Moreover, the proportion of H1 Keywords HILIC; linker histones; sequence variants; SNP; tumor cell lines Correspondence H.. This article evaluates the potential utility of HILIC as a mea
Trang 1H1.2 and H1.4 subtypes
Bettina Sarg1, Anna Gre´en2, Peter So¨derkvist2, Wilfried Helliger1, Ingemar Rundquist2
and Herbert H Lindner1
1 Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Austria
2 Division of Cell Biology, Linko¨pings Universitet, Sweden
The H1 histones are small basic proteins occurring in
all higher eukaryotes in multiple subtypes that differ
only slightly in their primary sequences H1 histones
consist of a central, highly conserved globular domain,
while the hydrophilic N- and C-terminal tails exhibit
less sequence conservation In addition to the hetero-geneity of their primary structures, the H1 tails are also extensively post-translationally modified (e.g phosphorylated or ADP-ribosylated) under various biological conditions Moreover, the proportion of H1
Keywords
HILIC; linker histones; sequence variants;
SNP; tumor cell lines
Correspondence
H H Lindner, Division of Clinical
Biochemistry, Biocenter, Innsbruck Medical
University, Fritz-Pregl-Strasse 3, A-6020
Innsbruck, Austria
Fax: +43 512 507 2876
Tel: +43 512 507 3521
E-mail: herbert.lindner@uibk.ac.at
(Received 10 March 2005, revised 10 May
2005, accepted 26 May 2005)
doi:10.1111/j.1742-4658.2005.04793.x
In humans, eight types of histone H1 exist (H1.1–H1.5, H1, H1t and H1oo), all consisting of a highly conserved globular domain and less con-served N- and C-terminal tails Although the precise functions of these iso-forms are not yet understood, and H1 subtypes have been found to be dispensable for mammalian development, it is now clear that specific func-tions may be assigned to certain individual H1 subtypes Moreover, micro-sequence variations within the isoforms, such as polymorphisms or mutations, may have biological significance because of the high degree of sequence conservation of these proteins This study used a hydrophilic interaction liquid chromatographic method to detect sequence variants within the subtypes Two deviations from wild-type H1 sequences were found In K562 erythroleukemic cells, alanine at position 17 in H1.2 was replaced by valine, and, in Raji B lymphoblastoid cells, lysine at position
173 in H1.4 was replaced by arginine We confirmed these findings by DNA sequencing of the corresponding gene segments In K562 cells, a homozygous GCCfiGTC shift was found at codon 18, giving rise to H1.2 Ala17Val because the initial methionine is removed in H1 histones Raji cells showed a heterozygous AAAfiAGA codon change at position 174 in H1.4, corresponding to the Lys173Arg substitution The allele frequency of these sequence variants in a normal Swedish population was found to be 6.8% for the H1.2 GCCfiGTC shift, indicating that this is a relatively fre-quent polymorphism The AAAfiAGA codon change in H1.4 was detected only in Raji cells and was not present in a normal population or in six other cell lines derived from individuals suffering from Burkitt’s lym-phoma The significance of these sequence variants is unclear, but increas-ing evidence indicates that minor sequence variations in linker histones may change their binding characteristics, influence chromatin remodeling, and specifically affect important cellular functions
Abbreviations
CE, capillary electrophoresis; HILIC, hydrophilic interaction liquid chromatography; HPCE, high performance capillary electrophoresis; RFLP, restriction fragment length polymorphism; SNP, single nucleotide polymorphism; TEAP, triethylammonium phosphate.
