Archives of Andrology: Journal of Reproductive Systems, 54:1–10, 2008Copyright r Informa Healthcare USA, Inc.ISSN: 0148-5016 print/1521-0375 onlineDOI: 10.1080/19396360701876849

Research Article

5Sperm Chromatin Released by Nucleases

In human spermatozoa, 15–20% of histones are retained in the nucleus to

coexist with protamines. Hypothetically, nucleohistone regions of sperm

chromatin mark DNA sequences for distinctive processing during fertilization

10and early embryogenesis. The structural organization and molecular composi-

tion of nucleohistones in human spermatozoa is poorly studied. Here, we

isolate and characterize fractions of sperm chromatin that are solubilized by

endogenous and micrococcal nucleases. Chromatin isolated by either nuclease

have a nucleosomal organization with the periodicity of B195bp (endogenous

15nuclease digest) and B189bp (micrococcal nuclease digest), which is similar to

that of somatic cells. A distinct feature of sperm nucleohistone is its specific

compact supra-nucleosomal organization that was demonstrated by two-

dimensional electrophoresis and by atomic force microscopy. The latter tech-

nique showed compacted fiber arrays composed of globular particles with the

20prevailing diameter of B16nm. A rough estimation indicates that histones may

cover continuous stretches of 450kbp of sperm DNA. This initial character-

ization of sperm chromatin solubilized by nucleases is important for our

understanding of the bipartite structural organization of the paternal genome.

KEYWORDS chromatin, endogenous nucleases, human sperm25

INTRODUCTION

In the nucleus of mammalian sperm, DNA is tightly packed by prota-

mines, forming a protected and inert conformation of chromatin ready for

30fertilization. Recent studies have demonstrated that an elementary structural

unit of the sperm nucleoprotamine contains B50 kb of DNA compacted by

protamines into a toroid with outer diameter of approximately 900 A, a

thickness of 200 A, and a central hole with a diameter of 150 A [Balhorn et al.

1999]. Human spermatozoa are unique among the mammals in that along

35with protamines, a significant amount (15–20%) of histones are retained in

the nucleus [Puwaravutipanich and Panyim 1975; Gusse et al. 1986;

Gatewood et al. 1990]. Accordingly, a small fraction of chromatin organized

into nucleosomes has been detected in human spermatozoa using digestion

with micrococcal nuclease [Banarjee et al. 1995; Zalenskaya et al. 2000].

40This dual organization of the chromatin in the human sperm has evoked

considerable interest. It has been proposed that the distribution of histones

Igor B. Nazarov

The Jones Institute for

Reproductive Medicine, Eastern

Virginia Medical School, 601

Colley Ave., Norfolk VA, 23507

Luda S. Shlyakhtenko and

Yuri L. Lyubchenko

The UNMC Nanoimaging Core

Facility, Department of

Pharmaceutical Sciences

College of Pharmacy, University

of Nebraska Medical Center,

986025 Nebraska Medical

Center, Omaha, NE 68198-6025

Irina A. Zalenskaya and

Andrei O. Zalensky

The Jones Institute for

Reproductive Medicine, Eastern

Virginia Medical School, 601

Colley Ave.,Q1 Norfolk VA, 23507

Abbreviations: AFM: atomic forcemicroscopy; EDTA: ethylenediaminetetraacetic acid; EN: endogenousnuclease; MN: micrococcal nuclease;PAGE: polyacrylamide gelelectrophoresis; PBS: phosphatebuffered saline; PMSF:phenylmethylsulphonylflouride; SDS:sodium dodecyl sulfate; TRIS:trishydroxymethylaminomethane.

Received 26 July 2007; accepted13 October 2007.

Address correspondence to Andrei O.Zalensky, The Jones Institute forReproductive Medicine, EasternVirginia Medical School, 601 ColleyAve., Norfolk VA, 23507. E-mail:zalensao@evms.edu

1

and protamines along paternal DNA is not random,

and this may provide a potential mechanism for

differential gene expression during fertilization and

45 early embryogenesis [Gatewood et al. 1987; Wykes

and Krawetz 2003; Miller et al. 2005]. This hypothesis

is corroborated by a detailed study of the histone

distribution among the members of the b-globin

gene family in the sperm chromatin, in which

50 histones were found to be associated only with

the genes that are expressed in the embryo

[Gardiner-Garden et al. 1998].

