ALBERT

Description: Introduction: Umbilical cord blood (UCB) is a salient source of primitive hematopoietic stem progenitor cells (HSPCs) for bone marrow (BM) reconstitution in patients with hematologic and non-hematologic malignancies. However, a relatively low number of HSPCs in UCB units and poor BM homing efficiency greatly hinders the clinical application of UCB CD34+ cells for transplantation. To overcome these hurdles, we developed two independent strategies that increase CD34+ cell numbers and improve BM homing efficiency of UCB HSPCs. First, we expanded UCB HSPCs by culturing them in decellularized Wharton's jelly matrix (DWJM), a biometric scaffold mimicking the 3-dimenstional (3D) microenvironment of BM. Second, we enhanced the in vitro transmigration and in vivo BM homing efficiency of UCB CD34+ cells by blocking EPO/EPOR signaling. Both approaches enhance UCB CD34+ cell migration toward stromal cell-derived factor 1 (SDF1). In this study, we employed RNA-Seq and RT-PCR approaches to analyze UCB HSPCs treated with EPO and co-cultured with DWJM, aiming to identify molecules that regulate UCB HSPC transmigration via EPO/EPOR signaling. Methods: CD34+ cells from highly enriched UCB units (〉90% purity) were treated with EPO for 24 hours and separately co-cultured with DWJM for 1 week. UCB CD34+ cells were collected and subjected to RNA-Seq and real-time PCR (RT-PCR) analyses. In vitro transmigration toward SDF-1 was assessed by transwell assay. To assess the involvement of RasGRP3 in UCB CD34+ cell mobility, cells were treated with 100 nM ingenol-3-angelate (I3A), a diacylglycerol (DAG) analog that specifically targets RAS Guanyl Releasing-Protein 3 (RasGRP3), for 16 hours followed by transwell assay. Anti-EPOR antibody-treated or EPO-treated cells were used as controls. In addition, RasGRP3 gene expression was examined in CD34+ cells from peripheral blood (PB) and BM samples collected from the same donor, and compared to RasGRP3 expression in UCB CD34+ cells. Unpaired, 2-tailed t-test was used to analyze results. Results: RasGRP3 was identified by RNA-Seq from the two independent approaches, EPO treatment and DWJM co-culture. EPO downregulated and DWJM upregulated RasGRP3 gene expression in UCB CD34+ cells. RasGRP3 expression was confirmed by qPCR. UCB CD34+ cells that migrated to the bottom chamber of the transwell assays, a population that has a higher mobility, showed an elevated RasGRP3 gene expression and a decreased EPOR cell surface expression. Activation of RasGRP3 by DAG analog I3A induced a significant increase in RasGRP3 gene expression (control: I3A treatment = 1: 202 ± 58, p=0.00012) that was associated with an enhanced transmigration capability (control: I3A = 41%+/-5: 54%+/- 3, p=0.032). Knocking-down of RasGRP3 in K562 cells, a known EPOR expressing cell line, impaired the transmigration capability of K562. CD34+ cells in peripheral blood (PB) showed a higher level of RasGRP3 gene expression compared to CD34+ cells in BM samples from the same healthy donors. RasGRP3 expression in PB CD34+ cells was significantly higher than BM and UCB CD34+ cells (qPCR signals relative to BM, BM: PB: UCB = 1: 431±65: 21±8, p=0.0012, 0.0023, and

Description: The maturation of a committed erythroid progenitor to a functional erythrocyte is characterized by a global decline in transcription and progressive nuclear condensation that ultimately culminates in enucleation. At the molecular level, erythroid maturation is driven by erythroid transcription factors and chromatin modifying enzymes that work in a coordinated manner to drive the expression of erythroid-specific genes, while silencing most other genes during the process of chromatin condensation and enucleation. Setd8 is the sole histone methyltransferase in mammals capable of mono-methylating histone H4 Lysine 20 (H4K20me1). In vitro studies suggest that Setd8 and H4K20me1 play critical roles in cell cycle progression, nuclear condensation, and DNA damage response (Reviewed in Beck et al, Genes and Development, 2012). In vivo studies on the function of Setd8 and H4K20me1 have been limited by the early embryonic lethality of constitutive Setd8 deletion (Oda et al, MCB, 2009). Although Setd8 is broadly expressed in tissues, there is a striking increase in Setd8 expression in CD71+ erythroid cells (Wu et al, Genome Biology, 