Alternative splicing of Bim and Erk-mediated Bim(EL) phosphorylation are dispensable for hematopoietic homeostasis in vivo

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim(EL), Bim(L) and Bim(S)) are generated by alternative splicing. Bim(EL), the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim(EL) through the DEF2 domain of Bim(EL) and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim(EL) degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim(EL) and Bim(L) are interchangeable, and that Bim(S) is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim(EL).     

Genomic organization of the human CD5 gene

CD5 is a member of the family of receptors which contain extracellular domains homologous to the type I macrophage scavenger receptor cysteine-rich (SRCR) domain. Here, we compare the exon/intron organization of the human CD5 gene with its mouse homologue, as well as with the human CD6 gene, the closest related member of the SRCR superfamily. The human CD5 gene spans about 24.5 kb and consists of at least 11 exons. These exons are conserved in size, number, and structure in the mouse CD5 homologue. No evidence for the biallelic polymorphism reported in the mouse could be found among a population of 100 individuals of different ethnic origins. The human CD5 gene maps to the Chromosome (Chr) 11q12.2 region, 82 kb downstream from the human CD6 gene, in a head-to-tail orientation, a situation which recalls that reported at mouse Chr 19. The exon/intron organization of the human CD5 and CD6 genes was very similar, differing in the size of intron 1 and the number of exons coding for their cytoplasmic regions. While several isoforms, resulting from alternative splicing of the cytoplasmic exons, have been reported for CD6, we only found evidence of a cytoplasmic tailless CD5 isoform. The conserved structure of the CD5 and CD6 loci, both in mouse and human genomes, supports the notion that the two genes may have evolved from duplication of a primordial gene. The existence of a gene complex for the SRCR superfamily on human Chr 11q (and mouse Chr 19) still remains to be disclosed.     

Chromosomal localization of SLC12A5/Slc12a5, the human and mouse genes for the neuron-specific K(+)-Cl(-) cotransporter (KCC2) defines a new region of conserved homology.

K(+)-Cl(-) cotransporters (KCCs) constitute a branch of the cation-chloride cotransporter (CCC) family. To date, four KCC isoforms (KCC1-KCC4) have been identified and they all mediate obligatorily coupled, electroneutral transmembrane movement of K(+) and Cl(-) ions. KCC2 (gene symbol SLC12A5) is expressed exclusively in neurons within the central nervous system and abnormalities in its expression have been proposed to play a role in pathological conditions such as epilepsy and neuronal trauma. Here we have determined chromosome location of both the human and the mouse genes encoding KCC2, which may assist in future efforts to determine the contribution of KCC2 to inherited human disorders. We assigned human SLC12A5 to 20q12-->q13.1 and its murine homolog, Slc12a5, to 5G2-G3 by fluorescence in situ hybridization (FISH). These mapping data are contradictory to the previously reported human-mouse conserved synteny relationships disrupting an exceptionally well-conserved homology segment between human Chr 20 and mouse Chr 2. We hence suggest the first region of conserved homology between human Chr 20 and mouse Chr 5.     

Cloning and chromosomal mapping of the human p53-related KET gene to Chromosome 3q27 and its murine homolog Ket to mouse Chromosome 16

KET is a member of the newly discovered family of proteins that is related to the tumor suppressor p53. Here we describe the molecular cloning of a human cDNA of 4846 bp encoding a protein of 680 amino acids. The human KET protein shares 98% identity with the previously characterized rat homolog. The remarkably high degree of conservation lends support to the notion that KET proteins have important basic functions in development and differentiation. Using the GeneBridge 4 radiation hybrid panel, we have mapped KET to human Chromosome (Chr) 3q27. KET is located between the somatostatin gene SST (proximal) and the apolipoprotein D gene APOD (distal) in a region of conserved synteny to mouse Chr 16. This chromosomal region is deleted in early stages of tumorigenesis of mouse islet cell carcinomas and contains the hitherto unidentified Loh2 gene, a putative suppressor of angiogenesis. The murine homolog Ket was mapped in an interspecific backcross panel and falls into this region of loss of heterozygosity. From our mapping data we infer that KET might act as a tumor suppressor and is considered as a candidate for Loh2. Received: 30 April 1998 / Accepted: 17 July 1998     

Cloning, expression and chromosomal location of NKX6B to 10q26, a region frequently deleted in brain tumors