Trang 2subtypes varies in a tissue- and species-specific manner,
and the expression of each subtype varies throughout
development and differentiation [1–3]
In the human genome, genes encoding eight different
subtypes of histone H1 have been identified The genes
encoding H1.1, H1.2, H1.3, H1.4, H1.5 and H1.t are
located on the short arm of chromosome 6, while H1
is located on chromosome 22 [4] and H1oo on
chro-mosome 3 [5] Only one copy of each gene is present
[6] Within these H1 genes several single nucleotide
polymorphisms (SNPs) have been reported (NCBI
Single Nucleotide Polymorphism Database) To our
knowledge, no acquired mutations have been reported
to date in any human H1 gene
Studies of the structure of H1 histones, their
inter-action with the nucleosome and their roles in
control-ling gene activity, indicate that these proteins have
both an essential architectural function and an
important task in regulating transcription [7,8] The
precise functions of these multiple H1 subtypes and
their modifications are not yet fully understood, but
it has been reported that distinct H1 histone variants
are preferentially localized to particular chromosomal
domains [9–11] Although individual H1 subtypes are
dispensable for mammalian development [12], it now
seems clear that linker histones, in general, are essential
for proper development [13] and it was recently found
that one subtype, H1.2, had a specific role in DNA
damage-induced apoptosis [14] It is useful therefore to
examine the properties and expression of these variants
as this furthers a better understanding of the relevance
of this diversity for particular cellular activities
The sequence similarity between histones H1.1–H1.5
requires highly efficient analytical methods for their
resolution To date, the most widely utilized
proce-dures for the study of human H1 proteins have been
PAGE and low-pressure ion-exchange
chromatogra-phy Two-dimensional gel electrophoresis allows the
separation of several H1 variants [15,16], and four H1
subtypes were obtained by using BioRex 70 column
chromatography [17–19] Both PAGE and
low-pressure ion-exchange chromatography, however, are
laborious, time-consuming and their resolution is
unsatisfactory
Recently, we described rapid and simple methods
for the separation of rat and mouse H1 histones by
using RP-HPLC [20,21] and high performance
capil-lary electrophoresis (HPCE) [22–24] Furthermore, by
applying hydrophilic interaction liquid
chromatogra-phy (HILIC) excellent fractionations of various
post-translationally modified core [25,26] and linker [27,28]
histones were obtained in both the analytical and the
semipreparative scale
This article evaluates the potential utility of HILIC
as a means of investigating the occurrence of sequence variations within linker histone subtypes from various human tumor cell lines By using HILIC we detected amino acid substitutions in H1.2 and H1.4 at the pro-tein level In addition, sequencing of the corresponding gene segments confirmed these findings at the genome level Furthermore, we also screened DNA from 103 healthy individuals to obtain the allele frequency for these variants
Results
HILIC is an excellent technique for using to separate histone proteins and their modified forms [25–29] This study aimed to apply and optimize the HILIC tech-nique in order to detect the occurrence of micro-sequence variations of H1 subtypes isolated from various human tumor cell lines As the level of histone H1 phosphorylation is lower in nondividing than in proliferating cells, we isolated H1 histones from cell cultures in the stationary phase, thus making it pos-sible to reduce the occurrence of additional peaks caused by phosphorylated forms of the parent pro-teins A typical separation pattern using a PolyCAT A column and a two-step sodium perchlorate gradient (0–0.68 m) in the presence of 70% (v⁄ v) acetonitrile and 0.015 m triethylammonium phosphate (TEAP) (pH 3.0) is shown in Fig 1A The CCRF-CEM H1 sample was separated into four peaks Analysis of sev-eral other cell lines showed the same pattern, but the relative concentrations of the subtypes varied (data not shown) H1 samples from Raji (Fig 1B) and K562 (Fig 1C) cells, however, showed different patterns, and additional peaks were detected, namely peak 3a in Fig 1B and peak 1a in Fig 1C
To identify the individual peaks of the chromato-grams we digested the various subfractions with chymo-trypsin, a protease that specifically hydrolyzes peptide bonds at the C terminus of Tyr, Phe and Trp As human H1 histones contain only one Phe residue, it was expected that cleavage with chymotrypsin should produce two peptide fragments In fact, when the digested proteins were separated by RP-HPLC, two main fractions were obtained An example is shown in Fig 2, where a digest of fraction 3 isolated by HILIC separation of H1 from CCRF-CEM cells (Fig 1A) was analyzed by RP-HPLC The purity and homogen-eity of the two fractions was assessed by capillary elec-trophoresis (CE) (data not shown) To identify the fractions, amino acid sequencing was performed Fraction 1 contained the C-terminal region starting after Phe at amino acid 105 No sequence data were
Trang 3obtained from fraction 2 because the first amino acid was blocked, indicating that fraction 2 consisted of the N-terminal region of this H1 subtype By using protein sequence data, the four H1 peaks from CCRF-CEM cells (Fig 1A) were identified as follows: peak 1, his-tone H1.2 (SWISS-PROT P16403); peak 2, hishis-tones H1.3 (SWISS-PROT P16402) + H1.5 (SWISS-PROT P16401); peak 3, histone H1.4 (SWISS-PROT P10412); and peak 4, histone H1.5
Identification of the Raji H1 fractions (Fig 1B) yielded the same result for peaks 1, 2 and 4, while peaks 3a and 3b contained histone H1.4 In order to exclude the presence of phosphorylated H1.4, which would be a reasonable explanation for this diversity, the Raji H1 proteins were incubated with alkaline phosphatase and subjected to HILIC Peak 4 showed a dramatic decrease in size, whereas the two H1.4 peaks were not affected by the phosphatase (data not shown) Therefore, peak 4 was identified as a phos-phorylated form of H1.5 Surprisingly, the two H1.4 subfractions (Fig 1B, peaks 3a and 3b) were not sep-arated either by HPCE or RP-HPLC or by different gel-electrophoretic methods (data not shown) To examine the structural difference between these two
A
B
C
Fig 1 Hydrophilic interaction liquid chromatography (HILIC)
separ-ation of H1 histones isolated from human tumor cell lines H1
his-tone samples from (A) CCRF-CEM cells, (B) Raji cells, and (C)
K562 cells were analyzed on a PolyCAT A column (4.6
mm · 250 mm) at 23 C, and at a constant flow of 1.0 mLÆmin)1,
by using a two-step gradient starting at solvent A ⁄ solvent B
(100 : 0) [solvent A: 70% (v ⁄ v) acetonitrile, 0.015 M
triethylammo-niumphosphate (TEAP, pH 3.0); solvent B: 70% (v⁄ v) acetonitrile,
0.015 M TEAP (pH 3.0) and 0.68 M NaClO4] The concentration of
solvent B was increased from 0 to 80% (v ⁄ v) during a time-period
of 5 min and from 80 to 100% (v ⁄ v) during a time-period of 60 min.
