 |
INTRODUCTION |
Development and maintenance of photoreceptor function in mammalian
retina requires the expression of photoreceptor-specific or
photoreceptor-enriched genes. Under- or over-expression of these genes,
such as the visual pigment rhodopsin (1, 2), can lead to a
developmental defect or photoreceptor degeneration. The molecular
mechanisms regulating photoreceptor gene expression involve
interactions of cis-acting elements in the promoter region with trans-acting transcription factors (3, 4). Among these factors, Crx (cone-rod
homeobox) (5, 6) and Nrl (neural retina leucine zipper) (7) are reported to be
essential for photoreceptor development and function (8, 9).
Crx is a member of the Otd/Otx homeodomain protein family expressed
predominantly in the rod and cone photoreceptors of the retina and
pinealocytes of the pineal gland (5, 6, 10). Crx regulates the
expression of several photoreceptor genes (5) as well as pineal genes
involved in melatonin synthesis (11) by binding to their promoters. It
acts synergistically with Nrl (5), a bZIP transcription factor
expressed specifically in rod photoreceptors (12). The Crx protein
includes a homeodomain (HD)1
near its N terminus, followed by a glutamine-rich (Gln) region, a basic
region, a WSP (SIWSPASESP) region, and an Otx tail
region that all share homology with corresponding regions of Otx1 and Otx2 (5). The Crx HD is of the K50 subtype (with lysine at its 50th residue) of the paired-like class (5), and it is responsible for binding to target DNA (5, 13), the nuclear localization of the Crx
protein (14), and mediating a physical and functional interaction with
Nrl (15). In vitro protein-DNA binding assays demonstrated
that the Crx HD binds to at least three target sites in the rhodopsin
promoter, all with a (C/T)TAATCC consensus sequence, including a high
affinity site called BAT-1 and two low affinity sites called Ret-1 and
Ret-4 (5). Transient transfection assays in HEK293 cells demonstrated
that the C-terminal region of Crx (between amino acids 107 and 284)
contains the transactivation domains AD-1 and AD-2, which are important
for its ability to activate promoters (13).
Mutant mice that are homozygous for a null allele of Crx
(Crx
/) fail
to develop outer segments of the
photoreceptors, which subsequently undergo progressive degeneration
(8). The expression levels of many photoreceptor genes are altered in
the Crx/ mouse retina, indicating that these
genes are either direct or indirect targets of Crx (8, 16, 17).
Consistent with these studies, over a dozen CRX2
mutations are associated with human retinal degenerative diseases, such
as autosomal dominant cone-rod dystrophy
(CORD2) (18-21), retinitis pigmentosa (20), and Leber congenital
amaurosis (20, 22-24). In vitro functional analyses of some
of these mutations demonstrated reduced binding to and/or
trans-activation of the rhodopsin promoter (13, 21, 23).
These results, combined with the mouse studies, provide strong evidence
that Crx is required for both the development and maintenance of
photoreceptors by acting as an important regulator of photoreceptor
gene expression.
Several studies have identified Crx-interacting proteins, such as Nrl
(15), CREB-binding protein/p300 (25), phosducin and phosducin-like
proteins (PhLP1 and PhLOP1) (26), the nonhistone high mobility group
protein HMGA1 (formerly HMG19Y) (27), and ataxin-7 (28). Among these,
Nrl, HMGA1, and CREB-binding protein/p300 enhance, whereas Phd (PhLPs)
and ataxin-7 repress, the transactivation activity of Crx. To further
enhance our understanding of Crx function, we carried out a
protein-protein interaction screen of a bovine retinal cDNA library
in yeast with Crx as bait. Here, we report the identification of
barrier-to-autointegration factor (Baf) as a Crx-interacting protein
and a detailed characterization of the physical and functional
interaction of Crx and Baf. Our studies suggest a novel cellular
function for Baf that is directly linked to transcriptional regulation
of tissue-specific genes in addition to its reported role in chromatin
decondensation and nuclear envelope assembly during mitosis
(29-31).
| |
EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screening and Interaction Assays--
The
two-hybrid assays in yeast (32) were carried out using the Matchmaker
Two-Hybrid System 2 (BD-Biosciences CLONTECH, Palo
Alto, CA) with a dual reporter strain, Y190, as described previously
(15, 28, 33). The "bait" construct Crx-HD-pAS2 contains the bovine
Crx homeodomain and its flanking sequences (amino acid residues
34-107) fused in frame with the Gal4-DNA binding domain (dbd) in the
pAS2-1 vector. A bovine retinal cDNA library (34) generated in
pACTII (the prey vector with a Gal4 activation domain) was used for
screening Crx-HD-interacting clones. The reporter strain Y190 was
transformed with the bait vector Crx-HD and tested for a basal
expression level of the dual reporter genes His3 and
lacZ using 3-amino-1,2,4-triazole (3-AT; a competitive inhibitor of the His3 protein) and a colony lift X-gal filter assay,
respectively, as described in the CLONTECH manual.
The bait-containing Y190 cells were subsequently transformed with 20 µg of DNA from the retinal cDNA library. Colonies that grew on
SD-Trp, Leu, His medium
supplemented with 15 mM 3-AT were considered as
"positives," and they were verified using X-gal filter assays. Y190
transformants containing the known interacting protein partners, Snf1
(in pAS1) and Snf4 (in pACTII), were used as controls for a
positive interaction (35). Yeast DNA harboring a mixture of the bait
and prey plasmids was prepared from each of the clones that tested
positive by the dual reporter assay. The prey plasmids in the positive
colonies were recovered by electroporation of the yeast DNA into
Escherichia coli strain DH5
, selection of E. coli colonies containing the plasmids, and subsequent
amplification and purification of plasmid DNA. False positives were
further eliminated by retransforming the prey DNA to the original bait
strain and a strain harboring the unrelated bait Snf1. Library clones
that were positive for interaction with Crx-HD but not with Snf1 were
sequenced and characterized.