Digestion of hamster sperm chromatin with

DNAse I [Sotolongo et al. 2003] or digestion of

55 mouse, hamster, and human sperm with endogen-

ous nucleases [Pittoggi et al. 1999; Sotolongo et al.

2003; 2005] revealed long stretches of sheltered DNA

40–50 kb long. It has been suggested that the

nuclease resistant regions of sperm chromatin corres-

60 pond to the microscopically observed nucleo-

protamine toroids, while the residual sperm histones

are associated with nuclease sensitive inter-toroid

linkers [reviewed in Ward and Ward 2004].

Major efforts have been directed to the portrayal

65 of limited DNA digests remaining in the sperm

nuclei pellet and characterization of endogenous

nucleases (EN) involved [Shaman et al. 2006].

Molecular and structural characterization of the

soluble chromatin has not been performed, and this

70 is the subject of the current study.

RESULTS

Soluble Chromatin Released byEndogenous Nucleases

Permeabilized human sperm cells were incubated

75 at 371C in the presence of divalent cations

(Caþþ þMgþþ ) for different periods of time, which

resulted in auto-digestion of the nuclear DNA.

Chromatin release in low ionic strength buffer was

not observed before 30min of incubation. Maximum

80 release (1–3% of total DNA) was at 2 h of incubation.

Electrophoretic separation of the liberated chromatin

(fractions S1 and S2, see scheme of experimental

design Fig. 1) under nondenaturing conditions

revealed a smear of chromatin with a low mobility

85 (Fig. 2A). In these conditions, S2 chromatin demon-

strated noticeably higher mobility than S1 chromatin.

Second dimension electrophoresis of S1/S2 chro-

matin in the presence of SDS exposed a nucleo-

some-like ladder of DNA fragments (thin arrows in

90Fig. 2B) emerging from the leading region of the

chromatin smear. We conclude that the chromatin in

S1 and S2 fractions have been cut into nucleosome-

like particles, which under conditions of native

electrophoresis were kept together in a large

95nucleoprotein complex. Hidden double-stranded

DNA cuts become detectable during electrophoresis

in the second dimension due to disruption of DNA-

protein interactions by SDS. Such behavior of sperm

chromatin is dissimilar to that of a fragmented

100chromatin isolated from somatic cells. High mole-

cular weight complex of sperm chromatin estimated

by electrophoretic mobility, may contain about 50

nucleosome-like particles.

In contrast, the trailing component of the chro-

105matin smear in the 1st dimension does not show

periodical DNA fragmentation in the 2nd dimension,

but produces an extended DNA streak (thick arrows

in Fig. 2B). Thus, EN revealed two types of the

soluble sperm chromatin that differ in their structural

110characteristics.

To estimate the periodicity of DNA fragments in

nucleosomal-type chromatin, DNA isolated from S1

and S2 fractions was separated in 1.5% agarose gel

(Fig. 2C). The values for the nucleosomal DNA

115repeat lengths were obtained from the slope of the

regression line in a plot of fragment size versus band

number. 100 bp DNA ladder was used as the

standard. The average from 10 experiments is

FIGURE 1 Experimental scheme of soluble chromatin frag-

ment isolation from human sperm.

I. B. Nazarov et al. 2

195.3� 3.5SD bp (p<0.0001), a value close to the

120 nucleosomal repeat in human somatic cells.

Chromatin protein composition was analyzed

using SDS electrophoresis. Separation of proteins

from S1 and S2 chromatin did not reveal noticeable

differences between them, therefore only data for

125 the S2 fraction are provided. Figure 3A shows

proteins of the S2 fraction separated by the 2D

electrophoresis (first dimension: native gel; second:

15% PAGE-SDS gel). This gel demonstrated the

presence of proteins with the range of molecular

130 weights corresponding to histones, shown by a

bracket in Fig. 3A. Histones present in the S2

fraction were confirmed by immunoblotting with

antibodies against core histones H4 and H3 after

one-dimensional SDS-PAGE separation (Fig. 3B).

