The Role of Histone H4 Biotinylation in the Structure of
Nina A. Filenko1, Carol Kolar2, John T. West4, S. Abbie Smith4, Yousef I. Hassan3, Gloria E. O. Borgstahl2,
Janos Zempleni3, Yuri L. Lyubchenko1*
1Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska, United States of America, 2The Eppley Institute for Research in
Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, United States of America, 3Department of Nutrition and Health Sciences, University
of Nebraska, Lincoln, Nebraska, United States of America, 4The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America
Background: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene
transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with
repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and
that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation.
Methodology/Principal Findings: To test this hypothesis we used atomic force microscopy to directly analyze structures of
nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in
the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant
(p,0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the
classical 147 bp length of nucleosomal DNA.
Conclusions/Significance: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with
histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene
silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the
effects of various epigenetic modifications of nucleosomes, in addition to biotinylation.
Citation: Filenko NA, Kolar C, West JT, Smith SA, Hassan YI, et al. (2011) The Role of Histone H4 Biotinylation in the Structure of Nucleosomes. PLoS ONE 6(1):
Editor: Vladimir Uversky, University of South Florida College of Medicine, United States of America
Received September 21, 2010; Accepted December 15, 2010; Published January 27, 2011
Copyright: ? 2011 Filenko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was funded by the National Science Foundation-Experimental Program to Stimulate Competitive Research EPS-0701892. Additional
support was provided by Nebraska Research Initiative (to YLL), the University of Nebraska Agricultural Research Division (Hatch Act), and National Institutes of
Health grants DK063945, DK077816, and DK082476 (all to JZ) and National Cancer Institute Eppley Cancer Center Support Grant [P30CA036727] (to GEOB and
YLL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Modifications of histones are among the epigenetic marks that
influence gene expression. Distinct histone modifications of one or
more tails have been proposed to act sequentially or in
combination to form a ‘histone code’ that is read by other
proteins to bring about distinct downstream events . Posttrans-
lational modifications of histone tails include methylation ,
phosphorylation , acetylation , ubiquitination  and
biotinylation . Our understanding of the molecular and
structural mechanisms of how these modifications impact
transcriptional activity remains inadequate. The demonstration
that acetylation of histones affects chromatin compaction at the
mononucleosomal  and trinucleosomal  levels provided
initial mechanistic insight into the relationship between nucleo-
some structure and gene expression. Using fluorescence resonance
energy transfer (FRET) analysis, Gansen et al. demonstrated that
histone acetylation decreased stability of mononucleosomes .
Histone H4 acetylation at lysine 16 (K16Ac-H4) was shown to
impact chromatin structure by inhibiting the formation of compact
30-nanometer–like fibers and to impede the ability to form cross-
fiber interactions . In addition, K16Ac-H4 inhibits the
chromatin assembly process and interferes with the function of
the ATP-dependent chromatin assembly and remodeling factor,
ACF. Recently, single-pair FRET was used to probe conforma-
tional changes in mononucleosomes induced by DNA methylation
. These studies showed that CpG methylation leads to the
compaction of nucleosomes and nucleosome structural rigidity.
Most recently, a novel posttranslational modification of
histones, biotinylation, was discovered by one of the co-authors
[6,12,13] and independently confirmed in another laboratory
. More recently, using LC/MS/MS, a third laboratory
detected large quantities of biotinylated histone H4 in Candida
albicans . Initially, a mechanism for enzymatic catalysis of
histone biotinylation by biotinidase was proposed by Wolf and co-
workers based on in vitro studies . However, recent studies used
recombinant histones and holocarboxylase synthetase (HCS) to
unambiguously demonstrate that HCS has histone biotinyl ligase
activity , and it is now evident that biotinylation of histones is
mediated preferentially by HCS . Biotinylated histones have
been detected in human cells  and distinct histone biotinyla-
tion sites were defined using peptide and in vivo studies , .
PLoS ONE | www.plosone.org1January 2011 | Volume 6 | Issue 1 | e16299
Ten distinct histone biotinylation sites have been identified: five in
histone H2A, three in histone H3 and two in histone H4. Histone
H4 can be biotinylated at amino terminal lysines 8 (K8Bio-H4)
and 12 (K12Bio-H4) .
Several lines of evidence suggest a functional role for histone
biotinylation in gene silencing, cellular responses to DNA damage,
and cell proliferation as reviewed elsewhere . Briefly, K8bio-
H4 and K12bio-H4 localize to alpha-satellite repeats in pericen-
tromeric regions, as well as to transcriptionally repressed
chromatin loci . K12bio-H4 is highly enriched in telomeric
repeats from human lung IMR-90 fibroblasts, where one out of
three H4-histones is biotinylated at K12 . Low abundance of
biotinylation marks has been linked with cleft palate in mice 
and genome instability in humans .
Based on the biochemical evidence above, we hypothesized that
H4 biotinylation alters the structure of nucleosomes and reduces
the accessibility of DNA to transcriptional machinery. Biophysi-
cally testing this concept was a major goal of this paper. We have
recently shown that high-resolution AFM imaging can detect the
subtle conformational changes in nucleosomes and reveal their
dynamic character [23,24]. In the current work, the same AFM
technology was employed to quantify histone biotinylation-
dependent changes in nucleosome structure. We report that
K12-biotinylation in histone H4 causes a significant change in
nucleosome structure leading to a ,15% increase in the amount
DNA wrapped around nucleosomes. We propose that this effect
provides a partial mechanistic explanation for the correlation
between histone biotinylation and gene silencing.
Similar to previous studies [23,24], the DNA template designed
for this work was a fragment of 353 bp DNA containing the
147 bp nucleosome positioning 601 sequence , flanked by two
arms of different lengths (79 bp and 127 bp). Differential arm
lengths enables mapping of the nucleosome position .
Depending on the number of DNA turns around the histone
core, the nucleosome will adopt one of several different
morphologies shown schematically in Fig. 1.The initial design
corresponds to the complex with one turn and the four other
conformations correspond to complexes with 1.25, 1.5, 1.75 and 2
turns. For clarity, Fig. 1 shows rotation of the long arm only,
although uniform wrapping of both arms occurs starting at a
position in the center of the 147 bp region, so the length of the
arms gradually decreases upon DNA wrapping. In addition, DNA
wrapping is accompanied by changes in the interarm angle. We
assigned a rotation angle of zero to the position of the long arm for
the complex with one turn. The conformation with 1.25 DNA
turns is characterized by a 90u rotation angle, and the complexes
with 1.5, 1.75 and 2 turns have the rotation angles 180u, 270u and
360u, respectively. These parameters were used in the procedure
of assigning of the nucleosome core particle (NCP) conformation.
Previous studies have demonstrated that histone proteins
produced in E. coli are competent to form nucleosomes with
DNA in vitro. For AFM studies we required significant quantities of
purified H4 with and without the biotin mark at K12. In addition,
we needed to be able to verify that biotinylation of H4 was only
present at the twelfth residue and not at alternate or additional
sites. Previous studies suggested that the E. coli HCS ortholog BirA
has histone biotinyl ligase activity and that recombinant histones
produced in bacteria could be biotinylated . Since the N-
terminal tail of histone H4 is solvent-exposed and contains several
lysine residues  that could be biotinylated by BirA, we used
site-directed mutagenesis to convert the codon for K12 to that for
Cys. Importantly, this mutation introduces the only Cys in the
entire recombinant H4 sequence. After expression in E. coli, the
undesired BirA-biotinylated minor fraction of recombinant histone
could then be removed from lysates by avidin chromatography,
and the unbiotinylated major fraction was subjected to sulfhydryl-
specific biotinylation of cysteine-12 in K12C-H4 in a chemical
reaction with maleimide-PEG2-biotin (K12Cbio-H4). The level of
chemical biotinylation of K12C-H4 was assessed by Western
blotting with streptavidin conjugates (Fig. S1, panel a) and anti-
biotin antibodies (Fig. S1, panel b). A faint biotin signal is
detectable in K12C-H4 purified from E. coli prior to avidin
chromatography (Fig. S1b, lane 1), consistent with low-level E. coli
BirA biotinylation of the heterologous H4 protein. No band was
observed in the purified fraction of histone K12C-H4, whereas a
strong signal was produced by chemical biotinylation with
maleimide-PEG2-biotin (Fig. S1, lane 3, both panels a and b).
Protein identity was confirmed using anti-H4 (Fig. S1, panel c).
Nucleosomes were formed with the 601 positioning sequence
(described above) in the presence of biotinylated K12CBio-H4 or
alternately with K12C-H4 that was not subjected to the maleimide
reaction. The other histone components, H2A, H2B, and H3 were
derived from E. coli and purchased from NEB. Wild-type or K12-
H4 was also acquired from NEB and was used in nucleosome
preparations to control for structural changes induced by the Cys
AFM imaging of mononucleosomes
As previously , nucleosomes were deposited on APS-mica,
rinsed, dried and imaged with AFM in air (Fig. 2 and Fig. S2). In
AFM images, the nucleosomes are visible as bright globules with
the DNA arms extending from both sides of the particles. Samples
prepared with WT histones and those with the use of K12C H4
were very similar. The yield of nucleosome sample in these
samples was 70–80% with the rest being the naked DNA. The
morphology of NCP is different when assembled from biotin-free,
native histone H4 as compared to those with biotinylated histone,
K12Cbio-H4. For instance, the number of molecules with 1.7–
1.75 turns with crossed DNA arms is 17% (2 molecules out of 12
total) and 31% (5 molecules out of 16 total), for the nucleosome
samples with native H4 and K12Cbio-H4, respectively. This is
further illustrated in Fig. 3 where enlarged images for the native
and biotinylated nucleosomal samples are shown. These images
can be interpreted in terms of different number of DNA turns
around the histone core, the number of DNA turns are marked
Figure 1. Schematics for various stages of the nucleosome
unwrapping. Nucleosome conformations are shown with different
number of turns, rotation angle and length of wrapped DNA.
AFM of Nucleosomes
PLoS ONE | www.plosone.org2 January 2011 | Volume 6 | Issue 1 | e16299
next to the nucleosome particles. The analysis of the images as
described below enabled us to characterize the structure of
nucleosomes in a number of the nucleosomal DNA turns (see
Materials and Methods S1, section II for details). The molecule
with 1.31 turn has a 110urotation angle, (110u+360u)/360u =1.31.
The molecule with 1.41 turn has almost parallel arms and rotation
angle of 147u. The arms of the molecules with 1.79 and 1.76 turns
are crossed with a rotation angle of 283u and 275u, respectively.
The arms form almost a straight line with rotation angle of 380u in
particle with 2.06 turns of DNA. Therefore, biotinylated
nucleosomes (Fig. 3b) compared to native nucleosome population
(Fig. 3a) is enriched with complexes with a large number of turns
with the mean value of turns being 1.61 and 1.72 for native and
biotinylated nucleosomes, respectively.
Analysis of AFM data
To assess the effect of biotin on the structure of nucleosome we
measured the following parameters of nucleosomes over a large
number of AFM images: (1) the length of the two protruding DNA
arms and (2) the angle between the DNA arms [23,24]. Then the
number of DNA turns around the histone core octamer was
calculated, The length of nucleosomal DNA wrapped around
octamer (nsDNA) was calculated by subtracting the lengths of both
free DNA arms from total length of uncomplexed DNA (see Fig.
Figure 4 compares the distribution of nsDNA length in
K12Cbio-H4 nucleosomes to those in native H4 and K12C-H4
controls. Each dataset was in the range of 100–110 complexes.
While lengths of nsDNA were similar in nucleosomes with native
H4 (panel a) and K12C-H4 (panel b), 49.861.5 nm, 48.861.4 nm
respectively, the length of nsDNA was greater in sample K12Cbio-
H4 (panel c), 56.661.1 nm. The difference between samples
native H4 and K12Cbio-H4 equaled 6.862.6 nm, which is larger
then the sum of standard errors and is statistically significant
(p,0.001), with degree of freedom of 198). The length difference
of nsDNA is ,20 bp in the length and is equivalent to extra 0.2
turns of nsDNA or to the increase of the number of turns per
octamer from 1.75 to almost 2 turns.
The effect of the histone H4 biotinylation on nucleosome
structure was reproducible (Fig. S4). Mean values for the nsDNA
lengths for native nucleosomes and K12Cbio-H4 NCP were
47.561.3 and 54.261.2 nm, respectively for second independent
set of samples. In this set of measurements the average difference
in the length of nsDNA was 6.762.5 nm, which is statistically
identical to the 6.862.6 nm obtained for the first set.
We confirmed our findings by using an alternative approach in
which the value of angle between the arms is used to calculate the
number of DNA turns [23,24]. Table 1 summarizes the results
based on angle measurements for two independent experiments.
The number of DNA turns in nucleosomes was greater for
K12Cbio-H4 compared with H4 and K12C-H4 controls. The
proportion of molecules with more than 1.5 turns was 55, 56 and
73% in samples H4, K12C-H4 and K12Cbio-H4, respectively.
Table 2 shows the differences in number of nucleosomal turns for
DNA between the native and biotinylated nucleosomes calculated
with both methods. The difference is 0.2 turns of nsDNA for both
sets of native and biotinylated nucleosomes when calculated based
on angle measurements. Thus, both procedures reproducibly
yielded similar results: biotinylation increases the length of DNA
Figure 2. Representative AFM scans of nucleosome core
particles. Nucleosomes were reconstituted using native histone H4
(a) or biotinylated histone K12Cbio-H4 (b). Images were acquired with
NanoScope IIId AFM system operating in Tapping mode. Scan sizes are
Figure 3. Representative enlarged AFM scans of NCP. Nucleo-
somes were reconstituted using non-biotinylated native histone H4 (a)
and biotinylated K12C histone H4 (b). The complexes are labeled with
the number of DNA turns around histone octamers. Scans sizes are
AFM of Nucleosomes
PLoS ONE | www.plosone.org3 January 2011 | Volume 6 | Issue 1 | e16299
associated directly with the nucleosome by ,13% that leads to the
increase in the mean number of DNA turns in nucleosomes from
1.75 (native) to about 2 (biotinylated mutant).
