Chromosome Research

, Volume 24, Issue 3, pp 309–323 | Cite as

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

  • Inna S. Kuznetsova
  • Dmitrii I. Ostromyshenskii
  • Alexei S. Komissarov
  • Andrei N. Prusov
  • Irina S. Waisertreiger
  • Anna V. Gorbunova
  • Vladimir A. Trifonov
  • Malcolm A. Ferguson-Smith
  • Olga I. Podgornaya
Article

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.

Keywords

Mouse genome Heterochromatin Tandem repeat LINE Bioinformatics analysis Fluorescent in situ hybridization (FISH) 

Abbreviations

CEN

Centromere

ChrmC

Chromocenters isolated by the biochemical approach

DAPI

4′, 6-diami-dino-2-phenylindole

DOP-PCR

PCR with DOP primer described in the Material and methods section

FISH

Fluorescent in situ hybridization

GPG

Golden Path Gap, 3 Mb empty region around each centromere in assembled genome

MdCP

Microdissected centromeric DNA

MEF

Mouse embryo fibroblast from C3H line

MiSat and MaSat

Centromeric minor and pericentromeric major satellites

MS3 and MS4

Mouse satellite 3 and 4, respectively

LINE

Long interspersed nuclear element

periCEN

Pericentromeric heterochromatin

satDNA

Satellite DNA

SINE

Short interspersed nuclear element

TE

Transposable elements

TR

Tandem repeat

Introduction

A prominent feature of mouse nuclei is the presence of well-defined zones of heterochromatin, termed chromocenters. These are formed by centromeres from different chromosomes that coalesce during interphase to form dense clusters that colocalize with specific heterochromatin protein markers (Guenatri et al. 2004; Papait et al. 2008; Andrey et al. 2010). Genes within chromocenters are generally repressed and characterized by specific epigenetic marks that are responsible for a transcriptionally repressed state of chromatin. In contrast, transcriptionally active domains are located in the nucleoplasm and enriched in epigenetic marks associated with an open chromatin conformation (Probst and Almouzni 2011). Bright 4, 6-diami-dino-2-phenylindole (DAPI) staining is the characteristic feature of chromocenters as well as staining with immunohistochemical markers for centromeric heterochromatin such as heterochromatin protein 1 (Guenatri et al. 2004; Snapp et al. 2013).

There has been progress in recent years in revealing the protein repertoire of chromocenters (Elgin and Reuter 2013; Shatskikh and Gvozdev 2013) although chromocenter-specific DNA content of constitutive heterochromatin is still not fully characterized (Saksouk et al. 2015). At the chromosomal level, constitutive heterochromatin may present on different parts of chromosomes, but mostly in pericentromeric and subtelomeric regions. The periCEN heterochromatin is believed to contribute to protection and strength to the CEN chromatin structures (Yunis and Yasmineh 1971). From the first time when heterochromatin was described (Heitz 1929), it was widely accepted that the centromere, periCEN and subtelomere are the regions of constitutive heterochromatin. However, more recent studies demonstrated that interstitial heterochromatic blocks were also found in different species.

Heterochromatic regions are enriched with tandem repeats originally defined as satellite DNA (satDNA). Although historically relegated as “junk DNA”, tandem repeats (TRs) were considered as important elements after the realization of the fact that tandem organization provides potentially unique functional characteristics. Tandemly repeated DNA is organized as multiple copies of a homologous DNA sequence of a certain size (repeat unit or monomer) that are arranged in a head-to-tail pattern to form tandem arrays. These arrays represent a distinct type of sequence organization shared by most eukaryotes. Centromeres from different species ranging from fission yeast to humans contain TRs. PeriCEN regions enriched in TRs appear to be critically important for establishing heterochromatin formation and proper chromosome segregation (Morris and Moazed 2007).

In house mouse (Mus musculus), there are two well-studied, highly conserved TR sequences known as CEN minor and periCEN major satellites (MiSat and MaSat, respectively, SATMIN and GSAT_MM in RepBase nomenclature). MiSat arrays are formed by an AT-rich, 120-bp monomer that occupies 300–600 kb of the terminal region of all mouse telocentric (single-armed) chromosomes. These regions are the sites of kinetochore formation and spindle microtubule attachment (Wong and Rattner 1988; Kipling et al. 1991). MaSat formed by a 234-bp heterotetrameric unit is the most abundant TR that lies adjacent to MiSat (Broccoli et al. 1990, 1991; Guenatri et al. 2004). MaSat and MiSat are routinely used to mark the corresponding regions periCEN and CEN, respectively.

The vast improvement of genome sequencing technologies still failed to assemble the heterochromatic regions due to their enrichment with TRs. These regions cannot be assembled by current bioinformatics methods and algorithms. The overwhelming majority of genome assemblies contain a 3-Mb (Golden Path Gap, GPG) gap around each CEN.

Here, we performed an attempt to determine the DNA composition of mouse heterochromatic regions. We used two sources of the heterochromatic material: biochemically isolated chromocenters and microdissected CEN regions and uncovered an enrichment of a precise fragment of long interspersed nuclear element (LINE) within chromocenters.

