From the Human Cytogenetics Laboratory, Cancer
Research, UK London Research Institute, Lincoln's Inn Fields
Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX,
§ Wellcome Trust Sanger Institute, Genome Campus,
Hinxton Cambridge CB10 1SA, and
Centre for Complex Fluids
Processing, Department of Chemical and Biological Process Engineering,
University of Wales Swansea, Singleton Park,
Swansea SA2 8PP, United Kingdom
Received for publication, July 3, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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Sequences containing the matrix recognition
signature were identified adjacent to the
LMP/TAP gene cluster in the human and mouse
major histocompatibility complex class II region. These sequences were
shown to function as nuclear matrix attachment regions (MARs). Three of
the five human MARs and the single mouse MAR recruit heterogeneous
nuclear ribonucleoprotein A1 (hnRNP-A1) in vivo during
transcriptional up-regulation of the major histocompatibility complex
class II genes. The timing of this recruitment correlates with a rise
in mature TAP1 mRNA. Two of the human MARs bind
hnRNP-A1 in vitro directly within a 35-bp sequence that
shows over 90% similarity to certain Alu repeat sequences. This study
shows that MARs recruit and bind hnRNP-A1 upon transcriptional
up-regulation.
Matrix attachment regions
(MARs)1 are short DNA
sequences that bind to the ribonucleoprotein network that is generally
referred to as the nuclear matrix or scaffold (1). A subset of MARs is
believed to mediate the organization of chromatin into a higher order
structure consisting of multiple topologically constrained loops
attached at their bases to the matrix (2). A role for MARs in DNA
replication has been proposed because many MARs contain origins of
replication, and newly replicated DNA is anchored to the nuclear matrix
(3). The mapping of MARs to 5'-regulatory regions of certain genes, to
actively transcribing genes, and to sites for binding activator,
repressor, and demethylation proteins suggests that MARs also play a
role in gene regulation (1, 4). In addition, MARs flanking individual
genes or gene clusters in several species have been shown to act as
insulator or boundary elements shielding genes within the domain from
adjacent regulatory elements (5).
MARs are identified biochemically by their selective retention in
nuclear matrix preparations, which are derived from nuclei depleted of
histones and most of the DNA. A set of characteristic motifs has been
associated with MARs, including DNA unwinding elements, AT tracts, and
DNase I-hypersensitive sites (1, 6). Binding of MARs to the matrix has
not been assigned to unique DNA sequences but rather to sequences
dispersed over several hundred base pairs. Recently, a unique bipartite
sequence element was found in a large proportion of MARs, called the
"MAR/SAR recognition signature" (MRS) (7). The MRS was reported to
predict correctly the positions of MARs/SARs in several species from
their genomic sequence alone.
In this study, we examined the role of MARs adjacent to the
LMP/TAP gene cluster in the class II region of
the human MHC. This genomic region was chosen as a model as both its
sequence, transcription, and function have been well characterized (8, 9). The cluster consists of four genes, LMP2 and
TAP1 at the centromeric end which share a bidirectional
promoter, and LMP7 and TAP2 at the telomeric end
(10). The LMP gene products ("low molecular mass
polypeptide") enhance the degradation of cytosolic antigens into
peptide fragments in the multisubunit proteasome in preparation for
binding at the cell surface to MHC class I molecules (11). The
TAP gene products ("transporter associated with antigen
processing") are involved in the transport of these peptides to the
endoplasmic reticulum where they bind newly synthesized MHC class I
molecules (12). Expression of the LMP and TAP
genes can be up-regulated by interferon- We used the MRS in conjunction with a pattern finding and clustering
program to predict the positions of five MARs in non-coding DNA
centromeric to the LMP/TAP gene cluster.
Biochemical analysis confirmed that they do indeed bind the nuclear
matrix and that three of these MARs recruit the mRNA processing
protein hnRNP-A1 in vivo during transcriptional
up-regulation. We identified a 35-bp sequence in two of these MARs that
is able to bind hnRNP-A1 directly in vitro. Similar findings
in the homologous region of the mouse genome suggest a conserved role
for these MARs in regulation of gene expression.
MAR Prediction Using the MRS--
The MRS proposed recently (7)
as a recognition signature for MARs has a pattern of two nucleotide
motifs separated by 200 bp or less. The match required for MAR
prediction is 15 of 16 bases in motif 1, AWWRTAANNWWGNNNC, and 8 of 8 bases in motif 2, AATAAYAA (IUB-IUPAC incompletely specified
nucleotide code (www.chem.qmw.ac.uk/iubmb/misc/naseq.html): W = A
or T; R = A or G; Y = C or T; N = A C G or T). Each
motif can be present in either forward or reverse orientation, and
clusters of more than one example of either motif within the constraint
of the pattern are regarded as a single MRS. An iteration of the
Nucleotide Interpretation Program (14) was used to identify such MRS
patterns, the output being parsed by a simple PERL script, pars_nip, to
identify clusters of motifs. Entering the output into the chromosome 6 data base (6ace) allowed visualization of the MRS patterns within their genomic environment and the determination of the positions shown in
Figs. 1 and 9. This procedure successfully confirmed the seven MRS
patterns described in the human globin locus (GenBankTM
accession number U01317) (7).
Cells and Treatments--
MRC5 human lung fibroblast cells were
grown as a monolayer to confluence in RPMI 1640 supplemented with 10%
fetal calf serum at 37 °C in a 5% CO2 atmosphere. In
experiments with up-regulated MRC5 cells, 200 units/ml IFN-
The B-lymphoblastoid cell line AHB, which constitutively expresses the
classical MHC and the LMP/TAP genes at high
levels, was grown in suspension in RPMI 1640 medium supplemented with 10% fetal calf serum at 37 °C in a 5% CO2 atmosphere.
In experiments to inhibit MHC class II gene expression,
PGE2 was added into the culture medium for 20 h as
described previously (16).
PCR Assay--
The nuclear matrix-attached (M) and the
matrix-independent or loop (L) DNA fractions from MRC5 cells, IFN- Nuclear Extract Preparation--
Nuclear extracts from MRC5
cells, IFN- Pull-down of Proteins and Mass Spectrometry
Identification--
The pull-down of proteins specifically binding the
MARs of interest was carried out as described previously (19) with
slight modifications. The MARs were amplified from total genomic DNA by
PCR using sets of specific primers (Table I) where the reverse primers
were previously biotinylated by Biotin-Chem-Link kit (Roche Diagnostics). Approximately 80 pmol of each MAR was immobilized onto
400 µg of streptavidin magnetic particles (Roche Diagnostics) following the protocol supplied by the manufacturer. The pull-down was
performed in a final volume of 550 µl containing 100 µg of nuclear
protein extract, 10% glycerol, 20 mM HEPES, pH 7.9, 1 mM MgCl2, 1 mM DTT, 50 mM NaCl, 0.1 µg/µl poly[d(I-C)] (Roche Diagnostics),
and 1 µg/µl sonicated salmon sperm DNA, protease inhibitor (Sigma).
