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Sequencevariantsofchickenlinkerhistone H1.a
Ewa Go
´
rnicka-Michalska
1
, Jan Pałyga
1
, Andrzej Kowalski
1
and Katarzyna Cywa-Benko
2
1 Department of Genetics, Institute of Biology, Akademia S
´
wie˛ tokrzyska, Kielce, Poland
2 Department of Animal Production, Faculty of Biology and Agriculture, Rzeszo
´
w University, Poland
In eukaryotes, the arrays of nucleosomes are linked
together by DNA to form chromatin fibers with a
diameter of 10 nm. In bulk chromatin, one molecule
of histone H1 binds to a linker DNA sealing off two
turns of DNA around a core histone octamer. Thus,
histone H1 plays a direct role in stabilizing nucleo-
somal and higher-order chromatin structures.
A histone H1 family in higher eukaryotes is repre-
sented by several primary-sequence subtypes (nonallelic
variants) encoded by different, albeit closely related
genes [1,2]. Disruption of a linkerhistone gene in both
protista and fungi demonstrated [3] that it is not essen-
tial for cell survival. On the other hand, elimination of
three but not one or two H1 subtypes in mouse leads
to embryonic lethality due to about 50% reduction in
an H1 to nucleosome ratio [4].
It has been shown that the expression patterns of
multiple H1 genes can change during development and
tissue differentiation [3]. The particular H1 variants
can also be nonrandomly distributed in chromatin,
differ in their turnover rates and in the extent of
post-translational phosphorylation [5]. Such differences
suggest that linkerhistonevariants could play a dis-
tinct role in the regulation of chromatin activity. Regu-
latory function of the histone H1 may include a
selective control of individual gene expression [3,6],
inhibition of DNA repair by homologous recombina-
tion [7], and transmission of apoptotic signals from the
nucleus to the mitochondria [8], as well as targeting
and activation of a major apoptotic nuclease
DFF40 ⁄ CAD during terminal stages of apoptosis [9].
Heritable developmental defects accompanied by
Keywords
allelic isoforms; chicken; genetic
polymorphism; histone H1; peptide
microsequencing
Correspondence
E. Go
´
rnicka-Michalska, Department of
Genetics, Institute of Biology, Akademia
S
´
wie˛ tokrzyska, ul. S
´
wie˛ tokrzyska 15;
25-406 Kielce, Poland
Fax: +48 41 3496292
Tel: +48 41 3496333
E-mail: egorn@pu.kielce.pl or
jpalyga@pu.kielce.pl
(Received 22 August 2005, revised 16
January 2006, accepted 19 January 2006)
doi:10.1111/j.1742-4658.2006.05147.x
Two allelic isoforms (H1.a1 and H1.a2) ofhistoneH1.a were identified
within two conservative flocks (R11 and R55) of Rhode Island Red chick-
ens. These proteins form three phenotypes: a1, a2 and a1a2. Birds with
phenotype a1 were most common (frequency 0.825–0.980) while the a1a2
chickens appeared relatively rarely (0.017–0.175). The third phenotype a2,
not detected in the tested populations, has only been revealed in progeny
of the purpose-mated a1a2 birds. The polymorphism ofhistoneH1.a was
observed in all examined chicken tissues, so that the H1 preparations isola-
ted from the lung, spleen, kidney and testis from the same individual exhib-
ited identical phenotypes (a1, a2, or a1a2). This finding, together with
inheritance data, supports the genetic nature of the H1.a polymorphism.
As indicated by cleavages with a-chymotrypsin and protease V8, the H1.a1
and H1.a2 are two highly related proteins which differ within N-terminal
part of their C-terminal tails. Only a single nonconservative amino acid
substitution between both H1.a allelic isoforms was detected by Edman
degradation: glutamic acid present at position 117 in histone H1.a1 was
replaced by lysine in histone H1.a2. Furthermore, using microsequencing
techniques we have found a sequence homology between the N- and C-ter-
minal parts of an unknown minor protein H1.y, present in the phenotype
a2, and similar regions ofhistone H1.b.
Abbreviations
2-ME, 2-mercaptoethanol; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; RBS, 0.01
M Tris ⁄ HCl, pH 7.4, 0.01 M NaCl,
0.003
M MgCl
2
; NaCl ⁄ Cit, 0.15 M NaCl, 0.015 M sodium citrate; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.
1240 FEBS Journal 273 (2006) 1240–1250 ª 2006 FEBS
changes in DNA methylation following a knockdown
of histone H1 genes in Arabidopsis [10], as well as dis-
turbances in chromatin structure leading to reduced
DNA methylation at specific regulatory sites and ⁄ or
altered expression of specific genes in triple-H1 null
mouse embryonic stem cells [11] suggest that linker
histones could operate as epigenetic regulators of
higher-order chromatin conformations.
Several studies support the idea that individual H1
subtypes could exhibit functional specialization. For
example, histone B4, a Xenopus embryonic linker his-
tone, may confer a totipotency to embryonic cells [12]
while H1b, a specific subtype of mouse histone H1
complement, can restrain muscle differentiation by
forming a complex with a homeoprotein Msx1 [13]. It
has been demonstrated [14] that H1.1, one out of seven
linker histones in Caenorhabditis elegans, took part in
chromatin silencing in a germ line. In addition, this
protein was also able to enhance a telomeric position
effect in budding yeast [15]. The authors suggested that
specific structural features of the H1.1 variant could
dedicate this protein to epigenetic silencing.
