From the Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, December 4, 2000, and in revised form, March 5, 2001
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ABSTRACT |
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MACROH2As are core histones that have a
unique hybrid structure consisting of an amino-terminal domain
that closely resembles a full-length histone H2A followed by a large
nonhistone region. The human MACROH2A1 gene, on
chromosome 5, encodes two MACROH2A subtypes, MACROH2A1.1 and
MACROH2A1.2, produced by alternate splicing. Here we report the
identification of MACROH2A2, a new MACROH2A subtype encoded by a
separate gene on human chromosome 10, MACROH2A2. The amino
acid sequence of human MACROH2A2 is 68% identical to human
MACROH2A1.2. We show by immunofluorescence on mouse tissue sections
that MACROH2A2, like MACROH2A1.2, is concentrated in the inactive
X chromosome. However, MACROH2A2 has a very different pattern of expression in the cell types present in the liver and kidney. When MACROH2A2 and MACROH2A1.2 are present in the same nucleus,
they have a similar, though nonidentical, pattern of localization, with
both subtypes present in the inactive X chromosome. Our results suggest
a developmental role for MACROH2A subtypes.
The MACROH2A core histones were first observed as two 42-kDa
proteins that remained associated with rat liver mononucleosomes in 0.5 M NaCl (1). The sequences of cDNAs that encode these proteins revealed that they have a unique hybrid structure consisting of an amino-terminal domain that closely resembles a full-length histone H2A followed by a large nonhistone domain. MACROH2As are released from the nucleosome core at the same salt concentration as
conventional H2As and appear to be released as a heterodimer with H2B
(1). This indicates that the H2A region of MACROH2A replaces
conventional H2A in the nucleosome core. It was estimated that 1 in 30 nucleosomes in rat liver would contain MACROH2A, assuming one MACROH2A
per nucleosome (1). The H2A region (amino acids 1-122) is followed by
a short linker region (10 amino acids) and then a region that is rich
in basic amino acids (28 amino acids) (see Fig. 1). This basic region
resembles basic tails present in other histones and most likely binds DNA.
The nonhistone region of MACROH2As is a feature not found in other
known core histones. It constitutes 57% of the protein and contains a
putative leucine zipper motif consisting of four heptad repeats (1).
The majority of the nonhistone region appears to have evolved from a
gene of unknown function that originated prior to the appearance of
eukaryotes (2). Among the sequences that are similar to the nonhistone
region is a conserved domain present in proteins involved in the
replication of RNA viruses (2).
We identified two distinct MACROH2A cDNA sequences (1) and used
specific antibodies to assign these sequences to the two MACROH2A
proteins that were resolved by electrophoresis (3). We named these
subtypes MACROH2A1.1 and MACROH2A1.2. The nucleotide sequences of
cDNAs that encode MACROH2A1.1 and MACROH2A1.2 differed only in a
single internal segment that encodes part of the nonhistone region (1,
3), and these subtypes are formed by alternate splicing of a single
gene, macroH2A1, on mouse chromosome 13 (4). Western blots
of nuclear extracts from adult and fetal tissues revealed distinct
tissue expression patterns that change during development (3). Both
subtypes are highly conserved in mammals and birds (2). These results
suggest that MACROH2A1.1 and MACROH2A1.2 differ in some aspect of their
function (3).
We demonstrated that the inactive X chromosome of female mammals can be
distinguished in interphase nuclei as a large MACROH2A-dense domain
called a macrochromatin body
(MCB)1 (5). The preferential
association of MACROH2A1.2 with the inactive X chromosomes of
differentiating female mouse embryonic stem cells is a
relatively late event, occurring several days after gene silencing (6).
In female embryos, however, association of MACROH2A1.2 with one of the
X chromosomes begins as early as the 12-cell stage (7), a time when the
trophectoderm cells begin to differentiate. This indicates that
MACROH2A1.2 associates with the inactive X chromosome at or near the
time of initiation of inactivation in these cells. The only other known
feature of X inactivation in preimplantation embryos that occurs before
MACROH2A accumulation is the accumulation of Xist RNA (8), a
cis-acting RNA transcribed from the X inactivation center of
the inactive X chromosome (9-13). The Xist gene is required
for the initiation of X inactivation (14-17), but it is not known how
Xist RNA functions in the inactivation process. The homology
of the nonhistone region of MACROH2A to a viral protein involved in RNA
replication, together with these results, lead us to hypothesize a
possible functional role for Xist RNA in localizing
MACROH2A1.2 to the inactive X chromosome (2, 5). A connection between
Xist and MACROH2A1 was established in a mouse fibroblast
model in which deletion of part of the Xist locus from the
inactive X chromosome leads to the loss of MACROH2A1 association
(18).
