The Role of Thymine-DNA Glycosylase In Transcriptional Regulation

Precise control over transcriptional regulation is required for normal cell function. Errors in transcriptional regulation underpin many diseases including cancer. Thymine DNA Glycosylase (TDG) is a base excision repair protein and a coregulator that has been implicated in a diverse set of fundamental biological processes including embryonic development, nuclear receptor signaling and Wnt signaling. Importantly, TDG has been shown to play an important role in transcriptional regulation in a wide variety of systems. Details surrounding the mechanism through which TDG acts remain unclear. In this thesis we explore the role of TDG in Estrogen Receptor (ER)-dependent signaling and in cellular senescence. To characterize the role of TDG in ER mediated signaling we first mapped β-Estradiol (E2)-dependent DNA binding of TDG in the MCF7 breast cancer cell line using ChIP-Seq. Using bioinformatics in conjunction with more traditional biochemistry techniques I established that a significant component of TDG binding occurs at enhancers, where it was able to mediate the production of enhancer RNA (eRNA) and 3-dimensional reorganization of transcriptional units. Knockdown of TDG disrupts E2-mediated upregulation of ER-targets and inhibits growth. Remarkably, in addition to behavior mimicking that of an oncogene, I find that TDG knockdown and depletion result in a much more aggressive phenotype, revealing its role as a potential potent tumor suppressor. To explore the role of TDG in cellular senescence we induced senescence in IMR90 human fibroblasts using hydrogen peroxide (H2O2) and monitored markers of senescence, including proliferation and β-galactosidase staining. I found that while senescence was readily inducible in this cell line using H2O2, knockdown of TDG was able to significantly impede the process. Using ChIP, I found that TDG was recruited to a CpG island overlapping the CDKN2A promoter, a tumor suppressor important for senescence. Further studies including ChIP, bisulfite sequencing and conventional assays revealed that TDG is required for H2O2-mediated transcription of CDKN2A in a CBP-dependent and active-demethylation independent manner. Collectively, these studies extend the role of TDG in transcriptional regulation, implicating it as a mediator of cellular senescence and as a mediator of eRNA transcription and 3-dimensional re-organization in hormone signaling

TBX3 promotes progression of pre-invasive breast cancer cells by inducing EMT and directly up-regulating SLUG

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial–mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Characterization of Mutational Status, Spheroid Formation, and Drug Response of a New Genomically-Stable Human Ovarian Clear Cell Carcinoma Cell Line, 105C

Ovarian clear cell carcinoma (OCCC) is a rare subtype of gynecological cancer for which well-characterized and authenticated model systems are scarce. We provide an extensive characterization of ‘105C’, a cell line generated from an adenocarcinoma of the clear cell histotype using targeted next-generation sequencing, cytogenetic microarrays, along with analyses of AKT/mTOR signaling. We report that that the 105C cell line is a bona fide OCCC cell line, carrying PIK3CA, PTEN, and ARID1A gene mutations, consistent with OCCC, yet maintain a stable genome as reflected by low copy number variation. Unlike KOC-7c, TOV-21G, and RMG-V OCCC lines also mutated for the above genes, the 105C cells do not carry mutations in mismatch repair genes. Importantly, we show that 105C cells exhibit greater resistance to mTOR inhibition and carboplatin treatment compared to 9 other OCCC cell lines in 3D spheroid cultures. This resistance may be attributed to 105C cells remaining dormant in suspension culture which surprisingly, contrasts with several other OCCC lines which continue to proliferate in long-term suspension culture. 105C cells survive xenotransplantation but do not proliferate and metastasize. Collectively, we show that the 105C OCCC cell line exhibits unique properties useful for the pre-clinical investigation of OCCC pathobiology.Medicine, Faculty ofNon UBCObstetrics and Gynaecology, Department ofReviewedFacultyResearche

En cournè det houéc : journal des cours d'adultes du département des Hautes-Pyrénées / édité par la Société bigourdane d'entr'aide pédagogique

19251925 (N6)-1926.Appartient à l’ensemble documentaire : MidiPyren

MOESM11 of Genome-wide analysis reveals a role for TDG in estrogen receptor-mediated enhancer RNA transcription and 3-dimensional reorganization

Additional file 11. MAB-Seq of GREB1 enhancer region. Cytosines in the GREB1 enhancer region which are mostly unmodified, regardless of TDG status or E2 treatment

MOESM6 of Genome-wide analysis reveals a role for TDG in estrogen receptor-mediated enhancer RNA transcription and 3-dimensional reorganization

Additional file 6. Motif analysis performed on sites of E2 mediated TDG localization that do not overlap with ER binding, revealed enrichment only for single motif

MOESM2 of Genome-wide analysis reveals a role for TDG in estrogen receptor-mediated enhancer RNA transcription and 3-dimensional reorganization

Additional file 2. External file sources used in study

TBX3 promotes progression of pre‐invasive breast cancer cells by inducing EMT and directly up‐regulating SLUG

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial–mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland