LINErelated component of mouse heterochromatin an

Authors: Inna S Kuznetsova Dmitrii I Ostromyshenskii Alexei S Komissarov Andrei N Prusov Irina S Waisertreiger Anna V Gorbunova Vladimir A Trifonov Malcolm A FergusonSmith Olga I Podgornaya

Publish Date: 2016/04/26

Volume: 24, Issue: 3, Pages: 309-323

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Abstract

Chromocenters are interphase nuclear landmark structures of constitutive heterochromatin The tandem repeat TRenriched 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 DOPPCR 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 LINErelated 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 LINEderived clones to the RepBase L1 records Most of them grouped in a ∼2kb 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 fulllength probe did not hybridize at detectable levels to the heterochromatic region on any chromosome the 2kb fragment found is definitely a part of these regions The precise LINE ∼2kb 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

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