The reaction mixture was preincubated for 10 min at room temperature;
400 µg of streptavidin magnetic particles with attached MARs was
added and the mixture incubated for a further 25 min on a rotator.
After magnetic separation, the particles were washed three times with
400 µl of washing buffer (50 mM KCl, 20 mM
HEPES, pH 7.9, 1 mM MgCl2, 0.5 mM
DTT). The first washing buffer included 0.1 µg/µl poly[d(I-C)].
The magnetic particles were resuspended in 30 µl of Laemmli sample
buffer containing 7 M urea, and the eluted proteins were
separated in a 12% Tris glycine pre-cast gel (Invitrogen). The gels
were either silver-stained (Silver Xpress, Invitrogen) or
Coomassie-stained, and proteins of interest were cut out and processed
further for mass spectrometry as described previously (20).
hnRNP-A1 and p21 Cloning and Expression--
By using specific
primer pairs (Table II) for RT-PCR,
hnRNP-A1 and the cdk inhibitor, p21, were amplified from their start to
stop codons from cytoplasmic RNAs isolated from MRC5 fibroblasts and
MRC5 treated with 50 ng/ml phorbol 12-myristate 13-acetate for 1 h, respectively. Phorbol 12-myristate 13-acetate treatment of
fibroblasts was necessary to up-regulate the p21 expression level in
MRC5. The amplified cDNAs were cloned in the pcDNA4/HisMax-TOPO expression vector (Invitrogen). They were then transfected transiently into MRC5 fibroblast cells by SuperFect Transfection Reagent (Qiagen Ltd., UK) as suggested by the supplier. The expressed recombinant hnRNP-A1 was purified by Xpress System Protein Purification
(Invitrogen), and the N-terminal fusion tag was removed by Enterokinase
Max (Invitrogen).
Western Blot--
Nuclear protein extracts and nuclear matrix
protein extracts from hnRNP-A1 transfected MRC5 cells were isolated at
24-h intervals up to 96 h. The proteins were separated in a 12%
Tris glycine pre-cast gel (Invitrogen) and transferred onto a
nitrocellulose membrane using an electroblotting apparatus XCell
SureLockTM (Invitrogen) according to the manufacturer's
protocols. Further procedures for detecting the recombinant hnRNP-A1
containing Xpress tag followed instructions of the
Anti-XpressTM-horseradish peroxidase antibody supplier
(Invitrogen). Signal detection was performed by membrane incubation in
Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology, Inc.).
Chromatin Immunoprecipitation (CHIP) and MAR Sequence
Detection--
The MRC5 cells were transfected transiently with
rec-hnRNP-A1 as described above. After 48 h of growth in culture,
half of the cells were treated with IFN- Detection of Pre-mRNAs and Mature mRNAs--
Cytoplasmic
RNAs isolated from different cell types (MRC5 and NIH3T3, MRC5 and
NIH3T3 IFN- Real Time Quantitative PCR/RT-PCR--
Cytoplasmic
and nuclear RNAs were isolated from MRC5 cells and MRC5 transfected
with p21 for 20 h and treated with IFN-
hnRNP-A1 recruitment in vivo to human MAR4 was examined by
CHIP at 0, 2, 4, 6, 8, and 24 h after the start of IFN- AFM Scanning and EMSA--
Direct hnRNP-A1 binding to the MARs
was investigated by examining DNA-protein complexes by AFM topography
imaging as described previously (23) and by EMSA. Sets of specific
primer pairs were used for amplification of the probes for EMSA (Table
I). Probes 1-5 were labeled with horseradish peroxidase using the
North2South Direct horseradish peroxidase labeling and detection kit
(Pierce). Each probe (80 ng) was incubated with 100, 200, or 300 ng of
hnRNP-A1 for 50 min in binding buffer (20 mM Tris-HCl, pH
7.4, 0.1 mM EDTA, 0.1% Triton X-100, 1%
2-mercaptoethanol, 2 mM MgCl2, 5% glycerol), containing either 0.1 µg/µl poly[d(I-C)] or 0.1 µg/µl
poly[d(I-C)] combined with 1 µg/µl sonicated salmon sperm DNA.
Labeled probes 1-5 and those from the incubation reactions were
separated in a 2% agarose gel. The DNA signal was detected by chemiluminescence.
Nucleotide Sequences--
The coordinates for each human MAR and
gene CDS (Fig. 1) in the nucleotide sequence GenBankTM
accession number X87344 are as follows: MAR1 (81997-82190), MAR2
(84791-84899), MAR3 (90295-90323), MAR4 (90593-90800), MAR5 (93872-93977), LMP2 (94885-100219), TAP1
(100813-109220), LMP7 (110036-113708), and TAP2
(116217-127095). The schematic of the mouse
Lmp/Tap region (Fig. 9A) is derived
from GenBankTM accession numbers U35323 (24) and AF027865.
The single MAR is located 168 bases downstream of Lmp2 which
is transcribed in the negative orientation (telomere to centromere).
Sequences used to design primers for cloning of hnRNP-A1 and the cdk
inhibitor, p21, from their start to stop codons were NM_002136 and
NM_000389, respectively.
Identification of MARs in the Human LMP/TAP Gene
Region--
A total number of 323 clustered MRS-containing genomic
fragments were found in the MHC (Fig. 1).
Five of these fragments immediately centromeric of the
LMP/TAP gene cluster in the MHC class II region were then studied in detail. The ability of these five fragments and
two random fragments from the same region, which do not contain the
MRS, to bind to the nuclear matrix and therefore to function as MARs
was tested by PCR (18). The level of relative binding (R) of
each DNA fragment to the nuclear matrix was determined as described
previously (17). Cell types with different profiles of MHC class II
gene expression were used to determine whether transcriptional status
in the region is relevant to the functioning of these MAR sequences.
The human fibroblast cell line MRC5 expresses the
LMP/TAP genes at low levels and does not express
the classical MHC class II genes. Expression of all these genes is
greatly increased when MRC5 cells are treated with IFN-
We found that all five genomic fragments containing the MRS from the
LMP/TAP gene region bound to the nuclear matrix,
regardless of the extraction method used (Fig.
