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Biomedical subjects

R Jaenisch

Publications and source records attributed to R Jaenisch.

At least 37 records · Page 2Linked to original sources
PubMed2001

Conditional deletion of brain-derived neurotrophic factor in the postnatal brain leads to obesity and hyperactivity.

Brain-derived neurotrophic factor has been associated previously with the regulation of food intake. To help elucidate the role of this neurotrophin in weight regulation, we have generated conditional mutants in which brain-derived neurotrophic factor has been eliminated from the brain after birth through the use of the cre-loxP recombination system. Brain-derived neurotrophic factor conditional mutants were hyperactive after exposure to stressors and had higher levels of anxiety when evaluated in the light/dark exploration test. They also had mature onset obesity characterized by a dramatic 80-150% increase in body weight, increased linear growth, and elevated serum levels of leptin, insulin, glucose, and cholesterol. In addition, the mutants had an abnormal starvation response and elevated basal levels of POMC, an anorexigenic factor and the precursor for alpha-MSH. Our results demonstrate that brain derived neurotrophic factor has an essential maintenance function in the regulation of anxiety-related behavior and in food intake through central mediators in both the basal and fasted state.

Animals↗
PubMed2001

Non-imprinted Igf2r expression decreases growth and rescues the Tme mutation in mice.

In the mouse the insulin-like growth factor receptor type 2 gene (Igf2r) is imprinted and maternally expressed. Igf2r encodes a trans-membrane receptor that transports mannose-6-phosphate tagged proteins and insulin-like growth factor 2 to lysosomes. During development the receptor reduces the amount of insulin-like growth factors and thereby decreases embryonic growth. The dosage of the gene is tightly regulated by genomic imprinting, leaving only the maternal copy of the gene active. Although the function of Igf2r in development is well established, the function of imprinting the gene remains elusive. Gene targeting experiments in mouse have demonstrated that the majority of genes are not sensitive to gene dosage, and mice heterozygous for mutations generally lack phenotypic alterations. To investigate whether reduction of Igf2r gene dosage by genomic imprinting has functional consequences for development we generated a non-imprinted allele (R2Delta). We restored biallelic expression to Igf2r by deleting a critical element for repression of the paternal allele (region 2) in mouse embryonic stem cells. Maternal inheritance of the R2Delta allele has no phenotype; however, paternal inheritance results in biallelic expression of Igf2r, which causes a 20% reduction in weight late in embryonic development that persists into adulthood. Paternal inheritance of the R2Delta allele rescues the lethality of a maternally inherited Igf2r null allele and a maternally inherited Tme (T-associated maternal effect) mutation. These data show that the biological function of imprinting Igf2r is to increase birth weight and they also establish Igf2r as the Tme gene.

Alleles↗
PubMed2000

X-Chromosome inactivation in cloned mouse embryos.

To study whether cloning resets the epigenetic differences between the two X chromosomes of a somatic female nucleus, we monitored X inactivation in cloned mouse embryos. Both X chromosomes were active during cleavage of cloned embryos, followed by random X inactivation in the embryo proper. In the trophectoderm (TE), X inactivation was nonrandom with the inactivated X of the somatic donor being chosen for inactivation. When female embryonic stem cells with two active X chromosomes were used as donors, random X inactivation was seen in the TE and embryo. These results demonstrate that epigenetic marks can be removed and reestablished on either X chromosome during cloning. Our results also suggest that the epigenetic marks imposed on the X chromosomes during gametogenesis, responsible for normal imprinted X inactivation in the TE, are functionally equivalent to the marks imposed on the chromosomes during somatic X inactivation.

Alleles↗
PubMed2000

Dynamic relocalization of histone MacroH2A1 from centrosomes to inactive X chromosomes during X inactivation.