This work shows directly and unambiguously that biotinylation
of histone H4 at K12 leads to a statistically significant increase in
the length of DNA wrapped around the histone core octamer.
This change of the nucleosome structure is shown schematically in
Fig. 5. Compared to 147 bp length of nsDNA wrapped around
nucleosomes formed with non-biotinylated wt H4 or with K12C-
H4, biotinylation at position 12 increases the length of nsDNA to
an average of 167 bp, which corresponds to adding to nsDNA of
almost 0.2 nucleosomal turns. Such a substantial increase of the
length of wrapped DNA should lead to elevated stability of
nucleosomes and is congruent with functional studies demonstrat-
ing a role for histone biotinylation in transcriptional repression.
The conclusion on elevated stability of nucleosomes with increased
number of turns is supported by our recent time-lapse AFM
imaging data [23,24] on the dynamics of nucleosomes. These data
showed that nucleosomes with 2 turns are much more stable than
those with 1.7 turns. Therefore, biotinylation of H4 at position 12
leads to stabilization of nucleosomes, suggesting that this structural
change contributes to regulation of gene expression.
Similar to previous studies [23,24], individual nucleosomes
containing control H4 and K12Cbio-H4 vary in the number of
DNA turns around each histone core suggesting that biotinylated
nucleosomes, similar to controls, dynamically undergo transient
unwrapping-wrapping processes. However, comparison of the
histograms from all samples (Fig. 4) reveals that biotinylation
causes a uniform shift towards more condensed nucleosomal
structures across the entire histogram without a preferable shift to
any particular conformation. This observation suggests that
biotinylation of H4 does not lead to the formation of nucleosomes
with a particular number of turns, but rather that biotin-
containing nucleosomes maintain the ability to undergo conden-
sation and decondensation with a more condensed average
Based on crystallography data, the well-ordered domains in
histones mediate the strong interactions of the histone core with
DNA, but the N-terminus of histone H4 is unstructured and does
not contribute to DNA binding. We propose that biotinylation
stabilizes the structure of the N-terminus of histone H4, leading
to the formation of novel contacts with DNA and the other
histones that accommodate two additional DNA pitches in the
nucleosomal body. The magnitude of this effect is surprisingly
high, given that only one residue (C12) in one histone protein
(H4) was biotinylated. Indeed, biotin is capable of forming of
stable complexes with proteins and complexes of biotin with
avidin and streptavidin are among the strongest noncovalent
molecular associations (Kd,10215M). According to crystallo-
graphic data for biotin-avidin complexes, an array of polar and
aromatic residues in avidin is involved in the tight binding .
Several aromatic residues such as tryptophan, phenylalanine and
tyrosine, in the biotin-binding site of avidin form a ‘‘hydrophobic
box’’, in which the biotin molecule resides. As histones also
possess aromatic and polar amino acids, similar attractive
interactions can be formed between biotin and histone molecules
within the nucleosome particle. There are a large number of
potential candidates for such interactions, and crystallography
studies are needed to test this model. Apparently the significant
change in nucleosome structure can increases nucleosome
stability and thus provides additional contacts for binding of
DNA leading to increasing of the stability of nucleosomes or
alternately provides novel binding sites for repressive epigenetic
factors. Therefore we speculate that the elevated stability of
nucleosomes due to the increase of the length of nsDNA is at least
partially responsible for silencing of genes reported in previous
biological activity studies ,,.
We used a K12C mutant of histone H4 in our studies for
targeted biotinylation of position 12, while in vivo only lysine
residues are biotinylated. Our AFM studies suggest that nucleo-
somes composed of native H4 and non-biotinylated K12C-H4 had
similar conformations, implying that the K12C substitution does
Figure 4. Histograms for lengths of nucleosomal DNA (nsDNA)
wrapped around histone cores. Nucleosomes were reconstituted
using native H4 histone (a), K12C-H4 mutant (b) or K12Cbio-H4 histone
(c). It can be seen that in nucleosomes made with K12Cbio-H4 wDNA is
shifted towards higher value compared to samples reconstituted using
non-biotinylated native H4 or K12C-H4 mutant. The mean values of
nsDNA indicated with arrows were 49.8 nm 61.5 nm, 48.8 nm 61.4 nm
and 56.6 nm 61.1 nm for NCP containing native histone H4, K12C-H4
mutant, and K12Cbio-H4, respectively.