Although MACROH2A1.2 is not solely localized to the inactive X
chromosome, even in female cells (5), that association provides a
framework for understanding the function of MACROH2A proteins. In our
current working model, one aspect of their function is to establish
and/or maintain transcriptionally silent chromatin domains. Here we
present the discovery of a new MACROH2A subtype, MACROH2A2, encoded by
a second MACROH2A gene, MACROH2A2. Analysis of the sequence,
tissue distribution, and nuclear distribution of MACROH2A2 suggests a
developmental role for MACROH2A subtypes.
MACROH2A2 Identification--
A Blast search of expressed tagged
sequences (dbEST) (19) with MACROH2A1 nonhistone domain sequences was
performed, and a unique but clearly related sequence was found. Three
representative clones were obtained, and one (clone
identification number 50058 from Soares infant brain 1NIB
library made from the whole brain of a 73-day-old human female) was
sequenced using an ABI 373A sequencer with Taq FS dye
terminator chemistry and found to contain the entire open reading frame
(GenBankTM accession number AF151534). The amino acid
sequence was aligned to the available published MACROH2A1 sequences
(1-3, 20, 21) using ClustalW (22).
MACROH2A Genes--
The rat macroH2A1 gene was cloned
from a rat Antibodies--
The nonhistone region of the
MACROH2A2 cDNA was amplified with the forward tailed
primer GGAAGGATCCCAAAGGACAGCGATAAA and the reverse tailed primer
CGAAGAATTCCCCTGCTGGAAAGTGCGG, digested with BamHI and
EcoRI, and cloned into pGEX-2TK (Amersham Pharmacia Biotech). The GST-MACROH2A2 nonhistone region fusion protein was expressed in bacteria, and the thrombin-cleaved protein was isolated as
described (5). Antisera were raised in rabbits (Cocalico Biologicals),
bound to a fusion protein affinity column, eluted, and passed through a
GST-MACROH2A1.2 nonhistone region affinity column to remove
cross-reacting antibodies (5). The rabbit antibody against the
nonhistone region of MACROH2A1.2 has been described (5). Direct
antibody labeling was accomplished using the FluorReporter Texas Red-X,
Fluorescein-EX, Alexa Fluor 488, and Alexa Fluor 594 protein labeling
kits from Molecular Probes as suggested by the manufacturer.
Antibodies against connexin 26 (N-19) and connexin 32 (C-20) were
obtained from Santa Cruz Biotechnology, Inc.
Western Blot--
To compare the relative levels of the MACROH2A
subtypes in mouse tissue, nuclei were isolated from adult mouse liver
and kidney following the procedure of Blobel and Potter (23), except
that the nuclear isolation buffer was 0.4 M mannitol, 60 mM KCl, 15 mM NaCl, 0.15 mM
spermine, 0.5 mM spermidine, 2 mM EDTA, 0.5 mM EGTA, 15 mM triethanolamine, pH 7.4, containing 0.3 mM phenylmethanesulfonyl fluoride and 6 µg/ml aprotinin, and the nuclei were centrifuged through a lower
layer that contained 2 M, rather than 2.3 M,
sucrose. Nuclear extracts were prepared (2), and equivalent amounts of
the extracts (in terms of DNA content, as determined by absorbance at
260 nm) were loaded, except for the MACROH2A2 blot, where 2.5 times
more liver extract was loaded to detect a band. Proteins were separated
by SDS gel electrophoresis and transferred onto polyvinylidene
difluoride membranes (3). The blocked membranes were incubated
overnight with the primary antibody followed by a secondary
peroxidase-conjugated donkey anti-rabbit IgG, and the signal was
detected using SuperSignal West Femto maximum sensitive substrate (Pierce).
To compare the specificity of labeled versus unlabeled
MACROH2A antibodies, nuclei were isolated from adult mouse kidneys, and
proteins were separated and transferred as described above. One strip
was stained directly with Coomassie Brilliant Blue, the others were
incubated overnight with the primary antibody followed by a secondary
alkaline phosphatase-conjugated mouse anti-rabbit IgG, and the signal
was detected using the 1-Step NBT/BCIP detection system (Pierce).
Tissue Immunofluorescence--
Immunofluorescence on adult mouse
frozen tissue sections was performed as described (5).
Immunofluorescence/Fluorescence in Situ
Hybridization--
Immunofluorescence/fluorescence in situ
hybridization on adult mouse frozen tissue sections was performed as
described (5).
Sequence Analysis of Histone MACROH2A2--
We identified
cDNAs in dbEST (19) that encode a new MACROH2A subtype that we
named MACROH2A2. A full-length human cDNA clone was sequenced, and
the encoded amino acid sequence was aligned with the published
MACROH2A1 sequences (Fig. 1a).
