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1.  It's All about the Talent 
Molecular Biology of the Cell  2010;21(22):3823.
PMCID: PMC2982123  PMID: 21079031
2.  Phosphoenolpyruvate carboxykinase and the critical role of cataplerosis in the control of hepatic metabolism 
The metabolic function of PEPCK-C is not fully understood; deletion of the gene for the enzyme in mice provides an opportunity to fully assess its function.
The gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC (PEPCK-C) was deleted in mice by homologous recombination (PEPCK-C-/- mice) and the metabolic consequences assessed.
PEPCK-C-/- mice became severely hypoglycemic by day two after birth and then died with profound hypoglycemia (12 mg/dl). The mice had milk in their stomachs at day two after birth and the administration of glucose raised the concentration of blood glucose in the mice but did not result in an increased survival. PEPCK-C-/- mice have two to three times the hepatic triglyceride content as control littermates on the second day after birth. These mice also had an elevation of lactate (2.5 times), β-hydroxybutyrate (3 times) and triglyceride (50%) in their blood, as compared to control animals. On day two after birth, alanine, glycine, glutamine, glutamate, aspartate and asparagine were elevated in the blood of the PEPCK-C-/- mice and the blood urea nitrogen concentration was increased by 2-fold. The rate of oxidation of [2-14C]-acetate, and [5-14C]-glutamate to 14CO2 by liver slices from PEPCK-C-/- mice at two days of age was greatly reduced, as was the rate of fatty acid synthesis from acetate and glucose. As predicted by the lack of PEPCK-C, the concentration of malate in the livers of the PEPCK-C-/- mice was 10 times that of controls.
We conclude that PEPCK-C is required not only for gluconeogenesis and glyceroneogenesis but also for cataplerosis (i.e. the removal of citric acid cycle anions) and that the failure of this process in the livers of PEPCK-C-/- mice results in a marked reduction in citric acid cycle flux and the shunting of hepatic lipid into triglyceride, resulting in a fatty liver.
PMCID: PMC1325233  PMID: 16300682
3.  Functional Characterization of a Novel Ku70/80 Pause Site at the H19/Igf2 Imprinting Control Region 
Molecular and Cellular Biology  2005;25(10):3855-3863.
The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg2+, implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.
PMCID: PMC1087736  PMID: 15870260
4.  Functional Characterization of a Testis-Specific DNA Binding Activity at the H19/Igf2 Imprinting Control Region 
Molecular and Cellular Biology  2003;23(22):8345-8351.
The DNA methylation state of the H19/Igf2 imprinting control region (ICR) is differentially set during gametogenesis. To identify factors responsible for the paternally specific DNA methylation of the ICR, germ line and somatic extracts were screened for proteins that bind to the ICR in a germ line-specific manner. A specific DNA binding activity that was restricted to the male germ line and enriched in neonatal testis was identified. Its three binding sites within the ICR are very similar to the consensus sequence for nuclear receptor extended half sites. To determine if these binding sites are required for establishment of the paternal epigenetic state, a mouse strain in which the three sites were mutated was generated. The mutated ICR was able to establish a male-specific epigenetic state in sperm that was indistinguishable from that established by the wild-type ICR, indicating that these sequences are either redundant or have no function. An analysis of the methylated state of the mutant ICR in the soma revealed no differences from the wild-type ICR but did uncover in both mutant and wild-type chromosomes a significant relaxation in the stringency of the methylated state of the paternal allele and the unmethylated state of the maternal allele in neonatal and adult tissues.
PMCID: PMC262389  PMID: 14585991
5.  Optimizing the detection of nascent transcripts by RNA fluorescence in situ hybridization 
Nucleic Acids Research  2001;29(8):e42.
An unusual feature of the mammalian genome is the number of genes exhibiting monoallelic expression. Recently random monoallelic expression of autosomal genes has been reported for olfactory and Ly-49 NK receptor genes, as well as for Il-2, Il-4 and Pax5. RNA fluorescence in situ hybridization (FISH) has been exploited to monitor allelic expression by visualizing the number of sites of transcription in individual nuclei. However, the sensitivity of this technique is difficult to determine for a given gene. We show that by combining DNA and RNA FISH it is possible to control for the hybridization efficiency and the accessibility and visibility of fluorescent probes within the nucleus.
PMCID: PMC31326  PMID: 11292856
6.  Expression and Functional Analysis of Uch-L3 during Mouse Development 
Molecular and Cellular Biology  2000;20(7):2498-2504.
Mice homozygous for the s1Acrg deletion at the Ednrb locus arrest at embryonic day 8.5. To determine the molecular basis of this defect, we initiated positional cloning of the s1Acrg minimal region. The mouse Uch-L3 (ubiquitin C-terminal hydrolase L3) gene was mapped within the s1Acrg minimal region. Because Uch-L3 transcripts were present in embryonic structures relevant to the s1Acrg phenotype, we created a targeted mutation in Uch-L3 to address its role during development and its possible contribution to the s1Acrg phenotype. Mice homozygous for the mutation Uch-L3Δ3-7 were viable, with no obvious developmental or histological abnormalities. Although high levels of Uch-L3 RNA were detected in testes and thymus, Uch-L3Δ3-7 homozygotes were fertile, and no defect in intrathymic T-cell differentiation was detected. We conclude that the s1Acrg phenotype is either complex and multigenic or due to the loss of another gene within the region. We propose that Uch-L3 may be functionally redundant with its homologue Uch-L1.
PMCID: PMC85452  PMID: 10713173
7.  Multiple Mechanisms Regulate Imprinting of the Mouse Distal Chromosome 7 Gene Cluster 
Molecular and Cellular Biology  1998;18(6):3466-3474.
Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57Kip2, as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57Kip2 during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57Kip2, and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57Kip2 have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57Kip2 is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).
PMCID: PMC108927  PMID: 9584186
8.  The Absence of Enhancer Competition between Igf2 and H19 following Transfer into Differentiated Cells 
Molecular and Cellular Biology  1998;18(4):1903-1910.
H19 and Igf2 are reciprocally imprinted genes that lie 90 kb apart on mouse chromosome 7. The two genes are coexpressed during development, with the H19 gene expressed exclusively from the maternal chromosome and Igf2 from the paternal chromosome. It has been proposed that their reciprocal imprinting is governed by a competition between the genes for a common set of enhancers. The competition on the paternal chromosome is influenced by extensive allele-specific methylation of the H19 gene and its 5′ flank, which acts to inhibit H19 transcription and thus indirectly leads to the activation of the Igf2 gene. In contrast, no allele-specific methylation has been detected on the maternal chromosome, and the basis for the preference for H19 transcription on that chromosome is unresolved. In this investigation, the mechanism controlling the silencing of the Igf2 gene on the maternal chromosome was explored by studying the transcriptional activity of a yeast artificial chromosome (YAC) containing Igf2 and H19 following transfer into differentiated tissue culture cells. Contrary to expectations, both H19 and Igf2 were expressed from a single integrated copy of the YAC. Furthermore, Igf2 expression appeared to be independent of the H19 locus, based on deletions of the H19 gene promoter and its enhancers. These results suggest that an active process is responsible for the transcriptional bias toward H19 on the maternal chromosome and that the hypomethylated state of this chromosome cannot be viewed as a “default” state. Moreover, the active process is not reproduced in a differentiated cell and may require passage through the female germ line.
PMCID: PMC121419  PMID: 9528761

Results 1-8 (8)