135 Importantly, chromatin fractions solubilized by EN

were free of protamines, which remained associated

with the nuclear pellet, as was demonstrated by

electrophoresis in acetic acid/urea gel (Fig. 3C).

Imaging of Chromatin Solubilized by140 EN using Atomic Force Microscopy

To acquire more information about structural

organization of the sperm chromatin released by

EN, we performed imaging of soluble sperm

chromatin using atomic force microscopy (AFM).

145Figure 4A demonstrates typical images of the S2

fraction in low ionic strength buffer (10 mM TE, Q21 mM

EDTA), Fig. 4B presents matching three-dimensional

images. AFM revealed compact chromatin fibers of

different length. The fibers are 27� 8 SD nm thick

150(range15–42nm) and composed of single (Fig. 4A,

B-5) or double (Fig. 4A, B-1, 2) rows of spherical

globular particles. A majority of the globes have a

mean diameter of 16� 2nm. The globes are tightly

packed and no linker DNA is visualized in the AFM

155images of sperm chromatin, in contrast to relaxed

reference chromatin of chicken erythrocytes imaged

using the same conditions (Fig. 4C).

Spheres, located at the less condensed areas of the

sperm chromatin (arrows in Fig. 4A, B-4) have dia-

160meters of B8 nm and lower in height. We propose

that these smaller particles are nucleosomes since

they are similar to the B8–10 nm diameter globules

in erythrocyte chromatin (thin arrows in Fig. 4C).

Figure 5A illustrates a cross-section examination

165of the chromatin fragments. Measurements of

center-to-center distance of adjacent particles along

the chromatin fiber path demonstrated a pronounced

periodicity within a fiber. Frequency distribution

histogram of the ‘‘center-to-center’’ distance

170(Fig. 5B) showed a Gaussian distribution with the

FIGURE 2 Characterization of chromatin solubilized by endogenous nucleases. Sperm nuclei were incubated in the presence of 2 mM

CaCl2, 2 mM MgCl2 during 2 h at 371C. A – Electrophoresis of chromatin in native conditions: 0.6% agarose gel on 0.5�TBE buffer;

Ethidium Bromide staining. Molecular weight markers (two left lanes): 100 bp Ladder (Bioline) and l/Hind DNA digest. B – Two-

dimensional electrophoresis. First dimension (top), native gel as in A; second dimension, 1.5% agarose gel. Ethidium Bromide staining.

Thin arrows show fragments of nucleosomal DNA, thick arrows – DNA lacking nucleosomal periodicity. C – Typical electrophoretic

patterns of DNA isolated from S1 and S2 fractions, which were used to determine nucleosomal repeat lengths: 1.5% agarose gel; DNA

marker is 100 bp Ladder (Bioline).

3 Sperm Chromatin Solubilized by Nucleases

major peak corresponding to 21.7� 0.5 nm and

smaller satellite peaks at 14nm and 31nm.

Sperm Chromatin Accessible toMicrococcal Nuclease

175 We have demonstrated that in human sperm, EN

action became noticeable after an extended lag

period, about 30 min–1 h in our experimental con-

ditions. This is in agreement with the timing of the

nuclease resistant DNA accumulation in sperm

180 reported earlier [Sotolongo et al. 2005]. At the next

step, we were interested in comparing the EN

pattern of digestion with that of micrococcal

nuclease (MN), traditionally used for analysis of

chromatin structure in somatic nuclei. In contrast to

185 EN, MN activity manifested almost immediately.

After the first minute of sperm nuclei incubation

with nuclease, a smear of high molecular weight

DNA was registered, which was quickly processed

into a nucleosomal-like DNA ladder after five

190 minutes (Fig. 6A, left panel). DNA of only mono-

nucleosomal size was present (Fig. 6A) after more

prolonged treatment with MN (30 min–1 h). The

amount of DNA in the soluble chromatin after

treatment with MN was 1–4% of total nuclear DNA

195 and varied between donors (lanes D1 and D2 in Fig.