The isolated protein fractions (designated 1–4) were desalted by
using RP-HPLC.
Fig 2 RP-HPLC analysis of peptide fractions of chymotrypsin-digested H1 from CCRF-CEM cells Peak 3 from H1 histones iso-lated with hydrophilic interaction liquid chromatography (HILIC) (Fig 1A) was digested with chymotrypsin, as described in the Experimental procedures The digest (containing 100 lg of protein) was injected onto a Nucleosil 300-5 C 18 column (250 mm · 3 mm) Analysis was performed at a constant flow of 0.35 mLÆmin)1with a multistep acetonitrile gradient starting at sol-vent A ⁄ solvent B (85 : 15) (solvent A: water containing 0.1% (v ⁄ v) trifluoroacetic acid; solvent B: 85% (v ⁄ v) acetonitrile and 0.1% (v ⁄ v) trifluoroacetic acid) The concentration of solvent B was increased linearly from 15 to 23% during a time-period of 25 min, from 23 to 70% during a time-period of 45 min and from 70 to 100% during a time-period of 5 min.
Trang 4proteins, we analyzed the two main fractions 1 and 2
obtained from chymotrypsin digestion and RP-HPLC
separation (as shown for CCRF-CEM cells in Fig 2)
of peaks 3a and 3b under HILIC conditions We
found that fraction 2 derived from peaks 3a and 3b
had the same elution time, while fraction 1 clearly
dif-fered Therefore, the C-terminal domain should be
responsible for the microheterogeneity of histone H1.4
In order to elucidate the nature of this alteration,
the C-terminal peptides were further cleaved with
endoproteinase Glu-C Digests were analyzed by
RP-HPLC by using a Nucleosil 300-5 C18 column
(250· 3 mm; Fig 3) Fragmentation yielded three
main peptides (I–III), which were identified by Edman
degradation: fraction I (eluting at 27 min) consisted of
a peptide starting at residue 105, fraction II (43 min)
consisted of a peptide starting at residue 115, and
frac-tion III (60 min) consisted of a peptide starting at
resi-due 150 The purity of the fractions obtained was
confirmed by CE (data not shown) Again, all peptides
were analyzed under HILIC conditions, and the result
showed that only the two fractions III contained
peptides with different elution times Further peptide
sequencing of fraction III revealed that the two H1.4 HILIC peaks differed from one another by a single amino acid substitution – lysine at position 173 (peak 3b) was replaced by arginine (peak 3a) This result was further confirmed by subjecting the two H1.4 subfrac-tions obtained by HILIC (Fig 1B) to electrospray ion-ization mass spectrometry analysis For peak 3a we found a mass of 21802.4 Da and for peak 3b a mass
of 21774.8 Da, the latter being in close agreement with the wild-type H1.4 mass, which was calculated to be 21776.1 Da The mass difference observed between the two peaks was 27.6 Da, and this corresponds to the mass difference between lysine and arginine This microsequence variant H1.4 Lys173Arg, found in Raji cells in the same concentrations as the wild-type H1.4, was detected neither in CCRF-CEM and K562 cells nor in several other human cell lines (e.g U937, HL60) or in human tissue (placenta, testis)
A further microheterogeneity was found when ana-lyzing the K562 H1 sample by HILIC (Fig 1C) Peak
1, which was a single fraction in CCRF-CEM and Raji cells, and identified as histone H1.2, was separated into two subfractions (peaks 1a and 1b) in K562 cells RP-HPLC separation after chymotrypsin digestion of the two fractions 1a and 1b yielded two main frag-ments each (similar to Fig 2) HILIC analysis revealed that fraction 2 from 1a had a shorter elution time than did fraction 2 from 1b, whereas no differences were observed between the fraction 1 samples Further clea-vage of the fraction 2 samples with endoproteinase Glu-C followed by peptide sequencing showed that the proteins differed by one amino acid out of a total of 212: peak 1a contained valine in position 17, while peak 1b contained wild-type alanine Histone H1.2 from all cell lines and tissues investigated contained only alanine at this location
