Epigenetic remodeling and deregulation of SAPAP4/DLGAP4 is linked with early-onset cerebellar ataxia. Homo sapiens

Epigenetic effects at the breakpoints of disease-associated balanced chromosomal rearrangements are not fully understood. To elucidate and characterize epigenetic effects of such a rearrangement, we studied the familial translocation t(8:20)(p12;q11.23) inherited across five generations together with early-onset non-progressive cerebellar ataxia. This translocation disrupts DLGAP4 and a DLGAP4 CpG island at 20q11.23, a locus encoding a brain-specific DLGAP4 mRNA (isoform a) and a gene-desert at 8p12. We find describe translocation-dependent, monoallelic epigenetic changes at DLGAP4 affecting its expression in cis. We found hypermethylation of the disrupted DLGAP4 CpG island and a delay in replication timing of this locus in multiple carriers. Hypermethylation of CpGs at the disrupted DLGAP4 CpG island is re-established in the somatic cells of each translocation carrier after being erased in their germ cells. Brain-specificity of DLGAP4 mRNA isoform a is maintained in carriers. However, this rearrangement leads to increased mono-allelic expression of another DLGAP4 mRNA isoform b from the truncated DLGAP4 locus. Furthermore, there is a translocation dependent enrichment of the conserved histone variant H2A.Z at the undisrupted DLGAP4 CpG island. Functional analyses reveal a novel intronic non-coding RNA, transcribed from the DLGAP4 promoter region and the DLGAP4 mRNA isoform a, both of which are and disrupted by the translocation, to be and highly expreed in the human brain. Using in situ hybridization we found this ncRNA to be bi-directionally expressed in human and mouse cerebellum, relative to elevated expression of DLGAP4 mRNA isoform a only in Purkinje neurons. These data provide a basis for the understanding of the roles epigenetic regulatory mechanisms transmitted through germ cells have in controlling expression of disrupted gene loci highly expressed in the human brain. Overall design: Examination of hypermethylation (MBD-Seq using a his-MBD2B antibody) and hypomethylation (MRE-Seq using HpaII restriction enzyme digestion) in translocation t(8;20)(p12;q11.23) carriers and normal controls. ChIP-Seq using H2A.Z, H3K4me3 and H3K27me3 antibodies in EBV-transformed peripheral blood lymphocytes obtained from translocation carriers and controls.

与疾病相关的平衡性染色体重排断点处的表观遗传(epigenetic)效应尚未完全阐明。为阐明并表征此类重排的表观遗传效应，我们对跨五代遗传的家族性t(8;20)(p12;q11.23)染色体易位(translocation)以及早发性非进展性小脑共济失调进行了研究。该易位会破坏20q11.23区域的DLGAP4基因及其CpG岛(CpG island)，该区域编码脑特异性DLGAP4 mRNA（亚型a），同时8p12区域为基因荒漠区。我们发现并描述了易位依赖性的单等位基因表观遗传改变，该改变发生于DLGAP4基因处，并顺式影响其表达。我们在多名易位携带者中发现，被破坏的DLGAP4 CpG岛存在高甲基化，且该位点的复制时序出现延迟。被破坏的DLGAP4 CpG岛的CpG高甲基化，在生殖细胞中被清除后，会在每名易位携带者的体细胞中重新建立。携带者体内DLGAP4 mRNA亚型a的脑特异性表达得以维持。然而，该染色体重排会导致截短的DLGAP4位点来源的另一型DLGAP4 mRNA（亚型b）的单等位基因表达水平升高。此外，未被破坏的DLGAP4 CpG岛处，存在易位依赖性的保守组蛋白变体H2A.Z富集。功能分析显示，一段由DLGAP4启动子区域及DLGAP4 mRNA亚型a转录而来的新型内含子非编码RNA（ncRNA），虽被该易位破坏，但在人脑内呈高表达。通过原位杂交(in situ hybridization)实验，我们发现该ncRNA在人和小鼠小脑内呈双向表达，而DLGAP4 mRNA亚型a仅在浦肯野神经元(Purkinje neurons)中表达升高。本研究数据为理解通过生殖细胞传递的表观遗传调控机制，如何调控人脑内高表达的受损基因位点的表达，提供了理论基础。
实验总体设计：对t(8;20)(p12;q11.23)易位携带者与正常对照者的超甲基化（采用带his标签的MBD2B抗体的甲基结合域测序(MBD-Seq)）与低甲基化（采用HpaII限制性酶消化的甲基化敏感限制性内切酶测序(MRE-Seq)）水平进行检测；采用H2A.Z、H3K4me3及H3K27me3抗体，对易位携带者与对照者的爱泼斯坦-巴尔病毒(Epstein-Barr virus, EBV)转化的外周血淋巴细胞进行染色质免疫共沉淀测序(ChIP-Seq)。