1 Georg-August University of Göttingen, Third Department of Zoology
Developmental Biology, Humboldtallee 34A, 37073 Göttingen, Germany
2 Georg-August University of Göttingen, Institute for X-ray Physics,
Geiststraße 11, 37073 Göttingen, Germany
3 Hannover Medical School, Neuroanatomy, Carl-Neuberg-Str.1, 30625 Hannover,
Germany
* Author for correspondence (e-mail: eschulz{at}gwdg.de )
Accepted 1 May 2002
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Summary |
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Key words: Caenorhabditis elegans, Histone H1, Chromatin, Intermediate filaments, Linker histone
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Introduction |
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We used C. elegans as a model system to investigate this question.
C. elegans possesses eight different linker histone variants, the
same number as human (Albig et al.,
1997; Yamamoto and Horikoshi,
1996
) or mouse (Drabent et
al., 1995
), and it allows us to dissect the function of individual
linker histone genes with RNA interference (RNAi) and cytological techniques.
In previous work we characterized the function of the major histone H1 variant
in C. elegans, H1.1 (Jedrusik and
Schulze, 2001
). Depletion of H1.1 leads to a loss of the
germ-line-specific chromatin silencing and consequently also to hermaphrodite
sterility. Although the depletion of H1.1 was created with relatively low
penetrance (11%) we believe that H1.1 is essential for the reproductive growth
of C. elegans. Interestingly, however, the biological function of
H1.1 is not a general housekeeping activity, such as maintaining the basic
structures of chromatin, the nucleosome and the 30 nm fiber would suggest, but
instead a specific contribution to a developmental program, the silencing and
the maintenance of the germ line.
In this study we address the function of H1.X, a linker histone-like protein and a member of the C. elegans linker histone gene family. Although H1.X does follow the canonical three-domain structure of the tripartite linker histones, it violates further principles of linker histone structure. In contrast to all regular linker histones, H1.X does not contain lysine in its N-terminal domains. Additionally, and also in contrast to regular linker histones, H1.X contains the highly hydrophobic amino acid residues tyrosine and leucine in both terminal domains. These drastic changes of the biochemical properties of its terminal domains suggest that histone H1.X has a function different from that of a typical linker histone. The work presented here confirms this assumption. RNA interference suggests that H1.X contributes to muscle function and development. H1.X is the first linker histone-like protein with a prominent cytoplasmic localization and function.
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Materials and Methods |
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cDNA cloning and RNA interference
A cDNA encoding H1.X (pC18/19) was cloned into pUC18 after reverse
transcribed PCR amplification with the primers ESMG19
5'-GGATCCAGCATATGACCACTTCGCTCATCCACATGG-3' and ESMG18
5'-CCATCGATGGTGGGAAATCTAAACTACAGGTGTC-3' from total RNA of
wild-type C. elegans using Superscript II polymerase (GibcoBRL)
according to the manufacturer's instructions. For dsRNA synthesis the insert
was transferred as a BamHI-ClaI restriction fragment to
pBluescript II SK (+) (Stratagene). Later, a second cDNA clone (yk480h7)
encoding the same protein was kindly provided by Yuji Kohara, Mishima. The
phenotype of H1.X depletion was generated with RNA-mediated interference
(Fire et al., 1998). For the
synthesis of double-stranded RNA (dsRNA), the plasmid template DNAs were
separately digested with BamHI and ClaI. The restriction
enzymes were deactivated at 80°C for 20 minutes. The digested DNA
templates were mixed together and transcribed in a single reaction using T7
and T3 RNA polymerases (Megascript T7 kit, Ambion). RNA integrity was
determined by gel electrophoresis; concentrations were determined by ethidium
bromide staining in the gel. Uncapped dsRNA H1.X with a concentration of 5
mg/ml was injected into hermaphrodite gonads of the wild-type strain.
Unrelated dsRNA (4 mg/ml GFP dsRNA) or phosphate buffer M9
(Sulston and Hodgkin, 1988
)
were injected in control experiments. The F1 progeny derived from 0 to 48
hours after injection were scored for embryonic lethality, morphology and
behavior. The injected animals were cultured on RNA feeding plates
(Timmons and Fire, 1998
). For
RNAi feeding experiments, H1.X cDNA was cloned between the T7 promoters of the
L4440 feeding vector and transformed into Escherichia coli
HT115(DE3). Feeding plates were prepared from HT115 cells induced with 0.4 mM
isopropyl-ß-D-thiogalacto-pyranosid (IPTG) for 4 hours at 37°C.
Bacterial expression of truncated and full length recombinant H1.X
proteins
Recombinant proteins were produced with the T7 polymerase expression system
(Rosenberg et al., 1987) in
E. coli BL21. For the expression of full-length H1.X the cDNA clone
yk480h7 was amplified with the primers ESMG18 and ESMG19 (see above), cut with
NdeI and ClaI and cloned into pET3a. For the expression of
the truncated H1.X protein (C-terminal 101 amino acid residues), the
corresponding region of the cDNA clone pC18/19 was amplified with the primers
ESMG54 5'-GGAATTCCATATGTCAGAAGTTCGTCAGAA-3' and ESMG18
5'-CCATCGATGGTGGGAAATCTAAACTACAGGTGTC-3', cut with NdeI
and ClaI and cloned into pET3a. Protein expression and extraction
were performed as described before
(Wisniewski and Schulze,
1994
).
Antibodies
Two different rabbit antisera were generated. For the first
(H1.X-11), a synthetic peptide corresponding to the C-terminal eleven
amino acids of H1.X (Glu-Leu-Arg-Thr-Gly-Thr-Arg-Lys-Ser-Tyr-Cys) was
synthesized and coupled to the carrier protein hemocyanin, which then was used
as the antigen. This peptide was chosen because it is unrelated to any other
C. elegans protein, including the remaining seven linker histones.
The second antigen was recombinant truncated H1.X (the C-terminal 101
amino-acid residues). 500 mg of antigen were used for the immunization of each
rabbit in a series of three injections. Antigen injections and resulting
antiserum collections for both sera were performed by Charles River (Kissleg,
Germany). The IgG fractions of the sera were obtained with a protein G column
using the MAbTrap GII Kit (Pharmacia Biotech).
H1.X-11 and
H1.X-101 were affinity-purified on SulfoLink (Pierce) columns, onto
which 1 mg of the corresponding HPLC purified antigen (Zorbax 300SB-C18 HPLC
column, Hewlett Packard) had been coupled. The antibody directed against
H1.1-H1.5 and H1.Q has been described previously
(Jedrusik and Schulze, 2001
).
This antibody is not reactive with H1.X.
Western blot analysis
The lysates of C. elegans were prepared by boiling living worms in
SDS sample buffer and subsequently separating the lysates on a 12%
SDS-polyacrylamide gel. Western blot analysis was performed according to the
method of (Towbin et al.,
1979). After transfer onto a nitrocellulose membrane the blot was
blocked for 1 hour at room temperature with 0.1% Tween-20 and 5% dry milk
powder in TBS (150 mM NaCl, 10 mM KCl, 10 mM Tris-HCl pH 7.6) and then washed
with TBS. The blot was incubated with 0.3 µg/ml
H1.X-11 or 14 ng/ml
H1.X-101 in TBS overnight at 4°C and washed with 0.1% Tween-20 in
TBS at room temperature. The detection step was performed with the Phototope
HRP Detection Kit (New England Biolabs) with a secondary anti-rabbit antibody
diluted 1:5000. The blotting membrane was exposed to a Kodak blue Xomat Xb-1
film.
Immunofluorescence
General procedures were performed as described previously
(Miller and Shakes, 1995). The
immunolabeling of embryos was done with the freeze-cracking procedure
(Strome and Wood, 1982
).
Embryos were fixed for 20 minutes in -20°C methanol and transferred to
-20°C acetone for a further 10 minutes. The slides were air dried and
incubated with 1.5% BSA in TBS before the first antibody was applied. Labeling
of adult C. elegans was done according to Finney and Ruvkun
(Finney and Ruvkun, 1990
).
Worms were suspended in ice-cold RFB buffer (160 mM KCl, 40 mM NaCl, 20 mM
Na2EGTA, 10 mM spermidine-HCl, 30 mM PIPES, pH 7.4 and 50%
methanol). Fixation was done with 1% formaldehyde in RFB in three freeze-thaw
cycles. The sample was incubated for 40 minutes on ice with agitation and then
washed two times with TTB (100 mM Tris HCl, pH 7.4, 1% Triton X-100, 1 mM
EDTA). The worms were resuspended in TBS with 1% beta-mercaptoethanol,
incubated for 2 hours on a shaker at 37°C and then washed in 10 volumes of
borate buffer (1M H3BO3, 0.5 M NaOH, pH 9.5) with 0.01%
Triton X-100. The worms were incubated for 15 minutes in borate buffer with 10
mM DTT and 0.01% Triton X-100 and washed again in 10 volumes of borate buffer
with 0.01% Triton X-100. The samples were then incubated in borate buffer with
0.3% H2O2 and 0.01% Triton X-100 for 15 minutes at room
temperature and once again washed with 0.01% Triton X-100 in borate buffer.
