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First published online 12 January 2005
doi: 10.1242/dev.01612

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Lsh controls silencing of the imprinted Cdkn1c gene

¹ Laboratory of Molecular Immunoregulation, Basic Research Program, SAIC-Frederick, National Cancer Institute, Frederick, MD 21702-1201, USA
² Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, MD 21702-1201, USA

View larger version (61K): [in a new window]   Fig. 1. Detection of lacZ gene expression in mouse embryos. (A) Summary of the staining for ß-galactosidase activity in embryos with different genotypes. (B) Representative staining for ß-galactosidase activity in day 13.5 embryos. Neither Lsh–/– nor Zac1 mutants show any overt morphological abnormalities (Geiman et al., 2001) (C.L.S. and S.V.K., unpublished). lacZ gene expression was widely distributed in Lsh+/+ and Lsh–/– embryos when the lacZ gene was inherited from the paternal side. No expression was observed when the lacZ gene was propagated from the maternal side.

View larger version (37K): [in a new window]   Fig. 2. Genomic DNA polymorphism analysis. (A) Schematic representation indicating the distinct imprinted genes examined. The filled boxes represent exons. The open boxes represent the position of differentially methylated regions as examined in this study. The lines under the graph indicate the position of the primers used for genomic DNA polymorphism analysis. The arrows indicate the position of the genomic polymorphisms. (B) Genomic DNA from F2 hybrids was amplified at specific regions using primers presented in Table 1B. PCR products were digested with BglI for H19, DdeI for Igf2, BglII for Igf2r, NheI for Meg9, and TfiI for Cdkn1c. Parent-of-origin alleles are distinguished by the size of the DNA fragments generated by digestion and visualized by ethidium bromide stain after agarose gel electrophoresis. Mice that were homozygous for 129 alleles and homozygous for Czech alleles served as controls. Only embryo #2 but not embryo #1 showed appropriate polymorphisms at the maternally expressed H19, Igf2r, Meg9, and Cdkn1c genes and at the paternally expressed Igf2 gene.

View larger version (68K): [in a new window]   Fig. 3. Effect of Lsh on expression of different imprinted genes. RT-PCR analysis for the indicated genes was performed using total RNA derived from F2 hybrid offspring. PCR products were subjected to RFLP analysis by using restriction enzymes listed in Table 1. Omission of reverse transcriptase and uncut PCR products served as controls.

View larger version (37K): [in a new window]   Fig. 4. Methylation analysis of DMRs at different imprinted loci in the absence of Lsh. The methylation status of individual DNA alleles derived from Lsh+/+ and Lsh–/– embryos are represented in the graph. The gene fragments were amplified for H19, Igf2r, Zac1 DMRs from sodium bisulphite-modified DNA and subjected to sequence analysis. (Top) Open boxes correspond to DMRs of each gene. The arrow indicates the transcription start site. Each row corresponds to an individual sequenced strand of DNA, and each circle represents a CpG on the strand, red circles and blue circles indicate methylated and unmethylated sites, respectively. Maternal (M) and paternal (P) alleles were distinguished by a DNA polymorphism in the DMR sequence (129/Czech: Igf2,1654A/G, U19619; Igf2r, 851C/G, L06446). *Parent-of-origin determined by the methylation pattern.

View larger version (54K): [in a new window]   Fig. 5. Methylation analysis at three DMRs of the Cdkn1c locus in the absence of Lsh. The methylation status was determined as outlined in Fig. 4. The arrow indicates the transcription start site. Each row corresponds to an individual sequenced strand of DNA, and each circle represents a CpG on the strand, red circles and blue circles indicate methylated and unmethylated sites, respectively. Maternal (M) and paternal (P) alleles were distinguished by a DNA polymorphism in the DMR sequences (129/Czech: 5' DMR Cdkn1c (ICG5) 34012 T/G; KvDMR1: ICG8a 101803 A/G and ICG8b 103181 G/C).

View larger version (103K): [in a new window]   Fig. 6. Binding of Lsh to the DMR of Cdkn1c. Chromatin immunoprecipitation assays were performed in 3T3 cells that had stably integrated the Flag-Lsh expression plamids. Specific antibodies directed against the Flag-tagged form of Lsh and murine control antibodies of the same isotype were used. Aliquots of chromatin taken before immunoprecipitation were used as `Input' controls. The immunoprecipitates obtained were PCR amplified using the primers specific to the DMRs of indicated genes.