age-related association of rdna and telomeres with the

Age-related association of rDNA and telomeres with thenuclear matrix in mouse hepatocytesAlberto S. Moraes12*,1, Mateus Mondin1{, Marcelo E. Beletti11, Margarida L.R. Aguiar-Perecin{, Ana M.A. Guaraldo{ andMaria Luiza S. Mello** Department of Cell Biology, Institute of Biology, State University of Campinas (UNICAMP), 13083-863 Campinas, Sao Paulo, Brazil{ Department of Parasitology, Institute of Biology, State University of Campinas (UNICAMP), 13083-863 Campinas, Sao Paulo, Brazil{ Department of Genetics, Superior Agriculture School Luiz de Queiroz, University of Sao Paulo, 13418-900 Piracicaba, SP, Brazil1

Department of Morphology, Institute of Biomedical Sciences, Federal University of Uberlandia, 38405-320 Uberlandia, MG, Brazil

AbstractTranscribed sequences have been suggested to be associated with the nuclear matrix, differing from non-transcribing

sequences, which have been reported to be contained in DNA loops. However, although a dozen of genes have their

expression level affected by aging, data on chromatin–nuclear matrix interactions under this physiological condition are still

scarce. In the present study, liver imprints from young, adult and old mice were subjected to FISH (fluorescence in situ

hybridization) for 45S rDNA and telomeric sequences, with or without a lysis treatment to produce extended chromatin

fibres. There was an increased amount of 45S rDNA sequences located in DNA loops as the animals grow older, while

telomeric sequences were always observed in DNA loops irrespective of the animal age. We assume that active rRNA

genes associate with the nuclear matrix, while DNA loops contain silent sequences. Transcription of each 45S rDNA repeat

unit is suggested to be dependent on its interaction with the nuclear matrix.

Keywords: aging; chromatin extensibility; development; nuclear matrix; rDNA; telomere

1. Introduction

A prevailing theory on aging states that it results from an

accumulation of tissue damage, similar to rust. This presumes

that aging results inevitably from accumulated damage to cells as

promoted by toxins, free-radical molecules, DNA-damaging

radiation, disease and stress (Harman, 1998). Nevertheless, there

is increasing evidence that specific genetic instructions and

structural modifications drive the process making it an active

process rather than a passive one (Oberdoerffer and Sinclair,

2007).

Studies by several authors, conducted in yeast, worms, flies

and mice, have confirmed substantial changes in gene expression

with aging (Burzynski, 2005). Among most important changes

reported is the silencing of tumour suppressors and other genes

involved in the control of cell cycle, apoptosis, detoxification and

cholesterol metabolism (Cao et al., 2001).

The activity of rRNA genes in mouse hepatocytes gradually

increases after birth, reaches a peak at 14 days of postnatal age

and then decreases drastically as the animals grow older (Ma and

Nagata, 1990). These changes are accompanied by an increase in

protein synthesis at about 1 month postnatally, followed by a

decrease in this synthesis along the adult life and low synthesis

levels in aged animals (Ma et al., 1991).

The rRNA genes, which in most eukaryotic species are

repeated ,100–5000 times per haploid genome, are located at

one or a few chromosomal sites called NORs (nucleolar organizer

regions) (Gaubatz et al., 1976; Long and Dawid, 1980). The rRNA

coding regions are organized as head-to-tail repeats, with the

transcribed regions separated by segments of non-transcribed

spacers (Fedoroff, 1979; Pan and Zhang, 2008). In rat hepato-

cytes, these sequences appear enriched in the purified nuclear

matrices after DNase I digestion, thus indicating that the high

transcription rates of rRNA genes and nuclear matrix interaction

are interconnected (Pardoll and Vogelstein, 1980).

The nuclear matrix is an operationally defined nuclear skeletal

structure assumed to be involved in many nuclear functions,

including DNA replication, transcription and repair and pre-mRNA

processing/transport. It is also a passenger concourse for

proteins going in and out of the nucleus through the nuclear pore

complex (Tsutsui et al., 2005). The nuclear matrix comprises a

protein framework formed by the nuclear pore/nuclear lamina

complex, a residual nucleolus and a residual internal fibrillar–

globular mesh (Berezney and Coffey, 1974; Gasser and Laemmli,

1986; Berezney, 1991). Until relatively recently, the nuclear matrix

was considered a rigid and static structure, but new theories now

consider it as a dynamic structure (Tsutsui et al., 2005; Barboro

et al., 2009).