To confirm the H1.2 Ala17Val sequence variation, a
183 bp PCR product was amplified in the 5¢-UTR, and the start of the coding sequence of the H1.2 gene cor-responding to the N-terminal tail of the protein PCR products from K562, Raji and wild-type blood donors were sequenced K562 DNA contained a homozygous CfiT substitution at nucleotide position 578 in the H1.2 gene, resulting in a change, in codon 18, from GCC to GTC (Fig 4), encoding alanine and valine, respectively This change in codon 18 corresponds to the substitution at amino acid position 17 in the H1.2 protein, as the initiating methionine is removed after translation in all H1 histones Traces of the wild-type
C in position 578 were also detected (Fig 4)
To obtain the population frequency of the H1.2 g578 CfiT substitution, 103 healthy individuals were screened by using a restriction fragment length
Fig 3 RP-HPLC analysis of peptide fractions of endoproteinase
Glu-C-digested fraction 1 from Raji H1.4 Peak 3a from Raji H1
histones isolated with hydrophilic interaction liquid chromatography
(HILIC) (Fig 1B) was digested with chymotrypsin and isolated by
RP-HPLC (Fig 2) The fraction 1 obtained was further digested with
endoproteinase Glu-C, as described in the Experimental
proce-dures, and the digest (containing 30 lg of protein) was injected
onto a Nucleosil 300-5 C18column (250 mm · 3 mm) Analysis was
performed at a constant flow of 0.35 mLÆmin)1 with a multistep
acetonitrile gradient starting at solvent A ⁄ solvent B (95 : 5) [solvent
A: water containing 0.1% (v ⁄ v) trifluoroacetic acid; solvent B: 85%
(v ⁄ v) acetonitrile and 0.1% (v ⁄ v) trifluoroacetic acid] The
concentra-tion of solvent B was increased linearly from 5 to 20% during a
time-period of 65 min and from 20 to 100% during a time-period of
25 min.
Trang 5polymorphism (RFLP) assay The wild-type H1.2 PCR
product was cleaved into three fragments by BsuRI,
while a PCR product containing the g578 CfiT
substi-tution was cleaved into two fragments (Fig 5) We
found 10 individuals to be heterozygous and two
homozygous for the g578 CfiT substitution, resulting
in an allele frequency of 6.8% in this population The
presence of the g578 CfiT substitution in samples
dis-playing the 130 bp fragment in the RFLP analysis was
confirmed by DNA sequencing
To detect the H1.4 Lys173Arg substitution, a 217 bp
fragment of the H1.4 gene, corresponding to the C
ter-minus of the H1.4 protein, was amplified by PCR Raji,
K562 and wild-type blood donor PCR products were
subjected to DNA sequencing Raji cells were
hetero-zygous for an AAA to AGA codon change at position
174 (Fig 6), resulting in a Lys173Arg substitution in
histone H1.4 To determine the allele frequency of this
g1250 AfiG substitution in the H1.4 gene, a
denaturat-ing HPLC method was developed (Fig 7)
Hetero-duplex and mutant homoduplex formation was
resolved after mixing all PCR products with wild-type PCR products of H1.4 No G alleles were detected in the normal population of 206 alleles studied As the Raji cell line was derived from an individual suffering from Burkitt’s lymphoma, six other cell lines established from Burkitt’s lymphoma were screened for the g1250 AfiG substitution However, none of these cell lines contained this sequence variation
Discussion
A major question concerning the expression of indi-vidual H1 subtypes is whether they have coevolved with functional differences Although the precise func-tion of H1 isoforms has yet to be determined, several observations suggest distinct and nonoverlapping roles for individual H1 variants Estimation of the rates of nucleotide substitution for mammalian H1 subtypes
Fig 5 Restriction fragment length polymorphism (RFLP) analysis
of H1.2 PCR products Lanes 1 and 2, blood donors, heterozygous
for g578 CfiT Lane 3, wild-type blood donor Lane 4, K562,
homo-zygous for g578 CfiT Lane 5, uncleaved control Lane 6, 100 bp
ladder.
