Thyroid Hormone Receptor SS

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2017 Thyroid Hormone Receptor SS (trß) Regulation Of Runt-Related Transcription Factor 2 (runx2) In Thyroid Tumorigenesis: Determination Of The Trß Nuclear Protein Complexes That Associate With The Runx2 Gene. Thomas Howland Taber University of Vermont

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Recommended Citation Taber, Thomas Howland, "Thyroid Hormone Receptor SS (trß) Regulation Of Runt-Related Transcription Factor 2 (runx2) In Thyroid Tumorigenesis: Determination Of The rT ß Nuclear Protein Complexes That Associate With The Runx2 Gene." (2017). Graduate College Dissertations and Theses. 820. https://scholarworks.uvm.edu/graddis/820

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THYROID HORMONE RECEPTOR ß (TRß) REGULATION OF RUNT-RELATED TRANSCRIPTION FACTOR 2 (RUNX2) IN THYROID TUMORIGENESIS: DETERMINATION OF THE TRß NUCLEAR PROTEIN COMPLEXES THAT ASSOCIATE WITH THE RUNX2 GENE.

A Thesis Presented

Thomas Howland Taber

The Faculty of the Graduate College

The University of Vermont

In Partial Fulfillment of the Requirements for the Degree of Master of Science Specializing in Pharmacology

October, 2017

Defense Date: August 15th, 2017 Thesis Examination Committee:

Frances E. Carr, Ph.D., Advisor Jeanne Harris, Ph.D., Chairperson George Wellman, Ph.D. Karen M. Lounsbury, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College

ABSTRACT

Thyroid Tumorigenesis is typically a well understood process, with well delineated oncogenic factors. Follicular and papillary thyroid cancers are typically survivable, with 5- year survival rates being >95% for Stage I-III of both cancer types. Anaplastic thyroid cancer, in contrast, lacks this prognosis, and is the most lethal of all endocrine-related cancers. The median survival time after a diagnosis is generally between 6-8 months, with a 5-year survival rate of <10%. Current treatment for anaplastic thyroid cancers routinely meet roadblocks, as resistance is quickly developed. Even non-discriminatory kinase inactivators, such as sorafenib, which are generally considered a drug of last resort, are unable to effect survival rates. As such, there is a clear need for further investigation of the causes of anaplastic thyroid cancer mechanisms. Previous work in the Carr lab revealed a novel regulatory pathway of an oncogene that is associated with several other endocrine-related cancers, as well as other non- endocrine-related cancers. Specifically, the Runt-related transcription factor 2 (Runx2) was found to be suppressed via direct binding of the thyroid hormone receptor beta 1 isoform (TRß1) to its proximal promotor. Runx2 was previously shown to be associated with increasing malignancy, with Runx2 occurring at low-levels in indolent cell lines, whilst occurring at high-levels in more malignant cell lines. TRß1, conversely, exhibited the opposite relationship. Endogenous levels of TRß1 were found to be high in indolent cell lines and were depleted in malignant cell lines. These findings were further confirmed via tissue microarrays. Restoration of TRß1 in malignant cell lines diminished Runx2 mRNA and protein levels, which was corroborated by evidence from electrophoretic mobility-shift assays, and chromatin immunoprecipitations that TRß1 was able to directly bind Runx2 promotor 1. Current studies have investigated the nuclear protein profile that associates with TRß1 to alter Runx2 transcription. Through EMSA-to-Mass Spectrometry methodologies, as well as novel DNA pulldown techniques, binding partners have been elucidated. Findings have also been confirmed via classical immunoprecipitations. Specifically, our findings show that TRß1 complexes with the brahma-related gene 1 (BRG1) protein, the nuclear co-repressor (NCOR), and BRG1-associated protein 60 (BAF60). BRG1 functions by preferentially recruiting histone deacetylases (HDAC), with BRG1 and the HDAC’s acting to alter chromatin, and thus transcription. Future studies aim at examining whether other proteins complex with TRß1 to alter Runx2 transcription, and whether these complexes are altered in aggressive cell lines.

ACKNOWLEDGEMENTS

I would like to first acknowledge my mentor of the past three years, Dr. Frances E.

Carr. As challenging of a student as I may have been, Dr. Frances E. Carr has been an incredibly effective and thoughtful mentor to me throughout my studies at the University of Vermont. Not only has she shown a willingness to accommodate whatever issues I may be going through in life, she has always treated me with the highest respect and dignity.

Dr. Frances E. Carr truly serves as an archetype of the definition of what a mentor is. Dr.

Frances E. Carr truly embodies the selflessness and dedication that academia represents.

I would also like to acknowledge all the members of the Carr lab during my tenure as both an undergraduate and graduate student at the University of Vermont. Jeffery White,

Jennifer Tomczak, Michael Barnum, Noelle Gillis, Eric Bolf, Katherine Amidon and

Caitlin Beaudet have all been excellent colleagues and all strive to be as helpful as possible, whenever possible. Even though I have been the definition of a train wreck, they have all provided excellent conversation, advice and are truly skilled at denigrating me in the most humorous of manners.

I should also acknowledge my family, as I wouldn’t exist without them - literally.

Thanks mom and dad. Humor aside, I don’t think I could have made it through the past two years without your love, dedication and support. You guys are truly the real MVP’s.

ii TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ii CHAPTER 1: COMPREHENSIVE LITERATURE REVIEW ...... 1 1.1 Introduction ...... 1 1.2 Thyroid Cancer ...... 1 1.2.1 Thyroid Cancer Classifications and Statistics ...... 1 1.2.2 Pathways Associated with Thyroid Tumorigenesis: RAS/PTEN ...... 3 1.2.3 Pathways Associated with Thyroid Tumorigenesis: EGFR ...... 5 1.3 Runt-related transcription factor 2 (Runx2)’s role in thyroid tumorigenesis ...... 8 1.3.1 Runx2’s role in skeletal development and maintenance ...... 8 1.3.2 Runx2’s role in tumorigenesis ...... 10 1.3.3 Runx2’s emerging impact in thyroid tumorigenesis ...... 11 1.4 Thyroid Hormone Receptor ß Isoform 1’s role as a tumor suppressor ...... 14 1.4.1 Structure and function ...... 14 1.4.2 Isoforms and generalized actions ...... 16 1.4.3 Actions of cognate ligand: T3 and T4 ...... 18 1.4.4 TRß1 as a tumor suppressor ...... 20 1.4 Ancillary Effectors ...... 21 1.4.1 Nuclear co-repressors ...... 21 1.4.2 Nuclear co-activators ...... 23 1.4.3 Brahma-related gene 1 ...... 24 1.4.4 Histone deacetylases ...... 25 CHAPTER 2: MATERIALS AND METHODS ...... 28 2.1 Research Goals ...... 28 2.2 Experimental Protocols ...... 29 2.2.1 Cell Culture and Nuclear Protein Extraction ...... 29 2.2.2 Oligonucleotide Biotinylation ...... 29 2.2.3 Electrophoretic Mobility Shift Assays (EMSA’s) ...... 31 2.2.4 Immunoblot ...... 32 2.2.5 Immunoprecipitation ...... 33 2.2.6 DNA Pulldown Assay ...... 33 2.2.7 DNAse I Hypersensitivity Assay ...... 34 2.2.8 Sample Preparation for Mass Spectrometry Analysis ...... 35 2.2.9 Analysis of Mass Spectrometry Results ...... 36 CHAPTER 3: RESULTS ...... 38 3.1 Experiment 1: Electrophoretic Mobility Shift Assays (EMSA’s) ...... 38 3.2 Experiment 2: DNA Pulldowns ...... 40 3.3 Experiment 3: Immunoprecipitations ...... 44 3.4 Experiment 4: Promotor Accessibility (DNAse I Hypersensitivity) ...... 48 3.5 Experiment 5: Mass Spectrometry ...... 50 CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS ...... 56 CHAPTER 5: BIBLIOGRAPHY ...... 62

iii

APPENDICIES ...... 74

LIST OF TABLES

Table ...... Page Chapter 3 Filtered protein list from mass spectrometry analysis of DNA pulldown ...... 52 Appendices Table 2.1: Mass Spectrometry Results from No Oligo Control reaction ...... 74 Table 2.2: Mass Spectrometry Results from RUNX2A reaction ...... 80 Table 2.3: Mass Spectrometry Results from 10x Competition reaction ...... 93

LIST OF FIGURES

Figure ...... Page Chapter 1 Figure 1.1 RAS-dependent thyroid tumorigenesis pathway ...... 5 Figure 1.2 EGFR signal transduction is integrated PI3K/AKT and MAPK ...... 8 Figure 1.3 Model of Bone cell development ...... 10 Figure 1.4 Domain schematic of nuclear hormone receptors detailing DNA and ligand binding domains ...... 16 Figure 1.5 Schematic of canonical histone deacetylation via NCOR1-Sin3 binding ....23 Chapter 2 Figure 2.1 Runx2 P1 promotor diagram and oligonucleotide schematic ...... 31 Figure 2.2 DNA Pulldown Schematic ...... 34 Chapter 3 Figure 3.1 EMSA’s illustrating functional TRß1 binding to the three identified TRE’s proximal to the Runx2 P1 promotor ...... 39 Figure 3.2 Dot blot analysis of oligonucleotide binding to beads ...... 41 Figure 3.3 DNA Pulldown using Runx2A and Nthy-ORI nuclear extract ...... 43 Figure 3.4 DNA Pulldown identifying major regulatory proteins that natively bind Runx2A ...... 44 Figure 3.5 Bidirectional immunoprecipitation of BRG1 and TRß1 ...... 46 Figure 3.6 Dot blot analysis of probe binding in in TRß1 immunoprecipitation ...... 47 Figure 3.7 Runx2 promotor accessibility in Nthy-ORI and SW1736 cell lines ...... 49 Figure 3.8 DNAse I hypersensitivity assay of the Runx2 promotor in Nthy-ORI cells using an anti-BRG1 drug PF1-3 ...... 50 Figure 3.9 SDS-PAGE image of DNA pulldown samples analyzed via mass spectrometry ...... 53 Figure 3.10 Interaction map of selected proteins identified via mass spectrometry ...... 54 Figure 3.11 Interaction map of selected proteins that could putatively form a multiprotein complex with TRß1 ...... 55 Chapter 4 Figure 4.1 Molecular switch diagram detailing possible protein complexes that could regulate Runx2 P1 ...... 61

v CHAPTER I: COMPREHENSIVE LITERATURE REVIEW 1.1 Introduction

The mechanisms that underlie the progression of indolent cancers to aggressive and metastatic cancers are typically well studied and are thought to be well understood.

However, current mechanisms and models are either insufficient or cannot be effectively translated into markers or prognostic indicators in the context of thyroid cancer. Survival rates for stage 1-3 thyroid carcinomas are generally excellent, yet stage 4, anaplastic thyroid cancer is the deadliest endocrine system cancer. As such, there exists a clear deficit between the comprehension and understanding of thyroid cancer and its tumorigenic mechanisms and the eventual prognosis of an individual’s specific cancer. Current information, however, suggests that epigenetic events are a primary driver of tumorigenesis and disease state progression in thyroid cancer. Furthermore, histone deacetylases, histone acetylases, DNA methyl transferases, and chromatin ATPases are increasingly being explored as potential therapeutic targets in the emerging field of precision medicine.

1.2 Thyroid Cancer

1.2.1 Thyroid Cancer Classifications and Statistics

Thyroid cancer can be classified into the following four subtypes: follicular, papillary, anaplastic and medullary. Papillary, follicular and anaplastic thyroid cancers (PTC, FTC,

ATC) all arise from the follicular tissue of the thyroid and are diagnosed based on cell morphology. Specifically, PTC is defined as exhibiting nuclear enlargement, crowding and grooves and exhibiting Orphan Annie Eye nuclear inclusions1. FTC is a well differentiated cancer type that most closely resembles normal follicular thyroid tissue ATC represents

1 the most dedifferentiated cell type, and thus lacks normal follicular cell characteristics.

Medullary thyroid cancer (MTC), in contrast, develops from the parafollicular c-cells of the thyroid that are chiefly involved in the production calcitonin2. MTC is well understood in terms of mechanism and is definitively linked to germline mutations. Specifically owing to aberrations and mutations in the RET proto-oncogene, MTC is almost always a familial disease3. Overall, survival outcomes are generally favorable, especially when accounting for its 10-year survival rate of 70% for its stage IV form4.

Unfortunately, the non-familial forms of thyroid cancer (PTC, FTC and FTC) diverge from the prognosis that a diagnosis of MTC typically enjoys. These cancers, whilst distinctly different, represent a spectrum of thyroid cancer, with ATC being considered the last stage. Although prognosis is generally favorable for lower stage PTC and FTC, ATC represents the deadliest of all endocrine system cancers. PTC and FTC 5-year survival statistics are typically >95%. Upon transition to ATC, however, the 5-year survival statistics plummets to <10%. In fact, the median survival time for ATC is approximately

6-months5. The aggressiveness of ATC has led to the automatic classification of ATC as

American Joint Committee on Cancer (AJCC) TNM system as stage IV6. Metastasis of the primary ATC is also observed in about half of patients at the time of diagnosis, with a large majority developing metastatic tumors despite treatment. Preferred areas of metastasis are primarily bone, lung and brain7. The preferences for these sites are ultimately believed to contribute to the deadliness of ATC; metastasis to lung is marked by multiple tumors preventing clinicians from effectively resecting the tumors or using targeted radiation.

Further complicating treatment approaches is the fact that patients with lung metastases

2 suffer from pulmonary hemorrhage that demands immediate intervention, potentially delaying treatment6.

Resistance to normally employed chemotherapeutics has been identified as a primary mechanism behind the lethality of ATC. Doxorubicin, the mainstay of ATC treatment, rapidly loses effectiveness as ATC gains resistance to the drug8. Experimental treatments that use histone-deacetylase inhibitors (HDACi) have also been controversial in the treatment of ATC. Current studies suggest that HDACi can have potent synergistic effects leading to apoptosis in cell culture experiments, however, results are highly dependent on cell line, intimating that ATC also exhibits great variability in molecular pathology9.

Generating further concern for a diagnosis is the rapid development of resistance towards sorafenib, a newer pharmaceutical agent that indiscriminately targets a broad range of receptor tyrosine kinases10. Overall, the resistance of ATC to a broad range of medications, including medications of last resort, prompts a growing concern over how best to treat and care for ATC. Furthermore, these facets make a compelling case for the identification of new pathways involved in the development of ATC.

1.2.2 Pathways Associated with Thyroid Tumorigenesis: RAS/PTEN

Although there clearly exists a deficit of both effective treatment and of a general understanding of ATC, there exists a plethora of data concerning the genesis of FTC and

PTC in early stages. Beginning with FTC, tumorigenesis is most often associated with alterations to RAS, PTEN, and PIK3CA – all of which alter the normal function of the

PI3K-AKT signaling system11,12. RAS normally functions as a kinase that activates both the MAPK pathway, as well as the PI3K-AKT pathway, which both lead to the downstream

3 activation of proto-oncogenic factors13,14. Specifically, RAS is a kinase that contains a self- terminating GTPase – conversion of GTP to GDP locks RAS into an inactive state that is ultimately unable to phosphorylate, and thus activate, downstream effectors. PTEN feeds into this pathway as well, and is generally understood to act as a counter to aberrations in

RAS function15. Knockout of PTEN leads to an amplified RAS effect. This mechanism is furthered by the fact that RAS mutation alone is typically not sufficient to transform cells to a malignant form; coincident PTEN inactivation or deletion is necessary. Furthermore, the most recent studies concerning RAS as a prognostic factor have concluded that RAS aberrations are not enough to inform a clinician as to whether thyroidectomy is necessary16.

Figure 1.1: RAS-dependent thyroid tumorigenesis pathway. RAS activates

Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and thus protein kinase B

(AKT) to induce proto-oncogenic genes, whilst phosphatase and tensin homolog

(PTEN) counters and suppresses tumorigenesis12.

1.2.3 Pathways Associated with Thyroid Tumorigenesis: EGFR

Epithelial growth factor receptor (EGFR), in contrast to PTEN/RAS and

AKT/PI3K, represents a better clinical prognostic factor, and is closely correlated to thyroid tumor stage. Furthermore, aberrant expression of EGFR is almost universally seen 5 in ATC and is statistically correlated to the probability of a cancer’s propensity for metastasis15. The action of EGFR is partly carried out via the RAS pathway as a downstream effector, and therefore represents another avenue by which proto-oncogenic factors become activated via the RAS-MAPK pathway. Evidence also suggests concurrent activation of PI3K/AKT pathways12.

EGFR essential action is that of a receptor tyrosine kinase (RTK). Introduction of epithelial growth factor (ligand) in the extracellular environment leads to the activation of

EGFR, which undergoes a conformational change that induces downstream effectors17.

Specifically, epithelial growth factor induces the recruitment of two EGFR subunits which dimerize. Dimerization of EGFR leads to its auto-phosphorylation at numerous tyrosine residues, which in turn recruits Src-homology 2 (SH2) domain-containing proteins, which act as downstream effectors. SH2-containing proteins bind RTK at motifs exhibiting kinase activity and become phosphorylated themselves18. SH2-containg proteins, such as RAS, then dissociate from EGFR and begin a phosphorylation cascade, with MAPK and

PI3K/AKT serving as prime examples of common targets19. A generalized schematic of these mechanisms is highlighted in Figure 1.2. Typically, these signals are sequestered via ancillary phosphatases, however, they may also be self-terminated in the case of SH2- domain containing tyrosine phosphatase20.

Current studies have revealed that EGFR is the subject of numerous genetic aberrations that result in unregulated signal transductions. In-frame deletions in exon 19 that alter amino acids spanning 747-750, and point-mutations at amino acid 858 in exon 21 of the EGFR gene represent >90% of EGFR mutations in cancers21. These mutations have been shown to alter signal transduction by allowing for spontaneous, ligand-independent

6 activation, as well as resisting signal sequestration. Evidence also suggests that alterations in EGFR retards the internalization of the receptor that normally occurs with higher than normal EGFR signaling22. Such an alteration is worrisome, as it represents a loss of cellular tumor suppressive compensation.

Inhibition of EGFR through pharmacotherapies that allosterically alter EGFR at areas other than amino acids 747-750 and areas flanking amino acid 858 also have been shown to be effective in PTC and FTC23. However, current pharmacotherapies are still inadequate in the treatment of late-stage, aggressive thyroid cancers such as ATC. The most recent clinical trials that incorporate the use of EGFR inhibitors show effects similar to traditional therapies such as cisplatin, and fail to effectively increase 5-year survival rates24. As such, EGFR is likely to only play an ancillary role in the maintenance of ATC, and poorly-differentiated PTC and FTC. Compensatory mechanisms, such as RAS/PTEN aberrations could possibly serve as a redundancy in the EGFR signaling pathway.

Figure 1.2: EGFR signal transduction is integrated PI3K/AKT and MAPK. EGFR

has been shown to be a primary player in thyroid tumorigenesis25.

1.3 Runt-related transcription factor 2 (Runx2)’s role in thyroid tumorigenesis

1.3.1 Runx2’s role in skeletal development and maintenance

The Runt-related transcription factor 2 (Runx2) is a transcription factor that has been extensively described as a crucial factor in both osteogenesis and skeletal maintenance. Considered a master regulatory protein, Runx2 integrates signals from a variety of pathways such as BMP/TGFß as well as Src. Knockout of Runx2 (-/-) in mouse models results in embryonic lethality, suggesting that Runx2 is an integral transcription factor in development26. Furthermore, knockout of Runx2 (-/-) is sufficient to block osteogenesis in embryonic mouse models. However, disruption after osteoblast cell fate commitment occurs does not alter embryonic survival26,27. These combined facets suggest 8 that regulation of Runx2 is well orchestrated, and that molecular switches exist to sequester

Runx2 activity after osteoblast commitment.

Attenuation of Runx2 activity is required for the normal development of bone.

Following the differentiation of progenitor mesenchymal cells into osteoblasts and chondrocytes, Runx2 is repressed, and relegated to playing a secondary role in osteoblast maintenance28. However, Runx2 becomes re-expressed and is pivotal in chondrocyte development. Specifically, Runx2 functions to further develop chondrocytes once hypertrophy is required for skeletal development29,30. The role of Runx2 in chondrocyte maturation is further supported by the fact that overexpression of Runx2 results in skeletal malformation31. Runx2 is specifically required for the ossification of chondrocytes and acts as a switch that halts secretion of proteoglycans, as well as collagen. This process, endochondral ossification, results in the eventual mineralization of the chondrocyte tissue, thus leading to skeletal development 32.

Figure 1.3 Model of Bone cell development. Runx2 is a major gatekeeper protein in

the development of chondrocytes and osteoblasts33.

1.3.2 Runx2’s role in tumorigenesis

Runx2’s role in osteogenesis is well documented and is generally well understood, however, Runx2 is considered an oncogenic factor in many cancer types. Although required for skeletal maintenance after bone development, Runx2 has been shown to become activated in several malignant cancer types, such as breast, prostate, lung and thyroid34,35. Furthermore, emerging evidence suggests that Runx2 acts to promote a panel of pro-invasive, pro-metastatic genes in a variety of tissues36,37. Oncogenic targets include genes such as SDF-1 and BSP – targets that have been identified in prostate cancer as pro- metastatic factors that are involved in metastasis and subsequent attachment to bone38.