The isolation of the nuclear matrix requires nuclease and high-

salt treatments that remove chromatin and other soluble nuclear

proteins from the cell nuclei (Nickerson, 2001). An ordinary method

for nuclear matrix isolation involves the extraction of nuclei with

highly concentrated solutions of monovalent salts such as 2 M NaCl

(Berezney and Coffey, 1977). After that, the nuclei adopt a so-called

nuclear halo configuration, in which nuclear DNA sticks out from the

residual structure (nuclear matrix) as negatively supercoiled loops

attached at their base by MARs (matrix attachment regions). If

1 These authors contributed equally to this work.2 To whom correspondence should be addressed (email [email protected]).Abbreviations: DAPI, 49,6-diamidino-2-phenylindole; e, erythroblast; ECF, extended chromatin fibre; FISH, fluorescence in situ hybridization; MAR, matrixattachment region; n, nucleoli.

Cell Biol. Int. (2010) 34, 925–931 (Printed in Great Britain)

Research Article

E The Author(s) Journal compilation E 2010 Portland Press Limited Volume 34 (9) N pages 925–931 N doi:10.1042/CBI20090457 N www.cellbiolint.org 925

nuclei are treated vertically, under the action of gravity, formation of

ECFs (extended chromatin fibres) may occur, as they are strongly

affected by nuclear matrix proteins, thus contributing to the higher-

order chromatin structure (Gerdes et al., 1994; Haaf and Ward,

1994; Davie, 1995; Cremer et al., 2000; Vidal, 2000). An increased

number of interactions between chromatin and nuclear matrix can

render or even prevent the ECF formation, since there are salt-

resistant interactions of MARs with the nuclear matrix (Moraes et

al., 2005, 2007). After this extraction, the nuclear DNA can be

fractionated into loop DNA and matrix-associated DNA; the latter

accounts for 1% of total nuclear DNA and is enriched in rRNA

sequences (Pardoll and Vogelstein, 1980). When this treatment is

followed by FISH (fluorescence in situ hybridization) assay,

transcriptionally inactive sequences produce long strings of signal

extending out on to the DNA halo or loop, whereas active

sequences remain tightly condensed as single spots within the

residual nucleus, thus suggesting that the association of genes with

the nuclear matrix may be a factor responsible for transcription

activation (Gerdes et al., 1994).

Taking into account that differences have previously been

reported for the changes in the activity of rRNA genes with

advancing age, in the present work, we aimed at comparing FISH

signals of active and silenced 45S rDNA and repetitive silenced

telomeric sequences in relation to their attachment to nuclear

matrix after ECF formation was induced in young, adult and old

mice hepatocytes.

2. Experimental procedures

2.1. Animals

Male mice from the inbred strain A/Uni, obtained from the

Multidisciplinary Center for Biological Investigation (CEMIB) of

the State University of Campinas were reared under normal

conditions and fed extruded chow (Purinaj) ad libitum. The

animals used were 1–2 (young), 15–20 (adult) and 61–100 (aged)

weeks old. At least three animals of each group were used.

Animals were killed by decapitation. Their livers were then

immediately removed and placed in cold 0.9% NaCl solution.

Liver slices were imprinted on histological slides. All protocols

involving animal care and use were approved by the Institutional

Committee for Ethics in Animal Experimentation (CEEA/IB/

UNICAMP – 378-1).

2.2. Treatments

Freshly prepared imprints were fixed in a mixture of absolute

ethanol and acetic acid (3:1, v/v, respectively) for 1 min and then

rinsed in 70% ethanol for 5 min. The preparations were positioned

vertically using a protocol previously described for ECF invest-

igation (Moraes et al., 2005). Briefly, the lysis solution consisted of

2 M NaCl plus 1% Triton X-100 in Tris/HCl buffer (25 mM, pH 7.4).

Treatment lasted for 5 h at 25uC, after which, the volume of

solution was completed with absolute ethanol to a final concen-

tration of 50%, and treatment was prolonged for another 10 min.

The slides were then carefully removed from the lysis solution and

transferred to 70% ethanol for 30 min. Fixed preparations not

subjected to the lysis protocol were used as controls.

2.3. FISH

The plasmid HM 456, which contains part of the 18S and 28S

rDNA of Xenopus laevis (Meunier-Rotival et al., 1979) was kindly

provided by Dr A.M. Vianna-Morgante (IB-USP, Sao Paulo, Brazil),

and the telomere-like oligonucleotide DNA sequence was syn-

thesized by Invitrogen. Probes were labelled by nick translation

with biotin-16dUTP (Bionick Labeling Kit, Life Technologies, Inc.).

The protocol used was previously described by Mondin et al.

