Jeffrey Malik

Ontogeny of erythroid gene expression

Erythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H2O2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H2O2 exposure. We have created a user-friendly Web site (http://www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community.

Primitive erythropoiesis in the mammalian embryo

Erythropoiesis in adult mammals is characterized by the progressive maturation of hematopoietic stem cells to lineage-specific progenitors, to morphologically identifiable precursors which enucleate to form mature erythrocytes. In contrast, primitive erythropoiesis is characterized by the appearance within the yolk sac of a transient, lineage-restricted progenitor population which generates a wave of erythroid precursors. These precursors undergo progressive maturation in the bloodstream, characterized by nuclear condensation and embryonic hemoglobin accumulation. This process is dependent on erythropoietin signaling through its cognate receptor, as well as the function of several erythroid-specific transcription factors, including GATA1 and EKLF. Targeted disruption of genes in the mouse that result in failure of the emergence or maturation of the primitive erythroid lineage leads to early fetal death, indicating that the primitive erythroid lineage is necessary for survival of the mammalian embryo. While it was thought for over a century that primitive erythroid cells were uniquely nucleated mammalian red cells, it is now recognized that they, like their definitive erythroid counterparts, enucleate to form reticulocytes and pyrenocytes. This surprising finding indicates that the primitive erythroid lineage is indeed mammalian, rather than non-mammalian, in character.

Erythropoietin critically regulates the terminal maturation of murine and human primitive erythroblasts

Primitive erythroid cells, the first red blood cells produced in the mammalian embryo, are necessary for embryonic survival. Erythropoietin and its receptor EpoR, are absolutely required for survival of late-stage definitive erythroid progenitors in the fetal liver and adult bone marrow. Epo- and Epor-null mice die at E13.5 with a lack of definitive erythrocytes. However, the persistence of circulating primitive erythroblasts raises questions about the role of erythropoietin/EpoR in primitive erythropoiesis. Using Epor-null mice and a novel primitive erythroid 2-step culture we found that erythropoietin is not necessary for specification of primitive erythroid progenitors. However, Epor-null embryos develop a progressive, profound anemia by E12.5 as primitive erythroblasts mature as a synchronous cohort. This anemia results from reduced primitive erythroblast proliferation associated with increased p27 expression, from advanced cellular maturation, and from markedly elevated rates of apoptosis associated with an imbalance in pro- and anti-apoptotic gene expression. Both mouse and human primitive erythroblasts cultured without erythropoietin also undergo accelerated maturation and apoptosis at later stages of maturation. We conclude that erythropoietin plays an evolutionarily conserved role in promoting the proliferation, survival, and appropriate timing of terminal maturation of primitive erythroid precursors.

Histone Methyltransferase Setd8 Represses Gata2 Expression and Regulates Erythroid Maturation

ABSTRACT Setd8 is the sole histone methyltransferase in mammals capable of monomethylating histone H4 lysine 20 (H4K20me1). Setd8 is expressed at significantly higher levels in erythroid cells than any other cell or tissue type, suggesting that Setd8 has an erythroid-cell-specific function. To test this hypothesis, stable Setd8 knockdown was established in extensively self-renewing erythroblasts (ESREs), a well-characterized, nontransformed model of erythroid maturation. Knockdown of Setd8 resulted in impaired erythroid maturation characterized by a delay in hemoglobin accumulation, larger mean cell area, persistent ckit expression, incomplete nuclear condensation, and lower rates of enucleation. Setd8 knockdown did not alter ESRE proliferation or viability or result in accumulation of DNA damage. Global gene expression analyses following Setd8 knockdown demonstrated that in erythroid cells, Setd8 functions primarily as a repressor. Most notably, Gata2 expression was significantly higher in knockdown cells than in control cells and Gata2 knockdown rescued some of the maturation impairments associated with Setd8 disruption. Setd8 occupies critical regulatory elements in the Gata2 locus, and knockdown of Setd8 resulted in loss of H4K20me1 and gain of H4 acetylation at the Gata2 1S promoter. These results suggest that Setd8 is an important regulator of erythroid maturation that works in part through repression of Gata2 expression.