2). These fragments were thus designated
MAR1-5. In all three cell types extracted with lithium 3,5-diiodosalicylate (LIS) and ammonium sulfate, MARs-1, -3, -4, and -5 were detected in nuclear matrix fraction only (R = 100%). In contrast, MAR2 showed substantially different levels of
matrix binding in cells with different levels of MHC gene expression. Around 40% of MAR2 sequences were found in the nuclear matrix fraction
isolated from MRC5 cells when ammonium sulfate was used, and 60% when
LIS was used. However, all the MAR2 sequences were bound to the nuclear
matrices isolated from IFN- MARs in the Human LMP/TAP Region Recruit hnRNP-A1 in
Vivo--
The five MARs from the LMP/TAP gene
region were 3'-biotinylated and attached to streptavidin-coated
magnetic particles. They were then used to pull-down proteins using
nuclear extracts from cells with different expression levels of the
adjacent genes. SDS-PAGE analysis showed that each MAR bound multiple
proteins (Fig. 3A). However,
one protein between 31 and 36 kDa showed substantial quantitative
differences in these cells. Far more of this protein bound to MARs
using nuclear extracts from IFN-
By using specific primers for RT-PCR (Table II), the cDNA for
hnRNP-A1 was cloned in full in the pcDNA4/HisMax-TOPO expression vector. This construct was then used to transiently transfect MRC5
cells. Western blot detection of the recombinant protein in nuclear
protein extracts and nuclear matrix protein extracts isolated from the
transfected cells revealed maximum expression and incorporation into
the nuclear matrix 72 h after transfection (Fig.
4). In all subsequent experiments
employing recombinant hnRNP-A1, protein binding to MARs and
LMP/TAP gene expression was examined 72 h
after transfection.
In vivo binding of hnRNP-A1 to MARs 1-5 in MRC5,
IFN-
CHIP analysis was then performed on AHB cells following down-regulation
of MHC class II gene expression, including LMP and TAP genes, by prostaglandin E2
(PGE2) (16) (Figs. 5, C and D, and
6). Substantially less hnRNP-A1 bound to MARs-1, -2, and -4 in these
cells (4-, 8-, and 11-fold, respectively) than in untreated AHB cells,
whereas binding to MARs-3 and -5 was unaffected. These findings confirm
the correlation between MHC class II gene expression and the level of
hnRNP-A1 binding to MARs-1, -2, and -4. Control experiments for the
CHIP background were carried out as described for Fig.
5A, using AHB cells. The intensity of the control signal was
less than 7% of the intensity of corresponding bands for
PGE2-treated AHB cells using anti-hnRNP-A1 antibody.
In addition to up-regulating MHC class II genes, IFN-
To investigate further the relationship between increased hnRNP-A1
binding to MARs-1, -2, and -4 and expression of the adjacent LMP2 and TAP1 genes, semi-quantitative RT-PCR was
performed (Fig. 6A). Cytoplasmic RNAs isolated from MRC5 and
IFN-
Quantitative analysis of the kinetics of hnRNP-A1 recruitment (Fig.
7A, bars c) showed
significantly increased binding of this protein to MAR4 4 h after
the start of IFN- hnRNP-A1 Binds Directly to Human MAR2 and MAR4 in Vitro--
The
protein pull-down and CHIP experiments showed that hnRNP-A1 bound to
all five MARs in the human LMP/TAP gene region
(Figs. 3A and 5). To determine whether the protein binds
directly to the MAR sequences, atomic force microscopy (AFM) scanning
of MAR·hnRNP-A1 complexes was performed. In the AFM images shown in
Fig. 8A, height is coded by
color, with low regions depicted in dark brown and higher
regions in increasingly lighter yellow tones. Only MAR2 and
MAR4 showed direct binding of hnRNP-A1 (Fig. 8A). The
biotinylated 3'-end of DNA appears as a small light spot (black
arrows). The hnRNP-A1, shown as a lighter area over the DNA
(white arrows), bound to a different position in MAR2 and
MAR4. However, for each of these two MARs, the position of protein
binding was consistent in all molecules examined. The hnRNP-A1-binding
sites in DNA were measured using line profiles of 100 DNA-protein
complexes for each MAR, and the results are summarized in Table
IV. Experimentally measured DNA lengths
are in good agreement with the theoretical range of lengths for A- or
B-form conformations. These results showed that hnRNP-A1 binds MAR2 and
MAR4 directly within the 35-bp sequence
GGAGGATCGCYTGAGGCCAGGAGTTCAAGACCAGC (Y = T or C). This binding was
confirmed by EMSA (Fig. 8, B and C), where
hnRNP-A1 bound within probes 2 and 4 only, corresponding to the 35-bp
sequence identified from AFM images. The 35-bp sequence is not present in MARs-1, -3, and -5. Further computational analysis showed this motif
to be over 90% similar to the oldest types of Alu repeat sequences,
AluJo and FLAM_C (Fig. 8D) and to contain a polymerase III
promoter element (B box) and several hormone-response elements (HREs),
which were shown previously to function as transcription factor binding
sites (28).
Conservation of the MRS and hnRNP-A1 Recruitment--
We
identified a single MRS-containing sequence 5' of the mouse
Lmp/Tap genes (Fig.
9A). A PCR assay with L- and
M-fractions as templates isolated after LIS extraction of mouse NIH3T3
fibroblast cells demonstrated that around 50% of the single mouse MAR1
sequences bound to the nuclear matrix in NIH3T3 cells and 60% in
IFN-
In vivo binding of hnRNP-A1 to mouse MAR1 was compared using
CHIP of soluble chromatin fragments from NIH3T3 and IFN- Regulation of gene expression in eukaryotes occurs at
many levels, including interactions of DNA with transcriptional
activators and repressors, DNA methylation, histone modifications, and
alternative splicing of mRNA (29). By using the MHC as a model, we
previously demonstrated rapid alterations in large scale chromatin
structure upon transcriptional activation with IFN- Our experiments showing the MRS contained within MARs in the
LMP/TAP genomic region of the human and mouse
genomes confirms and extends the findings of van Drunen et
al. (7) that the MRS is a conserved property of MARs, implying an
essential function for these sequences. A further level of conservation
is seen in the finding that these MARs recruit hnRNP-A1 during
transcriptional up-regulation in human and mouse cells, indicating a
conserved role for these MARs in mRNA processing.
The Nuclear Matrix--
The nuclear matrix can be defined as the
non-chromatin fibrogranular ribonucleoprotein network in the nucleus
that is readily observed in unextracted cells using the electron
microscope (31, 32). This network fills the nuclear interior and is
connected to the nuclear lamina. Much debate has focused on whether the nuclear matrix is a structural and functional entity in itself, or
whether it is induced during extraction procedures or cell fixation
prior to electron microscopy examination (15, 33). It has been
suggested recently that, irrespective of the nature of the matrix
in vivo, certain elements of the isolated matrix may reflect
a significant "chemical footprint" of protein-protein and
protein-nucleic acid interactions occurring during nuclear metabolism
(15). Our finding of recruitment in vivo of hnRNP-A1 to MAR
sequences in the MHC during up-regulation of MHC gene expression supports the view that we have discovered meaningful interactions of
MARs with hnRNP-A1, which is a major component of the nuclear matrix.