Although MACROH2A2 has the same basic architecture as the MACROH2A1
subtypes, it is not encoded by the MACROH2A1 gene, because
its cDNA and amino acid sequences differ from the MACROH2A1
subtypes along its entire length. Overall, the amino acid sequence of
MACROH2A2 is 68% identical to that of MACROH2A1.2. The H2A region of
MACROH2A2 is significantly more similar to the H2A region of the
MACROH2A1 subtypes, 84% identical, than it is to conventional H2A,
66% identical to a human H2A (Fig. 1b). The amino acid
sequence of the basic region of MACROH2A2 is only 25% identical to
that of the MACROH2A1 subtypes, although its size and basic character
are very similar.
The nonhistone region of MACROH2A2 is 64% identical to MACROH2A1.2.
The region corresponding to the alternately spliced exons in
MACROH2A1.1 and MACROH2A1.2 (underlined in Fig.
1a) is more similar to MACROH2A1.2 both in length (33 amino
acids) and sequence (48% identical). We have no evidence that the
transcript from the MACROH2A2 gene is alternately spliced.
The essential elements of the putative leucine zipper region are
conserved except in the third repeat, where there is a threonine in the
"d" position (Fig. 1a). On the basis of studies of other
leucine zipper proteins, the presence of a threonine in this position
would not necessarily preclude a coiled-coil interaction (24). The
bacterial and viral proteins that are similar to the nonhistone region
of MACROH2A1 subtypes (2) show a similar degree of homology to the
nonhistone region of MACROH2A2 (data not shown).
MACROH2A Gene Structure--
The gene for mouse
MACROH2A1 contains 10 exons and is located on chromosome 13 (4). Here
we report a description of the rat and human MACROH2A1 genes as well as
the human gene for MACROH2A2 (Fig. 2).
The rat gene was identified from five overlapping clones from a rat
One peculiarity of the MACROH2A1 gene is the 5' splice site
selection of exon 4, which encodes the very end of the basic region and
the beginning of the nonhistone region. We noticed that the two
reported cDNA sequences for human MACROH2A1 (20, 21) differ here,
with the basic region of one ending with three lysines and the other
with just two. Examination of the human gene sequence revealed that the
first lysine comes from the 3' end of E3 and that the next two come
from an alternate splice site selection of a tandem pair of lysine
codons at the 5' end of E4 as shown in Scheme
I. A survey of dbEST suggests that in the
human the second site is twice as likely to be selected as the first
(33 of 47 informative sequences). For the mouse the splice site
selections are equally represented (6 of 12 informative sequences). The
5' end of E4 in the rat also shows this potential; however, rat
sequences from this region are not represented in dbEST. The only rat
sequences available from this part of the cDNA use the second site
(1, 3). The significance of this phenomenon is unknown.
Whereas MACROH2A1 has been mapped to human chromosome 5 (21), we found the MACROH2A2 gene in sequences from
chromosome 10. Most of the MACROH2A2 locus is available,
including all the known exons and more than half of the known introns.
MACROH2A2 is organized identically to MACROH2A1
(Fig. 2). It also starts with a noncoding exon and ends with a long 3'
untranslated region. The only difference is in exon 5. Whereas
MACROH2A1 has two alternately spliced exon 5s, which we
refer to here as E5.2 and E5.1, MACROH2A2 has only one,
which happens to be slightly more similar to E5.2 of
MACROH2A1. No other spliced variants of MACROH2A2 have been
found in dbEST, and a BLAST search of dbEST with the complete intron
sequences between E4 and E6 of MACROH2A2 did not reveal any
other exon candidates.
MACROH2A1.2 and MACROH2A2 Have Distinct but Overlapping Patterns of
Expression--
We raised antibodies against the nonhistone region of
MACROH2A2 to examine the pattern of MACROH2A2 expression and its
distribution within the nucleus. Antibodies were affinity-purified and
absorbed against a GST-MACROH2A1.2 nonhistone region fusion protein to eliminate cross-reaction with MACROH2A1 subtypes (5). The
specificity of these purified antibodies and the MACROH2A1.2 antibodies
was tested on a Western blot of mouse liver and kidney nuclear
extracts. The MACROH2A2 antibodies detected a single band that runs
slightly slower than MACROH2A1.2 (Fig.
3). The MACROH2A1.2 content of mouse liver and kidney is similar. However, the MACROH2A2 content of the
kidney extract was higher than that of the liver, even when we loaded
2.5 times more liver extract (Fig. 3).
We examined the distributions of MACROH2A1.2 and MACROH2A2 in different
cell types and their distributions within the nucleus by
immunofluorescence on frozen tissue sections of mouse liver and kidney.