Histone variant macroH2A1 (macroH2A1) contains an NH(2)-terminal domain that is highly similar to core histone H2A and a larger COOH-terminal domain of unknown function. MacroH2A1 is expressed at similar levels in male and female embryonic stem (ES) cells and adult tissues, but a portion of total macroH2A1 protein localizes to the inactive X chromosomes (Xi) of differentiated female cells in concentrations called macrochromatin bodies. Here, we show that centrosomes of undifferentiated male and female ES cells harbor a substantial store of macroH2A1 as a nonchromatin-associated pool. Greater than 95% of centrosomes from undifferentiated ES cells contain macroH2A1. Cell fractionation experiments confirmed that macroH2A1 resides at a pericentrosomal location in close proximity to the known centrosomal proteins gamma-tubulin and Skp1. Retention of macroH2A1 at centrosomes was partially labile in the presence of nocodazole suggesting that intact microtubules are necessary for accumulation of macroH2A1 at centrosomes. Upon differentiation of female ES cells, Xist RNA expression became upregulated and monoallelic as judged by fluorescent in situ hybridization, but early Xist signals lacked associated macroH2A1. Xi acquired macroH2A1 soon thereafter as indicated by the colocalization of Xist RNA and macroH2A1. Accumulation of macroH2A1 on X chromosomes occurred with a corresponding loss of centrosomal macroH2A1. Our results define a sequence for the loading of macroH2A1 on the Xi and place this event in the context of differentiation and Xist expression. Furthermore, these results suggest a role for the centrosome in the X inactivation process.

Animals↗
PubMed2000

V(D)J recombination is not activated by demethylation of the kappa locus.

V(D)J recombination is thought to be regulated by changes in the accessibility of target sites, such as modulation of methylation. To test whether demethylation of the kappa locus can activate recombination, we generated two recombinationally active B cell lines in which the gene for maintenance of genomic DNA methylation, Dnmt1, was flanked with loxP sites. Transduction with a retrovirus expressing both the cre recombinase and green fluorescent protein allowed us to purify recombinationally active cells devoid of methylation. Loss of methylation of the kappa locus was not sufficient to activate recombination, although transcription was activated in one line. It appears that demethylation of the kappa locus is not the rate-limiting step for altering accessibility and thus regulated demethylation does not generate specificity of recombination.

Animals↗
PubMed2000

Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a.

Current evidence indicates that methylation of cytosine in mammalian DNA is restricted to both strands of the symmetrical sequence CpG, although there have been sporadic reports that sequences other than CpG may also be methylated. We have used a dual-labeling nearest neighbor technique and bisulphite genomic sequencing methods to investigate the nearest neighbors of 5-methylcytosine residues in mammalian DNA. We find that embryonic stem cells, but not somatic tissues, have significant cytosine-5 methylation at CpA and, to a lesser extent, at CpT. As the expression of the de novo methyltransferase Dnmt3a correlates well with the presence of non-CpG methylation, we asked whether Dnmt3a might be responsible for this modification. Analysis of genomic methylation in transgenic Drosophila expressing Dnmt3a reveals that Dnmt3a is predominantly a CpG methylase but also is able to induce methylation at CpA and at CpT.

5-Methylcytosine↗
PubMed2000

DNA methyltransferase contributes to delayed ischemic brain injury.

DNA methylation is important for controlling the profile of gene expression and is catalyzed by DNA methyltransferase (MTase), an enzyme that is abundant in brain. Because significant DNA damage and alterations in gene expression develop as a consequence of cerebral ischemia, we measured MTase activity in vitro and DNA methylation in vivo after mild focal brain ischemia. After 30 min middle cerebral artery occlusion (MCAo) and reperfusion, MTase catalytic activity and the 190 kDa band on immunoblot did not change over time. However, [(3)H]methyl-group incorporation into DNA increased significantly in wild-type mice after reperfusion, but not in mutant mice heterozygous for a DNA methyltransferase gene deletion (Dnmt(S/+)). Dnmt(S/+) mice were resistant to mild ischemic damage, suggesting that increased DNA methylation is associated with augmented brain injury after MCA occlusion. Consistent with this formulation, treatment with the MTase inhibitor 5-aza-2'-deoxycytidine and the deacetylation inhibitor trichostatin A conferred stroke protection in wild-type mice. In contrast to mild stroke, however, DNA methylation was not enhanced, and reduced dnmt gene expression was not protective in an ischemia model of excitotoxic/necrotic cell death. In conclusion, our results demonstrate that MTase activity contributes to poor tissue outcome after mild ischemic brain injury.