6B). In a parallel experiment using the same sample

of sperm nuclei, a nucleosome-like ladder generated

by EN appeared much later, after 1 h of incubation

(Fig. 6A, right panel).

200 Nucleosomal DNA repeat lengths calculated from

several independent experiments were 189� 5 bp

and 195� 5 bp for chromatin solubilized by MN and

EN, respectively. While these values of nucleosomal

repeat are very close, it is quite clear from the

205electrophoretic analysis (Fig. 6B) that the mobility of

the equivalent particles (e.g. di- tri- nucleosomes,

etc.) in MN and EN digested chromatin are different.

DNA of the corresponding chromatin fragments

released by digestion with EN was consistently

210longer, which may indicate that EN trims the ends of

nucleosomal fiber to a less extent.

DISCUSSION

In the early 1970s, it was found that in various

types of somatic cells Caþþ/Mgþþ dependent

215endogenous nucleases cleave chromosomal DNA

in a periodical manner: the cleavage sites being

separated by B200 bp [Hewish and Burgoyne 1973].

The same results were obtained using micrococcal

nuclease [reviewed in Felsenfeld 1978]. These

220periodical particles, or nucleosomes, appeared

under electron microscopy as beads on a string.

The nucleosome contains core and linker domains

that are structurally distinct. In the core, DNA of

invariable 146 bp lengths is wrapped around histone

225octamer. Linker DNA is associated with histones

belonging to the H1 family and its length varies

depending on participating histone H1 variant

[reviewed in Woodcock et al. 2006]. Chromatin

fragmented by EN or MN, as well as DNA isolated

230from the fragments, can be resolved by electrophor-

esis and visualized as nucleosomal particles or a

DNA nucleosomal ladder [Albright et al. 1980;

Pennings 1997].

FIGURE 3 Proteins in chromatin solubilized by endogenous nucleases. A – Chromatin proteins separated using two-dimensional

electrophoresis. First dimension – native agarose gel; second dimension – 15% SDS PAGE. Silver staining. B – Immunoblotting of

chromatin proteins separated in 15% SDS PAGE using antibodies against histones H3 and H4. C – Separation of chromatin proteins in

acetic acid/urea PAGE. Coomassie staining.

I. B. Nazarov et al. 4

EN activity in sperm cells has been demonstrated

235 for several mammalian species [Maione et al. 1997;

Pittogi et al. 1999; Sotolongo et al. 2003; 2005]. In

contrast to somatic, most of the human sperm chro-

matin is cleaved by the EN into large solubilization-

resistant fragments that contain DNA about 45–50 kb

240 long [Sotolongo et al. 2003; 2005].

Here we have demonstrated that up to 5% of

human sperm DNA was solubilized by EN activity as

a nucleoprotein that did not show a somatic type

organization periodicity on non-denaturating chro-

245matin electrophoresis and appeared as a large low

mobility complex (Fig. 2A). However, after dis-

sociation of the proteins, DNA released from this

complex was visualized as a nucleosomal ladder

(Fig. 2B, C; Fig. 6). Similar soluble complexes were

250produced by exogeneous MN (data not shown).

Immunoblotting data showed the presence in the

released chromatin of a complete compliment of

the core histones, thus supporting its nucleosomal

FIGURE 4 Typical AFM images of human sperm chromatin fibers obtained by digestion with endogenous nucleases. Chromatin was

deposited on mica and imaged in air. Heights are indicated by varying shades of color with low regions in dark reddish brown and higher

regions in increasingly lighter tones toward white. A – two-dimensional presentation; scale bar 120 nm. B – three-dimensional images of

the fibers 1–6 shown in A. C – comparison of chicken erythrocyte and sperm chromatin fibers. Chicken erythrocyte chromatin was

obtained using MN treatment of erythrocyte nuclei. Scale bar 20 nm.

