Most human cancers show evidence of genome-wide epigenetic lesions. One common epigenetic alteration is loss of imprinting (LOI) which is defined as either biallelic expression or silencing of normally monoallelically expressed genes. Recently, several studies have provided compelling evidence that LOI has a causal role in cellular transformation. A large proportion of patients with Beckwith-Wiedemann syndrome (BWS), a cancer predisposition condition, exhibit loss of methylation (LOM) at the differentially methylated region in human chromosome 11p15.5 known as KvDMRI. This epimutation, which is also observed in several adult cancers, is associated with LOI (i.e. silencing) of the potential tumor suppressor gene CDKN1C. During the last grant period, we have shown by targeted mutation of the orthologous region in the mouse, distal chromosome 7, that KvDMRI is an imprinting control region (ICR) with its deletion resulting in biallelic expression of a least 8 paternally repressed genes. Genetic analysis has shown that the placental and embryonic growth deficiency exhibited in this mutant can be attributed to the overexpression of different genes. We have shown that this locus acts as an enhancer-blocker in cell culture, that the insulator- associated protein CTCF binds to KvDMRI in vivo in an allele-specific manner, and that mutation of the CTCF binding sites significantly abrogates enhancer-blocking activity. In addition, we have identified both promoter and enhancer-like activities at this locus which are physically separable from the insulator properties. The current proposal consists of 4 specific aims intended to further our understanding of the mechanism(s) of KvDMRI function: (1) The KvDMRI locus will be evaluated by comprehensive mutagenesis and in vivo footprinting to identify additional DNA binding sites for proteins regulating the function of KvDMRI; (2) The proteins binding to these sites will be identified by either a """"""""candidate"""""""" approach based on the sequence of the putative binding site, or by conventional protein purification techniques and mass spectrometry; (3) We propose to assess each of the 3 putative regulatory elements (insulator, promoter, enhancer) identified within KvDMRI for their contribution to gene silencing in vivo by constructing a series of conditionally mutant mice; (4) The potential role of the noncoding RNA transcript Kcnq1ot1 will be tested by its premature termination and truncation. Since other imprinted domains containing growth regulating genes may be controlled in a similar fashion as the KvDMRI subdomain, and it is likely that there are many CTCF mediated chromatin insulators in the mammalian genome, information derived from these studies will have widespread relevance to the epigenetics of cancer. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA089426-05
Application #
7263034
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Okano, Paul
Project Start
2000-12-01
Project End
2008-11-30
Budget Start
2007-08-01
Budget End
2008-11-30
Support Year
5
Fiscal Year
2007
Total Cost
$258,173
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
824771034
City
Buffalo
State
NY
Country
United States
Zip Code
14263
Singh, Vir B; Sribenja, Sirinapa; Wilson, Kayla E et al. (2017) Blocked transcription through KvDMR1 results in absence of methylation and gene silencing resembling Beckwith-Wiedemann syndrome. Development 144:1820-1830
Asahara, Shun-ichiro; Etoh, Hiroaki; Inoue, Hiroyuki et al. (2015) Paternal allelic mutation at the Kcnq1 locus reduces pancreatic ?-cell mass by epigenetic modification of Cdkn1c. Proc Natl Acad Sci U S A 112:8332-7
Krueger, Christel; King, Michelle R; Krueger, Felix et al. (2012) Pairing of homologous regions in the mouse genome is associated with transcription but not imprinting status. PLoS One 7:e38983
Golding, Michael C; Magri, Lauren S; Zhang, Liyue et al. (2011) Depletion of Kcnq1ot1 non-coding RNA does not affect imprinting maintenance in stem cells. Development 138:3667-78
Wood, Michelle D; Hiura, Hitoshi; Tunster, Simon J et al. (2010) Autonomous silencing of the imprinted Cdkn1c gene in stem cells. Epigenetics 5:214-21
Oh-McGinnis, Rosemary; Bogutz, Aaron B; Lee, Kang Yun et al. (2010) Rescue of placental phenotype in a mechanistic model of Beckwith-Wiedemann syndrome. BMC Dev Biol 10:50
Shin, Jong-Yeon; Fitzpatrick, Galina V; Higgins, Michael J (2008) Two distinct mechanisms of silencing by the KvDMR1 imprinting control region. EMBO J 27:168-78
Fitzpatrick, Galina V; Pugacheva, Elena M; Shin, Jong-Yeon et al. (2007) Allele-specific binding of CTCF to the multipartite imprinting control region KvDMR1. Mol Cell Biol 27:2636-47
Diaz-Meyer, N; Yang, Y; Sait, S N et al. (2005) Alternative mechanisms associated with silencing of CDKN1C in Beckwith-Wiedemann syndrome. J Med Genet 42:648-55
Salas, Martha; John, Rosalind; Saxena, Anjana et al. (2004) Placental growth retardation due to loss of imprinting of Phlda2. Mech Dev 121:1199-210