Neurodevelopmental abnormalities associated with severe congenital neutropenia due to the R86X mutation in the HAX1 gene.

OBJECTIVE: Severe congenital neutropenia (SCN), also known as Kostmann syndrome (SCN3, OMIM 610738), includes a variety of haematological disorders caused by different genetic abnormalities. Mutations in ELA2 are most often the cause in autosomal dominant or sporadic forms. Recently, mutations in HAX1 have been identified as the cause of some autosomal recessive forms of SCN, including those present in the original pedigree first reported by Kostmann. We sought to determine the relationship between HAX1 gene mutations and the clinical characteristics of Japanese cases of SCN. METHODS: The genes implicated in SCN (ELA2, HAX1, Gfi-1, WAS, and P14) were analysed in 18 Japanese patients with SCN. The clinical features of these patients were obtained from medical records. Immunoblotting of HAX1 was performed on cell extracts from peripheral blood leucocytes from patients and/or their parents. RESULTS: We found five patients with HAX1 deficiency and 11 patients with mutations in the ELA2 gene. In HAX1 deficiency, a homozygous single base pair substitution (256C>T), which causes the nonsense change R86X, was identified in three affected individuals. Two sibling patients showed a compound heterozygous mutation consisting of a single base pair substitution (256C>T) and a 59 bp deletion at nucleotides 376-434. There was no detectable phenotype in any heterozygous carrier. All patients with HAX1 deficiency had experienced developmental delay. Three patients carrying R86X also suffered from epileptic seizures. In contrast, no SCN patient with heterozygous mutations in the ELA2 gene suffered from any neurodevelopmental abnormality. CONCLUSIONS: These findings suggest that the R86X mutation in the HAX1 gene is an abnormality in Japanese SCN patients with HAX1 deficiency and may lead to neurodevelopmental abnormalities and severe myelopoietic defects.

Genetic and epigenetic alterations in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a clonal disorder characterized by dyshematopoiesis and high susceptibility to acute myeloid leukemia (AML). As patients with MDS have widely variable prognosis, we need to stratify them according to chromosomal abnormalities, genetic alterations, and epigenetic deregulations associated with progression to AML in order to treat these patients appropriately. Recently, evidence has been accumulating on the molecular mechanism underlying self-renewal of stem cells. Specifically, we have been focusing on Polycomb-group (PcG) genes, which play an important role in supporting self-renewal. There is emerging evidence indicating that the PcG complexes are indispensable for sustaining stem cell activity and cancer stem cells. We have reported that the expression of BMI1, a member of PcG, in hematopoietic stem cells or progenitor cells predicts the prognosis of patients with MDS and progression to acute leukemia. And recent genome-wide analyses showed that major transcriptional regulators governing development are under the regulation of PcG complexes. Thus PcG not only provides a molecular marker for monitoring disease progression of MDS, but also provides a clue for elucidating a molecular mechanism underlying the disease progression, which may help in the development of a new therapeutic strategy against MDS. Herein, we describe cytogenetic, genetic and molecular aberrations in MDS, focusing on epigenetic alterations through PcG.

Expression of vinexin alpha in the dorsal half of the eye and in the cardiac outflow tract and atrioventricular canal.

Vinexin, a recently identified cytoskeletal protein, contains three SH3 domains and plays important roles in regulation of cytoskeletal organization and signal transduction. Using whole-mount in situ hybridization, we showed here that expression of vinexin alpha, the longer vinexin transcript, is strictly regulated, although the shorter transcript, vinexin beta, is expressed almost ubiquitously during embryonic development in mice. Expression of vinexin alpha was limited to within part of the eye and heart in 10.5 dpc embryos. Analysis of cryosections of 10.5 dpc embryos showed that vinexin alpha was expressed in a dorsal half of the retinal pigment epithelium and in the outflow tract and atrioventricular canal of the heart. Furthermore, we also found that vinexin alpha was expressed in the gonad and in a ventral part of the pons of 12.5 dpc embryos. These results indicated that the expression of vinexin alpha is strictly regulated in a temporally and spatially restricted manner.

Lack of the Polycomb-group gene rae28 causes maturation arrest at the early B-cell developmental stage.