After this step, the worms were washed with AbB (1x PBS, 0.1% BSA, 0.5%
Triton X-100, 0.05% sodium azide and 1mM EDTA) for 15 minutes. The
affinity-purified polyclonal
H1.X antibodies were used with a
concentration of 6 µg/ml (
H1.X-11) or with 10.75 µg/ml
(
H1.X-101) in AbA (AbB with 1% BSA), 10% goat serum and 0.1% Triton
X-100. The samples were incubated overnight at 4°C. Fibrillarin was
colocalized with ascites fluid of the mouse monoclonal antibody P2G3
(Christensen and Banker, 1992
),
diluted 1:3000. The samples were washed for 2 hours with several changes of
AbB. The green fluorescent secondary antibody [Cy2-conjugated goat anti-rabbit
IgG F(ab')2 fragment, Jackson ImmunoResearch Laboratories]
was diluted 1:800 with AbA; the red fluorescent antibody anti-mouse IgG-Cy3
conjugate (Sigma, C-2181) was diluted 1:100 with AbA and both were incubated
overnight at 4°C.
As an accessibility control, a mouse monoclonal antibody [IFA
(Bartnik et al., 1986)]
directed against intermediate filament proteins was applied in combination
with the
H1.X-11 antibody as a 1:1 diluted cell culture supernatant.
IFA was detected with anti-mouse IgG-Cy3 conjugate (Sigma, C-2181) diluted
1:100. The specimens were stained either after the antibody incubations or
directly after fixation with 1.6 µM
2'-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1
H-benzimidazole (Hoechst No. 33342) in PBS to visualize DNA.
Microscopy
Conventional and confocal light microscopy were performed with a Zeiss
Axioplan 2 microscope equipped with a Zeiss confocal laser-scanning module LSM
510, Zeiss laser scanning software LSM 510 Release 2.01, a Spot RT CCD camera
(Diagnostic Instruments, Sterling Heights, MI), Nomarski differential
interference contrast and epifluorescence optics. Green fluorescent images
were acquired with an excitation wavelength of 488 nm and an emission filter
band pass 505-550 nm; Hoechst and DAPI DNA staining images were acquired with
an excitation wavelength of 365 nm and an emission filter band pass 395 nm.
Fig. 2A-E,
Fig. 4,
Fig. 5 and
Fig. 6 are laser-scanning
micrographs, whereas Fig. 2F,
Fig. 3,
Fig. 7,
Fig. 8 and
Fig. 9 were recorded using a
conventional microscopic setup. Fig.
2A is a projection calculated from a stack of 71 individual two
dimensional laser scannings recorded with a resolution of 1024x1024,
which corresponded to 0.13 µm per pixel.
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H1.X::GFP expression in HeLa cells
For the expression of H1.X::GFP in human HeLa cells (ATCC CCL-2), the H1.X
cDNA was amplified with the primers ESMG73
5'-GGGGTACCACCATGGTGACCACTTCGCTCATCCACATGG-3' and MJ12
5'-CGGGATCCCGACAATAGCTCTTTCTGGTTCCGG-3' from the cDNA clone
yk480h7 and cloned with KpnI and BamHI into the vector
pEGFP-N1 (Clontech). The resulting plasmid expresses H1.X as an unfused
protein from the CMV promotor and was used for the transfection of HeLa cells
with lipofectamine (GibcoBRL) according to the manufacturer's instruction. The
cells were seeded onto coverslips and exposed for 24 hours to the DNA-loaded
liposomes in Optimem medium and cultured a further one or two days in DMEM
with 10% FCS before microscopy was performed. Microscopy of living cells was
done in PBS. Alternatively the cells were washed two times in PBS, fixed for 5
minutes with 4% formaldehyde in PBS (pH 7.6) at 25°C, washed with PBS and
extracted with 1% Triton TX-100 in PBS for 20 seconds.
