini et al, 2006)
Ihh, like Dhh, is expressed in few tissues. It is most prominently involved in the differentiation and development of bones and primitive endoderm. Specifically, expression of the
Ihh signaling molecules occurs within the prehypertrophic chondrocytes of bone growth plates and is involved in the differentiation and maturation of osteoblasts (Varjosalo and Taipale, 2008).
It has also been reported to have a role in matrix mineralization (Han et al, 2009). Concordant with bone development, Ihh expression is responsible for proper skeletal muscle formation. It has been implicated in the promotion of fetal myoblast survival and in the development of myofibrils
(Bren-Mattison, Hausburg and Olwin, 2011). In consequence, Ihh null mutants have many skeletal-related defects including inefficient bone growth, aberrant bone formation at growth plates and general short stature. Abnormal muscle mass formation is also visible in Ihh mutants by 14.5 dpc and loss in hindlimb muscle is visible by 18.5 dpc (Bren-Mattison, Hausburg and
Olwin, 2011). In regards to its function in primitive endoderm, if Ihh is absent, only half of organisms will survive to maturation due to malformed fetal yolk sacs (Varjosalo and Taipale,
2008).
Noticeably, Hh signaling is an extensive developmental system. Just as the absence of a ligand can result in major developmental defects, a mutation in other components of the pathway can have huge ramifications, especially in disease. A prominent example of this is with basal cell carcinoma, a skin cancer that is also the most prevalent type of cancer in the human population
(McMillan and Matsui, 2012). About 90% of spontaneous cases have been traced back to mutations in Hh signaling components. 15-30% of medulloblastoma cases, a malignant form of 4 brain cancer, are also connected to these same mutations, showing how serious the dysregulation of this pathway can be (McMillan and Matsui, 2012).
In addition to mutations affecting the transduction of the pathway, mutations resulting in overexpression of Hh ligands are also implicated in carcinogenesis. Overexpressed ligands can cause the rapid spread of cancer cells by acting in an autocrine or paracrine signaling fashion while in or near other cancer cells; tumors that form as a result are referred to as “ligand- dependent ” (McMillan and Matsui, 2012; Coni, Infante and Gulino, 2013). Studies involving pancreatic cancer, for instance, have found that high expression of Hh in the pancreas can result in the proliferation of pancreatic cancer cells (Hao et al, 2013). Analogously, when Hh signaling is repressed, cancer cell proliferation decreases and metastasis is less likely to occur. Other cancers that appear to be “ligand-dependent” include melanoma, glioblastoma, colorectal and metastatic prostate carcinomas (McMillan and Matsui, 2012).
Hedgehog signaling is also implicated in the proliferation of cancer stem cells (CSCs).
Cancer stem cells are cells that are able to give rise to all cell types found in a particular type of cancer (Quintana et al, 2008). It has been reported that Hh determines the developmental fate of these cells and regulates their self-renewal and tumor initiating properties (McMillan and Matsui,
2012). It is hypothesized that Hh accomplishes this by regulating a stemness-determining gene known as Nanog, which is overexpressed in cancers (Coni, Infante and Gulino, 2013). These cancer stem cells are implicated in a variety of cancers including colon, lung, brain, and even metastatic breast cancer (Coni, Infante and Gulino, 2013). Their integrality in such a myriad of cancers leads researchers to believe that cancer stem cells may just be part of a general mechanism of cancer instead of specific to certain types. Overall, it is due to Hh’s recognized role in cancer that Hh inhibitors are now being looked into as novel treatments; more in-depth knowledge of Hh pathway function is therefore crucial to drug development (Rudin et al, 2009). 5 1.2 Our current understanding of Hh signaling in mammals
The actual mechanism of Hh signaling and the resulting expression of genes are relatively similar between organisms. It is a highly conserved pathway throughout evolution; however, there are a few major differences, especially between Drosophila and mammals.
In Drosophila, Hh signaling involves a single Hh ligand (Hh), a 12-transmembrane protein receptor named Patched (Ptc), a 7-transmembrane protein named Smoothened (Smo), and a Hedgehog Signaling complex (HSC) (Van den Brink and Gijs, 2007). This complex has multiple components including a serine/threonine kinase named Fused (Fu), a kinesin-like molecule named Costal2 (Cos2), and a regulator protein named Suppressor of Fused (Sufu) (Van den Brink and Gijs, 2007). There are also several other kinases associated with the HSC, including protein kinase A (PKA), protein kinase CK1 (CK1) and glycogen kinase synthase 3
(GKS3). A transcription factor named Cubitus interruptus (Ci) is a final major component of the pathway (Van den Brink, 2007). Figure 1-2 below shows a simplified version of Hh signaling in
Drosophila.
Figure 1-2. The Drosophila Hh signaling pathway mechanism; both inactive and active states are represented (Hooper, 2013)
6 In the absence of Hh ligands, the pathway is inactive. Ptc represses Smo activity (Van den Brink and Gijs, 2007). Sufu constrains the nuclear translocation of Ci. PKA, CK1, GSK3 and Fu, which are bound by Cos2, then mediate the phosphorylation, processing and proteolysis of Ci. A repressor form of Ci (CiR) is formed and in turn represses transcription of the target genes (Van den Brink and Gijs, 2007).
To the contrary, activation of this pathway occurs in the presence of a Hh ligand. Hh binds and inhibits Ptc, conceding Smo to modulate the HSC (Van den Brink and Gijs, 2007).
Cos2, which binds PKA, CK1, GSK3, and Fu are then recruited by Smo to inhibit Sufu activity.
As a result, Ci is permitted to travel into the nucleus to activate the target gene expression.
The Hh signaling mechanism in mammals has slight variations from that of Drosophila.
As previously mentioned, mammals have three different Hh ligands: Shh, Dhh and Ihh (Varjosalo and Taipale, 2008). Certain components of the Drosophila HSC, such as Cos2, Fu and certain kinases, are not active in mammalian Hh signaling. A protein known as Kif7 is present, however, and serves a similar purpose as Cos2, while a variety of mammalian kinases such as DRK and
MAP3K10 replace Fu (Endoh-Yamagami et al, 2009). The transcription factor in mammals is also different; it is part of a Glioma associated zinc-finger protein family known as Glia (Hui and
Angers, 2011). There are three forms of this protein, Gli1, Gli2, and Gli3, which will be discussed in more detail later (Varjosalo and Taipale, 2008).
One of the main differences involves not the components of the mammalian pathway, but where signaling transpires. Hh signaling in vertebrates is dependent upon cilia, small cellular projections found on many vertebrate cells (Goetz, Ocbina and Anderson, 2009). It is in the cilia where Ptc and the HSC are located and where Gli regulation takes place (McMillan and Matsui,
2012). Figure 1-3 below displays mammalian Hh signaling as is transduced in the primary cilium.
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Figure 1-3. The inactive and active states of mammalian Hh pathway in cilia (Berbari et al, 2009).
From Figure 1-3, it is clear that in the absence of Hh, Ptch suppresses cytoplasmic Smo activity (Van den Brink and Gijs, 2007). As a result, Smo does not travel into the cilia to activate
Gli; Sufu is thus free to repress Gli nuclear translocation. The expression of the target Hh genes are consequently repressed.
