Interplay between Notch signalling and transcription factors Ebf1 and Pax5 in Lymphoid cell fate determination

Examensarbete för masterexamen

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Type: Examensarbete för masterexamen
Title: Interplay between Notch signalling and transcription factors Ebf1 and Pax5 in Lymphoid cell fate determination
Authors: Tingvall Gustafsson, Johanna
Abstract: It is established that Notch signalling and the B cell specific transcription factors Ebf1 and Pax5 are essential for T and B cell commitment respectively. The importance of the interplay between these factors in the regulation of lymphoid cell fate determination is supported by the discovery that heterozygous loss of Ebf1 and Pax5 enables Notch mediated T-lineage conversion in progenitor B cells. The aim of this project was to investigate the interplay between Notch signalling and Ebf1/Pax5 in the regulatory mechanism governing B versus T lineage decision, using different Next generation sequencing (NGS) technologies, including ChIP-seq, SLAM-seq and ATAC-seq. Investigation of the nascent mRNA expression in fetal liver WT and Ebf1+/-Pax5+/- pro-B cells, using SLAM-seq, revealed upregulation of several known Notch target genes as well as genes associated with T cell fate in response to Notch signalling. In lack of reliable data from CSL/ICN ChIP-seq, ATAC-seq was conducted for evaluation of the impact of Notch activity on the cells’ chromatin state. The result of the ATAC-seq showed that the pre-existing differences between WT and Ebf1+/-Pax5+/- pro-B cells prior to Notch stimulation accounts for most of the variance in the data even if small changes in chromatin accessibility upon Notch stimulation were observed. Gene ontology of differentially accessible regions between WT and Ebf1+/-Pax5+/-pro-B cells, annotated using PLAC-seq, showed an enrichment of biological processes associated with T cell fate. Increased accessibility of genes associated with T cell development in Ebf1+/-Pax5+/- cells in combination with the differential gene expression upon Notch stimulation suggest that high levels of Ebf1 and Pax5 is required for maintained B cell identity and that heterozygous loss of these factors increase the responsiveness to Notch mediated T lineage conversion. One of the most significant differences in chromatin accessibility between WT and Ebf1+/-Pax5+/- is a region associated with the promoter of Cdk6, a cell cycle regulator involved in the Notch induced T cell development. The differences in chromatin accessibility, as a consequence of heterozygous loss of Ebf1 and Pax5, is also reflected in gene transcription, with an over 10-fold increase in Cdk6 expression in Ebf1+/-Pax5+/- cells. To investigate the regulatory mechanisms governing Notch mediated lineage conversion in closer detail, regions with CSL occupancy associated with transcriptional response to Notch signalling was identified by integration of footprinting analysis on ATAC-seq data and available ChIP-seq data, in regions with PLAC-seq confirmed interaction to the promoter of differentially expressed genes. The analysis identified several regions associated with factors involved in Notch signalling, including the Notch transitionally activating complex associated protein PCAF as well as the known Notch target Hes1. Common for these regions, as well as for the Cdk6 locus, are binding of Ebf1/Pax5 to enhancer regions, suggesting that these transcription factors are involved in a more direct regulatory mechanism in the establishment and maintenance of B cell identity, in addition to their impact on the cells’ chromatin state.
Keywords: Lymphoid cell fate,;lineage commitment;lineage conversion;cellular plasticity;pro-B cells;Notch signalling;Ebf1;Pax5;ChIP-seq;SLAM-seq;ATAC-seq;PLAC-seq
Issue Date: 2019
Publisher: Chalmers tekniska högskola / Institutionen för biologi och bioteknik
Collection:Examensarbeten för masterexamen // Master Theses

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