Computer software
Database searches in GenBank were done with the BLAST program suite version
2.2.1; database searches in the C. elegans databases were done with
BLAST version 2.0a13MP (Altschul et al.,
1990). The profile hidden Markow model database Pfam 6.5 and the
software HMMER 2.1.1 (Bateman et al.,
2000
) were used for the protein domain analysis. Protein fold
prediction was done with 3D-PSSM (Kelley
et al., 2000
). A final procession of micrographs was performed
with the spot camera software 3.1 and Adobe PhotoShop 5.5.
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Results |
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The globular domain of H1.X is flanked by two terminal domains, whose size is comparable to that of the remaining linker histones of C. elegans. Consequently, H1.X is not the smallest nor the largest member of the linker histones gene family in C. elegans. The terminal domains of H1.X, however, contain features that are distinctly different from normal linker histone proteins. Usually both terminal domains are highly charged and contain about 40% lysine residues as well as high amounts of alanine, valine, proline, some residues of all other charged or polar amino acids, but no leucine, cysteine, histidine and strictly no aromatic amino residues. In contrast to this, the N-terminal domain of H1.X does not contain any lysine, but possesses one tyrosine, five leucine and five histidine residues. A BLASTP search in GenBank revealed that the N-terminal domain of H1.X is not only dissimilar to any other linker histone but also that it is dissimilar to any protein represented in the database.
In contrast to the N-terminal domain, the C-terminal domain of H1.X is relatively rich in lysine (22% compared with 28%-39% in H1.1-H1.5). It contains seven leucine residues, one tyrosine residue, two cysteine residues and two histidine residues. A BLASTP search performed on the C. elegans database Wormpep reveals that this C-terminal domain is related to the C-terminal domains of the remaining linker histones. Although the C. elegans linker histone genes encoding H1.1-H1.6 contain a single intron located at a conserved position in the globular domain, the gene encoding H1.X possesses this conserved intron as well as three additional introns located in all three domains of the protein. Therefore the structure of the gene encoding H1.X is clearly related to the remaining linker histones genes of C. elegans, but it also contains distinct and unusual structural differences.
The histone H1.X-encoding gene produces a single protein in C.
elegans
Two independent polyclonal antibodies were raised either against a
synthetic peptide or against a truncated recombinant H1.X protein. The
synthetic peptide (the C-terminal 11 amino-acid residues of H1.X) was designed
to prevent crossreactivity of the resulting antibodies with linker histone
proteins. It does not contain lysine and arginine and is dissimilar to all
other predicted C. elegans proteins. The truncated recombinant H1.X
protein was designed as a larger and therefore more potent antigen which,
however, bears the possibility of creating H1 crossreactive antibodies.
Fig. 1 shows a western blot
analysis of a C. elegans lysate with these two antibodies. Both
antibodies recognize a common band of 31 kDa. In SDS-PAGE linker histones
typically appear at molecular masses above their physical molecular masses
because the high positive net charge is not completely shielded by the ionic
detergent. The apparent size of H1.X (31 kDa) corresponds to that of the
C. elegans linker histones H1.1, H1.3 and H1.5
(Jedrusik and Schulze, 2001),
which have physical molecular masses of 21-23 kDa. The C. elegans H1
proteins H1.4 and H1.2 have an apparent molecular mass different from that of
H1.X and are not detected by
H1.X-101 in the western blot experiment.
This indicates that
H1.X-101, which was raised against the C-terminal
domain of H1.X, does not show a general crossreactivity with C.
elegans linker histones. This is confirmed by the observation that
H1.X-101 does not stain interphase chromatin or condensed chromosomes,
like a normal anti-H1 antibody does.
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The second antibody, H1.X-11, is also not crossreactive with C.
elegans linker histones but shows crossreactivity with two non-histone
proteins of C. elegans. One of these bands appears at 22 kDa and
represents an unidentified protein, whereas the other band at (60 kDa) is
identified as lamin by direct comparison with recombinant C. elegans
lamin protein (Liu et al.,
2000
) in the western blot. This comparison was performed because
H1.X-11 stains the nuclear lamina of all C. elegans nuclei in
immunocytological preparations. Both antibodies do not react with intermediate
filament proteins A1 and B1. As the 31 kDa band is exclusively recognized by
both antibodies, and as this band co-migrates with the recombinant H1.X
protein, we conclude that this band represents H1.X in C.
elegans.