Activated mammalian Hh signaling begins when Hh binds and inhibits Ptch, allowing
Smo to travel to the cilia to modulate the HSC (Van den Brink and Gijs, 2007). Sufu is inhibited and Kif7, an integral part of the HSC that is found in the cilia, prevents the proteolysis of Gli
(Endoh-Yamagami et al, 2009). In the prescence of Hh, Kif7 is also the chief factor in ensuring
Gli3 localization in the cilia where it is processed proceeding its translocation to the nucleus; it is then that target genes are expressed (Endoh-Tamagami et al, 2009). Because mammalian Hh signaling is not yet completely understood, there are most likely additional proteins that contribute to this pathway that have yet to be discovered.
Unlike Drosophila where Ci serves as both transcriptional activator and repressor, Gli1,
Gli2, and Gli3 each serve a different primary function in mammals. In mammalian Hh signaling, 8 Gli2 acts as the primary transcriptional activator while Gli3 functions as a primary repressor (Jia et al, 2009). The expression of Gli1 is dependent on the presence of Hh and is responsible for providing positive feedback for Hh signaling. Not only does each Gli protein have a different primary function but the absence of them also has varied effects (Varjosalo and Taipale, 2008).
Gli1 null mutants can develop normally, however embryos lacking Gli2 are not viable. Limb malformations are the most common consequence in Gli3 null mutants (Varjosalo and Taipale,
2008).
1.3 The roles and remaining questions of Sufu
One Hh component that has a regulatory role in Drosophila and mammals alike is Sufu.
We currently know that Sufu is not as incorporated in the fly pathway as it is mammals; however it is still not completely clear as to what extent. In mammals, Sufu null mutants exhibit unregulated Hh signaling activation (Cooper et al, 2005). Mutations in Sufu also have implications in Gorlin’s syndrome, which is characterized by a predisposition to cancer
(McMillan and Matsui, 2012).
Sufu can physically interact with all three Gli proteins (Jia et al, 2009). It is also known that Sufu is responsible for negatively regulating Gli-transcriptional activity in the absence of Hh
(Varjosalo and Gijs, 2008). It accomplishes this through sequestration of Gli proteins in the cytoplasm; in doing so, Sufu prevents Gli nuclear translocation. Sufu has also been found to negatively regulate Hh signaling through the delivery of a histone acetylation complex to the downstream Hh genes themselves (Jia et al, 2009).
Studies have shown that the majority of Hh signaling components are localized to the primary cilium, including Sufu. However, Sufu functioning is not dependent on cilia (Jia et al, 9 2009). In the absence of cilia, Sufu still sequesters Gli proteins in the cytoplasm and negatively regulates their transcriptional activity. Furthermore, when the level of Sufu within the cell is reduced in the absence of cilia, Hh signaling is still activated (Jia et al, 2009).
Sufu appears to be involved in Gli3 processing by recruiting GSK3beta to the cilia to form a Sufu-GSK3b-Gli3 trimolecular complex (Kise et al, 2009; Chen et al, 2011). This complex is responsible for the processing and phosphorylation of Gli3, and therefore the generation of a transcriptional repressor for target Hh genes. Analogously, in the presence of
Shh, this complex is dissociated and Gli3 processing is inhibited (Kise et al, 2009).
Studies in various Eukaryote species suggest that Sufu has two evolutionarily conserved domains, one found on the carboxy-terminal (C-terminal) end and another found on the amino- terminal (N-terminal) end (Merchant et al, 2004). The C-terminal domain is responsible for binding to the amino terminal ends of Gli proteins, while the N-terminal domain of Sufu is responsible for binding to the carboxy terminal ends of Gli proteins. Although each domain binds to Gli independently, concurrent binding is necessary for cytoplasmic tethering and repression of
Gli to occur (Merchant et al, 2004).
Despite the fact that all of this information is known about Sufu, the underlying biology of how Sufu performs its functions is still not completely understood. For instance, although we are aware that Sufu sequesters Gli proteins in the cytoplasm, it is not clear as to whether it interacts with the cytoskeleton or other cytoplasmic components to achieve this. Additionally, the mechanism as to how Sufu and Gli are separated in the presence of Hh is not apparent; it is possible that there are other proteins that act within the Hh pathway that are integral to this process. These same proteins could also be involved in the activation of Gli in the presence of
Hh. The nature of activated Gli is still an enigma, so there could be other proteins involved in activating Gli. It is questions such as these that interest us most and motivate us to perform further studies with Sufu. 10
To gain new information about how Sufu functions in the Hh signaling pathway, we set out to identify proteins that interact with Sufu. We confirmed that three proteins, transmembrane
131-like precursor (T131L), COP9 signalosome complex subunit one isoform (Gps1) and hypothetical Protein LOC67513 interact with Sufu in mammalian cells. Furthermore, we specifically discovered that they interact with both Sufu’s N and C terminal domains. Finally, we found that when overexpressed with Gli in mammalian cells, the proteins generally enhanced Gli activity. Ultimately we want to know how these Sufu-interacting proteins function within the Hh pathway; knowing this could help us further understand Sufu’s overall function. 11
2. Materials & Methods
2.1 Construction of Plasmids
Phusion PCR Reactions & PCR Purification
Many plasmids utilized throughout these experiments, as listed in table 1-1, were constructed into a desired vector in order to further test interactions and functions. Although each construction involved different enzymes, buffers and antibiotics, they followed the same general process. The first step was amplification of the desired sequences from DNA templates. We received partial cDNA (C-terminal only) of Ran-Binding Protein 9 (RanBP9), transmembrane 131-like precursor
(T131L), COP9 signalosome complex subunit 1 isoform (GPS1), and hypothetical protein
LOC67513 (LOC67513) from Dr. Yang. The other templates needed were already available in our lab or were purchased from Life Technologies Corporation. The latter was the case for the full-length T131L and GPS1 cDNA. Full-length LOC67513 was not available for order; long primers were therefore designed in order to amplify the 22 amino acid sequence that was missing in the partial construct. The sequences were amplified using Phusion Polymerase Chain Reaction
(PCR) technology. The set-up for these 50 µl PCR reactions involved 20ng of sample and can be found in table 5-1 in the Appendix. Each reaction involved a 98°C heat initiation step that lasted for three minutes. 35 thermal cycles of denaturation, annealing and extension followed, according to the times and temperatures listed in table 2-1. A final elongation step that involved a
10 minute long 72°C cycle culminated the reaction. The resulting amplified insert sizes as well as the unique primers that were used to complement the 3’ ends of the sense and anti-sense strands of each DNA template can also be found in table 2-1. 12
DNA Amplified Primers Denaturation Annealing Extension step template Insert step step Size T131L –PMV 1535 aa 1. 5'-GAA TTC GGCAT 98°C/0’10” 67°C/0’30” 72°C/2’18” SPOC6 (FL) GTT CCC ACAGTA GCT GGA GT-3’ (TM = 66.65°C) 2. 5’-AATCGG CTGAG GAA CAA GATCGC TGC-3’ (TM =66.46°C) GPS1 – PMV 235 aa 1. 5’GGATCAGCGTA 98°C/0’10” 67°C/0’30” 72°C/0’45” SPOC6 (FL) GCACCACCT-3’ 2. 5’AATTCCTTGCCC ATCAGCAG-3’ LOC67513 434 aa 1. 5’-GGG AGG CTG 98°C/0’10” *1. 67°C/0’30” *1. 72°C/0’18” (FL) AGC CGT CGG AGG *2. 65°C/0’30” *2. 72°C/0’18” CCG CCC GCG GGG GGC GG GCC CCC TAA CTG CCG C- 3’ (TM = 67.3°C) 2. 5’-GAT CCG AAT TCA GAG GAG GAG CGG AGA CTG CG G GGG AGGCTGAG C CGTC-3’ (TM = 65.45°C) 3. 5’-GGA TCCGGTAC CTCACCACACCAC AT CTTCGG-3’ (TM = 67.61°C) PEGFPC3- 178 aa 1. 5’-GA TCC AAGCT 98°C/0’10” 67°C/0’30” 72°C/0’30” mSufu (Sufu-N) TGCG GAG CTG CGG CCT A-3’ (TM = 69.47°C) 2. 5’-GAATTC GGTAC C TTA CTC AAATA TGGT TTC TCCCC G C-3’ (TM = 67.626°C) PEGFPC- 222 aa 2. 5’-GA TCC AAGCT 98°C/0’10” 68°C/0’30” 72°C/0’30” mSufu (Sufu-C) TGAT CCG CACCT G CAA GAG AGA-3’ (TM = 69.31°C) 2. 5’-GAATTC GGTAC C CTA GTG CAGTG G ACT GTC GAA CAC-3’ (TM = 67.37°C)
* LOC67513 was not available for order from INVITROGEN, so long primers were designed to amplify the lacking N-terminal (22aa); a primer that allowed one-step cloning was too long, so two reactions were needed.