In the liver, MACROH2A1.2 antibodies stained hepatocytes brightly and,
to a lesser extent, cells of the bile ducts (Fig. 4, a and b), as
reported (5) (hepatocytes are labeled H, and bile ducts are
labeled D). In contrast, hepatocytes were only faintly
stained with the MACROH2A2 antibodies, whereas the bile ducts, certain
cells around the central vein, and a small number of parenchymal cells
were brightly stained (Fig. 4, c and d). The
parenchymal cells that stain brightly for MACROH2A2 do not appear to be
hepatocytes, on the basis of their nuclear morphology.
To see which individual cells stained for MACROH2A1.2 or MACROH2A2, we
used direct immunofluorescence with a fluorescein-labeled MACROH2A1.2
antibody and a Texas Red-labeled MACROH2A2 antibody. We used female
mouse liver, because inactive X chromosome-associated MCBs are readily
apparent in hepatocytes and ductal cells stained for MACROH2A1.2 (Figs.
4a and 5a). In
several hepatocytes, two inactive X chromosome-associated MCBs were
seen (Fig. 5a), and we have shown that such nuclei are
polyploid (5). In the bile ducts both subtypes stained the ductal
epithelial cells (Fig. 5, DE), but only MACROH2A2 antibodies
stained the periductal cells significantly (Fig. 5, PD).
MacroH2A2 containing MCBs were apparent in the ductal epithelium. These
MCBs were only present in females (data not shown) and colocalized with
MACROH2A1.2 MCBs (Fig. 5, a-c). This indicates that these
MCBs involve the inactive X chromosome. Those cells around the
central vein (Fig. 5, V) and the non-hepatocyte parenchymal
cells (Fig. 5, NHP) that stain with the MACROH2A2 antibody
do not stain with the MACROH2A1.2 antibody. MCBs were not evident in
many of these nuclei, although they may have been obscured by the
relatively high level of MACROH2A2 seen throughout these nuclei.
These subtypes also showed distinct expression patterns in the
kidney (Fig. 6). The cortex of the kidney
contains several prominent structures including the proximal and distal
convoluted tubules and the glomeruli (the site of blood filtration).
Proximal convoluted tubules (Fig. 6, P) were
identified by their staining with an antibody to the gap junction
protein connexin 32 (25) (shown in green in b and
d). MACROH2A1.2 and MACROH2A2 were both found in the
proximal convoluted tubules (Fig. 6, a and b for MACROH2A1.2; c and d for MACROH2A2). We
identified distal tubules (Fig. 6, D) by their appearance
and lack of connexin 32 staining. They stained more brightly for
MACROH2A1.2 than for MACROH2A2. Glomeruli (Fig. 6, G) showed
very little staining for MACROH2A1.2 (a and b)
but were stained for MACROH2A2 (c and d). The
strongest MACROH2A2 signal was in the parietal layer of Bowman's
capsule (Fig. 6d, B), a structure that forms the
outer layer of the glomerulus. In the medulla of the kidney there was
less overlap in the staining patterns. In this part of the kidney
MACROH2A1.2 was detected at a high level in the straight proximal
tubules that we identified by costaining with antibodies to connexin 26 (25) (Fig. 6, e and f). Very little MACROH2A1.2
staining was seen in the cells between these tubules. In contrast,
MACROH2A2 staining was very low in the connexin 26 positive tubules but
was present at high levels in the cells between them (Fig. 6,
g and h).
A distinct expression pattern was also seen in the adrenal gland.
MACROH2A2 staining predominated in the outer cells of the capsule, and
MACROH2A1.2 staining predominated in the inner cells of the cortex and
medulla (Fig. 7).
The staining patterns of different cell types presented here are
summarized in Table I. We also stained
frozen sections of dog liver and kidney for MACROH2A1.2 and MACROH2A2,
and the staining patterns were virtually identical to those in the
mouse (data not shown).
MACROH2A1.2 and MACROH2A2 Have Similar although Nonidentical
Nuclear Distributions--
To confirm that the female-specific MCBs
observed with MACROH2A2 antibodies involve the inactive X chromosome,
we performed immunofluorescence with MACROH2A2 antibodies followed by
fluorescence in situ hybridization with an X chromosome
paint probe. This analysis was done on a section of female mouse
kidney, because female-specific MACROH2A2 MCBs are easily detected in
the nuclei of the cells of the proximal convoluted tubule. The
MACROH2A2 MCBs in these cells colocalized to one of the X chromosomes
(Fig. 8).
