Knockdown of ZNF268, which Is Transcriptionally Downregulated by GATA-1, Promotes Proliferation of K562 Cells

The human ZNF268 gene encodes a typical KRAB-C2H2 zinc finger protein that may participate in hematopoiesis and leukemogenesis. A recent microarray study revealed that ZNF268 expression continuously decreases during erythropoiesis. However, the molecular mechanisms underlying regulation of ZNF268 during hematopoiesis are not well understood. Here we found that GATA-1, a master regulator of erythropoiesis, repressed the promoter activity and transcription of ZNF268. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that GATA-1 directly bound to a GATA binding site in the ZNF268 promoter in vitro and in vivo. Knockdown of ZNF268 in K562 erythroleukemia cells with specific siRNA accelerated cellular proliferation, suppressed apoptosis, and reduced expression of erythroid-specific developmental markers. It also promoted growth of subcutaneous K562-derived tumors in nude mice. These results suggest that ZNF268 is a crucial downstream target and effector of GATA-1. They also suggest the downregulation of ZNF268 by GATA-1 is important in promoting the growth and suppressing the differentiation of K562 erythroleukemia cells

Altered translation of GATA1 in Diamond-Blackfan anemia

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA)[superscript 1, 2], congenital asplenia[superscript 3] and T cell leukemia[superscript 4]. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type– and tissue-specific defects remains unknown[superscript 5]. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.National Institutes of Health (U.S.) (Grant P01 HL32262)National Institutes of Health (U.S.) (Grant U54 HG003067-09

p53-mediated differentiation of the erythroleukemia cell line K562

The tumor suppressor gene p53 can mediate both apoptosis and cell cycle arrest. In addition, p53 also influences differentiation. To further characterize the differentiation inducing properties of p53, we overexpressed a temperature-inducible p53 mutant (ptsp53Val135) in the erythroleukemia cell line K562. The results show that wild-type p53 and hemin synergistically induce erythroid differentiation of K562 cells, indicating that p53 plays a role in the molecular regulation of differentiation. However, wild-type p53 did not affect phorbol 12-myristate 13-acetate-dependent appearance of the megakaryocyte-related cell surface antigens CD9 and CD61, suggesting that p53 does not generally affect phenotypic modulation. The cyclin-dependent kinase inhibitor p21, a transcriptional target of p53, halts the cell cycle in G1 and has also been implicated in the regulation of differentiation and apoptosis. However, transiently overexpressed p21 did neither induce differentiation nor affect the cell cycle distribution or viability of K562 cells, suggesting that targets downstream of p53 other than p21 are critical for the p53-mediated differentiation response