Table 2-1. Phusion PCR environments used to amplify sequences for plasmid construction. 13
100ng of amplified cDNA underwent gel electrophoresis on a 25ml 0.8% agarose gel (25mL
TBE, 0.2g agarose, 0.5µl EtBr) at 95V for 20 minutes to check whether the correct size inserts were amplified from the template; 1µ of 6x loading buffer (see table 5-2 in the Appendix) was added to the samples which were run against 5µl of a 1kb ladder. After confirming that Phusion
PCR was successful, PCR purification, according to the March 2008 QIAquick PCR Purification
Kit Protocol (pg.19-20) from QIAGEN was performed yielding 30 µl DNA product.
Digestion of insert and vectors
20 µl test digestions were performed in order to ensure the enzymes cut according to supposed restriction sites. This reaction included 300ng of desired vector, 0.5-1µl of enzymes, 2µl of enzyme-indicated buffer, and water. It was incubated in a 37°C water bath for two hours. 100ng of digested sample was then run on 25ml 0.8% agarose gel to check for the correct digested band sizes. The restriction enzymes and buffers required for each plasmid digestion can be found in table 2-2 below. Once the restriction sites were confirmed, a 40 µl vector digestion and a 40 µl insert digestion, each containing 3µg of sample, 1-2µl of enzymes, 2 µl of enzyme-indicated buffer and H2O was performed and incubated o/n. In the case that two enzymes were required but their buffer environments were not compatible, the digestion was performed over two days; this was the case in the construction of Flag-SufuN and Flag-SufuC. The first enzyme digestion reaction, which needed buffer 2, was incubated overnight. The next day, the first enzyme was heat inactivated in a 65°C/20 minutes hold in the PCR machine and the second enzyme reaction was set-up in the same tube. 10XB2-3was utilized to change the environment from buffer 2 to buffer 3, which was needed by the second enzyme; the sample was incubated o/n. Another 25 ml
0.8% agarose gel, holding 100ng of both the digested vector and insert, was run at 95V for 40 minutes to ensure complete digestion. 14
Vector/Insert Being Vector Restriction Insert Restriction Digested Enzymes & buffers Enzymes & Buffers PEGFPC3/RanBP9 ∆N Xho1 (buffer 2) HindIII (buffer 2) HindIII (buffer 2) SalI (buffer 3) +10xB2-3 PEGFPC3/GPS1 ∆N Xho1 (buffer 2) HINDIII (buffer 2) HINDIII (buffer 2 ) SalI (buffer 3) +10xB2-3 PEGFPC3/LOC67513 ∆N Xho1 (buffer 2) HINDIII (buffer 2) HINDIII (buffer 2) SalI (buffer 3) +10xB2-3 PEGFPC3/T131L ∆N RI (buffer 2) RI (buffer 2) SalI (buffer 3) XhoI (buffer 2) +10xB2-3 PEGFPC3/GPS1 FL HINDIII (buffer 2) HINDIII (buffer 2) BamHI (buffer 3) BamHI (buffer 3) +10xB2-3 +10xB2-3 PEGFPC1/LOC67513 FL ECORI (buffer 3) ECORI (buffer 3) Acc651 (buffer 3) Acc651 (buffer 3) PEGFPN1/T131L FL BamHI (buffer 3) BamHI (buffer 3) XhoI (buffer3) XhoI (buffer 3) pFlag-cmv2/SufuN HINDIII (buffer 2) HINDIII (buffer 2) Acc651 (buffer 3) Acc651 (buffer 3) + 10xB2-3 + 10xB2-3 Flag/SufuC HINDIII (buffer 2) HINDIII (buffer 2) Acc651 (buffer 3) Acc651 (buffer 3) + 10xB2-3 + 10xB2-3 Table 2-2. Vector and Insert Digestion Information.
Gel Extraction & Ligation
1µl of Calf intestinal alkaline phosphatase (CIP) was added to the digested vector and it was incubated in 37°C water bath for one hour. This prevented the two ends from reattaching before ligation. A 60ml 0.8% agarose gel (60ml TBE, 0.48g agarose, 1.2µl EtBr) was made in preparation for gel extraction. 6µl of 6x loading buffer was added to each 50µl vector and insert sample and was run in the gel with 10µl 1kb ladder. The gel was run for 3 hours; afterwards the
October 2009 E.Z.N.A gel extraction spin protocol (pg. 6-7) from OMEGA Bio-Tek was followed, yielding 30µl volumes of vector and insert samples. Gel electrophoresis was performed using a 25ml 0.8% gel and 0.5 µl of each sample in order to ensure the success of the gel 15
extraction. Once confirmed, the ligation ratio of insert and vector was calculated to determine what volume of each should be added to the 10µl ligation system; this ratio is based on the concentration of each and the goal is a 3:1 insert to vector ratio. In addition to the insert and vector, the ligation system also included 1µl 10x ligation buffer, 1µl ligase enzyme, and enough ddH2O to complete the 10µsystem. A control reaction containing only the vector was also set- up. After mixing, the reactions were put in a 16°C hold in the PCR machine o/n.
Transformation & Picking Colonies
Competent cells were transformed according to the protocol in TOPO TA Cloning (pg. 6-13).