AFM of Nucleosomes
PLoS ONE | www.plosone.org4 January 2011 | Volume 6 | Issue 1 | e16299
not alter the conformation of H4. Thus it is likely that changes in
NCP conformation are solely due to the biotinylation mark. Note
that chemical biotinylation scheme used in this work (via PEG
linker) is different from the in vivo biotinylation in which biotin is
bound to the epsilon amino group of lysine. The difference in the
linker may contribute to the structural change of the nucleosome,
but the finding that biotin is required for the observed effect
suggests that biotinylation per se rather than the chemical bond is
critical in the nucleosome structural change.
In conclusion, we should add that studies during the past decade
have dramatically changed our view of the structure of chromatin
and of its key unit, the nucleosome, in particular. A static picture is
currently being replaced with a dynamic one, and single-molecule
techniques were instrumental in characterizing these dynamic
properties of nucleosomes. AFM is capable of characterizing
complex molecular system at the nanoscale level making it possible
to visualize directly the unwrapping process of nucleosomes. The
current work highlights the ability of AFM to identify structural
changes in nucleosomes induced by a local modification, such as
biotinylation, and thus paves the way for studies of effects of other
epigenetic modifications of nucleosomes.
Materials and Methods
Preparation of mutant histone H4
Amino acid lysine at position 12 (K12) in histone H4 was
mutated to cysteine (K12C-H4), using Quick-change mutagenesis
(Stratagene) according to manufacturer’s instructions, to generate
a target for subsequent chemical biotinylation with a sulfhydryl-
reactive reagent. The coding sequence of H4 histone from Xenopus
laevis in a pET3a vector system was used as a template. The
primers were  59-GGTAAAGGTGGTAAA GGTCTGGGT
TGCGGTGGTGCTAAACGTCAC-39 and (antisense) 59-GTG
CC-39 (corresponding to protein sequence KGGKGLGCGG
AKRH). pET3a-transformed E. coli strain BL21(DE3) (Novagen)
was grown to abs600=0.8 in 2XYT medium, and the expression
of K12C-H4 was induced with 0.4 mM IPTG at 37uC for 90 min.
The cell pellet was lysed by Emulsiflex in wash buffer (50 mM Tris
HCl, pH 7.5; 100 mM NaCl, 1 mM 2-mercapthoethanol) and
centrifuged at 23,000 g for 10 min at 4uC. The inclusion body
pellet was washed in wash buffer containing 1% Triton X-100.
The pellet was suspended in 1 ml dimethyl sulfoxide, stirred
30 min at RT and wash buffer containing 6 M guanidine
hydrochloride was added. K12C-H4 was purified on Superdex200
HiLoad 16/60 column, Prep Grade (GE Healthcare).
Biotin-depletion of H4 histone
Previous studies suggested that microbial BirA has enzymatic
activity to biotinylate recombinant histones, albeit at low levels
. Endogenously biotinylated K12C-H4 was removed using
avidin agarose resin (Pierce). Briefly, 3 mg of K12C-H4 in PBS
buffer were added to 2 ml of 50% resin slurry in PBS (equivalent
to 1 ml of settled gel) and incubated overnight at 4uC with shaking.
The sample was centrifuged for 1 min at 50006 g and the
supernatant, containing biotin-depleted histone, was used for
subsequentstudies in amount
of1 mg atconcentration
Chemical biotinylation of K12C-H4
K12C-H4 was biotinylated at C12 residue to produce
K12Cbio-H4 by using the sulfhydryl-reactive reagent Malei-
mide-PEG2-Biotin according to the manufacturer’s instructions
(Thermo Scientific). Note that histone H4 contains no cysteine
residues other than the C12 inserted by mutation. Before
biotinylation, any C12-C12 disulfide bonds between two K12C-
H4 molecules were reduced with 5 mM tris(2-carboxyethyl)pho-
sphine (TCEP) for 30 min at RT. After TCEP removal with
Microcon centrifugal filters (Millipore), molecular weight cutoff
3,000, a 20-fold molar excess of Maleimide-PEG2-Biotin was
added and samples were incubated at 4uC overnight. The protein
was purified from nonreacted Maleimide-PEG2-Biotin using
Microcon filters with molecular weight cutoff 3,000.
Preparation of nucleosomal DNA
DNA for nucleosome assembly was generated by PCR using
plasmid pGEM3Z-601 as a template, which codes for a high-
affinity nucleosome positioning sequence . The PCR reaction
(33 cycles of 94uC/30 s, 54uC/30 s, 72uC/30 s) was conducted in
buffer containing 2.5 mM MgCl2, 0.15 mM dNTPs and 0.016
U/ml of Taq DNA polymerase with the following primers: forward
primer 59-GEMf CGGCCAGTGAATTGTAATACG-39; reverse
primer GEMr 59-CGGGATCCTAATGACCAAGG-39.