Animals↗
PubMed2000

Association of the Bloom syndrome protein with topoisomerase IIIalpha in somatic and meiotic cells.

Bloom syndrome (BS) is characterized by genomic instability and cancer susceptibility caused by defects in BLM, a DNA helicase of the RecQ-family (J. German and N. A. Ellis, The Genetic Basis of Human Cancer, pp. 301-316, 1998). RecQ helicases and topoisomerase III proteins interact physically and functionally in yeast (S. Gangloff et al., Mol. Cell. Biol., 14: 8391-8398, 1994) and in Escherichia coli can function together to enable passage of double-stranded DNA (F. G. Harmon et al., Mol. Cell, 3: 611-620, 1999). We demonstrate in somatic and meiotic human cells an association between BLM and topoisomerase IIIalpha. These proteins colocalize in promyelocytic leukemia protein nuclear bodies, and this localization is disrupted in BS cells. Thus, mechanisms by which RecQ helicases and topoisomerase III proteins cooperate to maintain genomic stability in model organisms likely apply to humans.

Adenosine Triphosphatases↗
PubMed2000

Severe peripheral sensory neuron loss and modest motor neuron reduction in mice with combined deficiency of brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4/5.

Neurotrophins are a family of structurally and functionally related proteins that regulate neuronal survival during development. In the peripheral nervous system (PNS), both in vitro and in vivo studies have shown that neurotrophins are potent factors for the survival of various sensory neurons and sympathetic neurons. However, it is not clear whether all PNS neurons are neurotrophin-dependent. In the central nervous system (CNS), studies using injury models show that neurotrophins promote the survival of CNS neurons. But mice lacking individual neurotrophins or a combination of BDNF and NT4 did not show significant CNS neuronal loss. Here we derived mice lacking three neurotrophins, brain-derived growth factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4) to study the effect of triple neurotrophin deficiency on peripheral and central neurons. These triple-deficient mice did not nurse and died within 12 hours after birth. Neuronal cell counts showed that triple mutant pups lacked most of their peripheral sensory neurons and had a statistically significant reduction of motor neurons in several motor nuclei. Our results suggest that neurotrophins are essential for the survival of most peripheral sensory neurons and affect the survival of a small portion of motor neurons during embryogenesis.

Animals↗
PubMed2000

Formation of a full complement of cranial proprioceptors requires multiple neurotrophins.

Inactivation of neurotrophin-3 (NT3) completely blocks the development of limb proprioceptive neurons and their end organs, the muscle spindles. We examined whether cranial proprioceptive neurons of the trigeminal mesencephalic nucleus (TMN) require NT3, brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT4) for their development. Complements of TMN neurons and masticatory muscle spindles were decreased by 62% in NT3 null mutants, 33% in BDNF null mutants, and 10% in NT4 null mutant mice at birth. The extent of proprioceptive deficiencies differed among different masticatory muscles, particularly in NT3 null mice. Masticatory muscles of embryonic mice heterozygous for the NT3(lacZneo) or BDNF(lacZ) reporter genes expressed both NT3 and BDNF, consistent with target-derived neurotrophin support of TMN neurons. Although more than 90% of TMN neurons expressed TrkB as well as TrkC receptor proteins by immunocytochemistry in wild-type newborns, TrkC or TrkB null mice exhibited only partial proprioceptive deficiencies similar to those present in NT3 or BDNF;NT4 null mice. Thus, in terms of the survival outcome, two main subpopulations of TMN neurons may exist during embryogenesis, one dependent on TrkC/NT3 functioning and the other utilizing TrkB/BDNF signaling. The differential dependence of TMN neurons on neurotrophins may reflect differential accessibility of the neurons to limiting amounts of NT3, BDNF, or NT4 in target tissues, especially if the tissue distribution or levels of BDNF, NT3, and NT4 were dynamically regulated both spatially and temporally.

