Iqcg is essential for sperm flagellum formation in mice.

Mammalian spermatogenesis comprises three successive phases: mitosis phase, meiosis phase, and spermiogenesis. During spermiogenesis, round spermatid undergoes dramatic morphogenesis to give rise to mature spermatozoon, including the condensation and elongation of nucleus, development of acrosome, formation of flagellum, and removal of excessive cytoplasm. Although these transformations are well defined at the morphological level, the mechanisms underlying these intricate processes are largely unknown. Here, we report that Iqcg, which was previously characterized to be involved in a chromosome translocation of human leukemia, is highly expressed in the spermatogenesis of mice and localized to the manchette in developing spermatids. Iqcg knockout causes male infertility, due to severe defects of spermiogenesis and resultant total immobility of spermatozoa. The axoneme in the Iqcg knockout sperm flagellum is disorganized and hardly any typical ("9+2") pattern of microtubule arrangement could be found in Iqcg knockout spermatids. Iqcg interacts with calmodulin in a calcium dependent manner in the testis, suggesting that Iqcg may play a role through calcium signaling. Furthermore, cilia structures in the trachea and oviduct, as well as histological appearances of other major tissues, remain unchanged in the Iqcg knockout mice, suggesting that Iqcg is specifically required for spermiogenesis in mammals. These results might also provide new insights into the genetic causes of human infertility.

Functional and molecular features of the calmodulin-interacting protein IQCG required for haematopoiesis in zebrafish.

We previously reported a fusion protein NUP98-IQCG in an acute leukaemia, which functions as an aberrant regulator of transcriptional expression, yet the structure and function of IQCG have not been characterized. Here we use zebrafish to investigate the role of iqcg in haematopoietic development, and find that the numbers of haematopoietic stem cells and multilineage-differentiated cells are reduced in iqcg-deficient embryos. Mechanistically, IQCG binds to calmodulin (CaM) and acts as a molecule upstream of CaM-dependent kinase IV (CaMKIV). Crystal structures of complexes between CaM and IQ domain of IQCG reveal dual CaM-binding footprints in this motif, and provide a structural basis for a higher CaM-IQCG affinity when deprived of calcium. The results collectively allow us to understand IQCG-mediated calcium signalling in haematopoiesis, and propose a model in which IQCG stores CaM at low cytoplasmic calcium concentrations, and releases CaM to activate CaMKIV when calcium level rises.

Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia.

Abnormal epigenetic regulation has been implicated in oncogenesis. We report here the identification of somatic mutations by exome sequencing in acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5). We discovered mutations in DNMT3A (encoding DNA methyltransferase 3A) in 23 of 112 (20.5%) cases. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 in vitro. Notably, there were alterations of DNA methylation patterns and/or gene expression profiles (such as HOXB genes) in samples with DNMT3A mutations as compared with those without such changes. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset and of promonocytic as well as monocytic predominance among AML-M5 individuals. Screening other leukemia subtypes showed Arg882 alterations in 13.6% of acute myelomonocytic leukemia (AML-M4) cases. Our work suggests a contribution of aberrant DNA methyltransferase activity to the pathogenesis of acute monocytic leukemia and provides a useful new biomarker for relevant cases.

Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML.

Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (As2O3) exerts its therapeutic effect by promoting degradation of an oncogenic protein that drives the growth of APL cells, PML-RARalpha (a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor alpha). PML and PML-RARalpha degradation is triggered by their SUMOylation, but the mechanism by which As2O3 induces this posttranslational modification is unclear. Here we show that arsenic binds directly to cysteine residues in zinc fingers located within the RBCC domain of PML-RARalpha and PML. Arsenic binding induces PML oligomerization, which increases its interaction with the small ubiquitin-like protein modifier (SUMO)-conjugating enzyme UBC9, resulting in enhanced SUMOylation and degradation. The identification of PML as a direct target of As2O3 provides new insights into the drug's mechanism of action and its specificity for APL.

AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec.

To explore the genetic abnormalities that cooperate with AML1-ETO (AE) fusion gene to cause acute myeloid leukemia (AML) with t(8;21), we screened a number of candidate genes and identified 11 types of mutations in C-KIT gene (mC-KIT), including 6 previously undescribed ones among 26 of 54 (48.1%) cases with t(8;21). To address a possible chronological order between AE and mC-KIT, we showed that, among patients with AE and mC-KIT, most leukemic cells at disease presentation harbored both genetic alteration, whereas in three such cases investigated during complete remission, only AE, but not mC-KIT, could be detected by allele-specific PCR. Therefore, mC-KIT should be a subsequent event on the basis of t(8;21). Furthermore, induced expression of AE in U937-A/E cells significantly up-regulated mRNA and protein levels of C-KIT. This may lead to an alternative way of C-KIT activation and may explain the significantly higher C-KIT expression in 81.3% of patients with t(8;21) than in patients with other leukemias. These data strongly suggest that t(8;21) AML follows a stepwise model in leukemogenesis, i.e., AE represents the first, fundamental genetic hit to initiate the disease, whereas activation of the C-KIT pathway may be a second but also crucial hit for the development of a full-blown leukemia. Additionally, Gleevec suppressed the C-KIT activity and induced proliferation inhibition and apoptosis in cells bearing C-KIT N822K mutation or overexpression, but not in cells with D816 mC-KIT. Gleevec also exerted a synergic effect in apoptosis induction with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.

Cloning, expression, purification and crystallization of NHR3 domain from acute myelogenous leukemia-related protein AML1-ETO.

The t(8;21) translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia. This translocation creates a fusion between the acute myelogenous leukemia 1 (AML1, a transcription factor) gene on chromosome 21 and the eight-twenty-one (ETO, a zinc finger nuclear protein) gene on chromosome 8, leading to the repression of certain AML1 target genes. We cloned NHR3 domain coding fragment into vector pGEX-6p-1 using PCR and obtained recombinant plasmid pGEX-6p-1-NHR3, which can be induced to stably overexpress fusion protein in E. coli. Through the purification on GST affinity chromatography column and PreScission protease cleavage, a large amount of NHR3 protein with high purity was obtained. In order to avoid possible interference of some strong negative charged molecules, NHR3 protein was further purified by Mono Q anion exchange chromatography. The NHR3 crystals were grown with hanging drop/vapor diffusion method and the first crystals appeared after four weeks at 18 degrees in 0.2 M Tris-sodium citrate dihydrate, 0.1 M sodium cacodylate, pH 6.5, and 30% iso-propanol (V/V). ESI mass spectrum showed that the molecular weight of this domain was in agreement with its primary structure sequence prediction, and circular dichroism spectral data (190-250 nm) showed that NHR3 had a well-defined secondary structure of 25.9% alpha-helix, 23.2% random coil and 50.9% turn, which was consistent with GOV4 software prediction.