Biomedical subjects

Nancy A Jenkins

Publications and source records attributed to Nancy A Jenkins.

At least 19 recordsLinked to original sources

PubMed2025

The common murine retroviral integration site activating Hhex marks a distal regulatory enhancer co-opted in human early T-cell precursor leukemia.

The Hhex gene encodes a transcription factor that is important for both embryonic and post-natal development, especially of hematopoietic tissues. Hhex is one of the most common sites of retroviral integration in mouse models. We found the most common integrations in AKXD (recombinant inbred strains) T-ALLs occur 57-61kb 3' of Hhex and activate Hhex gene expression. The genomic region of murine leukemia virus (MLV) integrations has features of a developmental stage-specific cis regulatory element (CRE), as evidenced by ATAC-seq in murine progenitor cells and high H3K27 acetylation at the syntenic CRE in human hematopoietic cell lines. With ChIP-exonuclease, we describe occupancy of LIM domain binding protein 1 (LDB1), the constitutive partner of the LIM Only-2 (LMO2), GATA1, and TAL1 transcription factors at GATA sites and at a composite GATA-E box within the CRE. With virtual 4C analysis, we observed looping between this +65kb CRE and the proximal intron one enhancer of HHEX in primary human ETP-ALLs and in normal progenitor cells. Our results show that retroviral integrations at intergenic sites can mark and take advantage of CREs. Specifically, in the case of HHEX activation, this newly described +65kb CRE is co-opted in the pathogenesis of ETP-ALL by the LMO2/LDB1 complex.

Humans↗

PubMed2003

Copper-binding-site-null SOD1 causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a common feature.

Cu/Zn superoxide dismutase (SOD1), a crucial cellular antioxidant, can in certain settings mediate toxic chemistry through its Cu cofactor. Whether this latter property explains why mutations in SOD1 cause FALS has been debated. Here, we demonstrate motor neuron disease in transgenic mice expressing a SOD1 variant that mutates the four histidine residues that coordinately bind Cu. In-depth analyses of this new mouse model, previously characterized models and FALS human tissues revealed that the accumulation of detergent-insoluble forms of SOD1 is a common feature of the disease. These insoluble species include full-length SOD1 proteins, peptide fragments, stable oligomers and ubiquitinated entities. Moreover, chaperones Hsp25 and alphaB-crystallin specifically co-fractionated with insoluble SOD1. In cultured cells, all 11 of the FALS variants tested produced insoluble forms of mutant SOD1. Importantly, expression of recombinant peptide fragments of wild-type SOD1 in cultured cells also produced insoluble species, suggesting that SOD1 possesses elements with an intrinsic propensity to aggregate. Thus, modifications to the protein, such as FALS mutations, fragmentation and possibly covalent modification, may simply act to augment a natural, but potentially toxic, propensity to aggregate.

Amyotrophic Lateral Sclerosis↗

PubMed2003

Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1).

Hermansky-Pudlak syndrome (HPS; MIM 203300) is a genetically heterogeneous disorder characterized by oculocutaneous albinism, prolonged bleeding and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules. In mice, at least 16 loci are associated with HPS, including sandy (sdy; ref. 7). Here we show that the sdy mutant mouse expresses no dysbindin protein owing to a deletion in the gene Dtnbp1 (encoding dysbindin) and that mutation of the human ortholog DTNBP1 causes a novel form of HPS called HPS-7. Dysbindin is a ubiquitously expressed protein that binds to alpha- and beta-dystrobrevins, components of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells. We also show that dysbindin is a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1; refs. 9-11), which regulates trafficking to lysosome-related organelles and includes the proteins pallidin, muted and cappuccino, which are associated with HPS in mice. These findings show that BLOC-1 is important in producing the HPS phenotype in humans, indicate that dysbindin has a role in the biogenesis of lysosome-related organelles and identify unexpected interactions between components of DPC and BLOC-1.

Animals↗

PubMed2003

Identification and characterization of a new pair of immunoglobulin-like receptors LMIR1 and 2 derived from murine bone marrow-derived mast cells.