Histone octamer assembly and purification
Histone octamers were assembled as follows . Procedure of
histone octamer preparation is given in supplementary materials.
Table 1. Comparison of number of turns of nucleosomal DNA (nsDNA) obtained from two independent samples of native H4 and
biotinylated H4 (K12Cbio-H4) nucleosomes.
Native H4 ncp, #1Native H4 ncp, #2K12Cbio-H4, sample #1
Number of turns of nsDNA1.6260.051.6160.041.8260.041.8160.06
Table 2. Differences in the number of nucleosomal turns for DNA between nucleosomes assembled using native H4 and
The number of turns of nsDNAK12Cbio-H4 - native H4, sample set #1K12Cbio-H4 - native H4, sample set #2
Based on the length of nsDNA0.2360.090.2360.08
Based on the angle measurements0.2060.090.2060.10
AFM of Nucleosomes
PLoS ONE | www.plosone.org5January 2011 | Volume 6 | Issue 1 | e16299
Octamers were separated from tetramer and dimer fractions with
size-exclusion chromatography (SEC) with Superdex 200 PC 3.2/
30 column (GE Healthcare) at 4uC. SEC fractions were analyzed
for purity and histone stoichiometry using SDS-PAGE. The gel
was stained using Coomassie Blue stain. Fractions containing
histones H2A, H2B, H3 and H4 in approximately equal ratios
were pooled and concentrated by centrifugation at 10,000 g. See
specifics in Materials and Methods S1, section I.
Nucleosomes were prepared as described . Briefly, histone
octamers and DNA containing the nucleosome positioning
sequence were mixed in equimolar concentrations in 2 M NaCl
and kept for 30 min at RT. A dilution series was prepared using
10 mM Tris HCl to produce final concentrations of 1 M, 0.67 M,
and 0.5 M NaCl. Diluted samples were kept at 4uC for 1 h before
dialysis against one change of volume of 0.2 M NaCl overnight.
Nucleosomes were concentrated using Microcon centrifugal filter
device, MWCO 10,000 at 7,000 g for 10 min at 4uC and dialyzed
against one change of 200 ml of buffer containing 10 mM Hepes-
NaCl, pH 7.5, and 1 mM EDTA for 3 h at 4uC.
Atomic force microscopy
Freshly cleaved mica was modified with 167 mM solution of 1-
(3-aminopropyl)-silatrane (APS) for 30 min at room T to make
APS-mica as described previously in . Other AFM works on
the chromatin in addition to APS functionalization used mica
coated with poly-lysine  or spermidine . The nucleosome
stock solution was diluted into 10 mM Tris-HCl, pH 7.5, 4 mM,
MgCl2buffer and 5 ml of the solution were deposited on APS-
treated mica for 3 minutes, washed with deionized water and dried
under argon flow. AFM images were collected on NanoScope IIId
system (Veeco/Digital Instruments, Santa Barbara, CA) as
described in  and .
Measurement of nucleosome parameters
The samples deposited on APS mica were analyzed with
Femtoscan software. The following 5 initial parameters were
measured: length of each DNA arm, angle between arms
(interarm angle), height of nucleosome core particle and diameter
as width of nucleosome core particle at half height. The length of
DNA was measured with FemtoScan software using parameter
‘‘curve’’. The length of wrapped DNA was measured by
subtracting sum of both DNA arms from length of uncomplexed
DNA. Importantly, the analysis of one set of native and
biotinylated nucleosomes was performed blindly without disclos-
ing whether the nucleosome contained biotinylated on non-
biotinylated H4. The errors of the calculated mean values are
standard errors of the mean (SEM).
Assumptions for estimation of the number of DNA turns
The calculations of DNA turns wrapped around histone
octamers were based on the following assumptions. (1) Based on
crystallographic data, 147 bp of DNA are wound around histone
octamer in 1.7 turns , i.e., 1 turn contains 86 bp of DNA. (2)
As long as in B form of DNA one base pair corresponds to
0.34 nm, the expected length value for 1 turn is 29 nm. How
specific number of turns was assigned is explained in detail in
Materials and Methods S1.
histone for biotinylation state. K12C-H4 was purified after
overexpression in E.coli (lane1), depleted for fraction possibly
biotinylated in vivo at lysines (lane 2) and biotinylated in vitro at
cysteine 12 with Maleimide-PEG2-Biotin (lane 3). The level of
chemical biotinylation was assessed by Western blotting with
streptavidin conjugates (panel a) and anti-biotin antibodies (panel
b). Control western blot with anti-H4 antibodies (panel c)
demonstrates that all three samples in lanes 1-3 are histone H4.
M - marker.