To confirm the interaction of Crx and the product of the Baf
gene identified by the yeast two-hybrid screening, an insert swap
between the bait and prey was performed. The Baf insert was PCR-amplified and cloned into the pAS2 bait vector at the
NdeI site (filled in) with the predicted open reading frame
fused in-fame with Gal4-dbd. The full-length coding region of bovine
Crx was cloned in-frame with Gal4 activation domain in
pACTII at the BamHI (5') and XhoI (3') site. The
resulting Baf bait and Crx prey constructs were co-transformed into the
yeast Y190 for 3-AT and X-gal assays as described above.
Constructs for Recombinant Protein Expression--
To express
and purify the bovine Baf (bBaf) protein from E. coli, a
PCR-amplified cDNA corresponding to the open reading frame of
bBaf was cloned in frame with the His6 tag of
pTrcHisA (Invitrogen) at the BamHI (5') and EcoRI
(3') site. For mammalian expression and in vitro
transcription/translation, a PCR-amplified cDNA containing the
bBaf coding region fused in frame with an N-terminal Myc tag was generated and cloned into pcDNA3.1(+) (Invitrogen) at the HindIII and XbaI site
(bBaf-pcDNA3.1(+)/myc). All of the PCR amplifications were performed using the high fidelity Pfu DNA polymerase
(Stratagene, La Jolla, CA). The frame of each fusion protein was
confirmed by sequencing using an ABI Prism DNA sequencing kit and ABI
Prism 310 Genetic Analyzer (PerkinElmer Life Sciences). The bacterial expression vector Crx-HD-GST containing the Crx homeodomain fused with
the GST tag in pGEX-4T-2 (Amersham Biosciences), the mammalian expression vectors carrying the coding cDNA of the human
(hCRX) and bovine (bCrx) Crx and its
deletion series, in frame with the Xpress tag in pcDNA3.1/HisC
(Invitrogen), and the hCRX constructs carrying missense
mutations in the HD were described previously (5, 15).
Purification of Bacterially Expressed Recombinant Proteins and
Antibody Collection--
The GST and Crx-HD-GST proteins were
expressed in E. coli and purified using
glutathione-Sepharose beads as described previously (5). The
His6-tagged Baf (Baf-His) and the His6 tag
alone (His) were expressed in the E. coli BL21 strain
(Stratagene) and purified using Ni2+-NTA-agarose resin
(Qiagen, Valencia, CA) under denaturing conditions with 6 M
guanidine-HCl according to the manufacturer's instructions with some
modifications. In brief, bacterial cells were lysed in a lysis buffer
(0.1 M NaH2PO4, 10 mM
Tris-Cl, 6 M guanidine HCl, pH 8.0), bound to the
Ni2+-NTA resin using the batch method, washed with the
lysis buffer supplemented with 5 mM imidazole, and eluted
with an imidazole gradient (20-700 mM) in the lysis
buffer. The fractions containing either Baf-His or His were pooled
after SDS-PAGE analysis of each fraction. The affinity-purified
proteins were renatured by dialysis against a storage buffer containing
25 mM Hepes, pH 7.6, 60 mM KCl, 0.1 mM EDTA, 2 mM dithiothreitol, 10% glycerol,
and 0.1 mM phenylmethylsulfonyl fluoride. The resulting
protein preparations were quantified using Bio-Rad Protein Assay Kit II
and analyzed by SDS-PAGE and immunoblots with the anti-polyhistidine
monoclonal antibody (Sigma-Aldrich) and an anti-Baf antibody (see
below). Each protein preparation was also analyzed for possible DNA
contamination by UV spectrum measurement (at 260 and 280 nm) and
agarose gel electrophoresis (1% with ethidium bromide). P261, a rabbit
polyclonal antibody to Crx, was described previously (28). A polyclonal antibody against human BAF was generated in rabbits using purified recombinant human BAF expressed in E. coli. This antibody
and its control serum (preimmune) were kindly provided by Robert Craigie.
Immunoblots with Whole Cell Extracts--
Crude protein lysates
were prepared by homogenization of frozen tissue samples or cell
pellets in a 3-fold volume of a sample buffer containing 62.5 mM Tris-HCl, pH 6.8, 4% SDS, 200 mM
dithiothreitol, 10% glycerol, and 0.001% bromphenol blue using a
Pro250 homogenizer (PRO Scientific Inc., Monroe, CT), followed by
immediately boiling for 10 min. Eight µl of each protein sample were
resolved by SDS-PAGE (15% gel) and transferred to a polyvinylidene
difluoride membrane (Bio-Rad, Hercules, CA), which was probed with the
primary antibody against Baf at a 1:1000 dilution, and the signal was
detected by a horseradish peroxidase-conjugated anti-rabbit-IgG
secondary antibody at a 1:1000 dilution and the ECL kit (Amersham Biosciences).
Co-immunoprecipitation and Pull-down
Assays--
Co-immunoprecipitation assays with in vitro
translated proteins were carried out essentially as described
previously (28) with minor modifications. 1% Triton X-100 was included
in the wash buffer (50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% Triton X-100), and 2-4 µl of a specific
antibody were used for co-immunoprecipitation. Antibodies used for
co-immunoprecipitation include anti-Crx P261 (28), anti-Myc (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), anti-Chx10 (a gift of Dr. Connie
Cepko), and anti-Nrl (12). Co-immunoprecipitated proteins were resolved
by SDS-PAGE and quantified using a Storm 860 PhosphorImager system and
ImageQuant 5.0 analytic program (Amersham Biosciences). For
co-immunoprecipitation assays with tissue extracts, whole cell extracts
were prepared by homogenizing tissue samples in a 3-fold volume of a
whole cell lysis buffer (50 mM Tris-Cl (pH 7.5), 450 mM NaCl, 1% Triton X-100, and 10% glycerol with a mixture
of protease inhibitors (Roche Molecular Biochemicals)). After a brief
centrifugation for 5 min at 10,000 × g, 200 µl of
the supernatants were incubated with 2 µl of the anti-Crx P261
antibody for 2 h at 4 °C, followed by the addition of 45 µl
of 50% Protein A-Sepharose beads and gentle mixing on a rotator at
4 °C overnight. After being washed five times with the wash buffer
(50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1%
Triton X-100), the bound proteins were eluted and analyzed by SDS-PAGE and immunoblots with the anti-Baf antibody.
