Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified 1, but others remain elusive. Ten of twenty-seven members from a Maltese family presented with HPFH. A genome-wide SNP scan followed by linkage analysis revealed a candidate region on chromosome 19p13.12–13. Sequencing identified a nonsense mutation in the KLF1 gene, p.K288X, ablating the DNA binding domain of this key erythroid transcriptional regulator 2. Only HPFH family members were heterozygote carriers of this mutation. Expression profiling on primary erythroid progenitors revealed down-regulation of KLF1 target genes in HPFH samples. Functional assays demonstrated that, in addition to its established role in adult globin expression, KLF1 is a critical activator of the BCL11A gene, encoding a suppressor of HbF expression 3. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.

The translocation (6;9)(p23;q34) in acute nonlymphocytic leukemia results in the formation of a highly consistent dek-can fusion gene. Translocation breakpoints invariably occur in single introns of dek and can, which were named icb-6 and icb-9, respectively. In a case of acute undifferentiated leukemia, a breakpoint was detected in icb-9 of can, whereas no breakpoint could be detected in dek. Genomic and cDNA cloning showed that instead of dek, a different gene was fused to can, which was named set. set encodes transcripts of 2.0 and 2.7 kb that result from the use of alternative polyadenylation sites. Translocations are the best-studied nonrandom chromosomal aberrations associated with specific subtypes of leukemia. As a result of a translocation, an oncogene can be activated through alterations in regulatory DNA sequences that leave the encoded protein intact (e.g., myc) or through formation of a fusion gene, encoding a chimeric protein (e.g., bcr-abl). The t(9;22) associated with chronic myeloid leukemia, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (29) results in the expression of a chimeric BCR-ABL protein with enhanced tyrosine kinase activity (16,19,27,38,45). Pendergast et al. showed that defined sequences encoded by the first exon of bcr interact with the SH2 domain of ABL (33). This interaction is essential for the activation of the ABL tyrosine kinase activity and for the transforming capacity of BCR-ABL. More recently, other fusion genes have been isolated. t(1;19), occurring in childhood pre-B-cell acute leukemia, fuses the E2a gene, encoding transcription factors E12 and E47, to a novel homeobox gene, PBX1 (26, 32). t(15;17), strongly associated with acute promyelocytic leukemia, fuses part of the retinoic acid receptor type a gene (RARt) to a novel gene on chromosome 15 named PML, which is predicted to be a transcription factor (9, 25). bcr-abl, E2A-pbx, andpml-RARao seem to be highly consistent partners.Previously we reported the cloning of t(6;9) breakpoints (43). t(6;9) is the hallmark of a specific subtype of AML characterized by a poor prognosis and a young age of onset. It is classified in the French-American-British system mostly as M2/M4 and rarely as Ml or refractive anemia with excess of blast cells (RAEB) (2,36,39). On chromosome 9, break-* Corresponding author.points take place in a specific intron, icb-9, of a large gene (>140 kb) named Cain (can) (43). On chromosome 6, breakpoints also occur in a single intron, icb-6, of a gene named dek (42). The result of t(6;9) is the formation of a dek-can fusion gene on chromosome 6p-, which is transcribed into an invariable, 5.5-kb, leukemia-specific dek-can mRNA (39). The fusion transcript encodes a 165-kDa chimeric protein, which derives from the in-frame fusion of dek and can open reading frames (ORFs). Sequence comparison of DEK and CAN with entries in the EMBL data base shows no homology to any known protein sequences. CAN contains several putative dimerization motifs, and the C-terminal part may function as an ancillar...

The process of erythropoiesis must be efficient and robust to supply the organism with red bloods cells both under condition of homeostasis and stress. The microRNA (miRNA) pathway was recently shown to regulate erythroid development. Here, we show that expression of the locus encoding miR-144 and miR-451 is strictly dependent on Argonaute 2 and is required for erythroid homeostasis. Mice deficient for the miR-144/451 cluster display a cell autonomous impairment of late erythroblast maturation, resulting in erythroid hyperplasia, splenomegaly, and a mild anemia. Analysis of gene expression profiles from wild-type and miR-144/451–deficient erythroblasts revealed that the miR-144/451 cluster acts as a “tuner” of gene expression, influencing the expression of many genes. MiR-451 imparts a greater impact on target gene expression than miR-144. Accordingly, mice deficient in miR-451 alone exhibited a phenotype indistinguishable from miR-144/451–deficient mice. Thus, the miR-144/451 cluster tunes gene expression to impart a robustness to erythropoiesis that is critical under conditions of stress.