Testing of different samples of K12C H4
particles reconstituted with K12C H4 mutant. Nucleo-
somes were made with K12C-H4 histone mutant. The sample was
prepared and imaged as described for Figure 2. The image
represents nucleosomes with different amount of DNA wrapped
around the core particle. K12Cbio-H4 nucleosome conformation
is similar to nucleosomes made with native histone H4. Scan size is
Representative AFM scan of nucleosome core
bution of length of uncomplexed DNA used for reconstitution of
nucleosome core particles. The length of DNA was measured with
FemtoScan software using parameter ‘‘curve’’. The data were
plotted as statistical histogram and fitted with Gaussian distribu-
tion. The most probable value of 117.560.1 nm was taken as
length of full DNA molecule in subsequent calculations of length of
DNA wrapped around nucleosome.
Length of uncomplexed DNA. Shown is distri-
two independent samples of native and biotinylated H4
nucleosomes. Nucleosomes were reconstituted using native H4
histone (a) or biotinylated K12Cbio-H4 histone (b). Length of free
DNA was measured using Femtoscan software. The length of
wrapped DNA was calculated by subtracting the sum of both free
DNA hands from total length of DNA. Data from two
independent experiments are overlapped. It can be seen that in
nucleosomes made with K12Cbio-H4 wDNA is shifted towards
higher value compared to samples reconstituted using non-
biotinylated native H4. Mean values for native NCP wDNA were
49.861.5 and 47.561.3 nm, respectively. Mean values for wDNA
54.261.2 nm, respectively.
Comparison of wrapped DNA length from
Figure 5. Model of the effect of biotinylation on conformation
of nucleosome. Both front and top views are shown. The segment of
the DNA arm that contributes to additional wrapping is shown in red.
AFM of Nucleosomes
PLoS ONE | www.plosone.org6January 2011 | Volume 6 | Issue 1 | e16299
Materials and Methods S1 Download full-text
Supplement to Materials and
We would like to thank M. Ryan Brodie for technical assistance,
Subhashinee S. Wijeratne for help with testing histone H4 samples for
biotinylation state and Dmytro B. Palets for help with AFM data analysis.
Conceived and designed the experiments: YLL JZ GEOB JTW.
Performed the experiments: NAF CK SAS YIH. Analyzed the data:
NAF CK SAS YIH YLL JZ GEOB JTW. Contributed reagents/
materials/analysis tools: NAF CK SAS YIH YLL JZ GEOB JTW. Wrote
the paper: NAF CK SAS YIH YLL JZ GEOB JTW.
1. Strahl BD, Allis CD (2000) The language of covalent histone modifications.
Nature 403: 41–45.
2. Martin C, Zhang Y (2005) The diverse functions of histone lysine methylation.
Nat Rev Mol Cell Biol 6: 838–849.
3. Li J, Lin Q, Yoon HG, Huang ZQ, Strahl BD, et al. (2002) Involvement of
histone methylation and phosphorylation in regulation of transcription by
thyroid hormone receptor. Mol Cell Biol 22: 5688–5697.
4. Verdone L, Caserta M, Di Mauro E (2005) Role of histone acetylation in the
control of gene expression. Biochem Cell Biol 83: 344–353.
5. Osley MA, Fleming AB, Kao CF (2006) Histone ubiquitylation and the
regulation of transcription. Results Probl Cell Differ 41: 47–75.
6. Camporeale G, Shubert EE, Sarath G, Cerny R, Zempleni J (2004) K8 and K12
are biotinylated in human histone H4. Eur J Biochem 271: 2257–2263.
7. Toth K, Brun N, Langowski J (2006) Chromatin compaction at the
mononucleosome level. Biochemistry 45: 1591–1598.
8. Bussiek M, Toth K, Schwarz N, Langowski J (2006) Trinucleosome compaction
studied by fluorescence energy transfer and scanning force microscopy.
Biochemistry 45: 10838–10846.
9. Gansen A, Toth K, Schwarz N, Langowski J (2009) Structural variability of
nucleosomes detected by single-pair Forster resonance energy transfer: histone
acetylation, sequence variation, and salt effects. J Phys Chem B 113: 2604–2613.
10. Shogren-Knaak M, Ishii H, Sun JM, Pazin MJ, Davie JR, et al. (2006) Histone
H4-K16 acetylation controls chromatin structure and protein interactions.
Science 311: 844–847.
11. Choy JS, Wei S, Lee JY, Tan S, Chu S, et al. (2010) DNA methylation increases
nucleosome compaction and rigidity. J Am Chem Soc 132: 1782–1783.
12. Kobza K, Camporeale G, Rueckert B, Kueh A, Griffin JB, et al. (2005) K4, K9
and K18 in human histone H3 are targets for biotinylation by biotinidase. Febs J
13. Stanley JS, Griffin JB, Zempleni J (2001) Biotinylation of histones in human cells.
Effects of cell proliferation. Eur J Biochem 268: 5424–5429.