We have identified and characterized two mouse cDNAs in a mouse antigen-stimulated bone marrow-derived mast cell cDNA library, both of which encode type I transmembrane proteins. The genes were closely mapped in the distal region of mouse chromosome 11 and expressed not only in mast cells but also widely in leukocytes. The extracellular domains of their encoded proteins contain a single variable immunoglobulin (Ig) motif sharing about 90% identity with amino acids, showing that they comprise a pair of molecules and belong to the Ig superfamily. We named these molecules leukocyte mono-Ig-like receptor1 and 2 (LMIR1 and 2). The intracellular domain of LMIR1 contains several immunoreceptor tyrosine-based inhibition motifs (ITIMs). When cross-linked, the intracellular domain was tyrosine phosphorylated and capable of recruiting tyrosine phosphatases, SHP-1 and SHP-2 and inositol polyphosphate 5-phosphatase, SHIP. LMIR2, on the other hand, contains a short cytoplasmic tail and a characteristic transmembrane domain carrying two positively charged amino acids associated with three kinds of immunoreceptor tyrosine-based activation motif (ITAM)-bearing molecules, DAP10, DAP12, and FcRgamma. These findings suggest that a new pair of ITIM/ITAM-bearing receptors, LMIR1 and 2, regulate mast cell-mediated inflammatory responses through yet to be defined ligand(s).

Amino Acid Sequence↗

PubMed2003

Stat5 synergizes with T cell receptor/antigen stimulation in the development of lymphoblastic lymphoma.

Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that mediate a wide range of actions induced by cytokines, interferons, and growth factors. We now report the development of thymic T cell lymphoblastic lymphomas in transgenic mice in which Stat5a or Stat5b is overexpressed within the lymphoid compartment. The rate of lymphoma induction was markedly enhanced by immunization or by the introduction of TCR transgenes. Remarkably, the Stat5 transgene potently induced development of CD8+ T cells, even in mice expressing a class II-restricted TCR transgene, with resulting CD8+ T cell lymphomas. These data demonstrate the oncogenic potential of dysregulated expression of a STAT protein that is not constitutively activated, and that TCR stimulation can contribute to this process.

Animals↗

PubMed2003

Identification of beta-carotene 15, 15'-monooxygenase as a peroxisome proliferator-activated receptor target gene.

Beta-carotene 15,15'-monooxygenase (BCM) catalyzes the first step of vitamin A biosynthesis from provitamin A carotenoids. We wished to determine the factors underlying the transcriptional regulation of this gene. After cloning of the 40 kilobase pair (kbp) mouse Bcm gene and determination of its genomic organization, analysis of the 2 kb 5'-flanking region showed several putative transcription factor binding sites including TATA box, a peroxisome proliferator response element (PPRE), AP2, and bHLH. The 2 kb fragment drove specific luciferase gene expression in vitro only in cell lines that express BCM (TC7, PF11, and monkey retinal pigment epithelium). Nucleotides -41 to +163, and -60 to +163 drove basal and specific Bcm transcriptional activity, respectively. Site-directed mutagenesis and gel shift experiments demonstrate that PPRE was essential for Bcm promoter specificity and that the peroxisome proliferator activated receptor (PPAR) gamma (PPARgamma) specifically binds to this element. Furthermore, cotransfection experiments and pharmacological treatments in vitro, using the specific PPARgamma agonists LY17883 and ciglitazone, demonstrate that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor-alpha heterodimer. Treatment of mice with the PPARalpha/gamma agonist WY14643 increases BCM protein expression in liver. Thus PPAR is a key transcription factor for the transcriptional regulation of the Bcm gene, suggesting a broader function for PPARs in the regulation of carotenoid metabolism metabolism that is consistent with their established role in neutral lipid metabolism and transport.

Animals↗

PubMed2003

Identification of Vangl2 and Scrb1 as planar polarity genes in mammals.

In mammals, an example of planar cell polarity (PCP) is the uniform orientation of the hair cell stereociliary bundles within the cochlea. The PCP pathway of Drosophila refers to a conserved signalling pathway that regulates the coordinated orientation of cells or structures within the plane of an epithelium. Here we show that a mutation in Vangl2, a mammalian homologue of the Drosophila PCP gene Strabismus/Van Gogh, results in significant disruptions in the polarization of stereociliary bundles in mouse cochlea as a result of defects in the direction of movement and/or anchoring of the kinocilium within each hair cell. Similar, but less severe, defects are observed in animals containing a mutation in the LAP protein family gene Scrb1 (homologous with Drosophila scribble). Polarization defects in animals heterozygous for Vangl2 and Scrb1 are comparable with Vangl2 homozygotes, demonstrating genetic interactions between these genes in the regulation of PCP in mammals. These results demonstrate a role for the PCP pathway in planar polarization in mammals, and identify Scrb1 as a PCP gene.