H1.X is tightly associated with the tonofilaments in the marginal
cells
The most prominent structures labeled with both antibodies as well as with
H1.X::GFP in transgenic animals are the marginal cells and the tonofilaments
therein. The signal appears in all larval stages and in adult C.
elegans. Fig. 2A shows a
projection calculated from a stack of 71 confocal images from a hermaphrodite
stained with H1.X-11; Fig.
2B shows one of these sections in differential interference
contrast. The antibody labeling procedure includes a detergent
extraction/reduction step. Using these conditions the only labeled structures
in the marginal cells are the tonofilaments. No further nuclear or cytoplasmic
signal appears besides the labeling of all nuclear lamina caused by the
anti-lamin crossreactivity of
H1.X-11. This result is confirmed by
independent antibody labeling (Fig.
2C) with
H1.X-101. In these preparations no nuclear signal
appeared in the nuclei of the marginal cells, although the cytoplasm of some
head neurons was stained (data not shown).
H1.X-101 did not label the
nuclear lamina. In H1.X::gfp transgenic animals
(Fig. 2D,F), the cytoplasm of
the marginal cells appeared brightly fluorescent, and the nuclei therein
became visible as the brightest fluorescent structures of the cells. The GFP
fluorescence was almost homogeneously distributed throughout the whole
cytoplasm, but the tonofilaments therein became clearly visible as bright
green dots when visualized in axial orientation
(Fig. 2F). A double labeling
experiment was performed with
H1.X-11 and the monoclonal antibody IFA,
which stains the intermediate filament proteins of the tonofilaments
(Bartnik et al., 1986
). Both
antibodies label identical structures - the tonofilaments in the marginal
cells (Fig. 3C,D). The
different pictures of the marginal cells created by H1.X antibody labeling and
H1.X::GFP fluorescence suggest that a fraction of H1.X::GFP fills the
cytoplasm as well as the nucleoplasm as a highly soluble protein, whereas a
further fraction tightly binds to the tonofilaments, a cytoskeletal structure
created by the intermediate filament system. This hypothesis was tested by
subjecting the H1.X::gfp animals to the fixation and detergent
extraction/reduction protocol used for antibody labeling. This experiment was
done in combination with the IFA antibody labeling and resulted in a double
labeling of the tonofilaments (data not shown) and no remaining further
signals in the cytoplasm or nucleoplasm. This observation confirms that a
major fraction of H1.X::GFP is a highly soluble protein in the cytoplasm as
well as in the nucleoplasm, which can be readily extracted with 1% Triton
X-100 and 1% ß-mercaptoethanol in two hours at 37°C.
H1.X::GFP is a soluble cytoplasmic and nucleoplasmic protein in
muscle cells, neurons and in excretory cells
The H1.X::gfp transgenic C. elegans lines revealed H1.X
expression in further cell types. H1.X::GFP is expressed in body-wall muscles
(Fig. 4A,C), as well as in the
vulva sex muscles (Fig. 4E). In
both cases the general appearance of the cells corresponds to the situation
already described for the marginal cells of the pharynx: the fluorescence
signal fills the cytoplasm, and the cells' nuclei appear as the brightest
fluorescent structure. H1.X presence in muscle cells was not detected with the
two different antibodies. We ascribe this to a complete extraction of H1.X
from these cells during the extraction/reduction step of the antibody labeling
procedure.
H1.X::GFP was also expressed in a limited number of head neurons
(Fig. 5A), in which the
fluorescence signal filled the total cytoplasm, including the neuronal
projections. The presence of H1.X in these cells was confirmed with the
H1.X-101 antibody (data not shown). Furthermore, H1.X::GFP was detected
in the cytoplasm of excretory cells (Fig.
5C).
H1.X is a nucleolar protein
In C. elegans embryos, H1.X::GFP expression starts with the
30-cell stage. Fig. 7A shows
the expression of H1.X::GFP in four cells in the periphery of an embryo with
more than 100 cells. This specimen is fixed. A shallow fluorescence signal
fills the nucleoplasm, and the nucleoli appear brightly fluorescent. In
Fig. 7B a comparable view of a
living embryo is presented. Here the cytoplasm is filled with a shallow
fluorescence signal, whereas the nuclei appear brightly fluorescent. The
prominent green spots in the nuclei are the nucleoli, which were identified by
differential interference contrast (DIC) microscopy (data not shown). These
observations suggest that a fraction of H1.X::GFP is a highly soluble protein
in the cytoplasm as well as in the nucleoplasm, whereas a different fraction
binds tightly to the nucleolus. Antibody labeling with H1.X-101
revealed a prominent labeling of the nucleoli in the polyploid gut nuclei
(Fig. 6B) of adult C.
elegans hermaphrodites, which colocalized with fibrillarin
(Fig. 6D), a structural
component of small nucleolar RNPs (snoRNPs) implicated in pre-rRNA processing.