Downregulation of Runx2 via microRNAs (miRNAs) have further implicated Runx2 as a crucial tumorigenic factor. miRNAs function as small RNA molecules that alter mRNA

10 translation in the cytoplasm – either enhancing or blocking translation. miR-466 was specifically found in prostate cancer lines to be under-expressed, with reconstitution impairing tumorigenesis. Attenuation of miR-466 induced tumorigenic properties in normal prostate cell lines, and was found to upregulate Runx2 target genes such as osteopontin, osteocalcin and members of the matrix metallopeptidase (MMP) family39. The model of miR-466 has been further expanded to cervical cancer, where miR-466 was found to be a predictive marker of lymph node invasion in patients with cervical cancer40.

Further evidence of Runx2’s critical role as a tumor promotor is suggested via the regulation of its promotor and the gene itself via epigenetic modification. As a master development factor, Runx2 is normally regulated and highly-repressed in non-skeletal tissues. Mutation or repression of factors critical to Runx2’s regulation results in re- expression of Runx2 and a resulting cancerous phenotype34. Reintroduction of such regulatory factors results in regulatory rescue. Polycomb chormobox 4 (CBX4) is a crucial factor in this regulation. Recruitment of CBX4 to the Runx2 proximal promotor results in epigenetic silencing of Runx2. Through attachment of histone deacetylase-3 (HDAC3) to

CBX4 and its nuclear protein complex, histone compaction occurs, with resultant repression41.

1.3.3 Runx2’s emerging impact in thyroid tumorigenesis

Runx2 is a prominent tumorigenic factor in a variety of tissues as described above.

Ranging from tissues such as prostate34, breast42, colorectal carcinomas41, and bone43,

Runx2 is considered to be an accurate marker of tumor stage and grade. Increasingly,

Runx2 is being found to play a prominent role in thyroid tumorigenesis in addition to the

11 aforementioned tissues37. As previously described, thyroid tumorigenesis in early stages is a generally well-understood process. However, transition of indolent cancers (such as FTC and PTC) into their later-stage varieties, and the subsequent loss of proliferative control – a hallmark of ATC – is harder to predict and is less understood in terms of mechanism.

Runx2 is emerging as an important target to be considered in the epithelial-to- mesenchymal transition (EMT) that coincides with an increase in malignancy37. Current studies have shown that Runx2 mRNA is overexpressed in FTC and PTC and generally coincides with tumor grade44. Furthermore, Runx2 mRNA was detected at higher levels in both serum, as well as circulating, non-hematopoietic cells in thyroid cancer patients.

Tissue microarray studies have also revealed that Runx2 is highly correlated to thyroid cancer type, as well as thyroid cancer staging. Runx2 was diminished in the least malignant tumors, whilst it was elevated in the most malignant tumors45.

Runx2 targets featured in other tissue types are also extended in the thyroid tumorigenesis narrative; SDF-1 and BSP reappear, and the MMP family, as well as the vascular endothelial growth factor (VEGF) are all upregulated and induced by Runx236,46.

In the context of PTC, the inhibitor of DNA binding 1 (Id1) has emerged as a candidate that drives Runx2 re-expression in thyroid tissue. Evidence also suggests that Id1’s action in re-expressing Runx2 leads to downstream changes in a number targets that are responsible for cellular morphology and are factors involved in EMT47. By re-expressing

Runx2, Id1 promotes over expression of cadherin-6 (CDH6), a factor that contributes to the loosening of the extracellular matrix that provides epithelial cells with their rigid tissue construct48,49. More specifically, CDH6 was found to be highly concentrated at the invasive front of thyroid tumors47,50.

12 The regulation of Runx2 is also governed through epigenetic modification. Recent data suggests that HDAC6 is a major regulator of Runx2 expression, at least in PTC.

Inhibition of HDAC6 via generalized HDAC inhibitors (HDACi) resulted in the downregulation of Runx2. The putative mechanism by which HDAC6 acts to encourage transcription of Runx2 is via alteration of DNA superstructure to allow an upstream enhancer region to upregulate Runx2 at its proximal promotor. This enhancer, the so-called

ENH3 region, is in turn governed via c-Jun binding. HDACi was sufficient to ablate the regulatory function of ENH3-c-Jun resulting in a marked decrease of Runx251. HDAC6 has also been shown to complex with the Runx2 protein itself, which has been previously identified as an auto-regulator, with confirmed Runx2 response elements (RREs) found throughout its proximal promotor43,52,53. An AP1 (c-Fos/c-Jun) site was also putatively described in this region, suggesting that the c-Fos and c-Jun proteins are major regulatory features of Runx2.

Runx2 regulation, however, is not fully elucidated. Although there is compelling evidence that Runx2 autoregulation is a major factor in its expression, attenuation of the

Runx2 gene via Id1 complicates this view. Id1, as previously described governs the Runx2 genetic program, however, Id1 is unable to bind DNA54. Id1 belongs to a class of proteins known as helix-loop-helix (HLH) proteins, however, Id1 lacks a DNA binding domain like other members of this class. Therefore, the effect that Id1 exerts on the Runx2 regulatory program can only be by blocking regulatory signals from other proteins and pathways36,55.

Although current data is insufficient to properly demonstrate the mechanism by which Id1 promotes RUNX2 expression, current immunoprecipitation data suggests the interaction of transcription factor GATA4 and Id1 as a potential driver, with GATA4 acting

13 to repress RUNX256,57. This model is further extended by the fact that Id1, as well as the other two members of family – Id2 and Id3 – can act directly to sequester the activity of

GATA457. Relevance of this model is also established by the fact that thyroid tissues have elevated levels of Id1 as revealed via RNA-seq58. Expression of Id1 was found to be lowest in normal thyroid tissue, with increasing levels correlated to the aggressiveness of a tumor.

Specifically, the highest levels of Id1, as shown via immunohistochemistry, were in hyperplastic, neoplastic and anaplastic thyroid tissues59. Current models suggest that Id1 expression is upregulated via the MAPK pathway and downregulated via bone morphogenic protein (BMP) – a common osteogenic regulator that drives cell fate determination60-62. Transient knockdown of Id1, as well as Id2 and Id3, via siRNA also showed a marked decrease in cell proliferation and invasion63. However, current evidence suggests regulation Id1 via thyroid hormone – T3, 3,3',5-triiodo-L-thyronine – suggesting tight regulation via thyroid hormone dependent processes, further implicating Id1 as a driver of thyroid cancer64.

1.4 Thyroid Hormone Receptor ß Isoform 1’s role as a tumor suppressor

1.4.1 Structure and function

Thyroid hormone receptors functions as canonical nuclear hormone receptors.

Including members such as the vitamin D receptor, retinoid receptors, as well as the steroid hormone receptors and the so-called “orphan” receptors that lack a known ligand, the nuclear hormone receptor family has a highly-conserved peptide structure and overall similar mechanistic action65. These receptors bind DNA as transcription factors and

14 generally recruit either co-activators or co-repressors based on the absence or presence of ligand. Through the recruitment of co-activators and co-repressors, these nuclear receptor dependent complexes enlist additional effectors; binding of the thyroid hormone receptor beta 1 (TRß1) with the nuclear co-repressor (NCOR) further recruits HDAC3. Ultimately this complex’s action is to repress transcription based on chromatin compaction66. Addition of T3 alters the proteins that bind TR’s. TRß1, which is known to bind a variety of corepressors, will preferentially bind nuclear coactivators such as NCOA1, 2, 3, 6 in the presence of T367.

The generalized structure of the nuclear hormone receptors consists of a variable

A/B domain, a conserved C domain that functions as the DNA binding domain, a linker D domain, a conserved E domain that functions as the ligand binding domain and also functions as a dimerization domain and a variable F domain68. Within the A/B domain exists an AF-1 region that has been postulated as the driver of ligand-independent activation. An AF-2 region exists within the E domain; however, it functions as a ligand- dependent activation domain65. The existence of two active regions that are mutually exclusive, one ligand-dependent and the other ligand-independent, contributes to the dual function usually observed in the nuclear hormone receptor family. Typical views of ligand- receptor functionality rest upon the idea that ligand is necessary for receptor function; however, nuclear hormone receptor action is simply altered via ligand binding giving rise to molecular switching. Furthermore, the thyroid hormone receptors, in contrast to other nuclear hormone receptors, do not rely upon ligand for nuclear translocation. Instead, the thyroid hormone receptors rely on signaling via phosphorylation to be trafficked to the nucleus69.

Figure 1.4 Domain schematic of nuclear hormone receptors detailing DNA and

ligand binding domains65.

1.4.2 Isoforms and generalized actions

Thyroid hormone receptors (TRs) come in two different flavors: α and ß; that are transcribed from separate genes. These two classes of thyroid hormone receptors are further divided, each with two distinct isoforms. Isoform α1 and α2 are alternative splice variants that differ at the c-terminus. The α2 variant is unable to bind its cognate ligand, and is therefore unable to be modulated by ligand. TRα2 is generally understood to act as a transcriptional antagonist in the perspective of thyroid hormone receptor action70. TRα1 functions, instead as a developmental regulator. Acting in a variety of tissues ranging from brain to lung to liver, TRα1 is nearly ubiquitous in human tissues71. Its function is denoted as having a highly metabolic regulatory role; deletion studies of TRα1 (-/-) in mice clearly show embryonic survival, however, cardiac function becomes impaired. Specifically,

TRα1 deficient mice had a statistically significant decrease in endurance during running studies and in maximum running speeds. TRα1 deletion, however, does not result in

16 circadian rhythm disruptions in the context of cardiac function, suggesting a targeted role in cardiac tissue72. Furthermore, studies have revealed that the heart rate of TRα1 deficient mice are on average 20% lower and exhibit abnormal electrocardiograms. Lowered body temperature and mild hypothyroidism is observed. This ultimately indicates that TRα1 functions as a cardio and metabolic regulator73.

Besides TRα1’s role as a metabolic regulatory protein, developmental studies in

Xenopus tropicalis have revealed that TRα1 is intricately involved in development. TRα1 is expressed before thyroidal genesis and is expressed before T3 zygotic synthesis. T3 secretion most likely acts as a molecular switch for development by altering TRα1 function

74. Indeed, resistance to TRα1 (RTRα1) is associated with growth retardation, psychomotor defects, anemia, and macrocephaly75-78. Most clinical presentations of RTRα1 are associated with mutations that result in truncated sequences of the TRα1 gene; the resultant protein structure almost entirely lacks a functional ligand binding domain 78. The lack of

T3 responsive due to a nonfunctional ligand binding domain typically leads to tissue- specific hypothyroidism. Even though high T3 serum levels are detectable, the circulatory pro-hormone T4 (Thyroxine, 3,5,3',5'-tetraiodothyronine), is severely depressed in patients suffering from RTRα178.

The ß isoform of the thyroid hormone receptor family is further divided, similar to the α variants. TRß1 and TRß2, however, serve more relegated roles; TRß1 is found at its highest levels in adipose tissue, the liver, kidneys, as well as the thyroid, whilst TRß2 is found in the anterior pituitary and select neural cells70. Of note, a third ß-isoform exists,

TRß3, however, it has only been established in rats79. Deletion of both TRß1 and TRß2 leads to alterations in the hypothalamus-pituitary-thyroid axis; goiter manifests, both T3

17 and T4 are elevated, as is thyrotropin80. Deafness is experienced in TRß-/- specimens, and the presentation of TRß-/- specimens mimic that of the most extreme examples of resistance to thyroid hormone syndrome81. Knockout of both TRα1/2 and TRß1/2 result in a combination of the aforementioned phenotypes, whilst also additionally conferring infertility to female offspring81.

1.4.3 Actions of cognate ligand: T3 and T4 effects

The majority of the nuclear hormone receptor superfamily have known cognate ligands and have been shown to exhibit altered structure when bound82. The thyroid hormone receptor family (TR’s), except for TRα2, have been demonstrated to exhibit this molecular switching. Given the effects of both hypo and hyperthyroidism, this provides the most succinct reasoning as to the effects of T3/T4 binding to the TR’s83. Indeed, the nuclear protein assembly that forms, as directed by TR’s, radically changes upon binding of T3/T4, transforming from a loose globular structure, to a highly ordered structure70. Binding of cognate ligand to the TR’s induces structural changes to reveal an AF-2 domain located in the E or ligand binding domain65. Without ligand, the AF-1 is the dominant controller of co-regulator binding and is defined as the amino acid regions 69-89 in the TRß1 protein

(A/B region, the DNA binding domain)84. Current evidence does not support an obfuscation of this domain upon ligand binding; instead it is thought that the AF-2 region, when functionally active, acts as another regulatory layer in the context of co-regulator recruitment85. Adding a further layer of regulation is the fact that binding of co-regulator, and thus the effects of TR binding, are governed by the nuclear protein environment which is ultimately dependent on tissue and cell type84.

18 Binding of cognate ligand induces a conformational change in the ligand binding domain or the E region of TR’s, and acts to reveal a functional AF-2 region. Specifically, helix 12 of the E region is amphipathic with an α-helical structure, and is unable to participate in co-regulator binding due to allosteric repulsion by a CORNR motif contained within helix 1. Binding of cognate ligand induces a structural change that prevents interference via this CORNR motif, allowing for helix 12 to bind and dock in the coactivator groove. This binding is associated with the unveiling of an LxxLL motif that allows for coregulator binding70,86,87. Typical mechanisms associate the AF-1 domain as the essential mediator of gene repression, whilst the AF-2 domain is understood to mediate gene activation. Current models suggest that the TR’s preferentially recruit such mediator proteins as NCOR, as well as SMRT. In turn, NCOR and SMRT act to recruit catalytic effectors; TR’s mediate transcriptional repression via the indirect recruitment of HDAC’s

(3, 5, 7), or via adaptor proteins such as Sin3, which in turn recruit separate HDAC’s (1,

2)65,70. These HDAC’s work to deacetylate histones at regions proximal to the promotor of target genes to induce chromatin compaction, and thus repress transcription.

Binding of cognate ligand to the TR’s, however, induces a conformational change, thereby destabilizing any protein complexes such as TR-NCOR1-Sin3-HDAC1. In turn, the AF-2 region preferentially recruits and binds drivers of transcription; the most classic complex being that of TRAP (thyroid receptor associated proteins), a TR specific Mediator complex70. Mediator complexes, such as that of TRAP, bind to activated transcription factors, such as TR, to recruit proteins necessary for transcription, such as topoisomerase, and eventually recruit RNA Polymerase II88. The TRAP/Mediator complex is most often described as being homologous to the activator complex found in yeast89. Indeed,

19 evolutionary studies posit that Mediator complexes such as TRAP are almost ubiquitous and universal in eukaryotes90,91. Composed of 25 unique subunits, the TRAP/Mediator complex includes members such as the TATA box binding protein (TBP)- factors in

TFIID92, as well as positive cofactors PC1, 2, 3, 4 and 5293,94.

1.4.4 TRß1 as a tumor suppressor

Although the delineation of the nuclear hormone receptors as either tumor suppressive or tumor promoting is inexact due to it being a binary classification, most information suggests that TRß1 functions mainly as a tumor suppressor45,95-98. This paradigm of tumor suppressive actions by TRß1 extends beyond the thyroid narrative45,99; information suggests a tumor suppressive role for TRß1 in the context of breast100, liver96, kidney98, and pancreas, amongst others83. Furthermore, aberrations in TRß, either through loss of expression or via mutation, is associated with the previously mentioned cancer types101-103. Reintroduction of TRß1 has been shown to induce phenotypic rescue, with marked decreases in cell proliferation and migration being observed104,105. The narrative of

TRß1 tumor suppression is also extended to prognosis; current data has associated TR immuno-positivity with increased patient survival in the context of BRCA1 mutant breast cancer. Conversely, TRα1 was negatively associated with survival, suggesting that TRß1 specifically functions as a factor critical to allaying EMT106.

Although ChIP-Seq and genome-wide binding assays have not been performed for

TRß1 in the context of cancer, regulation of proto-oncogenes by TRß1 have been shown.

Specifically, TRß1 has been shown to negatively regulate the activating protein 1 (AP1), canonically described as a heterodimerization between c-Fos and c-Jun107. AP1, itself a

20 transcription factor, is a known regulator of gene programs associated with cellular proliferation such as, but not limited to, CLCA2, BIRC3, NFATC2, MT1F and TIMP3108.

Knockdown of AP1 via siRNA has been documented to provide phenotypic rescue in a variety of cancers such as colorectal, prostate and breast109-111. TRß1-dependent regulation of AP1, however, does not adequately explain the tumor suppressive role entertained by

TRß1. Current data suggests that TRß1 exerts tumor suppressive effects via modulation of ras, BRCA1, tp53 and by transcriptional inhibition of cyclins D1, E and A2112,113.

1.5 Ancillary Effectors

1.5.1 Nuclear co-repressors

As previously described, TRß1 itself does not directly execute regulatory programs.

Instead, TRß1 acts to integrate molecular signals (cognate ligand, post-translational modifications, etc.) to recruit downstream effectors. By complexing with so-called

“ancillary effectors”, TRß1 acts to direct complex genetic events that have variety of implications in tumorigenesis. One such class of ancillary effectors that TRß1 canonically complexes with are the nuclear co-repressors. Specifically, evidence suggests that TRß1 acts to repress gene activation via the recruitment of either nuclear co-repressor 1 (NCOR1) or silencing mediator for retinoid or thyroid-hormone receptors (SMRT, NCOR2) in the presence of T3114-116. NCOR1 and SMRT contain three similar nuclear receptor interaction domains and are highly homologous. Furthermore, both NCOR1 and SMRT bind to TRß1, as well as other nuclear hormone receptors via similar mechanisms at specific residues117.

The seminal article that first identified NCOR1 as a crucial regulatory protein found that it bound TRß1 at amino acid residues 203-230 but that amino acid residues 230-260 act to

21 stabilize this interaction. This region was found to have specific homology to only one other nuclear receptor. This receptor, the vitamin D receptor, shows active binding to

NCOR1 in a ligand-independent manner similar to TRß1, suggesting a mechanism for ligand-independent repression by both receptors118. This furthers the concept that TRß1 action is highly dependent on cellular and tissue context.

NCOR1 and SMRT, however, do not possess catalytic activity themselves and are therefore unable to alter the nuclear environment. Much work has been performed to elucidate how TRß1 and the nuclear hormone receptors at large work to alter genetic programs. The current working model is that TRß1 or other nuclear hormone receptors either homodimerize or heterodimerize with RXR (or other factors) and then recruit

NCOR1 or SMRT. NCOR1 and SMRT then recruit either linker proteins or catalytic proteins directly. Canonically, NCOR1 and SMRT are understood to directly recruit and interact with Class II HDAC’s, and recruit Class I HDAC’s via linker proteins such as

Sin3a or Sin3b65,119-121.

Figure 1.5 Schematic of canonical histone deacetylation via NCOR1-Sin3 binding.

1.5.2 Nuclear co-activators

As previously described, TRß1 acts as a nuclear hormone receptor with a known ligand. Without ligand TRß1 acts to suppress gene activity; binding of ligand induces conformational changes in TRß1 leads to gene activation65,70. Although this binary model is considered canonical, emerging evidence suggests that TRß1 has a more nuanced and dynamic mechanism for gene regulation122. The dynamic nature of TRß1

23 activator/repressor further supports the idea that TRß1 action is primarily effected by the cellular- and tissue-specific context (i.e. the balance of different co-regulators). However, association of certain ancillary effectors – in contrast to NCOR1 and SMRT – are most often associated with gene activation. Indeed, the

1.5.3 Brahma-related gene 1

As described above, the actions of TRß1 are understood to arise from ancillary effectors that interact and bind with TRß1. Based upon the availability of cognate ligand –

T3 – TRß1 preferentially recruits either co-activators or co-repressors to exert its effects.

Furthermore, signal specificity is achieved by the variable distribution of proteins based off tissue type, cellular compartmentalization, as well as via the specific TRß1 response element (TRE). Although TRE’s are generally understood to be conserved throughout the genome, other response elements proximal to TRE’s lead to the preferential heterodimerization with TRß1 and are highly context dependent. Through variable heterodimerization, sequence specificity is achieved and proper epigenetic and regulatory programing is executed.

One such ancillary effector that is an emerging therapeutic target is the Bhrama- related gene 1 (BRG1, SMARCA4). Previously characterized as a member of the SWI/SNF complex, BRG1 enzymatically induces heterochromatinization via an ATP-dependent process123. Implicated as a driver of tumorigenesis, as well as a tumor suppressor, BRG1 is thought to exert its effects as an ancillary effector dependent on nuclear receptors.

Indeed, no evidence shows BRG1 directly binding to DNA, and evidence suggests that

BRG1 activity is modulated by a variety of nuclear receptor ligands, implying that its effects are nuclear receptor dependent. Also, associated with BRG1 is protein brahma

24 homologue (BRM, SMARCA2). BRG1 and BRM are typically understood to be differentially expressed and that they exist in a mutually exclusive state; BRG1 or BRM are solely expressed. Most information to date suggests that BRG1 is expressed in thyroid tissues, whilst BRM is not.

Limited evidence suggests BRG1 interaction with TRß1, however most to date have focused on BRG1 interactions with other nuclear hormone receptors such as the progesterone receptor (PR)124,125. Adding gravity to BRG1’s essential role in mediating epigenetic programs is the fact that dysregulation of BRG1 via mutation may happen in

20% of all human cancers126,127. Delineation of BRG1’s role in tumorigenesis remains murky at best; ample evidence suggests a role in promoting cancer growth, whilst ample evidence also suggests a role in suppressing cancer growth128-131. BRG1’s apparent dualistic role suggests that its function is directed by nuclear hormone receptors and other transcription factors. Supporting this notion is the fact that BRG1 does not directly bind

DNA and relies on protein-protein interactions to execute its program of chromatin remodeling.

