Interplay between Notch signalling and transcription factors Ebf1 and Pax5 in Lymphoid cell fate determination
Typ
Examensarbete för masterexamen
Program
Publicerad
2019
Författare
Tingvall Gustafsson, Johanna
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
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.
Beskrivning
Ämne/nyckelord
Lymphoid cell fate, , lineage commitment , lineage conversion , cellular plasticity , pro-B cells , Notch signalling , Ebf1 , Pax5 , ChIP-seq , SLAM-seq , ATAC-seq , PLAC-seq