Description of research activities
Our main research interests are aimed at elucidating the molecular pathways of red blood cell differentiation in physiological hematopoiesis and in hematological disease. To these ends, we using proteomic approaches in characterizing the protein complexes of key hematopoietic transcription factors, complemented by genomic, computational and functional approaches. Research projects in our lab can be summarized as follows:
1. Functional proteomic characterization of GATA-1 functions in hematopoiesis
The key hematopoietic transcription factor (TF) GATA-1 is essential for the differentiation of several hematopoietic lineages including the erythroid, megakaryocytic and eosinophilic lineages, of mast cells and of dendritic cells. GATA-1 is considered a master transcription factor of the erythroid lineage as it controls and coordinates, at the transcriptional level, all facets of erythroid differentiation. In addition, GATA-1 is of relevance to human health as GATA-1 mutations have been implicated in Trisomy 21-associated megakaryoblastic leukemia and in Diamond-Blackfan anemia. Fundamental questions addressed in our research relate as to how GATA-1 executes at the molecular level the parallel functions of activation and repression of different target gene sets, or how a single TF such as GATA-1 carries out essential functions in diverse hematopoietic lineages ranging from erythroid cells to eosinophils and mast cells. Our research efforts are spearheaded by the application of an in vivo biotinylation tagging approach coupled to streptavidin affinity purification, which we were the first to apply for the efficient, single step purification of TF protein complexes followed by their characterization by mass spectrometry (MS) (de Boer et al., PNAS 2003; Rodriguez et al., Methods Mol. Biol. 2006). This work suggested that the many functions of GATA-1 were carried out by distinct protein subcomplexes regulating specific subsets of target genes (Rodriguez et al., EMBO J. 2005). We have now taken this work in vivo by generating a transgenic mouse line carrying a biotinylatable tag knocked-in to the GATA-1 gene locus, resulting in all physiologically expressed GATA-1 protein being efficiently biotinylated in the erythroid lineage (Karkoulia and Strouboulis, unpublished). This unique mouse model allows us to functionally characterize GATA-1 protein complexes in vivo by employing quantitative proteomics to identify GATA-1 protein complexes in fetal liver erythropoiesis and in other hematopoietic lineages where GATA1 carries out essential functions.
2. Genomic and computational approaches in characterizing transcriptional and epigenomic regulatory networks in hematopoiesis
We have also combined our GATA-1 functional proteomic studies with genomic analyses of the GATA-1 genome-wide occupancies and of epigenetic profiling in erythroid differentiation. Specifically, we characterized the GATA-1 genome-wide occupancy profile in mouse fetal liver erythropoiesis by ChIPseq and identified approximately 3650 genes as being differentially bound to and regulated by GATA-1 (Papadopoulos, Karkoulia et al., Nucl. Acids Res. 2013). We have also developed novel computational machine learning approaches in integrating the GATA-1 ChIPseq data with publicly available genomic profiles for a number of epigenetic marks, resulting in the categorization of GATA-1 target genes into three classes, each with distinct epigenetic signatures and functional characteristics (Papadopoulos, Karkoulia et al., Nucleic Acids Res. 2013). Furthermore, using the computational tools we developed for integrating and visualizing multiple genomic TF occupancy and epigenetic profiles, we investigated the determinants of the erythroid versus megakaryocytic lineage specification stemming from a common progenitor cell and identified “bookmarking” functions for specific TFs and potentially novel epigenetic enzymatic activities which may be specifically involved in erythroid lineage specification (Papadopoulos and Strouboulis, in prep.).
3. Characterization of DNA methyltransferase 1 (DNMT1) functions in red blood cell differentiation
We have previously identified the maintenance DNA methyltransferase DNMT1 as a GATA-1 co-purifying protein in erythroid cells, raising the possibility that GATA-1 functions may be mediated through DNA methylation. We expressed biotin tagged DNMT1 in erythroid cells and characterized DNMT1 protein complexes by streptavidin pulldown coupled to mass spec. We confirmed the DNMT1 interaction with GATA-1 and further identified as DNMT1 interactors a number of transcription factors with known functions in hematopoiesis, including Zbp-89, Gfi-1b, Znf143, YY1 and FOG-1, all of which are also known to interact with GATA-1. Further work identified a small 17 aa domain near the N-terminus of DNMT1 as being necessary and sufficient for mediating DNMT1 interactions with the hematopoietic transcription factors. We are presently undertaking a dominant negative approach by overexpressing in erythroid cells the 17aa DNMT1 domain responsible for interactions with hematopoietic transcription factors, with the aim of specifically disturbing endogenous DNMT1 interactions with these transcription factors in order to reveal their in vivo functions in erythropoiesis (Papageorgiou, Karkoulia and Strouboulis, in prep.). We also undertook a lentiviral shRNA knockdown approach in investigating possible DNMT1 functions in terminal erythroid differentiation. This resulted in defective differentiation and a failure of cell cycle arrest at the G1/S phase. Expression profiling of DNMT1 knockdown cells showed that a number of cell cycle related genes were not properly silenced in the absence of DNMT1 during terminal differentiation. By contrast, the erythroid specific transcription program was unaffected by DNMT1 depletion. We are presently trying to establish whether DNA methylation is responsible for the repression of cell cycle related genes (Karkoulia, Papageorgiou and Strouboulis, in prep.).
In summary, our research activities combine proteomic, genomic, computational and functional approaches and have provided novel insight regarding the molecular basis of TF functions in hematopoiesis. In addition, the continuing combinatorial application of cutting edge approaches and the further development of novel methodologies promises to lead to further mechanistic insights regarding TF functions, for example, in the re-organization of chromatin looped domains in regulating gene expression in physiological and malignant hematopoiesis.