Materials and methods

Cell culture and metaphase chromosomes isolation

C3H mouse embryo fibroblast (MEF) with normal karyotype (less than 10 passages) was cultured in DMEM supplemented with 10 % fetal calf serum and gentamicin under standard conditions (Helgason and Miller 2005). Metaphase chromosomes were isolated from cells blocked in mitosis by 0.5 mg/ml colcemid (Gibco). Chromosome suspensions were fixed in methanol/acetic acid (3:1).

Metaphase preparation from mouse bone marrow

Three- to six-month-old male and female CBA mice were housed and maintained according to the approved standards in the Laboratory Animal Resources facility at Institute of Cytology RAS (St Petersburg, Russia). Metaphase chromosome preparations were made according to a standard cytogenetic protocol (Guo and Wu 2008).

Chromocenters and CEN isolation, cloning and sequencing

Chromocenters were isolated using a biochemical approach with high centrifugal force through gradients of sucrose from nuclei of mouse liver according to published methods (Prusov et al. 2002; Zatsepina et al. 2008). The resulting product yield was about 2 mg/ml of chromocenter DNA. Pericentromeric regions of four different chromosomes were microdissected and DNA amplified as described (Yang et al. 2009). Chromocenters (ChrmC) and microdissected centromeric DNA (MdCP) pools were reamplified with the degenerate oligonucleotide primer (DOP) (5′-CCGACTCGAGNNNNNNATGTGG-3′) under the following PCR conditions: (1) 92 °C, 1 min; (2) 56 °C, 2 min; (3) 72 °C, 2 min; repeat steps 1–3 for 32 more cycles and (4) 72 °C, 5 min (Arneson et al. 2008; Yang et al. 2009). PCR product sizes ranged from 200–1200 bp (Fig. 1a). PCR products were cloned into pCR2.1 TOPO using a TA Cloning Kit (Life Technologies). DNAs from the chromocenters were also digested with MspI (Life Technologies) and cloned into a pUC19 vector. Plasmids with insertions were transfected into DH5a Escherichia coli competent cells according to standard protocols (Sambrook and Russell 2001). Positive recombinant clones were sequenced by the Sanger method.
Fig. 1

Chromocenters and DOP-PCR-amplified centromeric fragments label the centromeres and chromocenters in nuclei and metaphase chromosomes. a Electrophoresis of DOP-PCR-amplified chromocenters (1) and microdissected centromere CEN (2). Marker fragments are shown at the left (bp). b Two-colour FISH with the DOP-PCR-amplified chromocenter probe (ChrmC, shown in green) and MaSat probe (shown red) on isolated liver nuclei: a merged image; b DOP-PCR-amplified chromocenters probe (ChrmC, green) and MaSat signals (red); c staining with DAPI (blue). c FISH with the DOP-PCR-amplified chromocenter a, b and centromere (c, d) probes (green): a metaphase plates from female mouse bone marrow; b metaphase plates from mouse embryonic fibroblasts (MEF); c metaphase plates from female mouse bone marrow; d metaphase plates from MEF. Nuclei and chromosomes were contrasted by DAPI (blue). Arrows indicate two chromosomes with less bright hybridization signals (Bar: 5 μm)

Plasmids and DNA probes for in situ hybridization

The following DNA fragments were used: a fragment (471 bp) of MaSat (Radic et al. 1987) cloned into pBluescriptIIKS+, the MiSat fragment (362 bp) inserted in pGEM7 vector (Kipling et al. 1995) and fragments of MS3 (300 bp) and MS4 (600 bp) cloned into pUC19 vector (Kuznetsova et al. 2005). All cloned chromocenters’ and centromere fragments as well as MaSat, MiSat, MS3 and MS4 were labelled with either digoxigenin-11-dUTP (DIG) or biotin16-dUTP (Roche, Germany). The labelled nucleotides were incorporated into fragments by PCR, using M13 forward and reverse primers. The same type of labelling was applied for both chromocenters and microdissected centromere DNA: probes were obtained using two rounds of amplification with the DOP primer (5′-CCGACTCGAGNNNNNNATGTGG-3′). The first round of amplification was made as described above. Dig-11-dUTP or biotin16-dUTP (Roche, Germany) was incorporated into the probes during the second round of amplification (Arneson et al. 2008; Yang et al. 2009). The products produced a smear in the 1 % agarose gel with an average size of 200–600 bp (Fig. 1a). FITC-labelled mouse Y-chromosome paint probe was purchased (Cambio, Cambridge, UK) and processed according to the manufacturer’s recommendations.

Cloned DNA quantification (dot-blot hybridization)

Ultrasonicated genomic DNA was dot-blotted onto positively charged nylon membranes (Hybond-N++, Amersham) in a series of dilutions ranging from 50 ng to 2 μg as the positive control. Filter-immobilized PCR products of cloned DNA fragments were hybridized with genomic DNA labelled with dig-dUTP using the Klenow enzyme (Sambrook and Russell 2001). The PCR products from the plasmids with the cloned fragments (MaSat, MiSat) were used to produce the calibration curve to estimate the proportion of newly cloned sequences in the genome. The trematode Himasthla elongata mariner fragment was used as a negative control (Galaktionov et al. 2014). Hybridization was performed at 42 °C in a solution containing 6× SSC, 0.5 % SDS, 5× Denhardt, 50 μg/ml salmon sperm, 50 % formamide, 0.01 mol/L EDTA and 20 ng/ml probe (Sambrook and Russell 2001). Densitometric quantification from the dot blots was performed using the Gel-Pro Analyzer Version 3.1.00.00. The hybridized DIG-labelled probe was detected with an antidigoxigenin alkaline phosphatase-conjugated antibody (Roche, Germany) according to the manufacturer’s protocol.