Human MARs 1-5 were retained in nuclear matrix fractions isolated by
LIS, ammonium sulfate, or NaCl treatment, followed by nuclease
digestion. No MAR sequences were found in the loop fraction in LIS- and
ammonium sulfate-extracted preparations except MAR2 in MRC5 cells, but
they were all found in the loop fraction in NaCl-extracted
preparations. Therefore, NaCl extraction releases these MARs from the
matrix fraction so that they are found in the loop fraction in a
proportion of the cells. These data are in agreement with the recent
report that 2 M NaCl extraction plus DNase I/RNase A
digestion disrupts chromosome territory architecture, in contrast to
extraction with ammonium sulfate at the same ionic strength (17,
34).
The difference between NaCl and ammonium sulfate extraction might be
explained by the greater blocking of electrostatic interactions between
DNA and nuclear matrix proteins by NaCl ions compared with that by
ammonium sulfate. Na+ and Cl Recruitment of hnRNPA1--
The hnRNP proteins are a major
component of the nuclear matrix, and of these, hnRNP-A1 is one of the
most abundant (33). Expression of recombinant hnRNP-A1 and Western blot
analysis of nuclear matrix extracts from MRC5 cells unambiguously
showed that it is a nuclear matrix protein. Our experiments confirmed
that hnRNP-A1 is a major protein binding to the MARs in the
LMP/TAP region.
hnRNP-A1 is an essential protein for mRNA maturation and interacts
with splicing factors to regulate alternative splicing (36). The
protein shuttles continuously between the nucleus and cytoplasm and is
also believed to function, together with other hnRNPs, as a carrier for
mRNA during export to the cytoplasm (37). Accumulation of hnRNP-A1
in the nucleus is transcription-dependent and is blocked by
RNA polymerase II inhibitors (38). Furthermore, it has been shown that
this accumulation can be triggered in early mouse embryos by the
appearance of nascent transcripts, to which it is proposed to bind
(39). IFN-
Our experiments revealed that MARs-1, -2, and -4 recruit hnRNP-A1 and
recombinant hnRNP-A1 in vivo during up-regulation of gene
expression (Fig. 5) increasing its concentration in the vicinity of the
LMP2 and TAP1 genes. However, pull-down
experiments in vitro (Fig. 3) showed substantially more of
this protein bound to all the MARs in up-regulated cells. This
discrepancy could be explained by the ability of all MARs to bind
hnRNP-A1 directly or through other proteins because CHIP background
controls showed signal less than 6% of the intensity of corresponding
bands for MRC5 cells (Fig. 5). Under in vitro conditions,
concentration of a particular protein is likely to be the major factor
for binding higher or lower amounts of it to a specific DNA sequence.
However, under in vivo conditions IFN-
Although the role of hnRNP-A1 in mRNA processing and transport has
been studied in detail (37), there are few reports of interactions of
the protein with DNA. hnRNP-A1 has been shown to bind an ATTT sequence
motif within the cell cycle regulatory unit in the human thymidine
kinase gene promoter region, suggesting a role for the protein in
regulating gene expression (43). hnRNP-A1 may also play a role in
telomere biogenesis, because it simultaneously binds single-stranded
telomeric DNA and telomerase RNA in vitro (44). In addition,
deficiency of hnRNP-A1 in a mouse erythroleukemia cell line leads to
short telomeres (45). This report is the first showing recruitment and
binding of hnRNP-A1 to MARs. We extended our investigation to the mouse
H2-m Identification of a Consensus Sequence for Direct Binding of
hnRNP-A1--
Although we showed that these MARs bound hnRNP-A1, it
was not clear whether binding is due to direct interactions or through some other protein(s). Formaldehyde used for fixation in CHIP analysis
is a tight (2-Å) cross-linking agent, efficiently producing both
protein-nucleic acid and protein-protein cross-links in vivo (46). To examine MAR/hnRNP-A1 binding more closely, we studied the
human MAR·hnRNP-A1 complexes in vitro using AFM
topographic imaging (Fig. 8). Only MAR2 and MAR4 were found to bind
hnRNP-A1 directly, suggesting that MARs-1, -3, and -5 bind the protein indirectly through one or more other proteins possibly from the hnRNP
family (47).
A 35-bp consensus motif for binding of hnRNP-A1 directly was identified
within MARs-2 and -4 by AFM and EMSA. A motif almost identical to this
35-bp motif was shown in vitro in double-stranded DNA from
human chromosome band 11q13 to bind hnRNP-A1 directly as
well (23). The genomic sequences on band 11q13 surrounding these motifs were identified as "potential MARs" using
"MAR-Finder" (48) (data not shown). The 35-bp binding motif does
not show any similarity to the hnRNP-A1-binding motif in the thymidine kinase gene promoter region (43) or to the 20-mer consensus sequence
identified in RNA for high affinity binding to hnRNP-A1 (49).
Subsequent sequence comparisons showed that the 35-bp hnRNP-A1-binding
motif maps to an ancient family of Alu repeat sequences (FLAM_C and
AluJo). Alu repeats are known to recruit regulatory proteins (28, 50),
and these particular forms of Alu are twice as abundant in the
LMP/TAP region as compared with the genome average (51). Furthermore, analysis of the human genome sequence reveals preferential retention of Alu repeats in G + C-rich (gene-rich) regions, suggesting a role for these repeats in gene expression (52).
The HREs in these Alu sequences have been shown to bind the retinoic
acid receptors in vitro (28), promoting assembly of an
active chromatin domain. It has been established that upon gene
activation, splicing factors are recruited from speckles and targeted
to sites of transcription (53). Our data showing hnRNP-A1 recruitment
in vivo by the same Alu sequences that recruit retinoic acid
receptors provide further evidence that so-called "junk DNA" has
biological function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IFN-
), resulting in an
increase in processing and presentation of MHC class I-associated
antigens (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(recombinant human IFN-
, R & D Systems, Oxon, UK) was added to the
medium for 24 h (15). In some experiments mouse embryo monolayer
NIH3T3 cells were used. These were grown and treated with IFN-
as
for MRC5 cells; however, E4 medium was used instead of RPMI 1640.
up-regulated MRC5 cells, AHB and NIH3T3 cells were isolated as
described previously (17). Extractions with 25 mM LIS, 0.65 M ammonium sulfate, or 2 M NaCl were employed
in these separations. A PCR assay was used to identify specific DNA
sequences in M- and L-fraction chromatin (18). The M- and L-DNA
fractions, 50 ng each as determined by spectrophotometric measurement,
were used as templates for amplification with corresponding PCR primer
pairs (Table I). In all assays, the
quantity of PCR products was maintained within the linear range
(increasing the concentration of template or the number of cycles
proportionally increased the signal). Aliquots of the PCR products were
then electrophoresed in a 1.5% agarose gel, stained with ethidium
bromide, and quantified densitometrically using Labworks 3.0 software.
