A subset of genes in mammals is regulated by genomic imprinting, a process that results in unequal expression of the maternal and paternal alleles of this class of genes. The existence of imprinted genes is hypothesized to explain why nuclear contributions from both parents are required for normal mammalian development. Furthermore, imprinting plays a role in the transmission of a number of human disorders, including Beckwith-Wiedemann Syndrome (BWS), Silver-Russell Syndrome (SRS), Prader-Willi Syndrome and Angelman Syndrome, in that the sex of the parent that transmits the affected gene(s) determines whether offspring will be affected. Aberrant imprinted gene expression is also involved in the onset or progression of cancers, such as Wilms tumors. The overall goal of our work is to elucidate the mechanism by which parental identity of imprinted genes is established and maintained. The studies will employ the conserved H19 and Igf2 locus. The imprinting of H19, which produces a non-coding RNA from the maternally-derived allele, and the linked and oppositely imprinted growth-promoting Igf2 gene is mediated through the 2 kb imprinting control region (ICR) and shared enhancers. The ICR, which is also designated the differentially methylated domain (DMD), acts as a methylation-senstitive, CTCF-dependent insulator. When unmethylated on the maternal allele, the insulator allows H19 exclusive access to shared enhancers. In contrast, a methylated paternal insulator enables Igf2 to engage the enhancers. This proposal will investigate the mechanism of H19/Igf2 imprinting through the following experiments. Individuals with BWS, sporadic Wilms tumors and SRS have been identified that have microdeletions and epimutations (hypermethylation) in the human ICR and aberrant imprinted regulation of H19 and Igf2.
Specific Aim 1 will generate mouse mutants and human iPS cells to elucidate the mechanisms governing the loss of imprinted gene regulation in these individuals. DNA hypermethylation of the paternal ICR allele is associated with repression of the H19 gene, but the precise sequences and mechanism mediating repression remain incompletely defined. We have shown that mutation of CpGs within the CTCF binding sites on the paternal allele causes demethylation and ectopic insulator function, whereas a similar number of CpG mutations outside of the CTCF binding sites but within the ICR has minimal effects. We propose that the CpG dinucleotides within the CTCF binding sites are critical for paternal- specific repression.
In Specific Aim 2, we will test this hypothesis by mutating all CpG outside of the CTCF binding sites but within the ICR and assessing function in vitro by repression assays and in vivo through gene targeting experiments. Finally, a novel highly expressed imprinted non-coding RNA has been identified between the H19 and Igf2 genes.
Specific Aim 3 will assess the role of this RNA by in vitro assays and in vivo gene targeting experiments. Together these experiments will allow a greater understanding of imprinted gene regulation at this important locus as well as serving for a model of epigenetic gene regulation in mammals. .

Public Health Relevance

Imprinted genes are critical for normal mammalian development, behavior and energy homeostasis. Genetic and epigenetic mutations have been identified in imprinted genes for a number of human syndromes and cancer. The experiments in this proposal will model such newly identified mutations in individuals with Beckwith-Wiedemann Syndrome and Silver-Russell Syndrome, providing a better understanding of the etiology of the disorders.

Agency
National Institute of Health (NIH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM051279-21
Application #
8706159
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Plasschaert, Robert N; Bartolomei, Marisa S (2014) Genomic imprinting in development, growth, behavior and stem cells. Development 141:1805-13
Weaver, Jamie R; Bartolomei, Marisa S (2014) Chromatin regulators of genomic imprinting. Biochim Biophys Acta 1839:169-77
Ideraabdullah, Folami Y; Thorvaldsen, Joanne L; Myers, Jennifer A et al. (2014) Tissue-specific insulator function at H19/Igf2 revealed by deletions at the imprinting control region. Hum Mol Genet 23:6246-59
Venkatraman, Aparna; He, Xi C; Thorvaldsen, Joanne L et al. (2013) Maternal imprinting at the H19-Igf2 locus maintains adult haematopoietic stem cell quiescence. Nature 500:345-9
Kelsey, Gavin; Bartolomei, Marisa S (2012) Imprinted genes ýýý and the number is? PLoS Genet 8:e1002601
Abramowitz, Lara K; Bartolomei, Marisa S (2012) Genomic imprinting: recognition and marking of imprinted loci. Curr Opin Genet Dev 22:72-8
Wu, Xin; Goodyear, Shaun M; Abramowitz, Lara K et al. (2012) Fertile offspring derived from mouse spermatogonial stem cells cryopreserved for more than 14 years. Hum Reprod 27:1249-59
Schmidt, Jonathan A; Abramowitz, Lara K; Kubota, Hiroshi et al. (2011) In vivo and in vitro aging is detrimental to mouse spermatogonial stem cell function. Biol Reprod 84:698-706
Bartolomei, Marisa S; Ferguson-Smith, Anne C (2011) Mammalian genomic imprinting. Cold Spring Harb Perspect Biol 3:
Choufani, Sanaa; Shapiro, Jonathan S; Susiarjo, Martha et al. (2011) A novel approach identifies new differentially methylated regions (DMRs) associated with imprinted genes. Genome Res 21:465-76