Nkx6-2 (former Gtx) is a murine-homeobox-containing gene localized distally on Chromosome (Chr) 7. Analysis of the expression pattern, together with DNA binding assays, suggests that this gene product might be important for differentiated oligodendrocyte function and in the regulation of myelin gene expression. We now report on the cloning and characterization of the human homolog (NKX6B). DNA sequence analysis of an 11-kb genomic fragment revealed that the complete human gene spans 1.2 kb and is composed of three exons. NKX6B is predicted to encode a polypeptide of 277 amino acids with 97% identity to mouse Nkx6-2. Northern blot experiments showed that NKX6B expression is tightly controlled in a tissue-specific fashion with the highest site of expression being the brain. Finally, using STS content mapping and RH analyis, we demonstrated that NKX6B maps to the 10q26, a region where frequent loss of heterozygosity has been observed in various malignant brain tumors. These results may implicate NKX6B as a candidate tumor suppressor gene for brain tumors, particularly for oligodendrogliomas. Received: 14 June 2000 / Accepted: 21 September 2000     

CD150 is a member of a family of genes that encode glycoproteins on the surface of hematopoietic cells

Human CD150 (SLAM) is a glycoprotein expressed on the surface of T, B, natural killer, and dendritic cells. The extracellular domain of CD150 is the receptor for measles virus and CD150 acts as a co-activator on T and B cells. We characterized the mouse and human CD150 genes, each of which comprises seven exons spanning approximately 32 kb. Mouse CD150 mRNA was detected in T cells and in most thymocyte subsets, except CD4-8- cells. Surprisingly, the CD4-8- thymocytes of CD3gammadeltanull mice, but not of Ragnull or severe combined immunodeficiency mice, expressed CD150. Whereas high levels of CD150 were found in Th1 cells, only small amounts were detectable in Th2 cells. CD150 expression was up-regulated upon in vitro activation of mouse T cells by anti-CD3. The complete mouse CD150 gene is highly homologous to its human orthologue in terms of nucleotide sequences and intron/exon organization. The human genomic sequences indicate that all isoforms detected so far have arisen from alternative splicing events. As judged by fluorescence in situ hybridization, mouse CD150 mapped to Chromosome (Chr) 1, band 1H2.2-2.3, and human CD150 was found on Chr 1q22. Human and mouse CD150 share sequence homologies with six other genes, five of which - CD84, CD229 (Ly-9), CD244 (2B4), CD48, and 19A - are localized in a 250-kb segment in close proximity to the human gene. Their location and their sequence similarities strongly suggest that the CD150 family of cell surface receptors arose via successive duplications of a common ancestral gene.     

Chromosome 5q deletion and epigenetic suppression of the gene encoding alpha-catenin (CTNNA1) in myeloid cell transformation

Interstitial loss of all or part of the long arm of chromosome 5, or del(5q), is a frequent clonal chromosomal abnormality in human myelodysplastic syndrome (MDS, a preleukemic disorder) and acute myeloid leukemia (AML), and is thought to contribute to the pathogenesis of these diseases by deleting one or more tumor-suppressor genes. Although a major commonly deleted region (CDR) has been delineated on chromosome band 5q31.1 (refs. 3-7), attempts to identify tumor suppressors within this band have been unsuccessful. We focused our analysis of gene expression on RNA from primitive leukemia-initiating cells, which harbor 5q deletions, and analyzed 12 genes within the CDR that are expressed by normal hematopoietic stem cells. Here we show that the gene encoding alpha-catenin (CTNNA1) is expressed at a much lower level in leukemia-initiating stem cells from individuals with AML or MDS with a 5q deletion than in individuals with MDS or AML lacking a 5q deletion or in normal hematopoietic stem cells. Analysis of HL-60 cells, a myeloid leukemia line with deletion of the 5q31 region, showed that the CTNNA1 promoter of the retained allele is suppressed by both methylation and histone deacetylation. Restoration of CTNNA1 expression in HL-60 cells resulted in reduced proliferation and apoptotic cell death. Thus, loss of expression of the alpha-catenin tumor suppressor in hematopoietic stem cells may provide a growth advantage that contributes to human MDS or AML with del(5q).     

Structure, expression, and chromosome mapping of LATS2, a mammalian homologue of the Drosophila tumor suppressor gene lats/warts

We have cloned and characterized LATS2, a novel mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2) mRNA, whereas expression of human LATS2 (HsLATS2) mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2 gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2 gene (by fluorescence in situ hybridization) to the 13q11-q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RB and BRCA2 have also been mapped.     

Genomic organization and mapping of the human and mouse neuronal β2-nicotinic acetylcholine receptor genes

As a first step in determining whether there are polymorphisms in the nicotinic acetylcholine receptor (nAChR) genes that are associated with nicotine addiction, we isolated genomic clones of the β2-nAChR genes from human and mouse BAC libraries. Although cDNA sequences were available for the human gene, only the promoter sequence had been reported for the mouse gene. We determined the genomic structures by sequencing 12 kb of the human gene and over 7 kb of the mouse gene. While the sizes of exons in the mouse and human genes are the same, the introns differ in size. Both promoters have a high GC content (60–80%) proximal to the AUG and share a neural-restrictive silencer element (NRSE), but overall sequence identity is only 72%. Using a 6-Mb YAC contig of Chr 1, we mapped the human β2-nAChR gene, CHRNB2, to 1q21.3 with the order of markers cen, FLG, IVL, LOR, CHRNB2, tel. The mouse gene, Acrb2, had previously been mapped to Chr 3 in a region orthologous to human Chr 1. We refined mapping of the mouse gene and other markers on a radiation hybrid panel of Chr 3 and found the order cen, Acrb2, Lor, Iv1, Flg, tel. Our results indicate that this cluster of markers on human Chr 1 is inverted with respect to its orientation on the chromosome compared with markers in the orthologous region of mouse Chr 3. Received: 26 January 1999 / Accepted: 10 May 1999     