The cells transformed with the ligated mixture and the cells transformed with the control were each plated on a pre-warmed LB plate with the correct antibiotic; the antibiotic ensured the death of any non-transformed cells or bacteria that were not resistant to the antibiotic. The antibiotics utilized in the plating of the partial and full-length RanBP9, T131L, GPS1, and LOC67513 was kanamycin (Kan); this is because the GFP vectors that they were cloned into are resistant to it.
The antibiotic utilized in the plating of SufuN and SufuC plasmids was ampicillin (Amp) due to the fact that the Flag vectors they were cloned into are resistant to it. The plates were put in 37°C incubator o/n. The colonies were counted for each plate, ensuring there were proportionally more colonies transformed with the ligated product than the vector; around a 10:1 ratio was desired. 6 colonies transformed with the ligated product were picked and grown in 1.5ml of LB medium with 4.5 µl of appropriate antibiotic. A control tube containing 1ml LB medium and 3µl of antibiotic ensured the antibiotic is effective in preventing the growth of unwanted bacteria. The colonies were placed in a 37°C incubator (shaking at 230rpm) for 8 hours.
16
Crude Miniprep, Reinoculation and PCR Purification
A plasmid crude miniprep of the six incubated LB cultures was performed and resulted in a 30µl collection of DNA solution. An enzyme screening digestion was performed with 0.5 - 2µl of the
DNA sample, along with a control. Gel electrophoresis of the digested samples followed, using a
40ml 0.8% gel (40ml TBE, 0.32g agarose, 0.8ul EtBr); this established that the selected colonies were transformed with our ligated product. Two of the six LB bacterial cultures were chosen for re-inoculation; this involved 200µl of each culture being added to 5ml LB plus 15µl antibiotic.
The culture was placed in 37° incubator (shaking at 230rpm) o/n. One of the cultures was chosen for PCR purification, again performed according to the QIAGEN protocol.
Concentration & Sequencing
The concentration of the PCR purified product was measured on the BIO-RAD Smart-Spec Plus
Spectrometer using 2.5µl DNA sample mixed with 97.5µl TE. The reported A260/A280 ratio also indicated the purity of the DNA sample; a measurement between 1.8 and 2.0 was preferable.
The DNA needed to be sent for sequencing in order to check for point mutations in our constructed plasmid. For sequencing, 1µM of each primer as well as 200-300µg/µl of DNA sample was needed. All sequencing requests were sent and performed by Penn State’s Genomics
Core Facility.
17
2.2 Co-Immunoprecipitation Assays
Figure 2-2 shows a basic mechanism for Co-IPs.
Figure 2-2. Basic mechanism for Co-Immuniprecipitation Assay (molecularsciences.com, 2006)
Cell Transfection & Extraction
293T cells stored at -80°C were plated in growth medium (without antibiotics) so that at the time of transfection, the cells would reach 90-95% confluence. At the time of transfection, the desired plasmids, including the DNA for both of the two hypothetical interactive plasmids, was diluted in
Opti-MEM according to the 24-well specifications found in table 5-3 in the Appendix; the same was done for lipofectamine 2000. After five minutes, the DNA sample and lipofectamine were combined and incubated at room temperature for 20 minutes. The DNA-lipofectamine complex was added to the cells while rocking the plate back and forth. The cells were incubated at 37°C,
5% CO2 for 24-48 hours before harvesting. At time of extraction, cells were rinsed once with 18
1ml cold PBS and a then scraped with 1.4ml PBS-EDTA. They were then spun down at 2000rpm for 5 minutes at 4°C. The PBS-EDTA was discarded and the pellet was resuspended in a volume of 1% Triton lysis buffer, with protease inhibitors, that was 4x the volume of the cell pellet; the recipe for the lysis buffer can be viewed in table 5-4 in the Appendix. The cell were incubated on ice for 20 minutes and spun at 4°C at top speed for 10 minutes. The supernatant was transferred to a new tube (kept on ice) and the concentration was measured.
Flag-Tagged Protein Immunoprecipitation
The lysates extracted were used in the Co-immunoprecipitation assays. Immunoprecipitation including the setup and addition of the beads was performed according to the SIGMA-ALDRICH
FlagGIPT-1 protocol.
SDS-Page & Western Blot
An SDS page gel was assembled using the 7.5% acrylamide separating and stacking gel recipes in tables 5-5 and 5-6 found in the Appendix. The separating gel was made first and sat for 15 minutes until ready; the stacking gel was poured on top and a comb was inserted in order to form
15 wells. 12µl of 5x SDS loading buffer and lysis buffer was added to each Co-IP supernatant sample and boiled for 5 minutes. When solidified, the comb was then removed from the gel and the wells were washed three times with ddH20. The SDS page BioRad minigel apparatus was assembled and filled with running buffer. 10µl samples were loaded into the wells and the gel was run at 20mA for 1.5 hours. When finished, the stacking gel was discarded and the remaining separating gel was marked. It was then soaked in Western transfer buffer (100ml 10x transfer buffer, 200ml methanol, 700ml ddH2O) for 10 minutes. During that time an 8.3x6cm NC membrane was wet with ddH2O and soaked in transfer buffer. The sandwich was assembled in 19
transfer buffer in a specific order: black cover down, sponge, paper, paper, gel, NC membrane, paper, paper, sponge, white cover above. The sandwich was placed in the BioRad tank filled with cold transfer buffer and an ice bucket. The transfer occurred at RT, 100V for 1 hour. When finished, the NC membrane was taken out and marked to identify where loading began.
The NC membrane was then rinsed once with PBS for 10 minutes and washed with PBS-0.1%
Tween20 three times, 5 minutes each. The membrane was then blocked with 5% milk in PBS-
0.1% Tween 20 (5g powder milk added to PBS-0.1% Tween 20 to make 20ml total) for 30 minutes. Afterwards, it was incubated o/n with the primary antibody (rabbit) diluted with 5%
BSA in PBS-0.1% Tween20. The next day, the membrane was rinsed twice with PBS-0.1%
Tween20 and then washed three times, 5 minutes each. Incubation with the secondary antibody diluted with 5% milk in PBS 0.1% Tween20 (1:5000 dilution) followed for 1 hour at room temperature. After rinsing the membrane twice and washing three times with PBS-0.1%
Tween20, it was washed once more with PBS. It was then ready for ECL development and film exposure in order to view the expression patterns of the Co-IPs, as is viewed in the results section below.
2.3 Dual-Luciferase Reporter Assays
Transfection & Harvesting
The transfection was performed in the same manner as the Co-IP experiments described above and in table 5-3 in the Appendix. The only difference was that Mef cells (both wild-type and
Sufu -/-) were used instead of 293T cells. A second difference is that also included in the 0.8µg 20
of plasmid used in the 24-well specification was 2ng of Renilla luciferase and 0.1µg of firefly luciferase per well as control. Each sample was transfected three times so multiple measurements could be taken and a standard deviation calculated. 24-48 hours after transfection, the cells were harvested. The medium was vacuumed and the cells were washed twice with 0.5ml of RT PBS.