Fig 6 DNA sequencing of H1.4 PCR products.
Fig 7 Denaturing HPLC of PCR-amplified H1.4 fragments (A) Wild-type blood donor (B) Raji, heterozygous for g1250 AfiG (C) Raji and wild-type PCR products in a 1 : 1 ratio.
Fig 4 DNA sequencing of H1.2 PCR products.
Trang 6H1a–H1e during evolution showed evidence of their
functional differentiation [30] The study revealed that
the rates of nucleotide substitution differed not only
among subtypes, but also among domains For all
subtypes, the synonymous substitution rate greatly
exceeded the nonsynonymous rate, and the terminal
domains were more variable than the central globular
domain We detected two nonsynonymous
substitu-tions in human H1 histones One caused alanine to
be substituted by valine in the N-terminal region
(position 17) of histone H1.2 in K562 cells, leading to
two subfractions using HILIC The H1.2 Ala17Val
variant constituted the major fraction of the H1.2
protein extracted, while a minor fraction of wild-type
H1.2 was present In agreement with this, K562
DNA was found to carry a homozygous GCC to
GCT change at codon 18 in the H1.2 gene There
were remains of the wild-type C at g578 (Fig 4),
explaining the minor wild-type H1.2 peak in the
HILIC chromatogram This result is probably
explained by the nondiploid karyotype of the K562
cells The H1.2 g578 CfiT sequence variant was also
found to be present in a normal population, with the
allele frequency 6.8%, and is therefore concluded
to be a polymorphism The H1.2 Ala17Val was
expressed in K562 cells and therefore probably also
in normal individuals carrying this gene variant
However, H1 histones show a high redundancy in
knockout organisms, and the deletion of one or more
subtypes causes increased expression of those
remain-ing [12,31] Therefore, it cannot be completely ruled
out that normal individuals carrying the H1.2 g578
CfiT are devoid of Ala17Val H1.2 expression This
polymorphic site in the H1.2 protein has previously
been recognized in human spleen [18] The
corres-ponding SNP was recently reported (NCBI SNP
data-base refSNP ID rs 2230653) In addition, three other
SNPs have been reported in H1.2, all leading to
synonymous changes (NCBI SNP) The role of the
N-terminal tail of H1 histones is unclear, but is
believed to be involved in positioning of the
glo-bular domain on the nucleosome [32] The N- and
C-terminal tails of histone H1 adopt a random coil in
solution [33] On binding of histone H1 to DNA,
significant parts of the N terminus are likely to take
on an a-helical structure [34], and this is probably
also the case for other H1 subtypes As the tails of
H1 histones do not adopt their native conformation
until they bind to chromatin, it is hard to predict the
structural changes that a single amino acid substitution
may trigger Substitution of valine for alanine may
affect the predicted a-helical structure of the tail as
valine has a less stabilizing effect on an a-helical
struc-ture If the structure is affected by the polymorphism, the positioning of H1.2 on the nucleosome, or the binding of H1.2 to chromatin, may be affected
A further nonsynonymous substitution prompted the replacement of lysine with arginine in the C-ter-minal tail (position 173) of histone H1.4 in Raji cells
By using HILIC, histone H1.4 was separated into two peaks: one wild-type H1.4; and one Lys173Arg H1.4 This microsequence variant was found for the first time and was present in stationary-phase cells in simi-lar amounts as wild-type H1.4 Genetic analysis of Raji cells showed a heterozygous H1.4 g1250 AfiG substitution, prompting alteration of codon 174 from AAA to AGA, in agreement with the Lys173Arg sub-stitution This sequence variant was not present in the
103 normal individuals that were screened, or in six other Burkitt’s lymphoma cell lines, implying that the Lys173Arg substitution is probably a mutation or a rare polymorphism detected, thus far, only in Raji cells Denaturing HPLC, used to screen for this genetic variant, provides a sensitive and highly specific method for investigating sequence variations [35], and the pos-sibility of false negative results is unlikely Histone H1.4 mRNA (GenBank NM_005321) has been repor-ted to contain a different polymorphism (NCBI SNP refSNP ID rs2298090), c455 AfiG, causing a Lys152Arg substitution (NP_005312.1)