5 Sperm Chromatin Solubilized by Nucleases

nature (Fig. 3B and unpublished results). Impor-

255 tantly, protamines remained in non-solubilized

chromatin fraction (Fig. 3C) associated with nuclease

resistant and insoluble toroids. In the sperm chro-

matin released by EN and MN nucleases, nucleoso-

mal DNA periodicity was estimated as 195 bp and

260 189 bp respectively, which is similar to the nucleo-

somal repeat of somatic cells. This DNA repeat value

strongly suggests that core particles are connected

by a linker DNA, which in somatic cells, is organized

by histones of the H1 family. Importantly, histone

265 H1 is involved in the formation of chromatin higher

order structure. However, no histone H1 has been

detected, in agreement with earlier reports on the

absence of H1 protein in mature human spermato-

zoa [Samuel 1978; Gatewood et al. 1990]. The

270 absence of histone H1 together with the fact that

DNA periodicity can be revealed only after removal

of the associated proteins are specific features of

sperm chromatin. The data suggest that in human

sperm, nucleosomes are held together within

275 large nucleoprotein complexes by yet unidentified

protein(s) that might be associated with inter-core

(linker) DNA.

Comparative kinetics of sperm chromatin diges-

tion with EN and MN (Fig. 5A) exhibited two modes

280of the chromatin release from the nuclei. MN pro-

duced soluble chromatin within several minutes

after application to sperm nuclei; EN activity became

noticeable later, after B30 min of incubation. These

differences may reflect the existence of two distinct

285chromatin domains associated with histones. It is

possible that MN preferentially attacks peripheral,

therefore more sensitive, areas of sperm chromatin.

Indeed, it has been shown that in sperm, MN attacks

telomeres but not central areas of chromosomes

290[Zalenskaya et al. 2000] and it is known that telo-

meres are localized at the sperm nucleus periphery

[Zalensky et al. 1995]. It is also possible that endo-

genous nucleases have specific intranuclear locali-

zation, which might be reflected in the pattern of

295digestion. Alternatively, different patterns of diges-

tion with EN and MN may be a consequence of the

existence in the human sperm of diverse types of

FIGURE 5 Analysis of sperm chromatin fibers AFM images. A – Trace of chromatin fiber sectioning used to determine center-to-center

distances between globular structures (left). Corresponding measurement of heights distribution (right). B – Cumulative frequency

distribution histogram of the center-to-center interglobular distances.

I. B. Nazarov et al. 6

nucleohistone structural organization. We might

expect that better understanding will be provided

300 after the nature of human sperm EN is established;

experiments are underway [Yamauchi et al. 2007].

Previous studies utilizing MN digestion of human

sperm, much shorter nucleosomal DNA repeat of

B150 bp [Banerjee et al. 1995] or the absence of the

305 periodical chromatin structure [Sakkas et al. 2002]

have been reported. Reasons for such discrepancies

might be connected with different procedures for

nuclei isolation and experimental conditions under

which MN action was executed. In addition, the

310 short nucleosomal repeat [Banerjee et al. 1995] was

established using Southern hybridization with the

DNA probe of unidentified nature isolated from

the core particle obtained by deep digestion with MN.

It is possible that the majority of sequences in

315 such probe originated from the telomere. Then,

the 150 bp repeat established by Banerjee and

co-authors [1995] is the same as the 146 bp

periodicity of the telomere chromatin in human

sperm [Zalenskaya et al. 2000]. Finally, our data

320are in agreement with 175–190 bp nucleosomal

periodicities determined in the mouse sperm [Pittogi

et al. 1999].

As can be seen from DNA electrophoresis (Fig. 6B),

DNA in di- and higher nucleosomal oligomers,

325resulting from EN activity is always longer than in

corresponding n-mers produced by MN. A lower

end trimming activity of EN as compared to that of

MN might explain this difference.

Further insight into the structural organization of

330the soluble sperm chromatin was obtained using

atomic force microscopy. This robust technique

provides a topographic picture of macromolecules

and in many cases is an approach of choice to study

chromatin organization [Bustamante et al. 1997; Lohr

335et al. 2007]. AFM imaging of the S2 chromatin

demonstrated compact fiber arrays, variable in width

(from 1 to 42 nm), and composed of globular parti-

cles (Fig. 4). These globules are mainly uniform in

size and have a prevailing diameter of about 16 nm.