To determine whether there is overlap in the distribution of MACROH2A2
and MACROH2A1.2, we again used direct immunofluorescence using
MACROH2A2 antibody labeled with Alexa 488 and a MACROH2A1.2 antibody
labeled with Alexa 594. In this case we first wanted to establish that
the labeling did not appreciably alter the specificity of the
antibodies. We tested them on a Western blot of kidney nuclear extract
and saw little or no change in specificity (Fig. 9). Immunofluorescence analysis of a cell
type that has relatively high levels of both MACROH2A subtypes, the
epithelial cells of the proximal convoluted tubule of the kidney,
showed a similar pattern of nuclear staining (Fig.
10). A distinct colocalizing inactive X
chromosome-associated MCB, plus relatively diffuse nuclear staining not
restricted to Hoechst bright domains, could be seen with both
antibodies (Fig. 10, a-c). Small regions where one subtype
predominated over the other were also present (Fig. 10d).
MACROH2A is a conserved family of core histone proteins. So far
the DNA data bases have revealed MACROH2As only in bony vertebrates including mammals, birds, and fish (data not shown). Here we have described a new member of the MACROH2A family, MACROH2A2.
MACROH2A2 is virtually identical in size and architecture to the
MACROH2A1 subtypes and has significant amino acid sequence homology
with those subtypes throughout its length. This suggests that the
fundamental functions of the H2A domain, the basic region, and the
nonhistone domain of MACROH2A2 are similar to their functions in the
MACROH2A1 subtypes. This idea is supported by our observation that,
like MACROH2A1.2, MACROH2A2 is concentrated in the inactive X
chromosome in certain cell types. The simplest interpretation of this
result is that both of these MACROH2A subtypes perform a similar
function in the inactive X.
On the other hand, MACROH2A2 differs from the MACROH2A1 subtypes
in several interesting ways. In primary structure it is only 68%
identical to MACROH2A1.2. This contrasts with the exceptional evolutionary conservation of both MACROH2A1 subtypes, which are ~95%
identical between mammals and birds (2). We do not have any other
complete MACROH2A2 sequences, but a comparison of the human MACROH2A2
sequence to partial mouse sequences from dbEST shows that mouse
MACROH2A2 is nearly identical to human MACROH2A2 (98% identical
covering 97% of the amino acid sequence (data not shown)). Thus,
the sequence differences between MACROH2A2 and the MACROH2A1 subtypes
are conserved. MACROH2A2 also has a very different pattern of
expression from that of MACROH2A1.2 (Table I), and our studies with dog
tissues (data not shown) indicate that these cell type differences are
conserved in evolution. When MACROH2A1 subtypes and MACROH2A2 are
present in the same nucleus, they have similar although nonidentical
distributions (Fig. 10). Our results suggest that the MACROH2A that is
not associated with the inactive X is also localized to specific
regions of chromatin and that the mechanism(s) involved in localizing
MACROH2A may have subtype specificity.
Our observation that MACROH2As have a preference for the inactive
X has been questioned in a recent report that examined the distribution
of MACROH2A1.2 and other core histones in primary human fibroblasts
(26). These authors suggested that the labeling of the inactive X
chromosome by MACROH2A antibodies or green fluorescent protein-tagged
MACROH2A may only reflect a higher density of chromatin in the inactive
X. This conclusion was based on their observations that the inactive X
was also labeled by antibodies against conventional core histones or
green fluorescent protein-H2A. Indeed the inactive X in many cells,
including human fibroblasts, is often readily identifiable by DNA
staining as the most prominent chromatin domain in the nucleus,
i.e. the Barr body. Our conclusion that MACROH2A preferentially associates with the inactive X is based on the relative
staining of the inactive X compared with other chromatin. Our studies
have focused on cells of mouse tissues where the inactive X cannot
be identified by DNA or chromatin stains due to the presence of other
large domains of similar or greater density (Fig. 10 and Refs. 5, 7,
and 27). However, in these cells the inactive X is readily identified
by MACROH2A staining (Fig. 10 and Refs. 5 and 7). Consistent with our
conclusions, Chadwick and Willard (28) found that myc epitope-tagged
MACROH2A was preferentially localized to the Barr body of
primary human fibroblasts, whereas myc epitope-tagged H2B showed no
such preference in comparison to other chromatin.