A prominent co-immunolabeling was further detected in the nucleoli of the germ
nuclei and many other cell types and tissues in C. elegans. The
nucleoli of the polyploid gut cells are comparatively deprived of DNA, shown
by the dark regions in the DAPIstained nucleus
(Fig. 6C). The nucleolar DNA
(rDNA) was occasionally visible as faintly DAPI-stained ring-like structure in
the confocal sections (data not shown). In all these cases the antibody
staining of H1.X colocalized with fibrillarin, and it was not associated with
the nucleolar DNA. In contrast to the other H1 proteins of C.
elegans, H1.X was never detected in condensed mitotic or meiotic
chromosomes or as a structural component of the interphase chromatin, as
revealed by antibody labeling (
H1.X-11) of embryos and meiotic oocytes.
H1.X::GFP was never found localized to condensed chromosomes. This is in
strict contrast to the properties of other H1 proteins in C. elegans.
H1.1-GFP fluorescence is readily observed in condensed mitotic chromosomes in
C. elegans embryos, and it is detected as a structural component of
interphase chromatin in antibody labeling of embryonic blastomeres and all
other cell types of C. elegans.
To investigate the relation of H1.X with cytoplasmic and nuclear substructures, H1.X::GFP was expressed in HeLa cells. Here H1.X::GFP does not appear in the cytoplasm but is a strictly nuclear protein (Fig. 8A). This observation indicates that the prominent cytoplasmic localization of H1.X::GFP in C. elegans is not caused by a general failure of the nuclear translocation of the fusion protein. When H1.X::GFP-expressing HeLa cells were fixed and Triton X-100 extracted, the GFP fluorescence of the nucleoplasm was strongly reduced and the nucleoli appeared brightly fluorescent (Fig. 8C). These results show that H1.X::GFP is a highly soluble protein in the nucleoplasm of HeLa cells and that it specifically binds to the nucleolus, like it does in C. elegans. In fixed as well as living HeLa cells H1.X::GFP is additionally localized in small spot-like structures, which may represent further nuclear bodies.
H1.X is involved in muscular function
RNA interference experiments were performed in order to analyze the
function of H1.X in C. elegans. 1351 F1 animals obtained from 42
dsRNA microinjected mothers (F0) were scored. 4.5% of these (61 animals)
showed a severe reduction of the final body size, a dumpy-like body appearance
and a slow and uncoordinated mode of locomotion
(Fig. 9A). 34 of these 61
animals were additionally defective in egg laying
(Fig. 9B). The average final
body length of these animals was 483±204 µm, whereas the average
body length of the control group (five animals measured) was 925±55
µm. Additionally a further independent phenotype was observed. 33 animals
(3% of the scored F1) possessed an elongated pharynx
(Fig. 9C). This group is not
overlapping with the small, dumpy-like and uncoordinated animals described
first. The average pharynx length was 141.4±8.4 µm, whereas it was
125.4±5.5 µm in the control group (236 F1 animals obtained from five
phosphate buffer injected mothers). All animals of the control group appeared
to be wild-type according to their mode of movement, reached the normal body
size and were never defective in egg laying. In order to assess the reduction
of H1.X in the phenotypic animals, immunocytology was performed. The phenotype
of the elongated pharynx was correlated with a severe reduction of the H1.X
content of the marginal cells. Fig.
3A shows the reduction of H1.X in such an animal, whereas
Fig. 3B presents a control
labeling with the IFA antibody to demonstrate antibody accessibility.
Fig. 3C,D present the
corresponding labeling of a control animal. RNA interference reduced the H1.X
content of the tonofilaments drastically, but a residual amount of the protein
is clearly detected. 231 F1 adult hermaphrodites from a H1.X RNA interference
experiment were analyzed with indirect immunofluorescence (H1.X-11).
Half of these animals showed a reduction of the intensity of the labeling of
the tonofilaments to about 50% of the signal intensity of the control group.
