|
|
Hematopoietic Stem cells have the capacity to differentiate along multiple lineages potentially giving rise to all cells present in the blood. This process is controlled by cell-specific and ubiquitously expressed transcription factors and cofactors. Defects in the transcriptional regulatory network of these cells can lead to leukemia. The major goal of our lab at the Sprott Centre for Stem Cell Research is to decipher the molecular mechanism of hematopoietic stem cell differentiation towards the erythroid lineage and to understand how deregulation of this process can cause leukemia. Towards this goal, we are using a systems biology approach including quantitative proteomics (isotope tagged methods), genomics (expression microarray, ChIP-sequencing), bioinformatics as well as molecular and cellular biology to understand the regulation of gene expression at the level of transcription, epigenetics and chromatin structure. Notably, we are employing a recently developed cell culture protocol to induce differentiation of red blood cells ex vivo from Hematopoietic Stem Cells that have been previously extracted from human blood, bone marrow or cord blood. This system provides us with large quantities of cells at various stages of hematopoietic differentiation to perform proteomics experiments.
Our approach involves the isolation of endogenous transcription factors at various stages of hematopoietic differentiation of healthy and/or leukemic cells and identifying their interacting partners by mass spectrometry. Notably, we are using Quantitative Proteomics (ICAT, iTRAQ) methods to pinpoint the dynamics of protein interactions within the transcriptional regulatory network (Methods Mol Biol. 359:17-35, 2007). We expect that understanding the dynamic changes in transcription factors’ interactions during cell differentiation will allow us to better control cell fate decisions. For example, we have shown that the bZIP protein MafK regulates ß-globin expression by exchanging its heterodimerization partner from the repressor Bach1 to the activator p45 during erythroid differentiation (Nature Structural and Molecular Biology, 11 (1): 73-80, 2004).
We are also using chromatin immunoprecipitation to study targeting of the identified transcription factors and cofactors to specific genes and/or genome wide as well as the resulting epigenetics modifications. Notably, the ß-globin locus is one of our major model systems. For example, we have shown that during erythroid differentiation, the hematopoietic activator NF-E2 is implicated in recruiting the H3K4 methyltransferase complex MLL2 to the ß-globin locus. Following its recruitment to a distal DNA regulatory region, MLL2 is transferred to the active ß-globin gene over a distance of 40 kb via a “spreading” mechanism (Molecular Cell, 27: 573-584, 2007).
Our most recent studies have focused on the regulation of ß-globin transcription by the histone methyltransferase G9a. We have shown that G9a is involved in maintaining the embryonic ß-globin gene in a repressed state while simultaneously activating the adult ß-globin genes in adult erythroid cells. While the repressive role of G9a relies on methylation of histone H3 at Lys9 and Lys27, its activating function is independent of its methyltransferase activity and involves cooperation with the H3K27 demethylase UTX. Importantly, we have shown that following its recruitment to the ß-globin LCR via interaction with the activator NF-E2, G9a spreads over the entire ß-globin locus in a manner similar to MLL2. (PNAS, 106 (43): 18303-18308, 2009) & (Epigenetics 5 (4), 2010).
|