Metazoan histone H1 proteins have a characteristic
tripartite structure which consists of a central globular
domain flanked by two basic N- and C-terminal tails
that are largely unstructured in a physiological solu-
tion [1]. However, these disordered regions can assume
a secondary structure when interacting with DNA
[16,17]. While the globular domain is the most highly
conserved region amongst somatic histone H1 variants,
the sequences of H1 tails are more variable, both in
length and amino acid composition, indicating that
they evolved by insertion or deletion and nucleotide
substitutions [18].
By expression of domain-swapping mutants, Brown
et al. [6] determined that the central globular domains
of H1
0
and H1c were responsible for their differential
effects on gene expression. Recently, using targeted
mutations, Hendzel et al. [19] showed that the C-ter-
minal domain is required for a high affinity binding of
the histone H1 to chromatin. Moreover, they discov-
ered differences in the binding affinity of the individual
histone H1 subtypes in vivo attributed to the subtype-
specific properties of the C-terminus [20].
Several nonallelic H1 variants may also be represen-
ted by two or more allelic isoforms in the chromatin
of several species [21–25] including humans [26]. Dif-
ferences in the amino acid sequences between allelic
variants of a given H1 subtype were demonstrated in
only a few instances. They were located mainly in ter-
minal domains of H1 molecule and usually originated
as a result of amino acid substitutions [25–28] or inser-
tions and deletions of repeated sequence motifs [21,24].
As in the case of the nonallelic variants [20], it seems
that the alterations in molecular structure of allelic H1
isoforms may affect their affinity to DNA and chroma-
tin, and their ability to interact with other chromatin
proteins. Most probably, this would result in certain
phenotypic effects [24,29,30].
In our laboratory we have revealed a polymorphism
for subtypes H1.a, H1.b, and H1.z in duck [23,31–33]
and quail [34,35]. In this work, in addition to well-
known allelic variantsof erythrocyte histone H5 [27]
distributed differently in distinct chicken breeds [36],
we also observed two allelic isoforms ofhistone H1.a
in the chickens with a rare histone H1.a2 in a Rhode
Island Red breed only. Herein, we have demonstrated
the genetic nature of the chickenhistoneH1.a poly-
morphism and have employed a limited enzymatic
cleavage and amino acid microsequencing to find a
difference in the primary structure of the H1.a1 and
H1.a2 allelic isoforms.
Results
Variability ofhistoneH1.a in Rhode Island Red
chickens
Histone H1 proteins were extracted with a diluted per-
chloric acid solution from erythrocyte nuclei of indi-
vidual chickens and analyzed in an acetic acid–urea
polyacrylamide gel. As seen in Fig. 1, the perchloric
acid-soluble proteins predominantly contained a fast
moving histone H5 and six slow migrating H1 sub-
types. They were designated according to the nomencla-
ture of [37] as H1.a,.a¢,.b,.c,.c¢ and .d. Two erythrocyte
isoforms ofhistoneH1.a (H1.11 L), H1.a1 and H1.a2,
were identified in the acetic acid–urea gel within Rhode
Island Red chicken populations. These variants form
three phenotypes: a1, a2 and a1a2 (Fig. 1). The animals
with phenotype a1 possess a high level of a single pro-
tein H1.a1. HistoneH1.a from the birds with phenotype
a1a2 was resolved into two well-separated bands, H1.a1
and H1.a2, with a slightly lower level of the H1.a2
isoform. The third phenotype a2 has only been found
following purposely mating the birds with phenotype
a1a2. In the a2 individuals, in addition to a high level of
the expected histone H1.a2, a low amount of either
H1.a1 or H1.a1-like protein (H1.y) was discerned
(Fig. 1). The differences in the electrophoretic mobility
between H1.a1 and H1.a2 in the acetic acid–urea gel
(Figs 1 and 2) and a lack of such differences in the poly-
acrylamide gel electrophoresis conducted in the presence
of SDS (Fig. 3) seem to indicate that both proteins
have a similar apparent molecular weight and can differ
from each other in a net charge.
E. Go
´
rnicka-Michalska et al. Variantsofchickenlinkerhistone H1.a
FEBS Journal 273 (2006) 1240–1250 ª 2006 FEBS 1241
A phenotypic variability ofhistoneH1.a was detec-
ted only in the Rhode Island Red chickens (Table 1).
In the R11 and R55 conservative flocks, we found two
phenotypes, a1 and a1a2. Birds with phenotype a1
were most common (frequency 0.825–0.980) while the
heterozygous a1a2 chickens were quite rare (frequency
0.017–0.175). Moreover, the frequency of the pheno-
type a1a2 was very similar in two different genetic
groups (R11 and R55) and varied widely between two
independent populations of the same conservative flock
R11 (Table 1).
A distribution of the phenotypes of erythrocyte his-
tone H1.a in a progeny from various matings of the
Rhode Island Red chickens is presented in Table 2. As
we were unable to detect animals with phenotype a2 in
all tested farm populations, we could only perform
three available types of crosses: a1 · a1, a1a2 · a1a2,
and a1 · a1a2. As expected, the third phenotype a2
was revealed after mating the a1a2 individuals. In the
offspring of such parents, we found three categories of
phenotypes: a1, a1a2 and a2 in a ratio 1 : 2 : 1. The
data obtained from all crosses support the assumption
that a single gene with two co-dominant alleles is
responsible for the observed polymorphism of histone
H1.a.