However, 10 animals (4.3% of the F1) showed a far stronger reduction of the
H1.X signal obtained from the tonofilaments. This group corresponded to the
animals that had an abnormally elongated pharnyx. We conclude from these
observations that H1.X contributes to the development of muscles and muscular
organs in C. elegans as well as to basic muscular functions, like
locomotion and egg laying. Alternatively some of these phenotypic properties
could also be caused by a loss of H1.X in neurons. This seems to be not very
probable, as RNAi-mediated depletion of proteins in neurons typically is not
very effective.
The C. elegans mutant unc-65(e351)
(Brenner, 1974) is slightly
uncoordinated, slowly moving and occasionally egg laying defective. Because
unc-65(e351) was mapped in very close vicinity to H1.X, we tested
whether unc-65(e351) is H1.X. This was done by PCR amplification of
the complete coding region of H1.X and subsequent DNA sequencing. This
revealed a wild-type H1.X sequence in unc-65(e351). Western blot
analysis and immunocytology of H1.X in unc-65(e351) also produced
data identical to the wildtype. We therefore conclude that unc-65 is
not H1.X.
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Discussion |
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In contrast to the linker histones and linker histone related proteins characterized so far, the linker histone-like protein H1.X resembles a tripartite linker histone that does not contain lysine in its N-terminal domain. By contrast, it contains multiple leucine, tyrosine, histidine and cysteine residues in its terminal domains. The DNA-binding forces of linker histones are generated by non-sequence specific electrostatic interactions between the numerous charged lysine residues of the linker histones with the phosphate di-ester groups of the DNA. Both terminal domains of normal linker histones are very rich in lysine residues and usually do not contain any highly hydrophobic amino acids, such as tyrosine, leucine, cysteine or histidine. These amino acids are prone to different types of side chain interactions, such as hydophobic interactions and the formation of covalent disulfide bonds. These types of interaction are not possible in normal linker histones and cannot directly serve the purpose of DNA binding. This suggests that the terminal domains of H1.X could be differently folded than the terminal domains of normal linker histones and that they could provide different types of chemical interactions, for example protein-protein interactions instead of DNA binding. Our cytological observations support this interpretation. H1.X is a prominent cytoplasmic protein in a limited number of cell types; these are the marginal cells, a set of nine epithelial cells in the pharynx (three of which form a syncytium), the body-wall muscle cells and the vulva muscles in the hermaphrodite. Additionally a limited number of head neurons and excretory cells also express H1.X.
The cytoplasm of the marginal cells is filled with thick bundles of intermediate filaments, the tonofilaments. H1.X is tightly associated with these tonofilaments. The presence of H1.X at the tonofilaments in the marginal cells was shown by indirect immunofluorescence with two independently raised polyclonal antibodies, H1.X::GFP fusion protein expression and a specific reduction of the immunofluorescence signal by H1.X RNA interference. Prominent and uniform cytoplasmic expression of H1.X was detected by indirect immunofluorescence in some head neurons. This observation was confirmed by a comparable expression of H1.X::GFP in these neurons. No visible filamentous structures were detected in the cytoplasm of these cells by confocal light microscopy. H1.X::GFP expression labeled additional cell types and produced a uniform fluorescence in the cytoplasm and in the nucleoplasm. Most of the H1.X::GFP fluorescence was removed by the extraction/reduction procedure necessary for antibody staining. This observation may explain why it was difficult to detect cytoplasmic H1.X expression in these cells using antibodies. Currently it can not be decided whether the differences in the distribution of antibody staining and H1.X::GFP fluorescence, as seen in the marginal cells, result from overexpression of H1.X::GFP (and therefore are artificial) or result from an extraction of native H1.X protein during the antibody staining procedure. Normal H1 proteins are strictly nuclear, and they are not removed from interphase chromatin or condensed chromosomes by the described extraction/reduction procedure.
Two different phenotypes were observed in the H1.X RNA interference
experiments. In the first phenotype, which correlated with an
immunocytologically detected depletion of H1.X in the pharynx, a
longitudinally elongation of this muscular organ was observed. Animals that
showed the second phenotype were small, slowly moving in an uncoordinated
fashion and defective in egg laying. We believe that the latter phenotype
results from a depletion of H1.X in sex and body-wall muscles. Interestingly,
RNAi with the intermediate filament protein C2 also results in a dumpy
phenotype with a slightly reduced motion at a penetrance of 10%
(Karabinos et al., 2001). The
expression pattern of intermediate filament protein C2 as well as some further
members of this gene family are as yet not known. It is therefore conceivable
that one of these proteins is expressed in body-wall and/or sex muscle cells.