On the basis of the association of MACROH2A1.2 with the inactive X
chromosome, we suggested that MACROH2As are involved in establishing
and/or maintaining transcriptionally silent chromatin domains (5). Our
present results demonstrate a new level of complexity and specificity
to MACROH2A protein utilization. One interesting possibility is that
each cell type has a specific complement of the genome associated with
MACROH2A-containing nucleosomes. This would require a mechanism to
localize MACROH2A to numerous specific chromosomal domains. MACROH2As
could be localized to specific chromatin domains by
cis-acting RNAs like Xist (5), and that targeting could
involve a direct interaction of the nonhistone region of MACROH2A with
RNA (2). This model predicts that cis-acting RNA genes like
Xist are strategically located throughout the genome. The
expression of such genes could be developmentally regulated and cell
type-specific. The cell type-specific expression of MACROH2A subtypes
demonstrated in the present work and in previous studies of the
MACROH2A1 subtypes (3) could provide another level of specificity and
control for such a system of transcriptional regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage library from which five overlapping clones were
isolated and mapped by polymerase chain reaction, and intron-exon
junctions were sequenced using primers derived from the cDNA
sequence (3). The mouse macroH2A1 gene was previously mapped
(4). The human MACROH2A genes were discovered in the data base from the
Human Genome Sequencing Project using the human cDNA sequences (20,
21) in a BLAST search. MACROH2A1 is in gi 12731492 from
human chromosome 5, and MACROH2A2 is in gi 12735407 from
human chromosome 10.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence comparison of MACROH2A subtypes.
a, the human MACROH2A2 amino acid sequence is shown aligned
with the other published MACROH2A sequences. Shown are the chicken
MACROH2A1 subtypes (GmH2A1.1, GenBankTM accession
number AF058445; and GmH2A1.2, GenBankTM accession number
AF058446) (2), the rat MACROH2A1 subtypes (RmH2A1.1,
GenBankTM accession number M99065; and RmH2A1.2,
GenBankTM accession number U79139) (1, 3), the mouse
MACROH2A1 subtypes (MmH2A1.1, GenBankTM accession number
AF171080; and MmH2A1.2, GenBankTM accession number
AF171081) (4), the human MACROH2A1 subtypes (HmH2A1.1,
GenBankTM accession number AF044286; and HmH2A1.2,
GenBankTM accession number AF041483) (20), and human
MACROH2A2 (HmH2A2, GenBankTM accession number AF151534).
d indicates "d" positions in the putative leucine
zipper. The underline indicates the region that is
alternately spliced in the MACROH2A1 subtypes. b, the H2A
regions of human MACROH2A1 and human MACROH2A2 are shown aligned with a
human H2A sequence (GenBankTM accession number
NP003507).
phage library. The human genes were identified from the sequence data
base from the human genome sequencing project. The
complete MACROH2A1 locus is available. The rat
macroH2A1 gene consists of nine exons spread over more than
60 kilobases. The human MACROH2A1 gene consists of 11 exons spread over almost 65 kilobases. The first exon of the
macroH2A1 gene of rat and mouse is noncoding. In the human
gene the first two exons are noncoding and alternately spliced. In fact
there is only one example of the first of the two being used (21), and
it has yet to show up in dbEST. The same situation may apply to rat and
mouse; however, there is no evidence for this in the data base yet. The
last exon of all known MACROH2A1s have long 3' untranslated
regions. The mouse MACROH2A1 message was reported with a short
3' untranslated region (4); however, a survey of mouse expressed
sequence tags indicates that the 3' untranslated region of mouse
macroH2A1 cDNAs is about 600 nucleotides (the
discrepancy is most likely due to an internal stretch of As being
mistaken for a poly(A) tail). One difference between the MACROH2A1
genes of rats, mice, and humans is in the exons encoding the histone
region. Exon 2 of the rat macroH2A1 gene is split into two
exons in the mouse and human genes, which we therefore here refer to as
E2 and E2a.
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Fig. 2.
Comparison of the exon structure of MACROH2A
genes. The human MACROH2A2 gene is from gi 12735407 from human chromosome 10. The human MACROH2A1 gene is from
gi 12731492 from human chromosome 5. There are apparently two optional
E1s, the first found in only one reported sequence (21). The mouse
macroH2A1 gene is on mouse chromosome 13 and was described
(4). The structure of the rat macroH2A1 gene is from this
report, and its chromosomal location is unknown. The transcription
initiation sites are not known, and, therefore, E1 sizes are minimums.
The coding regions are shaded. bp, base
pairs.
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Scheme I.
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Fig. 3.
Western blot of tissue nuclear extracts.
Blots of nuclear extracts from mouse liver (L) and kidney
(K) were stained with antibodies to MACROH2A1.2
(1.2) and MACROH2A2 (2). MACROH2A2 migrates
slightly slower on SDS-polyacrylamide gel electrophoresis gels with low
bisacrylamide (1). The liver lane stained with MACROH2A2 contained 2.5 times more extract than the other three lanes. Loadings were normalized
by their DNA content.
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Fig. 4.