(2007). Briefly, the preparations were treated with RNase A (Sigma

Chemical Co.), followed by treatment with 0.01 M HCl for 2 min,

washed in 26 SSC (16 SSC is 0.15 M NaCl/0.015 M sodium

citrate), treated with 5 mg/ml pepsin for 10 min at 37uC and fixed in

4% paraformaldehyde for 5 min, followed by denaturation with

26 SSC/50% formamide for 10 min at 80uC. Heat-denatured

probes were hybridized at 37uC for 20 h. After hybridization and

washes with 26 SSC/50% formamide and 26 SSC at 42uC,

probe detection used a two-step procedure for amplification.

Biotin was revealed using mouse anti-biotin and rabbit anti-mouse

TRITC conjugated antibodies. Coverslips were mounted in

Vectashield (Vector Laboratories) containing 0.5 mg/ml DAPI

(49,6-diamidino-2-phenylindole).

2.4. Microscopy

DNA FISH preparations were analysed under a Zeiss Axiophot 2

epifluorescence microscope equipped with an HBO-100 W

stabilized mercury lamp as the light source and appropriate

filters. Zeiss Plan 625/0.50, 640/0.75 and 6100/1.4 oil-

immersion objectives were used. Digital images were obtained

using a Sony CCD-IRIS/RGB Hyper HAD colour video camera and

ISIS (MetaSystems, Altlussheim) software. Selected images were

processed using CorelDrawj12 software.

2.5. Image processing

Images of DAPI-stained nuclei were segmented using supervised

threshold to mark the nuclear region. Areas with average

intensities above this threshold were set to 1 (nuclear region),

and all other areas were set to 0. The resulting images were called

DAPI mask.

FISH images for rDNA were converted into greyscale format

and were segmented using supervised threshold to determine

the region positive for this marker. The sum of pixel values of this

region was calculated and was called IGL. The sum of pixel values

with overlapping DAPI mask of value 1 was calculated, and its

relative IGL percentage was called %IGLin and corresponds to the

percentage of integrated grey levels of FISH signals inside nuclear

remnants. The sum of pixel values with overlapping DAPI mask with

value 0 was called %IGLout and corresponds to the percentual of

integrated grey levels of FISH signals outside nuclear remnants

(extended chromatin fibres and nuclear halos).

Aging and DNA/nuclear matrix

926 www.cellbiolint.org N Volume 34 (9) N pages 925–931 E The Author(s) Journal compilation E 2010 Portland Press Limited

2.6. Statistics

All calculations and statistical analyses were done with Minitab 14

(State College, PA). The Mann–Whitney test was used.

3. Results

3.1. rDNA sequences detach from the nuclear matrixwith development and aging

The variable number of FISH signals observed when nuclei from

animals of the three ages were compared with each other is a

consequence of the well-known hepatic polyploidy (Gupta,

2000). In nuclei from young mice, the DNA content of which is

mainly contained in the 2C class, only two rDNA spots were

clearly observed (Figure 1A), whereas in nuclei from adult and

old mice, more rDNA spots were visible according to the higher

ploidy levels observed in these animals (most nuclei with

DNA content 4C and 8C, respectively) (Figures 1B, 1C). The

rDNA FISH signals observed in young mice hepatocytes were

always condensed (Figure 1A). In contrast, the 45S rDNA sites

observed in hepatocytes from adult (Figure 1B) and old

(Figure 1C) mice were partially decondensed and appeared as

expanded clouds; they were always seen in the periphery of the

negative images of the n (nucleoli), and in association with

DAPI-stained dense clusters (chromocentres) (Figures 1B, 1C).

These variations can give us a clue of a different chromatin

organization of rDNA sequences when different ages were

compared and are probably a consequence either of a different

packaging of rDNA sequences at different ages or a result of the

different ploidy levels in young, adult and old mice. In the case

of adult and old animals, amplification of the signal could be the

result of increased numbers of rDNA copies in the polyploid

genome.

In fixed and lysed hepatocytes from young mice, which do

not properly form ECFs but uncommon nuclear halos instead,

the small dots of 45S rDNA detected by FISH remained inside the

nuclei after the lysis treatment (Figure 2A), whereas in fixed and

lysed preparations from adult and old mice, the FISH signals

spread throughout and outside the nuclei, such that many small

spots could be seen in the nuclear halos and ECFs (Figures 2B,

2C). The 45S rDNA signal intensity outside the nuclei was higher in

old mice when compared with adult mice (Figures 2B, 2C)

(Table 1). Moreover, the intensity of FISH signals inside the nuclei

was higher in adult hepatocytes than in old hepatocytes (Table 1),

thus arguing in favour of a higher number of rDNA sequences

attached to the nuclear matrix in nuclei from adult mice, when

compared with nuclei from old mice. These results were observed

in all the examined nuclei that formed ECFs.