The histone H1 preparations isolated from lung,
spleen, kidney and testis of three Rhode Island Red
Fig. 1. A comparison of three phenotypes of erythrocyte histone
H1.a in Rhode Island Red chickens. Histone H1 proteins from
erythrocytes nuclei were resolved in acetic acid–urea polyacryl-
amide gel. H1 and H5, histone H1 and H5; H1.a,.a¢,.b,.c,.c¢,.d, his-
tone H1subtypes; H1.a1, H1.a2, two isoforms ofhistone H1.a;
H1.y, H1.a1-like protein. The phenotypes ofhistoneH1.a were des-
ignated as a1, a2, a1a2.
Fig. 2. A comparison ofhistoneH1.a from erythrocytes (E), lung
(L), spleen (S), kidney (K) and testis (T) ofchicken individuals with
phenotype a1, a2 and a1a2. Proteins were separated in acetic acid–
urea polyacrylamide gel. All symbols are explained in the legend to
Fig. 1.
Fig. 3. Concentration and purification ofhistoneH1.a isoforms and
H1.y protein from chicken erythrocyte nuclei by a preparative SDS–
gel electrophoresis. The bands containing H1.a and H1.y were
excised from the gel, avoiding contamination with remnants of
H1.a¢.
Variants ofchickenlinkerhistoneH1.a E. Go
´
rnicka-Michalska et al.
1242 FEBS Journal 273 (2006) 1240–1250 ª 2006 FEBS
chicken phenotypes were also resolved in the acetic
acid-urea gel (Fig. 2) into six main subtypes [37],
which exhibited tissue specificity (Fig. 2). A poly-
morphism ofhistoneH1.a was observed in all exam-
ined chicken tissues (Fig. 2) so that H1 isolated from
the lung, spleen, kidney and testis of the same individ-
ual exhibited identical phenotypes (a1, a2 or a1a2).
These results, together with inheritance data (Table 2),
support the genetic nature of the H1.a polymorphism.
Limited proteolysis ofH1.a isoforms and protein
H1.y
The stained protein bands containing appropriate all-
elic forms of the chicken erythrocyte histoneH1.a or
protein H1.y were cut out from the acetic acid–urea
gel. After concentration and purification by prepara-
tive sodium dodecyl sulfate (SDS) electrophoresis
(Fig. 3), the proteins were recovered from gel strips by
electroelution and then precipitated from the solution.
Samples of the H1.a and H1.y prepared in such a way
were suitable for further analysis of the protein struc-
ture.
By using a limited digestion with a-chymotrypsin, we
revealed that the polymorphic isoforms ofhistone H1.a
differed in their C-terminal peptides (Fig. 4A). As his-
tone H1 molecule is cleaved with a-chymotrypsin on
the C-terminal side of a single phenylalanine [32], a
limited a-chymotrypsin digestion of the chicken histone
H1.a yielded two fragments [37]: a C-peptide that rep-
resented the C-terminal part of the H1.a (residue
110–224) and a slightly shorter N-peptide from the
N-terminus to Phe109 (Fig. 5). In the acetic acid–urea
gel, a relative migration of a-chymotrypsin generated
C
1
- and C
2
-peptides resembled that of the native pro-
teins H1.a1 and H1.a2 (Fig. 4A), while that of the
N-peptides was identical regardless of the H1.a iso-
form. Moreover, molecular masses of the chymotryptic
N-terminal peptides from H1.a1 and H1.a2 isoforms
determined by an electrospray ionization mass spectro-
metry were found to be identical except for a hetero-
geneity within the H1.a1 N-peptide that appeared to be
represented by a mixture of N-acetylated and N-un-
acetylated species (results not shown). Collectively,
these results clearly indicate that a putative difference
in the amino acid sequence between H1.a isoforms is
located in the region spanning amino acid residue 110
(arginine) and the C-terminus (Fig. 5). Similarly, one of
the two peptides obtained by H1.y digestion with
a-chymotrypsin migrated in the gel like H1.a1- or
H1.a2-derived N-peptide, whereas the second one, with
an electrophoretic mobility similar to H1.a C-peptides,
was almost undetectable in the gel (Fig. 4A).
To demonstrate which part of the C-terminal domain
is responsible for putative amino acid differences
between both isoforms, the H1.a1 and H1.a2 were
Table 1. A frequency of phenotypes and alleles of erythrocyte histoneH1.a in conservative flocks of four chicken breeds. Chickens from the
same genetic group, R11, were bred in two different farms in Szczytno near De˛blin and Chorzelo
´
w near Mielec.
Breed and genetic group Total number of birds
Number of birds with
phenotypes Frequency of phenotypes
Frequency of
alleles
a1 a1a2 a2 a1 a1a2 a2 a1 a2
Rhode Island Red
R55 50 49 1 – 0.980 0.020 0 0.99 0.01
R11 (Szczytno) 60 59 1 – 0.983 0.017 0 0.99 0.01
R11 (Chorzelo
´
w) 126 104 22 – 0.825 0.175 0 0.91 0.09
Leghorn G99 157 157 – – 1 0 0 1 0
Cornish CE2 61 61 – – 1 0 0 1 0
Greenleg Partridge Z11 52 52 – – 1 0 0 1 0
Table 2. Distribution of phenotypes of erythrocyte histoneH1.a in progeny from various matings of Rhode Island Red R11 chickens.
