1- Understand the molecular mechanism that controls erythropoiesis and its deregulation in ß-thalassemia
The production of functional hemoglobin-expressing
red blood cells from hematopoietic stem cells (HSCs) is a multi-step process guided by stage-specific transcription factors
(TFs). These TFs control the expression of genes that drive erythropoiesis while simultaneously counteracting alternative
cell fates, and they also ensure normal expression of ß-globin. Genetic defects that lead to decreased
transcription of the adult ß-globin gene are at the origin of the hemoglobin disorder ß-thalassemia.
Our goal here is to decipher the transcriptional regulatory network that controls
erythropoiesis in health and disease.
Our approach involves the isolation of endogenous TFs at various stages of
erythroid differentiation and identifying their interacting partners by mass spectrometry. Notably, we are using Quantitative
Proteomics (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 (The
EMBO Journal, 30 (3): 494-509, 2011).
locus is one of our major model systems in erythropoiesis. 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). Focusing on the H3K9 histone methyltransferase G9a/KMT1C, we showed that this enzyme 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. Importantly, we demonstrated that the dual function of G9a as a coactivator vs.
a corepressor entails its association within two distinct protein complexes, one containing the co-activator Mediator and
one containing the co-repressor Jarid1a/KDM5A. Functionally, the repressive role of G9a relies
on both methylation of histones H3K9/K27 and a coordinate action with the histone H3 lysine 4 (H3K4) demethylase
Jarid1a for the maintenance of gene repression. In contrast, the activating role of G9a is independent of its
methyltransferase activity and involves stabilization of the Mediator complex and cooperation with the H3K27 demethylase UTX.
(PNAS, 106 (43): 18303-18308, 2009) (PNAS, 109 (46): 18845-18859, 2012)
& (Epigenetics 5 (4), 2010).
Currently, we are
using a systems biology approach to build a network model of erythropoiesis based on dynamic changes
in TF protein levels during erythroid differentiation of human hematopoietic stem/progenitor cells. Using a combination of
relative (iTRAQ) and absolute (MRM) quantitative proteomics approaches, together with genomic approaches (RNAseq) in an ex
vivo erythropoiesis differentiation system that we have developed (Journal of
Visualized Experiments doi: 10.3791/2813), we aim
to identify and quantify the dynamic changes in the ensemble of TFs and cofactors that occur during erythroid differentiation.
Mathematical modeling will then allow us to integrate the dynamic and quantitative nature of the proteome into the
transcriptional network of erythropoiesis. This improved network model is expected to serve as the benchmark for healthy erythropoiesis
against which to compare erythroid-related disease states. This project is performed in collaboration with Jeff Ranish (ISB,
Seattle, WA) and Ted Perkins (OHRI, Ottawa, ON).