The clathrin-binding domain of CALM-AF10 alters the phenotype of myeloid neoplasms in mice.

The PICALM (CALM) gene, whose product is involved in clathrin-mediated endocytosis, has been identified in two recurring chromosomal translocations, involving either MLL or MLLT10 (AF10). We developed a mouse model of CALM-AF10(+) leukemia to examine the hypothesis that disruption of endocytosis contributes to leukemogenesis. Exclusion of the C-terminal portion of CALM from the fusion protein, which is required for optimal binding to clathrin, resulted in the development of a myeloproliferative disease, whereas inclusion of this domain led to the development of acute myeloid leukemia and changes in gene expression of several cancer-related genes, notably Pim1 and Crebbp. Nonetheless, the development of leukemia could not be attributed directly to interference with endocytosis or consequential changes in proliferation and signaling. In leukemia cells, full-length CALM-AF10 localized to the nucleus with no consistent effect on growth factor endocyctosis, and suppressed histone H3 lysine 79 methylation regardless of the presence of clathrin. Using fluorescence resonance energy transfer analysis, we show that CALM-AF10 has a propensity to homo-oligomerize, raising the possibility that the function of endocytic proteins involved in chimeric fusions may be to provide dimerization properties, a recognized mechanism for unleashing oncogenic properties of chimeric transcription factors, rather than disrupting the internalization of growth factor receptors.

Enhanced expression of FHL2 leads to abnormal myelopoiesis in vivo.

FHL2 is a multifunctional LIM domain protein that acts as a transcriptional modulator mediating proliferation and apoptosis in a tissue-specific manner. Upregulation of FHL2 has been detected in a variety of cancers. We demonstrate that upregulation of FHL2 is associated with a subset of acute myeloid leukemia with a characteristic gene-expression signature, and abnormalities of chromosome 5. In mice, expression of endogenous Fhl2 is downregulated coordinately during the differentiation of hematopoietic cells. Upregulation of FHL2 enhances proliferation of myeloid progenitor cells, and serial-replating efficiency of hematopoietic cells in vitro. Chimeric mice with enforced expression of FHL2 in bone marrow cells, are characterized by an expanded pool of myeloid progenitor cells, enhanced granulopoiesis and megakaryocytopoiesis. In addition, enhanced expression of FHL2 promotes cell-cycle entry of myeloid progenitor cells and increases the frequency of apoptosis of bone marrow cells in vivo. These results raise the possibility that deregulation of FHL2 contributes to the development of human myeloid disorders.

Transcript map and comparative analysis of the 1.5-Mb commonly deleted segment of human 5q31 in malignant myeloid diseases with a del(5q).

Loss of a whole chromosome 5, or a del(5q), are recurring abnormalities in malignant myeloid diseases. In previous studies, we defined a commonly deleted segment (CDS) of 1.5 Mb between D5S479 and D5S500 in patients with a del(5q), and we established a P1 artificial chromosome-based contig encompassing this interval. To identify candidate tumor suppressor genes (TSGs), we developed a transcript map of the CDS. The map contains 18 genes and 12 expressed sequence tags/UniGenes. Among the 18 genes are 10 genes that were previously cloned and 8 novel genes. The newly identified genes include CDC23, which encodes a component of the anaphase-promoting complex; RAB6KIFL, which encodes a kinesin-like protein involved in organelle transport; and KLHL3, which encodes a human homologue of the Drosophila ring canal protein, kelch. We determined the intron/exon organization of 14 genes and eliminated each gene as a classical TSG by mutation analysis. In addition, we established a single-nucleotide polymorphism map as well as a map of the mouse genome that is syntenic to the CDS of human 5q31. The development of a transcription map will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 5q.

cDNA cloning and genomic structure of three genes localized to human chromosome band 5q31 encoding potential nuclear proteins.

Loss of a whole chromosome 5, or a del(5q), is a recurring abnormality in malignant myeloid diseases. By cytogenetic and molecular analyses, we delineated previously a 1- to 1.5-Mb region that is deleted in all patients with a del(5q). In our efforts to identify a myeloid tumor suppressor gene within the commonly deleted segment (CDS), we have cloned and characterized the genes encoding three putative nuclear proteins, each of which contains a bipartite nuclear localization signal (NLS). In addition, C5ORF5 contains a putative rhoGAP domain at the N-terminus, C5ORF6 has a proline-rich sequence near the N-terminus, and C5ORF7 has a zinc-finger domain that partially overlaps the NLS. All three genes are ubiquitously expressed and encode novel proteins. The C5ORF5 cDNA is 5.47 kb encoding a protein of 915 amino acids (aa) with a predicted molecular mass of approximately 105 kDa. C5ORF5 has 23 exons spanning over 27 kb. The C5ORF6 transcript is 4.1 kb encoding a protein of 392 aa with a predicted molecular mass of approximately 43 kDa. C5ORF6 has 5 exons and spans approximately 11 kb. The C5ORF7 cDNA is 6.3 kb and encodes a protein of 1417 aa with a predicted molecular mass of approximately 155 kDa. C5ORF7 has 24 exons spanning approximately 64 kb. All three genes were localized to the distal half of the CDS between D5S1983 and D5S500. We evaluated each as a candidate tumor suppressor gene by the analysis of myeloid leukemia cells from patients with -5/del(5q), but no inactivating mutations were identified.

Molecular characterization of KLHL3, a human homologue of the Drosophila kelch gene.

The Drosophila kelch protein is a structural component of ring canals and is required for oocyte maturation. Here, we report the cloning and genomic structure of a new human homologue of kelch, KLHL3. At the amino acid level, KLHL3 shares 77% similarity with Drosophila kelch and 89% similarity with Mayven (KLHL2), another human kelch homolog. The approximately 6.5-kb mRNA has a single open reading frame encoding a protein of 587 amino acids with a predicted molecular mass of 650 kDa. Like kelch and KLHL2, the KLHL3 protein contains a poxvirus and zinc finger domain at the N-terminus and six tandem repeats (kelch repeats) at the C-terminus. At least three isoforms, which differ in the length of the N-terminus, are produced and may be the result of alternative promoter usage. We also identified alternative polyadenylation sites and alternative splicing; thus, as many as 12 mRNA variants and six putative protein isoforms could be produced. The KLHL3 gene is mapped to human chromosome 5, band q31, contains 17 exons, and spans approximately 120 kb of genomic DNA. KLHL3 maps within the smallest commonly deleted segment in myeloid leukemias characterized by a deletion of 5q; however, we detected no inactivating mutations of KLHL3 in malignant myeloid disorders with loss of 5q.

cDNA cloning, expression pattern, genomic structure and chromosomal location of RAB6KIFL, a human kinesin-like gene.

Kinesin-like proteins are microtubule-associated motors that play important roles in intracellular transport and cell division. We report here the characterization of a new human kinesin-like protein, Rabkinesin6 (RAB6KIFL). The composite cDNA sequence is 2957bp, and encodes a protein of 890 amino acids with a predicted molecular weight of 100kDa. It has high homology (93% similarity) with the mouse kinesin-like protein, Rab6kifl, indicating that it is the human homologue of the mouse gene. RAB6KIFL has all of the structural characteristics required to function as a microtubule-associated motor. Unlike the mouse gene which is ubiquitously expressed, RAB6KIFL expression appears to be tissue specific. It is widely expressed in fetal tissues, abundantly expressed in adult thymus, bone marrow and testis, and is expressed at low levels in heart, placenta and spleen. The RAB6KIFL gene is mapped to human chromosome 5, band q31, spans approximately 8.5kb of genomic DNA, and contains 19 exons. RAB6KIFL maps within the smallest commonly deleted segment in myeloid leukemias characterized by a deletion of 5q; however, we detected no mutations of RAB6KIFL in malignant myeloid disorders with loss of 5q. The description of this human kinesin-like protein may provide a better understanding of the diversity of this large family of proteins.

Human CDC23: cDNA cloning, mapping to 5q31, genomic structure, and evaluation as a candidate tumor suppressor gene in myeloid leukemias.

The transition from metaphase to anaphase and exit from mitosis involve the degradation of active cyclin B-CDC2 complexes by ubiquitin-mediated proteolysis. The anaphase-promoting complex (APC) catalyzes the formation of cyclin B-ubiquitin conjugates, thereby targeting cyclin B for degradation. The APC is composed of eight proteins, including four members of a family characterized by multiple tetratricopeptide repeats (TPR). We mapped two overlapping expressed sequence tag clones within a genomic contig on human chromosome 5, band q31. A search revealed high homology to Saccharomyces cerevisiae CDC23, a TPR protein component of the APC. We have isolated the human CDC23 cDNA containing the full-length predicted open reading frame. The approximately 3.3-kb message is ubiquitously expressed and encodes a protein with 591 amino acids (MW = 68,293 Da) and 9 TPR units. The protein has 30% identity and 51% similarity to the S. cerevisiae protein. The human CDC23 gene contains 16 exons and spans approximately 31 kb. CDC23 maps within the smallest commonly deleted segment in myeloid leukemias characterized by a deletion of 5q; however, we detected no mutations of CDC23 in leukemia cells with loss of 5q. Thus, CDC23 is unlikely to be involved in the pathogenesis of myeloid leukemias characterized by abnormalities of chromosome 5.