For pull-down assays, 100 ng of the purified Crx-HD-GST protein was
gently mixed with 45 µl of Ni2+-NTA beads coupled with
the Baf-His or His protein in 100 µl of a binding buffer (1× PBS,
0.01% Nonidet P-40) at 4 °C overnight. In a reciprocal approach, 50 ng of the purified Baf-His protein in 100 µl of the binding buffer
was incubated with glutathione-Sepharose beads coupled with Crx-HD-GST
or GST. Proteins bound to the beads were washed five times with the
wash buffer, eluted from the beads by boiling in 20 µl of a SDS-PAGE
loading buffer, resolved by SDS-PAGE (11-15% gel), and detected by
immunoblots with the anti-GST antibody (Sigma) (for assays with the His
protein beads) or anti-Baf antibody (for assays with the GST protein beads).
Electrophoretic Mobility Shift Assays (EMSAs)--
For EMSAs
with Crx-HD peptides, the GST tag was removed from the Crx-HD-GST
proteins by digestion with thrombin protease (Amersham Biosciences) at
a concentration of 2 units/µg of fusion protein (room temperature for
4 h). EMSAs with recombinant proteins and bovine retinal nuclear
extracts were performed as described (5). For supershift EMSAs, the
Crx-HD peptides or the bovine retinal nuclear extract were preincubated
with increasing amounts (in µl) of the Baf-His protein or the His tag
control in the reaction buffer for 10 min on ice prior to the addition
of the probes. The reactions were incubated on ice for an additional 30 min and resolved by native PAGE (5% gel).
Cell Culture and Transient Transfection Assays--
HEK293 cells
were cultured on 35-mm plates, transfected using the calcium phosphate
method, and analyzed using dual luciferase assays as described by Chen
et al. (13). Typically, a total of 3.0 µg of DNA was used
for each transfection, including 2 µg of the rhodopsin-luciferase
reporter pBR130-luc (36), 1 ng of the Renilla luciferase
reporter pRL-CMV (Promega, Madison, WI) as an internal control for
transfection efficiency, 100 ng of a mammalian vector expressing the
transcription activator Crx (bCrx-pcDNA3.1/HisC), and/or
Nrl (pMT-NRL), 50-800 ng of the Baf expression vector
(bBaf-pcDNA3.1(+)/myc), and various amounts of the
carrier DNA (pcDNA3.1/HisC) to keep the amount of total DNA
constant. Each sample was done in duplicate, and at least four
independent experiments were performed. The significance of the results
was calculated using Student's t test, and it was assumed
that each pair of samples under comparison had equal variances. For
analyzing the effect of Baf on transactivation activity of the Gal4
fusion proteins and c-Jun/c-Fos, two different luciferase reporters
were used: a Gal4-responsive luciferase construct pFR-luc (Stratagene)
for assays with Gal4dbd-Crx-(111-299) or Gal4-VP16 (13) and a
collagenase promoter-luciferase construct (36) for assays with
c-Jun/c-Fos.
Immunocytochemistry and Confocal Microscopy--
HEK293 cells
were cultured on poly-D-lysine (100 µg/ml; Sigma)-coated
glass coverslips and co-transfected with 1 µg of each of the
mammalian cell expression constructs bCrx-pcDNA3.1/HisC and bBaf-pcDNA3.1(+)/myc. At 24 h after
transfection, the cells were fixed with 4%
paraformaldehyde/phosphate-buffered saline (PBS, pH 7.4) for 15 min,
washed three times with PBS, permeabilized in a blocking buffer (5%
fetal calf serum, 0.5% Triton X-100, 0.5% glycine in PBS) for 30 min
at room temperature, and stained for Baf and Crx. The double staining
was performed by sequentially probing with the following antibodies in
PBS buffer with 1% bovine serum albumin: anti-Myc (1:400; Santa Cruz
Biotechnology), the monoclonal antibody to the expression tag of bBaf,
Alexa 488 goat anti-mouse secondary antibody (1:400; Molecular Probes,
Inc., Eugene, OR), anti-Crx antibody P261 (1:200), and Rhodamine Red goat anti-rabbit secondary antibody (1:1000; Molecular Probes). For
reciprocal double staining, rabbit anti-Baf (1:1000) and mouse anti-Xpress (1:1000), the expression tag of Crx, were used as the
primary antibodies. The coverslips were washed three times with the PBS
buffer containing 1% bovine serum albumin, mounted on Superfrost/Plus
slides (Fisher) with Vectashield mounting medium (Vector Laboratories
Inc., Burlingame, California), and examined using a scanning confocal
microscope (Model 410; Carl Zeiss, Thornwood, NY). The digitized images
were processed and analyzed using Adobe Photoshop version 6.0.
Preparation of Ocular Tissue Sections and
Immunohistochemistry--
Eyes collected from adult and postnatal day
0-5 (P0-P5) albino mice BALB/c were fixed in 4%
paraformaldehyde/PBS, pH 7.4, overnight and processed for routine
paraffin histology. Eyes were embedded for sagittal sectioning and cut
on a rotary microtome at 5 µm. Sections were mounted on
Superfrost/Plus slides and allowed to dry overnight before staining.
For embryonic eyes, the whole embryos of embryonic days 12.5, 14.5, and
18.5 were dissected from sacrificed timely pregnant BALB/c mothers,
treated as described above, embedded with the top of the head down in
the block, and serially sectioned. Sections containing the eyes were
saved for future staining. The pineal gland dissected from the brain of an adult Lewis rat (Charles River Laboratories, Wilmington, MA) was
processed in a similar manner. For immunostaining of Baf, sections were
deparaffinized in xylene and 100% alcohol, blocked with 3% hydrogen
peroxide (H2O2) in methanol for 30 min to
remove endogenous peroxidase, and rehydrated in 95 and 70% alcohol and distilled water, followed by heat-induced epitope retrieval.
