Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Peer reviewed journal publications; presentations at international meetings; patent applications filed. PARTICIPANTS: James R. Putnam, technician Chelsea Brideau, graduate student Nadia Drake, graduate student Patrick Murphy, graduate student Yoon Jung Park, graduate student Ruqian Zhao, visiting scientist TARGET AUDIENCES: Researchers in epigenetics and epigenomics PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Outcomes of these efforts are primarily in the realm of changes in knowledge. Some of these outcomes have been published; others are the subjects of manuscripts currently under review or in preparation. All changes in knowledge relate to mechanisms that control epigenetic phenomena in mammals as modeled using laboratory mice, insights into how these mechanisms control body growth, use of epigenomic signatures to identify novel imprinted genes and the development of new technologies to assess epigenetic state.
Publications
- Drake, N. M., Park, Y. J., Shirali, A. S., Cleland, T. A., and Soloway, P. D. (2009) "Imprint switch mutations at Rasgrf1 support conflict hypothesis of imprinting and define a growth control mechanism upstream of IGF1" Mamm Genome 20:(9-10), 654-663
- Fasano, S., D'Antoni, A., Orban, P. C., Valjent, E., Putignano, E., Vara, H., Pizzorusso, T., Giustetto, M., Yoon, B., Soloway, P., Maldonado, R., Caboche, J., and Brambilla, R. (2009) "Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) controls activation of extracellular signal-regulated kinase (ERK) signaling in the striatum and long-term behavioral responses to cocaine" Biological psychiatry 66:(8), 758-768
- Cipriany, B. R., Zhao, R., Murphy, P. J., Levy, S. L., Tan, C. P., Craighead, H. G., and Soloway, P. D. (2010) "Single Molecule Epigenetic Analysis in a Nanofluidic Channel" Anal Chem
- Kandalam, V., Basu, R., Abraham, T., Wang, X., Soloway, P. D., Jaworski, D. M., Oudit, G. Y., and Kassiri, Z. (2010) "TIMP2 Deficiency Accelerates Adverse Post-Myocardial Infarction Remodeling Because of Enhanced MT1-MMP Activity Despite Lack of MMP2 Activation" Circ Res
|
Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Outputs include peer reviewed publications listed elsewhere in this report; grant proposals submitted to federal agencies that were approved and awarded and public lectures. PARTICIPANTS: James R. Putnam, technician Chelsea McLean, graduate student Nadia Drake, graduate student Yoon Jung Park, graduate student Anders Lindroth, post doctoral fellow Paul D. Soloway, principal investigator TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcomes of these efforts are primarily in the realm of changes in knowledge. Some of these outcomes have been published; others are the subjects of manuscripts in preparation. All changes in knowledge relate to mechanisms that control epigenetic phenomena in mammals as modeled using laboratory mice. Highlights of the changes in knowledge include the following: two essential epigenetic modifications in the mammalian genome (DNA methylation and histone H3 lysine 27 methylation) were shown to operate in antagonism to one another; epigenomic data from ES cells, mouse embryo fibroblasts and neuronal progenitor cells were used successfully in a machine learning paradigm to identify previously unknown imprinted genes; transcriptome wide sequences was used successfully to identify additional previously unknown imprinted genes; imprinting of Rasgrf1 in mice during the perinatal period controls life long growth by early effects on the Ifg1 growth regulatory system; novel approaches to epigenomic analysis have been initiated by single molecule analysis on a nano-scale device.
Publications
- Lindroth AM, Park YJ, McLean CM, Dokshin GA, Persson JM et al. (2008) Antagonism between DNA and H3K27 Methylation at the Imprinted Rasgrf1 Locus. PLoS Genetics 4(8): e1000145.
- Lluri G, Langlois GD, Soloway PD, Jaworski DM (2008) Tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates myogenesis and beta1 integrin expression in vitro. Exp Cell Res 314(1): 11-24.
- Wang X, Sun Q, McGrath SD, Mardis ER, Soloway PD et al. (2008) Transcriptome-wide identification of novel imprinted genes in neonatal mouse brain. PLoS ONE 3(12): e3839.
|
Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Outputs completed during this reporting period include publications listed in the Publications section, presentations at international meetings including the biannual Gordon Conference on Epigenetics, media interviews and sharing of reagents with other scientists.
