Defects in E2F1/2 Expression Are Associated with Abnormalities in Cell Cycle and Differentiation in EKLF-Deficient Erythroid Cells.
Journal - ASH Annual Meeting Abstracts
Mice deficient in the erythroid transcription factor EKLF die~dE14 from severe anemia, attributed to decreased ß-globinexpression. Recent reports using microarray analyses indicatethat expression of numerous genes is perturbed in erythroidcells lacking EKLF. We performed flow cytometry of WT dE14 fetalliver (FL) cells with TER119 and CD71 and identified 5 previouslydescribed populations: R1+R2 composed of BFU-E and CFU-E, respectively,and R3+R4+R5 composed of more mature erythroblasts. The sameanalysis of dE14 EKLF-deficient FL cells showed R3+R4+R5 wereabsent, indicating a block in erythroid differentiation. Thisdifferentiation block introduced a bias into the previous microarraydata, as EKLF-deficient FL contain only immature erythroid cells(R1+R2), while WT FL cells are predominantly more differentiated(R3+R4+R5). Thus, expression of many genes was decreased dueto a loss of more mature erythroblasts, rather than due to theaction of EKLF or an intermediary. To obtain more rigorous comparativedata, we performed microarray analyses with EKLF-deficient FLcells and R1+R2 populations from WT FL cells. Expression ofnumerous genes was deregulated. Of note, many genes significantlydown regulated in previous microarray analyses were not downregulated when the 2 similar populations were compared. IngenuityPathway Analysis of the microarray data identified a biologicnetwork involved in cell cycle and DNA replication. At the centralnodes of the network were E2F1 and E2F2, transcription factorsinvolved in cell cycle control and differentiation. In quantitativeRT-PCR, E2F1 and E2F2 expression in EKLF-deficient mRNA wasdecreased to 40.5±1.6 and 7.6±1.6% of WT, respectively.Western blot analysis demonstrated comparable reductions inboth E2F proteins. Cell cycle analyses showed that EKLF-deficientR1 cells exhibit a significant delay exiting G0+G1 and enteringS phase (p<0.001). Both R1 and R2 cells exhibited a defectexiting S and entering G2+M (p<0.004). Colony forming assaysrevealed that EKLF-deficient FL cells had decreased frequencyof BFU-E in R1 (p<0.005), with a defect in ability to generateCFU-E, and decreased frequency of CFU-E in R2 (p<0.001) witha defect in ability to differentiate into more mature erythroblasts.Flow cytometry with annexin V staining revealed that EKLF-deficientcells were resistant to apoptosis, indicating apoptosis wasnot contributing to the block. Based on these data, we hypothesizedthat E2F1/2 were EKLF target genes. Thus we examined the chromatinat the E2F2 locus in WT and EKLF-deficient FL cells using ahigh throughput assay to identify DNase-I hypersensitive sites(HS). Three HS were identified in WT that were absent in EKLF-deficientchromatin, one in the promoter region, one in intron 2, andone in the 3' region. The E2F2 promoter HS region contains 2EKLF consensus binding sites, which were examined in gel mobilityshift assays for their ability to bind EKLF. One of these probesyielded a complex that co-migrated with a control ß-globinprobe complex. This complex was competed by an excess of E2F2or ß-globin probe and supershifted with an anti-EKLFMoAb. These results support the hypothesis that defects in E2F1/2expression are associated with abnormalities in cell cycle anddifferentiation, contributing to a failure of definitive erythropoiesisin EKLF-deficient mice.Footnotes* Corresponding authorDisclosure: No relevant conflicts of interest to declare.