Unit:
Τομέας ΙΙ [Οργανική Χημεία – Οργανική Χημική Τεχνολογία – Χημεία Τροφίμων]Library of the School of Science
Author:
Παπαγεωργίου Δημήτριος
Dissertation committee:
Καθηγήτρια Αθανασία Σιαφάκα-Καπάδαη (Επιβλέπουσα), Δρ. Ιώαννης Στρουμπούλης Ερευνητής Β΄, Αναπληρώτρια Καθηγήτρια Μαίρη Μαυρή
Original Title:
Πρωτεωμική προσέγγιση στην ανάλυση ρυθμιστικών σηματοδοτικών πορειών: ερυθροειδικοί μεταγραφικοί παράγοντες
Translated title:
Proteomic approach for the analysis of regulatory signaling pathways: erythroid transcriptions factors
Summary:
GATA-1 is a key hematopoietic transcription factor that is required for
erythrocyte differentiation. GATA-1 can positively or negatively regulate
erythroid genes through its ability to form multiple protein complexes. In this
thesis we sought to discover new GATA-1 protein complexes by using improved in
vivo biotinylation tagging coupled to mass spectrometry. In vivo biotinylation
involves the covalent attachment of a biotin group to a small peptide tag
(15-23 aa) fused to the protein of interest, by the bacterial biotin ligase
BirA and the very high affinity binding of the biotin-tagged protein by
streptavidin. We show that including Nonidet P-40 in the nuclear extract
preparation reduces background caused by the binding of endogenously
biotinylated proteins. We also show that the use of alternative nuclear extract
methods, results in more efficient nuclear protein extraction of GATA-1 protein
compared to the commonly used high salt extraction method. By using this
improved biotinylation tagging method we identify the novel interacting GATA-1
protein DNA methyltransferase I while being able to discard false positive
identified interactions. We present an example of the latter by showing how
GATA-1 interactions with TAF proteins as identified by mass spec, disappear
upon nuclease digestion of nucleic acids from the nuclear extracts. By applying
the biotinylation tagging approach for the reverse purification of DNMT1
protein complexes, we confirmed the interaction with GATA-1 and we further
identified novel interactions with key hematopoietic transcription factors such
as ZBP-89, ZNF143, Gfi-1b, FOG-1 and others. Validation of these interactions
further suggested that DNMT1 forms a core complex with ZBP-89 and ZNF143 and
subcomplexes with GATA-1, FOG-1 and Gfi-1b. In order to map the DNMT-1 domains
necessary for the interaction with transcription factors, DNMT1 deletion
mutants of the protein were used to show that the domain responsible for
interaction is the PCNA binding domain (PBD). This will be further used in
order to unravel the biological significance of the GATA-1 DNMT-1 interaction.
Keywords:
Erythropoiesis, In vivo biotinylation tagging, Mass Spectrometry, Transcription Factors, Protein-Protein Interactions
Number of index pages:
1-7
Number of references:
195