The rae28 gene (rae28) is a murine homologue of the Drosophila polyhomeotic gene, which is a member of the Polycomb-group genes. In this study, we examined the role of rae28 in lymphocyte development. Because homozygous rae28-deficient (rae28-/-) mice died in the perinatal period, we examined lymphocyte development by generating chimeric mice reconstituted with green fluorescence protein-labeled mutant fetal liver cells as well as in in vitro culture systems. We further examined RAE28 expression by reverse transcriptase polymerase chain reaction assay in human leukemic cells with B-lineage acute lymphoblastic leukemia (ALL). Severe B-cell maturation arrest was observed in rae28-/- between pro- and pre-B lymphocyte stages. B-cell development was also delayed in heterozygous neonates. Furthermore, interleukin-7-dependent colony-forming ability was impaired not only in homozygous lymphocytes but also in heterozygotes. Its human homologue, RAE28, is located on chromosome 12p13, which frequently is associated with chromosomal abnormalities and loss of heterozygosity in patients with hematologic malignancies. To determine whether a link exists between RAE28 and leukemia, we examined RAE28 expression in leukemic cells from pediatric patients with B-lineage ALL. RAE28 expression was not detected in four B-cell precursor ALL cases of a total of 43 examined, although RAE28 is normally expressed constitutively during the process of B-cell maturation as assessed in isolated cell populations. rae28 plays an important role in the early B-cell developmental stage in a gene dosage-dependent manner. Furthermore, the human RAE28 locus may provide a candidate gene causing the molecular pathogenesis of childhood B-cell precursor ALL.

Role of the beta isoform of 14-3-3 proteins in cellular proliferation and oncogenic transformation.

The 14-3-3 proteins are associated with proto-oncogene and oncogene products. Here, we generated NIH 3T3 cells overexpressing the beta isoform of the 14-3-3 proteins (14-3-3 beta) to examine the function of this isoform in cellular proliferation and oncogenic transformation. Overexpression of 14-3-3 beta in NIH 3T3 cells stimulated cell growth and supported anchorage-independent growth in soft agar medium and tumor formation in nude mice. To elucidate the molecular mechanisms of 14-3-3 beta-mediated NIH 3T3 transformation, we examined the activity of mitogen-activated protein kinase (MAPK) after serum stimulation. Overexpression of 14-3-3 beta augmented MAPK activity after serum stimulation, and MAPK activity correlated well with the amount of 14-3-3 beta expression. The colony-forming ability of NIH 3T3 cells overexpressing 14-3-3 beta in soft agar medium was efficiently abolished by exogenous expression of a dominant-negative mutant of MEK1 and 14-3-3 beta physically interacted with Raf-1 in these cells. These findings indicate that 14-3-3 beta has oncogenic potential, mainly through enhancement of Raf-1 activation and resultant augmentation of signaling in the MAPK cascade.

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes.

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.

Polycomb-group genes and hematopoiesis.

Although long-range chromatin organization during cell differentiation has not yet been determined, considerable evidence suggests that regulation of the chromatin structure may play a crucial part in transcriptional regulation. Drosophila genetics has introduced a unique system that can maintain genes in the on or off state after the initial gene expression decision has been established. This maintenance system is known to be mediated through the trithorax-group (trxG) and Polycomb-group (PcG) genes. The products from these 2 genes individually form multimeric complexes in the chromatin. The trxG genes are known to maintain the transcriptionally active states of the chromatin, and the PcG genes are thought to maintain the repressive states. The function of the PcG genes is defined in terms of anteroposterior patterning not only in Drosophila but also in mammals, whereas mammalian PcG genes have additional functions in higher order biological functions All PcG gene-deficient mice have provided evidence that these genes play a crucial role in hematopoiesis These findings should help shed new light on the roles of the chromatin regulatory system in hematopoiesis.

14-3-3 protein family members have a regulatory role in retinoic acid-mediated induction of cytokeratins in F9 cells.

We have found that the expression of five 14-3-3 protein isoforms is induced during the retinoic acid (RA)-mediated differentiation of mouse embryonal carcinoma F9 cells. The induced expression of the 14-3-3 proteins is presumed to have a role in enhancing the mitogen-activated protein kinase (MAPK) activity during RA-mediated F9 cell differentiation, because using genetically engineered budding yeast we showed that these isoforms enhanced the signaling in the MAPK cascade mainly through the interaction with Raf-1. Then we assessed the role of increased MAPK activity in F9 cell differentiation by interfering with signaling in the MAPK cascade in F9 cells. The exogenous expression of dominant-negative MEK1 efficiently abrogated RA-mediated induction of the cytokeratins EndoA and EndoC in the F9 cells. These results suggest that the 14-3-3 proteins play a role in the efficient induction of the cytokeratins during F9 cell differentiation through their signal enhancing activity in the MAPK cascade.

Structure and chromosomal localization of the RAE28/HPH1 gene, a human homologue of the polyhomeotic gene.

