Investigating the role of CDK1 in governing the transcriptional landscape in cancer cells
The family of cyclin-dependent protein kinases (CDKs) drives many aspects of eukaryotic cell function. The literature suggests a binary distinction between cell-cycle CDKs and transcriptional CDKs (tCDKs), but increasing evidence points to the functional overlap between these groups. Considering the...
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| Format: | Thesis |
| Language: | English |
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2024
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| _version_ | 1824459349110554624 |
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| author | Pluta, AJ |
| author2 | Murphy, S |
| author_facet | Murphy, S Pluta, AJ |
| author_sort | Pluta, AJ |
| collection | OXFORD |
| description | The family of cyclin-dependent protein kinases (CDKs) drives many aspects of eukaryotic cell function. The literature suggests a binary distinction between cell-cycle CDKs and transcriptional CDKs (tCDKs), but increasing evidence points to the functional overlap between these groups. Considering the need for temporal coordination of the cell cycle and the transcription cycle, it can be speculated that the CDKs might play a critical role in linking the two processes. Cyclin-dependent kinase 1 (CDK1) is a highly conserved essential regulator of mitosis, with recognised transcriptional functions revolving around the mitotic repression of gene expression. Recently, CDK1 was shown to act as a transcriptional repressor in early mouse embryos, where its heightened activity counteracts the differentiation of stem cells. CDK1 overexpression is also observed in several types of cancer. The work described in this thesis aimed to test the hypothesis that CDK1 dysregulation contributes to transcriptional re-programming during tumorigenesis by imposing a stem cell-like gene expression programme. An elegant method of examining this role is through the use of cell lines expressing an ATP analogue-sensitive CDK1 (AS-CDK1), which allows for rapid and specific inhibition of the kinase. This project details the characterization of the HeLa AS-CDK1 experimental model, and the development of two new cell lines for studying CDK1 function, HCT116 CDK1WT/AS and HEK293 AS-CDK1. A novel method of positive selection for AS-CDK1 clones is also described. HeLa AS-CDK1 was used for the initial analysis of the transcriptional effects of CDK1 inhibition in cancer cells. Using EU staining and chromatin-associated RNA-seq approaches, it was shown that the inhibition of HeLa AS-CDK1 cells with the ATP analogue 1-NM-PP1 results in the upregulation of gene expression. The examination of the two most dysregulated genes from the analysis revealed that their regulation also changes in unmutated HeLa cells, exposing previously unexplored off-target effects of 1-NM-PP1. The regulation of these genes by 1-NM-PP1 was then tested in the new cell lines, and through the use of other CDK1 inhibitors. Treatment of HEK293 AS-CDK1 cells with 1-NM-PP1 resulted in a global decrease in transcription but an upregulation of several dozen protein-coding genes. Subsequent analysis of the upregulated genes identified several transcription factors as candidates for involvement in mediating the transcriptional effects of CDK1 across different cell types, after their binding motifs were found to be enriched upstream of the transcription start site of the dysregulated genes. Together, the data collected in this study explored the potential of AS kinase technology in investigating CDK1 function in transcription, and provided crucial insight into the off-target effects of 1-NM-PP1, underlining the necessity of having wild-type controls while studying AS kinases. Although the extent of gene dysregulation following short CDK1 inhibition was limited, the gathered evidence pointed to previously unknown ways in which CDK1 could govern transcription. The newly identified motifs and their respective transcription factors indicate novel mediators of CDK1’s transcriptional functions, with potentially significant biological effects. The elucidation of the molecular mechanisms behind CDK1 regulation of these transcription factors will surely remain an intriguing avenue of research. |
| first_indexed | 2025-02-19T04:40:22Z |
| format | Thesis |
| id | oxford-uuid:f69a9e0d-03da-4086-b555-190b99f5bf8c |
| institution | University of Oxford |
| language | English |
| last_indexed | 2025-02-19T04:40:22Z |
| publishDate | 2024 |
| record_format | dspace |
