Congenital thrombocytopenia associated with a heterozygous variant in the MEIS1 gene encoding a transcription factor essential for megakaryopoiesis.

The transcription factor MEIS1 (myeloid ectotrophic insertion site 1) is crucial for the maintenance of hematopoietic stem cells and for megakaryopoiesis. Germline variants in MEIS1 are associated with restless-leg syndrome, but were not previously shown to cause cytopenias. This is the first report of a patient with congenital thrombocytopenia associated with a sequence variant in MEIS1, presenting with early onset severe thrombocytopenia and mild signs of bone marrow stress. Whole exome sequencing revealed a de novo monoallelic splice site variant in MEIS1, NM_002398.3:exon4:c.432 + 5 G > C, leading to a premature stop codon. We propose that heterozygous mutations in MEIS1 may cause congenital thrombocytopenia.

Cdan1 Is Essential for Primitive Erythropoiesis.

Congenital dyserythropoietic anemia type I (CDA I) is an autosomal recessive disease characterized by moderate to severe macrocytic anemia and pathognomonic morphologic abnormalities of the erythroid precursors, including spongy heterochromatin. The disease is mainly caused by mutations in CDAN1 (encoding for Codanin-1). No patients with homozygous null type mutations have been described, and mouse null mutants die during early embryogenesis prior to the initiation of erythropoiesis. The cellular functions of Codanin-1 and the erythroid specificity of the phenotype remain elusive. To investigate the role of Codanin-1 in erythropoiesis, we crossed mice carrying the Cdan1 floxed allele (Cdan fl/fl ) with mice expressing Cre-recombinase under regulation of the erythropoietin receptor promoter (ErGFPcre). The resulting CdanΔEry transgenic embryos died at mid-gestation (E12.5-E13.5) from severe anemia, with very low numbers of circulating erythroblast. Transmission electron microscopy studies of primitive erythroblasts (E9.5) revealed the pathognomonic spongy heterochromatin. The morphology of CdanΔEry primitive erythroblasts demonstrated progressive development of dyserythropoiesis. Annexin V staining showed increases in both early and late-apoptotic erythroblasts compared to controls. Flow cytometry studies using the erythroid-specific cell-surface markers CD71 and Ter119 demonstrated that CdanΔEry erythroid progenitors do not undergo the semi-synchronous maturation characteristic of primitive erythroblasts. Gene expression studies aimed to evaluate the effect of Cdan1 depletion on erythropoiesis revealed a delay of ζ to α globin switch compared to controls. We also found increased expression of Gata2, Pu.1, and Runx1, which are known to inhibit terminal erythroid differentiation. Consistent with this data, our zebrafish model showed increased gata2 expression upon cdan1 knockdown. In summary, we demonstrated for the first time that Cdan1 is required for primitive erythropoiesis, while providing two experimental models for studying the role of Codanin-1 in erythropoiesis and in the pathogenesis of CDA type I.

Codanin-1, the protein encoded by the gene mutated in congenital dyserythropoietic anemia type I (CDAN1), is cell cycle-regulated.

BACKGROUND: Congenital dyserythropoietic anemia type I is an inherited autosomal recessive macrocytic anemia associated with ineffective erythropoiesis and the development of secondary hemochromatosis. Distinct erythroid precursors with internuclear chromatin bridges and spongy heterochromatin are pathognomonic for the disease. The mutated gene (CDAN1) encodes a ubiquitously expressed protein of unknown function, codanin-1. Based on the morphological features of congenital dyserythropoietic anemia type I erythroblasts and data on a role in cell cycle progression of codanin-1 homolog in Drosophila we investigated the cellular localization and possible involvement of codanin-1 during the cell cycle. DESIGN AND METHODS: Codanin-1 localization was studied by immunofluorescence and immune electron microscopy. Cell cycle expression of codanin-1 was evaluated using synchronized HeLa cells. E2F proteins are the main regulator of G(1)/S transition. An E2F1-inducible cell line (U20S-ER-E2F1) enabled us to study codanin-1 expression following ectopic E2F1 induction. Direct binding of E2F1 to codanin-1 promoter was assessed by chromatin immunoprecipitation. We used a luciferase-reporter plasmid to study activation of CDAN1 transcription by E2F1. RESULTS: We localized codanin-1 to heterochromatin in interphase cells. During the cell cycle, high levels of codanin-1 were observed in the S phase. At mitosis, codanin-1 underwent phosphorylation, which coincided with its exclusion from condensed chromosomes. The proximal CDAN1 gene promoter region, containing five putative E2F binding sites, was found to be a direct target of E2F1. CONCLUSIONS: Taken together, these data suggest that codanin-1 is a cell cycle-regulated protein active in the S phase. The exact role of codanin-1 during the S phase remains to be determined. Nevertheless this represents the first step towards understanding the function of the proteins involved in congenital dyserythropoietic anemia.

EGF receptor activity is essential for adhesion-induced stress fiber formation and cofilin phosphorylation.

Integrin-mediated cell adhesion induces activation of the EGF receptor tyrosine kinase independently of the soluble growth factor ligand. EGFR activation is instrumental for subsequent activation of additional signaling pathways in adherent cells, including the Ras-MAP kinase pathway and the phosphatidylinositol 3-kinase/Akt pathway. We demonstrate here that integrin-dependent EGFR activation is also essential for adhesion-induced formation of actin stress fibers, focal adhesion localization and tyrosine phosphorylation of the adapter protein paxillin, as well as transcriptional activation of the serum response factor. All these events are known to be mediated by the small GTPase RhoA. EGFR activity was not found to regulate the activity status of RhoA, however. Instead, we found that EGFR activity is required for integrin-induced phosphorylation of cofilin. Cofilin is an actin-binding protein, which, when unphosphorylated, stimulates depolymerization and severing of actin filaments. Thus, in the absence of the kinase activity of the EGFR, cofilin remains dephosphorylated and depolymerizes actin filaments, rendering cells unable to respond to RhoA signaling. These studies demonstrate adhesion-dependent regulation of cofilin phosphorylation, and identify a novel role for EGFR in integrin signaling.

EGF receptor mediates adhesion-dependent activation of the Rac GTPase: a role for phosphatidylinositol 3-kinase and Vav2.

Organization of the actin cytoskeleton in eucaryotic cells is controlled by small GTPases of the Rho family. Rac becomes activated by growth factor stimulation and integrin-mediated cell adhesion to extracellular matrix and is known to have a crucial role in lamellipodia formation, cell spreading and migration. At present, the intracellular pathways that connect cell surface receptors to Rac activation are poorly characterized. It has been reported previously that integrin-mediated cell attachment induces activation of the EGF receptor (EGFR) in the absence of EGF. We demonstrate here that this activation is instrumental for integrin-dependent Rac activation. Thus, we found that cells in which EGFR activity had been inhibited failed to spread and form lamellipodia on fibronectin. Failure to spread coincided with inhibition of adhesion-induced GTP loading of Rac and also with inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Subsequent studies demonstrated that an activated form of PI 3-kinase restored Rac GTP loading in the presence of EGFR inhibition, while a dominant-negative form of PI 3-kinase blocked Rac GTP loading in fibronectin-adherent cells. Our further functional studies identified Vav2, a known exchange factor for Rac, as a crucial downstream component in EGFR- and PI 3-kinase-dependent Rac activation upon integrin-mediated cell adhesion. Our results provide a mechanistic insight into integrin-dependent Rac activation, and identify a novel role for EGFR, PI 3-kinase and Vav2 in this pathway.