The Polycomb group of (Pc-G) genes and trithorax group of genes are known to play a crucial role in the maintenance of the transcriptional repression state of Hox genes, probably through modification of the chromatin configuration. The rae28/mph1 gene is a mammalian homologue of the Drosophila polyhomeotic gene, which belongs to the Pc-G genes. As reported previously, we established mice deficient in the rae28/mph1 gene and showed that these homozygous animals displayed the developmental defects compatible with a human congenital disorder, CATCH22 syndrome. In this study we analyzed the structural organization of the human counterpart of the rae28/mph1 gene (RAE28/HPH1) and its processed pseudogene (psiPH), which are located on, respectively, human chromosome 12p13 and 12q13. The HPH1 gene consists of 15 exons spanning approximately 26 kb and its structural organization is well conserved between mouse and human. These genetic information of the RAE28/HPH1 gene may provide an important clue for further examination of its involvement in human congenital disorders related to CATCH22 syndrome.

The human homolog of Sex comb on midleg (SCMH1) maps to chromosome 1p34.

Polycomb group genes were originally identified in Drosophila as repressors required to maintain the silenced state of homeotic loci. About ten Polycomb group genes have been cloned in Drosophila, and mammalian homologs have been identified for most of these. Here, we isolate cDNAs encoding two isoforms of a human homolog of Drosophila Sex comb on midleg (Scm), named Sex comb on midleg homolog-1 (SCMH1). Overall, SCMH1 has 94% identity to its mouse counterpart Scmh1, and 41% identity to Scm, and contains two 1(3)mbt domains, and the SPM domain that are characteristic of Scm. SCMH1 is widely expressed in adult tissues, and maps to chromosome 1p34.

Characterization of cis-elements required for the transcriptional activation of the rae28/mph1 gene in F9 cells.

The rae28/mph1 gene is the mouse homologue of the Drosophila polyhomeotic gene, which plays a crucial role in the maintenance of the transcriptional repression state of Hox genes. Expression of the rae28/mph1 gene is induced during retinoic acid (RA)-mediated differentiation of embryonal carcinoma F9 cells. By transient-transfection experiments, we identified a pair of inverted differentiation response sequences (DRS(s)) in the 5' flanking region. Each of the DRS(s) contained the consensus sequence [5'-CCTCCCCXCXGCCCCCTCCXCXC-3'], which is also conserved in the human counterpart of the rae28/mph1 gene. Electrophoretic mobility shift assay and DNase I foot printing with nuclear extracts derived from F9 cells demonstrated the presence of novel DNA-binding factors which specifically interact with DRS(s). Nucleotide substitutions in the 3' DRS abrogated the factor binding and the transcriptional activation, suggesting that DRS(s) and DRS-binding factors play an important role in the transcriptional regulation of the rae28/mph1 gene.

A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro.

The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.

Sequence-specific DNA binding activity in the RAE28 protein, a mouse homologue of the Drosophila polyhomeotic protein.

The rae28 gene, a mouse homologue of the Drosophila polyhomeotic gene, is involved in the maintenance of the transcriptional repression states of Hox genes. In this study we synthesized the glutathione S transferase-RAE28 (GST-RAE28) fusion protein and examined sequence-specific DNA binding activity in the RAE28 protein by using the selected and amplified binding site method. After five rounds of enrichment, the eluted DNAs were amplified, cloned and sequenced. The sequences of individual oligonucleotides included the following consensus sequences; 5'-ACCA-3', 5'-ACCCA-3', 5'-CTATCA-3' and 5'-TGCC-3'. The oligonucleotides including these consensus sequences were show to have significant affinity with the GST-RAE28 fusion protein. The RAE28 protein was recently shown to form multimeric protein complexes with other members of mouse Pc-G proteins in the nucleus. These findings strongly suggest that the RAE28 protein constitutes a sequence-specific DNA binding domain in multimeric Pc-G protein complexes.

RAE28, BMI1, and M33 are members of heterogeneous multimeric mammalian Polycomb group complexes.

The Polycomb group loci in Drosophila encode chromatin proteins required for repression of homeotic loci in embryonic development. We show that mouse Polycomb group homologues, RAE28, BMI1 and M33, have overlapping but not identical expression patterns during embryogenesis and in adult tissues. These three proteins coimmunoprecipitate from embryonic nuclear extracts. Gel filtration analysis of embryonic extracts indicates that RAE28, BMI1 and M33 exist in large multimeric complexes. M33 and RAE28 coimmunoprecipitate and copurify as members of large complexes from F9 cells, which express BMI1 at very low levels, suggesting that different Polycomb group complexes can form in different cells. RAE28, BMI1 and M33 interact homotypically, and both RAE28 and M33 interact with BMI1, but not with each other. The domains required for interaction were localized. Together, these studies indicate that murine Polycomb group proteins are developmentally regulated and function as members of multiple, heterogeneous complexes.