Fluorescent in situ hybridization

The slides were denatured in 70 % formamide/2× SSC for 3 min at 72 °C. For each slide, 50 μl of hybridization solution (containing 1 μg of each labelled probe, 50 % of formamide, 2× SSC, 10 % dextran sulphate), was denatured for 10 min at 75 °C and allowed to prehybridize for 1 h at 37 °C. Hybridization took place for 16–18 h at 37 °C. A mixture of 10 μl each of Y chromosome paint FITC probe and biotin-labelled probes was applied onto the slide, and hybridization was performed at 42 °C overnight in a moist chamber. Slides were washed twice in 50 % formamide in 2× SSC, and twice in 2 SSC, 0.05 % Tween-20 for 5 min at 42 °C. Following a wash in PBST (PBS, 0.1 % Tween), slides were incubated with a streptavidin, Rhodamine-Red-X conjugate (LifeTechnology) and FITC-conjugated anti-DIG antibody (Roche). Finally, the slides were counterstained with 4, 6-diami-dino-2-phenylindole (DAPI) and mounted in an antifade solution (Vectashield, Vector laboratories, Burlingame, CA, USA). Images were captured with a Nikon (CCD) camera on a Zeiss/MetaMorph epifluorescence microscope and DMRXA fluorescent microscope (Leica Wetzlar GmbH, Germany) equipped with a FLUOTAR ×100/1.30 objective, a ×1.6 tube lens, a black-white CCD camera and appropriate filter cubes. Images were optimized using Adobe Photoshop CS2.

Sequence analysis

All sequence comparisons were performed using standard algorithms such as BLAST (Altschul et al. 1990). To check for the presence of repeat elements the sequence sets were searched against the RepBase database (Kohany et al. 2006) using CENSOR (Ver: 4.2.28) with the following parameters: default, nofilter, minsim 0.75, show_simple, bprg blastn, mode norm. Sequences were partially assembled by LinearCen1.0. For the detection of TRs, the Tandem Repeat Finder version 4.07 software (Benson 1999) was used with the following parameters: match 2, mismatch 7, delta 7, PM 80, PI 10, minscore 50, maxperiod 300. Sequences were compared with mouse TRs, (Komissarov et al. 2011). Clones were aligned against L1-like sequence records from RepBase using BLAST. The search of LINE fragments in human centromeres was done by CENSOR in partial assembly LinearCen1.1 (Miga et al. 2014, http://www.ncbi.nlm.nih.gov/assembly/GCA_000442335.2). Coordinates for the beginning and end of each fragment were determined in bp with respect to L1-like sequences and its ORFs (supplementary table S1). Clones were compared with the mouse reference genome build 38.3 (http://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.23); the unassembled contigs of two mouse WGS assemblies for projects AAHY (http://www.ncbi.nlm.nih.gov/nuccore/AAHY00000000.1/) and CAAA (http://www.ncbi.nlm.nih.gov/nuccore/CAAA00000000.1/) were obtained from the NCBI ftp site.

Results

Total probes

Microdissection of the CEN region yielded tiny amount of material. DNA from this material was amplified using degenerate oligonucleotide-primed-PCR (DOP-PCR) (see “Materials and methods” section) and successfully used as a FISH probes (Dernburg 2012). Microdissected and amplified CEN probes (MdCP) were hybridized to the periCEN regions using FISH techniques (Fig. 1).

Although the chromocenter preparation (ChrmC) produced DNA in amounts sufficient for cloning, we ran it through the same DOP-PCR procedure to have consistent results. The DOP-PCR products from both sources were quite similar (Fig. 1a) and had similar results after FISH hybridization (Fig. 1). In the interphase nuclei, the main ChrmC signal marked the chromocenters, although the correspondence with MaSat was not perfect, indicating the presence of sequences were different from MaSat in the ChrmC probe. The same picture was obtained using MdCP probe (Fig. 1a and Supplementary Fig.S1). We used interphase nuclei from different sources (liver, bone marrow, cell lines C3H and L929) and the main signal always label chromocenters. The correspondence in the rest of the nuclei was not as accurate as in the case of liver nuclei: some signals were observed between chromocenters (Supplementary Fig.S1). We suppose that these differences reflect the tissue specificity of the sequences involved in ChrmC formation rather than differences in treatment of biologic material, but this question requires additional investigation. Nonetheless, ChrmC and MdCP probes prepared from liver chromocenters labelled chromocenters of all cell types although to different extents.

Mouse embryonic fibroblasts (MEF) from C3H mice have a normal karyotype. In both cases (ChrmC and MdCP), the main probe signal labelled the CEN regions of chromosomes and covered all of them except one pair (Fig. 1c). It is known that sex chromosomes have a unique DNA repeat content (Pertile et al. 2009; Namekawa et al. 2010). This pair could be the sex chromosomes and the lack of probe labelling prompted us to check the separate clones on sex chromosomes (described below in the “FISH with the selected clones” section).