1.5.4 Histone deacetylases

Histone deacetylases (HDACs) are a class of proteins that are generally understood to alter the nuclear microenvironment by altering histone modifications. Specifically, these proteins act to deacetylate histones and associated with histone compaction132. HDACs are divided into four distinct classes based off homology to yeast equivalents133. Furthermore,

HDACs require zinc (except for Class III) and are referred to as Zn-dependent histone deacetylases133. These proteins, by removing acetyl groups at key lysine and arginine

25 residues on histones, act as the biological antagonist to the histone acetyl transferases

(HATs) which are understood to add acetyl groups to specific lysine residues on histones134,135. By compacting chromatin, HDACs generally repress the target gene, although recent studies have revealed that HDACs participate in complex chromatin reorganization events that may reveal downstream enhancer regions and may therefore act as positive drivers of transcription136. Given the complex genetic regulation that HDACs participate in, HDAC inhibitors (generally referred to as HDACi) are being explored as potential cancer therapeutics133. Evidence is promising, with current in vitro studies showing efficacy against both hematological and solid neoplasms137. In fact, HDACi – specifically Vorinostat (suberoylanilide hydroxamic acid; SAHA) - is currently approved as a monotherapy in T-cell lymphoma138.

HDACi therapies have a potential as thyroid cancer specific pharmaceuticals.

Considerable evidence has shown that HDACs preferentially bind with the nuclear hormone receptor superfamily, with specific evidence pointing towards TRß1 acting as a major binding factor51. The notion that HDACi could be a potential therapy for thyroid cancer is corroborated by the fact that HDACi is currently undergoing clinical trials for the treatment of a variety of endocrine-related cancers such as ovarian and breast cancers.

Furthermore, romidepsin (depsipeptide) is undergoing trials for radioactive iodine (RAI)- refractory thyroid cancer139. TRß1 has been shown to participate in direct binding with

HDAC2140 and HDAC3141. Furthermore, through binding of NCOR1 to TRß1, TRß1 can act to recruit HDAC3142-145, HDAC4143,144, HDAC5143,144,146, HDAC7144,145, HDAC9147.

Through interaction between TRß1 and SMRT, several other HDACs may be recruited.

Specifically, evidence shows that SMRT can bind to: HDAC1142,148-150, HDAC2142,148,151

26 HDAC3142,144,148,150,152-154, HDAC4151,153,154, HDAC5146,151,155, HDAC7144,145,155,156 and

HDAC10149. HDACs also participate in BRG1 complexes and work in concert with BRG1 to dynamically alter chromatin structure and thus influence gene expression of targets127,142.

27 CHAPTER 2: MATERIALS AND METHODS 2.1 Research Questions

The goal of these studies was to ascertain whether TRß1 interacts with the RUNX2 P1 promotor to actively repress RUNX2 (specific aim 1). Furthermore, these studies sought to establish the nuclear protein profile that associates with TRß1 in the context of RUNX2

P1 (specific aim 2). Secondary to these two aims was that the nuclear protein profile of

RUNX2 P1 in the context of a malignant cell line (SW1736) be elucidated as to shed light on putative mechanisms involved in thyroid tumorigenesis (stretch aim).

Initially, the RUNX2 P1 promotor was analyzed via ENCODE to establish whether known thyroid hormone receptor response elements (TRE’s) existed. Electrophoretic mobility shift assays and DNA pulldowns were then employed to ascertain direct TRß1 binding to the RUNX2 promotor. Furthermore, through immunoblot and mass spectrometry RUNX2 P1 binding factors were established, with specific TRß1 binding partners being ascertained via mutational analysis. Given the discovery of multiple TRE’s throughout the RUNX2 P1 proximal promotor, it was hypothesized that TRß1 bound to these sites to exert a repressive effect. It was further hypothesized that TRß1 exerted this repressive effect via the integration of numerous signaling pathways coalescing in dominant nuclear protein complexes. Ancillary to this central hypothesis, this study sought to establish an alternative protein profile that exists in conditions of diminished nuclear

TRß1, such as in anaplastic thyroid cancer.

28 2.2 Experimental Protocols

2.2.1 Cell Culture and Nuclear Protein Extraction

The cell line Nthy-ORI, representing “normal” thyroid follicular cells and SW1736, representing anaplastic thyroid cancer, were cultured in RPMI 1640 growth media with L- glutamine (300 mg/L), sodium pyruvate and nonessential amino acids (1%)

(Cellgro/Mediatech), supplemented with 10% fetal bovine serum (Life Technologies) and penicillin-streptomycin (200 IU/L) (Cellgro/Mediatech). Cells were maintained at 37°C,

5% CO2, and 100% humidity. Nthy-ORI were obtained from European Collection of Cell

Cultures (ECACC), whilst SW1736 was generously provided by Dr. John Copland III

(Mayo Clinic). SW1736 specifically has been determined to possess mutations in BRAF,

PI3KCA and AKT1157. Cell lines were validated (8/16/13) using short tandem repeat profiles and the Promega GenePrint10 System in the Advanced Genome Technologies

Core at the University of Vermont.

Cells intended for nuclear protein harvest were grown to >90% confluency and extracted using the NE-PER protocol per Thermo Scientific. Briefly, cells were washed 2x with 4°C PBS, harvested via scraping and pelleted by centrifugation at 800xg at 4°C. Cells were then lysed per the NE-PER kit protocol, with the addition of 100X HALT Protease

Inhibitor (Thermo Scientific).

2.2.2 Oligonucleotide Biotinylation

Oligonucleotides corresponding to so-called sites A, B and C which are proximal to the transcriptional start site of RUNX2 P1 were biotinylated for usage via EMSA as well as via DNA pulldown. Sites were chosen using previously characterized TRE’s and were

29 identified via ENCODE analysis45. Identified sites were designed to maintain native sequence approximately ±20 base pairs with final oligonucleotides ranging in length from

40-60 base pairs. Mutated sites, AM, BM and CM were also used for mutational analysis, wherein the TRE’s present were altered to ablate TRß1 binding. A schematic of the oligonucleotides developed are shown in Figure 2.1. Briefly, the reverse strand of each oligonucleotide was biotinylated at the 3’-OH terminus (ENZO). Reactions were formulated per ENZO instructions, with the reactions proceeding at 37°C for 30 minutes.

The reactions were quenched with the addition of 5 µL 0.2 M EDTA. Complementary forward oligo was then added to each reaction, with strands being annealed at 95°C for five minutes in a 400 mL H2O water bath. The reactions were allowed to cool to room temperature and then stored at -40°C for further use.

Figure 2.1 Runx2 P1 promotor diagram and oligonucleotide schematic.

2.2.3 Electrophoretic Mobility Shift Assays (EMSA’s)

TRβ binding in the Runx2 P1 promoter was characterized by EMSA. Biotinylated

DNA (15 fmol) was incubated with either 10 µg nuclear protein lysate from thyroid cells

(Nthy-ORI) or 100 ng recombinant TRβ protein (Active Motif) in binding buffer 31 (Invitrogen/Life Technologies) for 30 minutes at room temperature in the absence or presence of anti-TRβ antibody (Thermo; MA1-215). The resulting complexes were resolved on 6% native polyacrylamide gels (Invitrogen/LifeTechnologies) and transferred to Biodyne B modified nylon membranes (Thermo Scientific) by electroblot (Bio-Rad

Laboratories). The transferred DNA was then cross-linked directly to the blots via

Stratalinker and complexes were detected by enhanced chemiluminesence LightShift

Chemiluminescent kit according to the manufacturer's protocol (Thermo Scientific) and imaged with VersaDoc MP3000 (Bio-Rad Laboratories).

2.2.4 Immunoblot

Nuclear proteins and reactions from immunoprecipitations or DNA pulldowns were analyzed via immunoblot to detect specific proteins and elucidate specific protein-protein interactions. Briefly, proteins were resolved by polyacrylamide gel electrophoresis on 10% sodium dodecyl sulfate gels (Life Technologies) and immobilized onto nitrocellulose membranes (GE Healthcare) by electroblot (Bio-Rad Laboratories). Specific proteins were detected by immunoblotting with the following antibodies (TRß1: ab53170 – Abcam;

TRß1: MA1-216 – Thermo Scientific; BRG1: ab110641 – Abcam; HDAC3: 7G6C5 – Cell

Signaling; FOXA1: GTX100308 – Genetex) Immunoreactive proteins were detected by enhanced chemiluminesence (Thermo Scientific) and visualized by VersaDoc MP3000

(Bio-Rad Laboratories).

32 2.2.5 Immunoprecipitation

Nuclear extract was pre-cleared with Protein G DynaBeads. 200 µg of protein was incubated with either an isotype specific IgG control antibody (mouse: G3A1; rabbit:

DA1E – Cell signaling) or an anti-TRß (MA1-215 – Thermo Scientific) or anti-BRG1

(D1Q7F – Cell Signaling) antibody for one hour at 4°C. 50 µL of Protein G Dynabeads were added to the reaction, and incubated for one hour at 4°C. The beads were then washed three times with Immunoprecipitation Buffer (20 mM HEPES, 30 mM KCl, 1 mM EDTA,

10 mM (NH4)2SO4, 1 mM DTT and 0.2% (v/v) Tween-20, pH 7.6) and once with H2O.

Samples were eluted with Laemmli Sample Buffer (Bio-Rad) and dithiothreitol and analyzed via immunoblot or via mass spectrometry.

2.2.6 DNA Pulldown Assay

The proteins that bind to the native sequence of the A oligo of the RUNX2 P1 proximal promotor were characterized by DNA pulldown as previously described by

Sancisi et al51. Briefly, biotinylated oligonucleotide (.75 fmol) was added to 50 µL of streptavidin-coupled T1 Dynabeads (Invitrogen) with 200 µL of Immunoprecipitation

Buffer in a total reaction volume of 250 µL. The reactions proceeded at 4°C for one hour with constant agitation. Beads were then washed twice with 200 µL of the

Immunoprecipitation Buffer. Next, 350 µg of nuclear lysate was pre-cleared for 15 minutes with 120 µL of pre-washed Strepavadin-coupled T1 Dynabeads (Invitrogen) with 2 µg of sheared herring sperm DNA (Promega). Potassium chloride was added to each reaction to achieve a final concentration of 300 mM, and TENP-40 (20 mM Tris-HCl, 150 mM NaCl,

1 mM EDTA, .5% (v/v) NP-40, pH 7.5) was added to bring the final reaction volume to

33 750 µL. Reactions proceed for 1.5 hours at 4°C on with constant agitation. Beads were then washed three times with 350 µL of TENP-40, and washed once with H2O. Samples were eluted with Laemmli Sample Buffer (Bio-Rad) and dithiothreitol and analyzed via immunoblot or via mass spectrometry. An experimental design schematic follows:

Figure 2.2 DNA Pulldown schematic.

2.2.7 DNAse I Hypersensitivity Assay

Promoter accessibility was determined via DNase hypersensitivity assay. PF1-3 treated cells were treated at a concentration of 30 µM and were harvested after 6 hours of treatment. Chromatin samples were obtained from cultured human thyroid cells that were 34 cross-linked with 1% formaldehyde for 10 minutes, neutralized with 1.25M glycine, rinsed twice with PBS, pelleted, and frozen. Cells were then lysed in the presence of protease inhibitors (Roche) and chromatin was extracted and sonicated to 200–500 base pairs in size using a Covaris S220 Focused-ultrasonicator. 2 µg of chromatin were treated with 0.5 units of DNase I (Qiagen) for 15 minutes at room temperature in 1X DNase Incubation Buffer.

Reactions were terminated by the addition of 50 mM EDTA and vortexing. Crosslinking was reversed by incubating the samples at 95°C for 20 minutes. DNA fragments were purified using a PureLink PCR Clean-Up Column (Invitrogen). DNA was used as a template for quantitative real-time PCR using primers specific to the Runx2 P1 promoter.

2.2.8 Sample Preparation for Mass Spectrometry Analysis

Protein samples from immunoprecipitation or DNA pulldown reactions were analyzed via mass spectrometry. Samples were resolved via SDS-PAGE under denaturing conditions using 10% sodium dodecyl sulfate gels (Life Technologies). The gel containing the samples destined for mass spectrometry analysis were washed for 15 minutes, two times in ddH2O and depending on reaction size, were either stained via Coommassie Blue or via silver stain. Generally, reactions >1 mg were stained via Coommassie Blue, whilst reactions <1 mg were stained via silver stain. Following staining procedures, samples were imaged via VersaDoc MP3000. Bands of interested with excised and were chopped in >1 mm cubes and submitted to the Proteomics Core at UVM for mass spectrometry analysis.

35 Coomassie Blue

Gels destined for Coomassie blue were washed as previously described by

ThermoFisher. Briefly, gels were washed in 20 mL of water 3x5 minutes. Approximately

30 mL of GelCode Blue (Thermo) was added to each gel and allowed to incubate at 4°C overnight. The next day, the GelCode Blue stain was decanted, and the gels were distained by washing four times with 50 mL water for 30 minutes per wash.

Silver Stain

Gels destined for silver stain were washed as previously described above. Gels were fixed using 30% ethanol, 10% acetic acid. Gels were allowed to incubate, twice, for 15 minutes at room temperature with gentle agitation. Gels were then washed with 10% ethanol, twice, for 5 minutes at room temperature with gentle agitation. Gels were then washed with ddH2O, twice, for 5 minutes at room temperature. Gels were then silver stained per the manufacturer’s protocol for the silver stain (Thermo). Reactions were halted using 5% acetic acid.

2.2.9 Analysis of Mass Spectrometry Results

Proteins were identified via mass spectrometry. Briefly, product ion spectra were searched using the SEQUEST search engine on Proteome Discoverer 1.4 (Thermo Fisher

Scientific). SEQUEST results were then further analyzed by Scaffold 4.3 (Proteome

Software) to compare the unique peptide counts and filter the results. Cross-correlation

(Xcorr) significance filters were applied to limit the false positive rates to less than 1%.

The Xcorr values were as follows: (+1): 1.5, (+2): 2.2, (+3): 2.8, (+4): 3.5158. Other filters

36 applied were a minimum peptide cutoff of 2 as well as DeltaCN >0.1. Ultimately, the confidence parameters resulted in less than 1% false discovery.

37 CHAPTER 3: Results 3.1 Electrophoretic Mobility Shift Assay’s (EMSA)

Functional binding of TRß1 to the Runx2 P1 promotor was established via electrophoretic mobility shift assay’s (EMSA’s). Specifically, it was found that TRß1 bound as a purified protein as well as in the context of nuclear protein lysate. EMSA’s performed using purified TRß1 show two distinct bands above the free probe indicating binding as both a monomer and as a homodimer. It is important to note that all three binding motifs tested using this assay lack a TRE repetition and thus represent half-sites. EMSA’s performed using nuclear lysate show distinct complexes variably forming as shown through super shifts using an antibody specific to TRß1. Unlabeled, 10x competitor probe decreased signal and therefore indicates signal specificity. Replication of the experimental conditions using mutant probes showed no detectable binding or shift. The mutant probes were altered to disallow TRß1 binding without impacting the surrounding nucleotides.

Bands were completely ablated when using purified TRß1 as well as nuclear lysate (Figure

3.1).

Figure 3.1 EMSA’s illustrating functional TRß1 binding to the three identified

TRE’s proximal to the Runx2 P1 promotor. Reactions conditions were previously identified in Chapter 2.2.3. Panel A refers to reactions using purified, recombinant

TRß1 protein, whilst Panel B refers to reactions using nuclear extract. The arrows in Panel A refer to shifts produced by TRß1. The arrows in Panel B refer to shifted

bands occurring from the inclusion of an anti-TRß1 antibody, indicating specific

TRß1 binding.

39 3.2 DNA Pulldowns

DNA pulldowns were employed as a complimentary method to EMSA’s and allowed for the detection of proteins complexed with TRb1 via western blot. Furthermore, DNA pulldown materials were also more suitable for mass spectrometry. Furthermore, the DNA pulldown should have less background than EMSAs given the wash steps. Runx2A was specifically chosen as previous studies by the Carr lab found this site to be the most repressive via luciferase reporter assays. Binding of oligonucleotide to the mechanical support (T1 DynaBeads) was confirmed via dot blot shown in the figure that follows.

Figure 3.2 Dot Blot Analysis of Oligonucleotide Binding to Beads. Eluent from

binding reactions were reserved and assayed via dot blot where free oligo was

detected via strepavadin-HRP and enhanced chemiluminesence. The binding of both the Runx2A and Runx2Amut were observed to be >95%, indicating successful

material preparation for downstream usage.

It was confirmed that TRß1 binds to the Runx2A oligonucleotide and further probing revealed other proteins that bound in concert with TRß1. Specifically, a substantial amount of BRG1 was detected, as well as both Forkhead box protein A1 (FOXA1) and HDAC3.

Previous studies have shown binding of the androgen receptor (AR) with FOXA1, and

41 given the homology between TR and AR, it is not unlikely that FOXA1 and TRß1 participate in unison159,160.

HDAC3 has been well characterized as an ancillary effector of NCOR1 and has been shown to bind BRG1 as a multiprotein complex, suggesting its involvement in a primary regulatory complex directed by TRß1142,144,161. Previous data has shown concerted regulatory activity by RUNX1 and HDAC3, providing an alternative binding partner for

HDAC3162-164. Flanking the TRE in the Runx2A oligo is a Runx2 response element (RRE) which has significant homology to Runx1 response elements. Further supporting BRG1 binding being dependent on TRß1 is the fact that BRG1 signal (as well as TRß1) was ablated when using a mutant probe. Specificity of binding is also supported by a lack of signal when a 10x cold competitor probe was added to the reactions.

42 5% Input No Oligo RUNX2A nativeRUNX2A mutant10X Competition Immunoblot BRG1

TRß1

Figure 3.3 DNA Pulldown using Runx2A and Nthy-ORI nuclear extract. Samples

were reacted as previously described, with 1 mg reactions being employed. 5% of

the reaction was run, with the remaining 95% being reserved for mass

spectrometry. Results indicated that both BRG1 and TRß1 participate in binding

the RUNX2 oligo. Furthermore, results indicate that functional TRß1 binding is

essential for binding of BRG1,

Figure 3.4 DNA Pulldown identifying major regulatory proteins that natively bind

Runx2A. Samples were reacted with 350 µg of nuclear extract, with all sample being

run.

3.3 Immunoprecipitations

Further characterization of TRß1 binding partners and the proteins that participate in multi-protein complexes was further studied via immunoprecipitations and co- immmunoprecipitations. BRG1-TRß1 interaction was confirmed via co- immunoprecipitations using reconstituted complexes from Nthy-ORI nuclear extracts.

Bidirectional immunoprecipitations, wherein BRG1 was precipitated using an anti-BRG1 antibody, and TRß1 was precipitated using an anti-TRß1 antibody were employed. In both

44 circumstances both TRß1 and BRG1 were detected via immunoblot at levels well above background.

To further corroborate the specific binding of TRß1 to the Runx2A oligo, immunoprecipitations using Nthy-ORI nuclear extract and an anti-TR1 antibody were employed with the addition of biotinylated Runx2A oligonucleotide. The protocol outlined in the methods section of this tome was employed with the addition of probe during antigen-antibody binding. The final eluent was analyzed for protein (immunoblot) and for

DNA binding (dot blot). Dot blot samples were blotted and oligonucleotide was detected via strepavadin-HRP and enhanced chemiluminescence.

Figure 3.5 Bidirectional immunoprecipitation of BRG1 and TRß1.

Immunoprecipitations reactions that employed the use of anti-BRG1 and anti-TRß1 antibodies show specific BRG1-TRß1 interaction. When using TRß1 as a prey, and

an anti-TRß1 antibody as the bait, both TRß1 and BRG1 immunoprecipitate.

Conversely, when using BRG1 as a prey, and an anti-BRG1 antibody as the bait,

both BRG1 and TRß1 immunoprecipitate. 46

Figure 3.6 Dot blot analysis of probe binding in in TRß1 immunoprecipitation.

Labeled Runx2A probe was added to immunoprecipitation reactions and was eluted and assayed via dot blot. The immunoprecipitation eluent was assayed along with a standard - RUNX2A – at the same dilution used in the initial reaction. Density was

determined from the dot blot, with relative binding being adjusted for using the

standard. Results were then inverted and standardized to Nthy-ORI Nuclear

Extract (Rabbit IgG). n=1.

47 3.4 Promotor Accessibility (DNAse I Hypersensitivity)

Functional studies on TRß1’s role in Runx2 expression were previously accomplished by the Carr lab. It was specifically found that TRß1 had a surpressive effect on Runx2 expression via siRNA knockout, transient reintroduction of TRß1 and by luciferase activity45. The current study sought to investigate how TRß1 acts to suppress Runx2 expression in a more thorough and mechanistic manner. Nthy-ORI and SW1736 cells were initially assayed via DNAse I hypersensitivity to examine baseline Runx2 promotor accessibility. Nthy-ORI cells are have normal levels of TRß1, whilst SW1736 cells have depleted levels of TRß145. DNAse I hypersensitivity assays on both Nthy-ORI and

SW1736 revealed that the chromatin surrounding Runx2 was moderately compacted in the context of Nthy-ORI and open in the context of SW1736. Further assays using Nthy-ORI cells and involving the usage of the drug PF1-3165, which inhibits the catalytic functionality of BRG1 at its bromo domain to sequester lysine acetylation activity, resulted in a considerable alteration in the relative accessibility of the Runx2 promotor. Accessibility increased by approximately five-fold (p-value >.0001). The same experiment was performed in SW1736 cells and did not result in significant change (data not shown) insinuating a primary role for BRG1 in the regulation of Runx2 as directed by TRß1.

