Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule 4 (ICAM-4), an immunoglobulin superfamily member, participates in island formation. Earlier, we identified ␣ V integrins as ICAM-4 counterreceptors. Because macrophages express ␣ V , ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knock-out mice and developed quantitative, live cell techniques for harvesting intact islands and for re-forming islands in vitro. We observed a 47% decrease in islands reconstituted from ICAM-4 null marrow compared to wild-type marrow. We also found a striking decrease in islands formed in vivo in knock-out mice. Further, peptides that block ICAM-4/␣ V adhesion produced a 53% to 57% decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage ␣ V functions in island integrity. Importantly, we documented that ␣ V integrin is expressed in macrophages isolated from erythroblastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/␣ V adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis. IntroductionErythroid progenitors proliferate, differentiate, and enucleate within specialized bone marrow niches, termed erythroblastic islands. [1][2][3][4] These structural units are composed of developing erythroblasts surrounding a central macrophage. It is apparent from ultrastructural studies that extensive cell-cell interactions, both erythroblastmacrophage, as well as erythroblast-erythroblast, occur within these 3-dimensional structures. However, little is known regarding either the molecular nature or functional role of the specific adhesive interactions. We are exploring the potential function of erythroid ICAM-4, a recently characterized member of the immunoglobulin superfamily, in erythroblastic island formation. ICAM-4 expression is limited to erythroid and placental tissue 5 but, to date, there is no information on its role in erythropoiesis. We earlier identified ␣ 4 ␤ 1 and ␣ V family integrins as ICAM-4-binding partners. 6 Because macrophages express ␣ V and erythroblasts exhibit ␣ 4 ␤ 1 , ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast interactions via ICAM-4/␣ 4 ␤ 1 binding and regulating adhesion of erythroblasts to central macrophages via ICAM-4/␣ V binding.ICAM-4, which carries the Lansteiner Wiener (LW) blood group antigen system, has strong sequence homology with other members of the ICAM protein superfamily. 7,8 It is composed of 2 extracellular immunoglobulin-like domains, an N-terminal I set and a membrane proximal I2 set, and a single membr...

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.Alternative splicing of pre-mRNA leads to the synthesis of multiple protein isoforms from a single gene. It is an important mechanism for regulating gene expression and may be utilized by 40 -60% of human genes (1-4). Thus, the estimated 25,000 to 30,000 genes of the human genome can generate a much larger number of proteins. Regulation of alternative splicing occurs in both a tissue-and development-specific manner, resulting in alterations in the structure and function of critical proteins. Altered splicing regulation can also be of widespread importance in the etiology of human disease (5-7).The protein 4.1 gene family serves as an excellent model for investigating the regulation of alternative splicing. The four genes that comprise the family (4 .1R, 4.1G, 4.1B, and 4.1N) display a remarkable array of highly regulated, tissue-specific splicing events. These alternative splicing events facilitate expression of distinct isoforms of 4.1 protein in cells of erythroid, epithelial, neural, and muscle origin (8 -14); thus, they provide opportunities for understanding the mechanisms that regulate alternative splicing in several different cell types. To date, mechanistic studies have focused predominantly on erythroid cells, in which 4.1R protein is a structural component of the erythrocyte plasma membrane and is important for structural integrity and stability of the membrane skeleton. In differentiating erythroid progenitor cells, a dramatic switch in pre-mRNA splicing result...

Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation, chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally-regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentallyregulated architectural proteins in nucleated blood cells.