Type of mating Number of
Frequency of phenotypes in progeny
(observed ⁄ expected)
v
2
Male Female Families Progeny a1 a1a2 a2
a1 a1 6 25 25 ⁄ 25
a1a2 a1a2 2 67 15 ⁄ 16.75 36 ⁄ 33.5 16 ⁄ 16.75 0.404
a1a2 a1 2 54 26 ⁄ 27 28 ⁄ 27 0.074
E. Go
´
rnicka-Michalska et al. Variantsofchickenlinkerhistone H1.a
FEBS Journal 273 (2006) 1240–1250 ª 2006 FEBS 1243
digested with Staphylococcus aureus protease V8. After
histone H1.a treatment with the protease V8 that can
preferentially cleave a peptide bond following glutamic
acid, only one distinct peptide band, identified as C-ter-
minal tail ofH1.a (results not shown), was observed in
the gel (Fig. 4B). In the C-terminal domain of H1.a
molecule there are only two glutamic acid residues at
positions 117 and 120 [37]. Thus, a limited digestion of
H1.a with protease V8 generated a little shorter C-ter-
minal peptide ranging from residue 118 or 121 to the
C-terminus (Fig. 5). Moreover, the remaining part of
the histone H1.a, with several glutamic acid residues
(positions 2, 10, 46, 57 and 78), would have to undergo
further cleavages into a number of smaller fragments
[37] undetectable in the gel. As seen in Fig. 4B, the elec-
trophoretic mobilities of the C-terminal peptides
obtained from H1.a1 and H1.a2 isoforms following
digestion with protease V8 were similar. As we revealed
distinct differences in a migration between C
1
- and
C
2
-peptides derived from H1.a1 and H1.a2 proteins
after limited proteolysis with a-chymotrypsin, the uni-
form migration of protease V8-cleaved C-peptides
seemed to point out that differences between H1.a iso-
forms were confined to a short region spanning from
amino acid residue 110 to the glutamic acid at either
the position 117 or 120 (Fig. 5). As mentioned for H1.a
isoforms, a protease V8 digestion of the protein H1.y
also yielded only one peptide (Fig. 4B).
Tandem mass spectrometry
To find more precisely which site could be altered
between the two allelic H1.a isoforms, we applied a
tandem mass spectrometry for a sequence prediction of
the peptides generated by trypsin digestion. As a sim-
ilar set of peptides was detected in both proteins
(results not shown) we were unable to reveal any
meaningful alterations between the isoforms using this
procedure.
However, a primary structure of protein H1.y was
partially revealed by the tandem mass spectrometric
analysis of its trypsin products. Besides five pep-
tides present in almost all of the H1 subtypes, the
trypsin digestion generated one peculiar peptide
(AETAPVAAPDVAAAPTPAK), which showed a
high degree of similarity to histone H1.b (H1.03) (resi-
dues 1–19). This 19-amino acid peptide was a part of
Fig. 4. Limited proteolysis of H1.a1 and H1.a2 histones, and H1.y
(H1.a1-like protein) from chicken erythrocytes with a-chymotrypsin
(A) and Staphylococcus aureus V8 protease (B). The digestion prod-
ucts were resolved in the acetic acid–urea gel. C
1
,C
2
, chymotryptic
C-terminal peptides generated from the H1.a1 and H1.a2, respect-
ively; N, chymotryptic N-terminal peptides produced from the
H1.a1, H1.a2 and H1.y; C, V8 protease C-terminal peptides
obtained from the H1.a1, H1.a2 and H1.y; H1, undigested histone
H1 subtypes.
Fig. 5. The main peptides generated by a limited cleavage of the
chicken erythrocyte histoneH1.a isoforms with a-chymotrypsin and
Staphylococcus aureus protease V8. H1.a1 and H1.a2, isoforms of
histone H1.a; C and N, C- and N-termini ofhistone H1.a; (C
1
,C
2
)-
chym., chymotryptic C
1
-andC
2
-peptides generated from the H1.a
isoforms; N-chym., chymotryptic N-peptides produced from the
H1.a1 and H1.a2; C-V8, the possible protease V8 C-peptides
cleaved from the H1.a. Using a published sequence [37], we
assumed that limited proteolysis ofH1.a with the protease V8 cre-
ated a C-terminal peptide running from residue 118 or 121 to the
C-terminus. The expected difference between H1.a isoforms is
located at a short region spanning from Arg110 to the glutamic acid
either at position 117 or 120.
Variants ofchickenlinkerhistoneH1.a E. Go
´
rnicka-Michalska et al.
1244 FEBS Journal 273 (2006) 1240–1250 ª 2006 FEBS
the N-terminal domain, the most variable and specific
region for each H1 subtype [37]. As the individual ion
score for H1.y N-peptide was 75 and this value was
significantly higher than the threshold score of 45, it
strongly supports either the identity or very extensive
homology (P<0.05) between this peptide and the his-
tone H1.b (data not shown).
Partial microsequencing of chymotryptic
C-terminal fragments
We also applied automated Edman degradation for a
partial sequence determination in the H1.a1 and H1.a2
proteins in the region affected by a mutation. For this
purpose, the C-terminal fragments ofhistoneH1.a iso-
forms produced by a-chymotrypsin digestion have
been analyzed by amino acid sequencing after trans-
blotting the proteins from the SDS–polyacrylamide gel
to a polyvinylidene difluoride (PVDF) membrane. Fig-
ure 6 shows a partial sequence comparison of chicken
histone H1.a variants. In a region spanning the resi-
dues 110–120 in both H1.a1 and H1.a2 we revealed 10
identical amino acids out of 11 residues so that the all-
elic isoforms differed only by one amino acid. A gluta-
mic acid at the position 117 in the histone H1.a1 was
replaced by a lysine in the H1.a2 isoform. The partial
histone H1.a2 sequence data was deposited in the Uni-
Prot Knowledgebase (UniProtKB, http://www.ebi.
ac.uk) under the accession number P8451533.