Figure 3.7 Runx2 promotor accessibility in Nthy-ORI and SW1736 cell lines. Cells

were assayed using DNase I to establish baseline chromatin accessibility at the

Runx2 promotor. Statistical significance was p-value >0.005 as determined by an

unpaired t test.

Figure 3.8 DNAse I hypersensitivity assay of the Runx2 promotor in Nthy-ORI cells using an anti-BRG1 drug PF1-3. Cells were treated with PF1-3 at a concentration of

30 µM for 6 hours before harvest. Treatment with PF1-3, which blocks the catalytic activity of BRG1 led to a robust increase in chromatin accessibility. Significance was

p-value <0.0001 as determined by 2way ANOVA using an alpha value of 0.05.

3.5 Mass Spectrometry

To better understand the nuclear microenvironment and how it pertains to Runx2 regulation, mass spectrometry was employed to identify proteins. Briefly, reactions from

DNA pulldowns were separated via SDS-PAGE under denaturing conditions. Bands of interest were excised (Figure 3.9). Digestion of the bands and proteins, and subsequent

50 mass spectrometry analysis were handled and performed by the Proteomics Core at UVM.

Results from the control reactions (no oligonucleotide control and competition control) were subtracted from the experimental reaction (Runx2A oligonucleotide) with 225 unique proteins identified in total. Non-nuclear proteins, as identified via UniProt, as well as putative or uncharacterized proteins were excluded. Identified, unique proteins were initially analyzed via Reactome Pathway Database with IntAct interactors included.

Pathways with the highest hits found were the then selected, with primary actors being compiled into a selected list. Specifically, proteins involved in chromatin organization, disease and gene expression (transcription) were selected. Selected proteins are detailed in list 3.1 on the following page. Data was then collected from BioGRID3.4 to examine whether interactions between the proteins on the list had been previously documented

(Figure 3.10). Proteins were then filtered based on whether there was any evidence that they could form a multiprotein complex with TRß1 (Figure 3.11).

51 Table 3.1 Filtered protein list from mass spectrometry analysis of DNA pulldown.

Protein names Gene names Length (amino acids) Lysine-specific histone demethylase 1A KDM1A AOF2 852 KDM1 KIAA0601 LSD1 Abscission/NoCut checkpoint regulator ZFYVE19 471 ANCHR MPFYVE Insulin-like growth factor 2 mRNA-binding IGF2BP3 IMP3 579 protein 3 KOC1 VICKZ3 Nuclear factor 1 B-type NFIB 420 Nuclear factor 1 C-type NFIC NFI 508 Heat shock protein HSP 90-beta HSP90AB1 724 HSP90B HSPC2 HSPCB Heat shock 70 kDa protein 1A HSPA1A HSP72 641 HSPA1 HSX70 Transcriptional enhancer factor TEF-1 TEAD1 TCF13 426 TEF1 Elongation factor 1-delta EEF1D EF1D 281 Protein HIRA HIRA DGCR1 1017 HIR TUPLE1 Runt-related transcription factor 1 RUNX1 AML1 453 CBFA2 DNA-dependent protein kinase catalytic subunit PRKDC HYRC 4128 HYRC1 Transcription intermediary factor 1-beta TRIM28 KAP1 835 RNF96 TIF1B Mothers against decapentaplegic homolog 4 SMAD4 DPC4 552 Histone deacetylase 1 HDAC1 RPD3L1 482 Non-POU domain-containing octamer-binding NONO NRB54 471 protein Retinoblastoma-binding protein 5 RBBP5 RBQ3 538 DNA damage-binding protein 1 DDB1 XAP1 1140 Histone-binding protein RBBP7 RBAP46 425 General transcription factor 3C polypeptide 2 GTF3C2 911 SWI/SNF-related matrix-associated actin- SMARCE1 411 dependent regulator of chromatin subfamily E BAF57 member 1 Guanine nucleotide-binding protein-like 3 GNL3 E2IG3 NS 549 Thyroid hormone receptor-associated protein THRAP3 955 TRAP150 52

Figure 3.9 SDS-PAGE image of DNA pulldown samples analyzed via mass

spectrometry. DNA Pulldown samples were separated via SDS-PAGE under

denaturing conditions and then silver stained. The areas highlighted in red were excised and analyzed via mass spectrometry by the Proteomics facility at UVM. The

corresponding controls (no oligo and competition) were submitted for analysis.

Figure 3.10 Interaction map of selected proteins identified via mass spectrometry.

Proteins were selected for via Reactome Pathway Analysis and interactions were

mapped using BioGRID3.4.

Figure 3.11 Interaction map of selected proteins that could putatively form a

multiprotein complex with TRß1. Proteins that were previously identified were filtered based on whether there are published interactions, per BioGRID3.4, with the

addition of three ancillary effectors marked by an *.

CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS

The data presented in this thesis is mainly concerned with the nuclear environment in a non-malignant transformed cell system. Initially the goal of this research was to provide a protein profile comparison in the context of Runx2 P1 between a “normal” cell line (Nthy-ORI) and a malignant cell line (SW1736). Whilst this research will be continued, time constraints for this project did not make achieving the stretch aim feasible.

However, notwithstanding the stretch aim, the data presented here suggests a highly- regulated nuclear environment that primarily is concerned with chromatin machinery.

Indeed, the major factors identified either through immunoblot from DNA pulldowns or via mass spectrometry from DNA pulldown material identified several chromatin remodeling proteins or proteins that directly interact with chromatin machinery. Both

HDAC1 and HDAC3 were identified, although their direct functionality was not established or investigated. In conjunction to these two enzymes, BRG1 was identified as a primary actor in thyroid tumorigenesis based off its identification via immunoblot from

DNA pulldowns and immunoprecipitations. Furthermore, pharmacological inhibition of

BRG1 through the usage of PF1-3 and its resultant chromatin opening in the Nthy-ORI cell line adds gravity to the position that the major regulatory feature concerning Runx2 P1 is tightly controlled chromatin structure. In conjunction to this, the fact that BRG1 fails to bind with a mutant probe in a DNA pulldown implies that TRß1 acts as a major director of chromatin-related programs and is vital to chromatin stability.

Although this project was unable to progress to the stage where SW1736 was characterized in terms of DNA pulldown (and thus its nuclear protein profile), the relative

56 accessibility of the Runx2 P1 promotor in the SW1736 cell lines provides ample evidence that the nuclear protein profile is altered. Indeed, previous studies which characterized the levels of TRß1 in SW1736 revealed a greatly diminished amount of TRß1. Furthermore, the highest concentration of TRß1 was found to be outside of the nucleus and in the cytoplasm. This finding is concordant with previously described data wherein the levels of

TRß1 are diminished relative to tumor staging45. This working model, wherein TRß1 acts as a major regulatory feature of Runx2 P1 that represses Runx2 expression, is being extended to a breast cell line model. Current studies suggest that TRß1 acts as a global tumor suppressor in the context of breast, with TRß1 levels being correlated to survival in

BRCA1-positive cancers. TRß1 expression patterns also correlate with tumor staging in breast cancer. Malignant breast cancer lines also show high chromatin compaction at the

TRß1 promotor, suggesting that silencing of TRß1 through epigenetics is a major event in breast cancer tumorigenesis106,166.

Although a regulatory mechanism by which TRß1 suppresses tumorigenesis in breast cancer hasn’t been elucidated (as the previously cited studies examined TRß1 in the context of prognosis), expression patterns of chromatin-related proteins would suggest that

TRß1 directs an epigenetic program of tumor suppression. Global suppression of TRß1 also provides for a concise explanation as to why women who experience breast cancer are more likely to develop thyroid-related disorders and metachronous thyroid cancers167,168.

Future studies by the Carr lab seek to unravel whether there is any common causality given these correlations.

The specific mechanism by which TRß1 acts to direct an epigenetic program of repression of Runx2 P1 in the context of thyroid cancer remains to be established as well.

57 In conjunction to this, signaling events that lead to negative regulation by TRß1, as well as the nuclear hormone receptor family in general, are poorly understood. Emerging evidence in the field indicates chromatin remodeling as an essential mechanism by which nuclear hormone receptors, and thus TRß1, may act to suppress gene activity. Chromatin remodeling is considered an essential element in the framework of genetic regulation, wherein the availability of binding sites and upstream enhancer regions is dynamically controlled. To successfully interact with genomic regulatory elements and mediate oncogene repression, TRβ1 must induce reorganization of chromatin and local nucleosome structure resulting in changes in chromatin accessibility. Most of the seminal studies that have characterized TRβ-mediated chromatin remodeling have done so in the context of gene activation169-171. The mechanisms behind gene repression by TRβ are less well understood. There have been co-repressors (e.g. NCOR1, SMRT) identified as being essential for gene suppression by TRβ1172-174. Through recruitment of NCOR and SMRT,

HDACs are recruited to alter chromatin structure. The current model for TRβ-mediated chromatin reorganization is through a TRβ-NCOR-Sin3-HDAC complex, however there is a limited understanding of the signaling events that lead to TRβ-directed heterochromatinization or of how alternative TRβ-mediated complexes act to dynamically alter genetic programs.

The evidence provided by this work clearly indicates that BRG1 acts a major ancillary effector in the thyroid cancer system in conjunction to the actions of associated

HDACs. However, the exact mechanistic role of HDACs in the Runx2 P1 promotor context remain to be elucidated. Future studies that seek to unravel the mechanism of epigenetic regulation by TRß1 will ultimately examine the role that ancillary machinery plays in this

58 regulatory pathway; examining the roles of HDAC1 and HDAC3 in the regulation of

Runx2 P1 in the thyroid cancer context would address this. Furthermore, no published data exists on whether PF1-3 alters the protein complex that associates with TRß1-BRG1.

Preliminary data (not shown) indicate that inhibition of BRG1 via PF1-3 causes a dissociation between TRß1 and BRG1, and it is possible that PF1-3 may alter the chromatin machinery associated with BRG1, such as the HDAC’s. Given this fact, it is possible that alternative machinery associates with TRß1 (such a TRß1-NCOR1-Sin3A-HDAC (Class

I) complex) to induce alterations in the epigenetic profile of Runx2 P1 promotor.

Elucidating the exact role that BRG1 plays in the regulation of Runx2 is paramount.

Regardless of these questions that arise from the data presented in this thesis, putative models of regulation can be formed (Figure 4.1). Although these complexes were not investigated in a reconstituted system, publically available data from resources such as

BioGRID3.4 allow for the development of putative complexes. It is likely that TRß1 forms a complex with BRG1, with associated BAFs, as well as HDACs. Alternatively, complexes may form between TRß1 and FOXA1 and different roster of HDACs. Alternative to a

TRß1-centric complex, a multitude of other players may associate with the Runx2 P1 promotor. Previous studies have shown that RUNX1 associates with protein HIRA to positively regulate Runx1 itself. RUNX1, RUNX2 and RUNX3 share a commonality in their response elements, so it is not unlikely that RUNX1 may also positively regulate

Runx2 P1 through recruitment of protein HIRA175. Alternative to TRß1 suppression, it is also possible that TRß1 may associate with a different set of proteins that are associated with active Runx2 transcription. As previously described in this thesis, TRß1 may associate with co-regulators that are either activators or repressors. Although most of this discussion

59 has been focused on the role of repressive ancillary effectors, one canonical complex that is formed through TRß1 recruitment are the thyroid hormone receptor-associated proteins

(THRAP). THRAP3, a subunit of this complex was found via mass spec analysis from

DNA pulldown samples. THRAP is also alternatively referred to as vitamin D receptor interacting protein (DRIP) or as mediator. THRAP/DRIP/mediator act to recruit the RNA

Pol II holoenzyme complex, thereby encouraging active transcription of an active gene.

Speculatively, a TRß1-THRAP complex could form if key repressor elements are diminished. Furthermore, this study did not examine the role that cognate ligand plays in the regulation of the Runx2 P1 promotor. It is more than probable that alternative complexes form in reaction to the presence of T3.

Overall, the data presented in this thesis provides compelling evidence that TRß1 acts to execute a program concerned with chromatin stability and reorganization that ultimately acts to suppress Runx2 P1. Although the temporal confines of this project did not allow for the investigation of the nuclear regulatory environment in an aplastic thyroid cancer cell line, preliminary evidence via chromatin accessibility assays suggests that

Runx2 P1 expression is ultimately governed via chromatin organization. Furthermore, treatment of the Nthy-ORI cell line with BRG1 caused an increase in chromatin accessibility, suggesting that BRG1 plays a vital role in this regulation; this is consonant with the speculation that TRß1 executes a chromatin regulatory program. Future studies will investigate the differences in the nuclear regulatory environment in the context of

Runx2 P1 and will establish putative mechanisms by which TRß1-related dysregulation acts to secure Runx2 P1 expression.

Figure 4.1 Molecular switch diagram detailing possible protein complexes that could

regulate Runx2 P1. Complexes were postulated based off data from

immunoprecipitations and DNA pulldowns with the integration of protein-protein

interactions from Reactome Pathway Analysis and BioGRID3.4.

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73 APPENDICIES

Table 2.1 Mass Spectrometry Results from No Oligo Control reaction. Results are

sorted by relative abundance of the protein, with coverage and score shown.

Score # MW Accession Description ΣCoverage A2 AAs [kDa] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 36.96 53.10 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 28.89 20.42 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 30.82 36.87 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 22.85 26.00 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 13.05 9.52 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P04259 Keratin, type II cytoskeletal 6B OS=Homo sapiens 17.38 13.75 564 60.0 GN=KRT6B PE=1 SV=5 - [K2C6B_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 27.91 19.48 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 5.58 3.02 609 69.3 [ALBU_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 27.54 19.66 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] Q04695 Keratin, type I cytoskeletal 17 OS=Homo sapiens 16.20 15.92 432 48.1 GN=KRT17 PE=1 SV=2 - [K1C17_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 0.00 304 32.5 [TRY3_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 12.73 2.49 110 11.3 [DCD_HUMAN] Q5DNC6 Ser/thr protein kinase Emk1/MARK2 isoform EMK1A 24.39 0.00 41 4.5 (Fragment) OS=Homo sapiens PE=2 SV=1 - [Q5DNC6_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 52.95 122.21 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 48.15 56.46 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P08670 Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 - 43.56 40.60 466 53.6 [VIME_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 41.95 46.35 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 49.77 56.71 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 23.56 24.46 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 22.73 5.53 110 11.3 [DCD_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 26.22 25.06 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN] Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 5.26 2.88 285 282.2 [HORN_HUMAN] 0 P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 5.58 3.10 609 69.3 [ALBU_HUMAN] P13646 Keratin, type I cytoskeletal 13 OS=Homo sapiens 10.26 10.49 458 49.6 GN=KRT13 PE=1 SV=4 - [K1C13_HUMAN] P04259 Keratin, type II cytoskeletal 6B OS=Homo sapiens 32.80 32.70 564 60.0 GN=KRT6B PE=1 SV=5 - [K2C6B_HUMAN]

74 P02538 Keratin, type II cytoskeletal 6A OS=Homo sapiens 32.45 31.21 564 60.0 GN=KRT6A PE=1 SV=3 - [K2C6A_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 26.48 24.83 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] Q5T749 Keratinocyte proline-rich protein OS=Homo sapiens 3.45 2.78 579 64.1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 2.67 1.73 375 41.7 SV=1 - [ACTB_HUMAN] P05090 Apolipoprotein D OS=Homo sapiens GN=APOD PE=1 SV=1 3.70 0.00 189 21.3 - [APOD_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 2.29 304 32.5 [TRY3_HUMAN] Q14CN4 Keratin, type II cytoskeletal 72 OS=Homo sapiens 4.50 5.08 511 55.8 GN=KRT72 PE=1 SV=2 - [K2C72_HUMAN] Q7Z3Y8 Keratin, type I cytoskeletal 27 OS=Homo sapiens 4.58 1.80 459 49.8 GN=KRT27 PE=1 SV=2 - [K1C27_HUMAN] Q04695 Keratin, type I cytoskeletal 17 OS=Homo sapiens 12.73 9.32 432 48.1 GN=KRT17 PE=1 SV=2 - [K1C17_HUMAN] O43790 Keratin, type II cuticular Hb6 OS=Homo sapiens GN=KRT86 2.26 486 53.5 PE=1 SV=1 - [KRT86_HUMAN] Q7Z6R9 Transcription factor AP-2-delta OS=Homo sapiens 1.55 452 49.5 GN=TFAP2D PE=2 SV=1 - [AP2D_HUMAN] Q5D862 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 SV=1 - 0.50 239 247.9 [FILA2_HUMAN] 1 P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 37.73 74.18 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 37.08 27.78 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 31.34 31.31 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 31.14 37.27 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 12.20 6.73 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P04406 Glyceraldehyde-3-phosphate dehydrogenase OS=Homo 14.33 7.55 335 36.0 sapiens GN=GAPDH PE=1 SV=3 - [G3P_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 16.53 6.26 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 17.27 7.00 110 11.3 [DCD_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 3.61 2.96 609 69.3 [ALBU_HUMAN] P06748 Nucleophosmin OS=Homo sapiens GN=NPM1 PE=1 SV=2 - 5.44 294 32.6 [NPM_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 1.88 304 32.5 [TRY3_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 31.52 73.20 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 38.18 55.64 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 28.42 44.64 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 30.82 35.57 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 34.93 45.17 375 41.7 SV=1 - [ACTB_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 30.44 30.86 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 10.00 18.78 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 20.00 4.50 110 11.3 [DCD_HUMAN]

75 P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 3.61 2.65 609 69.3 [ALBU_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 2.24 304 32.5 [TRY3_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 19.28 16.76 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] Q04695 Keratin, type I cytoskeletal 17 OS=Homo sapiens 10.88 8.95 432 48.1 GN=KRT17 PE=1 SV=2 - [K1C17_HUMAN] K7EK59 Peroxisomal membrane protein PEX14 (Fragment) 3.75 0.00 160 17.4 OS=Homo sapiens GN=PEX14 PE=1 SV=1 - [K7EK59_HUMAN] P68104 Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 2.38 462 50.1 PE=1 SV=1 - [EF1A1_HUMAN] Q7Z6R9 Transcription factor AP-2-delta OS=Homo sapiens 1.55 452 49.5 GN=TFAP2D PE=2 SV=1 - [AP2D_HUMAN] Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 1.68 285 282.2 [HORN_HUMAN] 0 P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 47.67 105.80 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 39.38 55.86 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 42.22 78.91 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 45.70 57.95 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P05783 Keratin, type I cytoskeletal 18 OS=Homo sapiens 25.35 18.80 430 48.0 GN=KRT18 PE=1 SV=2 - [K1C18_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 44.82 48.09 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN] P68104 Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 11.90 8.24 462 50.1 PE=1 SV=1 - [EF1A1_HUMAN] P08123 Collagen alpha-2(I) chain OS=Homo sapiens GN=COL1A2 2.78 14.25 136 129.2 PE=1 SV=7 - [CO1A2_HUMAN] 6 Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 6.81 3.76 285 282.2 [HORN_HUMAN] 0 P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 11.86 19.17 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] Q08554 Desmocollin-1 OS=Homo sapiens GN=DSC1 PE=1 SV=2 - 3.91 2.56 894 99.9 [DSC1_HUMAN] P02452 Collagen alpha-1(I) chain OS=Homo sapiens GN=COL1A1 3.01 4.37 146 138.9 PE=1 SV=5 - [CO1A1_HUMAN] 4 P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 5.58 6.05 609 69.3 [ALBU_HUMAN] P04259 Keratin, type II cytoskeletal 6B OS=Homo sapiens 14.01 23.78 564 60.0 GN=KRT6B PE=1 SV=5 - [K2C6B_HUMAN] D3DTX7 Collagen, type I, alpha 1, isoform CRA_a OS=Homo sapiens 4.07 11.01 885 84.7 GN=COL1A1 PE=4 SV=1 - [D3DTX7_HUMAN] P39023 60S ribosomal protein L3 OS=Homo sapiens GN=RPL3 3.97 403 46.1 PE=1 SV=2 - [RL3_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 2.67 2.21 375 41.7 SV=1 - [ACTB_HUMAN] P36578 60S ribosomal protein L4 OS=Homo sapiens GN=RPL4 2.58 2.92 427 47.7 PE=1 SV=5 - [RL4_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 2.93 304 32.5 [TRY3_HUMAN] P06702 Protein S100-A9 OS=Homo sapiens GN=S100A9 PE=1 SV=1 11.40 3.23 114 13.2 - [S10A9_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 30.72 34.97 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] Q04695 Keratin, type I cytoskeletal 17 OS=Homo sapiens 14.58 10.80 432 48.1 GN=KRT17 PE=1 SV=2 - [K1C17_HUMAN] B4E3E7 cDNA FLJ58393 OS=Homo sapiens PE=2 SV=1 - 11.85 0.00 135 14.9 [B4E3E7_HUMAN] 76 P26641 Elongation factor 1-gamma OS=Homo sapiens GN=EEF1G 2.97 1.84 437 50.1 PE=1 SV=3 - [EF1G_HUMAN] P60842 Eukaryotic initiation factor 4A-I OS=Homo sapiens 2.46 406 46.1 GN=EIF4A1 PE=1 SV=1 - [IF4A1_HUMAN] P52597 Heterogeneous nuclear ribonucleoprotein F OS=Homo 4.10 415 45.6 sapiens GN=HNRNPF PE=1 SV=3 - [HNRPF_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 12.73 110 11.3 [DCD_HUMAN] Q5D862 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 SV=1 - 0.50 239 247.9 [FILA2_HUMAN] 1 Q5T749 Keratinocyte proline-rich protein OS=Homo sapiens 1.38 579 64.1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN] P14923 Junction plakoglobin OS=Homo sapiens GN=JUP PE=1 0.94 745 81.7 SV=3 - [PLAK_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 42.86 90.23 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 40.77 47.34 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 37.33 45.08 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 36.31 53.75 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P05787 Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 18.43 15.24 483 53.7 PE=1 SV=7 - [K2C8_HUMAN] P08729 Keratin, type II cytoskeletal 7 OS=Homo sapiens GN=KRT7 15.57 14.83 469 51.4 PE=1 SV=5 - [K2C7_HUMAN] P08670 Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 - 11.16 6.46 466 53.6 [VIME_HUMAN] P02538 Keratin, type II cytoskeletal 6A OS=Homo sapiens 16.67 18.96 564 60.0 GN=KRT6A PE=1 SV=3 - [K2C6A_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 11.02 12.00 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] Q8N532 TUBA1C protein OS=Homo sapiens GN=TUBA1C PE=2 7.69 2.41 325 36.6 SV=1 - [Q8N532_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 10.51 12.38 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P31943 Heterogeneous nuclear ribonucleoprotein H OS=Homo 3.56 0.00 449 49.2 sapiens GN=HNRNPH1 PE=1 SV=4 - [HNRH1_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 1.85 304 32.5 [TRY3_HUMAN] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 1.28 2.48 101 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] 4 P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 2.46 3.09 609 69.3 [ALBU_HUMAN] Q8IYW2 Cilia- and flagella-associated protein 46 OS=Homo sapiens 0.29 0.00 271 303.3 GN=CFAP46 PE=2 SV=3 - [CFA46_HUMAN] 5 Q6ZVL3 cDNA FLJ42424 fis, clone BLADE2004089, moderately 1.11 0.00 809 87.8 similar to Mus musculus PDZ domain actin binding protein Shroom mRNA (Fragment) OS=Homo sapiens PE=2 SV=1 - [Q6ZVL3_HUMAN] Q8IWP6 Class IVb beta tubulin OS=Homo sapiens PE=2 SV=1 - 4.04 0.00 445 49.7 [Q8IWP6_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 38.35 93.26 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 39.90 49.94 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 34.83 44.49 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 41.78 57.96 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 14.24 22.49 590 62.3 PE=1 SV=3 - [K2C5_HUMAN]