Both ChrmC and MdCP probes represent heterochromatic regions and suitable for further cloning.

Cloning and annotation

We obtained three libraries: (1) CEN region (MdCP, index C, 70 clones); (2) from chromocenters’ fraction (ChrmC, index X, 130 clones); (3) four clones with index “W” that were not amplified by DOP-PCR and were directly cloned from the chromocenter fraction after digestion with MspI. Clones with the inserts of ∼100 bp and above were selected and sequenced. The description of 38 fragments is shown in Table 1.
Table 1

Chromocenters’ clone annotation

No.

Clone name

Length

GC%

Repbase

Ref. Genome

WGS alignment length, bp

WGS contigs, thousands

Percent

FISH

GenBank ID

Similarity to MaSat fragments

 1

C1

347

49

GSAT_MM

C

94

4.7

  

KT283010

 2

C16

187

42

GSAT_MM

C

203

34

  

KT283016

 3

WC7

95

33

GSAT_MM

C

113

31

  

KT283045

Without similarity to any repetitive sequences

 4

C14

137

46

53

1<

  

KT283015

 5

C19

429

43

39

1<

>1

D

KT283017

 6

C20

833

51

73

1<

1

C

KT283018

 7

C26

161

41

46

1<

>1

 

KT283020

 8

C32

361

48

37

1<

>1

 

KT283022

 9

C38

152

36

47

1<

  

KT283023

 10

C44

137

46

53

1<

  

KT283025

 11

C58

231

52

29

1<

  

KT283033

 12

X72

321

52

35

1<

>1

C

KT283035

 13

X74

179

59

31

1<

 

D

KT283036

 14

X101

424

55

151

1<

>1

C

KT283039

 15

X114

584

50

40

1<

  

KT283040

 16

X135

208

40

Chr16

195

1<

  

KT283043

 17

WC8

104

30

102

1<

  

KT283046

 18

WC9

105

30

52

1<

  

KT283047

 19

WC15

112

56

Chr1

49

1<

  

KT283048

 20

X130

424

33

PB1D10 (frg)

Chr1

424

3.8

1

D

KT283042

Similarity to LINE

 21

C60

245

27

L1-2_Cgr

D

245

>200

1

C

KT283034

 22

C6

135

39

L1_MM

D

159

35

>1

 

KT283011

 23

C7

149

38

L1PA3

D

132

1.7

>1

C

KT283012

 24

C10

229

41

L1_MM

D

199

27

>1

C

KT283013

 25

C12

171

53

L1-63_XT(frg.)

45

1<

>1

C

KT283014

 26

C25

281

37

L1-2 Cja

D

98

1.5

5

C

KT283019

 27

C31

203

40

L1ME1

76

1<

  

KT283021

 28

C42

278

40

L1_MM

D

272

28

  

KT283024

 29

C45

164

30

LX7

154

66

5

C

KT283026

 30

C46

184

31

LX7

D

186

129

 

D

KT283027

 31

C47

144

49

LX7

D

176

12

  

KT283028

 32

C49

162

36

L1_MM/LX7

D

207

126

  

KT283029

 33

C52

185

43

L1MA5

D

189

2.5

  

KT283030

 34

C53

272

40

L1_MM/LX7

D

313

133

>1

 

KT283031

 35

C57

118

33

L1PREC2

88

136

  

KT283032

 36

X93

133

41

L1ME1

49

1<

  

KT283037

 37

X97

304

47

L1MB1

Chr1

309

1<

  

KT283038

 38

X119

282

37

L1-2_Cja

D

114

1.6

  

KT283039

The table is divided into three parts according to sequence similarity to (1) MaSat; (2) without any similarity; (3) LINE. “C” denotes clones obtained from the CEN probe (Fig. 1a (2)), letter “X” clones obtained from ChrmC (Fig. 1a, (1)) and W clones obtained from MspI-digested chromocenters. Fragment lengths are given in base pairs (bp). Letter “D” means dispersed; chromosome position given for clones with a unique position in the reference genome (X135–Chr16: 34408427–34408620; WC15–Chr1: 45728287–45728332; X130–Chr1:3784877–37849194 X97–Chr1: 15531115–15530813); C–MaSat in silico in reference genome position is close to the Golden Path Gap, i.e. centromeric. Two WGS columns reflect the clones’ presence in two mouse WGS databases: the length of the fragment aligned and the number of contigs that contain the aligned fragment. In situ results are shown in two columns: the approximate amount of clone estimated in percent; the position of clone determined by FISH; letter “C” and “D” have the same meaning as for the reference genome, i.e. centromeric and dispersed. Whole X130 exists only once in the reference genome (GRCm38.p3)—chrm1: 37848771–37849194. X130 is classified as “Without similarity to any repetitive sequences” for only ∼1/3 (“frg” in Table 1) of it possesses a similarity to part of SINE

The summary of successfully sequenced clones in the library, including a clone name, sequence length and GC content is provided in Table 1. In silico clone characterization was made by RepBase masking and by comparison with the mouse reference genome build 38.3. The results of in situ localizations of several clones are also shown in Table 1. When RepBase repeat masks were less than 80 % of the clone sequence by the program Censor, the case was marked “frg” (NN 20 (X130), 25(C12); Table 1). Clone X130 has a complex composition: 117 bp out of 424 bp clone length aligned to B1 (SINE)-related PB1D10. The full length of SINE is ∼170 bp, so most of the consensus SINE monomer was included in this clone.