Each well was scraped with 100µl of RT 1x Passive Lysis Buffer (PLB) (1vol 5X PLB + 4 vol ddH2O) and the cells were collected. Each sample was shortly centrifuged at 4°C and the top
20µl was collected and put aside for the assay.
Dual- Luciferase Reporter Assay
50µl of RT Luciferase Assay Reagent II (LARII) (Lyophilized Luciferase Assay substrate + 10ml
Luciferase Assay Buffer II) was added to a tube containing 20 µl of lysis buffer; it was pipetted up and down and then placed in the Turner BioSystems 20/20n Luminometer for the first reading to be taken. 50µl of RT Stop & Glo reagent (1 vol 50X Stop & Glo Substrate + 50 volStop &
Glo Buffer), prepared in a glass tube, was then added to the lysis buffer sample and vortexed before the second reading was taken. This measurement served as one of the two background measurements that were taken in order to have baseline data. The same process was followed for each sample in order to measure the activity expressed in each cell. Three trials were performed for each assay and the results were normalized; experimental error and statistical significance were calculated. The relative activity of each overexpressed protein was compared to the overexpression of Gli1. This is because overexpression of Gli1 has been previously reported to significantly activate the luciferase reporter in wild-type and Sufu -/- Mef cells (Yang et al, 2012).
The results were also compared to cells overexpressed with both Gli1 and Sufu, which has previously been reported to decrease the expression of the luciferase reporter (Yang et al, 2012).
This is concordant with Sufu’s function in inhibiting Gli1 in Hh signaling (Jia et al, 2009). 21
3. Results
3.1 Identification of Sufu-interacting proteins through a Yeast Two-Hyrbid Screen
To identify new Sufu-interacting proteins, our collaborators at U. Illinois performed a yeast-two hybrid (Y2H) screen that yielded about 100 different clones that showed interaction with Sufu in yeast. We extensively researched published information about these proteins in order to select interesting prospects to continue experimentation. Particularly, we searched for proteins that are localized within the nucleus or cytoplasm where Sufu has been known to function. We also excluded proteins that have been shown to carry out functions unrelated to Hh signaling. In the end, we decided to focus on four proteins: transmembrane protein 131-like precursor (T131L), ran-binding protein 9 (RanBP9), COP9 signalosome complex subunit 1 isoform (GPS1), and hypothetical protein LOC67513 (LOC67513).
From literature we found that RanBP9 is involved in the translocation of proteins and
RNA through the nuclear pore by binding to RAN, which is a small GTP-binding protein (U.S.
National Library of Medicine, 2013). The second protein, T131L, also is involved in the regulation of the nuclear export of proteins; it interacts with exportin7, which in turn interacts with Ran (Mingot et al, 2004). Because Sufu is involved in Gli cytoplasmic sequestration, we hypothesized that T131L and RanBP9 may have a role in this process due to their importance in protein translocation (Jia et al, 2009). The third protein, Gps1, is a subunit of COP9 signalosome, which has been implicated in Ci phosphorylation in Drosophila (Wu et al, 2011). The complex’s existing role in Hh signaling makes the Gps1 subunit an interesting prospect for our study. There is very little information currently available about LOC67513; however this protein was 22 represented by eight independent clones in the Y2H screen, suggesting a high likelihood that it strongly interacts with Sufu.
3.2 The interaction between Sufu and T131L, Gps1, Loc67513 fragments are confirmed in mammalian cells
We obtained the Y2H clones of T131L, Gps1, LOC67513 and RanBP9, each of which was a C-terminal fragment of the protein cloned in a pACT2 vector. These partial cDNA fragments were subcloned into PEGFPC3 vectors to make Green Fluorescent Protein (GFP)- tagged proteins. Co-IP experiments were then performed in 293T cells between each of these protein fragments and Flag-Sufu to corroborate the interactions detected in the Y2H screen. The positive control utilized in this experiment was GFP-Gli2 (1-967aa), which has been previously shown to interact with Sufu (Zeng, Jia and Liu, 2010).
A B
C D
Figure 3-1. T131L, Gps1, and LOC67513 protein fragments physically interact with Sufu in mammalian cells, while RanBP9 does not. (A-C) Sufu co-immunoprecipitates with T131L (A), Gps1 (B) and LOC67513 (C) when overexpressed in 293T cells. (D) Sufu does not co-immunoprecipitate with RanBP9 when overexpressed in 293T cells (N/A indicates that the straight lysates of RanBP9 and Sufu were not blotted together). 23
As seen in figure 3-1, the C-terminal fragments of T131L, Gps1 and LOC67513 interact with
Flag-Sufu in mammalian cells, confirming the results of the Y2H screen. Despite T131L showing a weaker transfected expression in the cells, there was just as strong of an interaction with Sufu as Gps1 and LOC67513. Unlike the other three proteins, RanBP9 did not interact with
Sufu, suggesting that the interaction seen in yeast might be false positive. Further studies were performed with T131L, Gps1 and LOC67513.
3.3 Full-length T131L, Gps1 and Loc67513 interact with Sufu in mammalian cells
Following the co-immunoprecipitation assays with the partial proteins, we set out to confirm the interactions with full-length proteins. Because full-length proteins may have different localization than partial proteins, they may be prevented from binding to Sufu.
Therefore, this assay will give us more confidence as to whether these proteins can actually interact with Sufu in their natural cell context. We ordered the full-length cDNAs of the three proteins that initially exhibited interactions with Sufu and cloned them into the desired expression vectors containing GFP tags, as was described in the Materials and Methods section. As T131L was predicted to be a transmembrane protein, we cloned it into pEGFPN1 vector to add the GFP tag to its C-terminus (Weizmann Institute of Science, 2013). This is because the T131L contains a signal at its N-terminus, and adding a GFP tag at the N-terminus may alter the subcellular localization of the protein (Berk, Lodish and Zipursky, 2000).
24
A B
C
Figure 3-2. Sufu physically interacts with full-length proteins Gps1, LOC67513 and T131L in mammalian cells. Sufu co-immunoprecipitates with Gps1 (A), LOC67513 (B) and T131L (C) when overexpressed in 293T cells.
Figure 3-2 shows that Sufu can physically interact with the full-length Gps1, LOC67513 and
T131L. As in the partial construct Co-IP, T131L showed lower expression than the other proteins, which could be due to its larger protein size. Despite this, it still showed equally strong interaction with Sufu. Gps1 and LOC67513 are most likely localized to the cytoplasm or nucleus in 293T cells, as was previous reported, where interactions with Sufu could occur (U.S. National
Library of Medicine, 2013; Mingot et al, 2004). T131L, as previously mentioned, is hypothesized to be a transmembrane protein and specifically a single-pass type 1 membrane protein, which means it spans the membrane only once (UniProt, 2013; Weizmann Institute of Science, 2013). 25 Its probable location implicates that Sufu may interact with the C-terminal end of T131L, which is believed to be present in the cytoplasm.
3.4 The three proteins interact with both the N- and C-terminal domains of Sufu
Next we investigated which part of Sufu interacts with T131L, GPS1, and LOC67513.