77 P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 12.92 12.04 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 5.58 3.55 609 69.3 [ALBU_HUMAN] P17844 Probable ATP-dependent RNA helicase DDX5 OS=Homo 3.26 5.16 614 69.1 sapiens GN=DDX5 PE=1 SV=1 - [DDX5_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 2.27 304 32.5 [TRY3_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 12.73 2.87 110 11.3 [DCD_HUMAN] Q8IYW2 Cilia- and flagella-associated protein 46 OS=Homo sapiens 0.29 0.00 271 303.3 GN=CFAP46 PE=2 SV=3 - [CFA46_HUMAN] 5 Q05D76 EEA1 protein (Fragment) OS=Homo sapiens GN=EEA1 13.83 0.00 311 35.2 PE=2 SV=1 - [Q05D76_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 36.80 71.81 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 46.06 50.57 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 38.34 41.46 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 35.31 26.60 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 10.95 14.50 101 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] 4 A1A4E9 Keratin 13 OS=Homo sapiens GN=KRT13 PE=1 SV=1 - 12.01 5.73 458 49.6 [A1A4E9_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 10.68 10.70 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 4.76 3.92 609 69.3 [ALBU_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 12.73 3.16 110 11.3 [DCD_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 16.28 13.28 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN] P04259 Keratin, type II cytoskeletal 6B OS=Homo sapiens 11.35 14.59 564 60.0 GN=KRT6B PE=1 SV=5 - [K2C6B_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 16.53 13.28 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 304 32.5 [TRY3_HUMAN] Q14568 Heat shock protein HSP 90-alpha A2 OS=Homo sapiens 3.50 343 39.3 GN=HSP90AA2P PE=1 SV=2 - [HS902_HUMAN] Q8TF72 Protein Shroom3 OS=Homo sapiens GN=SHROOM3 PE=1 0.45 199 216.7 SV=2 - [SHRM3_HUMAN] 6 P08123 Collagen alpha-2(I) chain OS=Homo sapiens GN=COL1A2 1.02 136 129.2 PE=1 SV=7 - [CO1A2_HUMAN] 6 Q92945 Far upstream element-binding protein 2 OS=Homo sapiens 1.55 711 73.1 GN=KHSRP PE=1 SV=4 - [FUBP2_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 41.15 98.34 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 40.58 58.52 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 16.67 43.86 101 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] 4 P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 35.21 60.18 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 28.25 32.62 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P01040 Cystatin-A OS=Homo sapiens GN=CSTA PE=1 SV=1 - 58.16 4.75 98 11.0 [CYTA_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 27.70 20.78 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN]

78 Q2TB90 Putative hexokinase HKDC1 OS=Homo sapiens GN=HKDC1 4.14 3.51 917 102.5 PE=1 SV=3 - [HKDC1_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 6.73 7.31 609 69.3 [ALBU_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 27.27 1.65 110 11.3 [DCD_HUMAN] P04406 Glyceraldehyde-3-phosphate dehydrogenase OS=Homo 9.25 2.76 335 36.0 sapiens GN=GAPDH PE=1 SV=3 - [G3P_HUMAN] P02538 Keratin, type II cytoskeletal 6A OS=Homo sapiens 17.73 30.29 564 60.0 GN=KRT6A PE=1 SV=3 - [K2C6A_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 12.37 26.66 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] Q3SY84 Keratin, type II cytoskeletal 71 OS=Homo sapiens 4.78 2.14 523 57.3 GN=KRT71 PE=1 SV=3 - [K2C71_HUMAN] P52789 Hexokinase-2 OS=Homo sapiens GN=HK2 PE=1 SV=2 - 2.29 1.70 917 102.3 [HXK2_HUMAN] O94906 Pre-mRNA-processing factor 6 OS=Homo sapiens 1.81 1.98 941 106.9 GN=PRPF6 PE=1 SV=1 - [PRP6_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 23.31 23.80 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 2.83 304 32.5 [TRY3_HUMAN] P10599 Thioredoxin OS=Homo sapiens GN=TXN PE=1 SV=3 - 12.38 2.07 105 11.7 [THIO_HUMAN] P31151 Protein S100-A7 OS=Homo sapiens GN=S100A7 PE=1 SV=4 10.89 0.00 101 11.5 - [S10A7_HUMAN] E9PFH7 C-Jun-amino-terminal kinase-interacting protein 3 3.23 0.00 133 146.6 OS=Homo sapiens GN=MAPK8IP3 PE=1 SV=1 - 0 [E9PFH7_HUMAN] P05089 Arginase-1 OS=Homo sapiens GN=ARG1 PE=1 SV=2 - 3.42 2.31 322 34.7 [ARGI1_HUMAN] Q7Z3Y8 Keratin, type I cytoskeletal 27 OS=Homo sapiens 6.54 8.98 459 49.8 GN=KRT27 PE=1 SV=2 - [K1C27_HUMAN] Q8IYW2 Cilia- and flagella-associated protein 46 OS=Homo sapiens 0.29 0.00 271 303.3 GN=CFAP46 PE=2 SV=3 - [CFA46_HUMAN] 5 Q6ZVL3 cDNA FLJ42424 fis, clone BLADE2004089, moderately 1.11 1.93 809 87.8 similar to Mus musculus PDZ domain actin binding protein Shroom mRNA (Fragment) OS=Homo sapiens PE=2 SV=1 - [Q6ZVL3_HUMAN] Q53H37 Calmodulin-like skin protein variant (Fragment) OS=Homo 5.48 1.96 146 15.9 sapiens PE=2 SV=1 - [Q53H37_HUMAN] M0R1V7 Ubiquitin-60S ribosomal protein L40 (Fragment) OS=Homo 14.29 1.89 63 7.1 sapiens GN=UBA52 PE=1 SV=1 - [M0R1V7_HUMAN] Q4JFL9 Protein S100 (Fragment) OS=Homo sapiens GN=FLG PE=2 9.78 0.00 92 11.0 SV=1 - [Q4JFL9_HUMAN] L0R5A1 Alternative protein CSF2RB OS=Homo sapiens GN=CSF2RB 7.41 0.00 108 11.6 PE=4 SV=1 - [L0R5A1_HUMAN] Q01469 Fatty acid-binding protein, epidermal OS=Homo sapiens 8.15 135 15.2 GN=FABP5 PE=1 SV=3 - [FABP5_HUMAN] P31944 Caspase-14 OS=Homo sapiens GN=CASP14 PE=1 SV=2 - 3.31 242 27.7 [CASPE_HUMAN] P06733 Alpha-enolase OS=Homo sapiens GN=ENO1 PE=1 SV=2 - 1.61 434 47.1 [ENOA_HUMAN] P04075 Fructose-bisphosphate aldolase A OS=Homo sapiens 2.20 364 39.4 GN=ALDOA PE=1 SV=2 - [ALDOA_HUMAN] Q5T749 Keratinocyte proline-rich protein OS=Homo sapiens 1.73 579 64.1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN]

Table 2.2 Mass Spectrometry Results from RUNX2A reaction. Results are sorted by

relative abundance of the protein, with coverage and score shown.

Score # MW Accession Description ΣCoverage A2 AAs [kDa] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 53.55 570.76 1014 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 47.83 110.33 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] A8K4Q9 Hexokinase OS=Homo sapiens PE=2 SV=1 - 22.25 50.41 917 102.4 [A8K4Q9_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 51.02 86.10 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 37.84 91.97 584 58.8 PE=1 SV=6 - [K1C10_HUMAN] Q00839 Heterogeneous nuclear ribonucleoprotein U OS=Homo 23.64 35.96 825 90.5 sapiens GN=HNRNPU PE=1 SV=6 - [HNRPU_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 25.84 35.10 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 32.88 38.44 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] Q15029 116 kDa U5 small nuclear ribonucleoprotein component 14.20 14.75 972 109.4 OS=Homo sapiens GN=EFTUD2 PE=1 SV=1 - [U5S1_HUMAN] Q9GZR7 ATP-dependent RNA helicase DDX24 OS=Homo sapiens 16.41 21.10 859 96.3 GN=DDX24 PE=1 SV=1 - [DDX24_HUMAN] P46087 Probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase 16.01 18.78 812 89.2 OS=Homo sapiens GN=NOP2 PE=1 SV=2 - [NOP2_HUMAN] Q16531 DNA damage-binding protein 1 OS=Homo sapiens GN=DDB1 9.30 13.24 1140 126.9 PE=1 SV=1 - [DDB1_HUMAN] P55265 Double-stranded RNA-specific adenosine deaminase 8.56 17.85 1226 136.0 OS=Homo sapiens GN=ADAR PE=1 SV=4 - [DSRAD_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 45.24 44.87 473 51.2 PE=1 SV=4 - [K1C16_HUMAN] P42285 Superkiller viralicidic activity 2-like 2 OS=Homo sapiens 8.45 12.49 1042 117.7 GN=SKIV2L2 PE=1 SV=3 - [SK2L2_HUMAN] P43243 Matrin-3 OS=Homo sapiens GN=MATR3 PE=1 SV=2 - 9.09 12.99 847 94.6 [MATR3_HUMAN] Q15459 Splicing factor 3A subunit 1 OS=Homo sapiens GN=SF3A1 10.09 13.24 793 88.8 PE=1 SV=1 - [SF3A1_HUMAN] Q96QC0 Serine/threonine-protein phosphatase 1 regulatory subunit 9.68 12.32 940 99.0 10 OS=Homo sapiens GN=PPP1R10 PE=1 SV=1 - [PP1RA_HUMAN] Q92598 Heat shock protein 105 kDa OS=Homo sapiens GN=HSPH1 12.12 15.16 858 96.8 PE=1 SV=1 - [HS105_HUMAN] Q16643 Drebrin OS=Homo sapiens GN=DBN1 PE=1 SV=4 - 14.33 16.51 649 71.4 [DREB_HUMAN] O14980 Exportin-1 OS=Homo sapiens GN=XPO1 PE=1 SV=1 - 7.00 6.57 1071 123.3 [XPO1_HUMAN] P53396 ATP-citrate synthase OS=Homo sapiens GN=ACLY PE=1 4.72 8.60 1101 120.8 SV=3 - [ACLY_HUMAN] 80 Q96T37 Putative RNA-binding protein 15 OS=Homo sapiens 7.37 6.45 977 107.1 GN=RBM15 PE=1 SV=2 - [RBM15_HUMAN] P52789 Hexokinase-2 OS=Homo sapiens GN=HK2 PE=1 SV=2 - 6.22 12.06 917 102.3 [HXK2_HUMAN] P04259 Keratin, type II cytoskeletal 6B OS=Homo sapiens 23.58 29.13 564 60.0 GN=KRT6B PE=1 SV=5 - [K2C6B_HUMAN] P78527 DNA-dependent protein kinase catalytic subunit OS=Homo 1.28 6.08 4128 468.8 sapiens GN=PRKDC PE=1 SV=3 - [PRKDC_HUMAN] Q1KMD3 Heterogeneous nuclear ribonucleoprotein U-like protein 2 7.63 3.83 747 85.1 OS=Homo sapiens GN=HNRNPUL2 PE=1 SV=1 - [HNRL2_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 7.06 8.94 609 69.3 [ALBU_HUMAN] Q9Y2L1 Exosome complex exonuclease RRP44 OS=Homo sapiens 5.85 6.48 958 108.9 GN=DIS3 PE=1 SV=2 - [RRP44_HUMAN] O43290 U4/U6.U5 tri-snRNP-associated protein 1 OS=Homo sapiens 6.13 7.74 800 90.2 GN=SART1 PE=1 SV=1 - [SNUT1_HUMAN] Q9BVJ6 U3 small nucleolar RNA-associated protein 14 homolog A 6.49 2.32 771 87.9 OS=Homo sapiens GN=UTP14A PE=1 SV=1 - [UT14A_HUMAN] Q7Z794 Keratin, type II cytoskeletal 1b OS=Homo sapiens 11.76 14.38 578 61.9 GN=KRT77 PE=2 SV=3 - [K2C1B_HUMAN] Q13263 Transcription intermediary factor 1-beta OS=Homo sapiens 3.95 3.96 835 88.5 GN=TRIM28 PE=1 SV=5 - [TIF1B_HUMAN] Q5T749 Keratinocyte proline-rich protein OS=Homo sapiens 5.87 6.12 579 64.1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN] O00410 Importin-5 OS=Homo sapiens GN=IPO5 PE=1 SV=4 - 3.56 3.59 1097 123.5 [IPO5_HUMAN] P46013 Antigen KI-67 OS=Homo sapiens GN=MKI67 PE=1 SV=2 - 2.21 4.00 3256 358.5 [KI67_HUMAN] P33176 Kinesin-1 heavy chain OS=Homo sapiens GN=KIF5B PE=1 3.84 1.89 963 109.6 SV=1 - [KINH_HUMAN] Q12906 Interleukin enhancer-binding factor 3 OS=Homo sapiens 3.91 6.25 894 95.3 GN=ILF3 PE=1 SV=3 - [ILF3_HUMAN] O95782 AP-2 complex subunit alpha-1 OS=Homo sapiens GN=AP2A1 2.97 2.07 977 107.5 PE=1 SV=3 - [AP2A1_HUMAN] Q12965 Unconventional myosin-Ie OS=Homo sapiens GN=MYO1E 2.80 3.90 1108 127.0 PE=1 SV=2 - [MYO1E_HUMAN] Q8WUA4 General transcription factor 3C polypeptide 2 OS=Homo 3.84 1.73 911 100.6 sapiens GN=GTF3C2 PE=1 SV=2 - [TF3C2_HUMAN] P05109 Protein S100-A8 OS=Homo sapiens GN=S100A8 PE=1 SV=1 23.66 2.07 93 10.8 - [S10A8_HUMAN] P53675 Clathrin heavy chain 2 OS=Homo sapiens GN=CLTCL1 PE=1 1.65 2.41 1640 186.9 SV=2 - [CLH2_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 12.73 5.13 110 11.3 [DCD_HUMAN] Q9Y2A7 Nck-associated protein 1 OS=Homo sapiens GN=NCKAP1 1.68 3.87 1128 128.7 PE=1 SV=1 - [NCKP1_HUMAN] Q9H0D6 5'-3' exoribonuclease 2 OS=Homo sapiens GN=XRN2 PE=1 3.05 5.30 950 108.5 SV=1 - [XRN2_HUMAN] O75369 Filamin-B OS=Homo sapiens GN=FLNB PE=1 SV=2 - 1.23 3.64 2602 278.0 [FLNB_HUMAN] Q9H0A0 N-acetyltransferase 10 OS=Homo sapiens GN=NAT10 PE=1 7.71 8.23 1025 115.7 SV=2 - [NAT10_HUMAN] O75152 Zinc finger CCCH domain-containing protein 11A OS=Homo 2.96 1.83 810 89.1 sapiens GN=ZC3H11A PE=1 SV=3 - [ZC11A_HUMAN] P21333 Filamin-A OS=Homo sapiens GN=FLNA PE=1 SV=4 - 1.36 4.27 2647 280.6 [FLNA_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 31.99 39.01 472 51.5 PE=1 SV=4 - [K1C14_HUMAN] P22531 Small proline-rich protein 2E OS=Homo sapiens GN=SPRR2E 43.06 2.25 72 7.8 PE=2 SV=2 - [SPR2E_HUMAN]

