Expression and Function in Erythropoiesis

New York Blood Center, New York NY

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Abstract

Description (provided by applicant) Studies of 4.1 gene expression and 4.1 protein function have revealed a remarkable richness in the complement of 4.1 proteins encoded in the human genome; and unexpected new roles for 4.1 proteins in various supramolecular structures in nucleated cells. Here we propose to explore two novel aspects of 4.1 expression in erythroid cells, using mouse and human models of erythropoiesis. The first focuses on 4.1R gene expression, which is modulated during progenitor differentiation so as to downregulate synthesis of 135 kDa isoforms containing the N-terminal headpiece, and upregulate 80 kDa isoforms with a functional spectrin-actin binding domain. Our recent results suggest that alternative splicing at exon 2', which controls 135 kDa 4.1R synthesis, is functionally coupled to alternative transcription initiation events at newly discovered alternative first exons far upstream of the coding exons. We will test the hypotheses that each first exon is associated with a distinct transcriptional promoter with tissue-specific expression patterns; that promoter choice determines 135 kDa 4.1R expression; and that transcriptional control mediates up-regulation of 4.1R expression during late erythropoiesis. These qualitative and quantitative changes in 4.1R expression are critical to proper red cell mechanical properties. A second major goal of this proposal is to test the hypothesis that the highly homologous 4.1G protein plays an important role in erythropoiesis, and may compensate for the loss of 4.1R in 4.1R knockout mice. This hypothesis derives from the findings that 4.1 appears to function in nuclear architecture and mitotic spindle assembly, yet 4.1R deficiency does not appear to affect early erythroid development in the 4.1R knockout mouse. Preliminary results show that 4.1G is expressed in early erythroid cells and that it functionally resembles 4.1R with respect to binding interactions with erythroid ligands including band 3 and glycophorin C. We propose to study 4.1G function in erythroid development via gene knockout experiments and by characterization of 4.1G binding partners in early erythroid cells. These studies also open the possibilities for a better understanding of the role of 4.1 proteins not only in erythropoiesis, but also in human disease processes in nonerythroid tissues, as recent results indicate that such defects may extend beyond the familiar erythroid disorders caused by 4.1R deficiency.