Biochemical and structural analyses suggested that Sufu consists of two domains, the Sufu-N and
Sufu-C (Merchant et al, 2004). We constructed truncated Sufu representing the two domains by
PCR and cloned them into the Flag-cmv2 vector. We then performed co-immunoprecipitation assays between the three proteins and Sufu-N and Sufu-C. The positive control utilized was the previously confirmed interaction between the full-length Gps1 construct and full-length Flag-
Sufu (fig. 3-2).
26 A B
C
Figure 3-3. Full-length proteins T131L, Gps1 and LOC67513 each physically interact with both Sufu-N and Sufu-C in mammalian cells. Sufu-N and Sufu-C each co-immunoprecipitate with Gps1 (A), LOC67513 (B) and T131L (C) when overexpressed in 293 T cells.
Figure 3-3 shows that positive interactions were detected between the three full-length proteins and both N-terminal and C-terminal domains of Sufu. Sufu shows similar binding to Gli proteins; concurrent binding with both domains is necessary for cytoplasmic tethering and resulting repression of Gli (Merchant et al, 2004). Binding of Gli with both domains of Sufu results in successful functioning of Sufu. It is possible that by interacting with both domains of
Sufu, these proteins (T131L, Gps1 and LOC67513) may interfere with the Sufu-Gli interaction. 27 3.4 The three proteins significantly activate Gli-dependent reporters in Sufu -/- Mef cells
Having confirmed their interaction with Sufu, we sought to address the roles of the three
Sufu-interacting proteins in Hedgehog signaling through luciferase reporter assays. In this reporter assay, cells were transfected with the effector proteins, a Gli-dependent luciferase reporter (8xGli-BS-LUC) and a Renilla luciferase reporter (pTK-RL), which was used as a control for transfection efficiency.
The first luciferase assay was performed utilizing Sufu -/- Mef cells, which do not contain endogenous Sufu. The proteins were overexpressed in cells along with Gli1. The figure below represents the relative activity measured from each of the samples.
Relative Activity of T131L, GPSI, &
LOC67513 in Sufu -/- MEF cells 14 * 12 10 * * 8 relative activity 6
4
2 Relative ReporterRelative Activity 0
Gli1 - + + + + + Sufu - - + - - - T131L - - - + - - GPSI - - - - + - LOC - - - - - + Figure 3-4. The measured reporter activity in Sufu -/- Mef cells. The pluses represent the plasmids that were transfected in each column’s samples. The ‘*’ represents statistically significant differences in levels of activity when compared to the activity elicited by Gli1-only transfected cells; statistical significance is correlative to a calculated p-value that is less than 0.05.
28 All three proteins, when overexpressed with Gli1, lead to higher reporter activation compared to
Gli1-only transfected cells. T131L activated the reporter the most with about an 11-fold activation of the reporter, significantly higher than that of the Gli1 treated cells (p=0.003). Gps1 also significantly enhanced Gli1 activity (p=0.006). Finally, the overexpressed LOC67513 cells elicited about an 8-fold relative activation and a statistically significant p-value calculated at about 0.002 when compared to the activity elicited by Gli1-only expressed cells. From this assay, it is clear that in the absence of Sufu, whether endogenous or expressed, the three proteins have a function in promoting Gli1 in activating its target gene expression.
In order to study the effect that overexpressed Sufu could have on the activity of these cells, a second luciferase assay was performed again utilizing Sufu -/- Mef cells but with additional Sufu transfected. The figure below shows the relative activities that were measured by the reporter.
Relative Activity in Sufu -/- MEF cells
8 7 6 5 4 relative activity 3 * 2 * * 1
0 Relative Relative Reporter Activity
Gli1 - + + + + + - Sufu - - + + + + - T131L - - - + - - - GPSI - - - - + - - LOC - - - - - + - Figure 3-5. The measured reporter activity in Sufu -/- Mef cells. The pluses represent the plasmids that were transfected in each column’s samples. The ‘*’ represents statistically significant differences in levels of activity when compared to the activity elicited by Gli/Sufu transfected cells; statistical significance is correlative to a calculated p-value that is less than 0.05.
29
The cells overexpressed with T131L, Gps1 and LOC67513, respectively, showed significant activation of the reporter in comparison to the Gli1 and Sufu transfected cells. The activations, however, were still lower than the expressed activity of the Gli1 only expressed cells. As in the prior luciferase assay, T131L had the largest-fold activation among the three proteins. The sample transfected with T131L had about a 2-fold relative activation (p=0.002), the sample transfected with Gps1 had about a 1.5-fold relative activation (p=0.035) and the sample transfected with LOC67513 had about a 1.8 fold relative activation (p=0.002). Because the proteins were co-transfected with Sufu and Gli1, it could be deduced from this assay that through their interactions with Sufu, the inhibition on Gli is released, thus showing an increase in activation of the Gli-dependent reporter.
3.5 T131L significantly activates Gli-dependent reporter activation in wild-type Mef cells
We wanted to see the effect of endogenous Sufu on the relative activity of T131L, Gps1 and LOC67513 in mammalian cells. This was accomplished by overexpressing our effector proteins in wild-type Mef cells. In comparison to Sufu -/- Mef cells, the transfection efficiency of the wild-type cells was much lower. Despite this, significant results were still obtained as are seen in figure 3-6.
30 Relative Activity in WT MEF cells
45 40 *
35 30 25 relative activity
20 *
15
Relative Reporter Activity 10 5
0 Gli1 - + + + + +
Sufu - - + - - - T131L - - - + - -
GPSI - - - - + - LOC - - - - - +
Figure 3-6. The measured reporter activity in wild-type Mef cells. The pluses represent the plasmids that were transfected in each column’s samples. The ‘*’ represents statistically significant differences in levels of activity when compared to the activity elicited by Gli-only transfected cells; statistical significance is correlative to a calculated p-value that is less than 0.05.
As in the previous two experiments, T131L significantly enhances the activity of Gli1 (p=0.006).
Unlike the overexpressed T131L cells, the Gps1 and LOC67513 transfected samples elicited a lower activation than the Gli1 only transfected cells. Of the two, only the overexpressed
LOC67513 cells were calculated as significantly activating the reporter, however it was significant in the respect that it elicited a lower activation than Gli1 only transfected cells. It had a 17 fold activation (p=0.013) in comparison to the 23 fold activation of Gli1 only expressed cells. Due to the higher relative activation, T131L could have a larger role in directly promoting the activation of Gli1 in the mammalian Hh pathway than LOC67513. LOC67513 also could be more sensitive to the presence of endogenous Sufu resulting in a lower relative activation. 31 We then wanted to measure the relative activity of T131L, Gps1 and LOC67513 in wild- type Mef cells with overexpressed Sufu. The results of this assay were as follows in figure 3-7.
Relative Activity in WT MEF cells 16 * 14 * 12 10
8 relative activity 6 4 * *
2 Relative ReporterActivity 0
Gli1 - + + + + + + + Sufu - - + + + + - - T131L - - - + - - + - GPSI - - - - + - - + LOC - - - - - + - -
Figure 3-7. The measured reporter activity in wild-type Mef cells. The pluses represent the plasmids that were transfected in each column’s samples. The ‘*’ represents statistically significant differences in levels of activity when compared to the activity elicited by Gli/Sufu transfected samples (if comparing sample that also is transfected with Sufu) or by Gli-only transfected samples (if comparing sample does not contain transfected Sufu); statistical significance is correlative to a calculated p-value that is less than 0.05. The two columns on the right were transfected with 0.4 µg of plasmid, double the amount of plasmid normally transfected.