The C-terminal tail of histone H1 is believed to be responsible for the condensation of chromatin [32,36], and the condensing property of rat H1d probably resides in a C-terminal 34 amino acid stretch [37] Different H1 subtypes may have different chromatin-condensing properties [38,39] On binding to DNA, the C-terminal tail probably adopts the structure of a seg-mented a helix [40] Replacement of lysine with argi-nine may affect the secondary structure of the C-terminal tail and the binding of H1.4 to chromatin,
as arginine offers additional hydrogen-bonding abilities
to DNA as compared to lysine Lysine 173 in H1.4 is situated in an SPKK motif, one of the known sites for H1 phosphorylation As far as we know, there are no differences in the phosphorylation behavior of SPKK and SPRK motifs
Identification of numerous linker histone variants in vertebrates suggests that these proteins may play spe-cialized roles Recent investigations using gene-targeting techniques, however, suggest that the specific timing of expression may have a greater functional significance than the nature of the individual H1 subtypes [2,41]
It is, however, not clear to what extent the function of H1 variants depends on their primary sequence or on the specific timing of their expression The literature presents arguments in favor of both possibilities [2,7]
Trang 7The selective effect of linker histones on
transcrip-tion of individual genes was demonstrated by using
an in vivo system for inducible overexpression of
dif-ferent H1 subtypes (H1, H1c) in mouse cells [42,43]
Subtype-specific effects were shown to be related to
differences in the structure of the globular domains
[44] As H1 subtypes differ not only in primary
sequence but also in turnover rate and extent of
phosphorylation, they have the potential to add a
great deal of flexibility to chromatin structure and
transcriptional activation
Both genes homologous to H1.2 and H1.4 have been
disrupted in mouse, each resulting in viable and fertile
knockout mice [12], indicating that these individual
sub-types are dispensable and that compensatory effects
reside between the subtypes, thus keeping H1
stoichio-metry intact Despite the dispensability of wild-type H1
subtypes, however, microsequence variants may have
biological significance Recent evidence reveals a highly
specific function for H1.2 in DNA damage-induced
apoptosis [14] As the somatic H1 subtypes H1.1–H1.5
show a high degree of sequence conservation, such
spe-cificity must rely on subtle differences in amino acid
sequence Therefore, as histone H1 is implicated in
chro-matin organization, cell differentiation, gene regulation
and apoptosis, these processes may be affected by minor
sequence variations, including SNPs
In conclusion, we have demonstrated the remarkably
high resolving power of HILIC by using this technique
to separate sequence variants within human linker
his-tone subtypes We were thus able to detect an
Ala17-Val substitution in histone H1.2 in K562 cells, as well
as a Raji-specific H1.4 Lys173Arg sequence variation
at the protein level These observations were confirmed
at the genetic level The significance of these variations
is unclear, but it seems increasingly clear that minor
sequence variations in linker histones may affect
important cellular functions in vivo
Experimental procedures
Chemicals
Sodium perchlorate (NaClO4), trifluoroacetic acid and
tri-ethylamine were purchased from Fluka (Buchs,
Switzer-land) All other chemicals were purchased from Merck
(Darmstadt, Germany), unless indicated otherwise
Cell lines and culture conditions
CCRF-CEM acute lymphoblastic leukemia cells, Raji cells
(originally derived from patients with Burkitt’s lymphoma)
and K562 erythroleukemic cells were cultured in
RPMI-1640 medium (Biochrom, Berlin, Germany) supplemented with 10% (v⁄ v) fetal bovine serum, penicillin (60 lgÆmL)1) and streptomycin (100 lgÆmL)1) in the presence of 5% (v⁄ v) CO2 The cells were seeded at a density of 8· 104
cellsÆmL)1and harvested after 7 days to accumulate cells in stationary phase
Preparation of H1 histones Human cells (6–7· 109) were collected by centrifugation
(800 g for 10 min) H1 histones were extracted with
per-chloric acid (5%, w⁄ v) according to the procedure of Lind-ner et al [28]