Three more clones were found to have unique sequences (N16 (X135), N19 (WC15), N37 (X97). Most of the L1-related clones expectedly showed dispersed patterns in the reference genome where almost all chromosome arms were covered by alignment hits. The MaSat-related clones were classed as pericentromeric (C), i.e. with several alignment hits near or in close proximity to a Golden Pass Gap and enriched in unplaced contigs. All MaSat-containing fragments existed in numerous contigs. If clones C16 and WC7 had approximately the same copy numbers in WGS, the C1 amount is ten times less. The different amounts of MaSat-based clones in unassembled contigs suggest that MaSat is not as uniform as was previously thought (Vissel and Choo 1989; Abdurashitov et al. 2009). Each MaSat array could possibly arise in different chromosomes during assembly, and we suspect that MaSat is not uniformly spread across chromosomes as it was considered previously. Mapping all different MaSat-containing clones and verifying chromosome specificity will be subjects for a future study.

All clones without similarity to known repetitive sequences (Table 1, NN 4-20) were also present in the WGS database. The amount of contigs with these clones was ∼10-fold lower than that for MaSat- and LINE-containing clones (Table 1, NN 1-3 and NN 21-38). These data suggest that clones do not contain unknown repetitive sequences. Two clones contained internal tandem repeats: X74 has 45 bp × 3.2 tandem repeat; X101 has two tandem repeats separated by spacer: 14 bp × 4.1 and 68 bp × 3. These fragments, when matched to WGS, appeared to be too short for sequence identification. The identity of these fragments remains to be elucidated; however, at least 3 of them definitely belong to heterochromatic CEN regions (Table 1, Fig. 3).

Most of the LINE-like clones (Table 1, NN 21-38) have dispersed distributions predicted in silico by comparison with the reference genome. In spite of this prediction, the distribution of most of them by FISH shows a strong heterochromatic location (Table 1, Fig. 3). There is also a contradiction between in silico and experimental data on the amount of LINE-based clones. For example, the in silico prediction for C25 is a relatively modest amount in the WGS dataset; despite this prediction, our experiments reveal a major amount of this fragment comparable to the amount of MaSat. The same is true for the C45 clone (Fig. 2). Both have strongly heterochromatic locations (Table 1, Fig. 3). A brief summary of our in situ experiments are shown in the “in situ” section in Table 1 and in the in situ experiments detailed below.
Fig. 2

Example of dot-blot hybridization. Amount of loaded DNA is shown at the left; clone names are shown on the top; densitometry results are shown in columns (In situ % (percentage), Table 1). Abbreviations: tDNA—total DNA loaded; positive control, for this DNA label used as the probe. pHs—trematode Himasthla elongata mariner fragment was amplified in the same way as the rest of the clone fragments and used as a negative control

Fig. 3

FISH with the selected clones. Images displayed in the same order as in Table 1. a C7 clone as an example of the set of double (two-colour) FISH for each clone in Table 2. I Double FISH of C7 (green) with red labelled probes of MaSat, MS4, MiSat, MS3 and C7 (red) and sex chromosomes (green). II Each clone indicated (green) was hybridized together with MiSat (red) on metaphase plates. C19 is shown as an example of staining counted as dispersed (Table 2). C101—single probe hybridization without MiSat. III MEF interphase nuclei; double FISH with clone indicated (green) and MaSat (red, upper row) or MiSat (red, lower row); Bar = 5 μm

Amount of each fragment in the genome

The approximate amount in the genome of each of the clones that we studied was calculated using reverse dot-blot hybridization, i.e. different dilutions of total genomic DNA was blotted onto nylon membranes and hybridized with labelled DNA from the various cloned fragments (Fig. 2, tDNA). Negative control did not give any specific signal (Fig. 2, pHs). The difference in density of signal between clones was observed as expected. The known cloned mouse TRs were used as the reference points. The proportion of MaSat in a total mouse DNA preparation was ∼8 %, and this is higher than the amount of satDNA found in total DNA preparations from rats and humans (Abdurashitov et al. 2009). The hybridizations show that ∼1 % of the mouse genome consists of MiSat. These results were in good agreement with previously published data (Wong and Rattner 1988; Moens and Pearlman 1990). The amount of recently cloned MS3 and MS4 happens to be lower than that previously established (Kuznetsova et al. 2005): ∼1 % for MS4 and >1 % for MS3. The amount of the clones’ insert DNA was calculated with respect to known TR amount. The amount of known TR varies in the literature (Kipling et al. 1994; Garagna et al. 1993); so, our calculations are very approximate.

The amount of each of the cloned insert DNAs in the genome calculated from five experiments is shown in Table 1 (In situ, %). Most of the clones are in low copy number in the genome, although not unique. The LINE-related clone N29 (C45, Table 1) is highly repetitive in accordance with the in silico prediction. But, the clone N26 (C25, Table 1) is highly repetitive in contrast to the in silico prediction.