Animals↗

PubMed2003

Bcl11a is essential for normal lymphoid development.

Bcl11a (also called Evi9) functions as a myeloid or B cell proto-oncogene in mice and humans, respectively. Here we show that Bcl11a is essential for postnatal development and normal lymphopoiesis. Bcl11a mutant embryos lack B cells and have alterations in several types of T cells. Phenotypic and expression studies show that Bcl11a functions upstream of the transcription factors Ebf1 and Pax5 in the B cell pathway. Transplantation studies show that these defects in Bcl11a mutant mice are intrinsic to fetal liver precursor cells. Mice transplanted with Bcl11a-deficient cells died from T cell leukemia derived from the host. Thus, Bcl11a may also function as a non-autonomous T cell tumor suppressor gene.

Animals↗

PubMed2003

Genome-wide single-nucleotide polymorphism analysis defines haplotype patterns in mouse.

The nature and organization of polymorphisms, or differences, between genomes of individuals are of great interest, because these variations can be associated with or even underlie phenotypic traits, including disease susceptibility. To gain insight into the genetic and evolutionary factors influencing such biological variation, we have examined the arrangement (haplotype) of single-nucleotide polymorphisms across the genomes of eight inbred strains of mice. These analyses define blocks of high or low diversity, often extending across tens of megabases that are delineated by abrupt transitions. These observations provide a striking contrast to the haplotype structure of the human genome.

Animals↗

PubMed2003

Ru2 and Ru encode mouse orthologs of the genes mutated in human Hermansky-Pudlak syndrome types 5 and 6.

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous disease involving abnormalities of melanosomes, platelet dense granules and lysosomes. Here we have used positional candidate and transgenic rescue approaches to identify the genes mutated in ruby-eye 2 and ruby-eye mice (ru2 and ru, respectively), two 'mimic' mouse models of HPS. We also show that these genes are orthologs of the genes mutated in individuals with HPS types 5 and 6, respectively, and that their protein products directly interact. Both genes are previously unknown and are found only in higher eukaryotes, and together represent a new class of genes that have evolved in higher organisms to govern the synthesis of highly specialized lysosome-related organelles.

Adaptor Protein Complex 3↗

PubMed2003

Isolation and characterization of the mouse ubiquitin-specific protease Usp15.

We have characterized the mouse ortholog of the human ubiquitin-specific protease USP15. Mouse Usp15 consists of 981 amino acids with a predicted molecular mass of 112 kDa, contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes, and is 98% identical/99% similar to human USP15. Usp15 shares 59.5% identity/75.5% sequence similarity with the mouse Unp(Usp4) oncoprotein. Recombinant Usp15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to glutathione S-transferase. Usp15 can also cleave the ubiquitin-proline bond, as can USP15 and Usp4. Alignment of mouse and human Usp15 and Usp4 protein sequences suggested that Usp15/USP15 may be alternately spliced in a manner analogous to Usp4. Sequence analysis of RT-PCR products from several human and mouse cell lines and tissues revealed alternate splicing in all cells studied. Northern blot analysis of both mouse and human Usp15 revealed two differently sized mRNAs in all tissues examined, owing to alternate polyadenylation sites spaced by 1.5 kb. Chromosomal mapping by interspecific backcross analysis localized the Usp15 gene to the distal region of mouse Chromosome (Chr) 10. This region is syntenic with human Chr 12q24, the location of human USP15, and a different location to Unp(Usp4) (Chr 9). Identification of the mouse Usp15 gene (>69.5 kb) and human USP15 gene (145 kb) sequences in genome databases reveals that both are composed of 22 exons with identical splice sites, and both have an exon/intron structure identical to the mouse Usp4 gene, including the alternately spliced exon. Phylogenetic studies suggest that a sequence currently identified as a chicken Usp4 ortholog is in fact a USP15 ortholog, while bona-fide chicken, cow, and rat Usp4 orthologs can be identified in EST databases.