2009), suggesting that Setd8 may have erythroid specific functions. Initial studies from our lab and others suggest that Setd8 regulates erythroid maturation and represses Gata2 expression (Malik et al MCB 2015; DeVilbiss et al MCB 2015). To delineate the function of Setd8 in vivo, we generated an erythroid-specific Setd8 deletion by crossing mice with flox sites flanking exon 7 of Setd8 (Setd8 fl/fl; Oda et al, MCB, 2009) with mice expressing a Cre-Recombinase GFP fusion protein under the control of the endogenous erythropoietin receptor promoter (ErGFPCre; Heinrich et al, Blood, 2004). Setd8Δ/Δ;ErGFPCre embryos demonstrated visible anemia starting at E9.5, with death occurring at E12.5 due to severe anemia. The early onset of anemia is consistent with a defect in primitive erythropoiesis. Cytospins and imaging flow cytometric analyses demonstrated a block in primitive erythroblast maturation and a profound impairment in nuclear condensation, with a nuclear area of 96 um2 in the Setd8 null erythroblasts and 56um2 in the littermate control erythroblasts at E10.5. Setd8 null erythroblasts also had abnormalities in cell cycle progression as well as increased staining for γH2AX, suggesting accumulation of DNA damage. There were similar numbers of Erythro-myeloid progenitors, (EMP; defined as defined as Ter119-negative, kit-hi,CD41-hi cells) in both the Setd8Δ/Δ;ErGFPCre and littermate control embryos, however the Setd8 null progenitors failed to differentiate into definitive erythroblasts. The role of Setd8 in transcriptional regulation is controversial, with some studies suggesting it is an activator and others suggesting it is a repressor. Global transcriptome analyses on sorted populations of Setd8 null and littermate control erythroblasts suggest that Setd8 acts primarily as a repressor in erythroid cells, with the majority of transcripts upregulated following Setd8 deletion. RNA-seq analyses further demonstrated a profound increase in the expression of cell cycle checkpoint enzymes such as P21 (fold change 73, p〈 e-53) and a significant increase in Gata2 expression (fold change 16, P

Description: The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during a time of hemoglobin accumulation, rapid cell division, and nuclear condensation. Disruption of this process is associated with myelodysplastic syndromes and congenital anemias. Congenital Dyserythropoietic Anemia type I (CDA-I), is an autosomal recessive disease that presents with severe macrocytic anemia early in infancy or early childhood. Patients with CDA-I have erythroid hyperplasia in the bone marrow. The erythroblasts in CDA-I are frequently binucleate, have chromatin bridging, and defective chromatin condensation (Renella 2011). CDA-I is most commonly caused by mutations in the protein codanin 1 (CDAN1). The function of CDAN1 is poorly understood, but it is thought to regulate histone incorporation into nascent DNA during cellular replication (Ask 2012). The study of CDA-1 has been limited by lack of in vitro models that recapitulate key features of the disease, and to date, the majority of studies on CDAN1 function have been done in non-erythroid cells. To model CDA-I we introduced a point mutation (PM) commonly observed in CDA-1 patients (R1042W) into HUDEP2 cells. In addition, we generated two HUDEP2 cell lines with heterozygous deletion of CDAN1 (del-R1042). All CDAN1 mutant cell lines had decreased viability in during both expansion and terminal maturation compared to control lines. Chromatin bridges and multinucleate cells were observed in all three mutant CDAN1 lines but were most prominent in the PM line. Intriguingly, global gene analysis demonstrated that all mutated lines had significantly elevated gamma-globin expression compared to controls, consistent with reports of elevated fetal globin expression in CDA-1 patients. Interestingly, the PM cell line had faster cell divisions than the del-R1042 or control lines, characterized by decreased doubling time and verified by quantifying dilution of a fluorescent dye. In contrast, the del-R1042 lines had slower cell doubling times than both the controls and the PM line. The PM line also had an increased median intensity of BrdU compared to controls and del-R1042 lines, suggesting an accelerated S-phase. KI67 staining of the PM line showed an increase percentage of mitotic