Heat-induced epitope retrieval was performed in citrate buffer (pH 6.0)
using a Decloaking Chamber (Biocare Medical, Walnut Creek, CA).
Heat-induced epitope retrieval-treated slides were rinsed in distilled
water and placed in PBS, blocked with 20% normal donkey serum for 30 min, and probed with rabbit anti-Baf (1:1000) or its control serum overnight at 4 °C. The Baf labeling was detected using Vectastain Elite Rabbit IgG ABC kit (Vector Laboratories Inc., Burlingame, CA) and
DAB peroxidase substrate (Sigma) according to the manufacturer's instructions. After color development, the slides were washed in PBS,
dehydrated in alcohol, cleared in xylene, and coverslipped with a
resinous mounting medium (Permount; Fisher). The results were examined
by light microscopy (Olympus BH-2) and photographed using a Spot SP100
Cooled Color Digital Camera (Diagnostic Instruments Inc.).
Immunostaining of Crx was performed similarly as described above except
using the Crx antibody P261 at a 1:800 dilution and hematoxylin (Harris
Formula; Surglpath Medical Industries, Inc.) for counterstaining of
nuclei of retinal sections (light blue). The
retinal sections used for Crx staining were made from an eye of the
adult C57BL/6 mouse (wild type) or a mutant mouse homozygous for a null
allele of Crx (Crx/) at 14 days
of age (kindly provided by Drs. Takahisa Furukawa and Connie Cepko).
Bioinformatics Analysis of the Human BAF Gene--
Similarity
searches were performed by using BLAST software (37) to scan the human
genome data base from NCBI (available on the World Wide Web at
www.ncbi.nlm.nih.gov) and from Celera Corp. (available on the World
Wide Web at www.celera.com). Matching output sequences from the BLAST
analyses as well as STS marker sequences were positioned on the genome
sequence using the resources provided by the NCBI human genome Web site
(www.ncbi.nlm.nih.gov/genome/guide/human/). The software ClustalW (38)
(available on the World Wide Web at www2.ebi.ac.uk/clustalw) was used
for both sequence alignment and for computing phylogenic relationships
among sequences, which were graphically elaborated by the program
DRAWTREE (39) (available on the World Wide Web at
bioweb.pasteur.fr/seqanal/interfaces/drawtree-simple.html).
Screening of Patients with Photoreceptor Degeneration for
Mutations in BAF--
Most of the patients were recruited from the
Berman-Gund Laboratory for the Study of Retinal Degenerations, where
they were diagnosed through eye examinations including funduscopy and
electroretinography. Patients with Bardet-Biedl syndrome, Leber
congenital amaurosis, and retinitis pigmentosa were included in this
study. Inheritance patterns were inferred from family history. Informed
consent was obtained from all participants, in accordance with the
tenets of the Declaration of Helsinki, before they donated 10-30 ml of blood for this research. DNA was purified from peripheral blood leukocytes by standard procedures. Blood samples were also obtained from control individuals with no visual symptoms of and no known blood
relatives with hereditary retinal degeneration.
The human BAF coding regions were PCR-amplified from the
leukocyte DNA samples from patients and from controls using the
following primers: exon 2, primer 4546 (5'-GCCCTAATCTGCCTTTTTTTTGGG-3') and 4547 (5'-GCACTAGGTACACGCAGCCACCCC-3'); exon 3, 4548 (5'-AGCAGCACGCTCCTTCCTTTTCCC-3') and 4549 (5'-TGGATGAGGGCTGGGGATTGAGAG-3'), according to previously published methods (40). 33P-Radiolabeled PCR products were
scanned for mutations by single-stranded conformation polymorphism
analysis in two nondenaturing acrylamide gels, one with and one without
10% glycerol. Samples with an abnormal single-stranded conformation
polymorphism analysis pattern were amplified a second time and analyzed
by direct sequencing in both sense and antisense directions.
| |
RESULTS |
Baf Interacts with Crx in Yeast Two-hybrid Assays--
To identify
Crx-interacting proteins, the yeast two-hybrid assay was employed using
a set of bait constructs that would produce fusion proteins containing
wild-type bovine Crx with or without N- and C-terminal deletions (13).
Upon transformation to the reporter yeast strain Y190, only the
homeodomain (Crx-HD) bait showed no autoactivation of the
His3 and lacZ reporter genes (data not shown),
suggesting that other regions of Crx, particularly the C-terminal
region, contain a transactivation domain(s). This is consistent with
the results of deletion analysis of Crx in mammalian cells (13,
41).
Crx-HD was subsequently used as bait for screening a bovine retinal
cDNA library (34). After screening 1.7 × 106
co-transformed yeast cells, eight clones grew on a His-minus medium
with 15 mM 3-AT (a competitive inhibitor of His3) and
produced a blue color on X-gal filters. Sequence analysis revealed that one of the dual positive clones encoded phosducin (Phd), a
photoreceptor protein already known to interact with Crx (26). Two
other clones that were positive in both reporter assays harbored an
identical cDNA insert of 0.6 kb with a predicted open reading frame
of 89 amino acids (accession number AF529228) plus an additional 28 amino acids derived from the 5'-untranslated region at its N terminus
fused in-frame with the Gal4 activation domain. Sequence comparison
showed that the open reading frame encoded a protein with a predicted
amino acid sequence identical to that of the human
barrier-to-autointegration factor BAF (42) (Fig.
1). Baf is highly conserved among
different mammalian species. At the amino acid level, bovine Baf shares
100, 97, and 97% homology, respectively, with its orthologs in humans,
mice, and rats. At the nucleotide level, bovine Baf shares
93% homology with human BAF in the coding region (data not
shown).
