LINE-related component of mouse heterochromatin and complex chromocenters’ composition

Abstract

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.

Mouse chromocenters DNA content: sequencing and in silico analysis

“…Obviously, heterochromatic probes specific for both sex chromosomes or only for the Y chromosome could be designed on the base of the newly found fragments. These kind of probes will be very useful for revealing heterochromatic regions of murine sex chromosomes, which are not labeled by any commercially available chromosome-specific probes (routinely used in molecular cytogenetic studies) [ 35 ] and, consequently, the probes will provide a possibility to determine sex chromosome associations.…”

“…Experimental validation of these findings by FISH was performed by cloning, sequencing and mapping of DOP amplified ChrmC DNA [ 35 ]. The clones we selected for FISH, cover the whole ~ 2 kb fragment of L1 ORF2 and Lx.…”

“…The CEN assembly of human CENs is available in databases (LinearCen 1.1, http://www.ncbi.nlm.nih.gov/assembly/GCA_000442335.2 ). We mapped all the LINE fragments found in two human CENs to the RepBase LINE consensus and the concentration of fragments in the same region at the end of the second ORF is clearly visible [ 35 ]. We conclude that the precise LINE ~ 2-kb fragment, but not the full-length LINE, is the component of mouse and human constitutive heterochromatin enriched with TRs.…”

“…chromocenters, are quite complicated structures far of being uniform. Not only TR of different families but precise LINE fragments are involved in their formation [ 35 ].…”

Function of Junk: Pericentromeric Satellite DNA in Chromosome Maintenance

“…First, in certain cases, eukaryotic chromosomes appear to be completely lacking satellite DNA (e.g., horse chromosome 11, orangutan chromosome 12) (Wade et al 2009;Piras et al 2010;Locke et al 2011) raising the question of if/how these chromosomes may be incorporated into chromocenters. Interestingly, a recent report has shown that transposable element (TE)-derived tandem repeats are a component of mouse chromocenters (Kuznetsova et al 2016). TE-derived sequences are abundant, heterochromatinized and typically interspersed throughout eukaryotic chromosomes (Saksouk et al 2015;Nishibuchi and Déjardin 2017), including pericentromeric heterochromatin, where they are present in complex islands within large satellite DNA tracts (Sun et al 1997(Sun et al , 2003.…”