cells. These data are consistent with the finding that multinucleate cells and chromatin bridges are more common in the PM line. Furthermore, electron microscopy suggests the PM cell line may have defects in heterochromatin formation. Together, these data imply a specific functional role for residue R1042, and suggest that within the context of R1042W, loss of the arginine residue and replacement thereof with a tryptophan may have different mechanistic consequences. Collectively, our preliminary data suggests that CDAN1 is important in the regulation of DNA replication and organization in maturing erythroblasts. Specific mutations may confer a change of function which results in dysplastic erythroid cells that are sensitive to dysregulated cell cycle mechanics due to the high rate of cell division. We hypothesize R1042W substitution may accelerate cell division at the expense of appropriate checkpoints and result in dysplastic erythroid cells and mimic the clinical presentation of increased multinuclearity, decreased cell viability, and increased gamma globin expression. Most importantly, generation of models with specific patient mutations will provide further mechanistic insight into CDA-I pathology. Disclosures No relevant conflicts of interest to declare.

Description: Maturation of erythroid progenitors is associated with significant changes in gene expression in the context of a nucleus that dramatically decreases in size in preparation for enucleation, and is regulated by the coordinated action of transcriptional regulators and epigenetic modifiers. In eukaryotes, all DNA is bound by histone proteins into chromatin. Posttranslational modifications of the N-terminal "tails" of these proteins are key regulators of chromatin structure and gene expression. We hypothesized that terminal erythroid maturation is associated with changes in the abundance of specific histone posttranslational modifications. To address this hypothesis, we utilized mass spectrometry to perform an unbiased assessment of the abundance histone post translational modifications in maturing erythroblasts. We cultured peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) down the erythroid lineage using a semi-synchronous culture system (as outlined in Gautier et al. Cell Reports 2016), and sent cells for mass spectrometry on day 7 of erythroid maturation, when the cells are predominately basophilic erythroblasts, and on day 12 of erythroid maturation, when they are predominately poly- and ortho- chromatic erythroblasts. The maturation stage of the cells was confirmed by both cytospins and imaging flow cytometric analyses. Two independent replicates were performed and key results confirmed by western blotting. Terminal erythroid maturation was associated with a dramatic decline in the abundance of multiple histone marks associated with active transcription elongation, including Histone H3 lysine 36 di- and tri-methylation (H3K36me2, H3K36me3), and Histone H3 Lysine 79 di-methylation (H3K79me2). Surprisingly, this was not accompanied by an increase in the abundance of repressive heterochromatin marks (H3K27me3, H3K9me3, and H4K20me3) or a global decline in histone acetylation. Histone H4 lysine 16 acetylation (H4K16Ac), associated with RNA polymerase II pause release (Kapoor-Vazirani MCB 2011) significantly declined, but multiple acetylation marks including H3K36Ac and H3K23Ac increased in abundance. As expected, the abundance histone H4 lysine 20 mono-methylation (H4K20me1), which is implicated both in erythroblast chromatin condensation (Malik Cell Reports 2017) and the regulation of RNA Polymerase II pausing (Kapoor-Vazirani MCB 2011) also significantly increased. Consistent with these data, integration of RNA-seq and ChIP-seq data identified 3,058 genes whose expression decreased from basophilic erythroblast to orthochromatic erythroblasts, which lost enrichment for H3K36me3 (mark of active elongation) without accumulating H3K27me3 (heterochromatin mark). Based on these data, we hypothesized that RNA polymerase II pausing is a critical regulator of gene expression in maturing erythroblasts. RNA Polymerase II (Pol II) pausing is a highly regulated mechanism of transcriptional regulation, whereby transcription is initiated, but pauses 30-60bp downstream of the transcription start site. For paused Pol II to be released into