340Partial unfolding observed in some parts of the

chromatin fibers revealed smaller globules with a

FIGURE 6 Periodical nucleosomal structure of soluble chromatin isolated from sperm due to digestion with endogenous or

micrococcal nucleases. A – Kinetics of nuclease action. DNA electrophoresis in 1.5% agarose gel, Ethidium Bromide staining. Incubation

time is shown on the top. DNA marker is 100 bp ladder (New England Biolabs). B – DNA electrophoresis in 1.5% agarose gel; D1 and

D2 – different donors.

7 Sperm Chromatin Solubilized by Nucleases

diameter B8 nm. These globules most likely corre-

spond to the core nucleosome particles. We propose

that large globes, such as presented in Fig. 4A, B-5,

345 are composed of 4–6 nucleosomes as visualized in

the areas where chromatin is unfolded (Fig. 4A, B-4).

In the compact fibrils of sperm chromatin, a

center-to-center distance of 21.7� 0.5 nm was

observed for adjacent globules (Fig. 5). In the fibers

350 of chicken erythrocyte chromatin, particle center-to-

center distance is 31.4 nm, of which B22 nm

belongs to internucleosomal linker DNA and

B10 nm is the diameter of the nucleosome core

particle [D’Erme et al. 2001]. Thus, histone contain-

355 ing chromatin of human sperm nuclei is organized

into much more compact structures as compared to

chromatin of somatic cells.

Chromatin fragments observed in AFM experi-

ments are heterogeneous in size and estimates sug-

360 gest that more than 50 nucleosomes may be

involved. This covers over 12 kbp of DNA, which

correlates well with the estimation of the size of the

EN-released complexes by electrophoretic mobility

on DNP electrophoresis (Fig. 2).

365 It is not known if the endogenous and exoge-

neous nucleases target the same areas of chromatin,

and the important question to be answered is

how the nuclease sensitive (nucleohistone) areas

are distributed along the human genome in

370 spermatozoa. Research using microarray profiling of

histone/protamine distribution in spermatozoa is in

progress (D. Miller, University of Leeds, personal

communication) and experiments using FISH

localization of the histone-associated sequences

375 are underway in our laboratory. It is anticipated that

the combination of these approaches will help to

define more accurately molecular organization of

DNA in spermatozoa and shed some light on its

significance for fertilization and early embryonic

380 development.

MATERIALS AND METHODS

Biological Material

Semen samples were collected from healthy

donors after at least 2 days of abstinence. Written

385 informed consent was obtained from all participants.

The samples were collected by masturbation and

allowed to liquefy at room temperature. The

samples were kept frozen (at �801C) until processed

for analyses.

390Isolation of Sperm Cells

All procedures were performed at 41C. Frozen

semen samples were thawed, diluted with ice cold

phosphate buffered saline (PBS) containing 0.5 mM

phenylmethylsulphonylflouride (PMSF), vortexed,

395filtered through 2 layers of Miracloth, and pelleted

by centrifugation (850� g for 10 min). The pellet

was washed with PBS two more times. Presence of

contaminating cells was evaluated by microscopic

analysis. Samples, which had more than 1% of

400somatic or immature germ cells, were excluded from

experiments.

Isolation of Chromatin Solubilizedby Nucleases

The general experimental scheme is outlined in

405Fig. 1. Washed sperm cells were resuspended in

PBS/PMSF containing 0.5% Triton X-100, incubated

on ice for 10 min, and washed twice with PBS/PMSF.

Partially demembranized cells were resuspended in

the reaction buffer comprised of 10 mM Tris-HCl, pH

4108.0, 2 mM CaCl2, 2 mM MgCl2, 10 mM dithiothreitol,

protease inhibitor cocktail (Roche) at a DNA

concentration of 3–5 mg/ml. Cells were incubated

at 371C for the times indicated, aliquots were

immediately cooled on ice, then centrifuged for

4155 min at 500 g, 401C. Supernatant (S1) was collected

and EDTA was added to 5 mM. The pellet was

resuspended in 2 volumes of TED buffer (10 mM

Tris-HCl, Q3pH 8.0, 5 mM EDTA, 10 mM dithiothreitol)

and extracted on ice for 0.5 h. The sample was

420centrifuged for 5 min at 500 g at 401C and super-

natant (S2) was collected.

