The Histone Methyltransferase Setd8 Represses Gata2 Expression and Regulates Erythroid Maturation

The maturation of a committed erythroid progenitor to a functional red blood cell is a complex process that involves significant changes in gene expression during a time of rapid cell division and nuclear condensation. Posttranslational modifications of histone proteins are critical determinates of erythroid gene expression, however the histone modifications required to execute the erythroid-specific transcriptional program during maturation are incompletely understood. Setd8 is the sole histone methyltransferase in mammals capable of generating mono-methylated histone H4 lysine 20 (H4K20me1). Setd8 and H4K20me1 are transcriptional regulators that also play an important role in mediating nuclear condensation, cell cycle progression and response to DNA damage (Beck, Genes Dev 2012). Setd8 is expressed throughout the maturation of murine and human erythroid cells (Kingsley et al, Blood 2012; An et al, Blood 2014), and erythroid precursors have significantly higher levels of Setd8 expression than any other cell- or tissue- type (biogps.org), suggesting that Setd8 may have an erythroid-specific function. Data regarding the function of Setd8 in erythroid cells is limited; with the only published study to date linking Setd8 to Gata1-mediated repression (DeVilbiss, PNAS 2013). We hypothesize that mono-methylation of H4K20 by Setd8 is a critical mediator of transcriptional repression during erythroid maturation.

To test this hypothesis, lentiviral mediated shRNA was utilized to generate stable knockdown of Setd8 in extensively self-renewing erythroblasts (ESREs). ESREs are a well-characterized, non-transformed model of erythroid maturation (Getman et al, Exp Hematol 2014), which are cultured from yolk sac or fetal liver. Once the culture is established, ESREs self-renew extensively at the proerythroblast stage, without losing the ability to mature and enucleate in approximately three days (England et al, Blood 2012). Knockdown of Setd8 was confirmed at the mRNA (~70% knockdown) and protein level (~50% knockdown), and persisted throughout maturation. Setd8 knockdown did not alter ESRE proliferation or viability, or result in accumulation of DNA damage. As expected, Setd8 knockdown was associated with a significant decline in H4K20me1 compared to scramble controls. Setd8 knockdown resulted in a significant delay in hemoglobin accumulation as determined by benzidine staining. Setd8 knockdown also significantly impaired several facets of erythroid maturation, resulting in larger cell area, persistent kit expression, incomplete nuclear condensation, and lower rates of enucleation than control cells.

To delineate the molecular mechanisms underlying these abnormalities in erythroid maturation, global gene expression was analyzed by RNA-seq in biological replicates of Setd8 knockdown and scramble samples. CuffDiff was used for differential expression analyses. 1149 genes were differentially expressed (p-value<10-3, False Discovery Rate<0.01). Consistent with the repressor function of Setd8, 780 genes were upregulated (including Gata2) and 369 genes were downregulated (including Setd8, Pklr, Alad, Tfrc, and Hbb-b1.) The top three networks identified by Ingenuity Pathway Analyses of the differentially expressed genes were Hematologic Development and Disease, Hereditary Disorder, and Cell Signaling/DNA Recombination and Repair. The increased Gata2 expression was validated using qPCR in independent Setd8 knockdown cultures.

Gata2 is a critical regulator of the balance between maturation and self-renewal in hematopoietic stem and progenitor cells, and Gata2 overexpression is sufficient to impair erythroid maturation (Ikonomi et al, Exp Hematol 2000). The Gata2 gene has both general (1G) and hematopoietic specific (1S) promoters, as well as several well-characterized enhancers that control its spatial and temporal expression (Grass et al, MCB 2006). Chromatin Immunoprecipitation experiments demonstrated Setd8 occupancy at the Gata2 1S promoter and at the critical +9.5 enhancer. Knockdown of Setd8 resulted in loss of Setd8 occupancy in the Gata2 locus and an increase in histone H4 acetylation at the Gata2 1S promoter. Taken together, these results indicate that Setd8 is an important transcriptional regulator of erythroid cells and suggest that Setd8 facilitates erythroid maturation by repressing Gata2 expression.