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Comparison of amino acid sequences of Baf
from cows, humans, mice, and rats. The predicted amino acid
sequence from an open reading frame (89 amino acids) of the bovine
cDNA clone described here is shown at the top. Residues
that are identical in all four species are shaded in
dark gray, whereas conservative changes are
shaded in light gray. A
nonconservative change is indicated by a white
box.
|
|
To test the specificity of Baf-Crx-HD interaction, Y190 cells were
co-transformed with the bBaf prey plasmid and each of the pAS bait
plasmids, Crx-HD, the empty vector, and Snf1 (unrelated bait as a
negative control), respectively. Double transformants were analyzed by
the dual reporter assays. Fig.
2A shows that expression of
the His3 and lacZ reporter gene was activated
only in yeast cells harboring both the Crx-HD bait and Baf prey or the
positive control partners Snf1 and Snf4 (35). The presence of either
the Snf1 or empty bait vector with the Baf prey did not yield a
positive result under identical conditions, suggesting that Baf does
not interact with Gal4-dbd and that the interaction of Baf and Crx-HD
is specific.
View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2.
Baf interacts with Crx in yeast two-hybrid
assays. A, using Crx-HD as bait; B, using
bovine Baf as bait. The indicated bait and prey vectors were
cotransformed into the yeast Y190 cells and analyzed for the
reporter activity of lacZ and His3 using
 -galactosidase (-gal) and 3-AT tests as described
under "Experimental Procedures." A positive interaction is scored
by growing on 3-AT medium and producing a blue
color on an X-gal filter.
|
|
To further establish the specificity of a Baf-Crx interaction, an
insert swap between the bait and prey plasmid was performed; the coding
region of bBaf was cloned in frame with Gal4-dbd in the pAS2
bait vector, whereas the full coding region of bovine Crx
(bCrx) was cloned in-frame with the Gal4 activation domain in the pACTII prey vector. The resulting bait and prey plasmids were
co-transformed into Y190 cells and analyzed using the dual reporter
assay. Fig. 2B shows that a positive interaction was detected with the Baf bait and Crx prey but not with the empty prey
vector, demonstrating that Baf interacts with the full-length Crx in yeast.
Baf Binds Directly to Crx in Vitro--
To test whether Baf binds
to Crx directly, bacterially expressed His6-tagged Baf
(Baf-His), His6 alone control (His), GST-tagged Crx-HD
(Crx-HD-GST), and GST alone control proteins were purified using
Ni2+-NTA and glutathione-Sepharose beads, respectively, and
used for pull-down assays. Purified Baf-His shows a doublet on an
SDS-PAGE gel with an apparent molecular mass of 10 kDa, which is
consistent with the calculated molecular mass for the fusion protein
with a His6 tag (3 kDa for the His tag alone) (Fig.
3A, lane
3 versus lane 2). It is
unclear if the doublet resulted from degradation or contamination of
other proteins. Fig. 3B shows that the Baf-His protein was
pulled down by the Crx-HD-GST beads but not the GST control beads
(lane 2 versus lane
1). In a reciprocal experiment, the Crx-HD-GST protein was
pulled down by the Baf-His beads but not the His control beads (Fig.
3C, lane 2 versus
lane 1). These results suggest that Baf interacts
with the Crx homeodomain directly.
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3.
Baf directly interacts with Crx in
vitro via the homeodomain. A, SDS-PAGE (12%
gel) demonstrating the purification of bacterially expressed bovine
Baf-His and His proteins. A proximal 100 ng of the indicated protein
and a prestained protein marker M (New England Biolabs) were loaded on
the gel and stained with Coomassie Blue. The apparent molecular mass of
each marker band in kilodaltons is shown on the left.
B, GST pull-down assays. 50 ng of the purified Baf-His
protein was incubated with glutathione-Sepharose beads coupled with
either the GST (lane 1) or Crx-HD-GST
(lane 2) protein. After stringent washes, bound
proteins were eluted from the beads, resolved by SDS-PAGE (12% gel),
and immunoblotted with an anti-Baf antibody. The presence of GST and
Crx-HD-GST protein on the beads was confirmed by SDS-PAGE and
immunoblots with an anti-GST antibody (Sigma) (lanes
3 and 4). C, His pull-down assays. 100 ng of the Crx-HD-GST protein was incubated with Ni2+-NTA
beads (Qiagen) coupled with either the His (lane
1) or Baf-His (lane 2) protein.
Proteins bound to the beads were treated as described for B
and analyzed with the anti-GST antibody. The presence of the purified
His and Baf-His protein on the beads was confirmed by SDS-PAGE and
immunoblots with an anti-polyhistidine antibody (Sigma)
(lanes 3 and 4). D,
co-immunoprecipitation (co-ip) assays using in
vitro translated Baf and Crx proteins and a Crx antibody. A
Myc-tagged bovine Baf, the full-length bovine Crx (Crx-(1-299)), and
its N-terminal deletion (Crx-(111-299)) were generated using a TnT T7
Quick Coupled Transcription/Translation kit (Promega) in the presence
(marked by an asterisk) and absence of
[35S]methionine. The indicated translation products were
used for co-immunoprecipitation by the anti-Crx antibody P261
(lanes 1-4). Lanes 5-7,
the input controls without co-immunoprecipitation (one-twentieth of the
volume used for co-immunoprecipitation). E,
co-immunoprecipitation of Baf with Chx10. Co-ip was performed using
in vitro translated Baf (35S-labeled;
asterisk), Chx10, and a Chx10 antibody. Lanes
1 and 2, are negative controls without the
addition of the Chx10 antibody or protein, respectively. F,
Baf does not co-immunoprecipitate with Nrl. The indicated
35S-labeled (asterisk) and unlabeled in
vitro translated proteins were analyzed by co-immunoprecipitation
assays with the anti-Nrl antibody (lanes 1-3).