PARTICIPANTS: National Cancer Institute, Univ of Vermont, Ontario Cancer Institute, Scripps Research Institute, Universite de Provence
Impacts
Ongoing research efforts seek to understand how epigenetic modifications are established, maintained and exchanged between chromosomal locations in the mammalian genome. The system studied in my lab is the Rasgrf1 locus, where we have identified the only known mammalian DNA methylation promoter, which functions to control local DNA methylation. This year, we have made several discoveries: (1) the DNA methylation promoter element controls not only DNA methylation, but histone methylation as well; (2) histone H3 methylation on lysine 27 antagonizes placement of DNA methylation and vise versa; (3) histone H3 methylation on lysine 9 supports placement of DNA methylation; (3) the DNA and histone methylation promoter is homologous to small 31 nt piRNA sized transcripts, which may be essential to the control of the methylation events; (4) epigenetically controlled gene expression on one allele of Rasgrf1 is affected by mutations on the homologous allele, providing an example
of trans chromosomal interactions; (5) disruptions in epigenetically controlled Rasgrf1 expression states leads to aberrant growth phenotypes in mice.
Publications
- Slape C, Chung YJ, Soloway PD, Tessarollo L, Aplan PD. 2007. Mouse embryonic stem cells that express a NUP98-HOXD13 fusion protein are impaired in their ability to differentiate and can be complemented by BCR-ABL. Leukemia 21(6):1239-1248.
- Lluri G, Langlois GD, Soloway PD, Jaworski DM. 2007. Tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates myogenesis and beta1 integrin expression in vitro. Exp Cell Res.
- Soloway PD. 2006. Gene nutrient interactions and evolution. Nutr Rev 64(5 Pt 2):S52-54; discussion S72-91.
- Soloway PD. 2006. Paramutable Possibilities. Nature 441:413-414.
- Lluri G, Langlois GD, McClellan B, Soloway PD, Jaworski DM. 2006. Tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates neuromuscular junction development via a beta1 integrin-mediated mechanism. J Neurobiol 66(12):1365-1377.
- Jaworski DM, Soloway P, Caterina J, Falls WA. 2006. Tissue inhibitor of metalloproteinase-2(TIMP-2)-deficient mice display motor deficits. J Neurobiol 66(1):82-94.
- Holmes R, Soloway PD. 2006. Regulation of imprinted DNA methylation. Cytogenet Genome Res 113(1-4)122-129.
- Holmes R, Chang Y, Soloway PD. 2006. Timing and Sequence Requirements Defined for Embryonic Maintenance of Imprinted DNA Methylation at Rasgrf1. Mol Cell Biol 26(24):9564-9570.
- English JL, Kassiri Z, Koskivirta I, Atkinson SJ, Di Grappa M et al. 2006. Individual Timp deficiencies differentially impact pro-MMP-2 activation. J Biol Chem 281(15):10337-10346.
- Crocker SJ, Whitmire JK, Frausto RF, Chertboonmuang P, Soloway PD et al. 2006. Persistent macrophage/microglial activation and myelin disruption after experimental autoimmune encephalomyelitis in tissue inhibitor of metalloproteinase-1-deficient mice. Am J Pathol 169(6):2104-2116.
- Chaillan FA, Rivera S, Marchetti E, Jourquin J, Werb Z et al. 2006. Involvement of tissue inhibition of metalloproteinases-1 in learning and memory in mice. Behav Brain Res 173:191-198.
|
Progress 01/01/05 to 12/31/05
Outputs
Efforts have focused on two issues: Understanding mechanisms of epigenetic regulation and mapping genes that control the effects of diet on colon cancer. In mammals, DNA is methylated at at CpGs and associated histones are methylated at lysines. Proper methylation is crucial for normal development and inappropriate methylation contributes to cancer. Little is known about how methylation is controlled. We previously identified a repeated DNA sequence that regulates establishment of DNA methylation in the male germ line at Rasgrf1. This year, we showed that the same sequence regulates maintenance of paternal allele DNA methylation in somatic tissue of fertilized embryos, but only during the first 5 days of embryogenesis in mice prior to the epiblast stage. Beyond this very early period of development, the methyl-regulating sequence is dispensable. In related studies, we have shown that in addition to regulating DNA methylation, the repeat sequence also regulates histone
H3 tri-methlation on lysine 27 of the maternal allele. Furthermore, DNA methylation is able to exclude histone H3 lysine 27 methylation. This work demonstrates that patterns of histone and DNA methylation at this locus are coordinately regulated and involve a common mechanism. In a second project, we are studying nutrient-genome interactions relevant to cancer. Many studies have shown that diet influences cancer rates. As with many environmental variables, the in vivo responses are affected by genetic factors. So which genes influence how diet affects cancer? Very little is known. We are using recombinant inbred (RI) strains of mice to map genomic locations of such genes. We previously showed that two strains, C57BL/6 and A/J, harbor sufficient variability in responses to diet that they can be used to map genetic loci controlling the effects of diet on cancer. Fourteen RI strains derived from C57BL/6 and A/J were obtained from Jackson Labs, the colony was expanded and mice from each
RI strain were divided into two groups. One was fed a high fat, low mineral and low vitamin western style diet. The other was fed a control diet. Both groups were given the carcinogen azoxymethyane (AOM) to induce colon cancer. After tumors had developed, we analyzed all mice for tumor number and identified three strains with diet-dependent tumor responses. The logistic regression analysis is ongoing to identify genetic loci controlling the diet-responsive phenotype.