As seen in figure 3-7, overexpression of Sufu significantly inhibited Gli1 activity. Co-expression of T131L (p=0.02) and Gps1 (p=0.001), respectively, led to slightly higher activity than the activity measured in cells co-expressing Gli1 and Sufu. LOC67513 overexpressed cell samples had almost identical measured relative activity in comparison to Gli1 and Sufu overexpressed cells. These results are similar to those from the Sufu -/- Mef cell reporter assay that involved overexpression of Sufu. Like that assay, these results suggest that both T131L and Gps1 could 32 have a role in either releasing the inhibition function of Sufu and/or directly promoting the activation of Gli1.
The final two samples that were measured in this assay support the latter hypothesis.
These cells were transfected without overexpression of Sufu, but with double the amount of plasmid that was transfected in the assay in figure 3-6. The overexpressed T131L cells show a relative 13 fold activation when co-transfected with Gli1 (p=0.002) and the overexpressed Gps1 cells show a relative 14 fold activation when co-transfected with Gli1 (p=0.001); this is in comparison to the Gli1 only expressed cells that showed a 6 fold activation. These results are significant in T131L’s case because for the first time the relative activity measured by the reporter in T131L transfected cells more than doubled the activity measured in Gli1 only transfected cells. This indicates that the amount of T131L present in a cell may affect the relative activity of Gli1. The activity measured by the reporter in Gps1 and Gli1 co-expressed cells in this assay shows different results than those in figure 3-6. Instead of having no significant activity measured by the reporter as in figure 3-6, there was double the activation of Gli1 in this assay when double the amount of Gps1 was transfected. This could indicate that Gps1 competes with endogenous Sufu’s inhibition activity and is sensitive to its presence, but when expressed at very high levels, outcompetes Sufu and promotes Hh signaling. Overall, these results support the idea that T131L and Gps1 may have a role in promoting the activation of Gli1 in the mammalian Hh pathway.
3.6 T131L activates Gli-dependent reporter in dose-dependent manner
Because cells co-expressed with T131L and Gli1 consistently activated the reporter in both Sufu -/- Mef cells and wild-type Mef cells, alluding to consistent activation of Hh signaling, 33 the final luciferase reporter assay tested whether different doses of T131L affected the relative activity of a mammalian cell, and therefore Hh signaling. This reporter assay was performed utilizing wild-type Mef cells and the results are shown below in figure 3-8.
Relative Activity in WT MEF cells 10.00 * 8.00
6.00 *
relative activity 4.00
2.00
Relative ReporterActivity 0.00
Gli1 - + - - + + T131L (0.2µg) - - + - + - T131L (0.4µg) - - - + - +
Figure 3-8. The measured reporter activity comparing in WT Mef cells comparing two different doses of T13L. 1x T131L is associated with the transfection of 0.2 micrograms of T131L and 2x T131L is associated with the transfection of 0.4 micrograms of T131L. The pluses represent the plasmids that were transfected in each column’s samples. The ‘*’ represents statistically significant differences in levels of activity when compared to the activity elicited by Gli-only transfected cells; statistical significance is correlative to a calculated p-value that is less than 0.05.
The results show that there is significant reporter activation of both doses of T131L when co- expressed with Gli1. The non-activation measured in the T131L only overexpressed cells ensures that T131L does not activate the Gli-dependent reporter on its own, which would discredit our luciferase reporter assay experiments. Gli1 co-expressed with a single dose of T131L shows over a 5 fold activation with a calculated p-value of 0.005 when compared to the activation measured in Gli1 only transfected cells; the double dose of T131L co-expressed with Gli1 shows about an 8 34 fold activation and a calculated p-value of about 0.035. These results signify that T131L has a dose-dependent role in Gli1 activation.
35 4. Discussion
Hh signaling is critical to the development of mammals. Gli transcription factors mediate all transcriptional responses to Hh (Berbari et al, 2009). Sufu is involved in the inhibition of Gli and therefore the repression of Hh signaling (Van den Brink and Gijs, 2007). It was the desire of discovering novel information about this enigmatic protein that drove our research. Specifically, we wanted to discover new proteins that interact with Sufu in mammalian cells and that have a role in its functioning.
The results of the yeast-two hybrid screen, the co-immunoprecipitation assays and the luciferase reporter assays adduced that we were successful in finding interactions with T131L,
GPS1 and LOC67513 in mammalian cells. On the contrary, although the yeast-two hybrid screen detected a preliminary interaction between Sufu and RanBP9, it was determined to be a false- positive through the partial construct Co-IP experiments (fig. 3-1).
The Co-IP experiments in mammalian cells were instrumental in our research. They not only confirmed the interaction between partial proteins and Sufu, but also informed us that the interaction occurred with the full-length proteins as well. This is important due to the fact that full-length proteins are more restrictive in their interactions and localization. Full-length T131L,
Gps1 and LOC67513 all interacted with Sufu supporting previously reported data that they all naturally localize in either the nucleus or cytoplasm where Sufu functions (Mingot et al, 2004;
U.S. National Library of Medicine, 2013; Wu et al, 2011). The Co-IPs performed using the truncations of Sufu also provided us with useful findings. It has been previously reported that
Sufu contains two very prominent terminal domains, both of which interact with Gli during cytoplasmic tethering (Merchant et al, 2004). Our results showed that T131L, Gps1 and
LOC67513 interact with both Sufu domains. This indicates that each of the proteins has more than one interaction interface with Sufu. 36 After learning of these protein interactions, the next goal was to decipher what possible role they had in Sufu functioning and Hh signaling. The luciferase reporter assays provided us with much information. First, we discovered that T131L, GPS1 and LOC67513 all activate
Hedgehog signaling in Sufu -/- Mef cells (figs. 3-4, 3-5). Because endogenous Sufu was not present in these cells and no additional Sufu was transfected (fig. 3-4), it could be deduced that these proteins not only interact with Sufu, but also have a function in directly promoting Gli activation and thus activating mammalian Hh signaling. This hypothesis could also be considered when looking at the results of the Sufu -/- Mef reporter assay with overexpressed Sufu (fig. 3-5).
When the three proteins were overexpressed with both Gli and Sufu, the reporter was significantly activated, however not nearly as much as when Sufu was not present. This could not only mean that the three proteins interact with Gli and directly activate the pathway in Sufu -/-
Mef cells, but it could also imply that they directly interact with Sufu and by doing so, release the inhibition function of it. The possibility of the proteins interacting with both Gli1 and Sufu is reasonable due to the fact that Gli1 and Sufu are in the same complex; Gli1, Sufu and the new proteins would therefore be in the same general vicinity and able to interact with each other.
Working with wild-type (WT) Mef cells allowed us to see the role that endogenous Sufu has in the functioning of the three proteins in Hh signaling. We found that T131L significantly activates Hh signaling in WT Mef cells in the presence and absence of overexpressed Sufu (figs.
