LINE-related component of mouse heterochromatin and complex chromocenters' composition
LINE-related component of mouse heterochromatin and complex chromocenters' composition
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DOI：
10.1007/s10577-016-9525-9
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发表时间：
2016-09-01
影响因子：
2.6
通讯作者：
Podgornaya, Olga I.
中科院分区：
文献类型：
--
作者：
Kuznetsova, Inna S.;Ostromyshenskii, Dmitrii I.;Podgornaya, Olga I.
染色质中心是异染色质组成的间期核标志性结构。不同染色体的串联重复序列（TR）富集部分在染色中心聚集在一起。近年来，在确定染色中心的蛋白质含量方面取得了进展，尽管尚不清楚除了TR之外，哪些DNA序列是构成异染色质的基础。目前的工作的目的是找出除了TRs的DNA序列参与了色中心的形成。用DOP-PCR扩增分离的染色中心和显微切割的着丝粒区域，然后克隆和测序。与Repbase比对，小鼠参考基因组和WGS数据库将来自两个文库的序列分成三组：（1）与着丝粒周围小鼠主要卫星相似的序列;（2）与任何重复序列不相似的序列;（3）与长散布核元件（LINE）相似的序列。LINE相关序列在计算机预测的染色体上具有分散模式分布。选择的克隆用于荧光原位杂交（FISH）。10个供试克隆与中期染色体的染色中心和着丝粒区杂交。将这些克隆与四个已知克隆的TR（satDNA，卫星DNA）和性染色体特异性探针进行双重FISH。探针结合不同的色中心区域而不重叠;因此，FISH结果揭示了复杂的色中心组成。我们将18个LINE衍生的克隆映射到RepBase L1记录。它们大多集中在第二个ORF末端的一个类似2-kb的区域和3'非翻译区（UTR）。因此，即使克隆数量有限，我们也可以确定L1元件的异染色质区域特异性。虽然L1全长探针没有杂交在任何染色体上的异染色质区在可检测的水平，发现的2-kb片段肯定是这些地区的一部分。与2kb片段相似的精确LINE是富含TR的小鼠和人组成型异染色质的组分。用于扩增来自异染色质材料的两个来源的探针的方法揭示了LINE的精确片段在染色中心内的富集。
Chromocenters are interphase nuclear landmark structures of constitutive heterochromatin. The tandem repeat (TR)-enriched parts of different chromosomes cluster together in chromocenters. There has been progress in recent years in determining the protein content of chromocenters, although it is not clear which DNA sequences underly constitutive heterochromatin apart from the TRs. The aim of the current work was to find out which DNA sequences besides TRs are involved in chromocenters' formation. Biochemically isolated chromocenters and microdissected centromeric regions were amplified by DOP-PCR, then cloned and sequenced. Alignment to Repbase, the mouse reference genome and WGS databases separated the sequences from both libraries into three groups: (1) sequences with similarity to pericentromere mouse major satellite; (2) sequences without similarity to any repetitive sequences; (3) sequences with similarity to long interspersed nuclear elements (LINEs). LINE-related sequences have a disperse pattern distribution on chromosomes predicted in silico. Selected clones were used for fluorescent in situ hybridization (FISH). The 10 clones tested hybridized to chromocenters and centromeric regions of metaphase chromosomes. These clones were used for double FISH with four known cloned TRs (satDNA, satellite DNA) and a probe specific for the sex chromosomes. The probes bind various chromocenters' regions without overlapping; so, FISH results reveal a complex chromocenter composition. We mapped 18 LINE-derived clones to the RepBase L1 records. Most of them grouped in a similar to 2-kb region at the end of the second ORF and 3' untranslated region (UTR). So, even the limited number of the clones allows us to determine the region of the L1 element that is specific for heterochromatic regions. Although the L1 full-length probe did not hybridize at detectable levels to the heterochromatic region on any chromosome, the 2-kb fragment found is definitely a part of these regions. The precise LINE similar to 2-kb fragment is the component of mouse and human constitutive heterochromatin enriched with TRs. The method used for amplification of the probes from two sources of the heterochromatic material uncovered the enrichment of a precise fragment of LINE within chromocenters.