Figure 1 DNA FISH of 45S rDNA on fixed hepatocyte nuclei from young (A), adult (B) and old (C) miceNuclei were counterstained with DAPI. FISH signals are seen around the nucleoli (n). Scale bars, 12 mm.

Cell Biol. Int. (2010) 34, 925–931

E The Author(s) Journal compilation E 2010 Portland Press Limited Volume 34 (9) N pages 925–931 N www.cellbiolint.org 927

3.2. Telomeric sequences are always located inchromatin loops

The behaviour of telomeric repetitive motifs, known to be hetero-

chromatic and non-genic was also observed, in order to test the

hypothesis that only transcribed sequences remain attached to the

nuclear matrix, after the lysis treatment here employed.

We observed that, in mouse hepatocyte nuclei, telomeres were

also detected as small dots (young and adult mice) (Figures 3A,

3C). In adult (Figure 3C) and old mice (not shown), telomeric

sequences were seen spread in all the analysed nuclei; due to the

increased extent of ploidy, several blocks could be seen, most of

them near the bright DAPI-stained chromocentres but not

associated with them. In young mice, these sequences were

Figure 2 45S rDNA differently associates with the nuclear matrix in hepatocytes of different agesrDNA FISH signals on fixed and vertically lysed hepatocyte nuclei from young (A), adult (B) and old (C) mice. Nuclei were counterstained with DAPI. Arrowsindicate direction of gravity and of ECFs. Scale bars, 30 mm.

Table 1 Image analysis of rDNA FISH signals after lysis treatment%IGLin, percentual of integrated gray levels of FISH signals inside nuclear remnants; %IGLout, percentual of integrated gray levels of FISH signalsoutside nuclear remnants (extended chromatin fibres and nuclear halos); in/out, proportion between integrated gray levels of FISH signals inside andoutside nuclear remnants; M, median.

Model Specimen %IGLin %IGLout In/out M

Adult 1 31.60 68.40 0.46 0.46*2 40.57 59.43 0.683 33.17 66.83 0.504 9.83 90.17 0.115 21.13 78.87 0.27

Old 1 7.04 92.96 0.08 0.10*2 9.40 90.60 0.103 5.77 94.23 0.064 3.72 96.28 0.045 11.68 88.32 0.136 9.09 90.91 0.107 18.43 81.57 0.238 8.69 91.31 0.10

* Statistical difference with P50.0104.



detected only in nuclei of e (erythroblasts) (Figure 3A). In these

cells, most of the signals appeared positioned as organized

features, concentrated in one part of the cell.

After lysis treatment, the telomeric sequences extended

outside the nuclei at all the ages studied, but with less intensity

in nuclei from young mice (Figure 3B), where they appeared at the

nuclear halos. In this case, a DNA mass distributed homogenously

out and inside the nuclei could be clearly observed. A similar

pattern of telomeric sequences inside the nuclei and spreading

out of them was observed in adult mice (Figure 3D). However, the

intensity of the signals in the halo and in the ECFs of the adult

mice was significantly brighter than those observed in the nuclei of

young mice. Moreover, some dense blocks were clearly seen in

the halo, a phenomenon not observed in nuclei of young mice. The

telomeric sequences that remained in the nuclei were seen also on

the bases of the DNA loop structures located at the nuclear

periphery, which is in accordance with the notion that telomeres

are often associated with the nuclear envelope (Pandita et al.,

2007).

4. Discussion

The 45S rDNA repeats, which are responsible for the production of

a large amount of rRNAs (Wellauer et al., 1974; Perry, 1976;

Kominami et al., 1978), were expected to differently bind to the

nuclear matrix with development and aging, since decreased

transcription levels have been reported under these conditions

(Ma and Nagata, 1990). Indeed, the present results indicate that,

as the animal ages, there is an increase in the amount of 45S rDNA

Figure 3 DNA FISH of telomeric DNA on fixed non-lysed (A, C) and fixed and vertically lysed (B, D) hepatocytes nuclei from young (A, B) and adult (C, D)miceRegardless of age, telomeres do not associate with the nuclear matrix. Nuclei were counterstained with DAPI. e, erythroblast; h, hepatocyte. The arrowindicates direction of gravity and the ECF flow. Scale bars, 12 mm.

Cell Biol. Int. (2010) 34, 925–931


repeats non-associated with the nuclear matrix and located in the

nuclear halos or ECFs, thus suggesting that the attachment of

these sequences to the nuclear matrix is related to gene

activation.