We also determined the first 11 amino acid residues
for the longer C-terminal chymotryptic peptide cleaved
off the chicken H1.y (Fig. 6). The analyzed fragment
corresponded exactly to the beginning of the C-ter-
minal domain of the histone H1.b (residues 109–119;
with serine at position 6 and aspartic acid at position
8 of the peptide) and that of the histone H1.a2 (with
proline at position 6 and lysine at position 8 of the
peptide). As the protein H1.y was isolated from the
chicken phenotype a2 enriched in the isoform H1.a2,
the bands used for a histone H1.y concentration were
cut out from the acetic acid-urea gel in which H1.y and
H1.a2 migrated close to each other (Fig. 1). Further
separation of H1.y protein from the histone H1.a2
could not be achieved by the preparative SDS–gel elec-
trophoresis due to almost identical migration of both
proteins (Fig. 3). Therefore, we conclude that our his-
tone H1.y preparation was sequenced as a mixture of
the histone H1.y and an accompanying histone H1.a2.
Thus, the automated Edman degradation of H1.y
chymotryptic C-peptide revealed a sequence homology
between the beginning of the C-terminal part of H1.y
and that of H1.b (residues 109–119; Fig. 6).
Discussion
Each of the six H1 genes (11L, 11R, 03, 0.10, 01 and
02) identified in the chicken genome [38] encodes for a
different H1 subtype [37]. Differences in amino acid
sequences among H1 subtypes could be connected with
their differential capacity for interaction with chroma-
tin [1,20]. In a current dynamic view [39], the H1 mole-
cules, with a residence time on the nucleosome
modulated by a phosphorylation, are continuously
exchanged between chromatin binding sites in a ‘stop-
and-go’ mode. A specificity in amino acid sequence
may also preferentially alter the chromatin residence
time of a unique variant at a specific locus or chroma-
tin region [20,39]. Thus, a microheterogeneity in the
primary structure may imply functional differences
between distinct H1 subtypes. The linkerhistone het-
erogeneity can further increase due to a genetic poly-
morphism and ⁄ or post-translational modifications
[36,40,41]. In this study, two allelic isoforms (H1.a1
and H1.a2) ofhistoneH1.a were identified within two
conservative flocks (R11 and R55) of Rhode Island
Red chickens. HistoneH1.a also exhibits a distinct
intrapopulation variability in other avian species such
as duck [31] and quail [34]. As in the chicken, the poly-
morphic H1.a subtype from duck erythrocytes was rep-
resented by two electromorphs, H1.a1 and H1.a2, with
similar apparent molecular weights and different net
charges [31]. The genetic polymorphism of histone
H1.a in Pharaoh quail was associated with a lack of
this proteins in some birds or its occurrence at either
an intermediate or high level in other individuals [34].
In both flocks of Rhode Island Red chickens
(Table 1), representing a typical multipurpose breed
with a brownish red plumage, a frequency of allele
a2 was quite low (0.01–0.09). Similarly, the allele a2,
which was also rare or absent in most genetic duck
groups, was enriched in the color-feathered duck popu-
Fig. 6. Alignment of the first 11 amino acid residues in the chymo-
tryptic C-peptides obtained from chicken histones H1.a1, H1.a2 and
protein H1.y. ChickenhistoneH1.a (residues 110–120) and H1.b
(residues 109–119) sequences designated with the asterisk [Uni-
Prot Knowledgebase (UniProtKB) accession numbers P08287 and
P08285, respectively] are presented for comparison.
E. Go
´
rnicka-Michalska et al. Variantsofchickenlinkerhistone H1.a
FEBS Journal 273 (2006) 1240–1250 ª 2006 FEBS 1245
lations [31]. Surprisingly, the number of homozygous
a2 chickens was not reduced and the proportion of
other phenotypes did not differ from the expected val-
ues in the offspring of purpose-mated heterozygous
birds. As the homozygous individuals a2 did not seem
to be less viable, it is likely that a lack of this pheno-
type in both conservative chicken flocks might be
either fortuitous or caused by breeding conditions. The
changes in histone H1 allele frequencies were observed
in quail populations divergently selected for a high or
low reduction in body mass following transient starva-
tion [30]. Only a1a1 homozygotes and a1a0 hetero-
zygotes were detected among individuals of those
populations, while rare homozygotes a0a0 were found
exclusively in a control random mating population
[30]. Thus, it was likely that a divergent selection in
quail could have acted against the allele a
0
. Berdnikov
et al. [42] analyzed the influence of environment on the
histone H1 allele frequencies in Pisum sativum. Using a
collection of 833 accessions of the cultivated pea ori-
ginating from different regions of the Old World they
revealed that the alleles of the specific H1 subtypes
(H1–5 and, possibly, H1–1) were subjected to climatic-
ally dependent natural selection under conditions of
primitive farming.