Amino Acid Sequence↗

PubMed2003

Interallelic complementation at the mouse Mitf locus.

Mutations at the mouse microphthalmia locus (Mitf) affect the development of different cell types, including melanocytes, retinal pigment epithelial cells of the eye, and osteoclasts. The MITF protein is a member of the MYC supergene family of basic-helix-loop-helix-leucine-zipper (bHLHZip) transcription factors and is known to regulate the expression of cell-specific target genes by binding DNA as homodimer or as heterodimer with related proteins. The many mutations isolated at the locus have different effects on the phenotype and can be arranged in an allelic series in which the phenotypes range from near normal to white microphthalmic animals with osteopetrosis. Previous investigations have shown that certain combinations of Mitf alleles complement each other, resulting in a phenotype more normal than that of each homozygote alone. Here we analyze this interallelic complementation in detail and show that it is limited to one particular allele, Mitf(Mi-white) (Mitf(Mi-wh)), a mutation affecting the DNA-binding domain. Both loss- and gain-of-function mutations are complemented, as are other Mitf mutations affecting the DNA-binding domain. Furthermore, this behavior is not restricted to particular cell types: Both eye development and coat color phenotypes are complemented. Our analysis suggests that Mitf(Mi-wh)-associated interallelic complementation is due to the unique biochemical nature of this mutation.

Animals↗

PubMed2003

A highly efficient recombineering-based method for generating conditional knockout mutations.

Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.

Animals↗

PubMed2003

Common sites of retroviral integration in mouse hematopoietic tumors identified by high-throughput, single nucleotide polymorphism-based mapping and bacterial artificial chromosome hybridization.

Retroviral insertional mutagenesis in mouse hematopoietic tumors provides a powerful cancer gene discovery tool. Here, we describe a high-throughput, single nucleotide polymorphism (SNP)-based method, for mapping retroviral integration sites cloned from mouse tumors, and a bacterial artificial chromosome (BAC) hybridization method, for localizing these retroviral integration sites to common sites of retroviral integration (CISs). Several new CISs were identified, including one CIS that mapped near Notch1, a gene that has been causally associated with human T-cell tumors. This mapping method is applicable to many different species, including ones where few genetic markers and little genomic sequence information are available. It can also be used to map endogenous proviruses.

Animals↗

PubMed2003

Genome-based identification of cancer genes by proviral tagging in mouse retrovirus-induced T-cell lymphomas.

The identification of tumor-inducing genes is a driving force for elucidating the molecular mechanisms underlying cancer. Many retroviruses induce tumors by insertion of viral DNA adjacent to cellular oncogenes, resulting in altered expression and/or structure of the encoded proteins. The availability of the mouse genome sequence now allows analysis of retroviral common integration sites in murine tumors to be used as a genetic screen for identification of large numbers of candidate cancer genes. By positioning the sequences of inverse PCR-amplified, virus-host junction fragments within the mouse genome, 19 target genes were identified in T-cell lymphomas induced by the retrovirus SL3-3. The candidate cancer genes included transcription factors (Fos, Gfi1, Lef1, Myb, Myc, Runx3, and Sox3), all three D cyclins, Ras signaling pathway components (Rras2/TC21 and Rasgrp1), and Cmkbr7/CCR7. The most frequent target was Rras2. Insertions as far as 57 kb away from the transcribed portion were associated with substantially increased transcription of Rras2, and no coding sequence mutations, including those typically involved in Ras activation, were detected. These studies demonstrate the power of genome-based analysis of retroviral insertion sites for cancer gene discovery, identify several new genes worth examining for a role in human cancer, and implicate the pathways in which those genes act in lymphomagenesis. They also provide strong genetic evidence that overexpression of unmutated Rras2 contributes to tumorigenesis, thus suggesting that it may also do so if it is inappropriately expressed in human tumors.

Animals↗

PubMed2002

Evi3, a common retroviral integration site in murine B-cell lymphoma, encodes an EBFAZ-related Krüppel-like zinc finger protein.