According to the model proposed by Heng et al. (2004), there

are two types of MAR sequences with ability to bind to the nuclear

matrix: (i) the constitutive MARs, which are found in the bases of

the DNA loops and outside transcribed sequences, presenting a

structural role; (ii) facultative MARs, which are located inside the

genes, being responsible for their transcriptional activity. Since

the MARs found until now on rDNA sequences are located only in

flanking sequences, it seems that they are of the constitutive type

(Chen et al., 1997). In fact, there is no description of facultative

MARs on rDNA genes.

In this regard, our present results confirm the existence of

MARs in rRNA genes, since many rDNA copies remained attached

to the nuclear matrix even after the lysis treatment. The

association of MARs with the nuclear matrix is salt-resistant

(Cook et al., 1976; Moraes et al., 2007).

Additionally, the results regarding the 45S rDNA genes suggest

that, in multicopy genes, there is a nuclear matrix-dependent

control of gene expression different from that of single copy

genes. In single copy genes, the adhesion to the nuclear matrix

determines their transcriptional level (Allen et al., 2000; Whitelaw

et al., 2000), while in the case of 45S rDNA, this interaction may

determine if each gene copy will be transcribed or not. Thus, the

amount of transcripts produced could be regulated by controlling

the production average of the thousands of gene copies rather

than by controlling the level of activity of each single repeating

unit.

In relation to the telomeric sequences, the results clearly show

that the association of the chromatin with the nuclear matrix is a

feature common to transcribing sequences. After the lysis

treatment, most telomeric sequences exit the nuclei, remaining

attached only to the nuclear envelope, and it is independent of the

animal developmental state or age. Since telomeric repetitive

motifs are devoid of genes, it is reasonable to conclude that non-

transcribing sequences do not associate with the nuclear matrix.

However, a question still remains on whether other heterochro-

matic sequences such as pericentric repeats have a similar

behaviour.

It is probable that the telomeric sequences in nuclei from

young mice are so packed that they were unavailable to telomeric

probes in fixed nuclei or that, in adult and old mice, there is

unpackaging of the telomeric structure, either as a natural

physiological event or as a by-product of the genomic instability

that is characteristic of the aging process. Nevertheless, after

lysis, which is responsible for solubilizing several histone and non-

histone proteins, the telomeres became accessible to the

telomeric probes, thus binding them and revealing the sequences

located at the nuclear halos. However, it is not to be overlooked

that the telomeric repetitive motifs were seen in control nuclei only

of polyploid adult and old hepatocytes and not in diploid nuclei

from young mice, where there are less telomeric repeats present

and, due to the sensitivity of the method, these sequences were

detectable only when stretched with the lysis treatment.

In the present study, we showed that, when 45S rDNA genes

are being highly transcribed, as in the case of young mice (Ma and

Nagata, 1990), all of these genes were found completely attached

to the nuclear matrix, while with development and aging, their

copies are progressively switched off and consequently detached

from the nuclear matrix. At the same time, the non-coding

telomeric sequences were found detached from the nuclear

matrix irrespective of the age or developmental state of the

animal, but associated with the nuclear envelope, thus supporting

the idea that non-coding regions probably do not associate with

the nuclear matrix.

Author contribution

Alberto Moraes carried out FISH experiments, microscopy, image

processing, statistics and drafted the manuscript. Mateus Mondin

and Margarida Aguiar-Perecin carried out FISH experiments and

drafted the manuscript. Marcelo Beletti carried out image

processing and image analysis and drafted the manuscript. Ana

Guaraldo provided animal facilities and carried out bioterism

services. Maria Luiza Mello co-ordinated the study and drafted the

manuscript.

Acknowledgements

We are grateful to Dr Nick Gilbert from the Cancer Research

Centre (Edinburgh, U.K.) for the critical reading of the manuscript

prior to submission.

Funding

This study was part of a thesis presented by A.M. to the Institute of

Biology, UNICAMP, in partial fulfillment of the requirements for a

Ph.D. degree and was supported by grants from the Brazilian

National Council for Research and Development (CNPq, grant

number. 470587/03-2), the Sao Paulo State Research Foundation

(FAPESP, grant number 2006/00066-8) and Brazilian Research

and Teaching Foundation (CAPES). A.M. and M.L.M. received

Ph.D. and research fellowships respectively, from CNPq. M.M.

received a Pro-Doc fellowship from CAPES.

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Received 8 December 2009/ 6 May 2010; accepted 2 June 2010

Published as Immediate Publication 2 June 2010, doi 10.1042/CBI20090457

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age-related association of rdna and telomeres with the

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