The similar molecular weights and different net
charges of the chicken H1.a1 and H1.a2 suggest that
one of these proteins might have arisen as a result of
amino acid substitution or post-translational modifica-
tion. In order to check whether phosphorylation is
responsible for H1.a heterogeneity, we performed a
digestion of H1 samples from the individuals differing
in the H1.a phenotype with alkaline phosphatase
(results not shown). As the enzyme treatment did not
change the patterns ofhistoneH1.a migration, we
believed that phosphorylation is not responsible for
the observed polymorphism. Thus, the most convin-
cing explanation of the intrapopulation variability of
H1.a is that the H1.a1 and H1.a2 represent allelic pro-
teins encoded by a gene with two codominant alleles at
a locus. Previously, we detected [36] two allelic electro-
morphs (a and b) of the chicken erythrocyte histone
H5. A limited proteolysis with a-chymotrypsin and
subsequent microsequencing (unpublished results)
revealed that both polymorphic variants differed in
their N-terminal tails and therefore they resembled two
chicken H5 sequencevariants Va and Vb with a gluta-
mine-to-arginine substitution at the position 15 [27].
Most of the allelic variants so far reported for the
polymorphic histone H1 subtypes differ in the N- or
C-terminal tails [23,24,27,32], though an amino acid
replacement in the globular domain was also detected
[25]. Specific site cleavages in H1.a1 and H1.a2 pro-
teins with a-chymotrypsin and protease V8 have
shown that a variable region of the H1.a isoforms is
located at the beginning of the C-terminal tail, between
amino acid residue 110 and glutamic acid either at
position 117 or 120. Relying on the sequence data for
the chickenhistone H1 subtypes [37] and their genes
[38], a limited amino acid variability is apparent in
the part of C-terminal tail adjacent to the globular
domain. In this region there are several conservative
(N ⁄ S, E ⁄ D, V ⁄ G) and nonconservative (V⁄ T, P⁄ S,
K ⁄ L) substitutions between paralogous H1 subtypes
including those involving charged residues.
The C-terminal domain ofhistone H1 is presumably
involved in the organization of the linker DNA and
may be responsible for stabilization of condensed chro-
matin fiber [19,43]. Vila et al. [16] have studied con-
formational properties of a C-terminal peptide CH-1,
which is placed adjacent to the central globular domain
in rodent histone H1
0
(residues 99–121), by a Fourier-
transform infrared spectroscopy. The authors showed
the presence of inducible helical and b-turn (S ⁄ TPKK)
elements in this peptide in both trifluoroethanol solu-
tion and in the complexes with DNA. In the presence
of helical inducers, the helical region has a marked am-
phipatic nature with all basic residues on one site of
the helix and all hydrophobic residues on the other one
[17]. The amino acid substitutions in this H1 region
may influence its binding to chromatin [19], including
binding to specific chromatin proteins, for histone H1
C-terminal region may mediate protein–protein interac-
tions [9,44]. Lu and Hansen [43] demonstrated that the
ability of the H1
0
to alter linker DNA conformation
and to stabilize condensed chromatin structure is not
distributed evenly throughout the entire domain. The
sequences that mediate folding were localized to two
specific subdomains within C-terminal tail (97–121 and
146–169). Interestingly, the two H1
0
S ⁄ TPKK motifs
were found in each of the C-terminal subdomain (resi-
dues 97–121 and 146–169). These sequences form
b-turns that bind the minor groove of DNA [45].
A comparison ofsequence data of the C-terminal
region responsible for H1.a variability indicates that
allelic isoforms of the chickenhistoneH1.a differ by
only one amino acid residue. At position 117, there is
a glutamic acid in histone H1.a1 and lysine in histone
H1.a2. At the nucleotide level, a transition from G to
A in the first position of glutamic acid codons (GAA
or GAG) might have resulted in the replacement of
Glu117 to Lys (codons: AAA or AAG) in H1.a2.
Thus, a charge difference between both H1.a allelic
isoforms was due to a nonconservative Glu117Lys
substitution which could in turn explain a different
mobility of H1.a1 and H1.a2 in the acetic acid–urea gel.
Variants ofchickenlinkerhistoneH1.a E. Go
´
rnicka-Michalska et al.
1246 FEBS Journal 273 (2006) 1240–1250 ª 2006 FEBS
The amino acid sequence variation among different
paralogous H1 subtypes might influence a positive
charge of the protein and thereby affect the interaction
between negatively charged DNA and positively
charged C-terminal tails. Recent studies have shown
[46,47] that a modulation of the coulombic interactions
between H1 and DNA by post-translational modifica-
tions or by amino-acid mutations is essential for gene
regulation. Thus, the substitution of a negatively
charged amino acid (Glu) in the chicken H1.a1 to a
positively charged one (Lys) in the H1.a2 is an essen-
tial sequence alteration with possible functional conse-
quences. The electrostatic DNA–histone interactions
are supplemented by the regulated interactions between
histones and effector proteins in the chromatin region
posed for gene transcription, as proposed by the
‘histone code’ hypothesis [48]. Histone covalent modifi-
cations, especially on core histone N-tails, act sequen-
tially or in a combination to form a nucleosomal
‘epigenetic code’ that can differentially interact with
structural, enzymatic and ⁄ or regulatory protein com-
plexes [48]. As suggested [10], the members of the his-
tone H1 family which could also harbor epigenetic
markings [40] may act themselves as epigenetic regula-
tors. Lately, some experiments seem to provide evi-
dence for functional specificity among H1 subtypes
[8,12,13,44].