81 P55884 Eukaryotic translation initiation factor 3 subunit B OS=Homo 2.83 2.24 814 92.4 sapiens GN=EIF3B PE=1 SV=3 - [EIF3B_HUMAN] Q5D862 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 SV=1 - 0.92 2.33 2391 247.9 [FILA2_HUMAN] Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 2.91 0.00 2850 282.2 [HORN_HUMAN] Q04695 Keratin, type I cytoskeletal 17 OS=Homo sapiens GN=KRT17 15.51 13.31 432 48.1 PE=1 SV=2 - [K1C17_HUMAN] O60341 Lysine-specific histone demethylase 1A OS=Homo sapiens 2.58 2.29 852 92.8 GN=KDM1A PE=1 SV=2 - [KDM1A_HUMAN] Q9P0K7 Ankycorbin OS=Homo sapiens GN=RAI14 PE=1 SV=2 - 2.14 0.00 980 110.0 [RAI14_HUMAN] P63010 AP-2 complex subunit beta OS=Homo sapiens GN=AP2B1 1.92 1.80 937 104.5 PE=1 SV=1 - [AP2B1_HUMAN] P11388 DNA topoisomerase 2-alpha OS=Homo sapiens GN=TOP2A 1.11 0.00 1531 174.3 PE=1 SV=3 - [TOP2A_HUMAN] P23490 Loricrin OS=Homo sapiens GN=LOR PE=1 SV=2 - 4.17 3.98 312 25.7 [LORI_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 4.80 2.38 375 41.7 SV=1 - [ACTB_HUMAN] P68104 Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 2.38 1.91 462 50.1 PE=1 SV=1 - [EF1A1_HUMAN] Q14974 Importin subunit beta-1 OS=Homo sapiens GN=KPNB1 PE=1 1.37 1.82 876 97.1 SV=2 - [IMB1_HUMAN] P52948 Nuclear pore complex protein Nup98-Nup96 OS=Homo 0.61 2.13 1817 197.5 sapiens GN=NUP98 PE=1 SV=4 - [NUP98_HUMAN] P31944 Caspase-14 OS=Homo sapiens GN=CASP14 PE=1 SV=2 - 3.31 1.79 242 27.7 [CASPE_HUMAN] Q9UL03 Integrator complex subunit 6 OS=Homo sapiens GN=INTS6 0.90 0.00 887 100.3 PE=1 SV=1 - [INT6_HUMAN] P61626 Lysozyme C OS=Homo sapiens GN=LYZ PE=1 SV=1 - 6.08 0.00 148 16.5 [LYSC_HUMAN] A1X283 SH3 and PX domain-containing protein 2B OS=Homo sapiens 0.99 1.73 911 101.5 GN=SH3PXD2B PE=1 SV=3 - [SPD2B_HUMAN] P53992 Protein transport protein Sec24C OS=Homo sapiens 1.19 2.73 1094 118.2 GN=SEC24C PE=1 SV=3 - [SC24C_HUMAN] Q02413 Desmoglein-1 OS=Homo sapiens GN=DSG1 PE=1 SV=2 - 0.95 1.77 1049 113.7 [DSG1_HUMAN] P06702 Protein S100-A9 OS=Homo sapiens GN=S100A9 PE=1 SV=1 11.40 2.90 114 13.2 - [S10A9_HUMAN] Q9Y2W1 Thyroid hormone receptor-associated protein 3 OS=Homo 0.94 1.90 955 108.6 sapiens GN=THRAP3 PE=1 SV=2 - [TR150_HUMAN] P25311 Zinc-alpha-2-glycoprotein OS=Homo sapiens GN=AZGP1 3.36 0.00 298 34.2 PE=1 SV=2 - [ZA2G_HUMAN] Q8N5C6 S1 RNA-binding domain-containing protein 1 OS=Homo 1.11 0.00 995 111.7 sapiens GN=SRBD1 PE=1 SV=2 - [SRBD1_HUMAN] P54198 Protein HIRA OS=Homo sapiens GN=HIRA PE=1 SV=2 - 1.18 0.00 1017 111.8 [HIRA_HUMAN] Q96DR4-2 Isoform 2 of StAR-related lipid transfer protein 4 OS=Homo 7.55 0.00 159 18.5 sapiens GN=STARD4 - [STAR4_HUMAN] Q8NF09 FLJ00394 protein (Fragment) OS=Homo sapiens 5.46 0.00 238 26.1 GN=FLJ00394 PE=2 SV=1 - [Q8NF09_HUMAN] Q4KMP7 TBC1 domain family member 10B OS=Homo sapiens 2.60 3.26 808 87.1 GN=TBC1D10B PE=1 SV=3 - [TB10B_HUMAN] P08195 4F2 cell-surface antigen heavy chain OS=Homo sapiens 2.06 1.81 630 68.0 GN=SLC3A2 PE=1 SV=3 - [4F2_HUMAN] Q5VZ66 Janus kinase and microtubule-interacting protein 3 1.54 0.00 844 98.5 OS=Homo sapiens GN=JAKMIP3 PE=2 SV=2 - [JKIP3_HUMAN] O00159 Unconventional myosin-Ic OS=Homo sapiens GN=MYO1C 12.70 15.91 1063 121.6 PE=1 SV=4 - [MYO1C_HUMAN] Q68E01 Integrator complex subunit 3 OS=Homo sapiens GN=INTS3 0.67 1.77 1043 118.0 PE=1 SV=1 - [INT3_HUMAN] 82 O00159-3 Isoform 3 of Unconventional myosin-Ic OS=Homo sapiens 13.22 17.70 1044 119.6 GN=MYO1C - [MYO1C_HUMAN] Q08211 ATP-dependent RNA helicase A OS=Homo sapiens GN=DHX9 0.94 2.31 1270 140.9 PE=1 SV=4 - [DHX9_HUMAN] Q8NE71 ATP-binding cassette sub-family F member 1 OS=Homo 1.30 0.00 845 95.9 sapiens GN=ABCF1 PE=1 SV=2 - [ABCF1_HUMAN] C9J9P7 RNA-binding protein 5 (Fragment) OS=Homo sapiens 10.00 0.00 120 14.5 GN=RBM5 PE=1 SV=1 - [C9J9P7_HUMAN] H0YF68 Serine/threonine-protein kinase TAO3 (Fragment) OS=Homo 7.03 1.89 185 20.9 sapiens GN=TAOK3 PE=1 SV=1 - [H0YF68_HUMAN] Q9UG16 Putative uncharacterized protein DKFZp564P0562 0.83 0.00 1325 152.0 (Fragment) OS=Homo sapiens GN=DKFZp564P0562 PE=2 SV=1 - [Q9UG16_HUMAN] Q05CW7 NAT10 protein (Fragment) OS=Homo sapiens GN=NAT10 12.64 8.85 554 62.3 PE=2 SV=1 - [Q05CW7_HUMAN] B3KQH1 cDNA FLJ90452 fis, clone NT2RP3001475, highly similar to 1.56 0.00 897 99.9 Splicing factor 3B subunit 3 OS=Homo sapiens PE=2 SV=1 - [B3KQH1_HUMAN] Q9Y2U8 Inner nuclear membrane protein Man1 OS=Homo sapiens 0.99 911 99.9 GN=LEMD3 PE=1 SV=2 - [MAN1_HUMAN] Q9BWU0 Kanadaptin OS=Homo sapiens GN=SLC4A1AP PE=1 SV=1 - 1.26 796 88.8 [NADAP_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 14.90 10.96 584 58.8 PE=1 SV=6 - [K1C10_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 14.45 9.15 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 8.48 22.71 1014 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 10.09 4.33 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] O43143 Pre-mRNA-splicing factor ATP-dependent RNA helicase 6.04 7.09 795 90.9 DHX15 OS=Homo sapiens GN=DHX15 PE=1 SV=2 - [DHX15_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 11.42 5.87 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] Q14974 Importin subunit beta-1 OS=Homo sapiens GN=KPNB1 PE=1 4.79 1.63 876 97.1 SV=2 - [IMB1_HUMAN] Q14684 Ribosomal RNA processing protein 1 homolog B OS=Homo 6.33 0.00 758 84.4 sapiens GN=RRP1B PE=1 SV=3 - [RRP1B_HUMAN] P08238 Heat shock protein HSP 90-beta OS=Homo sapiens 6.35 2.46 724 83.2 GN=HSP90AB1 PE=1 SV=4 - [HS90B_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 5.76 3.65 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] Q9NR30 Nucleolar RNA helicase 2 OS=Homo sapiens GN=DDX21 2.30 0.00 783 87.3 PE=1 SV=5 - [DDX21_HUMAN] Q00610 Clathrin heavy chain 1 OS=Homo sapiens GN=CLTC PE=1 1.37 0.00 1675 191.5 SV=5 - [CLH1_HUMAN] B4DVK5 cDNA FLJ54759, highly similar to DNA replication licensing 5.58 1.62 412 45.0 factor MCM5 OS=Homo sapiens PE=2 SV=1 - [B4DVK5_HUMAN] Q99959 Plakophilin-2 OS=Homo sapiens GN=PKP2 PE=1 SV=2 - 0.91 0.00 881 97.4 [PKP2_HUMAN] O43790 Keratin, type II cuticular Hb6 OS=Homo sapiens GN=KRT86 3.50 2.08 486 53.5 PE=1 SV=1 - [KRT86_HUMAN] Q12906 Interleukin enhancer-binding factor 3 OS=Homo sapiens 1.34 2.41 894 95.3 GN=ILF3 PE=1 SV=3 - [ILF3_HUMAN] P21333 Filamin-A OS=Homo sapiens GN=FLNA PE=1 SV=4 - 0.49 2.86 2647 280.6 [FLNA_HUMAN] Q12788 Transducin beta-like protein 3 OS=Homo sapiens GN=TBL3 1.61 0.00 808 89.0 PE=1 SV=2 - [TBL3_HUMAN] E5RGG6 Protein NDRG1 (Fragment) OS=Homo sapiens GN=NDRG1 90.48 0.00 42 4.7 PE=1 SV=6 - [E5RGG6_HUMAN]

83 L0R5A1 Alternative protein CSF2RB OS=Homo sapiens GN=CSF2RB 7.41 0.00 108 11.6 PE=4 SV=1 - [L0R5A1_HUMAN] A0A087WYY3 Cation-transporting ATPase OS=Homo sapiens GN=ATP13A3 3.99 0.00 701 77.3 PE=1 SV=1 - [A0A087WYY3_HUMAN] Q12768 WASH complex subunit strumpellin OS=Homo sapiens 1.21 1159 134.2 GN=KIAA0196 PE=1 SV=1 - [STRUM_HUMAN] Q8N1F7 Nuclear pore complex protein Nup93 OS=Homo sapiens 1.22 819 93.4 GN=NUP93 PE=1 SV=2 - [NUP93_HUMAN] O14787 Transportin-2 OS=Homo sapiens GN=TNPO2 PE=1 SV=3 - 1.11 897 101.3 [TNPO2_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 40.99 49.77 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 31.85 41.45 584 58.8 PE=1 SV=6 - [K1C10_HUMAN] P17844 Probable ATP-dependent RNA helicase DDX5 OS=Homo 27.36 27.01 614 69.1 sapiens GN=DDX5 PE=1 SV=1 - [DDX5_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 21.19 21.70 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P02545 Prelamin-A/C OS=Homo sapiens GN=LMNA PE=1 SV=1 - 19.58 20.35 664 74.1 [LMNA_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 28.33 43.25 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 14.79 28.15 1014 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] P05187 Alkaline phosphatase, placental type OS=Homo sapiens 23.18 33.53 535 57.9 GN=ALPP PE=1 SV=2 - [PPB1_HUMAN] Q9BVP2 Guanine nucleotide-binding protein-like 3 OS=Homo sapiens 17.12 7.76 549 62.0 GN=GNL3 PE=1 SV=2 - [GNL3_HUMAN] O76021 Ribosomal L1 domain-containing protein 1 OS=Homo 13.88 4.31 490 54.9 sapiens GN=RSL1D1 PE=1 SV=3 - [RL1D1_HUMAN] Q07666 KH domain-containing, RNA-binding, signal transduction- 16.03 7.35 443 48.2 associated protein 1 OS=Homo sapiens GN=KHDRBS1 PE=1 SV=1 - [KHDR1_HUMAN] P20700 Lamin-B1 OS=Homo sapiens GN=LMNB1 PE=1 SV=2 - 9.73 6.58 586 66.4 [LMNB1_HUMAN] P13797 Plastin-3 OS=Homo sapiens GN=PLS3 PE=1 SV=4 - 10.48 7.87 630 70.8 [PLST_HUMAN] Q9NVI7 ATPase family AAA domain-containing protein 3A OS=Homo 7.73 7.52 634 71.3 sapiens GN=ATAD3A PE=1 SV=2 - [ATD3A_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 8.21 16.89 609 69.3 [ALBU_HUMAN] P14866 Heterogeneous nuclear ribonucleoprotein L OS=Homo 8.49 0.00 589 64.1 sapiens GN=HNRNPL PE=1 SV=2 - [HNRPL_HUMAN] P61978 Heterogeneous nuclear ribonucleoprotein K OS=Homo 11.02 4.67 463 50.9 sapiens GN=HNRNPK PE=1 SV=1 - [HNRPK_HUMAN] P04843 Dolichyl-diphosphooligosaccharide--protein 9.06 0.00 607 68.5 glycosyltransferase subunit 1 OS=Homo sapiens GN=RPN1 PE=1 SV=1 - [RPN1_HUMAN] P61221 ATP-binding cassette sub-family E member 1 OS=Homo 6.34 7.07 599 67.3 sapiens GN=ABCE1 PE=1 SV=1 - [ABCE1_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 11.19 14.91 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 13.56 8.77 472 51.5 PE=1 SV=4 - [K1C14_HUMAN] Q53GS9 U4/U6.U5 tri-snRNP-associated protein 2 OS=Homo sapiens 6.73 5.88 565 65.3 GN=USP39 PE=1 SV=2 - [SNUT2_HUMAN] Q9NXV6 CDKN2A-interacting protein OS=Homo sapiens 8.28 1.70 580 61.1 GN=CDKN2AIP PE=1 SV=3 - [CARF_HUMAN] O00541 Pescadillo homolog OS=Homo sapiens GN=PES1 PE=1 SV=1 6.46 0.00 588 68.0 - [PESC_HUMAN] Q9Y2R4 Probable ATP-dependent RNA helicase DDX52 OS=Homo 5.01 4.06 599 67.5 sapiens GN=DDX52 PE=1 SV=3 - [DDX52_HUMAN]

84 Q13492 Phosphatidylinositol-binding clathrin assembly protein 4.14 4.12 652 70.7 OS=Homo sapiens GN=PICALM PE=1 SV=2 - [PICAL_HUMAN] Q9NSD9 Phenylalanine--tRNA ligase beta subunit OS=Homo sapiens 4.58 1.83 589 66.1 GN=FARSB PE=1 SV=3 - [SYFB_HUMAN] Q92841 Probable ATP-dependent RNA helicase DDX17 OS=Homo 10.97 6.01 729 80.2 sapiens GN=DDX17 PE=1 SV=2 - [DDX17_HUMAN] Q9Y5J1 U3 small nucleolar RNA-associated protein 18 homolog 6.83 4.76 556 62.0 OS=Homo sapiens GN=UTP18 PE=1 SV=3 - [UTP18_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 8.27 2.43 375 41.7 SV=1 - [ACTB_HUMAN] Q96HC4 PDZ and LIM domain protein 5 OS=Homo sapiens 3.52 0.00 596 63.9 GN=PDLIM5 PE=1 SV=5 - [PDLI5_HUMAN] Q9NZI8 Insulin-like growth factor 2 mRNA-binding protein 1 4.85 0.00 577 63.4 OS=Homo sapiens GN=IGF2BP1 PE=1 SV=2 - [IF2B1_HUMAN] P33240 Cleavage stimulation factor subunit 2 OS=Homo sapiens 3.47 1.61 577 60.9 GN=CSTF2 PE=1 SV=1 - [CSTF2_HUMAN] O60506 Heterogeneous nuclear ribonucleoprotein Q OS=Homo 3.37 2.15 623 69.6 sapiens GN=SYNCRIP PE=1 SV=2 - [HNRPQ_HUMAN] P27694 Replication protein A 70 kDa DNA-binding subunit OS=Homo 3.90 0.00 616 68.1 sapiens GN=RPA1 PE=1 SV=2 - [RFA1_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 22.73 0.00 110 11.3 [DCD_HUMAN] Q96RE7 Nucleus accumbens-associated protein 1 OS=Homo sapiens 4.74 1.88 527 57.2 GN=NACC1 PE=1 SV=1 - [NACC1_HUMAN] P08621 U1 small nuclear ribonucleoprotein 70 kDa OS=Homo 3.89 4.29 437 51.5 sapiens GN=SNRNP70 PE=1 SV=2 - [RU17_HUMAN] P0DMV8 Heat shock 70 kDa protein 1A OS=Homo sapiens 7.64 3.93 641 70.0 GN=HSPA1A PE=1 SV=1 - [HS71A_HUMAN] P08670 Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 - 4.08 1.73 466 53.6 [VIME_HUMAN] O00425 Insulin-like growth factor 2 mRNA-binding protein 3 4.84 0.00 579 63.7 OS=Homo sapiens GN=IGF2BP3 PE=1 SV=2 - [IF2B3_HUMAN] Q9Y262 Eukaryotic translation initiation factor 3 subunit L OS=Homo 3.19 2.27 564 66.7 sapiens GN=EIF3L PE=1 SV=1 - [EIF3L_HUMAN] Q08211 ATP-dependent RNA helicase A OS=Homo sapiens GN=DHX9 1.81 3.16 1270 140.9 PE=1 SV=4 - [DHX9_HUMAN] P49748 Very long-chain specific acyl-CoA dehydrogenase, 3.36 2.02 655 70.3 mitochondrial OS=Homo sapiens GN=ACADVL PE=1 SV=1 - [ACADV_HUMAN] Q9UHD8 Septin-9 OS=Homo sapiens GN=SEPT9 PE=1 SV=2 - 3.24 586 65.4 [SEPT9_HUMAN] Q15291 Retinoblastoma-binding protein 5 OS=Homo sapiens 1.30 0.00 538 59.1 GN=RBBP5 PE=1 SV=2 - [RBBP5_HUMAN] O15371 Eukaryotic translation initiation factor 3 subunit D OS=Homo 2.55 2.00 548 63.9 sapiens GN=EIF3D PE=1 SV=1 - [EIF3D_HUMAN] Q9Y2X3 Nucleolar protein 58 OS=Homo sapiens GN=NOP58 PE=1 2.27 2.25 529 59.5 SV=1 - [NOP58_HUMAN] P54652 Heat shock-related 70 kDa protein 2 OS=Homo sapiens 4.85 1.98 639 70.0 GN=HSPA2 PE=1 SV=1 - [HSP72_HUMAN] Q13283 Ras GTPase-activating protein-binding protein 1 OS=Homo 2.36 1.75 466 52.1 sapiens GN=G3BP1 PE=1 SV=1 - [G3BP1_HUMAN] Q13485 Mothers against decapentaplegic homolog 4 OS=Homo 1.09 0.00 552 60.4 sapiens GN=SMAD4 PE=1 SV=1 - [SMAD4_HUMAN] P53350 Serine/threonine-protein kinase PLK1 OS=Homo sapiens 1.49 2.03 603 68.2 GN=PLK1 PE=1 SV=1 - [PLK1_HUMAN] Q9BZR8 Apoptosis facilitator Bcl-2-like protein 14 OS=Homo sapiens 1.83 1.88 327 36.6 GN=BCL2L14 PE=1 SV=1 - [B2L14_HUMAN] Q9NZN4 EH domain-containing protein 2 OS=Homo sapiens 1.47 0.00 543 61.1 GN=EHD2 PE=1 SV=2 - [EHD2_HUMAN] 85 Q9BVL2 Nucleoporin p58/p45 OS=Homo sapiens GN=NUPL1 PE=1 1.34 0.00 599 60.9 SV=1 - [NUPL1_HUMAN] P01768 Ig heavy chain V-III region CAM OS=Homo sapiens PE=1 7.38 0.00 122 13.7 SV=1 - [HV307_HUMAN] Q6L981 Synphilin-1 OS=Homo sapiens GN=SNCAIP PE=2 SV=1 - 7.95 0.00 88 10.2 [Q6L981_HUMAN] Q5T749 Keratinocyte proline-rich protein OS=Homo sapiens 1.55 0.00 579 64.1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN] Q8ND61-2 Isoform 2 of Uncharacterized protein C3orf20 OS=Homo 1.79 0.00 782 88.0 sapiens GN=C3orf20 - [CC020_HUMAN] A2A2Z9 Ankyrin repeat domain-containing protein 18B OS=Homo 1.19 0.00 1011 118.2 sapiens GN=ANKRD18B PE=1 SV=1 - [AN18B_HUMAN] B0QYT6 Ran GTPase-activating protein 1 (Fragment) OS=Homo 18.92 0.00 37 3.9 sapiens GN=RANGAP1 PE=1 SV=1 - [B0QYT6_HUMAN] Q00839 Heterogeneous nuclear ribonucleoprotein U OS=Homo 0.85 0.00 825 90.5 sapiens GN=HNRNPU PE=1 SV=6 - [HNRPU_HUMAN] O00712 Nuclear factor 1 B-type OS=Homo sapiens GN=NFIB PE=1 2.62 0.00 420 47.4 SV=2 - [NFIB_HUMAN] B4DXW6 cDNA FLJ50285, highly similar to Arginyl-tRNA synthetase 2.20 1.72 454 52.3 (EC 6.1.1.19) OS=Homo sapiens PE=2 SV=1 - [B4DXW6_HUMAN] F8WE98 Filamin-A (Fragment) OS=Homo sapiens GN=FLNA PE=1 1.16 0.00 604 66.6 SV=2 - [F8WE98_HUMAN] H3BUW6 Abscission/NoCut checkpoint regulator (Fragment) 12.28 1.73 57 6.4 OS=Homo sapiens GN=ZFYVE19 PE=1 SV=1 - [H3BUW6_HUMAN] Q53T09 Putative uncharacterized protein XRCC5 (Fragment) 1.41 0.00 568 64.2 OS=Homo sapiens GN=XRCC5 PE=4 SV=1 - [Q53T09_HUMAN] A0A087WW53 Coronin OS=Homo sapiens GN=CORO1B PE=1 SV=1 - 3.08 0.00 260 28.9 [A0A087WW53_HUMAN] Q93070 Ecto-ADP-ribosyltransferase 4 OS=Homo sapiens GN=ART4 2.23 314 35.9 PE=2 SV=2 - [NAR4_HUMAN] Q16630 Cleavage and polyadenylation specificity factor subunit 6 1.63 551 59.2 OS=Homo sapiens GN=CPSF6 PE=1 SV=2 - [CPSF6_HUMAN] Q14134 Tripartite motif-containing protein 29 OS=Homo sapiens 1.53 588 65.8 GN=TRIM29 PE=1 SV=2 - [TRI29_HUMAN] Q9H211 DNA replication factor Cdt1 OS=Homo sapiens GN=CDT1 1.83 546 60.4 PE=1 SV=3 - [CDT1_HUMAN] Q96ME7 Zinc finger protein 512 OS=Homo sapiens GN=ZNF512 PE=1 1.94 567 64.6 SV=2 - [ZN512_HUMAN] Q15233 Non-POU domain-containing octamer-binding protein 1.70 471 54.2 OS=Homo sapiens GN=NONO PE=1 SV=4 - [NONO_HUMAN] Q8N5A5 Zinc finger CCCH-type with G patch domain-containing 2.07 531 57.3 protein OS=Homo sapiens GN=ZGPAT PE=1 SV=3 - [ZGPAT_HUMAN] P12956 X-ray repair cross-complementing protein 6 OS=Homo 2.30 609 69.8 sapiens GN=XRCC6 PE=1 SV=2 - [XRCC6_HUMAN] O75083 WD repeat-containing protein 1 OS=Homo sapiens 1.32 606 66.2 GN=WDR1 PE=1 SV=4 - [WDR1_HUMAN] Q9BW27 Nuclear pore complex protein Nup85 OS=Homo sapiens 1.22 656 75.0 GN=NUP85 PE=1 SV=1 - [NUP85_HUMAN] Q96PX9 Pleckstrin homology domain-containing family G member 4B 0.79 1271 139.6 OS=Homo sapiens GN=PLEKHG4B PE=2 SV=4 - [PKH4B_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 33.85 54.89 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P08670 Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 - 31.76 32.78 466 53.6 [VIME_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 23.46 55.48 584 58.8 PE=1 SV=6 - [K1C10_HUMAN] 86 P08729 Keratin, type II cytoskeletal 7 OS=Homo sapiens GN=KRT7 30.28 35.05 469 51.4 PE=1 SV=5 - [K2C7_HUMAN] P05787 Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 32.51 27.21 483 53.7 PE=1 SV=7 - [K2C8_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 26.13 50.89 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P15924 Desmoplakin OS=Homo sapiens GN=DSP PE=1 SV=3 - 3.17 5.35 2871 331.6 [DESP_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 18.14 14.58 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 9.27 18.93 1014 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 23.56 21.17 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] Q16658 Fascin OS=Homo sapiens GN=FSCN1 PE=1 SV=3 - 14.60 8.05 493 54.5 [FSCN1_HUMAN] Q9ULV4 Coronin-1C OS=Homo sapiens GN=CORO1C PE=1 SV=1 - 8.86 11.56 474 53.2 [COR1C_HUMAN] Q96CW1 AP-2 complex subunit mu OS=Homo sapiens GN=AP2M1 9.66 7.19 435 49.6 PE=1 SV=2 - [AP2M1_HUMAN] O43660 Pleiotropic regulator 1 OS=Homo sapiens GN=PLRG1 PE=1 9.14 3.95 514 57.2 SV=1 - [PLRG1_HUMAN] Q9GZS1 DNA-directed RNA polymerase I subunit RPA49 OS=Homo 9.56 6.06 481 53.9 sapiens GN=POLR1E PE=1 SV=2 - [RPA49_HUMAN] Q9BQE3 Tubulin alpha-1C chain OS=Homo sapiens GN=TUBA1C 9.13 7.04 449 49.9 PE=1 SV=1 - [TBA1C_HUMAN] P02538 Keratin, type II cytoskeletal 6A OS=Homo sapiens 16.13 23.16 564 60.0 GN=KRT6A PE=1 SV=3 - [K2C6A_HUMAN] P14923 Junction plakoglobin OS=Homo sapiens GN=JUP PE=1 SV=3 6.17 3.57 745 81.7 - [PLAK_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 6.24 5.83 609 69.3 [ALBU_HUMAN] Q9UMS4 Pre-mRNA-processing factor 19 OS=Homo sapiens 6.15 4.38 504 55.1 GN=PRPF19 PE=1 SV=1 - [PRP19_HUMAN] P31943 Heterogeneous nuclear ribonucleoprotein H OS=Homo 7.57 2.61 449 49.2 sapiens GN=HNRNPH1 PE=1 SV=4 - [HNRH1_HUMAN] P02545 Prelamin-A/C OS=Homo sapiens GN=LMNA PE=1 SV=1 - 4.97 1.61 664 74.1 [LMNA_HUMAN] Q02413 Desmoglein-1 OS=Homo sapiens GN=DSG1 PE=1 SV=2 - 3.24 2.06 1049 113.7 [DSG1_HUMAN] P42167 Lamina-associated polypeptide 2, isoforms beta/gamma 7.93 2.42 454 50.6 OS=Homo sapiens GN=TMPO PE=1 SV=2 - [LAP2B_HUMAN] P26599 Polypyrimidine tract-binding protein 1 OS=Homo sapiens 5.46 3.95 531 57.2 GN=PTBP1 PE=1 SV=1 - [PTBP1_HUMAN] Q04695 Keratin, type I cytoskeletal 17 OS=Homo sapiens GN=KRT17 7.18 1.78 432 48.1 PE=1 SV=2 - [K1C17_HUMAN] Q8N1N4 Keratin, type II cytoskeletal 78 OS=Homo sapiens 6.35 3.92 520 56.8 GN=KRT78 PE=2 SV=2 - [K2C78_HUMAN] Q5T749 Keratinocyte proline-rich protein OS=Homo sapiens 5.53 8.15 579 64.1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN] P19338 Nucleolin OS=Homo sapiens GN=NCL PE=1 SV=3 - 3.52 3.74 710 76.6 [NUCL_HUMAN] Q8NC51 Plasminogen activator inhibitor 1 RNA-binding protein 9.80 4.59 408 44.9 OS=Homo sapiens GN=SERBP1 PE=1 SV=2 - [PAIRB_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 5.33 3.87 375 41.7 SV=1 - [ACTB_HUMAN] Q969G3 SWI/SNF-related matrix-associated actin-dependent 5.60 1.77 411 46.6 regulator of chromatin subfamily E member 1 OS=Homo sapiens GN=SMARCE1 PE=1 SV=2 - [SMCE1_HUMAN] P08651 Nuclear factor 1 C-type OS=Homo sapiens GN=NFIC PE=1 4.33 0.00 508 55.6 SV=2 - [NFIC_HUMAN]