Fluorescent in situ hybridization with the selected clones

The FISH resolution did not allow obtaining precise probe localization—whether it is centromeric (CEN) or pericentromeric (periCEN). The only reasonable statement is that the probe is localized close to the primary constriction. We can only describe the position as being broadly centromeric for most of the probes. The exceptions are those probe sequences, whose positions have been determined previously by other methods as fibre-FISH (Kuznetsova et al. 2006) and by study of the prematurely condensed chromosomes of 1-day mouse embryos (Kuznetsova et al. 2007). The localization of the new clones in relation to CEN (MiSat and MS3) or periCEN (MaSat and MS4) sometimes suggests their relative position.

Each clone selected for FISH was used for two-colour FISH with four known cloned TRs (satDNA) and a probe specific for the sex chromosomes. The example for clone C7 is shown (Fig. 3a). C7 appeared to be a fragment of L1 (Fig. 4) and the position of this clone is predicted in silico to be dispersed (Table 1). However, the real distribution of this clone in situ appeared to be far from dispersed. We found that the probe was hybridized to the centromeric region on metaphase chromosomes and to chromocenters in interphase nuclei (Fig. 3a). Two-color FISH showed that chromocenters were not uniform—probes label different compartments. MaSat and MS4 were tested as periCEN while MiSat and MS3 were tested as CEN (Kuznetsova et al. 2006). It was observed that the C7 clone occupied an inner part of chromocenters but without overlapping with the other known probes. Even on chromosomes, when chromatin is condensed, the overlapping of probes was not exact. It appeared that C7 is localized to the periCEN region. The same is true for the rest of the clones mapped. None of the clones coincided with MiSat and all of them were localized mostly to the periCEN region of metaphase chromosomes (Fig. 3a, c). Three clones displayed the dispersed pattern, which could be expected for L1-derived (C46) or unknown sequences (C19, X74). One example of the dispersed pattern using FISH is shown in Fig. 3b (C19). These probes were excluded from further analysis.
Fig. 4

a Schematic diagram of the position of LINE-derived clones on the L1_MM and Lx consensus RepBase records. Precise positions are shown in Supplementary Table S1. b The LINE fragments found in two human chromosomes CEN (2 and 17) that are mapped to the LINE consensus. Seventeen fragments of L1 in total have been found in two human CENs

Clones C20, X72 and X101 were from the group “without similarity to any repetitive sequences” (Table 1). All these clones are present in the WGS database (Tables 1 and 2), and all of them map to the periCEN region. The tiny amount available of X101 is insufficient for the double FISH experiment, but C20 and X72 are present in the genome in amounts comparable to MiSat (∼1 %; Fig. 2 and Table 1). Two-colour FISH of these probes with MiSat allows us to expect the periCEN location of the corresponding sequences (Fig. 3b) with C20 being more peripheral from CEN than X72 is (Fig. 3b) due to the degree of overlapping signals. Despite being previously unknown, these sequences are definitely members of the CEN-periCEN compartment.
Table 2

Positions of selected clones in the mouse genome, interphase nuclei and chromosomes

 

Position

No.

Clone name

In silicoa

periCENb

Chromocenterc

Sex chromosomesd

X

Y

1

C19

WGS

   

2

C20

WGS

+

+

+

+

3

X72

WGS

+

+

+

4

X74

WGS

   

5

X101

WGS

+

   

6

C60

L1,D

+

+

+

7

C7

L1,D

+

+

+

8

C10

L1,D

+

+

+

+

9

C12

L1,D

+

+

+

10

C25

+

+

+

 

11

C45

LX7, WGS

+

+

+

12

C46

LX7,D

   

13

X130

WGS

+

+

+

+

For clone names, see Table 1. Probes with a dispersed pattern of hybridization were not analyzed further. Probe X101 could not be analyzed further due to its low content in the genome

aClone definition according to databases (RepBase; Reference Genome (38.3), Table 1)

bProbe located in the periCEN region of the chromosomes (+) or has a dispersed pattern (−)

cProbe located in the interphase chromocenters; sex chromosomes

dProbe located in the sex chromosomes (+) or not (−).

We investigated the presence of heterochromatic clones in the sex chromosomes. The unique DNA repeat content of sex chromosomes has been shown for TR (Cooke et al. 1984; 1985), but not for the unknown or LINE-derived sequences. Most of our clones labelled the X chromosome, but only three out of the ten checked map to the Y chromosome. So, the Y chromosome contains unique DNA repeats as well as TRs.

Most of the clones tested are relatives of LINEs for which a dispersed pattern had been expected. On the contrary, all of them show a pericentromeric location and, probably, are concentrated in the periCEN region for there is no perfect correspondence with the MiSat signal in double FISH (Fig. 3b). This indicates that LINE-based fragments might be an essential component of the heterochromatic region.

The position of L1-related fragments in RepBase records

We mapped LINE-like clones to the RepBase consensuses of L1_MM and Lx. All 18 LINE-related clones could be assigned readily to the map. Most of them grouped in a ∼2-kb region at the end of the second ORF and just close to this region in the murine-specific Lx family (Fig. 4a). Thus, even the limited amount of clones allowed us to map the region of the L1 element, which is specific for heterochromatic regions.

