RESUMEN
BACKGROUND: The H19/Igf2 imprinting control region (ICR) functions as an insulator exclusively in the unmethylated maternal allele, where enhancer-blocking by CTCF protein prevents the interaction between the Igf2 promoter and the distant enhancers. DNA methylation inhibits CTCF binding in the paternal ICR allele. Two copies of the chicken ß-globin insulator (ChßGI)(2) are capable of substituting for the enhancer blocking function of the ICR. Insulation, however, now also occurs upon paternal inheritance, because unlike the H19 ICR, the (ChßGI)(2) does not become methylated in fetal male germ cells. The (ChßGI)(2) is a composite insulator, exhibiting enhancer blocking by CTCF and chromatin barrier functions by USF1 and VEZF1. We asked the question whether these barrier proteins protected the (ChßGI)(2) sequences from methylation in the male germ line. METHODOLOGY/PRINCIPAL FINDINGS: We genetically dissected the ChßGI in the mouse by deleting the binding sites USF1 and VEZF1. The methylation of the mutant versus normal (ChßGI)(2) significantly increased from 11% to 32% in perinatal male germ cells, suggesting that the barrier proteins did have a role in protecting the (ChßGI)(2) from methylation in the male germ line. Contrary to the H19 ICR, however, the mutant (mChßGI)(2) lacked the potential to attain full de novo methylation in the germ line and to maintain methylation in the paternal allele in the soma, where it consequently functioned as a biallelic insulator. Unexpectedly, a stricter enhancer blocking was achieved by CTCF alone than by a combination of the CTCF, USF1 and VEZF1 sites, illustrated by undetectable Igf2 expression upon paternal transmission. CONCLUSIONS/SIGNIFICANCE: In this in vivo model, hypomethylation at the ICR position together with fetal growth retardation mimicked the human Silver-Russell syndrome. Importantly, late fetal/perinatal death occurred arguing that strict biallelic insulation at the H19/Igf2 ICR position is not tolerated in development.
Asunto(s)
Muerte Fetal/genética , Retardo del Crecimiento Fetal/genética , Impresión Genómica , Elementos Aisladores , Factor II del Crecimiento Similar a la Insulina/genética , ARN no Traducido/genética , Animales , Secuencia de Bases , Factor de Unión a CCCTC , Pollos , Metilación de ADN , Femenino , Muerte Fetal/metabolismo , Retardo del Crecimiento Fetal/metabolismo , Marcación de Gen , Células Germinativas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Largo no Codificante , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Globinas beta/genéticaRESUMEN
A approximately 2.4-kb imprinting control region (ICR) regulates somatic monoallelic expression of the Igf2 and H19 genes. This is achieved through DNA methylation-dependent chromatin insulator and promoter silencing activities on the maternal and paternal chromosomes, respectively. In somatic cells, the hypomethylated maternally inherited ICR binds the insulator protein CTCF at four sites and blocks activity of the proximal Igf2 promoter by insulating it from its distal enhancers. CTCF binding is thought to play a direct role in inhibiting methylation of the ICR in female germ cells and in somatic cells and, therefore, in establishing and maintaining imprinting of the Igf2/H19 region. Here, we report on the effects of eliminating ICR CTCF binding by severely mutating all four sites in mice. We found that in the female and male germ lines, the mutant ICR remained hypomethylated and hypermethylated, respectively, showing that the CTCF binding sites are dispensable for imprinting establishment. Postfertilization, the maternal mutant ICR acquired methylation, which could be explained by loss of methylation inhibition, which is normally provided by CTCF binding. Adjacent regions in cis-the H19 promoter and gene-also acquired methylation, accompanied by downregulation of H19. This could be the result of a silencing effect of the methylated maternal ICR.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/genética , ARN no Traducido/biosíntesis , ARN no Traducido/metabolismo , Proteínas Represoras/metabolismo , Animales , Sitios de Unión , Factor de Unión a CCCTC , Metilación de ADN , Regulación de la Expresión Génica/fisiología , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ratones , Mutación , Unión Proteica , ARN Largo no CodificanteRESUMEN
A Cre recombinase expression cassette was inserted into the X-linked Hprt locus by gene targeting in a mouse embryonic stem (ES) cell line isogenic to strain 129S1/SvImJ (129S1), then the transgene was introduced into 129S1 mice through ES cell chimeras. When females hemizygous for this transgene were mated to males carrying a neomycin selection cassette flanked by loxP sites, the cassette was always excised regardless of Cre inheritance and without detectable mosaicism. The usefulness of this "Cre-deleter" transgenic line is in its efficiency and defined genetic status in terms of mouse strain and location of the transgene.
Asunto(s)
Integrasas/genética , Ratones Transgénicos , Proteínas Virales/genética , Animales , Femenino , Hipoxantina Fosforribosiltransferasa/genética , Masculino , Ratones , Ratones EndogámicosRESUMEN
Imprinting of the mouse insulin-like growth factor 2 (Igf2) and H19 genes is regulated by an imprinting control region (ICR). The hypomethylated maternal copy functions as a chromatin insulator through the binding of CTCF and prevents Igf2 activation in cis, while hypermethylation of the paternal copy inactivates insulator function and leads to inactivation of H19 in cis. The specificity of the ICR sequence for mediating imprinting and chromatin insulation was investigated by substituting it for two copies of the chicken beta-globin insulator element, (Ch beta GI)(2), in mice. This introduced sequence resembles the ICR in size, and in containing CTCF-binding sites and CpGs, but otherwise lacks homology. On maternal inheritance, the (Ch beta GI)(2) was hypomethylated and displayed full chromatin insulator activity. Monoallelic expression of Igf2 and H19 was retained and mice were of normal size. These results suggest that the ICR sequence, aside from CTCF-binding sites, is not uniquely specialized for chromatin insulation at the Igf2/H19 region. On paternal inheritance, the (Ch beta GI)(2) was also hypomethylated and displayed strong insulator activity--fetuses possessed very low levels of Igf2 RNA and were greatly reduced in size, being as small as Igf2-null mutants. Furthermore, the paternal H19 allele was active. These results suggest that differential ICR methylation in the female and male germ lines is not acquired through differential binding of CTCF. Rather, it is likely to be acquired through a separate or downstream process.
Asunto(s)
Cromatina/fisiología , Impresión Genómica , Globinas/genética , Factor II del Crecimiento Similar a la Insulina/genética , ARN no Traducido/genética , Animales , Pollos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Estructura Terciaria de Proteína , ARN Largo no CodificanteRESUMEN
Ten test materials derived from petroleum or hydrotreated shale oils were applied 3 times/week for up to 105 weeks to the shaved skin of 25 male and 25 female C3H/HeN mice per group. Mineral oil and benzo(a) pyrene (0.15%) were control materials. Clinical observations were recorded during the study. At death, histopathologic examination was conducted on skin, internal organs and any gross lesions. Exposures to some materials were ended midway in the study due to severe irritation. Chronic toxicity of all materials was limited to inflammatory and degenerative skin changes. Significant increases over control incidence of skin tumors (squamous cell carcinoma and fibrosarcoma) occurred with both petroleum and shale-derived naphtha (21%, 50%), Jet A (26%, 28%), JP-4 (26%, 50%), and crude oils (84%, 54%). Severely hydrotreated shale oil and petroleum and shale-derived diesel distillates were not considered tumorigenic. Results indicate that toxicity of comparable petroleum and shale-derived fractions was qualitatively similar and confirm earlier findings that hydrotreating reduces or eliminates carcinogenicity of raw shale oil.