High-Level Setd8 Expression Is Important for Establishment of Normal Patterns of Erythroid Gene Expression and Is Essential for Stress Erythropoiesis
Authors: Couch Tyler A , Murphy Zachary C. , Lillis Jacquelyn , Getman Michael , Kingsley Paul D. , Kurita Ryo , Nakamura Yukio , Steiner Laurie A. 
Published: October 2019

Abstract
Setd8 is the sole histone methyltransferase capable of mono-methylating histone H4, lysine 20. Setd8 is expressed at basal levels in most cell types and is important for many basic cellular functions, including cell cycle progression, transcriptional regulation, and mitotic chromatin condensation. Setd8 is expressed ~10-fold higher in erythroblasts than any other cell type and during erythroid maturation of human CD34+ HSPC, Setd8 protein levels increase in parallel with Gata1 levels, suggesting that Setd8 may have an erythroid-specific function(s). Consistent with this hypothesis, erythroid-specific deletion of Setd8 was embryonic lethal, resulting in profound anemia. Setd8-null erythroblasts had cell cycle abnormalities, failure of transcriptional repression, and defective terminal erythroid maturation. (Malik et al., Cell Reports, 2017). These studies provided important insights into the function of Setd8 in erythroid cells, but were not able to clearly delineate the "housekeeping" functions of Setd8 from its specific functions in erythropoiesis.
To identify the erythroid-specific functions of Setd8, we sought to identify and disrupt the enhancer that drives high level Setd8 expression in erythroid cells. Using publically available ChIP-seq data sets, we identified a putative enhancer located in intron 1 of the SETD8 gene that was occupied by Gata1, Tal1, and H3K4me1 in human erythroblasts derived from culture of CD43+ HSPCs. This putative enhancer was able to drive luciferase expression in a reporter gene assay, and deletion of the Gata1:Tal1 site at the center of this region was sufficient to abrogate reporter gene activity. Based on these data, we hypothesized that this was the enhancer that drives high level expression of Setd8 in erythroid cells. To test this hypothesis, we used CRISPR/Cas9 genome editing to delete this region in HUDEP-2 cells. Briefly, Cas9 and guide RNA ribonucleoprotein complexes targeting the enhancer were delivered into the cells using electroporation (Gundry et al., Cell Reports, 2015). PCR and sequencing were used to confirm genome editing in monoclonal cell lines. Homozygous deletion of the enhancer (Δ/Δ) reduced SETD8 expression to 27.8% of WT (+/+) controls by RT-qPCR (n=3 for each genotype; p=0.0018). Decreased Setd8 protein levels and H4K20 mono-methylation was confirmed by Western blot. Further supporting an important function of Setd8 in erythropoiesis, deletion of the enhancer and exon 7 in CD34+ HSPCs resulted in a decreased efficiency of erythroid colony formation to 49.6% of control (n=5, p=0.0359).
To gain insights into Setd8 gene regulation in erythroid cells, we performed RNA-seq, comparing the Δ/Δ and +/+ enhancer lines. In total, there were 603 genes differentially expressed (p<0.05; fold change >1.5), including SETD8, FAS, and CDKN1A (p21Cip1). Pathway analyses identified numerous genes associated with apoptosis and cell death to be up-regulated. Intriguingly, multiple genes in important for stress erythropoiesis were differentially expressed in the Setd8 Δ/Δ and +/+ enhancer lines and were also differentially expressed in Setd8-null murine erythroblasts (Malik et al., Cell Reports, 2017). Most notably, both the Δ/Δ enhancer lines and the Setd8-null erythroblasts had significantly higher levels of Fas death receptor transcript than control cells. Down-regulation of Fas is essential for stress erythropoiesis (Liu et al., Blood, 2006). We therefore hypothesized that Setd8 is important for the stress erythropoiesis response. To test this hypothesis, we subjected EpoR-Cre+/-;Setd8fl/+ (Setd8Δ/+) and EpoRCre+/-;Setd8+/+ (Setd8+/+) mice to anemic stress by retro-orbital bleeding. Setd8Δ/+ and Setd8+/+ mice had similar hematocrit after anemic stress (26.6 vs 29.4%; p=0.216), but the Setd8Δ/+ had an impaired ability to mount a stress response, with a lower MCV (43.0 vs 45.1 fL, p=0.003) and reticulocyte count (8.05 vs 2.14%, p=0.031) Consistent with the transcriptomic data, Setd8Δ/+ mice had higher levels of Fas transcript in splenic erythroblasts than Setd8+/+ controls. Together, these data suggest that high level Setd8 expression is important for normal erythroid maturation and gene expression, and for regulating the stress erythropoiesis response.

Disclosures
No relevant conflicts of interest to declare.