Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent (∼45 to ∼85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 3'-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.

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Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.

Nuclear receptors TR2 and TR4 (TR2/TR4) were previously shown to bind in vitro to direct repeat elements in the mouse and human embryonic and fetal ␤-type globin gene promoters and to play critical roles in the silencing of these genes. By chromatin immunoprecipitation (ChIP) we show that, in adult erythroid cells, TR2/TR4 bind to the embryonic ␤-type globin promoters but not to the adult ␤-globin promoter. We purified protein complexes containing biotin-tagged TR2/TR4 from adult erythroid cells and identified DNMT1, NuRD, and LSD1/CoREST repressor complexes, as well as HDAC3 and TIF1␤, all known to confer epigenetic gene silencing, as potential corepressors of TR2/TR4. Coimmunoprecipitation assays of endogenous abundance proteins indicated that TR2/TR4 complexes consist of at least four distinct molecular species. In ChIP assays we found that, in undifferentiated murine adult erythroid cells, many of these corepressors associate with both the embryonic and the adult ␤-type globin promoters but, upon terminal differentiation, they specifically dissociate only from the adult ␤-globin promoter concomitant with its activation but remain bound to the silenced embryonic globin gene promoters. These data suggest that TR2/TR4 recruit an array of transcriptional corepressors to elicit adult stage-specific silencing of the embryonic ␤-type globin genes through coordinated epigenetic chromatin modifications.Regulatory pathways that control development through temporally specified gene activation and repression mechanisms have been recognized as "epigenetic" (i.e., heritable changes not involving alterations in the primary DNA code) for decades, although the molecules that elicit those developmental programs through epigenetic means have only been elucidated during the past several years. It is currently widely accepted that metazoan transcription factors (both activators and repressors) elicit their specific transcriptional responses through an enormous variety of cofactor molecules whose major purpose is to modulate chromatin structure (8, 31). Many such cofactors have been shown to chemically modify histones, transcription factors, and cofactors, as well as DNA, in order to elicit the required transcriptional responses.The ␤-globin locus has been extensively studied as a paradigm for epigenetic regulation of lineage-specific and developmentally specific gene expression (29), as well as for its clinical relevance to ␤-globin disorders such as sickle cell disease and ␤-thalassemia. The human ␤-globin locus is composed of ε-(embryonic), G␥-and A␥-(fetal), and ␦-and ␤-globin (adult) genes, which are spatially arranged from 5Ј to 3Ј and developmentally expressed in the same order (72). The elucidation of the molecular basis for ␥-globin silencing in the adult stage in particular has been the focus of intense investigation, since it has been observed that coinheritance of genetic conditions that confer elevated ␥-globin synthesis can significantly alleviate the symptoms of ␤-globin disorders (44, 56). Previously, several adul...

Transcription factor GATA-1 is essential for the development of the erythroid lineage. To ascertain whether strict control of GATA-1 expression level is necessary for achieving proper erythropoiesis, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of the GATA-1 gene hematopoietic regulatory domain. We examined the GATA-1 expression level by exploiting the transgenic mice and found 2 GFP-positive hematopoietic progenitor fractions in the bone marrow. One is the GFP high fraction containing mainly CFU-E and proerythroblasts, which coexpress transferrin receptor, while the other is the GFP low /transferrin receptor-negative fraction containing BFU-E. Since the intensity of green fluorescence correlates well with the expression level of GATA-1, these results indicate that GATA-1 is highly expressed in erythroid colony-forming unit (CFU-E) but low in erythroid burstforming unit (BFU-E), suggesting that the incremental expression of GATA-1 is required for the formation of erythroid progenitors. We also examined GFP-positive fractions in the transgenic mouse spleen and fetal liver and identified fractions containing BFU-E and CFU-E, respectively. This study also presents an efficient method for enriching the CFU-E and BFU-E from mouse hematopoietic tissues. ( IntroductionPluripotent hematopoietic progenitors are committed to and differentiated along the erythroid lineage by the control of various cytokines, growth factors, and signals from the microenvironment. 1 In hematopoietic progenitors, these signals are transduced to transcription factors in the nucleus, and the progenitor cells differentiate and mature into erythrocytes by changing their gene expression profiles. 2 GATA-1 is a zinc-finger transcription factor, which plays a central role in erythropoiesis. GATA-1 binds to the GATA factor-binding motifs (T/A)GATA(A/G) that have been identified in the regulatory sequences of many genes expressed in erythroid and megakaryocytic cells. [3][4][5] Expression of GATA-1 is restricted to erythroid, megakaryocytic, eosinophilic, basophilic, and mast cells within the hematopoietic system. 6 The Sertoli cells in the testis also express GATA-1. 7 Mutations in the GATA-1 gene cause defects in erythropoiesis, platelet formation, mast cell maturation, and eosinophil development. [8][9][10][11][12][13][14] GATA-1-null mutant embryonic stem (ES) cells could not differentiate into mature erythrocytes due to the arrest of erythroid maturation at the proerythroblast stage. 15,16 The arrest provoked rapid apoptosis of the proerythroblasts. 17 We previously established GATA-1 gene knockdown ES cells, in which the GATA-1 expression level was reduced to approximately 5% of that in wild-type ES cells. 10,18 Proerythroblast-like cells derived from the GATA-1 knockdown ES cells have an ability to proliferate vigorously, but a GATA-1 level of 5% cannot sustain the gene expression required for maturation of proerythroblasts. 18 McDevitt et al also demonstrated that a 4-to 5-fold decrease in...