Transcriptional regulation of endothelial KLF2 in vascular development
The development of a functional vascular system is critically dependent on shear stress, a hemodynamic force exerted onto the vascular endothelium by flowing blood. Krüppel-like factor 2 (KLF2) is the central transcriptional regulator of the endothelial shear stress response, yet the mechanisms by w...
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| Format: | Thesis |
| Language: | English |
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2022
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| _version_ | 1826309609460072448 |
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| author | Fleisinger, L |
| author2 | de Val, S |
| author_facet | de Val, S Fleisinger, L |
| author_sort | Fleisinger, L |
| collection | OXFORD |
| description | The development of a functional vascular system is critically dependent on shear stress, a hemodynamic force exerted onto the vascular endothelium by flowing blood. Krüppel-like factor 2 (KLF2) is the central transcriptional regulator of the endothelial shear stress response, yet the mechanisms by which shear stress signalling controls KLF2 expression remain controversial. Activation of KLF2 by shear stress was previously hypothesised to occur through a MEK5-ERK5-MEF2 pathway directly interacting with the KLF2 promoter, thereby inducing KLF2 expression. However, KLF2 is expressed in many different cell types in response to a variety of different upstream signals, suggesting cell type-specific KLF2 expression might be regulated by yet undescribed distal enhancers. Further, MEF2 factors not only control differentiation of many non-endothelial cell types, but also regulate the expression of endothelial genes independent of shear stress signalling, suggesting additional transcriptional regulators must interact with MEF2 to induce context-specific KLF2 expression. In this thesis, I identify and characterise two KLF2 enhancers able to drive reporter gene expression specifically in endothelial cells (ECs) and endocardial cushions (ECCs) independently of the KLF2 promoter, which is by itself unable to drive any reporter expression in development. Pharmacological inhibition of cardiac contractions during development resulted in a reduction of reporter expression driven by both enhancers, suggesting they might be regulated by shear stress-responsive transcription factors (TFs). Validated phylogenetic foot-printing combined with mutational analysis of both KLF2 enhancers implicated roles for MEF2 binding alongside a number of other TFs. Crucially, a previously unidentified MEF2-HBOX double motif was found to be required for the endothelial activity of both KLF2 enhancers, and was sufficient to identify a novel EC-specific enhancer for KLF4, a gene that compensates for KLF2 in vivo. This work demonstrates previously unappreciated complexity in the transcriptional regulation of KLF2, and provides an opportunity to better understand transcriptional dynamics at the locus of this disease-relevant gene. |
| first_indexed | 2024-03-07T07:38:20Z |
| format | Thesis |
| id | oxford-uuid:ab8a7b57-072b-401d-9156-68a06b282f2f |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:38:20Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:ab8a7b57-072b-401d-9156-68a06b282f2f2023-03-29T09:02:33ZTranscriptional regulation of endothelial KLF2 in vascular developmentThesishttp://purl.org/coar/resource_type/c_db06uuid:ab8a7b57-072b-401d-9156-68a06b282f2fVascular endotheliumGene regulatory networksDevelopmental biologyTranscription factorsEnglishHyrax Deposit2022Fleisinger, Lde Val, SNeal, AThe development of a functional vascular system is critically dependent on shear stress, a hemodynamic force exerted onto the vascular endothelium by flowing blood. Krüppel-like factor 2 (KLF2) is the central transcriptional regulator of the endothelial shear stress response, yet the mechanisms by which shear stress signalling controls KLF2 expression remain controversial. Activation of KLF2 by shear stress was previously hypothesised to occur through a MEK5-ERK5-MEF2 pathway directly interacting with the KLF2 promoter, thereby inducing KLF2 expression. However, KLF2 is expressed in many different cell types in response to a variety of different upstream signals, suggesting cell type-specific KLF2 expression might be regulated by yet undescribed distal enhancers. Further, MEF2 factors not only control differentiation of many non-endothelial cell types, but also regulate the expression of endothelial genes independent of shear stress signalling, suggesting additional transcriptional regulators must interact with MEF2 to induce context-specific KLF2 expression. In this thesis, I identify and characterise two KLF2 enhancers able to drive reporter gene expression specifically in endothelial cells (ECs) and endocardial cushions (ECCs) independently of the KLF2 promoter, which is by itself unable to drive any reporter expression in development. Pharmacological inhibition of cardiac contractions during development resulted in a reduction of reporter expression driven by both enhancers, suggesting they might be regulated by shear stress-responsive transcription factors (TFs). Validated phylogenetic foot-printing combined with mutational analysis of both KLF2 enhancers implicated roles for MEF2 binding alongside a number of other TFs. Crucially, a previously unidentified MEF2-HBOX double motif was found to be required for the endothelial activity of both KLF2 enhancers, and was sufficient to identify a novel EC-specific enhancer for KLF4, a gene that compensates for KLF2 in vivo. This work demonstrates previously unappreciated complexity in the transcriptional regulation of KLF2, and provides an opportunity to better understand transcriptional dynamics at the locus of this disease-relevant gene. |
| spellingShingle | Vascular endothelium Gene regulatory networks Developmental biology Transcription factors Fleisinger, L Transcriptional regulation of endothelial KLF2 in vascular development |
| title | Transcriptional regulation of endothelial KLF2 in vascular development |
| title_full | Transcriptional regulation of endothelial KLF2 in vascular development |
| title_fullStr | Transcriptional regulation of endothelial KLF2 in vascular development |
| title_full_unstemmed | Transcriptional regulation of endothelial KLF2 in vascular development |
| title_short | Transcriptional regulation of endothelial KLF2 in vascular development |
| title_sort | transcriptional regulation of endothelial klf2 in vascular development |
| topic | Vascular endothelium Gene regulatory networks Developmental biology Transcription factors |
| work_keys_str_mv | AT fleisingerl transcriptionalregulationofendothelialklf2invasculardevelopment |