Identification and characterization of the gene encoding a second proteolipid subunit of human vacuolar H(+)-ATPase (ATP6F).

The proteolipid domain of vacuolar H(+)-ATPase (V-ATPase) plays a major role in H+ transport in microvesicles and other acidic organelles. We have cloned the second human proteolipid of the V-ATPase (designated hATP6F), a homologue of the Saccharomyces cerevisiae proteolipid VMA16, which is an essential subunit of yeast V-ATPase. hATP6F is a hydrophobic protein with five putative transmembrane segments, having 61% amino acid identity and 83% similarity to the yeast protein, except in the N-terminus, and contains a conserved glutamic acid residue (Glu98) that is essential for H(+)-transporting activity. The gene for hATP6F (gene symbol, ATP6F), which consists of eight exons and spans approximately 3.5 kb, was isolated and mapped to human chromosome band 1p32.3 and the region 10.81 cR centromeric of the STS marker SHGC36789 (LOD = 6.75) by fluorescence in situ hybridization and radiation hybrid mapping, respectively. This is the first evidence in human of the existence of a second gene encoding a distinct V-ATPase proteolipid.

Comparison of the human and mouse genes encoding the telomeric protein, TRF1: chromosomal localization, expression and conserved protein domains.

Mammalian chromosome ends contain long arrays of TTAGGG repeats that are complexed to a telomere specific protein, the TTAGGG repeat binding factor, TRF1. Here we describe the characterization of genes encoding the human and mouse TRF1 proteins, hTRF1 and mTRF1. The mTRF1 cDNA was isolated based on sequence similarity to the hTRF1 cDNA and the mTRF1 mRNA was shown to be ubiquitously expressed as a single 1.9 kb polyadenylated transcript in mouse somatic tissues. High levels of a 2.1 kb transcript were found in testes. In vitro translation of the mTRF1 cDNA resulted in a 56 kDa protein that binds to TTAGGG repeat arrays. mTRF1 displayed the same sequence specificity as hTRF1, preferring arrays of TTAGGG repeats as a binding substrate over TTAGGC and TTGGGG repeats. Expression of an epitope-tagged version of mTRF1 showed that the protein is located at the ends of murine metaphase chromosomes. In agreement, conceptual translation indicated that mTRF1 and hTRF1 are similarly-sized proteins with nearly identical C-terminal Myb-related DNA binding motifs. In addition, comparison of the predicted mTRF1 and hTRF1 amino acid sequences showed that the acidic nature of the N-terminus of TRF1 is conserved and revealed a highly conserved novel domain of approximately 200 amino acids in the middle of the proteins. However, other regions of the proteins are poorly conserved (<35% identity) and the overall level of identity of the mTRF1 and hTRF1 amino acid sequences is only 67%. The TRF1 genes are not syntenic; the hTRF1 gene localized to human chromosome 8 band q13 while the mTRF1 gene localized to mouse chromosome 17 band E3. The data indicate that the genes for mammalian telomeric proteins evolve rapidly.

Sequence of human hexokinase III cDNA and assignment of the human hexokinase III gene (HK3) to chromosome band 5q35.2 by fluorescence in situ hybridization.

Complementary DNA clones encoding human hexokinase III were isolated from a liver cDNA library. There was 84.7% identity between the amino acid sequences of human and rat hexokinase III. RNA blotting showed the presence of hexokinase III mRNA in liver and lung. Fluorescence in situ hybridization localized the human hexokinase III gene (HK3) to chromosome 5, band q35.2.

Structural organization and mapping of the human mitochondrial glycerol phosphate dehydrogenase-encoding gene and pseudogene.

Mitochondrial glycerol phosphate dehydrogenase (mtGPD) is the rate-limiting enzyme in the glycerol phosphate shuttle, which is thought to play an important role in cells that require an active glycolytic pathway. Abnormalities in mtGPD have been proposed as a potential cause for non-insulin-dependent diabetes mellitus. To facilitate genetic studies, we have isolated genomic clones containing the coding regions of the human mtGPD-encoding gene (GPDM). The gene contains 17 exons and is estimated to span more than 80 kb. All splice junctions contain GT/AG consensus sequences. Introns interrupt the sequences encoding the leader peptide, the FAD-binding site, the calcium-binding regions, and a conserved central element postulated to play a role in glycerol phosphate binding. Fluorescence in situ hybridization was used to map this gene to chromosome 2, band q24.1. A retropseudogene was identified and mapped to chromosome 17.

The human gene CGT encoding the UDP-galactose ceramide galactosyl transferase (cerebroside synthase): cloning, characterization, and assignment to human chromosome 4, band q26.