Impacts
The first project will reveal mechanisms by which chromatin methylation patterns are controlled in the genome. It may identify therapeutic targets for manipulating these epigenetic events to control gene expression. The second project may reveal the genetic basis of how diet affects cancer outcome differently in different individuals.
Publications
- Jaworski, D.M., J. Boone, J. Caterina, P. Soloway and W.A. Falls. 2005. Prepulse inhibition and fear-potentiated startle are altered in tissue inhibitor of metalloproteinase-2 (TIMP-2) knockout mice. Brain Res 1051(1-2): 81-9.
- Jourquin, J., E. Tremblay, A. Bernard, G. Charton, F.A. Chaillan, E. Marchetti, F.S. Roman, P.D. Soloway, V. Dive, A. Yiotakis, M. Khrestchatisky and S. Rivera. 2005. Tissue inhibitor of metalloproteinases-1 (TIMP-1) modulates neuronal death, axonal plasticity, and learning and memory. Eur J Neurosci 22(10): 2569-2578.
- Ogier, C., R. Creidy, J. Boucraut, P.D. Soloway, M. Khrestchatisky and S. Rivera. 2005. Astrocyte reactivity to Fas activation is attenuated in TIMP-1 deficient mice: an in vitro study. BMC Neurosci 6(1): 68.
- Lee, M.M., B.J. Yoon, K. Osiewicz, M. Preston, B. Bundy, A.M. van Heeckeren, Z. Werb and P.D. Soloway. 2005. Tissue inhibitor of metalloproteinase 1 regulates resistance to infection. Infect Immun 73(1):661-5.
- Mohammed, F.F., C.J. Pennington, Z. Kassiri, J.S. Rubin, P.D. Soloway, U. Ruther, D.R. Edwards and R. Khokha. 2005. Metalloproteinase inhibitor TIMP-1 affects hepatocyte cell cycle via HGF activation in murine liver regeneration. Hepatology 41(4): 857-67.
- Kim, K.H., K. Burkhart, P. Chen, C.W. Frevert, J. Randolph-Habecker, R. C. Hackman, P.D. Soloway and D. K. Madtes. 2005. Tissue Inhibitor of MP-1 Deficiency Amplifies Acute Lung Injury in Bleomycin Exposed Mice. Am J Respir Cell Mol Biol.
- Yoon, B.-J., H. Herman, B. Hu, Y.J. Park, A.M. Lindroth, A. Bell, A.G. West, Y. Chang, A. Stablewski, J.C. Piel, D.I. Loukinov, V. Lobanenkov and P.D. Soloway. 2005. Rasgrf1 Imprinting is Regulated by a CTCF-dependent Methylation-Sensitive Enhancer Blocker. Mol Cell Biol 25(24): 11184-11190.
|
Progress 01/01/04 to 12/31/04
Outputs
Efforts have focused on two issues: Understanding mechanisms of epigenetic regulation and validating methods for mapping genes that control the effects of diet on colon cancer. In mammals, DNA is methylated at at CpGs and associated histones are methylated at lysines. Proper methylation is crucial for normal development and inappropriate methylation contributes to cancer. Little is known about how methylation is controlled. We previously identified a repeated DNA sequence that regulates establishment of DNA methylation in the male germ line at Rasgrf1. This year, we showed that the same sequence regulates maintenance of DNA methylation in somatic tissue of fertilized embryos, but only during the first 5 days of embryogenesis in mice prior to the epiblast stage. Beyond this very early period of development, the methyl-regulating sequence is dispensable. In related studies, we have shown that in addition to regulating DNA methylation, the repeat sequence also regulates
histone H3 tri-methlation on lysine 9. This work demonstrates that patterns of histone and DNA methylation at this locus are coordinately regulated and involve a common mechanism. In a second project, we are studying nutrient-genome interactions relevant to cancer. Many studies have shown that diet influences cancer rates. As with many environmental variables, the in vivo responses are affected by genetic factors. So which genes influence how diet affects cancer? Very little is known. We are using recombinant inbred (RI) strains of mice to map genomic locations of such genes. To identify which RI strains would be useful for this, we used 30 mice from each of two strains, C57BL/6 and A/J as well as 30 of their F1 progeny. Each group of 30 was divided into two groups. One was fed a high fat, low mineral and low vitamin western style diet. The other was fed a control diet. Both groups were given the carcinogen azoxymethyane (AOM) to induce colon cancer. After tumors had developed, we
analyzed all mice for tumor number and showed that the C57BL/6 mice were highly resistant to AOM: Only two tumors were observed, regardless of the diet used. A/J mice were susceptible to AOM with the majority of mice developing multiple tumors, but diet did not affect tumor number. The F1 mice were similar to the A/J parent, however, they developed a significantly larger tumor burden when fed the western diet. These data immediately revealed that there was no simple pattern of dominant, recessive or intermediate phenotypes typical of simple Mendelian inheritance. This also showed that there was an interaction between alleles of the two parental inbred genomes that influenced tumor outcome. The lack of simple Mendelian inheritance and the presence of allelic interactions that influenced a quantifiable phenotype are hallmarks of a complex, quantitative trait and indicates C57BL/6 and A/J are excellent tools to begin identifying the genetic loci controlling the influence of diet upon
tumor development. Work with RI strains has been initiated.