Retroviral insertional mutagenesis in inbred mouse strains provides a powerful method for cancer gene discovery. Here, we show that a common retroviral integration site (RIS) in AKXD B-cell lymphomas, termed Evi3, encodes a novel zinc finger protein with 30 Krüppel-like zinc finger repeats. Most integrations at Evi3 are located upstream of the first translated exon and result in 3' long-terminal repeat (LTR)-driven overexpression of Evi3. Evi3 is highly related to the early B-cell factor-associated zinc finger gene (Ebfaz), and all 30 zinc fingers found in EVI3 are conserved in EBFAZ. EBFAZ binds to and negatively regulates early B-cell factor (EBF) (also known as olfactory-1, OLF1), a basic helix-loop-helix (bHLH) transcription factor required for B-lineage commitment and the development of the olfactory epithelium. EBFAZ also binds to SMA- and MAD-related protein-1 (SMAD1) and SMAD4 in response to bone morphogenetic protein-2 (BMP2) signaling, which in turn activates the homeobox regulator of Xenopus mesoderm and neural development Xvent-2. Surprisingly, while Ebfaz and Evi3 are coexpressed in many tissues, and both proteins are nuclear, we could not detect Ebfaz expression in B cells by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas Evi3 expression could be detected at all stages of B-cell development. Our results suggest that EVI3, like EBFAZ, is a multifunctional protein that participates in many signaling pathways via its multiple zinc fingers. Furthermore, our results suggest that EVI3, not EBFAZ, is the member of this protein family that interacts with and regulates EBF in B cells.

Amino Acid Sequence↗

PubMed2002

Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease.

Mice that are homozygous with respect to a mutation (ax(J)) in the ataxia (ax) gene develop severe tremors by 2-3 weeks of age followed by hindlimb paralysis and death by 6-10 weeks of age. Here we show that ax encodes ubiquitin-specific protease 14 (Usp14). Ubiquitin proteases are a large family of cysteine proteases that specifically cleave ubiquitin conjugates. Although Usp14 can cleave a ubiquitin-tagged protein in vitro, it is unable to process polyubiquitin, which is believed to be associated with the protein aggregates seen in Parkinson disease, spinocerebellar ataxia type 1 (SCA1; ref. 4) and gracile axonal dystrophy (GAD). The physiological substrate of Usp14 may therefore contain a mono-ubiquitin side chain, the removal of which would regulate processes such as protein localization and protein activity. Expression of Usp14 is significantly altered in ax(J)/ax(J) mice as a result of the insertion of an intracisternal-A particle (IAP) into intron 5 of Usp14. In contrast to other neurodegenerative disorders such as Parkinson disease and SCA1 in humans and GAD in mice, neither ubiquitin-positive protein aggregates nor neuronal cell loss is detectable in the central nervous system (CNS) of ax(J) mice. Instead, ax(J) mice have defects in synaptic transmission in both the central and peripheral nervous systems. These results suggest that ubiquitin proteases are important in regulating synaptic activity in mammals.

Alleles↗

PubMed2002

New genes involved in cancer identified by retroviral tagging.

Retroviral insertional mutagenesis in BXH2 and AKXD mice induces a high incidence of myeloid leukemia and B- and T-cell lymphoma, respectively. The retroviral integration sites (RISs) in these tumors thus provide powerful genetic tags for the discovery of genes involved in cancer. Here we report the first large-scale use of retroviral tagging for cancer gene discovery in the post-genome era. Using high throughput inverse PCR, we cloned and analyzed the sequences of 884 RISs from a tumor panel composed primarily of B-cell lymphomas. We then compared these sequences, and another 415 RIS sequences previously cloned from BXH2 myeloid leukemias and from a few AKXD lymphomas, against the recently assembled mouse genome sequence. These studies identified 152 loci that are targets of retroviral integration in more than one tumor (common retroviral integration sites, CISs) and therefore likely to encode a cancer gene. Thirty-six CISs encode genes that are known or predicted to be genes involved in human cancer or their homologs, whereas others encode candidate genes that have not yet been examined for a role in human cancer. Our studies demonstrate the power of retroviral tagging for cancer gene discovery in the post-genome era and indicate a largely unrecognized complexity in mouse and presumably human cancer.

Animals↗