LINE-related component of mouse heterochromatin and complex chromocenters’ composition
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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 ∼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 ∼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.
KeywordsMouse genome Heterochromatin Tandem repeat LINE Bioinformatics analysis Fluorescent in situ hybridization (FISH)
Chromocenters isolated by the biochemical approach
PCR with DOP primer described in the Material and methods section
Fluorescent in situ hybridization
Golden Path Gap, 3 Mb empty region around each centromere in assembled genome
Microdissected centromeric DNA
Mouse embryo fibroblast from C3H line
- MiSat and MaSat
Centromeric minor and pericentromeric major satellites
- MS3 and MS4
Mouse satellite 3 and 4, respectively
Long interspersed nuclear element
Short interspersed nuclear element
The authors are entirely grateful to the anonymous reviewers for the very professional and helpful comments. This work was supported by the Russian Foundation for Basic Research (grant nos. 05-04-49156-а, 11-04-01700), the Russian Science Foundation (grant no.15-15-20026), Saint-Petersburg State University (grant no. 22.214.171.1244) and the granting program of “Molecular and Cell Biology” of the Presidium of Russian Academy of Sciences (no. 01.2.014571). We would like to thank Prof. Eugene D. Ponomarev (The Chinese University of Hong Kong) for the help with English corrections. Editing and publishing costs have been paid for by a grant from the Russian Science Foundation (grant no.15-15-20026).
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
Mice were housed and maintained according to the approved standards in the Laboratory Animal Resources facility at Institute of Cytology RAS (St Petersburg, Russia).
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