14. Takechi R, Taniguchi A, Ebara S, Fukui T, Watanabe T (2008) Biotin
deficiency affects the proliferation of human embryonic palatal mesenchymal
cells in culture. J Nutr 138: 680–684.
15. Ghosh (2009) Physiology, regulation and pathogenesis of nitrogen methabolism
in opportunistic fungal pathogen Candida albicans. PhD Thesis. Lincoln, NE:
University of Nebraska-Lincoln.
16. Hymes J, Fleischhauer K, Wolf B (1995) Biotinylation of histones by human
serum biotinidase: assessment of biotinyl-transferase activity in sera from normal
individuals and children with biotinidase deficiency. Biochem Mol Med 56:
17. Bao B, Pestinger V, Hassan YI, Borgstahl GE, Kolar C, et al. Holocarboxylase
synthetase is a chromatin protein and interacts directly with histone H3 to
mediate biotinylation of K9 and K18. J Nutr Biochem DOI # S0955-
18. Camporeale G, Giordano E, Rendina R, Zempleni J, Eissenberg JC (2006)
Drosophila melanogaster holocarboxylase synthetase is a chromosomal protein
required for normal histone biotinylation, gene transcription patterns, lifespan,
and heat tolerance. J Nutr 136: 2735–2742.
19. Zempleni J, Wijeratne SS, Hassan YI (2009) Biotin. Biofactors 35: 36–46.
20. Camporeale G, Oommen AM, Griffin JB, Sarath G, Zempleni J (2007) K12-
biotinylated histone H4 marks heterochromatin in human lymphoblastoma cells.
J Nutr Biochem 18: 760–768.
21. Wijeratne SS, Camporeale G, Zempleni J (2009) K12-biotinylated histone H4 is
enriched in telomeric repeats from human lung IMR-90 fibroblasts. J Nutr
Biochem 21: 310–316.
22. Chew YC, West JT, Kratzer SJ, Ilvarsonn AM, Eissenberg JC, et al. (2008)
Biotinylation of histones represses transposable elements in human and mouse
cells and cell lines and in Drosophila melanogaster. J Nutr 138: 2316–2322.
23. Lyubchenko YL, Shlyakhtenko LS (2009) AFM for analysis of structure and
dynamics of DNA and protein-DNA complexes. Methods 47: 206–213.
24. Shlyakhtenko LS, Lushnikov AY, Lyubchenko YL (2009) Dynamics of
Nucleosomes Revealed by Time-Lapse Atomic Force Microscopy. Biochemistry
25. Thastrom A, Lowary PT, Widlund HR, Cao H, Kubista M, et al. (1999)
Sequence motifs and free energies of selected natural and non-natural
nucleosome positioning DNA sequences. J Mol Biol 288: 213–229.
26. Kobza K, Sarath G, Zempleni J (2008) Prokaryotic BirA ligase biotinylates K4,
K9, K18 and K23 in histone H3. BMB Rep 41: 310–315.
27. Luger K, Rechsteiner TJ, Richmond TJ (1999) Preparation of nucleosome core
particle from recombinant histones. Methods Enzymol 304: 3–19.
28. Livnah O, Bayer EA, Wilchek M, Sussman JL (1993) Three-dimensional
structures of avidin and the avidin-biotin complex. Proc Natl Acad Sci U S A 90:
29. Gralla M, Camporeale G, Zempleni J (2008) Holocarboxylase synthetase
regulates expression of biotin transporters by chromatin remodeling events at the
SMVT locus. J Nutr Biochem 19: 400–408.
30. Lowary PT, Widom J (1998) New DNA sequence rules for high affinity binding
to histone octamer and sequence-directed nucleosome positioning. J Mol Biol
31. Dyer PN, Edayathumangalam RS, White CL, Bao Y, Chakravarthy S, et al.
(2004) Reconstitution of nucleosome core particles from recombinant histones
and DNA. Methods Enzymol 375: 23–44.
32. Shlyakhtenko LS, Gall AA, Filonov A, Cerovac Z, Lushnikov A, et al. (2003)
Silatrane-based surface chemistry for immobilization of DNA, protein-DNA
complexes and other biological materials. Ultramicroscopy 97: 279–287.
33. Suzuki Y, Higuchi Y, Hizume K, Yokokawa M, Yoshimura SH, et al. (2010)
Molecular dynamics of DNA and nucleosomes in solution studied by fast-
scanning atomic force microscopy. Ultramicroscopy 110: 682–688.
34. Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal
structure of the nucleosome core particle at 2.8 A resolution. Nature 389:
AFM of Nucleosomes
PLoS ONE | www.plosone.org7 January 2011 | Volume 6 | Issue 1 | e16299