A control ratio between the sum of intensities of the M- plus
L-fractions and the band intensity of PCR product using total genomic
DNA as an internal control with the same primer pair as used in the M-
and L-fractions was estimated. This ratio was always ~1 ± 0.021, confirming the equivalent efficiency of the PCR in the M- and
L-fractions and in total genomic DNA. All PCR experiments were carried
out in triplicate for three independent M- and L-fraction separations.
Similar results were obtained and summarized in histograms.
Primers, annealing temperature, and expected PCR products for amplified
probes
up-regulated MRC5 cells, and AHB cells were prepared.
The cells were collected and washed in Buffer A (146 mM
sucrose, 100 mM KCl, 10 mM Tris, pH 7.0, 1.5 mM MgCl2). The pellet was then resuspended in
Buffer A, and Nonidet P-40 was added to a final concentration of 0.5%. After 5 min of incubation on ice, the nuclei were collected by centrifugation. The pellet was then resuspended in Buffer C (5 mM HEPES, pH 7.9, 26% glycerol, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT) to
1 × 108 nuclei/ml, and NaCl was added to a final
concentration of 300 mM. After 30 min of incubation on ice,
the nuclear extracts were separated from the debris by centrifugation
at 24,000 × g for 20 min at 4 °C. The extracts were
aliquoted and stored at
80 °C.
Primers, annealing temperature, and expected RT-PCR products for
amplified probes
at 200 units/ml for a
further 24 h to up-regulated MHC gene expression. IFN-
-treated
and -untreated cells were collected (~5 × 108 cells
each) and fixed in tissue culture medium containing 1% formaldehyde
for 10 min at room temperature. All further steps of this assay were as
described previously (21). Chromatin sonication was performed to
produce DNA fragments in the range of 400-700 bp (electrophoretically
determined in 1.5% agarose). The immunoprecipitation was performed
with Anti-XpressTM antibody recognizing the Xpress tag in
rec-hnRNP-A1. CHIP was similarly performed on AHB cells, MRC5 cells,
IFN-
up-regulated MRC5 cells, and mouse NIH3T3 with and without
IFN-
treatment for 24 h, using goat polyclonal antibody against
endogenous hnRNP-A1 (Santa Cruz Biotechnology, Inc.). In control CHIP
experiments, normal goat IgG (Santa Cruz Biotechnology, Inc.) replaced
the anti-hnRNP-A1 antibody. The naked co-immunoprecipitated DNAs were then used as templates in semi-quantitative PCR assays (50 ng of
DNA/reaction) for MAR sequence detection. Primer pairs used for MAR
sequence detection are given in Table I. The quantity of PCR products
was again maintained within the linear range. Subsequently, aliquots of
the PCR products were electrophoresed in a 1.5% agarose gel, stained
with ethidium bromide, and quantified densitometrically using Labworks
3.0 software. The intensities of MAR fragments amplified from
co-immunoprecipitated DNA were divided by the intensity of MAR
fragments amplified from total genomic DNA (50 ng of DNA/reaction)
using the same primer pairs. The calculated ratio (relative binding)
indicates the enrichment of co-immunoprecipitated DNA in corresponding
MAR sequences, compared with their number in the same amount of genomic
DNA. All PCR experiments were carried out in quadruplicate for three
independent CHIP analyses. Similar results were obtained and summarized
in histograms. Control real time quantitative PCR experiments were
performed for human MAR4 and the single mouse MAR1 as described below.
Primers used in quantification experiments are given in Table
III.
Primer pairs used for real time quantitative PCR/RT-PCR
treated for 24 h, MRC5/rec-hnRNP-A1 transfected for
72 h, and MRC5/rec-hnRNP-A1 transfected for 72 h, and IFN-
treated for 24 h, and AHB and AHB cells PGE2-treated for 20 h) were used as starting templates for RT-PCR to monitor the levels of mature mRNAs. To assess the levels of pre-mRNAs, total nuclear RNAs isolated from the above human cell lines were examined. Cytoplasmic and total nuclear RNAs were isolated as described
previously (22). The subsequent semi-quantitative RT-PCR was performed
using specific primers shown in Table II and Qiagen® One-step RT-PCR
kit (Qiagen) following the supplier's recommendations. Control
experiments with
-actin were carried out using standard
primers (Promega). Aliquots of the RT-PCR products were separated in a
1.5% agarose gel, ethidium bromide-stained, and quantified
densitometrically using Labworks 3.0 software. All RT-PCR experiments
were carried out in triplicate for two independent RNA isolations.
Similar results were obtained and summarized in histograms. Each
relative expression value represents a ratio between the densities of
specific mRNA transcripts to corresponding
-actin transcripts.
for 0, 2, 4, 6, and
8 h. They were used as starting templates for real time
quantitative RT-PCR. Aliquots of the RNAs were reverse-transcribed using random hexamers and Multiscribe reverse transcriptase according to the manufacturer's instructions (Applied Biosystems). Primers were
designed using Primer Express software (Applied Biosystems), taking
into account intron/exon boundaries to ensure specific amplification of
cDNA (Table III). Primers were designed to
-actin as an internal
control for normalization of starting cDNA levels. Quantitative PCR
was performed using SYBR Green PCR Master Mix according to the
manufacturer's instructions (Applied Biosystems), with the exception
that 25-µl reaction volumes were used, with 45 cycles of
amplification. Each of the primer pairs was optimized to ensure
amplification of the specific product and absence of primer dimers
(Table III). PCR was performed on the Taqman 7700 (Applied Biosystems).
Following amplification, RT-PCR products were electrophoresed on
agarose gels to check for the correctly sized product. The real time
PCR results were analyzed using the sequence detection system software
version 1.9 (Applied Biosystems). Gene expression levels were
calculated using the comparative Ct method
(
Ct).
Ct validation experiments
showed similar amplification efficiency for all templates used
(difference between line slopes for all templates less than 0.1).
Expression levels of the genes were normalized to those in MRC5 cells
without IFN-
treatment. At least two independent experiments were
performed for each gene. Similar results were obtained and summarized
in histograms.
treatment of MRC5 cells and p21-transfected MRC5 cells.