HPLC The equipment used for HPLC consisted of two 114M pumps, a 421A system controller and a Model 165 UV-vis-ible-region detector (Beckman Instruments, Palo Alto, CA, USA) The effluent was monitored at 210 nm and the peaks were recorded by using Beckman System Gold software
HILIC Whole human H1 samples were analyzed on a PolyCAT A column (4.6 mm· 250 mm; 5 lm particle pore size; 30 nm pore size; ICT, Vienna, Austria) at 23C, and at a constant flow of 1.0 mLÆmin)1, by using a two-step gradient starting at solvent A⁄ solvent B (100 : 0) [solvent A: 70% (v ⁄ v) acetonit-rile, 0.015 m TEAP, pH 3.0; solvent B: 70% (v⁄ v) aceto-nitrile, 0.015 m TEAP (pH 3.0) and 0.68 m NaClO4] The concentration of solvent B was increased from 0 to 80% (v⁄ v) during a time-period of 5 min and from 80 to 100% (v⁄ v) during a time-period of 60 min The isolated protein fractions were desalted by using RP-HPLC Histone fractions obtained in this manner were collected and, after adding 0.01 m 2-mercaptoethanol, freeze-dried and stored at)20 C
RP-HPLC The peptides obtained by limited chymotrypsin digestion of human H1 histones were separated by using a Nucleosil 300-5 C18column (250 mm· 3 mm internal diameter; 5 lm particle pore size; end-capped; Macherey-Nagel, Du¨ren, Germany) Samples of 100 lg were injected onto the col-umn Chromatography was performed within 70 min at a constant flow of 0.35 mLÆmin)1with a multistep acetonitrile gradient starting at solvent A⁄ solvent B (85 : 15) [solvent A: water containing 0.1% (v⁄ v) trifluoroacetic acid; solvent B: 85% (v⁄ v) acetonitrile and 0.1% (v ⁄ v) trifluoroacetic acid] The concentration of solvent B was increased linearly from 15 to 23% during a time-period of 25 min, from 23 to 70% during a time-period of 45 min and from 70 to 100% during a time-period of 5 min
Trang 8Human H1 peptide fractions obtained by digestion
with endoproteinase Glu-C were separated by using the
same column and solvents as described above The
con-centration of solvent B was increased linearly from 5 to
20% during a time-period of 65 min and from 20 to
100% during a time-period of 25 min Fractions obtained
in this manner were collected and, after adding 20 lL of
2-mercaptoethanol (0.2 m), were lyophilized and stored at
)20 C
Chymotrypsin digestion
Histone H1 subfractions ( 100 lg), obtained from human
cell lines by HILIC fractionation, were digested with
a-chymotrypsin (EC 3.4.21.1) (Sigma type I-S, 1⁄ 150, w ⁄ w)
in 100 lL of 100 mm sodium acetate buffer (pH 5.0) for
30 min at room temperature The digest was subjected to
RP-HPLC
Endoproteinase Glu-C digestion
Histone H1.4 fractions ( 50 lg) were digested with
Sta-phylococcus aureus V8 Protease (Boehringer Mannheim,
Mannheim, Germany; 1 : 20, w⁄ w) in 50 lL of 50 mm
NH4HCO3buffer (pH 7.8) for 5 h at 37C Histone H1.2
fractions ( 50 lg) were digested in 50 lL of 25 mm
phos-phate buffer (pH 7.8) for 6 h at room temperature The
digests were subjected to RP-HPLC
Peptide sequencing
Peptide sequencing was performed on an Applied
Biosys-tems Inc (ABI, Foster City, CA, USA) Model 492 Procise
protein sequenator Typically, 5–100 pm of a peptide
sam-ple was run for 3–40 cycles, as required for an
unambigu-ous identification
Mass-spectrometric analysis
Determination of the molecular masses of the two histone
H1.4 subfractions obtained by the HILIC run was carried
out by electrospray ionization mass spectrometry using an
LCQ ion trap instrument (ThermoFinnigan, San Jose, CA,
USA) Samples (15 lg) were dissolved in 50% (v⁄ v)
aque-ous methanol, containing 0.1% (v⁄ v) formic acid, and
injected into the ion source
DNA samples
Genomic DNA from various cell lines was extracted by
using the DneasyTM tissue kit (Qiagen, Hilden, Germany)
and examined for sequence variations in codon 18 of H1.2
and in codon 174 of H1.4 To obtain the frequency of the
two polymorphisms in H1.2 and H1.4 in a normal
popula-tion, DNA samples collected randomly from 103 normal individuals in south-east Sweden were screened The indi-viduals were selected from a population register and were 22–77 years of age (mean age, 52 years; SD, 17 years; 47% men and 53% women) The design was approved by Linko¨-ping University Hospital Ethical Committee DNA was extracted from the blood samples by using the QIAamp DNA Blood Maxi kit (Qiagen)