| spelling | oxford-uuid:f69a9e0d-03da-4086-b555-190b99f5bf8c2025-02-18T09:53:51ZInvestigating the role of CDK1 in governing the transcriptional landscape in cancer cellsThesishttp://purl.org/coar/resource_type/c_db06uuid:f69a9e0d-03da-4086-b555-190b99f5bf8cCell cycleMolecular biologyCyclin-dependent kinasesTranscriptionEnglishHyrax Deposit2024Pluta, AJMurphy, SNorbury, CThe family of cyclin-dependent protein kinases (CDKs) drives many aspects of eukaryotic cell function. The literature suggests a binary distinction between cell-cycle CDKs and transcriptional CDKs (tCDKs), but increasing evidence points to the functional overlap between these groups. Considering the need for temporal coordination of the cell cycle and the transcription cycle, it can be speculated that the CDKs might play a critical role in linking the two processes. Cyclin-dependent kinase 1 (CDK1) is a highly conserved essential regulator of mitosis, with recognised transcriptional functions revolving around the mitotic repression of gene expression. Recently, CDK1 was shown to act as a transcriptional repressor in early mouse embryos, where its heightened activity counteracts the differentiation of stem cells. CDK1 overexpression is also observed in several types of cancer. The work described in this thesis aimed to test the hypothesis that CDK1 dysregulation contributes to transcriptional re-programming during tumorigenesis by imposing a stem cell-like gene expression programme. An elegant method of examining this role is through the use of cell lines expressing an ATP analogue-sensitive CDK1 (AS-CDK1), which allows for rapid and specific inhibition of the kinase. This project details the characterization of the HeLa AS-CDK1 experimental model, and the development of two new cell lines for studying CDK1 function, HCT116 CDK1WT/AS and HEK293 AS-CDK1. A novel method of positive selection for AS-CDK1 clones is also described. HeLa AS-CDK1 was used for the initial analysis of the transcriptional effects of CDK1 inhibition in cancer cells. Using EU staining and chromatin-associated RNA-seq approaches, it was shown that the inhibition of HeLa AS-CDK1 cells with the ATP analogue 1-NM-PP1 results in the upregulation of gene expression. The examination of the two most dysregulated genes from the analysis revealed that their regulation also changes in unmutated HeLa cells, exposing previously unexplored off-target effects of 1-NM-PP1. The regulation of these genes by 1-NM-PP1 was then tested in the new cell lines, and through the use of other CDK1 inhibitors. Treatment of HEK293 AS-CDK1 cells with 1-NM-PP1 resulted in a global decrease in transcription but an upregulation of several dozen protein-coding genes. Subsequent analysis of the upregulated genes identified several transcription factors as candidates for involvement in mediating the transcriptional effects of CDK1 across different cell types, after their binding motifs were found to be enriched upstream of the transcription start site of the dysregulated genes. Together, the data collected in this study explored the potential of AS kinase technology in investigating CDK1 function in transcription, and provided crucial insight into the off-target effects of 1-NM-PP1, underlining the necessity of having wild-type controls while studying AS kinases. Although the extent of gene dysregulation following short CDK1 inhibition was limited, the gathered evidence pointed to previously unknown ways in which CDK1 could govern transcription. The newly identified motifs and their respective transcription factors indicate novel mediators of CDK1’s transcriptional functions, with potentially significant biological effects. The elucidation of the molecular mechanisms behind CDK1 regulation of these transcription factors will surely remain an intriguing avenue of research. |
| spellingShingle | Cell cycle Molecular biology Cyclin-dependent kinases Transcription Pluta, AJ Investigating the role of CDK1 in governing the transcriptional landscape in cancer cells |
| title | Investigating the role of CDK1 in governing the transcriptional landscape in cancer cells |
| title_full | Investigating the role of CDK1 in governing the transcriptional landscape in cancer cells |
| title_fullStr | Investigating the role of CDK1 in governing the transcriptional landscape in cancer cells |
| title_full_unstemmed | Investigating the role of CDK1 in governing the transcriptional landscape in cancer cells |
| title_short | Investigating the role of CDK1 in governing the transcriptional landscape in cancer cells |
| title_sort | investigating the role of cdk1 in governing the transcriptional landscape in cancer cells |
| topic | Cell cycle Molecular biology Cyclin-dependent kinases Transcription |
| work_keys_str_mv | AT plutaaj investigatingtheroleofcdk1ingoverningthetranscriptionallandscapeincancercells |