87 P14868 Aspartate--tRNA ligase, cytoplasmic OS=Homo sapiens 3.59 1.81 501 57.1 GN=DARS PE=1 SV=2 - [SYDC_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 8.67 8.21 473 51.2 PE=1 SV=4 - [K1C16_HUMAN] Q9Y512 Sorting and assembly machinery component 50 homolog 2.99 0.00 469 51.9 OS=Homo sapiens GN=SAMM50 PE=1 SV=3 - [SAM50_HUMAN] Q9P258 Protein RCC2 OS=Homo sapiens GN=RCC2 PE=1 SV=2 - 4.41 2.52 522 56.0 [RCC2_HUMAN] P17844-2 Isoform 2 of Probable ATP-dependent RNA helicase DDX5 1.68 0.00 535 60.5 OS=Homo sapiens GN=DDX5 - [DDX5_HUMAN] P68371 Tubulin beta-4B chain OS=Homo sapiens GN=TUBB4B PE=1 11.46 5.81 445 49.8 SV=1 - [TBB4B_HUMAN] P07437 Tubulin beta chain OS=Homo sapiens GN=TUBB PE=1 SV=2 11.49 3.60 444 49.6 - [TBB5_HUMAN] P28347 Transcriptional enhancer factor TEF-1 OS=Homo sapiens 1.88 0.00 426 47.9 GN=TEAD1 PE=1 SV=2 - [TEAD1_HUMAN] P36578 60S ribosomal protein L4 OS=Homo sapiens GN=RPL4 PE=1 2.58 2.43 427 47.7 SV=5 - [RL4_HUMAN] Q9Y265 RuvB-like 1 OS=Homo sapiens GN=RUVBL1 PE=1 SV=1 - 2.63 0.00 456 50.2 [RUVB1_HUMAN] Q5T750 Skin-specific protein 32 OS=Homo sapiens GN=XP32 PE=1 6.00 0.00 250 26.2 SV=1 - [XP32_HUMAN] Q9NR12 PDZ and LIM domain protein 7 OS=Homo sapiens 1.53 1.70 457 49.8 GN=PDLIM7 PE=1 SV=1 - [PDLI7_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 10.00 1.99 110 11.3 [DCD_HUMAN] Q9BQ67 Glutamate-rich WD repeat-containing protein 1 OS=Homo 2.02 1.72 446 49.4 sapiens GN=GRWD1 PE=1 SV=1 - [GRWD1_HUMAN] Q15007 Pre-mRNA-splicing regulator WTAP OS=Homo sapiens 2.78 1.80 396 44.2 GN=WTAP PE=1 SV=2 - [FL2D_HUMAN] Q92734 Protein TFG OS=Homo sapiens GN=TFG PE=1 SV=2 - 2.50 0.00 400 43.4 [TFG_HUMAN] A6NMY6 Putative annexin A2-like protein OS=Homo sapiens 2.95 2.09 339 38.6 GN=ANXA2P2 PE=5 SV=2 - [AXA2L_HUMAN] Q7Z794 Keratin, type II cytoskeletal 1b OS=Homo sapiens 5.54 6.80 578 61.9 GN=KRT77 PE=2 SV=3 - [K2C1B_HUMAN] P22531 Small proline-rich protein 2E OS=Homo sapiens GN=SPRR2E 25.00 3.07 72 7.8 PE=2 SV=2 - [SPR2E_HUMAN] Q5D862 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 SV=1 - 0.42 0.00 2391 247.9 [FILA2_HUMAN] Q01196 Runt-related transcription factor 1 OS=Homo sapiens 1.99 0.00 453 48.7 GN=RUNX1 PE=1 SV=3 - [RUNX1_HUMAN] Q92599 Septin-8 OS=Homo sapiens GN=SEPT8 PE=1 SV=4 - 1.86 1.63 483 55.7 [SEPT8_HUMAN] Q14318 Peptidyl-prolyl cis-trans isomerase FKBP8 OS=Homo sapiens 1.94 1.88 412 44.5 GN=FKBP8 PE=1 SV=2 - [FKBP8_HUMAN] Q8WVV4 Protein POF1B OS=Homo sapiens GN=POF1B PE=1 SV=3 - 1.70 0.00 589 68.0 [POF1B_HUMAN] Q8IYW2 Cilia- and flagella-associated protein 46 OS=Homo sapiens 0.29 0.00 2715 303.3 GN=CFAP46 PE=2 SV=3 - [CFA46_HUMAN] P23490 Loricrin OS=Homo sapiens GN=LOR PE=1 SV=2 - 4.17 3.13 312 25.7 [LORI_HUMAN] B2RDL4 cDNA, FLJ96667 OS=Homo sapiens PE=2 SV=1 - 1.32 0.00 684 77.2 [B2RDL4_HUMAN] J3KSD2 Cytoplasmic dynein 1 light intermediate chain 2 (Fragment) 14.29 0.00 49 5.7 OS=Homo sapiens GN=DYNC1LI2 PE=1 SV=1 - [J3KSD2_HUMAN] L8ECB5 Alternative protein LARGE OS=Homo sapiens GN=LARGE 35.05 0.00 97 11.1 PE=4 SV=1 - [L8ECB5_HUMAN] A0A087WUG8 Tubulin polyglutamylase TTLL11 (Fragment) OS=Homo 12.12 0.00 66 7.6 sapiens GN=TTLL11 PE=4 SV=1 - [A0A087WUG8_HUMAN]

88 P68104 Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 2.38 462 50.1 PE=1 SV=1 - [EF1A1_HUMAN] P05388 60S acidic ribosomal protein P0 OS=Homo sapiens 2.21 317 34.3 GN=RPLP0 PE=1 SV=1 - [RLA0_HUMAN] Q15646 2'-5'-oligoadenylate synthase-like protein OS=Homo sapiens 2.14 514 59.2 GN=OASL PE=1 SV=2 - [OASL_HUMAN] Q08554 Desmocollin-1 OS=Homo sapiens GN=DSC1 PE=1 SV=2 - 1.68 894 99.9 [DSC1_HUMAN] Q15366 Poly(rC)-binding protein 2 OS=Homo sapiens GN=PCBP2 3.01 365 38.6 PE=1 SV=1 - [PCBP2_HUMAN] P78371 T-complex protein 1 subunit beta OS=Homo sapiens 2.99 535 57.5 GN=CCT2 PE=1 SV=4 - [TCPB_HUMAN] Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 1.68 2850 282.2 [HORN_HUMAN] P25705 ATP synthase subunit alpha, mitochondrial OS=Homo 1.27 553 59.7 sapiens GN=ATP5A1 PE=1 SV=1 - [ATPA_HUMAN] P26641 Elongation factor 1-gamma OS=Homo sapiens GN=EEF1G 3.20 437 50.1 PE=1 SV=3 - [EF1G_HUMAN] Q68E01-4 Isoform 4 of Integrator complex subunit 3 OS=Homo sapiens 6.85 0.00 555 63.5 GN=INTS3 - [INT3_HUMAN] A0A075B6Z2 Protein TRAJ56 (Fragment) OS=Homo sapiens GN=TRAJ56 38.10 0.00 21 2.2 PE=4 SV=1 - [A0A075B6Z2_HUMAN] B4DVQ0 cDNA FLJ58286, highly similar to Actin, cytoplasmic 2 15.32 8.05 333 37.3 OS=Homo sapiens PE=2 SV=1 - [B4DVQ0_HUMAN] B4E0A5 cDNA FLJ51425, highly similar to Deoxycytidine kinase (EC 10.11 0.00 188 22.5 2.7.1.74) OS=Homo sapiens PE=2 SV=1 - [B4E0A5_HUMAN] Q99062-2 Isoform 2 of Granulocyte colony-stimulating factor receptor 4.54 0.00 771 85.1 OS=Homo sapiens GN=CSF3R - [CSF3R_HUMAN] P04406 Glyceraldehyde-3-phosphate dehydrogenase OS=Homo 36.72 10.44 335 36.0 sapiens GN=GAPDH PE=1 SV=3 - [G3P_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 17.29 16.54 584 58.8 PE=1 SV=6 - [K1C10_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 15.53 17.83 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 14.61 12.18 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P29692 Elongation factor 1-delta OS=Homo sapiens GN=EEF1D 12.10 0.00 281 31.1 PE=1 SV=5 - [EF1D_HUMAN] P06748 Nucleophosmin OS=Homo sapiens GN=NPM1 PE=1 SV=2 - 11.90 0.00 294 32.6 [NPM_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 9.86 6.03 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P35249 Replication factor C subunit 4 OS=Homo sapiens GN=RFC4 7.16 0.00 363 39.7 PE=1 SV=2 - [RFC4_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 3.22 0.00 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P22626 Heterogeneous nuclear ribonucleoproteins A2/B1 OS=Homo 5.67 0.00 353 37.4 sapiens GN=HNRNPA2B1 PE=1 SV=2 - [ROA2_HUMAN] P40937 Replication factor C subunit 5 OS=Homo sapiens GN=RFC5 4.71 340 38.5 PE=1 SV=1 - [RFC5_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 10.00 1.79 110 11.3 [DCD_HUMAN] O00232-2 Isoform 2 of 26S proteasome non-ATPase regulatory subunit 2.52 0.00 436 50.5 12 OS=Homo sapiens GN=PSMD12 - [PSD12_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 7.20 5.87 472 51.5 PE=1 SV=4 - [K1C14_HUMAN] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 1.28 2.66 1014 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] B7Z4V7 cDNA FLJ50087 OS=Homo sapiens PE=2 SV=1 - 1.41 0.00 568 64.2 [B7Z4V7_HUMAN] Q5QPM0 RNA-binding protein Raly (Fragment) OS=Homo sapiens 5.26 0.00 171 18.6 GN=RALY PE=1 SV=6 - [Q5QPM0_HUMAN]

89 L0R5A1 Alternative protein CSF2RB OS=Homo sapiens GN=CSF2RB 7.41 1.61 108 11.6 PE=4 SV=1 - [L0R5A1_HUMAN] A0A087WWT3 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1 - 3.79 0.00 396 45.1 [A0A087WWT3_HUMAN] A0A075B6Z2 Protein TRAJ56 (Fragment) OS=Homo sapiens GN=TRAJ56 38.10 0.00 21 2.2 PE=4 SV=1 - [A0A075B6Z2_HUMAN] Q562R1 Beta-actin-like protein 2 OS=Homo sapiens GN=ACTBL2 4.79 376 42.0 PE=1 SV=2 - [ACTBL_HUMAN] P36873 Serine/threonine-protein phosphatase PP1-gamma catalytic 3.10 323 37.0 subunit OS=Homo sapiens GN=PPP1CC PE=1 SV=1 - [PP1G_HUMAN] Q99729 Heterogeneous nuclear ribonucleoprotein A/B OS=Homo 3.01 332 36.2 sapiens GN=HNRNPAB PE=1 SV=2 - [ROAA_HUMAN] P51991 Heterogeneous nuclear ribonucleoprotein A3 OS=Homo 2.38 378 39.6 sapiens GN=HNRNPA3 PE=1 SV=2 - [ROA3_HUMAN] P08670 Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 - 60.30 81.04 466 53.6 [VIME_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 45.65 71.20 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 32.86 38.86 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 28.08 26.58 584 58.8 PE=1 SV=6 - [K1C10_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 30.18 32.08 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P61978 Heterogeneous nuclear ribonucleoprotein K OS=Homo 21.38 4.26 463 50.9 sapiens GN=HNRNPK PE=1 SV=1 - [HNRPK_HUMAN] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 10.95 13.58 1014 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] Q15233 Non-POU domain-containing octamer-binding protein 14.01 12.14 471 54.2 OS=Homo sapiens GN=NONO PE=1 SV=4 - [NONO_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 6.44 8.10 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 4.76 4.29 609 69.3 [ALBU_HUMAN] P26599 Polypyrimidine tract-binding protein 1 OS=Homo sapiens 5.27 1.86 531 57.2 GN=PTBP1 PE=1 SV=1 - [PTBP1_HUMAN] P14618 Pyruvate kinase PKM OS=Homo sapiens GN=PKM PE=1 5.46 4.38 531 57.9 SV=4 - [KPYM_HUMAN] P48643 T-complex protein 1 subunit epsilon OS=Homo sapiens 5.18 541 59.6 GN=CCT5 PE=1 SV=1 - [TCPE_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 7.73 3.03 375 41.7 SV=1 - [ACTB_HUMAN] Q13547 Histone deacetylase 1 OS=Homo sapiens GN=HDAC1 PE=1 3.73 0.00 482 55.1 SV=1 - [HDAC1_HUMAN] P10809 60 kDa heat shock protein, mitochondrial OS=Homo sapiens 5.93 2.80 573 61.0 GN=HSPD1 PE=1 SV=2 - [CH60_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 2.09 304 32.5 [TRY3_HUMAN] O43172 U4/U6 small nuclear ribonucleoprotein Prp4 OS=Homo 1.92 0.00 522 58.4 sapiens GN=PRPF4 PE=1 SV=2 - [PRP4_HUMAN] P17987 T-complex protein 1 subunit alpha OS=Homo sapiens 1.80 2.26 556 60.3 GN=TCP1 PE=1 SV=1 - [TCPA_HUMAN] P49368 T-complex protein 1 subunit gamma OS=Homo sapiens 2.02 2.47 545 60.5 GN=CCT3 PE=1 SV=4 - [TCPG_HUMAN] B4DQJ6 cDNA FLJ51276, highly similar to Secretogranin-2 OS=Homo 2.31 0.00 477 54.2 sapiens PE=2 SV=1 - [B4DQJ6_HUMAN] B7Z2F4 T-complex protein 1 subunit delta OS=Homo sapiens PE=2 1.80 1.80 389 42.3 SV=1 - [B7Z2F4_HUMAN] M0QZ34 Glioma tumor suppressor candidate region gene 2 protein 4.00 1.84 200 23.5 (Fragment) OS=Homo sapiens GN=GLTSCR2 PE=1 SV=1 - [M0QZ34_HUMAN] 90 P68104 Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 2.38 462 50.1 PE=1 SV=1 - [EF1A1_HUMAN] P17844 Probable ATP-dependent RNA helicase DDX5 OS=Homo 1.95 614 69.1 sapiens GN=DDX5 PE=1 SV=1 - [DDX5_HUMAN] P07477 Trypsin-1 OS=Homo sapiens GN=PRSS1 PE=1 SV=1 - 2.43 247 26.5 [TRY1_HUMAN] P02545 Prelamin-A/C OS=Homo sapiens GN=LMNA PE=1 SV=1 - 1.51 664 74.1 [LMNA_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 10.00 110 11.3 [DCD_HUMAN] P14866 Heterogeneous nuclear ribonucleoprotein L OS=Homo 1.53 589 64.1 sapiens GN=HNRNPL PE=1 SV=2 - [HNRPL_HUMAN] P26368 Splicing factor U2AF 65 kDa subunit OS=Homo sapiens 2.11 475 53.5 GN=U2AF2 PE=1 SV=4 - [U2AF2_HUMAN] Q9UHX1 Poly(U)-binding-splicing factor PUF60 OS=Homo sapiens 1.79 559 59.8 GN=PUF60 PE=1 SV=1 - [PUF60_HUMAN] Q8NBI6 Xyloside xylosyltransferase 1 OS=Homo sapiens GN=XXYLT1 1.27 393 43.8 PE=1 SV=1 - [XXLT1_HUMAN] Q1ED39 Lysine-rich nucleolar protein 1 OS=Homo sapiens 2.40 458 51.6 GN=KNOP1 PE=1 SV=1 - [KNOP1_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 39.60 91.84 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 32.53 44.20 584 58.8 PE=1 SV=6 - [K1C10_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 23.92 38.99 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 34.27 48.10 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] Q9BQE3 Tubulin alpha-1C chain OS=Homo sapiens GN=TUBA1C 25.17 16.38 449 49.9 PE=1 SV=1 - [TBA1C_HUMAN] P09874 Poly [ADP-ribose] polymerase 1 OS=Homo sapiens 7.69 16.58 1014 113.0 GN=PARP1 PE=1 SV=4 - [PARP1_HUMAN] Q04695 Keratin, type I cytoskeletal 17 OS=Homo sapiens GN=KRT17 24.07 28.02 432 48.1 PE=1 SV=2 - [K1C17_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 18.93 10.08 375 41.7 SV=1 - [ACTB_HUMAN] P31943 Heterogeneous nuclear ribonucleoprotein H OS=Homo 19.60 19.87 449 49.2 sapiens GN=HNRNPH1 PE=1 SV=4 - [HNRH1_HUMAN] Q96CW1 AP-2 complex subunit mu OS=Homo sapiens GN=AP2M1 9.20 3.78 435 49.6 PE=1 SV=2 - [AP2M1_HUMAN] P36578 60S ribosomal protein L4 OS=Homo sapiens GN=RPL4 PE=1 13.58 11.98 427 47.7 SV=5 - [RL4_HUMAN] O00148 ATP-dependent RNA helicase DDX39A OS=Homo sapiens 12.18 9.77 427 49.1 GN=DDX39A PE=1 SV=2 - [DX39A_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 15.08 27.44 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] Q16181 Septin-7 OS=Homo sapiens GN=SEPT7 PE=1 SV=2 - 6.64 4.31 437 50.6 [SEPT7_HUMAN] P04350 Tubulin beta-4A chain OS=Homo sapiens GN=TUBB4A PE=1 7.43 5.32 444 49.6 SV=2 - [TBB4A_HUMAN] Q9H0S4 Probable ATP-dependent RNA helicase DDX47 OS=Homo 7.91 5.33 455 50.6 sapiens GN=DDX47 PE=1 SV=1 - [DDX47_HUMAN] Q16658 Fascin OS=Homo sapiens GN=FSCN1 PE=1 SV=3 - 7.91 2.68 493 54.5 [FSCN1_HUMAN] P06733 Alpha-enolase OS=Homo sapiens GN=ENO1 PE=1 SV=2 - 5.53 1.66 434 47.1 [ENOA_HUMAN] P05787 Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 12.01 14.88 483 53.7 PE=1 SV=7 - [K2C8_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 4.43 8.94 609 69.3 [ALBU_HUMAN] P68104 Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 4.55 5.50 462 50.1 PE=1 SV=1 - [EF1A1_HUMAN]

91 P06702 Protein S100-A9 OS=Homo sapiens GN=S100A9 PE=1 SV=1 24.56 2.56 114 13.2 - [S10A9_HUMAN] P02538 Keratin, type II cytoskeletal 6A OS=Homo sapiens 14.01 21.21 564 60.0 GN=KRT6A PE=1 SV=3 - [K2C6A_HUMAN] Q9P0V9 Septin-10 OS=Homo sapiens GN=SEPT10 PE=1 SV=2 - 4.85 4.18 454 52.6 [SEP10_HUMAN] Q16576 Histone-binding protein RBBP7 OS=Homo sapiens 4.47 0.00 425 47.8 GN=RBBP7 PE=1 SV=1 - [RBBP7_HUMAN] B3KRK8 cDNA FLJ34494 fis, clone HLUNG2005030, highly similar to 6.14 2.00 407 46.9 VIMENTIN OS=Homo sapiens PE=2 SV=1 - [B3KRK8_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 2.65 304 32.5 [TRY3_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 10.00 2.41 110 11.3 [DCD_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 15.43 14.78 473 51.2 PE=1 SV=4 - [K1C16_HUMAN] P08729 Keratin, type II cytoskeletal 7 OS=Homo sapiens GN=KRT7 5.54 4.87 469 51.4 PE=1 SV=5 - [K2C7_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 18.86 21.13 472 51.5 PE=1 SV=4 - [K1C14_HUMAN] Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 1.23 1.82 2850 282.2 [HORN_HUMAN] P26641 Elongation factor 1-gamma OS=Homo sapiens GN=EEF1G 2.97 2.44 437 50.1 PE=1 SV=3 - [EF1G_HUMAN] H0YJH9 Polyadenylate-binding protein 2 (Fragment) OS=Homo 11.46 1.93 96 11.1 sapiens GN=PABPN1 PE=1 SV=1 - [H0YJH9_HUMAN] Q68D69 Putative uncharacterized protein DKFZp779G1236 OS=Homo 0.89 1.91 1014 113.3 sapiens GN=DKFZp779G1236 PE=2 SV=1 - [Q68D69_HUMAN] P56182 Ribosomal RNA processing protein 1 homolog A OS=Homo 1.95 461 52.8 sapiens GN=RRP1 PE=1 SV=1 - [RRP1_HUMAN]

Table 2.3 Mass Spectrometry Results from 10x Competition reaction. Results are

sorted by relative abundance of the protein, with coverage and score shown.