Stat and interferon genes identified by network analysis differentially regulate primitive and definitive erythropoiesis

BackgroundHematopoietic ontogeny is characterized by overlapping waves of primitive, fetal definitive, and adult definitive erythroid lineages. Our aim is to identify differences in the transcriptional control of these distinct erythroid cell maturation pathways by inferring and analyzing gene-interaction networks from lineage-specific expression datasets. Inferred networks are strongly connected and do not fit a scale-free model, making it difficult to identify essential regulators using the hub-essentiality standard.ResultsWe employed a semi-supervised machine learning approach to integrate measures of network topology with expression data to score gene essentiality. The algorithm was trained and tested on the adult and fetal definitive erythroid lineages. When applied to the primitive erythroid lineage, 144 high scoring transcription factors were found to be differentially expressed between the primitive and adult definitive erythroid lineages, including all expressed STAT-family members. Differential responses of primitive and definitive erythroblasts to a Stat3 inhibitor and IFNγ in vitro supported the results of the computational analysis. Further investigation of the original expression data revealed a striking signature of Stat1-related genes in the adult definitive erythroid network. Among the potential pathways known to utilize Stat1, interferon (IFN) signaling-related genes were expressed almost exclusively within the adult definitive erythroid network.ConclusionsIn vitro results support the computational prediction that differential regulation and downstream effectors of STAT signaling are key factors that distinguish the transcriptional control of primitive and definitive erythroid cell maturation.

Erythropoietin Signaling Regulates Key Epigenetic and Transcription Networks in Fetal Neural Progenitor Cells

Erythropoietin (EPO) and its receptor are highly expressed in the developing nervous system, and exogenous EPO therapy is potentially neuroprotective, however the epigenetic and transcriptional changes downstream of EPO signaling in neural cells are not well understood. To delineate epigenetic changes associated with EPO signaling, we compared histone H3 lysine 4 dimethylation (H3K4me2) in EPO treated and control fetal neural progenitor cells, identifying 1,150 differentially bound regions. These regions were highly enriched near protein coding genes and had significant overlap with H4Acetylation, a mark of active regulatory elements. Motif analyses and co-occupancy studies revealed a complex regulatory network underlying the differentially bound regions, including previously identified mediators of EPO signaling (STAT5, STAT3), and novel factors such as REST, an epigenetic modifier central to neural differentiation and plasticity, and NRF1, a key regulator of antioxidant response and mitochondrial biogenesis. Global transcriptome analyses on neural tubes isolated from E9.0 EpoR-null and littermate control embryos validated our in vitro findings, further suggesting a role for REST and NRF1 downstream of EPO signaling. These data support a role for EPO in regulating the survival, proliferation, and differentiation of neural progenitor cells, and suggest a basis for its function in neural development and neuroprotection.

The Methyltransferase Setd8 Is Essential for Erythroblast Survival and Maturation

Erythropoiesis is a highly regulated process that generates enucleate red blood cells from committed erythroid progenitors. Chromatin condensation culminating in enucleation is a defining feature of this process. Setd8 is the sole enzyme that can mono-methylate histone H4, lysine 20 and is highly expressed in erythroblasts compared to most other cell types. Erythroid Setd8 deletion results in embryonic lethality from severe anemia due to impaired erythroblast survival and proliferation. Setd8 protein levels are also uniquely regulated in erythroblasts, suggesting a cell-type-specific role for Setd8 during terminal maturation. Consistent with this hypothesis, Setd8 Δ/Δ erythroblasts have profound defects in transcriptional repression, chromatin condensation, and heterochromatin accumulation. Together, these results suggest that Setd8, used by most cells to promote mitotic chromatin condensation, is an essential aspect of the transcriptional repression and chromatin condensation that are hallmarks of terminal erythroid maturation.