Silencing of transposons in mammalian germ cells

描述（由申请人提供）：异位基因沉默可能导致疾病。人类癌细胞经常包含m5C净损失的基因组，并且有许多肿瘤抑制基因序列正常但不表达的报道；这些基因有甲基化的启动子。基因组甲基化模式异常与癌症之间的许多关联已被报道。在许多常见肿瘤中，导致全局去甲基化和局灶性高甲基化的改变的性质是完全未知的，但必须涉及调节因子的获得或损失；没有DNA甲基转移酶被证明在癌症中发生突变或沉默。甲基化和异位基因沉默也被认为在基因型无关的表型多样性中起作用，如在同卵双胞胎中的不一致。这表明在配子体发生或发育早期靶向基因沉默的错误，同样是未知的调节因素。鉴定Dnmt3L是生殖细胞基因组甲基化模式建立的一个重要因素，这为分离调节新生甲基化的因素和定义将甲基化引导到特定序列的线索提供了第一个机会。我们将使用新的最小干扰相互作用筛选和最先进的蛋白质质谱鉴定来识别与Dnmt3L复合的因素，我们将应用新的遗传方法进行Ecat11的功能分析，Ecat11是一个在初步研究中发现与Dnmt3L相互作用的特别有趣的因素。肿瘤中控制从头甲基化的因子的异位沉默或激活可能是癌症基因组中常见的甲基化模式中断的原因。在哺乳动物（和人类）表观遗传学领域，确定调节新生甲基化的因素是一个迫在眉睫的重要问题。哺乳动物基因组的功能取决于配子体发生和早期发育过程中基因组甲基化模式的准确建立和维持。异常甲基化模式在许多恶性肿瘤中很常见；生殖细胞或胚胎甲基化调节因子的重新表达可能是肿瘤特异性甲基化异常的原因。生殖细胞中甲基化印记建立错误可导致Angelman综合征、Prader-Willi综合征或beckwithi - wiedeman综合征，而印记因子基因突变可导致复发性家族性双亲本包体痣，其中父本基因组印记被施加到母体基因组（Judson et al., 2002; Bestor and Bourc'his, 2006）。目前认为，基因组甲基化模式的建立或维持中的错误导致了基因型无关的表型变异，特别是在精神疾病中，同卵双胞胎的不一致性率通常超过40% （Meissner, 1965）。基因组甲基化模式的生物学重要性直到最近才变得清楚，但目前对它们建立的机制几乎一无所知。这里描述的研究几乎可以肯定地确定调节雄性生殖系从头甲基化的因素。

DESCRIPTION (provided by applicant): Ectopic gene silencing can contribute to disease. Human cancer cells frequently contain genomes that have undergone a net loss of m5C and there are many reports of tumor suppressor genes that are normal in sequence but not expressed; such genes have methylated promoters. Many associations between abnormalities of genomic methylation patterns and cancer have been reported The nature of the alterations that lead to global demethylation with focal hypermethylation in many common tumors is completely unknown, but must involve the gain or loss of regulatory factors; none of the DNA methyltransferases has been shown to be mutated or silenced in cancer. Methylation and ectopic gene silencing have also been attributed a role in genotype- independent phenotypic diversity, as in discordance in monozygotic twins. This suggests errors in targeting of gene silencing during gametogenesis or early in development, again by unknown regulatory factors. The identification of Dnmt3L as an essential factor in the establishment of genomic methylation patterns in germ cells provides the first opportunity to isolate factors that regulate de novo methylation and to define the cues that direct methylation to specific sequences. We will use a new and minimally perturbed interaction screen and state-of-the-art protein identification by mass spectrometry to identify factors that complex with Dnmt3L, and we will apply new genetic methods in the functional analysis of Ecat11, an especially interesting factor found to interact with Dnmt3L in preliminary studies. Ectopic silencing or activation in tumors of factors that control de novo methylation is a likely cause of the disrupted methylation patterns often seen in cancer genomes. The identification of factors that regulate de novo methylation is an issue of immediate importance in the field of mammalian (and human) epigenetics. The function of the mammalian genome depends on the accurate establishment and maintenance of genomic methylation patterns during gametogenesis and early development. Abnormal methylation patterns are common in many malignant tumors; it is possible that re-expression of germ-cell or embryonic methylation regulators is responsible for tumor-specific methylation abnormalities. Errors in the establishment of methylation imprints in germ cells can cause Angelman syndrome, Prader-Willi syndrome, or Beckwith-Wiedeman syndrome, and mutations in genes for imprinting factors cause recurrent familial biparental hydatidiform moles, in which paternal genomic imprints are imposed on the maternal genome (Judson et al., 2002; Bestor and Bourc'his, 2006). Errors in the establishment or maintenance of genomic methylation patterns are currently believed to contribute to genotype-independent phenotypic variation, especially in psychiatric illness where rates of discordance in monozygotic twins often exceeds 40% (Meissner, 1965). The biological importance of genomic methylation patterns has only recently become clear, but at this time almost nothing is known of the mechanisms by which they are established. The studies described here are almost certain to identify factors that regulate de novo methylation in the male germ line.