We have previously cloned the human UDP-galactose ceramide galactosyltransferase (CGT, E.C. 2.4.1.45) cDNA. Its open reading frame encodes the key enzyme in the biosynthesis of the glycosphingolipids, cerebrosides and sulfatides, essential constituents of the myelin membrane of the central nervous system (CNS) and PNS. Expression of the CGT gene and of the myelin-specific proteins in the terminal differentiated oligodendrocyte of CNS and in Schwann cells of PNS is cell-specific and highly time-regulated. The CGT gene therefore is important in the differentiation program of the oligodendrocyte lineage. Here we report the structural organization and the chromosomal localization of the human CGT gene. The coding sequence is separated into five exons, which are distributed over >40 kb. The CGT locus was mapped to the distal region of human chromosome 4, band q26. The organization of the CGT gene and of the UGT (uridylglucuronosyltransferases) gene family suggests a correlation to functional domains of the encoded proteins.

Human fructose-1,6-bisphosphatase gene (FBP1): exon-intron organization, localization to chromosome bands 9q22.2-q22.3, and mutation screening in subjects with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (EC 3.1.3.11) is a key regulatory enzyme of gluconeogenesis that catalyzes the hydrolysis of fructose-1,6-bisphosphate to generate fructose-6-phosphate and inorganic phosphate. Deficiency of fructose-1,6-bisphosphatase is associated with fasting hypoglycemia and metabolic acidosis because of impaired gluconeogenesis. We have cloned and characterized the human liver fructose-1,6-bisphosphatase gene (FBP1). FBP1, localized to chromosome bands 9q22.2-q22.3 by fluorescence in situ hybridization, consists of seven exons that span > 31 kb, and the six introns are in the same position as in the rat gene. FBP1 was screened for mutations in two subjects with fructose-1,6-bisphosphatase deficiency. Four nucleotide substitutions were identified, two of which were silent mutations in the codons for Ala-216 (GCT-->GCC) and Gly-319 (GGG-->GGA). The other substitutions were in intron 3, a C-->T substitution 7 nucleotides downstream from the splice donor site, and in the promoter region, an A-->T substitution 188 nucleotides upstream from the start of transcription. These nucleotide substitutions were also found in normal unaffected subjects and thus are not the cause of fructose-1,6-bisphosphatase deficiency in the two subjects studied. The molecular basis of hepatic fructose-1,6-bisphosphatase deficiency in these subjects remains undetermined but could result from unidentified mutations in the promoter that decrease expression or from mutations in another gene that indirectly lead to decreased fructose-1,6-bisphosphatase activity.

Localization of the glucagon receptor gene to human chromosome band 17q25.

The gene encoding the human glucagon receptor (GCGR) was mapped to chromosome band 17q25 by fluorescence in situ hybridization to metaphase chromosomes. An Alu variable poly(A) DNA polymorphism was identified in this gene. Studies in the CEPH families showed significant evidence of linkage between DNA polymorphism and markers localized to the distal long arm of chromosome 17.

Sequence-independent amplification and labeling of yeast artificial chromosomes for fluorescence in situ hybridization.

We have developed a method that allows reliable construction of high quality FISH probes from yeast artificial chromosomes (YACs) based on the separation of YACs by pulse-field gel electrophoresis and a rapid sequence-independent amplification procedure (SIA). These probes can be used to localize YACs on metaphase chromosomes and also with high efficiency, in interphase nuclei.

Analysis of deletions of the long arm of chromosome 11 in hematologic malignancies with fluorescence in situ hybridization.

We studied samples containing deletions of the long arm of chromosome 11 (11q) from patients with hematologic malignancies by using cytogenetic and fluorescence in situ hybridization (FISH) techniques. Cytogenetic analysis of 28 patients and of a cell line showed that all deletions included band 11q23. FISH analysis demonstrated that the proximal part of 11q23, including NCAM, was deleted in 13 of 15 patients and the cell line. Recurring chromosomal losses in human tumors have been regarded as evidence that the affected regions contain tumor-suppressor genes. These results suggest that the putative tumor-suppressor gene is proximal to the MLL gene which is also located in 11q23.

Do terminal deletions of 11q23 exist? Identification of undetected translocations with fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) was performed on bone marrow or peripheral blood cells thought to contain a del(11)(q23q25) from four patients who had acute leukemia or myelodysplasia. Cells from all patients were shown to contain translocations that involved chromosome 6 in three of them. Our data suggest that a large proportion of presumptive del(11)(q23) or del(11)(q23q25) chromosomes may represent previously unidentified translocations that can be detected by FISH.