A Short Isoform of Tensin1 Is a Novel Regulator of F-Actin Assembly in Human Erythroblasts That Promotes Enucleation

Mammalian red blood cells (RBCs) are generated via a terminal erythroid differentiation pathway culminating in cell polarization and enucleation. Actin filament polymerization is critical for enucleation, with F-actin assembling into a prominent structure at the rear of the nucleus termed the ‘enucleosome’, but the molecular regulatory mechanisms remain poorly understood. We utilized publicly available RNA-seq and proteomics datasets to mine for actin-binding proteins and actin-nucleation factors differentially expressed during human erythroid differentiation, and discovered that a focal adhesion and actin-binding protein-Tensin-1-shows a dramatic increase in expression late in terminal differentiation. The increase in TNS1 mRNA expression was confirmed by RT-PCR and TaqMan assays, as well as western blotting for TNS1, in human CD34+ cells induced to terminally differentiate into erythroid cells, with expression undetectable before day 7 and increasing dramatically by days 11-14 in culture. Remarkably, we found that these cells express a novel truncated form of Tensin-1 (eTNS1; apparent MW: ~125 kDa), missing the N-terminal actin-binding domain and half of the internal unstructured domain, but retaining the C-terminal SH2 and PTB domains, due to an internal mRNA translation start site resulting in a unique exon1. The region upstream of eTNS1 has features of an active erythroid promoter with an ATAC peak, H3K4 trimethylation, H3K27 acetylation, and GATA1 and TAL1 occupancy. This promoter demonstrates increasing chromatin accessibility during terminal erythroid differentiation paralleling increasing gene expression. In addition, an erythroid enhancer is present in intron 4 of the truncated transcript, indicated by an ATAC peak, H3K27 acetylation, and GATA1 and TAL1 occupancy. Sequence comparisons across various species revealed that the erythroid exon1 is not found in rodents and appears to be unique to primates. Confocal fluorescence microscopy demonstrated that eTNS1 localized to the cytoplasm during erythroid differentiation of CD34+ cells, appearing most abundant in polarized and enucleating cells, and was retained in the reticulocyte, with no apparent membrane association or focal adhesion formation. Knocking out eTNS1 had no effect on assembly of the spectrin membrane skeleton, based on western blotting for α1β1-spectrin and protein 4.1R, as well as confocal microscopy localizing α1β1-spectrin to the membrane of the incipient reticulocyte in polarized and enucleating cells. However, cells without eTNS1 had greatly reduced F-actin, failing to assemble F-actin into the enucleosome, instead forming aberrant F-actin cables or mislocalized foci in the cytoplasm or nucleus, or no detectable F-actin. Single cell analysis of eTNS1+ and eTNS1- cells with the Zeiss CD7 high-throughput confocal microscope showed that loss of eTNS1 impaired enucleation significantly, with ~30% reticulocytes generated as compared to eTNS+ cells. We conclude that eTNS1 is a novel regulator of F-actin assembly during human erythroid terminal differentiation that promotes efficient enucleation.

No relevant conflicts of interest to declare.