Recently, Berdnikov et al. [24] compared quantita-
tive traits in near isogenic lines of lentil and grasspea
carrying allelic variantsof the most abundant subtype
H1-1. In this experiment, small (1–8%) but significant
differences have been revealed for some quantitative
traits such as: the mean number of seeds per pod,
mean seed mass, the flower fresh mass and the number
of ovules in a carpel. These effects might have reflected
a direct association between histone H1 complement
and fitness, as evidenced for a Lys178Asn substitution
in the C-terminus of H1–5)1, a slow allelic variant of
pea histone H1–5 [25] that was most abundant in geo-
graphical region with a cold climate [42].
At present, the functional significance of allelic vari-
ation within the relevant H1 subtype is unknown.
Although no satisfactory assay for assessing their role
in chromatin is currently available [26], we [30] and
others [42] observed a differential susceptibility of
H1 allelic variants to artificial and natural selection,
respectively, in response to environmental stimuli that
most likely might challenge a genetic buffering capacity
of Hsp90 chaperone complexes and ⁄ or other capacitors
of phenotypic variation [49]. Recent observation [50]
that Hsp90 facilitates loading linker histones to a speci-
fic binding and transporting protein NASP seems to
support a putative role of the chaperone in buffering
phenotypic effects of genetic variation in H1 histones
under normal conditions.
Experimental procedures
Animals
In this study we used Rhode Island Red chickens main-
tained as the conservative flocks (R11 and R55) at the
breeding poultry farm in Szczytno near De˛blin and in
Chorzelo
´
w (R11) near Mielec, Poland. The Cornish chick-
ens were obtained from Poultry Research and Development
Centre in Zakrzewo near Poznan
´
. All the remaining chicken
breeds were from the farm in Szczytno near De˛blin.
Blood from wing vein of individual birds was separately
collected into NaCl ⁄ Cit solution (0.15 m NaCl, 0.015 m
sodium citrate) containing 0.1 mm phenylmethylsulfonyl
fluoride (PMSF). Further experiments were carried out using
lung, spleen, kidney and testis from three 5-month-old indi-
viduals of the Rhode Island Red R11 breed. After quick
decapitation, the tissues were immediately removed, perfused
or washed with ice-cold NaCl ⁄ Cit solution supplemented
with phenylmethylsulfonyl fluoride and stored at )20 °C,
but no longer than three weeks. These experiments were car-
ried out in accordance with the European Communities
Council Directive of November 24 1986 (86/609/EEC).
Isolation of erythrocytes, nuclei and histone H1
extraction
All procedures were performed at 0–4 °C. The blood mixed
with NaCl ⁄ Cit solution was centrifuged at 2000 g for 10 min
(MPW-360, MPW Med. Instruments, Warsaw, Poland). The
supernatant with a layer of leukocytes was aspirated, and
the erythrocytes were washed twice with NaCl ⁄ Cit solution
containing 0.5 mm phenylmethylsulfonyl fluoride. The pellet
of red blood cells was stored at )20 °C until used.
Nuclei were isolated by one of two methods: (a) Erythro-
cyte nuclei were prepared by a lysis in 0.1 m phosphate
buffer, pH 7.0, containing 0.9% (w ⁄ v) NaCl, 0.03% (w ⁄ v)
saponin and 1 mm phenylmethylsulfonyl fluoride [28].
(b) Nuclei from lung, spleen, kidney and testis were pre-
pared by a sucrose-Triton X-100 method [51].
Histone H1 proteins were extracted from the washed
nuclei first with 1 m and then with 0.5 m perchloric acid.
Protein from the combined supernatants were precipitated
with 20% (w ⁄ v) trichloroacetic acid, washed twice with aci-
dified acetone (acetone ⁄ concentrated HCl, 250 : 1, v ⁄ v) and
acetone, and then air dried.
Electrophoretic analysis
Histone H1 proteins were separated in an acetic acid–urea
gel containing 15% (w ⁄ v) acrylamide, 0.9 m acetic acid and
E. Go
´
rnicka-Michalska et al. Variantsofchickenlinkerhistone H1.a
FEBS Journal 273 (2006) 1240–1250 ª 2006 FEBS 1247
8 m urea and stained in Coomassie Blue R-250 [32]. For
preparative SDS gel electrophoresis, approximately 18
stained gel fragments of the acetic acid–urea gel containing
H1.a isoforms and H1.a1-like protein (H1.y) were sepa-
rately incubated in adaptation buffer [2% (w ⁄ v) SDS, 10%
(v ⁄ v) 2-mercaptoethanol (2-ME), 10% (v ⁄ v) glycerol,
0.125 m Tris ⁄ HCl, pH 6.8]. The gel pieces were then
stacked in a well of one-dimensional 15% (w ⁄ v) polyacryl-
amide gel containing 0.1% (w ⁄ v) SDS [35].
Isolation ofhistoneH1.a isoforms and protein
H1.y
The stained bands of the appropriate protein, purified and
concentrated during preparative electrophoresis, were
excised from the SDS gel to avoid neighboring contamina-
tions [35]. Three protein bands of the same kind (H1.a1,
H1.a2 or H1.y) were pooled into glass tubes, and put over a
dialysis membrane. Electroelution was performed in a buffer
containing 0.1% (w ⁄ v) SDS, 0.025 m Tris, and 0.192 m
glycine. Protein samples precipitated with 0.2 m KCl were
centrifuged at 10 000 g for 10 min (K24D, Janetzki, Leipzig,
Germany). Precipitated SDS was then removed by washing
the pellets with 20% (w ⁄ v) trichloroacetic acid. A small
amount of pellet remaining after centrifugation was washed
twice with acidified acetone and acetone, and dried.