Immunoprecipitated DNAs were used as templates in subsequent real time
quantitative PCR experiments. Levels of protein recruitment were
calculated according to the
Ct method. Similar
amplification efficiencies were shown for all templates used.
Recruitment levels to MAR4 were normalized to that in MRC5 cells
without IFN-
treatment. Two independent experiments were carried
out. Similar results were obtained and summarized in histograms.
Recruitment of hnRNP-A1 to the single mouse MAR1 was examined in NIH3T3
cells 0 and 24 h after IFN-
treatment. Results were analyzed as
for the human MAR4.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
for 24 h (25). The B-lymphoblastoid cell line AHB constitutively expresses
these genes at high levels.
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Fig. 1.
Scheme of the human MHC with enlarged
LMP/TAP gene region showing candidate
MAR sequences containing the MRS (black boxes
1-5). On the top is shown the distribution
of the clustered MRS-containing genomic fragments in the MHC.
-treated MRC5 and AHB cells by both
extraction procedures. In NaCl-extracted cells, a lower proportion of
MARs 1-5 sequences (45-65%) bound to the nuclear matrix than in LIS
and ammonium sulfate-extracted cells, and no significant differences
were found in cells with different levels of MHC gene expression.
Neither of the control sequences bound to the nuclear matrix in these
cell types using the three extraction procedures.
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Fig. 2.
PCR assay for testing the nuclear matrix
binding ability of five candidate human MARs and two control non-MAR
sequences in the LMP/TAP gene
region. A, L- and M-DNA fractions were isolated by 25 mM LIS, 2 M NaCl, and 0.65 M
ammonium sulfate extractions from MRC5, IFN- up-regulated MRC5, and
AHB cells. These fractions were used in the subsequent PCR assay as
templates. Positive and negative internal controls were performed with
total genomic DNA and pBR322 templates, respectively, for each
sequence. Two non-MAR sequences from the same gene region were tested
for their nuclear matrix binding ability as a control for the assay
specificity. The PCR products were separated in a 1.5% agarose gel.
B, R values for binding of the sequences to the
nuclear matrix was estimated as the band intensity in the M-fraction
(IM) divided by the sum of intensities in the matrix
(IM) plus the loop (IL) fractions
(R = IM/(IM + IL)). Values show the means ± S.E.
for three independent PCR assays.
up-regulated MRC5 cells and AHB
cells, which have higher expression levels of the adjacent
LMP2 and TAP1 genes and the classical MHC class
II genes, than untreated MRC5 cells. This protein was identified by
mass spectroscopy as the heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) (Fig. 3B). Subsequent investigations thus focused
on this protein, which is involved in mRNA processing and export from the nucleus.
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Fig. 3.
Pull-down of proteins binding to human
MARs. A, SDS-PAGE of proteins pulled down by MARs 1-5
from MRC5, IFN- up-regulated MRC5, and AHB nuclear extracts. The
proteins specifically bound to each MAR were run in a 12% Tris glycine
pre-cast gel and silver-stained. B, identification of
hnRNP-A1 by peptide mass fingerprinting.
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Fig. 4.
Kinetics of rec-hnRNP-A1 expression in
transiently transfected MRC5 cells. Nuclear protein extracts
(NE) and nuclear matrix protein extracts (NME)
from transfected cells were isolated at 24, 48, 72, and 96 h after
transfection, and SDS-PAGE was performed. Western blot analysis was
carried out to detect the Xpress epitope in the rec-hnRNP-A1. Nuclear
extract from MRC5 cells without transfection was used as a negative
control.
-treated MRC5, and AHB cells was then compared by CHIP. Live
cells were fixed with formaldehyde, DNA·hnRNP-A1 complexes
immunoprecipitated, and binding analyzed by semi-quantitative PCR (Fig.
5). Substantially more hnRNP-A1 bound to
MARs-1, -2, and -4 in IFN-
-treated MRC5 cells (about 4-, 27-, and
10-fold, respectively) and in AHB cells (6-, 31-, and 12-fold,
respectively) than in untreated MRC5 cells (Fig. 5, A and
B). No differences were observed for MAR3 and MAR5. These
experiments demonstrate significantly increased binding of hnRNP-A1 to
MARs-1, -2, and -4 in cells where MHC class II expression, including
the LMP/TAP genes, is up-regulated. Control experiments for the background signal as a result of nonspecific binding to antibody and protein A-Sepharose were performed by replacing
anti-hnRNP-A1 with normal IgG. The background for each MAR detected by
semi-quantitative PCR was less than 6% of the intensity of
corresponding bands obtained for MRC5 cells using anti-hnRNP-A1
antibody (Fig. 5A). Real time quantitative PCR for recruitment of this protein to MAR4 was performed to confirm the accuracy of the semi-quantitative experiments (Fig. 5B,
striped bars). Around 11.6-fold higher binding of hnRNP-A1
to MAR4 was detected in IFN-
-treated MRC5 cells compared with
untreated cells. This result correlates well with that obtained by
semi-quantitative PCR for MAR4 (around 10-fold).
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Fig. 5.
CHIP analysis of in vivo
recruitment of hnRNP-A1 to human MARs 1-5. A,
recruitment of endogenous hnRNP-A1 to MARs 1-5 in MRC5 cells, IFN-
up-regulated MRC5 cells, and AHB cells. 1.5% agarose electrophoresis
of semi-quantitative PCR products using primer pairs specific for the
five human MARs and goat anti-hnRNP-A1 antibody co-immunoprecipitated
DNA fractions from MRC5, IFN-
up-regulated MRC5, and AHB cells as
templates. Amplification of the MARs from genomic DNA was carried out
as internal controls. CHIP using MRC5 cells and normal goat IgG instead
of specific antibody was performed as a background control.
B, histograms of relative binding of endogenous hnRNP-A1 to
MARs 1-5. Values show means ± S.E. for three independent CHIP
experiments each analyzed in quadruplicate. Black bars,
which represent results from the semi-quantitative PCR, are related to
the left-hand side scale. Striped bars show
hnRNP-A1 recruitment to MAR4 measured by real time quantitative PCR
(QPCR, see the right-hand side scale).
C, recruitment of endogenous hnRNP-A1 to MARs 1-5 in AHB
cells and PGE2 down-regulated AHB cells. Similar internal
and background controls were used as in A. D,
histograms of relative binding of endogenous hnRNP-A1 to the MARs.
