Orchestrating Developmental Signaling to Balance Erythroblast Proliferation and Differentiation

The highly conserved exosome complex mediates the degradation and processing of multiple classes of RNAs. How this post-transcriptional RNA- and gene-regulatory machine impacts cell fate decisions and differentiation is poorly understood. Previously, we demonstrated that exosome complex subunits confer an erythroid maturation barricade, and the erythroid transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the cognate genes. While dissecting requirements for the maturation barricade in mice, we discovered that the exosome complex is a vital determinant of a developmental signaling transition that dictates proliferation versus differentiation of erythroid precursor cells. Using shRNAs to downregulate exosome complex subunits, we developed conditions that disrupt exosome complex integrity and analyzed functional consequences. We discovered that erythroid precursor cells derived from E14.5 murine fetal liver exhibited significantly decreased survival when erythropoietin (Epo) was removed from the culture media. Live cells decreased from 66% in the control to 24% (p = 2 x 10^-5) in the Exosc8-knockdown condition. The maturation-associated increase (1.5 fold, p = 0.003) in the Ter119⁺CD71⁺ (R3: early/late basophilic erythroblast) population upon Exosc8 knockdown did not occur without exogenous Epo. Thus, the survival and differentiation of erythroid precursors after downregulating Exosc8 is critically dependent on Epo. We tested whether Exosc8 downregulation impaired stem cell factor (SCF)-mediated proliferation/amplification of erythroid precursors. Using a phospho-flow cytometry assay, we quantitated the capacity of SCF or Epo to instigate cell signaling using the shared downstream substrate Akt. SCF induced maximal Akt phosphorylation in immature Ter119^-CD71^high erythroblasts (5.5 fold, p = 3 x 10^-6). As erythroid maturation progressed to Ter119⁺CD71^high, the SCF response was diminished. Exosc8 downregulation abrogated SCF-mediated induction of phospho-Akt. Epo induced maximal Akt phosphorylation in Ter119⁺CD71^high erythroblasts, and Exosc8 downregulation accelerated acquisition of this signaling response. Whereas Epo did not affect Akt phosphorylation in control Ter119^-CD71^high erythroblasts, Epo increased phospho-Akt 4 fold (p = 0.003) upon Exosc8 downregulation. Thus, downregulating Exosc8 abrogated SCF signaling that supports erythroid precursor proliferation/amplification, while precociously inducing Epo-dependent pro-differentiation signaling. As downregulating Exosc8 abrogated SCF signaling, we tested whether the exosome complex or individual subunits confer Kit expression or promote signaling via a post-receptor mechanism. Downregulating Exosc8 or Exosc9 reduced Kit surface expression in the R2 (proerythroblast and early basophilic) cell population [14.6 (p = 3.6 x 10^-4) and 6 fold (p= 1.3 x 10^-6) decrease, respectively]. Exosc8 downregulation also reduced Kit mRNA and primary transcript levels at all erythroid maturation stages. By 24 h post-infection with shExosc8-expressing retrovirus, Kit protein decreased 10 fold (p = 0.046) in Ter119^- erythroid precursor cells. We tested whether the Exosc8 requirement for Kit primary transcript, mRNA and protein expression involved alterations in the distribution of transcriptionally-competent serine 5-phosphorylated RNA polymerase II (Pol II) at Kit. Using quantitative ChIP analysis with control and Exosc8-knockdown Ter119^- cells, Exosc8 downregulation reduced phospho-Ser5 Pol II occupancy within the coding region (+5 kb) and 3' UTR, but not at the promoter. Furthermore, Exosc9 occupied multiple regions of the Kit locus. These results support a model in which the exosome complex confers Kit expression and stem cell factor/Kit signaling via a transcriptional mechanism. Functioning as a gatekeeper of the SCF - Epo developmental signaling transition, the exosome complex controls the massive production of erythroid cells that ensures organismal survival in homeostatic and stress contexts. Studies are underway to establish the full repertoire of exosome complex targets in the developing erythroblast, how these targets relate to exosome complex targets in hematopoietic stem and progenitor cells and exosome complex-dependent strategies for translational applications.