Score # MW Accession Description ΣCoverage A2 AAs [kDa] H6VRG2 Keratin 1 OS=Homo sapiens GN=KRT1 PE=3 SV=1 - 41.93 114.59 644 66.0 [H6VRG2_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 36.31 63.92 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 31.34 54.84 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 34.03 42.07 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 20.34 31.87 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 20.34 21.34 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 22.73 4.76 110 11.3 [DCD_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 3.61 3.79 609 69.3 [ALBU_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 2.68 304 32.5 [TRY3_HUMAN] Q08554 Desmocollin-1 OS=Homo sapiens GN=DSC1 PE=1 SV=2 - 1.68 2.54 894 99.9 [DSC1_HUMAN] P15924-2 Isoform DPII of Desmoplakin OS=Homo sapiens GN=DSP - 0.44 0.00 2272 260.0 [DESP_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 13.11 15.93 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN] P47929 Galectin-7 OS=Homo sapiens GN=LGALS7 PE=1 SV=2 - 8.09 2.06 136 15.1 [LEG7_HUMAN] Q5D862 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 SV=1 - 0.50 2.34 2391 247.9 [FILA2_HUMAN] Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 1.23 0.00 2850 282.2 [HORN_HUMAN] Q8N1N4 Keratin, type II cytoskeletal 78 OS=Homo sapiens 3.08 3.77 520 56.8 GN=KRT78 PE=2 SV=2 - [K2C78_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 2.67 375 41.7 SV=1 - [ACTB_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 36.18 80.24 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 45.54 83.28 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 26.37 49.42 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 15.73 19.58 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P08670 Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 - 15.24 7.83 466 53.6 [VIME_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 13.39 30.93 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 5.58 5.79 609 69.3 [ALBU_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 11.02 9.89 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] 93 P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 2.57 304 32.5 [TRY3_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 7.61 4.81 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN] Q5D862 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 SV=1 - 0.50 2.23 2391 247.9 [FILA2_HUMAN] Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 1.68 0.00 2850 282.2 [HORN_HUMAN] Q5T749 Keratinocyte proline-rich protein OS=Homo sapiens 1.38 0.00 579 64.1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN] F8WEK9 Serotransferrin OS=Homo sapiens GN=TF PE=1 SV=1 - 10.43 0.00 115 12.5 [F8WEK9_HUMAN] Q59GD4 Collagen, type V, alpha 3 preproprotein variant (Fragment) 0.84 0.00 1658 162.7 OS=Homo sapiens PE=2 SV=1 - [Q59GD4_HUMAN] K7EK59 Peroxisomal membrane protein PEX14 (Fragment) 3.75 0.00 160 17.4 OS=Homo sapiens GN=PEX14 PE=1 SV=1 - [K7EK59_HUMAN] P68104 Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 2.38 462 50.1 PE=1 SV=1 - [EF1A1_HUMAN] P32119 Peroxiredoxin-2 OS=Homo sapiens GN=PRDX2 PE=1 SV=5 5.56 198 21.9 - [PRDX2_HUMAN] Q8TF72 Protein Shroom3 OS=Homo sapiens GN=SHROOM3 PE=1 0.45 1996 216.7 SV=2 - [SHRM3_HUMAN] P25311 Zinc-alpha-2-glycoprotein OS=Homo sapiens GN=AZGP1 3.36 298 34.2 PE=1 SV=2 - [ZA2G_HUMAN] P04406 Glyceraldehyde-3-phosphate dehydrogenase OS=Homo 2.39 335 36.0 sapiens GN=GAPDH PE=1 SV=3 - [G3P_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 43.63 61.93 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 33.39 23.38 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 27.57 25.55 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 28.17 16.67 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 9.96 5.68 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 6.27 4.63 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 10.00 1.88 110 11.3 [DCD_HUMAN] Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 1.23 0.00 2850 282.2 [HORN_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 304 32.5 [TRY3_HUMAN] H6VRF8 Keratin 1 OS=Homo sapiens GN=KRT1 PE=3 SV=1 - 10.25 4.08 644 66.0 [H6VRF8_HUMAN] E9PIQ7 HCLS1-associated protein X-1 OS=Homo sapiens GN=HAX1 16.56 0.00 151 17.2 PE=1 SV=1 - [E9PIQ7_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 1.56 2.10 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] B4DWU0 cDNA FLJ56791, highly similar to Keratin, type I 8.89 2.00 135 15.7 cytoskeletal 16 OS=Homo sapiens PE=2 SV=1 - [B4DWU0_HUMAN] K7EQQ3 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 4.10 0.00 390 39.5 PE=1 SV=1 - [K7EQQ3_HUMAN] Q562L5 Actin-like protein (Fragment) OS=Homo sapiens GN=ACT 17.48 0.00 103 11.5 PE=3 SV=1 - [Q562L5_HUMAN] A0A075B6Z2 Protein TRAJ56 (Fragment) OS=Homo sapiens GN=TRAJ56 38.10 0.00 21 2.2 PE=4 SV=1 - [A0A075B6Z2_HUMAN] H6VRF8 Keratin 1 OS=Homo sapiens GN=KRT1 PE=3 SV=1 - 21.74 39.01 644 66.0 [H6VRF8_HUMAN]

94 P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 14.77 14.91 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 13.53 16.72 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 11.89 14.36 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] C9JKR2 Albumin, isoform CRA_k OS=Homo sapiens GN=ALB PE=1 5.28 2.98 417 47.3 SV=1 - [C9JKR2_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 5.93 5.96 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] Q86W20 Protease serine 1 (Fragment) OS=Homo sapiens 15.48 1.65 84 9.2 GN=PRSS1 PE=3 SV=1 - [Q86W20_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 56.06 122.26 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 45.91 89.04 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 52.11 105.06 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 52.23 78.01 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 25.25 47.68 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 35.73 38.58 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN] P01040 Cystatin-A OS=Homo sapiens GN=CSTA PE=1 SV=1 - 48.98 2.57 98 11.0 [CYTA_HUMAN] P05787 Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 18.84 20.47 483 53.7 PE=1 SV=7 - [K2C8_HUMAN] Q3SY84 Keratin, type II cytoskeletal 71 OS=Homo sapiens 8.60 11.31 523 57.3 GN=KRT71 PE=1 SV=3 - [K2C71_HUMAN] Q7Z3Y8 Keratin, type I cytoskeletal 27 OS=Homo sapiens 10.46 11.45 459 49.8 GN=KRT27 PE=1 SV=2 - [K1C27_HUMAN] Q5T749 Keratinocyte proline-rich protein OS=Homo sapiens 5.70 7.60 579 64.1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 5.91 9.07 609 69.3 [ALBU_HUMAN] P31944 Caspase-14 OS=Homo sapiens GN=CASP14 PE=1 SV=2 - 7.85 4.48 242 27.7 [CASPE_HUMAN] P06702 Protein S100-A9 OS=Homo sapiens GN=S100A9 PE=1 24.56 2.24 114 13.2 SV=1 - [S10A9_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 17.27 6.12 110 11.3 [DCD_HUMAN] P04259 Keratin, type II cytoskeletal 6B OS=Homo sapiens 29.79 51.89 564 60.0 GN=KRT6B PE=1 SV=5 - [K2C6B_HUMAN] P08729 Keratin, type II cytoskeletal 7 OS=Homo sapiens GN=KRT7 11.94 9.65 469 51.4 PE=1 SV=5 - [K2C7_HUMAN] P08670 Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 - 6.01 2.42 466 53.6 [VIME_HUMAN] P14923 Junction plakoglobin OS=Homo sapiens GN=JUP PE=1 3.36 1.69 745 81.7 SV=3 - [PLAK_HUMAN] P05109 Protein S100-A8 OS=Homo sapiens GN=S100A8 PE=1 11.83 2.25 93 10.8 SV=1 - [S10A8_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 3.17 304 32.5 [TRY3_HUMAN] P04406 Glyceraldehyde-3-phosphate dehydrogenase OS=Homo 4.48 3.21 335 36.0 sapiens GN=GAPDH PE=1 SV=3 - [G3P_HUMAN] P02538 Keratin, type II cytoskeletal 6A OS=Homo sapiens 26.60 51.04 564 60.0 GN=KRT6A PE=1 SV=3 - [K2C6A_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 25.85 22.82 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] Q5D862 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 SV=1 - 0.46 0.00 2391 247.9 [FILA2_HUMAN]

95 Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 1.68 3.80 2850 282.2 [HORN_HUMAN] Q04695 Keratin, type I cytoskeletal 17 OS=Homo sapiens 14.81 13.66 432 48.1 GN=KRT17 PE=1 SV=2 - [K1C17_HUMAN] I3L1U1 Misshapen-like kinase 1 (Fragment) OS=Homo sapiens 4.35 0.00 161 18.1 GN=MINK1 PE=4 SV=1 - [I3L1U1_HUMAN] S4R3R4 Protein lin-7 homolog B (Fragment) OS=Homo sapiens 5.22 0.00 115 12.9 GN=LIN7B PE=4 SV=1 - [S4R3R4_HUMAN] Q02413 Desmoglein-1 OS=Homo sapiens GN=DSG1 PE=1 SV=2 - 0.95 1049 113.7 [DSG1_HUMAN] P25311 Zinc-alpha-2-glycoprotein OS=Homo sapiens GN=AZGP1 3.36 298 34.2 PE=1 SV=2 - [ZA2G_HUMAN] Q9BRR6 ADP-dependent glucokinase OS=Homo sapiens GN=ADPGK 1.01 497 54.1 PE=1 SV=1 - [ADPGK_HUMAN] P0CG48 Polyubiquitin-C OS=Homo sapiens GN=UBC PE=1 SV=3 - 21.02 685 77.0 [UBC_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 37.25 39.31 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 27.61 32.01 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 31.16 24.53 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 31.68 52.20 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 15.08 9.15 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 12.92 10.20 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - 3.61 2.34 609 69.3 [ALBU_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 0.00 304 32.5 [TRY3_HUMAN] P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2 - 10.00 0.00 110 11.3 [DCD_HUMAN] P09923 Intestinal-type alkaline phosphatase OS=Homo sapiens 1.70 0.00 528 56.8 GN=ALPI PE=1 SV=2 - [PPBI_HUMAN] Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 - 1.23 0.00 2850 282.2 [HORN_HUMAN] Q6UWE9 SFVP2550 OS=Homo sapiens GN=UNQ2550 PE=2 SV=1 - 7.77 0.00 103 11.6 [Q6UWE9_HUMAN] A0A0A0MR98 NAD kinase OS=Homo sapiens GN=NADK PE=1 SV=1 - 3.62 0.00 414 45.2 [A0A0A0MR98_HUMAN] L0R5A1 Alternative protein CSF2RB OS=Homo sapiens GN=CSF2RB 7.41 0.00 108 11.6 PE=4 SV=1 - [L0R5A1_HUMAN] Q12912 Lymphoid-restricted membrane protein OS=Homo sapiens 1.44 555 62.1 GN=LRMP PE=1 SV=3 - [LRMP_HUMAN] H6VRF8 Keratin 1 OS=Homo sapiens GN=KRT1 PE=3 SV=1 - 8.70 6.48 644 66.0 [H6VRF8_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 8.14 1.72 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 5.99 1.65 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] H7BZ93 Histone-lysine N-methyltransferase SETD2 (Fragment) 1.01 0.00 1290 145.5 OS=Homo sapiens GN=SETD2 PE=1 SV=1 - [H7BZ93_HUMAN] Q08378-4 Isoform 3 of Golgin subfamily A member 3 OS=Homo 2.47 0.00 1134 126.1 sapiens GN=GOLGA3 - [GOGA3_HUMAN] F8VRN8 LIM domain and actin-binding protein 1 OS=Homo sapiens 3.61 0.00 388 43.2 GN=LIMA1 PE=1 SV=1 - [F8VRN8_HUMAN] K7EQQ3 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 4.10 0.00 390 39.5 PE=1 SV=1 - [K7EQQ3_HUMAN] A0A087WWT3 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1 - 3.79 0.00 396 45.1 [A0A087WWT3_HUMAN] 96 A0A075B6Z2 Protein TRAJ56 (Fragment) OS=Homo sapiens GN=TRAJ56 38.10 0.00 21 2.2 PE=4 SV=1 - [A0A075B6Z2_HUMAN] P15924 Desmoplakin OS=Homo sapiens GN=DSP PE=1 SV=3 - 19.78 130.38 2871 331.6 [DESP_HUMAN] P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 54.50 252.06 644 66.0 PE=1 SV=6 - [K2C1_HUMAN] P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 69.86 202.62 584 58.8 GN=KRT10 PE=1 SV=6 - [K1C10_HUMAN] P35908 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens 70.58 203.27 639 65.4 GN=KRT2 PE=1 SV=2 - [K22E_HUMAN] P14923 Junction plakoglobin OS=Homo sapiens GN=JUP PE=1 26.17 52.65 745 81.7 SV=3 - [PLAK_HUMAN] P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 41.86 107.12 590 62.3 PE=1 SV=3 - [K2C5_HUMAN] P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 31.78 53.20 623 62.0 PE=1 SV=3 - [K1C9_HUMAN] P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 43.43 54.77 472 51.5 GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] Q5T749 Keratinocyte proline-rich protein OS=Homo sapiens 13.47 31.56 579 64.1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN] P47929 Galectin-7 OS=Homo sapiens GN=LGALS7 PE=1 SV=2 - 44.12 17.62 136 15.1 [LEG7_HUMAN] Q5D862 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 SV=1 - 7.03 12.76 2391 247.9 [FILA2_HUMAN] P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 14.67 10.29 375 41.7 SV=1 - [ACTB_HUMAN] Q7Z794 Keratin, type II cytoskeletal 1b OS=Homo sapiens 14.36 29.66 578 61.9 GN=KRT77 PE=2 SV=3 - [K2C1B_HUMAN] Q8WVV4 Protein POF1B OS=Homo sapiens GN=POF1B PE=1 SV=3 - 7.98 10.33 589 68.0 [POF1B_HUMAN] P07339 Cathepsin D OS=Homo sapiens GN=CTSD PE=1 SV=1 - 4.61 4.75 412 44.5 [CATD_HUMAN] Q5T750 Skin-specific protein 32 OS=Homo sapiens GN=XP32 PE=1 9.20 4.02 250 26.2 SV=1 - [XP32_HUMAN] Q02413 Desmoglein-1 OS=Homo sapiens GN=DSG1 PE=1 SV=2 - 2.67 12.48 1049 113.7 [DSG1_HUMAN] Q96QA5 Gasdermin-A OS=Homo sapiens GN=GSDMA PE=1 SV=4 - 4.94 5.34 445 49.3 [GSDMA_HUMAN] P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 24.95 37.02 473 51.2 GN=KRT16 PE=1 SV=4 - [K1C16_HUMAN] P02538 Keratin, type II cytoskeletal 6A OS=Homo sapiens 21.63 65.08 564 60.0 GN=KRT6A PE=1 SV=3 - [K2C6A_HUMAN] P22531 Small proline-rich protein 2E OS=Homo sapiens 36.11 5.63 72 7.8 GN=SPRR2E PE=2 SV=2 - [SPR2E_HUMAN] Q8N1N4 Keratin, type II cytoskeletal 78 OS=Homo sapiens 4.23 5.44 520 56.8 GN=KRT78 PE=2 SV=2 - [K2C78_HUMAN] P23490 Loricrin OS=Homo sapiens GN=LOR PE=1 SV=2 - 4.17 9.36 312 25.7 [LORI_HUMAN] P0CG48 Polyubiquitin-C OS=Homo sapiens GN=UBC PE=1 SV=3 - 23.65 3.61 685 77.0 [UBC_HUMAN] P27348 14-3-3 protein theta OS=Homo sapiens GN=YWHAQ PE=1 2.86 2.07 245 27.7 SV=1 - [1433T_HUMAN] P67936 Tropomyosin alpha-4 chain OS=Homo sapiens GN=TPM4 4.03 2.56 248 28.5 PE=1 SV=3 - [TPM4_HUMAN] P58107 Epiplakin OS=Homo sapiens GN=EPPK1 PE=1 SV=2 - 1.18 2.40 5090 555.3 [EPIPL_HUMAN] P35030 Trypsin-3 OS=Homo sapiens GN=PRSS3 PE=1 SV=2 - 4.28 3.04 304 32.5 [TRY3_HUMAN] P35321 Cornifin-A OS=Homo sapiens GN=SPRR1A PE=1 SV=2 - 17.98 1.81 89 9.9 [SPR1A_HUMAN] P35900 Keratin, type I cytoskeletal 20 OS=Homo sapiens 4.72 2.91 424 48.5 GN=KRT20 PE=1 SV=1 - [K1C20_HUMAN]

97 Q09666 Neuroblast differentiation-associated protein AHNAK 1.68 2.13 5890 628.7 OS=Homo sapiens GN=AHNAK PE=1 SV=2 - [AHNK_HUMAN] P04792 Heat shock protein beta-1 OS=Homo sapiens GN=HSPB1 7.80 4.34 205 22.8 PE=1 SV=2 - [HSPB1_HUMAN] P00338 L-lactate dehydrogenase A chain OS=Homo sapiens 3.61 1.86 332 36.7 GN=LDHA PE=1 SV=2 - [LDHA_HUMAN] P04406 Glyceraldehyde-3-phosphate dehydrogenase OS=Homo 4.48 2.64 335 36.0 sapiens GN=GAPDH PE=1 SV=3 - [G3P_HUMAN] Q9NR96-2 Isoform 2 of Toll-like receptor 9 OS=Homo sapiens 4.31 0.00 975 109.6 GN=TLR9 - [TLR9_HUMAN] Q6ZVX7 F-box only protein 50 OS=Homo sapiens GN=NCCRP1 4.00 3.47 275 30.8 PE=1 SV=1 - [FBX50_HUMAN] Q6KB66 Keratin, type II cytoskeletal 80 OS=Homo sapiens 4.65 5.94 452 50.5 GN=KRT80 PE=1 SV=2 - [K2C80_HUMAN] H0YB44 PWWP domain-containing protein 2A (Fragment) 37.14 0.00 35 3.9 OS=Homo sapiens GN=PWWP2A PE=4 SV=1 - [H0YB44_HUMAN] Q45KI0 Trypsin I (Fragment) OS=Homo sapiens GN=PRSS1 PE=3 23.81 2.56 84 9.2 SV=1 - [Q45KI0_HUMAN] A0A096LP31 Uncharacterized protein OS=Homo sapiens PE=4 SV=1 - 50.00 0.00 62 7.5 [A0A096LP31_HUMAN] P68104 Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 1.52 462 50.1 PE=1 SV=1 - [EF1A1_HUMAN] P20930 Filaggrin OS=Homo sapiens GN=FLG PE=1 SV=3 - 0.44 4061 434.9 [FILA_HUMAN] P32119 Peroxiredoxin-2 OS=Homo sapiens GN=PRDX2 PE=1 SV=5 5.56 198 21.9 - [PRDX2_HUMAN] Q13835 Plakophilin-1 OS=Homo sapiens GN=PKP1 PE=1 SV=2 - 1.47 747 82.8 [PKP1_HUMAN]