Abstract
Hundreds of sterile α-motif (SAM) domains have predicted structural similarities and are reported to bind proteins, lipids, or RNAs. However, the majority of these domains have not been analyzed functionally. Previously, we demonstrated that a SAM domain-containing protein, SAMD14, promotes SCF/proto-oncogene c-Kit (c-Kit) signaling, erythroid progenitor function, and erythrocyte regeneration. Deletion of a Samd14 enhancer (Samd14–Enh), occupied by GATA2 and SCL/TAL1 transcription factors, reduces SAMD14 expression in bone marrow and spleen and is lethal in a hemolytic anemia mouse model. To rigorously establish whether Samd14–Enh deletion reduces anemia-dependent c-Kit signaling by lowering SAMD14 levels, we developed a genetic rescue assay in murine Samd14–Enh−/− primary erythroid precursor cells. SAMD14 expression at endogenous levels rescued c-Kit signaling. The conserved SAM domain was required for SAMD14 to increase colony-forming activity, c-Kit signaling, and progenitor survival. To elucidate the molecular determinants of SAM domain function in SAMD14, we substituted its SAM domain with distinct SAM domains predicted to be structurally similar. The chimeras were less effective than SAMD14 itself in rescuing signaling, survival, and colony-forming activities. Thus, the SAMD14 SAM domain has attributes that are distinct from other SAM domains and underlie SAMD14 function as a regulator of cellular signaling and erythrocyte regeneration.
Highlights
Hundreds of sterile ␣-motif (SAM) domains have predicted structural similarities and are reported to bind proteins, lipids, or RNAs
Samd14 genetic rescue assay in Samd14 enhancer–mutant progenitor cells
Loss of the Samd14-enhancer (Samd14 –Enh) prevented anemia-induced Samd14 expression in spleen and attenuated c-Kit signaling in erythroid progenitors (Fig. 1A)
Summary
A composite E-box–GATA cis-element confers Samd expression in spleen and bone marrow hematopoietic cells [4, 19]. To test the role of Samd SAM in Samd14-mediated cellular survival, we utilized a Samd14 –EnhϪ/Ϫ spleen erythroid progenitor culture system and quantified the percentage of live (annexinVϪDraq7Ϫ), early apoptotic (EA; annexinVϩDraq7Ϫ), and late apoptotic cells (LA; annexinVϩDraq7ϩ) cells (Fig. 5F). Compared with Samd, Samd14 ⌬SAM-expressing CD71ϩTer119ϩ cells contained a 5.8-fold (p ϭ 0.016) higher percentage of late apoptotic cells (Fig. S5B). SAM domain chimeric proteins were expressed in spleen erythroid progenitors isolated from Samd14 –EnhϪ/Ϫ mice using our genetic rescue assay (Fig. 6C). Among GFPϩCD71ϩTer119ϩ and GFPϩCD71ϩTer119Ϫ, the percentage of late apoptotic cells was lower in S14-mc versus S14-Neb cells (Fig. 7D) These results demonstrate that the first and second ␣-helices of the Samd SAM domain uniquely contain sequences that regulate cellular signaling and survival in erythroid progenitors and are lacking in Neb
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