We found the same fragments in the human genome. The human genome sequence remains incomplete, with multimegabase-sized gaps representing the endogenous CENs and other heterochromatic regions. The same is true for the genomes with less advanced assembly including mouse genome (Chinwalla et al. 2002). Centromeric regions are omitted from most of ongoing genomic studies. In the recent study, a special effort has been made to identify two satellite array variants in both X and Y centromeres, as determined by array length and sequence composition (Miga et al. 2014). As a result, 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 (Fig. 4b). The concentration of fragments in the same region in the end of the second ORF is evident.

Discussion

Repetitive DNA sequences may account for more than two thirds of the mammalian genome (de Koning et al. 2011), yet their regulatory and architectural role remains largely enigmatic, partly because of technical difficulties using current molecular biology techniques. Certain subclasses of DNA repeats have a propensity to aggregate to form visually recognizable structures in interphase nuclei–chromocenters (Wijchers et al. 2015), and we have made an attempt here to clarify the DNA content of chromocenters.

Biochemically isolated chromocenters (ChrmC) and centromeric regions (MdCP) taken through DOP-PCR amplification procedures showed in fluorescent in situ hybridization (FISH) that the resulting products are part of the CEN region of chromosomes and chromocenters. Among sequenced clones, three clones represented mouse major satellite (MaSat). On the other hand, 16 clones were absent from the mouse-assembled genome, but were found in the raw WGS database. Finally, 18 clones represent LINE-like fragments. From the clones selected for FISH, three clones displayed the dispersed distribution across chromosomes. The remaining ten clones mapped to the CEN regions of chromosomes and to the chromocenters in interphase nuclei. Among these ten clones, six were LINE fragments and four - unknown sequences. There was no overlapping of signals inside the chromocenters in double FISH, arguing for a complex chromocenters’ composition. When aligned against the full-length LINE from RepBase records, the majority of our newly identified LINE-derived clones were found to be grouped at the end of the second ORF and 3′UTR flanking regions (3′UTR). The majority of the LINE-based clones, when aligned against the full-length LINE from RepBase records, are found to be grouped at the end of the second ORF and 3′UTR. Facultative heterochromatin is enriched by full-length LINEs as shown by bioinformatics data (Waterston et al. 2002) and experimentally by FISH (Boyle et al. 1990; Solovei et al. 2009), but LINE is absent in constitutive heterochromatin.

Degenerate oligonucleotide primer

At the beginning of the current work, we used the DOP-PCR primer as it was initially developed. This primer is based on a partially degenerate sequence and is used in a PCR protocol with two different annealing temperatures. The procedure, termed degenerate oligonucleotide-primed-PCR (DOP-PCR), allows essentially random amplification of DNA from any source. The DOP-PCR technique is based on the principle that at a sufficiently low annealing temperature, only the six specific bases of the 3′ end of the oligonucleotide will prime the reaction, theoretically priming every 46 (∼4 kb) base pairs along the starting DNA. Therefore, specified six-nucleotide sequence is likely to occur at a frequency allowing a highly diverse amplification to take place. In a separate study, DOP-PCR has been found to amplify DNA from sources ranging in size from 2.4 kb up to complete genomes, using the same primer and protocol in all cases (Telenius et al. 1992). Thus, this method for amplifying DNA should have implications for chromosome labelling and cloning. This method can be applied to many species including the plants and non-mammalian groups where interspersed repeats are not easily available for general amplification (Telenius et al. 1992).

The great enthusiasm for this new method was soon followed by a realization of its limitations. This was noticed first in the reverse chromosome labelling technique. Although producing a relatively even signal for euchromatin, the DOP-PCR amplification often fails to label highly repetitive sequences in the acrocentric short arms, centromere and heterochromatic regions. This effect is caused by either (1) failure of these sequences to amplify (the DOP-PCR amplification is specific for the most 3′ six base sequence) or (2) because of suppression of the signal by the Cot 1 DNA, or (3) by the inclusion of a probe preannealing step in the hybridization protocol. However, it is somewhat unpredictable whether these repetitive regions hybridize (Carter et al. 1992). It seems likely that the DOP primer that we used prefers to amplify transposable elements (TEs) rather than TRs, suggesting that our library is depleted of TRs.

L1 derivate as tandem repeat

It is known that LINE elements can be repeated tandemly giving rise to TR, but this involves a full-length element (Ahmed and Liang 2012). In the process of classifying mouse large TR, we already observed the L1 fragment-based TR (Komissarov et al. 2011). L1-related TR included part of the ORF2 and 3′-end (3′UTR) in their monomers. TE-related arrays were mapped to the reference genome in silico. Most of the loci found for the TR-L1 family do not exceed 5 kb. All loci were built of monomers from ∼1 up to ∼2 kb. All the monomers are similar to the one found in the current work, i.e. they are fragments of the ∼2 kb of ORF2 end and 3′UTR. All loci were displayed on banded chromosomes. The TR-L1 family is definitely enriched in heterochromatic bands and their concentration on the X chromosome was shown. At the same time, no TE-related TR was found on the Y chromosome (Komissarov et al. 2011). We expected that validation of these findings by FISH would be technically challenging, because other retroelements may obscure the results, but current work shows that the precise L1 fragment definitely exists in heterochromatic regions and that most of it is absent from the Y chromosome (Table 2). Experimental data about the structure of L1-derived clones correspond to the in silico prediction (Komissarov et al. 2011), although facultative heterochromatin along chromosome arms is not visible by FISH. The ∼2 kb L1-derived fragment enriches constitutive heterochromatic regions, which are absent in genome assemblies. This absence limited our in silico predictions.