Immunofluorescence of mouse
liver. Consecutive female mouse liver sections were stained for
MACROH2A1.2 (a and b) or MACROH2A2 (c
and d). In b and d MACROH2A staining
is shown merged with red propidium iodide DNA stain. V,
central vein; D, bile duct; H, hepatocyte;
NHP, nonhepatocyte parenchymal cell. Bar, 50 µm.
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Fig. 5.
Close-up of a single female mouse liver
section. The section was stained for MACROH2A1.2 (a)
and MACROH2A2 (b). The merged image is shown in
c. D, bile duct; PD, periductal cell;
DE, ductal epithelium; V, cell type lining the
central vein; H, hepatocyte; NHP, nonhepatocyte
parenchymal cell. Bar, 20 µm.
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Fig. 6.
Immunofluorescence of mouse
kidney. Female mouse kidney sections were stained for MACROH2A1.2
(a, b, e, and f) or
MACROH2A2 (c, d, g, and h). Sections
in a--d were from the cortex, and those in
e--h were from the medulla. The panels on the
right show MACROH2A staining merged with images showing
proximal tubule staining (marked P) with antibodies to
connexin 32 (green, b and d) and
connexin 26 (green, f and h).
D, distal tubule; G, glomerulus; B,
parietal layer of Bowman's capsule. Bar, 50 µm.
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Fig. 7.
Immunofluorescence of mouse adrenal
gland. A female mouse adrenal gland section is shown stained with
Hoechst 33258 (a), Alexa 488-labeled antibody to MACROH2A2
(b), and Alexa 594-labeled antibody to MACROH2A1.2
(c). A merge of a and b is shown
(d).
Cell type-specific expression of MACROH2A subtypes
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Fig. 8.
Colocalization of MACROH2A2 MCBs to an
X chromosome. Three nuclei of a proximal convoluted tubule from a
female mouse kidney section are shown stained for MACROH2A2
(red, a) and X chromosomes (green,
b). The merged image showing colocalization of the MCBs with
one of the X chromosomes is shown in c. Bar, 5 µm.
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Fig. 9.
Specificity of labeled antibodies. A
Western blot of mouse kidney nuclear extracts is shown. Lane
assignments are as follows: lane 1, a strip stained directly
with Coomassie Brilliant Blue; lane 2, MACROH2A1.2
primary antibody; lane 3, MACROH2A1.2 primary antibody
labeled with Alexa 594; lane 4, MACROH2A2 primary antibody;
lane 5, MACROH2A2 primary antibody labeled with Alexa 488;
lane 6, MACROH2A1.1 primary antibody. The positions of
MACROH2A1.2 (1.2), MACROH2A1.1 (1.1), and
MACROH2A2 (2) are labeled.
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Fig. 10.
Internuclear distribution of MACROH2A
subtypes. Direct immunofluorescence detection of MACROH2A subtypes
in nuclei of cells of a proximal convoluted tubule from a female mouse
kidney section is shown stained with Hoechst 33258 (a),
Alexa 488-labeled antibody to MACROH2A2 (b), and Alexa
594-labeled antibody to MACROH2A1.2 (c). A merge of
a and b is also shown (d).
Bar, 5 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank E. Holzbaur, O. Jacenko, and J. Wolfe for making their resources available and S. Karki and S. Maitra for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Grant GM49351 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF151534.
To whom correspondence should be addressed. Tel.: 215-898-0454;
Fax: 215-573-5189; E-mail: pehrson@vet.upenn.edu.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M010919200
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ABBREVIATIONS |
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The abbreviations used are: MCB, macrochromatin body; dbEST, Expressed Sequence Tags data base; GST, glutathione S-transferase.
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REFERENCES |
---|
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---|
1. | Pehrson, J. R., and Fried, V. A. (1992) Science 257, 1398-1400[Medline] [Order article via Infotrieve] |
2. | Pehrson, J. R., and Fuji, R. N. (1998) Nucleic Acids Res. 16, 2837-2842[CrossRef] |
3. | Pehrson, J. R., Costanzi, C., and Dharia, C. (1997) J. Cell. Biochem. 65, 107-113[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Rasmussen, T. P.,
Huang, T.,
Mastrangelo, M. A.,
Loring, J.,
Panning, B.,
and Jaenisch, R.
(1999)
Nucleic Acids Res.
27,
3685-3689 |
5. | Costanzi, C., and Pehrson, J. R. (1998) Nature 393, 599-601[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Mermoud, J. E.,
Costanzi, C.,
Pehrson, J. R.,
and Brockdorff, N.
(1999)
J. Cell Biol.
147,
1399-1408 |
7. |
Costanzi, C.,
Stein, P.,
Worrad, D. M.,
Schultz, R. M.,
and Pehrson, J. R.