Lanes 4-6, the input controls (one-twentieth of
the volume used for co-immunoprecipitation).
|
|
To determine whether other regions of Crx could mediate Crx-Baf
interaction, co-immunoprecipitation assays with in vitro
translated proteins were performed using a 35S-labeled-Baf,
a polyclonal antibody P261 against Crx (28), and either a full-length
Crx or its deletion mutant Crx-(111-299), where the homeodomain and
its flanking 38 amino acids at the N terminus are removed (13). Fig.
3D shows that Baf was co-immunoprecipitated with the
full-length Crx (Crx-(1-299)) by the Crx antibody (lane 1), which did not occur in the absence of either the Crx
protein (lane 2) or the Crx antibody
(lane 3). This result, consistent with that
obtained from the yeast two-hybrid assays, provides further evidence
that Baf interacts directly with the full-length Crx. In contrast, Baf
was not co-immunoprecipitated with the N-terminal truncation mutant
Crx-(111-299), (lane 4), suggesting that the homeodomain (plus its N-terminal flanking sequence) is necessary for
Crx to bind to Baf.
Baf Binds to Several Homeodomain Transcription Factors, but Not the
bZIP Protein Nrl--
Since the homeodomain of Crx is highly
homologous to that of Otx1 (88%) and Otx2 (86%) (5), it seemed likely
that Baf could interact with Otx1 or Otx2. To test this possibility, we
performed co-immunoprecipitation assays with in vitro
translated Otx1 or Otx2 (35S-labeled) and a Myc-tagged Baf
using an anti-Myc antibody. As predicted, both Otx1 and Otx2 can be
co-immunoprecipitated with Baf (data not shown), suggesting that Baf
can interact directly with Otx1 and Otx2.
Since the homeodomain of Otd/Otx family belongs to the K50
subtype of the paired-like class, we decided to evaluate whether Baf
could interact with Chx10, a Q50 subtype of the paired-like homeodomain protein expressed in the retina (43). Fig. 3E
shows that Baf was co-immunoprecipitated with Chx10 by an antiserum to
Chx10, suggesting that Baf also interacts directly with Chx10. In
addition, in vitro co-immunoprecipitation assays also
detected an interaction of Baf with Pax-6, an S50 subtype
of the paired-like homeodomain (44) (data not shown). Thus, Baf
interacts with all three subtypes of paired-like homeodomain proteins.
To determine the specificity of Baf-homeodomain interactions, we
searched for evidence that Baf might bind to the Maf family neural
retina leucine zipper protein Nrl (7), which is specifically expressed
in the rod photoreceptor cells (12). Nrl contains a basic motif-leucine
zipper domain that is thought to mediate its binding to DNA (45) and
interaction with Crx (15). As shown in Fig. 3F, an anti-Nrl
antibody failed to bring down Baf with Nrl in a co-immunoprecipitation
assay (lane 3), although it co-immunoprecipitated
Crx with Nrl in a control experiment under similar conditions
(lane 1). Furthermore, no interaction between Baf
and Nrl was detected using a yeast two-hybrid assay (data not shown).
Baf and Crx Are Co-localized in the Nucleus and Can Be
Co-immunoprecipitated from a Retinal Extract--
To gain insights
into a possible relevance of a Baf-Crx interaction in vivo,
the expression pattern of the Baf protein was examined using
immunoblots of whole cell extracts probed with a polyclonal antibody
against the human BAF. Fig. 4A
shows that the Baf antibody recognized a doublet with an apparent
molecular mass of ~7 kDa in protein extracts made from all tissues
(lanes 1-11) and cell lines (including those
with retinal and nonretinal origins; lanes
12-15) examined, including retina, retinal pigment epithelium, and iris of the eye. The size of the detected protein is
consistent with the predicted molecular weight of Baf and with the
reported molecular weight of purified human BAF (42). Although the
doublet can be observed in all of the lanes, it is more
apparent with cultured cell lines that contain a mixture of cells in
various stages of the cell cycle. It is unclear whether this doublet
results from a post-translational modification of Baf or protein
degradation. The ubiquitous expression of Baf suggests that it may be
essential for cellular function in both dividing and nondividing
(terminally differentiated) cells. To examine Baf expression in the
retina, immunohistochemical studies were performed using the Baf
antibody and retinal sections from adult and embryonic mouse eyes. As
shown in Fig. 4B, the anti-Baf antiserum stained all of the
nuclear layers of the adult retina. A control serum (preimmune) did not produce any signal under the same conditions, suggesting that the
staining by the anti-Baf antiserum is specific. In contrast, the
anti-Crx antibody P261 stained intensely the outer nuclear layer
(ONL) of the wild-type mouse retina, where the photoreceptor nuclei are localized, and also stained lightly the outer part of the
inner nuclear layer (INL), where bipolar cells reside (Fig. 4C). For a negative control, P261 did not stain the retina
of a mutant mouse (Crx/) that is homozygous
for a null allele of Crx (8), suggesting that the staining
observed for the wild-type retina by P261 is specific to Crx.
Furthermore, double labeling with P261 and a Chx10 antibody confirmed
that those inner nuclear layer cells positive for Crx expression are
actually bipolar cells (data not shown). These results demonstrated
that Crx and Baf are co-expressed in the nuclei of photoreceptor cells
as well as in bipolar cells of the retina. The Baf immunoreactivity was
also found in the nuclei of the developing mouse retina at all of the
developmental stages tested (as early as embryonic day 12.5; data not
shown), suggesting that Baf may play an important role in the
development and maintenance of the retina by acting in the nucleus. In
addition, to examine whether Baf is co-expressed with Crx by
pinealocytes in the pineal gland, we performed immunostaining of Baf
and Crx using paraffin sections from a rat pineal gland. As shown in
Fig. 4D, both the anti-Baf antiserum and anti-Crx antibody
stained pinealocytes intensely. The staining patterns are very similar and appear to be nuclear, suggesting that Baf may also interact with
Crx in the pineal gland and play a role in pineal function.
