XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells.
XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells.
复制标题
DOI：
10.1186/1756-8935-6-23
复制
发表时间：
2013-08-01
影响因子：
3.9
通讯作者：
Brown CJ
中科院分区：
文献类型：
--
作者：
Minks J;Baldry SE;Yang C;Cotton AM;Brown CJ
通过X染色体失活建立兼性异染色质需要长的非编码RNA XIST/Xist。然而，RNA实现染色体范围的基因沉默的分子机制仍不清楚。小鼠Xist被证明具有冗余的结构域用于顺式定位，并需要一系列保守的串联‘A’重复序列来沉默。我们以前描述了一种人类可诱导的XIST转基因，它能够在体细胞中顺式定位并抑制下游报告基因，现在我们利用这些细胞来剖析对人类XIST依赖的基因沉默至关重要的序列。我们证明了可诱导的全长XIST基因的表达能够抑制邻近的两个报告基因以及整合位点上长达3MB的内源基因的表达。仅包含XIST重复A区的诱导性构建物就可以沉默侧翼报告基因，但不能沉默更远端的内源基因。报告基因沉默也可以通过由9个共识重复A序列组成的合成结构来完成，这与之前在小鼠身上的研究一致。逐渐变短的结构显示重复数和RNA的沉默能力之间存在线性关系。只包含两个Repeat A单位的构建体仍然能够部分沉默报告基因，因此可以用于定点突变，以证明重复序列的两个回文核心内的序列对于沉默是必不可少的，并且它很可能第一个回文序列折叠形成发夹，与在真核序列中观察到的补偿突变一致。邻近报告基因的沉默只需94个碱基的XIST就可以实现，包括A重复序列的两个“单体”。这一区域包括一对进化上保守的基本回文序列，其中第一个很可能形成重复内发夹结构。为了将沉默扩散到染色体上的内源基因，需要额外的序列。
The establishment of facultative heterochromatin by X-chromosome inactivation requires the long non-coding RNA XIST/Xist. However, the molecular mechanism by which the RNA achieves chromosome-wide gene silencing remains unknown. Mouse Xist has been shown to have redundant domains for cis-localization, and requires a series of well-conserved tandem ‘A’ repeats for silencing. We previously described a human inducible XIST transgene that is capable of cis-localization and suppressing a downstream reporter gene in somatic cells, and have now leveraged these cells to dissect the sequences critical for XIST-dependent gene silencing in humans. We demonstrated that expression of the inducible full-length XIST cDNA was able to suppress expression of two nearby reporter genes as well as endogenous genes up to 3 MB from the integration site. An inducible construct containing the repeat A region of XIST alone could silence the flanking reporter genes but not the more distal endogenous genes. Reporter gene silencing could also be accomplished by a synthetic construct consisting of nine copies of a consensus repeat A sequence, consistent with previous studies in mice. Progressively shorter constructs showed a linear relationship between the repeat number and the silencing capacity of the RNA. Constructs containing only two repeat A units were still able to partially silence the reporter genes and could thus be used for site-directed mutagenesis to demonstrate that sequences within the two palindromic cores of the repeat are essential for silencing, and that it is likely the first palindrome sequence folds to form a hairpin, consistent with compensatory mutations observed in eutherian sequences. Silencing of adjacent reporter genes can be effected by as little as 94 bp of XIST, including two ‘monomers’ of the A repeat. This region includes a pair of essential palindromic sequences that are evolutionarily well-conserved and the first of these is likely to form an intra-repeat hairpin structure. Additional sequences are required for the spread of silencing to endogenous genes on the chromosome.
登录
查看更多内容
影响因子：
16
作者：
Kanhere A;Viiri K;Araújo CC;Rasaiyaah J;Bouwman RD;Whyte WA;Pereira CF;Brookes E;Walker K;Bell GW;Pombo A;Fisher AG;Young RA;Jenner RG
通讯作者：
Jenner RG
影响因子：
5.2
作者：
Hoffman, LM;Hall, L;Carpenter, MK
通讯作者：
Carpenter, MK
影响因子：
56.9
作者：
Duret, L;Chureau, C;Avner, P
通讯作者：
Avner, P
影响因子：
16.8
作者：
通讯作者：
--
影响因子：
14.9
作者：
Duszczyk MM;Zanier K;Sattler M
通讯作者：
Sattler M