The translocation (6;9) is associated with a specific subtype of acute myeloid leukemia (AML). Previously, it was found that breakpoints on chromosome 9 are clustered in one of the introns of a large gene named Cain (can). cDNA probes derived from the 3' part of can detect an aberrant, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. cDNA cloning of this mRNA revealed that it is a fusion of sequences encoded on chromosome 6 and 3' can. A novel gene on chromosome 6 which was named dek was isolated. In dek the t(6;9) breakpoints also occur in one intron. As a result the dek-can fusion gene, present Defined karyotypic aberrations are associated with specific subtypes of leukemia. Detailed molecular characterization of these aberrations may identify genes involved in leukemogenesis and in the precise regulation of proliferation and differentiation in the hematopoietic system. Translocations are the best-studied chromosomal abnormalities. As the result of a translocation, the function or activity of oncogenes located at or near the translocation breakpoint is altered. In myeloid leukemia three translocation breakpoints have been cloned and analyzed at the molecular level.The two best studied, t(9;22) in chronic myeloid leukemia (27, 43) and t(15;17) in acute promyelocytic leukemia (2,8,12), result in the formation of chimeric genes that encode fusion proteins. In chronic myeloid leukemia this is a BCR-ABL protein that has an enhanced tyrosine kinase activity (34, 49) directly responsible for its in vivo tumorigenic potential (14,25). In acute promyelocytic leukemia a PMLRARa fusion protein that represents an altered transcription factor (16, 33) is found.The third translocation is the t(6;9) (p23;q34), found in a specific subtype of acute myeloid leukemia (AML) (1,39,41). This leukemia is characterized by a poor prognosis, affects young adults, and is classified mostly as M2 or M4 and rarely as Ml (according to the French-American-British classification of AML). A region on chromosome 9 situated 360 kb downstream of the c-abl gene was cloned and analyzed. It was found that breakpoints were clustered in a region of 8 kb in five patients, four with t(6;9) AML and one with acute undifferentiated leukemia (AUL) (47). Through cDNA cloning this region could be identified as one of the introns of a large gene (>100 kb) encoding a 7-kb transcript. This intron was named icb-9; the intron containing the breakpoints on chromosome 9 and situated in the middle of * Corresponding author. a gene named Cain (can). The 3' part of can is translocated to the 6p-chromosome, and only 3' can probes detect an additional, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. No additional transcripts were detected with 5' can probes. The breakpoint region on chromosome 6p23 was isolated from a genomic XEMBL3 library constructed of bone marrow DNA from one of the t(6;9) patients. An area of 40 kb of chromosome 6 DNA was cloned in overlapping phages. Southern blot analysis sh...

Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Krüppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in ␤-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis.

Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA. IntroductionDiamond-Blackfan anemia (DBA) presents as normochromic, macrocytic anemia with reduced erythroid precursors in the BM. 1 Approximately half of DBA patients have skeletal abnormalities such as thumb malformations and growth retardation. 2 DBA is mostly diagnosed in infants less than 1 year of age, but in recent years, nonclassic cases of DBA are being diagnosed in adult patients. 1 DBA is associated with mutations in genes encoding ribosomal proteins in 55% of patients. 3 The most prominently mutated gene (in 25% of patients) is RPS19, 4 but mutations in RPS7, RPS10, RPS17, RPS24, and RPS26 in the small ribosomal subunit and in RPL5, RPL11, and RPL35A in the large ribosomal subunit have also been found. 3 The mutations cause haploinsufficiency of ribosomal proteins and lead to loss of ribosome function; this reduces general translation, as observed in lymphocytes derived from DBA patients. 5 Knockdown of RPS19 in hematopoietic progenitors either from human BM or cord blood decreases the colony-forming capacity of erythroid progenitors, whereas it affects the colony-forming capacity of myeloid progenitors to a far lesser extent. 6 Knockdown of Rps19 in mouse fetal liver-derived erythroblasts impairs their proliferation, but the differentiation of cells that survive the knockdown is not affected. 7 Because ribosome synthesis consumes up to 25% of a cell's energy, a disbalance in the synthesis of ribosomal proteins activates p53 and inhibits cell proliferation. 8 Free Rpl11 and Rpl5 bind and inhibit Mdm2, which reduces p53 ubiquitination and leads to its stabilization. Erythroid cells may ...

Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes vErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal dierentiation under appropriate conditions, were established from fetal livers of p537/7 mice. Expression and activation of the EGF-receptor (HER-1/ c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal dierentiation. Upon inhibition of ErbB function, dierentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5-and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms. Oncogene (2001) 20, 3651 ± 3664.

The Philadelphia (Ph) chromosome, the product of t(9:22), is the cytogenetic hallmark of chronic myelogenous leukemia. The c-abl oncogene on chromosome 9 is translocated to the Ph chromosome and linked to a breakpoint cluster region (bcr), which is part of a large bcr gene. This results in the formation of a bcr-c-abl fusion gene, which is transcribed into an 8.5 kb chimeric mRNA encoding a 210 kd bcr-c-abl fusion protein. The Ph chromosome is also found in acute lymphoblastic leukemia (Ph+ ALL). Although the c-abl is translocated and a new 190 kd c-abl protein has been identified, no breakpoints are observed in the bcr (Ph+bcr- ALL). Here we show that in Ph+bcr- ALL, breakpoints in chromosome 22 occur within the same bcr gene, but more 5' of the bcr. Cloning of a chimeric bcr-c-abl cDNA demonstrates that the fusion gene is transcribed into a 7 kb mRNA, encoding a novel fusion protein.