In the micrococcal nuclease (MN) experiments,

20 U of the enzyme was added per mg of DNA,

incubation was at 401C. The reaction was stopped by

425the addition of EDTA, digested nuclei were cen-

trifuged, and supernatant (S1þ S2) was collected.

DNA concentrations in cell suspensions and S1/S2

extracts were determined by spectrometry measur-

ing OD at 260 nm of the alkali-denatured samples

430and by fluorimetry using emission at 458 nm in the

presence of Hoechst 33258.

I. B. Nazarov et al. 8

One-dimensional DNAElectrophoresis and Determination

of Nucleosomal DNA Repeat435 Length

DNA was isolated by a standard phenol-

chloroform extraction. In brief, samples were treated

at 371C with 100mg/ml proteinase K in the presence of

0.5% SDS overnight, followed by phenol/chloroform

440 extraction and ethanol precipitation. DNA was

dissolved in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA

(TE buffer) and subjected to electrophoreses in 1.5%

agarose gel in 1�TAE buffer (40 mM Tris-HClQ4 , pH

8.0, 20 mM acetic acid, 1 mM EDTA). DNA was

445 stained with ethidium bromide; DNA fluorescence

was recorded using Kodak Image station 440. Sizes

of DNA fragments were determined from calibration

curve generated using 100 bp DNA ladder. Calcula-

tion of nucleosomal periodicity was described in

450 Results.

Protein Electrophoresis andWestern-blotting

Proteins were analyzed using two electrophoretic

systems: 15% SDS-PAGE according to Laemmli [1970]

455 or 15% acetic acid/urea PAGE according to a

modified method of Panyim and Chalkley [Hurley

1977]. The latter allows visualizing protamines that

precipitate in the presence of SDS and therefore

cannot be resolved in SDS-PAGE. Proteins were

460 stained with Coomassie R-250. For identification of

histones, the proteins separated in 15% SDS-PAGE

were transferred to a PVDF membrane. Immunode-

tection of histone fractions was performed using

polyclonal monospecific antibodies as described

465 [Zalensky et al. 2002].

Native Electrophoresis of SolubleChromatin

Native electrophoresis of chromatin was per-

formed using 0.6% agarose gel on 0.5�TBE bufferQ5 .

470 Samples were prepared by adding 6�DNA loading

buffer to fractions of soluble chromatin. The gel was

run at 6 V per cm of length for 3 h. DNA was stained

with ethidium bromide and image acquisition was

performed as described above.

475Two-dimensional Electrophoresis

After electrophoresis, agarose strips were cut out,

incubated in 1X TAE containing 1% SDS during

15 min at room temperature, and were placed on the

start of the 1.5% agarose/1X TAE gel. Electrophoresis

480was performed at 8 V/cm during 1–2 h, and DNA

was stained with ethidium bromide.

Protein analysis used 15% SDS PAGE for separa-

tion in the second dimension. Proteins were silver

stained using SilverQuest staining kit (Invitrogen

485Inc). Images of DNA or proteins were acquired using

Kodak Image Station 440.

Atomic Force Microscopy

Samples were prepared using mica surface (APS

mica), modified with 1-(3-aminopropyl) silatrane as

490described earlier [Shlyakhtenko et al. 2003]. The

chromatin samples were diluted 10 to 20 fold in TE

and 5 mL of the sample was deposited on APS mica.

After 2 min, the sample was rinsed with deionized

water and dried with argon. Images were acquired

495in air using a MultiMode SPM NanoScope IV system

(Veeco/Digital Instruments, Santa Barbara, CA)

operating in tapping mode. Tapping Mode Etched

Silicon Probes (TESP; Veeco/Digital Instruments,

Inc.) with a spring constant of about 42 N/m and a

500resonant frequency between 270–320 kHz were

used. Image processing and cross-section measure-

ments were performed using Femtoscan (Advanced

Technologies Center, Moscow, Russia).

ACKNOWLEDGEMENTS

505This work has been supported by an EVMS

Institutional Grant and in part by National Institutes

of Health Grant HD-042748 to A. O. Z. We thank

Dr. S. Gitlin for proofreading the manuscript.

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