(2000)
Development
127,
2283-2289 |
8. | Sheardown, S. A., Duthie, S. M., Johnston, C. M., Newall, A. E. T., Formstone, E. J., Arkell, R. M., Nesterova, T. B., Alghisi, G., Rastan, S., and Brockdorff, N. (1997) Cell 91, 99-107[CrossRef][Medline] [Order article via Infotrieve] |
9. | Borsani, G., Tonlorenzi, R., Simmler, M. C., Dandolo, L., Arnaud, D., Capra, V., Grompe, M., Pizzuti, A., Muzny, D., Lawrence, C., and Ballabio, A. (1991) Nature 351, 325-329[CrossRef][Medline] [Order article via Infotrieve] |
10. | Brockdorff, N., Ashworth, A., Kay, G. F., Cooper, P., Smith, S., McCabe, V. M., Norris, D. P., Penny, G. D., Patel, D., and Rastan, S. (1991) Nature 351, 329-331[CrossRef][Medline] [Order article via Infotrieve] |
11. | Brockdorff, N., Ashworth, A., Kay, G. F., McCabe, V. M., Norris, D. P., Cooper, P. J., Swift, S., and Rastan, S. (1992) Cell 71, 515-527[Medline] [Order article via Infotrieve] |
12. | Brown, C. J., Ballabio, A., Rupert, J. L., Lafreniere, R. G., Grompe, M., Tonlorenzi, R., and Willard, H. F. (1991) Nature 349, 38-44[CrossRef][Medline] [Order article via Infotrieve] |
13. | Brown, C. J., Hendrich, B. D., Rupert, J. L., Lafreniere, R. G., Xing, Y., Lawrence, J., and Willard, H. F. (1992) Cell 71, 527-542[Medline] [Order article via Infotrieve] |
14. | Herzing, L. B. K., Romer, J. T., Horn, J. M., and Ashworth, A. (1997) Nature 386, 272-275[CrossRef][Medline] [Order article via Infotrieve] |
15. | Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S., and Brockdorff, N. (1996) Nature 379, 131-137[CrossRef][Medline] [Order article via Infotrieve] |
16. | Lee, J. T., and Jaenisch, R. (1997) Nature 386, 275-279[CrossRef][Medline] [Order article via Infotrieve] |
17. | Marahrens, Y., Panning, B., Dausman, J., Strauss, W., and Jaenisch, R. (1997) Genes Dev. 11, 156-166[Abstract] |
18. | Csankovszki, G., Panning, B., Bates, B., Pehrson, J. R., and Jaenisch, R. (1999) Nat. Genet. 22, 323-324[CrossRef][Medline] [Order article via Infotrieve] |
19. | Boguski, M. S., Lowe, T. M., and Tolstoshev, C. M. (1993) Nat. Genet. 4, 332-333[Medline] [Order article via Infotrieve] |
20. | Lee, Y., Hong, M., Kim, J. W., Hong, Y. M., Choe, Y.-K., Chang, S. Y., Lee, K. S., and Choe, I. S. (1998) Biochim. Biophys. Acta 1399, 73-77[Medline] [Order article via Infotrieve] |
21. |
Mao, M.,
Fu, G.,
Wu, J.-S.,
Zhang, Q.-H.,
Zhou, J.,
Kan, L.-X.,
Huang, Q.-H.,
He, K.-L.,
Gu, B.-W.,
Han, Z.-G.,
Shen, Y.,
Gu, J., Yu, Y.-P.,
Xu, S.-H.,
Wang, Y.-X.,
Chen, S.-J.,
and Chen, Z.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8175-8180 |
22. | Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract] |
23. | Blobel, G., and Potter, V. R. (1966) Science 154, 1662-1665[Medline] [Order article via Infotrieve] |
24. | Hu, J. C., O'Shea, E. K., Kim, P. S., and Sauer, R. T. (1990) Science 250, 1400-1403[Medline] [Order article via Infotrieve] |
25. |
Sainio, K.,
Gilbert, S. F.,
Lehtonen, E.,
Nishi, M.,
Kumar, N. M.,
Gilula, N. B.,
and Saxen, L.
(1992)
Development
115,
827-837 |
26. | Perche, P., Vourc'h, C., Konecny, L., Souchier, C., Robert-Nicoud, M., Dimitrov, S., and Khochbin, S. (2000) Curr. Biol. 10, 1531-1534[CrossRef][Medline] [Order article via Infotrieve] |
27. | Moore, K. L., and Barr, M. L. (1953) J. Comp. Neurol. 98, 213-231[Medline] [Order article via Infotrieve] |
28. |
Chadwick, B. P.,
and Willard, H. F.
(2001)
J. Cell Biol.
152,
375-384 |