View larger version (74K):
[in this window]
[in a new window]
|
Fig. 4.
Baf is ubiquitously expressed, including
neurons in the retina and pinealocytes of the pineal gland that express
Crx. A, immunoblot analysis. 8 µl of whole cell
extracts were separated by SDS-PAGE (15% gel), transferred to
polyvinylidene difluoride membranes (Bio-Rad), and probed with a rabbit
polyclonal antibody against Baf at a 1:1000 dilution. Lanes
1-8, extracts made from the indicated tissues of a rat
(Brown Norway; Charles River Laboratories). Lanes
9-11, extracts made from the indicated tissues dissected
from a bovine eye; RPE, retina pigment epithelium.
Lanes 12-15, extracts made from cultured cell
lines: HEK293 (human embryonic kidney; ATCC), Y79 (human
retinoblastoma; ATCC), Weri-Rb-1 (human retinoblastoma; ATCC), and 661w
(SV40 T-antigen-transformed 661 mouse photoreceptor cell line) (71).
B, immunostaining of Baf on retinal sections from adult mice
(BALB/c) using the anti-Baf or its control (preimmune) serum. The cell
layer labels are as follows. RPE, retinal pigment
epithelium; OS, outer segments; ONL, outer
nuclear layer; INL, inner nuclear layer; GCL,
ganglion cell layer. C, immunostaining of Crx
(brown color) on retinal sections from a
wild-type (C57BL/6) and a homozygous Crx knock-out mouse
(Crx/) using anti-Crx antibody P261. The sections were
counterstained with hematoxylin to show nuclei (light
blue). D, immunostaining of Baf (D1, D2) and Crx
(D4, D5), respectively, on pineal gland sections from an adult Lewis
rat. D3 received the control serum for Baf (preimmune), whereas D6 did
not receive any primary antibody. D2, D3, D5, and D6 represent a higher
magnification (×400) of D1 and D4 (×100).
|
|
Since both anti-Baf and anti-Crx antibodies were generated in rabbits,
double staining of Crx and Baf in the retinal or pineal sections is
technically difficult to perform. To confirm that Crx and Baf are
co-localized in the nucleus of cells that express both proteins, HEK293
cells were co-transfected with constructs overexpressing an
Xpress-tagged Crx and a Myc-tagged Baf. The subcellular localization of
the two recombinant proteins in co-transfected cells was examined using
immunocytochemistry with double labeling and confocal microscopy. Two
pairs of rabbit/mouse antibodies to the recombinant Crx and Baf were
used for double labeling, either rabbit anti-Crx/mouse anti-Myc (Baf)
or rabbit anti-Baf/mouse anti-Xpress (Crx). Double staining with both
pairs of antibodies yielded very similar results. As shown in Fig.
5A, Crx (red) and Baf (green) are co-localized in the nucleus of the
co-transfected cells, although a small amount of Baf is also seen in
the cytoplasm.
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Baf and Crx are co-localized in the nucleus
of co-transfected 293 cells and can be co-immunoprecipitated from a
bovine retinal extract. A, immunostaining of Crx
(red) and Baf (green) in HEK293 cells
co-transfected with their expression vectors using antibodies to Xpress
(the expression tag of Crx) and Baf. B,
co-immunoprecipitation (Co-ip) of Baf with Crx from a
retinal extract. 200 µl of whole cell extracts made from bovine
retina (Ret) and spleen (Spl) were incubated with
the anti-Crx antibody P261, precipitated by Protein A beads, and
immunoblotted with the anti-Baf antibody (lanes 1 and 2). Lanes 3-6, input controls
(without immunoprecipitation) loaded with 10 µl of the indicated
extracts and immunoblotted with either anti-Baf (lanes
3 and 4) or anti-Crx P261 antibody
(lanes 5 and 6).
|
|
To further examine Crx-Baf interaction, co-immunoprecipitation
assays were performed using the Crx antibody P261 and protein extracts
made from the bovine retina and other tissues. The presence of Baf in
the immunoprecipitated protein complex was revealed by immunoblots with
the anti-Baf antibody. Fig. 5B shows that the Crx antibody
co-immunoprecipitated Baf from the retinal extract (lane
1) but not from the spleen extract (lane
2) that does not contain Crx (demonstrated by the immunoblot
with anti-Crx, lane 6), despite a higher amount
of Baf detected in the spleen than in the retinal extract
(lane 4 versus lane
3). These results, combined with the co-localization of both
proteins in the nucleus of co-transfected cells and retinal
photoreceptor cells, suggest that Baf and Crx interact in
vivo.
Baf Represses the Transactivation Activity of Crx--
To address
the physiological relevance of a Baf-Crx interaction, we examined
whether overexpression of Baf has any effect on the transactivation
activity of Crx using transient transfection assays. It has been
established that Crx is able to trans-activate the
expression of a rhodopsin promoter-luciferase reporter (pBR130-luc), either on its own or in combination with Nrl, in transiently
transfected HEK293 cells (5). We then included a mammalian vector
expressing the Baf, in various ratios to the vector(s) expressing Crx,
Nrl, and Crx plus Nrl, respectively, in the transactivation assays. The
resulting activities of transactivation were compared with those
obtained from samples receiving the empty vector (100%). Consistent
with the previous reports (5), the rhodopsin reporter alone had little
if any activity in HEK293 cells (data not shown). The promoter activity
was increased by 3-6-fold when co-transfected with either
Crx or Nrl alone and by 60-80-fold with both together. In contrast,
co-transfection of the Baf expression vector alone with the reporter
did not produce any detectable effect on rhodopsin promoter activity
(data not shown), suggesting that Baf, on its own, does not regulate
rhodopsin promoter activity. However, when both the Crx and Baf
expression vectors were included in the co-transfection assays, Baf
significantly decreased the Crx-dependent transactivation activity in a concentration-dependent manner, ranging from
34% (p = 0.00025) at a Baf/Crx
ratio of 0.5:1 to 60% (p = 5.4 × 107) at a ratio of 4:1 (Fig.