active elongation, pTEFb must hyper-phosphorylate Serine 2 of the Pol II c-terminal domain (CTD). Importantly, pTEFb can be directed to specific loci through interaction with transcription factors, including GATA1 (Elagib Blood 2008; Bottardi NAR 2011). Hexim1 is a key regulator of Pol II pausing that sequesters pTEFb and inhibits its action. Consistent with a central role for Pol II pausing dynamics in the regulation of terminal erythroid maturation, Hexim1 is highly expressed in erythroid cells compared to most other cell types and its expression increases during terminal erythroid maturation. Conversely, the expression of CCNT1 and CKD9, the components of pTEFb, decline during terminal maturation, and the level of elongation competent (Ser2 and Ser2/Ser5 CTD phosphorylated) Pol II also decreases dramatically. To gain insights into the function of Pol II pausing in maturing erythroblasts, we induced Hexim1 expression in HUDEP2 cells (Kurita PLoS One 2013) using hexamethane bisacetamide (HMBA). HMBA treatment increased Hexim1 levels a dose dependent manner and was associated with gene expression and phenotypic changes suggestive of accelerated erythroid maturation. Together, these data suggest that RNA Pol II pausing dynamics are an important regulator of terminal erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Description: Chromatin condensation culminating in enucleation is a hallmark of erythropoiesis, however the mechanisms driving this process are incompletely understood. Setd8 is the sole enzyme that can mono-methylate histone H4, lysine 20 (H4K20me1) and is an important regulator of cell cycle progression, higher order chromatin structure, and genome stability. (Reviewed in Beck, Genes and Development, 2010) Setd8 and H4K20me1 are unique among epigenetic regulators in that their expression is dynamically regulated during the cell cycle. Setd8 expression peaks during G2/M, where it promotes mitotic chromatin condensation, and becomes undetectable during S-phase due to ubiquitin dependent destruction. (Oda, Mol Cell, 2010) The presence of H4K20me1 mirrors that of Setd8, peaking in G2/M, and reaching a nadir during S-phase due to removal by the histone demethylase PHF8. (Liu, Nature, 2010) Interestingly, Setd8 is expressed at levels 8- to 10- fold higher in CD71+ erythroblasts than in any other cell type, (Wu Genome Biology,2009) suggesting that it has an erythroid-specific function. We hypothesize that Setd8 drives chromatin condensation in maturing erythroblasts. In cell lines, forced accumulation of H4K20me1 during S-phase due to perturbation of either Setd8 or PHF8 results in pre-mitotic chromatin condensation (Centore Mol Cell 2010; Liu Nature 2010). We demonstrate that primary erythroblasts express Setd8 and accumulate H4K20me1 throughout the cell cycle, suggesting that Setd8 and H4K20me1 in promote chromatin condensation during terminal maturation. We further demonstrate that Setd8 is essential for erythropoiesis, with erythroid-specific Setd8 deletion resulting in profound anemia that is lethal by E12.5. The early onset of anemia indicates a defect in the primitive erythroid lineage, which emerges from the yolk sac at E8.5, and proliferates, matures, and enucleates in the circulation as a semi-synchronous cohort. (Kingsley, Blood, 2004) Detailed analyses of Setd8-null erythroblasts revealed severe defects in cell cycle progression, increased DNA content suggesting loss of genomic integrity, accumulation of DNA damage, and a modest increase in the rate of apoptosis. Global transcriptome analyses demonstrated that Setd8-null erythroblasts had activation of checkpoint genes such as CDKN1a and Gene Set Enrichment Analyses identified significant enrichment of cell cycle and p53 signaling pathways. Despite evidence of p53 activation, concomitant p53 deletion was not able to rescue the Set8-null phenotype, indicating that Setd8 has an essential role in promoting erythroid proliferation and survival that is independent of the p53 pathway. Consistent with our hypothesis that Setd8 drives chromatin condensation in maturing erythroblasts, the nuclear area of Setd8-null cells was nearly twice that of controls at E11.5 (119 and 69 um2, respectively p