Impacts
The first project will reveal mechanisms by which chromatin methylation patterns are controlled in the genome. It may identify therapeutic targets for manipulating these epigenetic events to control gene expression. The second project may reveal the genetic basis of how diet affects cancer outcome differently in different individuals.
Publications
- No publications reported this period
|
Progress 01/01/03 to 12/31/03
Outputs
Many of our genomics efforts over the past year have focused on understanding mechanisms of epigenetic regulation. We have been specifically interested in discovering how patterns of DNA methylation are established and propagated in the genome and how these regulate transcription and have studied this in two related projects. In mammals, DNA is methylated at cytosines within CpG dinucleotides. Properly regulated methylation is crucial for normal development. Inappropriate methylation may contribute to tumorigenesis by silencing tumor-suppressor genes or by activating growth-stimulating genes. Although many genes have been identified that acquire methylation and whose expression is methylation-sensitive, little is known about how DNA methylation is controlled. We have identified a DNA sequence that regulates establishment of DNA methylation in the male germ line at Rasgrf1. In mice, the imprinted Rasgrf1 locus is methylated on the paternal allele within a
differentially methylated domain (DMD) 30 kbp 5 prime of the promoter. Expression is exclusively from the paternal allele in neonatal brain. Methylation is regulated by a repeated sequence, consisting of a 41-mer repeated 40 times, found immediately 3 prime of the DMD. The repeats are needed for both establishment of methylation in the male germ line and maintenance of methylation after fertilization. In addition, DMD methylation is required for imprinted Rasgrf1 expression. Together the DMD and repeat element constitute a binary switch that regulates the locus. We are now positioned to identify repeat interacting factors to elucidate the trans acting components that regulate DNA methylation. At Igf2r, an intronic sequence called region 2, has been shown by others to control methylation of the active maternal allele at that locus. We have shown that replacing the Rasgrf1 repeats on the paternal allele with Igf2r region 2 allows both methylation and expression of the paternal copy of
Rasgrf1 demonstrating that methylation controlling sequences can function ectopically. Surprisingly, paternal transmission of the mutant allele also induced methylation and expression in trans of the normally unmethylated and silent wildtype maternal allele. Once activated, the wildtype maternal Rasgrf1 allele maintained its activated state in the next generation, independently of the paternal allele. Furthermore, the influence of the region 2 insertion at Rasgrf1 is not limited to the Rasgrf1locus on chromosome 9, instead, the mutation can exert its effects on the Igf2r locus on chromosome 17 demonstrating that trans effects are not restricted to homologous chromosomes. These results recapitulate in mice the epigenetic phenomena of paramutation described in plants and trans allele methylation and quelling described in fungi. Furthermore, this may serve as a model system for characterizing the mechanisms underlying related phenomena in humans and sheep in which susceptibility to
diabetes and muscle development respectively are controlled by trans allele interactions. We have begun to ask if dietary perturbations applied to this system can produce multigenerational epigenetic alterations.
Impacts
This work will reveal mechanisms by which normal and aberrant DNA methylation patterns are established and maintained in the genome. It may identify therapeutic targets for manipulating these epigenetic events to control gene expression.
Publications
- Herman, H., Lu, M., Anggraini, M., Sikora, A., Chang, Y., Yoon, B.-J. and Soloway, P.D. 2003. Trans-Allele Methylation and Paramutation-like Effects In Mice. Nature Genetics 34: 199-202.
|
|