Animals↗
PubMed2000

The putative Drosophila methyltransferase gene dDnmt2 is contained in a transposon-like element and is expressed specifically in ovaries.

Several organisms, including Drosophila melanogaster, are apparently devoid of DNA methylation. This might reflect a highly restricted activity of DNA methyltransferases, a loss of methyltransferase activity during evolution or the dispensability of DNA methylation due to an efficient substitute mechanism. Vestiges of a Drosophila DNA methylation system have been identified recently. We show here that the putative DNA methyltransferase gene, dDnmt2, is the component of a transposon-like element. This element also contains a second, novel open reading frame with homologies to a yeast protein involved in RNA processing. Both open reading frames are coordinately expressed and transcripts are present specifically in ovarian nurse cells as well as during early stages of embryonic development.

Amino Acid Sequence↗
PubMed2000

A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation.

Xist is required for X inactivation. To study the initiation of X inactivation, we have generated a full-length mouse Xist cDNA transgene and an inducible expression system facilitating controlled Xist expression in ES cells and differentiated cultures. In ES cells, transgenic Xist RNA was stable and caused long-range transcriptional repression in cis. Repression was reversible and dependent on continued Xist expression in ES cells and early ES cell differentiation. By 72 hr of differentiation, inactivation became irreversible and independent of Xist. Upon differentiation, autosomal transgenes did not effect counting, but transgenic Xist RNA induced late replication and histone H4 hypoacetylation. Xist had to be activated within 48 hr of differentiation to effect silencing, suggesting that reversible repression by Xist is a required initiation step that might occur during normal X inactivation in female cells.

Animals↗
PubMed2000

Knocking the NT4 gene into the BDNF locus rescues BDNF deficient mice and reveals distinct NT4 and BDNF activities.

To directly compare biological activities of the neurotrophins NT4 and BDNF in vivo, we replaced the BDNF coding sequence with the NT4 sequence in mice (Bdnfnt4-ki). Mice expressing NT4 in place of BDNF were viable, in contrast with BDNF null mutants, which die shortly after birth. Although the Bdnfnt4-ki/nt4-ki and wild-type Bdnf+/+ alleles yielded similar levels of NT4 and BDNF proteins, NT4 supported more sensory neurons than BDNF and promoted functional synapse formation in cultured hippocampal neurons. Homozygous Bdnfnt4-ki/nt4-ki mice showed reduced body weight, infertility and skin lesions, suggesting unique biological activities of NT4 in vivo. The distinct activities of NT4 and BDNF may result partly from differential activation of the TrkB receptor and its down-stream signals.

Animals↗
PubMed2000

Ischemic brain damage in mice after selectively modifying BDNF or NT4 gene expression.

The neurotrophins and the tyrosine kinase (Trk) B receptor may play a protective role in the pathophysiology of cerebral ischemia. In this study, the authors investigated whether reducing endogenous expression of TrkB-binding neurotrophins modifies the susceptibility to ischemic injury after 1-hour middle cerebral artery occlusion followed by 23 hours of reperfusion in a filament middle cerebral artery occlusion model. Mice lacking both alleles for neurotrophin-4 (nt4-/-) or deficient in a single allele for brain-derived neurotrophic factor (bdnf+/-) exhibited larger cerebral infarcts compared to wild-type inbred 129/SVjae mice (68% and 91%, respectively, compared to controls). Moreover, lesions were larger (21%) in nt4-/- mice after permanent middle cerebral artery occlusion. Hence, expression of both NT4 and BDNF, and by inference the TrkB receptor, confers resistance to ischemic injury.

Animals↗
PubMed2000

Retroviral expression in embryonic stem cells and hematopoietic stem cells.

Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

Adoptive Transfer↗
PubMed2000

Mutations in the WRN gene in mice accelerate mortality in a p53-null background.

Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN(-/-);p53(-/-) mice show an increased mortality rate relative to WRN(+/-);p53(-/-) animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.

4-Nitroquinoline-1-oxide↗