Values show the means ± S.E. for three independent CHIP
experiments each analyzed in quadruplicate. E, recruitment
of rec-hnRNP-A1 to MARs 1-5 in transiently transfected MRC5 cells and
MRC5-transfected and IFN-
up-regulated. CHIP with anti-rec-hnRNP-A1
antibody was performed on untransfected MRC5 cells as a background
control. Amplification of the MARs from genomic DNA was carried out as
an internal control. F, the values of relative rec-hnRNP-A1
recruitment to the MARs are shown as means ± S.E. for three
independent CHIP experiments, each analyzed in quadruplicate.
treatment of
MRC5 cells led to higher expression of hnRNP-A1 (Fig. 6). To test whether increased binding of
hnRNP-A1 to MARs-1, -2, and -4 is a result of the generally increased
hnRNP-A1 concentration in the nucleus following transcriptional
up-regulation or whether this is a specific recruitment, CHIP was
performed against recombinant hnRNP-A1 transfected into MRC5 cells.
IFN-
up-regulation of transfected cells did not affect the
expression level of rec-hnRNP-A1 (Fig. 6). Substantially
more of this protein bound to MARs-1, -2, and -4 in
IFN-
-treated MRC5 cells (about 3-, 24-, and 8-fold,
respectively) compared with untreated cells (Fig. 5, E and
F), suggesting that the increased binding is not simply due
to the increased concentration of hnRNP-A1 in the nucleus. CHIP with
anti-rec-hnRNP-A1 was carried out in untransfected MRC5 cells as a
control. The intensity of control bands was less than 3% of the
intensity of corresponding bands in transfected cells.
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Fig. 6.
Quantitation of gene expression by
RT-PCR. A, cytoplasmic RNA (for monitoring levels of
mature mRNAs) and nuclear RNA (for monitoring pre-mRNAs) were
extracted from MRC5, IFN- up-regulated MRC5, hnRNP-A1-transfected
MRC5, hnRNP-A1-transfected, and IFN-
up-regulated MRC5, AHB, and
PGE2-treated AHB cells, co-amplified by semi-quantitative
RT-PCR and electrophoresed in a 1.5% agarose gel. B,
histogram of ratios between the densities of specific bands and of the
-actin in corresponding cells. The results are means ± S.E. for two independent RNA isolations each analyzed in
triplicate.
-treated MRC5 cells were used as starting templates to
quantitate mature LMP2 and TAP1 mRNAs,
whereas nuclear RNAs were used to quantitate pre-mRNAs. The results
are summarized in Fig. 6B. Higher levels of hnRNP-A1 from
rec-hnRNP-A1 transfection led to 4-5-fold less LMP2 and
TAP1 pre-mRNAs and higher levels of the corresponding
mature mRNAs (Fig. 6). IFN-
treatment resulted in a slight
increase of LMP2 and TAP1 pre-mRNAs, whereas
the levels of mature mRNAs in cytoplasm were increased 3-fold for
LMP2 and 5-6-fold for TAP1. Taken together, these data thus demonstrate that both transfection of hnRNP-A1 and
IFN-
treatment facilitate the production of mature LMP2
and TAP1 mRNAs.
treatment of MRC5 cells. This time correlates with
a rise in mature LMP2 and TAP1 mRNAs
(bars d and e, respectively). However, we were
only able to detect an increase in their pre-mRNAs 6 h after
the start of IFN-
treatment (Fig. 7A, bars a
and b, respectively). To find an explanation for this
discrepancy, we transiently transfected MRC5 cells with the cdk
inhibitor, p21, which is reported to inhibit splicing in
vitro (26). Levels of LMP2 and TAP1
mRNAs (Fig. 7B, bars d and e,
respectively) were found to rise 4 h after the start of IFN-
treatment; however, the increase was about 20-fold less than that in
cells without transfected p21 (Fig. 7A). This time again
correlates with the recruitment of hnRNP-A1 to MAR4 (bars
c). Binding of hnRNP-A1 to MAR4 was, however, approximately half
that in untransfected cells. Crucially, in the p21-transfected cells,
we found a rise in the LMP2 and TAP1
pre-mRNAs in 4 h from the start of IFN-
treatment
(bars a and b, respectively). Taken together,
these data indicate that the apparent rise in mRNA levels prior to
pre-mRNA reflects the rapid activation of the splicing machinery
and the coupled mRNA export (27), suggesting a relationship between
hnRNP-A1 recruitment and mRNA processing.
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Fig. 7.
Kinetics of hnRNP-A1 recruitment to MAR4 in
relation to LMP2 and TAP1 gene
expression. A, hnRNP-A1 recruitment in vivo
to MAR4 (bars c) was examined by CHIP at 0, 2, 4, 6, and
8 h after the start of IFN- treatment of MRC5 cells. The
appearance of LMP2 and TAP1 pre-mRNAs
(bars a and b, respectively) and their mRNAs
(bars d and e, respectively) was monitored
simultaneously by real time quantitative RT-PCR using nuclear and
cytoplasmic RNA preparations, respectively. Expression levels were
calculated using the
Ct method. The recruitment level
of the protein to MAR4 and the expression level of the
LMP2/TAP1 genes were normalized to that in MRC5
cells without IFN-
treatment. Bars a-c are related to
the left-hand side scale and bars d and
e to the right-hand side scale. The
results are mean values from two independent CHIP experiments and RNA
isolations. B, kinetics of hnRNP-A1 recruitment to MAR4 and
LMP2, TAP1 gene expression as in A,
using MRC5 cells transfected with p21, 20 h before IFN-
treatment. Bars c-e are related to the left-hand side
scale, and bars a and b to the
right-hand side scale.
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Fig. 8.
In vitro binding of hnRNP-A1.
A, AFM topography images of human MARs 1-5 after incubation
with hnRNP-A1 and fixation of the complexes onto mica. In the images,
height is coded by color, with low regions depicted by dark
brown and higher regions in increasingly lighter tones in
yellow from 0 Å. Only MAR2 and MAR4 showed formation of
complexes with hnRNP-A1 in vitro. The black and
white arrows mark the positions of 3'-end biotinylation and
protein binding, respectively. B, scheme of the probes
amplified for EMSA by PCR. Probes 2 and 4 (35 bp each) correspond to
the 35-bp sequence for binding hnRNP-A1 identified from AFM
experiments. C, probes 1-5 labeled with horseradish
peroxidase (lanes a), probes incubated with 100, 200, or 300 ng of hnRNP-A1 in the presence of 0.1 µg/µl poly[d(I-C)]
(lanes b, d, and f, respectively),
probes incubated in the presence of 0.1 µg/µl poly[d(I-C)], and 1 µg/µl sonicated salmon sperm DNA (lanes c, e,
and g, respectively) were separated in a 2% agarose gel.
The DNA signal was detected by chemiluminescence. D, an
alignment of the 35-bp hnRNP-A1 binding motif (hnRNP-A1B)
with FLAMs and Alu left ends. Positions are numbered with respect to
the FLAM_C sequence and matches to hnRNP-A1B shown in red or
blue (to degenerate code). The B box consensus of the
promoter for polymerase III is also shown with the invariant bases
underlined. The four HREs are superimposed in
purple.
