Histones to the cytosol: exportin 7 is essential for normal terminal erythroid nuclear maturation.

Global nuclear condensation, culminating in enucleation during terminal erythropoiesis, is poorly understood. Proteomic examination of extruded erythroid nuclei from fetal liver revealed a striking depletion of most nuclear proteins, suggesting that nuclear protein export had occurred. Expression of the nuclear export protein, Exportin 7 (Xpo7), is highly erythroid-specific, induced during erythropoiesis, and abundant in very late erythroblasts. Knockdown of Xpo7 in primary mouse fetal liver erythroblasts resulted in severe inhibition of chromatin condensation and enucleation but otherwise had little effect on erythroid differentiation, including hemoglobin accumulation. Nuclei in Xpo7-knockdown cells were larger and less dense than normal and accumulated most nuclear proteins as measured by mass spectrometry. Strikingly,many DNA binding proteins such as histones H2A and H3 were found to have migrated into the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in Xpo7-knockdown cells. Thus, terminal erythroid maturation involves migration of histones into the cytoplasm via a process likely facilitated by Xpo7.

Homeodomain-interacting protein kinase 2 plays an important role in normal terminal erythroid differentiation.

Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic development because Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis, whereas the single knockouts do not. These serine-threonine kinases phosphorylate and consequently modify the functions of several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation (explained in part by impaired cell-cycle progression as well as increased apoptosis) and terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, such as alpha-globin and mitoferrin 1, demonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.

ID1 promotes expansion and survival of primary erythroid cells and is a target of JAK2V617F-STAT5 signaling.

The discovery of JAK2V617F as an acquired mutation in the majority of patients with myeloproliferative disorders (MPDs) and the key role of the JAK2-STAT5 signaling cascade in normal hematopoiesis has focused attention on the downstream transcriptional targets of STAT5. Despite evidence of its vital role in normal erythropoiesis and its ability to recapitulate many of the features of myeloid malignancies, including the MPDs, few functionally validated targets of STAT5 have been described. Here we used a combination of comparative genomics and chromatin immunoprecipitation assays to identify ID1 as a novel target of the JAK2-STAT5 signaling axis in erythroid cells. STAT5 binds and transactivates a downstream enhancer of ID1, and ID1 expression levels correlate with the JAK2V617F mutation in both retrovirally transfected fetal liver cells and polycythemia vera patients. Knockdown and overexpression studies in a well-characterized erythroid differentiation assay from primary murine fetal liver cells demonstrated a survival-promoting action of ID1. This hitherto unrecognized function implicates ID1 in the expansion of erythroblasts during terminal differentiation and suggests that ID1 plays an important role in the pathogenesis of polycythemia vera. Furthermore, our findings contribute to an increasing body of evidence implicating ID proteins in a wider range of cellular functions than initially appreciated.

BRCA1 haploinsufficiency for replication stress suppression in primary cells.

BRCA1-a breast and ovarian cancer suppressor gene-promotes genome integrity. To study the functionality of BRCA1 in the heterozygous state, we established a collection of primary human BRCA1(+/+) and BRCA1(mut/+) mammary epithelial cells and fibroblasts. Here we report that all BRCA1(mut/+) cells exhibited multiple normal BRCA1 functions, including the support of homologous recombination- type double-strand break repair (HR-DSBR), checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. In contrast, the same cells were defective in stalled replication fork repair and/or suppression of fork collapse, that is, replication stress. These defects were rescued by reconstituting BRCA1(mut/+) cells with wt BRCA1. In addition, we observed 'conditional' haploinsufficiency for HR-DSBR in BRCA1(mut/+) cells in the face of replication stress. Given the importance of replication stress in epithelial cancer development and of an HR defect in breast cancer pathogenesis, both defects are candidate contributors to tumorigenesis in BRCA1-deficient mammary tissue.