Characterising the functional role of GATA1short isoform in normal and malignant haematopoiesis
ML-DS and its precursor disease TAM are genetically simple models of acute myeloid leukaemia to study how two or more genetic changes synergize to induce maturation arrest and leukaemic transformation. Both TAM and ML-DS are driven by mutations in the gene encoding the X-linked erythroid-megakaryocy...
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| Format: | Thesis |
| Language: | English |
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2021
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| _version_ | 1797070127047376896 |
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| author | Cruz Hernandez, D |
| author2 | Vyas, P |
| author_facet | Vyas, P Cruz Hernandez, D |
| author_sort | Cruz Hernandez, D |
| collection | OXFORD |
| description | ML-DS and its precursor disease TAM are genetically simple models of acute myeloid leukaemia to study how two or more genetic changes synergize to induce maturation arrest and leukaemic transformation. Both TAM and ML-DS are driven by mutations in the gene encoding the X-linked erythroid-megakaryocyte transcription factor GATA1 that result in truncation of 83 N-terminal amino acids to produce GATA1short (GATA1s). Through single cell RNA sequencing of trisomy 21 haematopoietic primitive, progenitor and precursor cells derived from cord blood, I identified 5 cell clusters with robust GATA1 expression that were named: EMMPPs, cycling-EMMPPs, megakaryocyte progenitor, erythroid progenitor, and basophil/eosinophil/mast cell progenitor. I showed that in TAM, GATA1s mutations resulted in aberrant gene expression in all GATA1 expressing cell compartments and led to maturation defects of the erythroid and megakaryocytic lineages. These defects were most likely explained by sustained expression of GATA2, RUNX1 and NFE2 among other genes. I optimized and employed a CRISPR-Cas9 system to recreate GATA1s mutation and showed that the erythroid maturation defect and sustained GATA2 expression in TAM can be recapitulated in vitro. This experimental approach prioritised cell compartments where trisomy 21 and GATA1s have the greatest synergy and would allow me to determine the contribution of trisomy 21 to TAM and ML-DS. Moreover, I optimized CUT&TAG to specifically profile genome wide GATA1 biding sites in low cell number. I showed biding of GATA1s at the -77kb enhancer of GATA2 in an ML-DS derived cell line. We hypothesize that sustained GATA2 expression in TAM and in ML-DS might be driven by GATA1s biding at the -77kb GATA2 enhancer. Finally, I developed a highly sensitive flow cytometric assay to detect wild type cells expressing both GATA1 isoforms and mutant TAM and ML-DS cells with exclusive expression of GATA1s. I envision this assay can substitute for next generation sequencing of GATA1 mutational profiling when not available to help diagnose TAM and ML-DS in clinical settings. |
| first_indexed | 2024-03-06T22:34:37Z |
| format | Thesis |
| id | oxford-uuid:59753454-5fb2-4b5b-bf96-674b62480eaa |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T22:34:37Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | oxford-uuid:59753454-5fb2-4b5b-bf96-674b62480eaa2022-03-26T17:09:52ZCharacterising the functional role of GATA1short isoform in normal and malignant haematopoiesisThesishttp://purl.org/coar/resource_type/c_db06uuid:59753454-5fb2-4b5b-bf96-674b62480eaaCancer in childrenEnglishHyrax Deposit2021Cruz Hernandez, DVyas, PRoberts, IML-DS and its precursor disease TAM are genetically simple models of acute myeloid leukaemia to study how two or more genetic changes synergize to induce maturation arrest and leukaemic transformation. Both TAM and ML-DS are driven by mutations in the gene encoding the X-linked erythroid-megakaryocyte transcription factor GATA1 that result in truncation of 83 N-terminal amino acids to produce GATA1short (GATA1s). Through single cell RNA sequencing of trisomy 21 haematopoietic primitive, progenitor and precursor cells derived from cord blood, I identified 5 cell clusters with robust GATA1 expression that were named: EMMPPs, cycling-EMMPPs, megakaryocyte progenitor, erythroid progenitor, and basophil/eosinophil/mast cell progenitor. I showed that in TAM, GATA1s mutations resulted in aberrant gene expression in all GATA1 expressing cell compartments and led to maturation defects of the erythroid and megakaryocytic lineages. These defects were most likely explained by sustained expression of GATA2, RUNX1 and NFE2 among other genes. I optimized and employed a CRISPR-Cas9 system to recreate GATA1s mutation and showed that the erythroid maturation defect and sustained GATA2 expression in TAM can be recapitulated in vitro. This experimental approach prioritised cell compartments where trisomy 21 and GATA1s have the greatest synergy and would allow me to determine the contribution of trisomy 21 to TAM and ML-DS. Moreover, I optimized CUT&TAG to specifically profile genome wide GATA1 biding sites in low cell number. I showed biding of GATA1s at the -77kb enhancer of GATA2 in an ML-DS derived cell line. We hypothesize that sustained GATA2 expression in TAM and in ML-DS might be driven by GATA1s biding at the -77kb GATA2 enhancer. Finally, I developed a highly sensitive flow cytometric assay to detect wild type cells expressing both GATA1 isoforms and mutant TAM and ML-DS cells with exclusive expression of GATA1s. I envision this assay can substitute for next generation sequencing of GATA1 mutational profiling when not available to help diagnose TAM and ML-DS in clinical settings. |
| spellingShingle | Cancer in children Cruz Hernandez, D Characterising the functional role of GATA1short isoform in normal and malignant haematopoiesis |
| title | Characterising the functional role of GATA1short isoform in normal and malignant haematopoiesis |
| title_full | Characterising the functional role of GATA1short isoform in normal and malignant haematopoiesis |
| title_fullStr | Characterising the functional role of GATA1short isoform in normal and malignant haematopoiesis |
| title_full_unstemmed | Characterising the functional role of GATA1short isoform in normal and malignant haematopoiesis |
| title_short | Characterising the functional role of GATA1short isoform in normal and malignant haematopoiesis |
| title_sort | characterising the functional role of gata1short isoform in normal and malignant haematopoiesis |
| topic | Cancer in children |
| work_keys_str_mv | AT cruzhernandezd characterisingthefunctionalroleofgata1shortisoforminnormalandmalignanthaematopoiesis |