PCR amplification and RFLP analysis of H1.2
A 183 bp fragment of the H1.2 gene (HIST1H1C, GenBank X57129) was amplified by using the PCR primers 5¢-CCCAGGCGCTGCTTC-3¢ (nucleotides 469fi483 of the H1.2 gene) and 5¢-CTCTGACACCGGGGGAC-3¢ (nucleotides 651fi635 of the H1.2 gene) The PCR was per-formed with 50 ng of DNA in a 20 lL reaction, containing
1 mm MgCl2, 0.025 UÆmL)1TaqDNA polymerase, 20 mm Tris⁄ HCl, pH 8.4, 50 mm KCl, 1 lm of each primer (all Life Technologies, Gaithersburg, MD, USA) and 200 lm of each dATP, dCTP, dGTP and dTTP (Amersham Pharma-cia Biotech, Piscataway, NJ, USA) After an initial denatur-ation at 94C for 2 min, amplification was performed for
35 cycles with denaturation at 94C for 1 min, annealing
at 57C for 1 min and extension at 72 C for 1 min, in a thermal cycler (PTC-200; MJ Research, Watertown, MA, USA) The reaction was completed with an extension step
at 72C for 7 min RFLP analysis was carried out by digesting the 183 bp PCR product with 10 U BsuRI (HaeIII) (MBI Fermentas, St Leon-Rot, Germany), at
37C overnight BsuRI recognizes the sequence 5¢-GGCC-3¢, and the PCR product from wild-type DNA was digested into three fragments of 21, 53 and 109 bp Digestion of the PCR product from DNA containing the g578 CfiT substi-tution in the recognition sequence produced two fragments, one of 53 bp and one of 130 bp The digested PCR products were analyzed on Tris⁄ borate ⁄ EDTA-agarose gels containing 1% (w⁄ v) agarose (BioRad, Hercules, CA, USA), 3% (w⁄ v) NuSieve GTG Agarose (FMC BioProd-ucts, Rockland, ME, USA) and ethidium bromide (0.5 lgÆmL)1) The fragments were visualized under UV transillumination and photographed by using a Polaroid camera
PCR amplification of H1.4 and polymorphism detection by using denaturing HPLC
A 217 bp fragment of the H1.4 gene (HIST1H1E, GenBank M60748) was PCR amplified by using the primers 5¢-GA AGAGCGCCAAGAAGACC-3¢ (nucleotides 1173fi1191
of the H1.4 gene) and 5¢-CTACTTTTTCTTGGCTGCCG (nucleotides 1389fi1370 of the H1.4 gene), using the same conditions as described above For mutation analysis, a denaturating HPLC system (WAVE Nucleic Acid
Trang 9Frag-ment Analysis System; Transgenomic, Crewe, UK) was
used All samples were mixed with wild-type PCR product,
which had previously been subjected to DNA sequence
ana-lysis, in a 1 : 1 ratio to ensure detection of g1250 AfiG
homozygous mutants Before analysis, the samples were
denaturated at 95C for 4 min and then gradually
cooled, by 1CÆmin)1, until 25C was reached, to allow
heteroduplex formation
The optimal melting temperature for the fragment was
calculated by using the wave software, and a temperature
of 63.3C was used for analysis The flow rate was
0.9 mLÆmin)1 and the total run time 7.2 min Samples
(20 lL) were injected onto the DNA Sep Column at 54%
buffer A (0.1 m triethylammonium acetate, pH 7.0) and
46% buffer B [0.1 m triethylammonium acetate, pH 7.0,
and 25% acetonitrile (v⁄ v)] and heteroduplexes were
separ-ated by using a gradient starting at 49% buffer A and 51%
buffer B, and gradually increasing to 40% buffer A and
60% buffer B
DNA sequencing
Direct cycle sequencing of H1.2 and H1.4 PCR products
was performed with the corresponding forward PCR
pri-mer, using Thermo Sequenase radiolabeled terminator cycle
sequencing kit (USB Corporation, Cleveland, OH, USA),
and labeling with33P dideoxy nucleotides (Amersham
Phar-macia Biotech), according to the manufacturers’
recommen-dations Prior to sequencing, the PCR products were
purified and concentrated by using GFX PCR DNA and
the Gel band purification kit (Amersham Pharmacia
Bio-tech) The labeled products from the sequencing reaction
were separated on 6% (w⁄ v) polyacrylamide gels,
contain-ing 6 m urea, in a gel apparatus (OWL) at 70 W constant
power After electrophoresis, the gel was dried and exposed
to X-ray film
Acknowledgements
We thank A Devich, A Molbaek and S Gstrein for
their excellent technical assistance This work, as part
of the European Science Foundation EUROCORES
Programme EuroDYNA, was supported by funds from
the Austrian Science Foundation (project I23-B03) and
the EC Sixth Framework Programme under Contract
no ERAS-CT-2003-980409 and in part by the Swedish
Cancer Society
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