3-6, 3-7, and 3-8). The lack of significant results for Gps1 and Loc67513 in reporter assays presented in figure 3-6 and 3-7 makes the function of the two proteins in mammalian wild-type cells less clear. Gps1 showed significant results when overexpressed Sufu was present, however did not show significance when it was not; LOC67513 had the opposite results. When the amount of Gps1 was doubled in the absence of over-expressed Sufu, however, there was significant activation (fig. 3-7). This could indicate that Gps1 competes with endogenous Sufu and only at high levels can outcompete its functioning and significantly promote Gli activation. 37 Overall, unlike T131L which shows unmistakable activation in both Sufu -/- Mef and WT cells, the GPS1 and LOC67513’s function is not as clear. Any discrepancy in significant results could be due to experimental error. Transfection efficiency was low during the experimental process, and although the Renilla reporter was used as a control, transfection would need to occur in enough cells to get conclusive results. Furthermore, many cells may have died before harvesting the transfected cells which could also skew the results and cause bigger error bars. Despite these discrepancies, the reporter assay experiments show that T131L has an overall function in promoting Hh signaling in mammalian cells through the activation of Gli or through binding to
Sufu, and in doing so, releasing the inhibition of Gli.
Not only did we find that T131L consistently activates the Gli-dependent reporter in Sufu
-/- Mef cells and WT Mef cells, but does so in a dose-dependent manner. This means that different doses of the protein have different effects on Hh signaling. In T131L’s case, there was significantly higher activation measured when the amount of T131L expressed in wild-type mammalian cells was doubled (fig. 3-8). Therefore mammalian Hh signaling occurs at a higher rate when T131L is increased in vitro. This could have multiple implications. Hh signaling is responsible for the development of body areas in specific patterns. Therefore, the amount of
T131L expressed in a cell at any given time could have some role as to how much Hh signaling occurs for certain patterning events.
Now that interactions have been confirmed, there are many additional questions that we would be interested in answering. For instance, we are interested in whether these protein interactions are necessary for Sufu functioning and Hh signaling or if they only occurred because we happened to overexpress these proteins together. We also want to confirm the specific function that these proteins have within mammalian cells and signaling; the reporter assays implied possibilities, but the specifics are still unknown. Other functional analyses, such as
RNAi knockdown, could be performed. Experiments such as these could show us whether 38 T131L, Gps1 and LOC67513 are required for Hh signaling. These knockdown experiments are beginning to be researched in our lab.
Overall, our experiments and results may serve as a framework for future students to further investigate Sufu interacting proteins. It is clear that these interactions do occur and may have a large impact on Hh signaling and mammalian development. Learning about how Sufu specifically functions in this pathway could open many doors and answer many important questions about how we as humans grow and develop. Just taking small experimental steps such as we have could eventually provide some insight on how certain human diseases develop and progress, especially cancers that are so problematic in our society.
39 Appendix
Final conc 10 l (test PCR) 50 µl (real PCR) 5x HF buffer 1x 2 l 10 l 10mM dNTP 0.2mM 0.2 l 1 l Primer A (5 M) 0.5 M 1 l 5 l Primer B (5M) 0.5 M 1 l 5 l template 10 ng? 1 l 2 l H2O 4.7 l 26.5 l (DMSO) Phusion 0.02U/l 0.1 l 0.5 l Table 5-1. Phusion PCR reaction set-up
Recipe Stock For 10ml 0.35M 0.5M Tris, pH6.8 7ml 0.35M SDS 1g 30% Glycerol 3ml 0.6M DTT 0.93g 0.175mM Powder 1.2mg Bromophenol blue
*1ml aliquot stored at -20C Table 5-2. Recipe for 6x loading buffer
Culture vessel Relative area DNA/total Vol Lipo/total Vol Medium 24 well 1 0.8ug/50ul 2.0ul/50ul 500ul 12 well 2 1.6ug/100ul 4.0/100ul 1ml 35 mm 5 4.0ug/250ul 10/250ul 2ml 60 mm 10 8.0ug/0.5ml 20/0.5ml 5ml 100 mm 30 24ug/1.5ml 60/1.5ml 15ml Table 5-3. Transfection set-up.
40 Recipe Stock For 500ml 1% Triton X-100 5ml 150mM NaCl 5M 15ml 20mM Hepes pH7.5 1M 10ml 10% Glycerol 1M 50ml 1mMEDTA 0.5M 1ml H2O 419 Table 5-4. Recipe for 1% Triton lysis buffer
% Acrylamide 5 6 7 7.5 8 9 10 11 12
30% Acrylamide 0.833 1.0 1.165 1.25 1.335 1.5 1.665 1.835 2.0 1.5 M Tris pH 8.8 1.265 1.265 1.265 1.265 1.265 1.265 1.265 1.265 1.265 dH2O 2.822 2.655 2.49 2.405 2.32 2.155 1.99 1.82 1.655 20% SDS 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025
10% APS (1: 100) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 TEMED (1: 1000) 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 Table 5-5. Recipe for 5ml SDS Separating Gel (in ml)
% Acrylamide 5 6 7 7.5 8 9 10 11 12
30% Acrylamide 0.414 0.5 0.575 0.625 0.675 0.75 0.825 0.925 1.0 0.5 M Tris pH 6.8 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 dH2O 3.256 3.17 3.1 3.05 3.00 2.92 2.85 2.75 2.67 20% SDS 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul
10% APS (1: 100) 50 ul 50 ul 50 ul 50 ul 50 ul 50 ul 50 ul 50 ul 50 ul TEMED (1: 1000) 5 ul 5 ul 5 ul 5 ul 5 ul 5 ul 5 ul 5 ul 5 ul Table 5-6. Recipe for 2.5ml SDS Stacking Gel (in ml, unless otherwise noted) 41
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ACADEMIC VITA Emily Valerio 520 E. Calder Way Apt. 416 State College, PA 16801 [email protected] ______Education
The Pennsylvania State University, University Park, PA (Class of 2013) Eberly College of Science Schreyer Honors College B.S. Candidate in Biology: Genetics and Developmental Biology Option Expected Graduation: May 2013
Archbishop John Carroll High School (Class of 2009) Valedictorian
Research Experience
Undergraduate Research, Liu Developmental Biology Lab, Fall 2011 to present Researched/performed a variety of experiments regarding the interactions between proteins and components of Hedgehog signaling pathway Utilized and amalgamation of lab equipment and performed wet-lab techniques such as DNA cloning, agarose gel imaging, luciferase reporter assays, Western blots and co-immunoprecipitation assays Participated/presented in lab meetings and undergraduate research poster exhibition
Honors/Awards
Dean’s List, every semester fall 2010 to present University Libraries Award for Information Literacy, 2nd Place (Undergraduate Research Exhibition) – April 2012
Activities Teaching Assistant, Biology Department, January 2012 to present SHOtime Mentor, 2011 to 2012 THON 2011 OPPerations Committee Member Member of Springfield THON, 2009 to present Intramural Co-Ed Flag Football, 2009-2010