Description: Setd8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. Setd8 mRNA is expressed ~10-fold higher in erythroid cells than any other cell type (biogps.org) and Setd8 protein levels increase in concert with GATA1 levels during erythroid differentiation of CD34+ HSPCs, suggesting Setd8 may have a role regulating the erythroid transcriptome. Consistent with this hypothesis, erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.5 (E11.5) due to profound anemia and global transcriptomic analyses of sorted populations of E10.5 Sed8 null and control erythroblasts demonstrated a profound defect in transcriptional repression, with 340/345 differentially expressed genes (DEG) expressed at higher levels in the Setd8 null cells than controls (Malik Cell Reports 2017). Primitive erythroblasts mature and enucleate in a semi-synchronous manner in circulation. To better understand the function of Setd8 in regulating the erythroid transcriptome, we extended our transcriptomic analyses by performing RNA-seq in sorted E9.5 Sed8 null (EpoRCre+; Setd8 Δ/Δ) and control (EpoRCre+; Setd8 Δ/+) erythroblasts. The Setd8 null cells failed to repress 20/137 (15%) of the genes that are down regulated in control cells from E9.5 to E10.5. Although relatively few genes were impacted, those genes were enriched for the pathway "Oxidative Stress" (adjusted p-value 0.009) suggesting that Setd8 may regulate specific functions during terminal erythroid maturation. We next compared the DEG in Setd8 null erythroblasts to transcriptomic changes that occur as a cell transcends the hematopoietic hierarchy, gaining lineage specificity while suppressing the multi-lineage transcriptome (GSE14833). A large fraction, 105/345 (~30%), of genes up-regulated in Setd8 null erythroblasts, are also up-regulated in multipotent progenitors compared to proerythroblasts. In contrast, only 16/345 (5%) were also up-regulated in granulocyte-monocyte progenitors suggesting that Setd8 does not repress other lineage restricted signatures. Together, these results suggest that Setd8 regulates repression of the multi-lineage transcriptome during erythroid differentiation from multipotent progenitor cells. To gain insights into how Setd8 regulates the erythroid transcriptome, we performed ATAC-seq (Buenrostro Nature Methods 2013) on sorted populations of erythroblasts from E10.5 Sed8 null and control embryos. Cell number for the Setd8 null samples was limited due to anemia, with ~1000 cells used for each replicate. Setd8 and control replicates were aggregated and accessible regions were identified using MACS2. Regions more accessible in Setd8 null cells were identified by computing a log2 ratio between Setd8 null and control samples using deepTools bamCompare. In addition, we utilized ChIPmentation (Schmidl Nature Methods 2015) to assay H3K27me3 occupancy across the genome of WT E10.5 erythroblasts to identify regions of heterochromatin in maturing erythroblasts. Two replicates were performed using 2.5-5x105 cells per assay, and peak called was done using MACS2. A total of 157 genes were identified that had more accessible chromatin in Setd8 null cells and contained an enrichment for H3K27me3 in WT cells suggesting that these genes should be repressed during normal erythropoiesis. Among these were several DEG that were up-regulated in the Setd8 null cells including Hhex, Cd63, and Gata2. Genomic data integration also identified several additional transcriptional regulators that are active in earlier hematopoietic progenitors but typically silenced during erythroid differentiation including Notch1 and Cebpa. Pathway analysis of the 157 genes identified several stemness-related pathways including "Transcriptional regulation of pluripotent stem cells" and "OCT4, SOX2, NANOG repress genes related to differentiation" (adjusted p-values 0.005 and 0.008, respectively). The chromatin regions that were more accessible in the Setd8 null cells were enriched for the DNA binding motifs of the transcription factor ERG (p-value 1-257), SCL (p-value 1e-193), and NRF1 (p-value 1e-101). Taken together, these data suggest that Setd8 works in concert with erythroid transcription factors to repress the transcriptional network in stem and progenitor cells and establish appropriate patterns of gene expression during erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

Description: 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 (p1.5), 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.