Mapping hnRNP-A1 binding to human MARs according to the line profiles
of AFM topography images
-treated NIH3T3 (Fig. 9, B and C). This
sequence thus functions as a real MAR. Control random sequences from
the same gene region, which do not contain the MRS, did not show any
binding to the nuclear matrix (Fig. 9, B and C).
These observations confirm that the MRS is a conserved property of MARs
in different species (7) (Table V).
RT-PCR analysis revealed that IFN-
treatment increased expression of
the Lmp2 and Tap1 mouse genes around 4-5-fold
(Fig. 10, A and
B).
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Fig. 9.
Scheme of the
Lmp/Tap region of the mouse MHC.
A, candidate MAR in the Lmp/Tap gene
region containing the MRS pattern (black box 1).
B, L- and M-DNA fractions were isolated from NIH3T3 and
IFN- up-regulated NIH3T3 mouse cells by 25 mM LIS
extraction. These fractions were used in the subsequent PCR assay as
templates. Positive and negative internal controls were carried out
with total genomic DNA and pBR322 templates, respectively, for each
sequence. Two non-MAR sequences from the same gene region were tested
for their nuclear matrix binding ability as a control for assay
specificity. The PCR products were separated in a 1.5% agarose gel.
C, R values for the sequences binding to the
nuclear matrix were estimated as in Fig. 2B. Values show the
means ± S.E. for three independent PCR assays.
An example for conservation of MRS pattern between human and mouse
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Fig. 10.
Correlation between recruitment of hnRNP-A1
and expression of mouse Lmp/Tap
genes. A, expression levels of Lmp2
and Tap1 in NIH3T3 and IFN- -treated NIH3T3 cells,
monitored by RT-PCR. B, histogram of ratios between the
densities of specific bands and of the
-actin in
corresponding cells. The results are means ± S.E. for two
independent RNA isolations each analyzed in triplicate. C,
1.5% agarose electrophoresis of semi-quantitative PCR-products using
primer pair specific for the mouse MAR and anti-hnRNP-A1 antibody
co-immunoprecipitated DNA fragments from NIH3T3 and IFN-
up-regulated NIH3T3 cells as templates. Amplification of mouse MAR from
genomic DNA was performed as an internal control. In background control
CHIP experiments normal goat IgG replaced the antibody. D,
the values of relative hnRNP-A1 recruitment to the MAR are shown as
means ± S.E. for three independent CHIPs each analyzed in
quadruplicate. Black bars, which represent results from the
semi-quantitative PCR, are related to the left-hand side
scale. Striped bars show the hnRNP-A1 recruitment to
MAR1 assessed by real time quantitative PCR (see the right-hand
side scale).
up-regulated NIH3T3 cells by anti-hnRNP-A1 antibody (reactive against
both human and mouse hnRNP-A1). Control experiments for the CHIP
background were carried out as described for Fig. 5; however, NIH3T3
cells were used in this assay. The background found was less than 8% of the intensity of corresponding bands for NIH3T3 cells obtained by
anti-hnRNP-A1. Relative binding of hnRNP-A1 to the mouse MAR was
assessed by semi-quantitative (black bars) and quantitative (striped bars) PCR (Fig. 10, C and D)
as described for human MARs. Approximately 10-12-fold higher in
vivo binding of the protein to mouse MAR1 was found in both PCR
assays in IFN-
-treated cells compared with that in untreated cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(30). This work
examines interactions of non-coding DNA in the MHC with proteins in
cells having different levels of MHC class II gene expression. We show substantial recruitment in vivo of a major mRNA
processing protein hnRNP-A1 to certain MARs in the MHC during
transcriptional activation of the MHC class II genes. Although the
nuclear matrix is known to contain hnRNP-A1, this is the first evidence
that MARs recruit this protein.
are smaller than
NH
up-regulates the expression of over 200 genes (25),
including hnRNP-A1 (Fig. 6). We would thus expect a substantially
increased concentration of nuclear hnRNP-A1 in fibroblasts treated with
IFN-
and in AHB cells where all the MHC class II genes are
expressed, compared with untreated fibroblasts.
treatment of MRC5
cells transiently transfected with hnRNP-A1 did not influence
transcription of the recombinant gene (Fig. 6). This suggests that
recruitment of hnRNP-A1 in vivo to the MARs is not simply
due to an increase in the concentration of the protein but is more
likely to be related to transcriptional up-regulation of the adjacent
genes and the simultaneous requirement for mRNA processing
machinery. Recently, it was found that hnRNP-A1 plays an important role
in the negative regulation of splicing by binding to exonic silencer
elements and counteracting the positive activity of SR proteins (40).
The timing of recruitment of this protein to MARs (Fig. 7) suggests
that they may serve to enhance splicing by sequestration of the
protein. Nuclear speckles are likely to be a depot for hnRNP-A1,
because they serve as transient storage and assembly sites for
pre-mRNA splicing factors that associate with sites of active
transcription (41, 42).
1 gene region and found similar recruitment by one of
two MARs theoretically predicted by the MRS and experimentally shown to
function as MARs (results not shown). Further studies need to be
performed to determine whether MARs in other regions of the MHC or
other up-regulated genomic regions bind hnRNP-A1.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Frank Uhlmann for the helpful discussion and comments on the paper, Petros Takousis for useful advice on real time PCR, and Dinah Rahman for protein sequencing.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Cancer Research UK and the UK Engineering and Physical Sciences Research Council. Cancer Research UK London Research Institute comprises the Lincoln's Inn Fields and Clare Hall Laboratories of the former Imperial Cancer Research Fund following the merger of the ICRF with the Cancer Research Campaign in February 2002.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.
¶ Supported by the Wellcome Trust.
** To whom correspondence should be addressed: Human Cytogenetics Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. Tel.: 44-20-7269-3220; Fax: 44-20-7269-3655; E-mail: Denise.Sheer@cancer.org.uk.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M206621200
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ABBREVIATIONS |
---|
The abbreviations used are:
MARs, matrix
attachment regions;
MHC, major histocompatibility complex;
MRS, the
matrix recognition signature;
hnRNP, heterogeneous nuclear
ribonucleoprotein;
RT, reverse transcriptase;
DTT, dithiothreitol;
CHIP, chromatin immunoprecipitation;
IFN-, interferon-
;
EMSA, electrophoretic mobility shift assay;
HREs, hormone-response elements;
LIS, lithium 3,5-diiodosalicylate;
PGE2, prostaglandin
E2;
rec, recombinant;
AFM, atomic force microscopy.
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