Genomic structure, mapping, and expression analysis of the mammalian Lunatic, Manic, and Radical fringe genes

The three members of the mammalian fringe gene family, Manic fringe (Mfng), Radical fringe (Rfng), and Lunatic fringe (Lfng), were identified on the basis of their similarity to Drosophila fringe (fng) and their participation in the evolutionarily conserved Notch receptor signaling pathway. Fringe genes encode pioneer secretory proteins with weak similarity to glycosyltransferases. Both expression patterns and functional studies support an important role for Fringe genes in patterning during embryonic development and an association with cellular transformation. We have now further characterized the expression and determined the chromosomal localization and genomic structure of the mouse Mfng, Rfng, and Lfng genes; the genomic structure and conceptual open reading frame of the human RFNG gene; and the refined chromosomal localization of the three human fringe genes. The mouse Fringe genes are expressed in the embryo and in adult tissues. The mouse and human Fringe family members map to three different chromosomes in regions of conserved synteny: Mfng maps to mouse Chr 15, and MFNG maps to human Chr 22q13.1 in the region of two cancer-associated loci; Lfng maps to mouse Chr 5, and LFNG maps to human Chr 7p22; Rfng maps to mouse Chr 11, and RFNG maps to human Chr 17q25 in the minimal region for a familial psoriasis susceptibility locus. Characterization of the genomic loci of the Fringe gene family members reveals a conserved genomic organization of 8 exons. Comparative analysis of mammalian Fringe genomic organization suggests that the first exon is evolutionarily labile and that the Fringe genes have a genomic structure distinct from those of previously characterized glycosyltransferases. Received: 19 February 1999 / Accepted: 22 February 1999     

PHF2, a novel PHD finger gene located on human Chromosome 9q22

We have isolated and characterized a novel PHD finger gene, PHF2, which maps to human Chromosome (Chr) 9q22 close to D9S196. Its mouse homolog was also characterized and mapped to the syntenic region on mouse Chr 13. The predicted human and mouse proteins are 98% identical and contain a PHD finger domain, eight possible nuclear localization signals, two potential PEST sequences, and a novel conserved hydrophobic domain. Northern analysis shows widespread expression of PHF2 in adult tissues, while in situ hybridization on mouse embryos reveals staining in the neural tube and dorsal root ganglia significantly above a ubiquitous low level expression signal. From its expression pattern and its chromosomal localization, PHF2 is a candidate gene for hereditary sensory neuropathy type I, HSN1. Received: 9 July 1998 / Accepted: 16 October 1998     

A linkage map of mouse chromosome 19: definition of comparative mapping relationships with human chromosomes 10 and 11 including the MEN1 locus.

A linkage map of mouse Chromosome (Chr) 19 was constructed using an interspecific cross and markers defined by restriction fragment length variants. The map includes 20 markers, 9 of which had not been mapped previously in the mouse. The data further defined the relationship between genes on mouse Chr 19 and those on the long arm of human Chr 10 and the pericentric region of the long arm of human Chr 11. The comparative mapping analysis suggests that the proximal segment of mouse Chr 19 may contain the MEN1 locus and that the current study has identified additional genes that may be useful for positional cloning of this putative tumor suppressor gene.     

Murine Stim1 maps to distal Chromosome 7 and is not imprinted

STIM1 (GOK) maps to a region of human Chromosome (Chr) 11p15.5 that is implicated in several embryonal tumors, and some evidence indicates that STIM1 may have a growth suppressor role in rhabdomyosarcoma. In this study we have mapped the murine homolog, Stim1, to the same position as Hbb on distal mouse Chr 7. This region is separated by 20 cM from the region of distal Chr 7 that contains Igf2, H19, and other imprinted genes. Using strain-specific polymorphisms, we have shown that Stim1 is expressed from both parental alleles in fetal and neonatal mouse tissues. Similar analyses of human Wilms' tumor and normal kidney tissues demonstrated biallelic expression of STIM1 in the majority of samples. These data demonstrate that Stim1 expression is not regulated by genomic imprinting in either mouse or human tissues. Thus, if STIM1 is a tumor suppressor at 11p15.5, loss of expression is not due to imprinting effects. Received: 23 January 1998 / Accepted: 10 April 1998