In the Ascaris lumbricoides intermediate filament, proteins are
present in body-wall, pharyngeal and uterine muscle cells
(Bartnik et al., 1986
). These
represent three differently striated muscle cell types. H1.X expression was
furthermore detected in excretory cells and in some head neurons. In C.
elegans, the intermediate filament proteins B1 and A4 are expressed in
the excretory cells, and RNAi with the intermediate filament proteins A2 and
A3 results in uneven excretory canals, body muscle displacement and paralysis
(Karabinos et al., 2001
). A
neuronal expression has been reported for the intermediate filament protein A1
(Karabinos et al., 2001
). It
is therefore conceivable that the adult expression of H1.X is always
associated with co-expression of intermediate filament proteins. H1.X,
however, was never detected in the hypodermis, a tissue in which the
intermediate filament system is of essential importance
(Karabinos et al., 2001
). We
therefore propose that H1.X could be a facultative component of the
intermediate filament system in C. elegans.
Linker histones can be seen as a facultative and non-essential assembly
factor of the 30 nm chromatin fiber. The location of H1 in the filament, its
role in filament formation and the structure of the 30 nm filament itself have
all been controversial (for a review, see
Ramakrishnan, 1997). A
cytoplasmic- and filament-associated function of histone H1 has been suggested
before. A somatic chromatin type histone H1 was shown to be associated with
the sea urchin sperm flagellum, and histone H1 was recognized as a component
of the cilia and their basal bodies in the ciliate Paramecium and in
the green algae Chlamydomonas reinhardtii
(Multigner et al., 1992
).
Unfortunately no further investigations have followed this initial report.
A recent theoretical analysis performed on all available linker histone
gene structures demonstrated a correlation of the presence of flagellated
gametes with the presence of tripartite non-polyadenylated linker histones in
plants as well as in animals (Kaczanowski
and Jerzmanowski, 2001). It is speculated by these authors that an
evolutionarily early function of H1 might have been that of a mediator between
specialized microtubules and chromosomes during mitosis. Interestingly we
noted a significantly increased occurrence of males during the first
generations of the H1.X::GFP expressing transgenic animals. Male genotypes
(X0) originate from hermaphrodite mothers (XX) by non-disjunction of the
X-chromosome. Germline expression of repetitive transgenes is typically lost
after a few generations. This could indicate that H1.X::GFP expression
interferes with normal chromosome segregation during meiosis.
Histone H1.X is a divergent member of the linker histone gene family of
C. elegans. Current sequence data suggest that it is derived from
normal linker histones in the line of nematodes and that it has no counterpart
in other phyla. This protein, however, demonstrates that relatively few
changes are required to convert a linker histone into a cytoplasmic
fiber-associated protein. Interestingly, cdc2 kinase, which is also known as
histone H1 kinase (Arion et al.,
1988), extensively regulates the structure of the cytoplasmic as
well as of the nuclear intermediate filament system by phosphorylating
vimentin (Tsujimura et al.,
1994
) and lamin (Heald and
McKeon, 1990
) in a cell-cycle-dependent manner. In addition cdc2
kinase also phosphorylates caldesmon
(Yamashiro et al., 1991
), a
molecule that binds to tubulin (Ishikawa
et al., 1992
) and is an established regulator of the actin
filament system (Sobue et al.,
1981
). Whereas the phosphorylation of the intermediate filament
proteins by cdc2 kinase results in disassembly of the filaments, the role of
H1 phosphorylation during mitosis is not understood. The coregulation of
cytoplasmic filaments and chromatin by cdc2 kinase supports the idea of a
present or past functional relation between the cytoskeleton and linker
histones or linker histone-related proteins.
In many cells of C. elegans H1.X is associated with the nucleolus,
where it co-localizes with fibrillarin, but not with rDNA. A linker histone
variant, which is exclusively associated with the nucleolus, and which
strongly binds to rDNA in vitro, has been described in a number of plants
(Tanaka et al., 1999).
Recently an unexpected extracellular function of mammalian histone H1 in
muscle regeneration and proliferation has been described
(Henriquez et. al., 2002
).
Our present data suggest that H1.X is the first example of a protein resembling the general structure of a tripartite linker histone that probably is not a chromatin component and that interacts with other nuclear and cytoplasmic structures instead. We therefore think that the unusual facts presented here will promote the analysis of linker histone function and linker histone evolution in a general way.
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