Digestion with a-chymotrypsin and protease V8
from Staphylococcus aureus
Histone H1.a isoforms and protein H1.y isolated by elec-
troelution were dissolved in: (a) 10 lL 0.05 m Tris ⁄ HCl,
pH 8.0 and a-chymotrypsin (45 UÆmg
)1
, Serva Electrophor-
esis, Heidelberg, Germany) was added to a concentration
of 0.2 ngÆlL
)1
; (b) 10 lL 0.1 m Tris ⁄ HCl, pH 7.5 and pro-
tease V8 (660 UÆmg
)1
, Sigma, St. Louis, MO, USA) was
added to final enzyme concentration 7 ngÆlL
)1
. In both
cases, the proteolysis was performed at room temperature
for 15 and 45 min, respectively. The reaction was stopped
by boiling the samples for 2 min. Then appropriate volumes
of 8 m urea and concentrated acetic acid, 2-ME and gly-
cerol were added to receive 2.5 m urea, 0.9 m acetic acid,
5% (v ⁄ v) 2-ME and 10% (v ⁄ v) glycerol in the reaction
solution. The proteolytic degradation products were
resolved in the acetic acid–urea polyacrylamide gel.
Identification of protein resolved in the gel
by mass spectrometry
The strips of the acetic acid–urea gel containing H1.a iso-
forms and protein H1.y, stained with Coomassie blue
R-250, were equilibrated with SDS-adaptation buffer. Only
one gel piece per well was loaded into the wells of 15%
(w ⁄ v) polyacrylamide gel containing SDS. After electro-
phoresis, the protein bands were excised, in-gel reduced,
S-alkylated and digested with modified trypsin (Promega,
Madison, WI, USA). Tryptic peptides, washed out from
the gels, were resolved by RP-HPLC using LC Packings
(Sunnyvale, CA, USA) C
18
precolumn (300 lm · 5 mm)
and C
18
column (75 lm · 15 cm). Peptides were eluted at
0.2 lLÆmin
)1
with a linear gradient of acetonitrile from 0%
to 25% (v ⁄ v) in 25 min in the presence of 0.05% (v ⁄ v) for-
mic acid. The column outlet was coupled to an electrospray
mass spectrometer (Micromass, Manchester, UK).
Molecular mass analysis was performed using the nano-
Z-spray ion source of quadrupole-time of flight electrospray
mass spectrometer working with the regime of data depend-
ent on MS to tandem MS switch, allowing for a 3-s sequen-
cing scan for each detected peptide. The data were analyzed
using the mascot program (http://www.matrixscience.com).
All electrospray experiments were carried out at the mass
spectrometry facility of the Institute of Biochemistry and
Biophysics of the Polish Academy of Sciences in Warsaw,
Poland.
In-gel digestion, electroblotting and amino acid
analysis
Stained bands containing concentrated and purified iso-
forms ofhistoneH1.a and protein H1.y were cut out from
the SDS-preparative gel and soaked in adaptation buffer
[0.1% (w ⁄ v) SDS, 1 mm EDTA, 10% (v ⁄ v) glycerol,
0.125 m Tris ⁄ HCl, pH 6.8]. Next, the samples were loaded
into the separate wells of 15% (w ⁄ v) polyacrylamide gel
containing 0.1% (w ⁄ v) SDS and overlayered with 4 lLof
the adaptation buffer containing 1 lgÆlL
)1
of a-chymotryp-
sin (45 UÆmg
)1
, Serva Electrophoresis). After electrophor-
esis, the proteins and polypeptides were electroblotted for
1.5 h at 90 V onto PVDF membrane (Roche, Mannheim,
Germany) using 0.01 m 3-cyclohexylamino-1-propane-
sulfonic acid, 10% (v ⁄ v) methanol, pH 11 as a transfer buf-
fer. The PVDF membrane was washed in MilliQ (Millipore,
Bedford, MA, USA) deionized water, stained with 0.1%
(w ⁄ v) Coomassie Blue R-250 in 40% (v ⁄ v) methanol and
1% (v ⁄ v) acetic acid. After destaining in 50% (v ⁄ v) meth-
anol, the filter was rinsed in MilliQ deionized water and air
dried. Finally, the bands of chymotryptic C-peptides derived
from H1.a isoforms and protein H1.y were excised from the
blots and analyzed by automated Edman sequencing.
The N-terminal amino acid sequence analysis was per-
formed on a gas-phase sequencer (Model 491, Perkin
Elmer-Applied Biosystems, Foster City, CA, USA) at Bio-
Center (Jagiellonian University, Krako
´
w, Poland). The
phenylthiohydantoin derivatives were analyzed by on-line
gradient high performance liquid chromatography on
Microgradient Delivery System Model 140C equipped with
Programmable Absorbance Detector Model 785 A (both
from Perkin Elmer-Applied Biosystems).
Variants ofchickenlinkerhistoneH1.a E. Go
´
rnicka-Michalska et al.
1248 FEBS Journal 273 (2006) 1240–1250 ª 2006 FEBS
Acknowledgements
This work was supported by grant KBN 6P06D03520
from the Polish Ministry of National Education and
Science.
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Ewa Go
´
rnicka-Michalska
1
, Jan Pałyga
1
, Andrzej Kowalski
1
and Katarzyna Cywa-Benko
2
1 Department. that our his-
tone H1. y preparation was sequenced as a mixture of
the histone H1. y and an accompanying histone H1. a2 .
Thus, the automated Edman degradation