The clones we selected for FISH cover the whole ∼2-kb fragment of L1 ORF2 and Lx (Fig. 4b). Thus, the whole fragment of ∼2 kb is part of the heterochromatic TR arrays. The clones are quite short in length (∼200 bp, Table 1) and the fact that the bright FISH signal is comparable to the signal of MaSat and MiSat argues for the tandem organization of the ∼2-kb fragment inside the heterochromatic regions. The appearance of most clones on FISH (Fig. 3) is consistent with their tandem organization. This could be verified by fibre-FISH, which will be the subject of a future study.

On the other hand, LINE fragments are found in assembled human CENs as distinct units and not as arrays (Miga et al. 2014). These findings could be due to assembly errors. The possibility of the existence of L1-related arrays in the human genome could be checked by mining the human genome WGS database.

Full-length long interspersed nuclear elements do not colocalize with heterochromatin on FISH

There is cytological evidence that the major interspersed repeat families of the mouse, the LINE L1 element and the SINEs occupy discrete positions on metaphase chromosomes that correspond to G bands and R bands, respectively (Boyle et al. 1990). The precise repetitive sequences characterize the C, G and R bands of mouse chromosomes: centromeric and subtelomeric regions (constitutive heterochromatin, which is localized to the chromocenters and enriched with TR), gene-poor mid-late replicating non-centromeric heterochromatin (L1-rich heterochromatin) and gene-dense early-replicating chromatin (euchromatin, SINE-rich). The probes routinely used to identify different regions are probes for MaSat (C bands), L1 (the major class of the long interspersed repetitive sequences, LINE; G bands) and B1 (the major class of the short interspersed repetitive sequences related to human Alu sequences, SINE; R bands) (Solovei et al. 2009). Genome sequencing confirms and underlines the most notable features of repeat elements, namely the contrasting genomic distribution of LINEs and SINEs. Whereas LINEs are strongly biased towards (A + T)-rich regions, SINEs are strongly biased towards (G + C)-rich regions. The contrast is all the more notable because both elements are thought to be inserted into the genome through the action of the same endonuclease. It seemed that with the availability of two mammalian genomes, it would become possible to extend this analysis to explore whether (A + T) and (G + C) content are truly causative factors or merely reflections of an underlying biological process (Waterston et al. 2002). Nowadays, with ∼5000 eukaryotic genomes available and the distribution of SINEs and LINE confirmed for most of them, the mystery still remains. On the other hand, if the LINEs and SINEs are retroposons that were dispersed through the genome by reintegration of reverse transcriptase products, why are the heterochromatic TR-rich regions depleted of them?

The chromosomal distribution of the SINE and LINE probes were confirmed by FISH on metaphase chromosome arms, which perfectly corresponds to Giemsa banding patterns. Most Giemsa banding protocols also stain the centromeric heterochromatin, but the L1 probe does not hybridize at detectable levels to this region on any chromosome (Boyle et al. 1990). Neither L1 nor B1 hybridize to centromeric heterochromatin, indicating that there are few or no interspersed repetitive sequences in these chromosomal regions. However, we have found the precise L1 fragment that is characteristic for the chromosome periCEN region and chromocenters (Figs. 3 and 4).

It has been shown that no significant G-negative bands appear on the X chromosome by the hybridization method. Thus, the X chromosome is exceptionally rich in LINE sequences (Boyle et al 1990). We found that the L1 fragment is also present in X periCEN but not on the Y (Fig. 1, Table 2).

Therefore, the precise LINE ∼2-kb fragment, but not the full-length LINE, is the component of mouse and human constitutive heterochromatin enriched with TRs.

Notes

Acknowledgments

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. 1.37.153.2014) 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.

Ethical standards

Mice were housed and maintained according to the approved standards in the Laboratory Animal Resources facility at Institute of Cytology RAS (St Petersburg, Russia).

Supplementary material

10577_2016_9525_MOESM1_ESM.doc (2 mb)
Fig. S1(DOC 2012 kb)
10577_2016_9525_MOESM2_ESM.doc (102 kb)
Table S1(DOC 102 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Inna S. Kuznetsova
    • 1
    • 2
    • 3
  • Dmitrii I. Ostromyshenskii
    • 1
  • Alexei S. Komissarov
    • 1
    • 2
  • Andrei N. Prusov
    • 4
  • Irina S. Waisertreiger
    • 1
  • Anna V. Gorbunova
    • 1
  • Vladimir A. Trifonov
    • 5
  • Malcolm A. Ferguson-Smith
    • 6
  • Olga I. Podgornaya
    • 1
    • 2
    • 7
  1. 1.Institute of Cytology RASSt PetersburgRussia
  2. 2.St. Petersburg State UniversitySt PetersburgRussia
  3. 3.School of Biomedical SciencesThe Chinese University of Hong KongShatinHong Kong
  4. 4.A.N. Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  5. 5.Institute of Molecular and Cellular Biology SB RAS, NovosibirskRussia; Novosibirsk State UniversityNovosibirskRussia
  6. 6.Cambridge Resource Centre for Comparative GenomicsUniversity of CambridgeCambridgeUK
  7. 7.Far Eastern Federal UniversityVladivostokRussia

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