6A). In contrast, Baf did not
significantly (p > 0.05) repress the
Nrl-dependent activity under similar conditions, even at
the higher Baf/Nrl ratio tested (4:1) (Fig.
6A). To examine whether the repression effect of Baf is
dependent on the Crx homeodomain, we used a fusion protein construct,
Gal4dbd-Crx-(111-299), lacking the homeodomain in transfection assays.
This construct can transactivate a Gal4-responsive promoter-luciferase
reporter in 293 cells (13). The addition of increasing amounts of the
Baf expression vector with this Gal4dbd-Crx fusion construct did not
repress Crx-mediated transactivation (data not shown), suggesting that
the homeodomain is needed for Baf to repress Crx. Furthermore, Baf
failed to show any effect on transactivation activity of Gal4-VP16 for
the Gal4-responsive promoter-luciferase reporter or on the activity of
c-Jun and c-Fos for a collagenase promoter-luciferase reporter (data
not shown). These results suggest that Baf is not a general repressor
of transcription and that its repression effect on Crx requires an
interaction with the homeodomain. Consistent with this, Baf also
decreased the synergistic activity of Crx plus Nrl in a
concentration-dependent manner, ranging from 26%
(p = 0.025) at a Baf/activator ratio of 1:1 to 64%
(p = 0.00028) at a ratio of 4:1 (Fig.
6A).
View larger version (38K):
[in this window]
[in a new window]
|
Fig. 6.
Baf binds to Crx-DNA complexes and represses
Crx-mediated transactivation of the rhodopsin promoter in HEK293
cells. A, Baf represses the ability of Crx, but not
Nrl, to transactivate a rhodopsin-luciferase reporter. HEK293 cells
were co-transfected with a total of 3 µg of DNA, including 2 µg of
pBR130-luc containing the bovine rhodopsin promoter ( 130 to +70 bp)
fused to a luciferase reporter (36); an internal control plasmid
pRL-CMV (Promega) for transfection efficiency; 100 ng of expression
vector carrying the coding region of the transcription activator Crx
(bCrx-pcDNA3.1/HisC, solid black),
Nrl (pMT-hNRL, horizontal lines), or
Crx plus Nrl (hatched pattern); and the indicated
amount (presented as a ratio relative to the total amount of the
activator DNA with 0:1 for the samples without Baf) of the Baf
expression vector bBaf-pcDNA3.1(+)/myc. Transactivation
activity (-fold activation) for each transfection was calculated by
comparing the relative luciferase activity (after normalized with the
internal control) with that obtained from the pBR130-luc reporter alone
(0-fold). The data are presented as mean values of percentage of
transactivating activity relative to the values obtained from those
samples without the Baf vector (100%). The error
bars represent the S.E. from four independent experiments
(n = 4). B, Baf binds to Crx-HD-DNA
complexes. EMSA reactions were carried out using a Crx-HD peptide and
three 32P-labeled oligomer probes harboring Crx target
sites from the bovine rhodopsin promoter, BAT-1 (lanes
1-8), Ret-4 (lanes 9-16), and Ret-1
(lanes 17-24) (5). The indicated amount (µl)
of the Baf-His (lanes 3-5, 11-13,
and 19-21) or the His control (lanes
6-8, 14-16, and 22-24) protein was
preincubated with the Crx-HD peptide in the reaction buffer for 10 min
on ice before mixing with a 1 nM concentration of the
indicated probes. The reactions were then incubated on ice for an
additional 30 min and resolved by native PAGE (5% gel). The
concentration of each purified protein was about 40 ng/µl for Baf-His
and His alone and 10 ng/µl for Crx-HD. C, Baf binds to a
native Crx-DNA complex. EMSAs were carried out using 5.0 µg of a
bovine retinal nuclear extract and 0.5 nM
32P-labeled Ret-4 oligomer as the probe. The presence of
protein-DNA complexes is indicated by the arrows, marked Crx and U for the binding activity of Crx
and a ubiquitously expressed protein, respectively, as demonstrated
previously (5). The indicated amount (in µl) of the Baf-His
(lanes 4-7) or the His control (lane
8) protein was preincubated with the retinal nuclear extract
in the reaction buffer for 10 min on ice prior to the addition of the
probe. A supershift containing the Baf-Crx-DNA complex observed with
lanes 4-7 is marked by a double
arrow. Lane 1 did not receive any
protein, whereas lanes 2 and 3 received only the Baf-His protein or the retinal nuclear extract,
respectively.
|
|
Baf Does Not Abolish the DNA Binding Activity of Crx--
Since
overexpression of Baf did not show any effect on either the basal
activity of the rhodopsin promoter or the transactivating activity of
Nrl and Gal4-VP16, the mechanism by which Baf represses Crx is likely
to be specific to Crx. One such mechanism could be to block the ability
of Crx to bind to its targets, especially considering that Baf
interacts with the Crx homeodomain. We decided to test this possibility
using EMSAs with recombinant Crx and Baf proteins. Due to
technical difficulties in the expression and purification of a
full-length recombinant Crx from E. coli, we carried out
EMSAs using the purified Crx-HD peptides and DNA probes carrying three
Crx target sites (BAT-1, Ret-1, and Ret-4) of the rhodopsin promoter,
in the absence and presence of a His6-tagged recombinant
Baf protein (Baf-His) (Fig. 6B). In the absence of Baf,
Crx-HD produced a band shift(s) with each probe, as reported previously
(13) (lanes 2, 10, and 18).
However, preincubation of Crx-HD with increasing amounts of recombinant
Baf resulted in supershifts of Crx-HD/DNA bands (lanes
3-5 versus lane 2;
lanes 11-13 versus lane
10; lanes 19-21 versus
lane 18). The supershifts did not occur with the
His control under the same conditions (lanes 6-8, 14-16, and 22-24). In
addition, the Baf protein alone, even at the highest concentration
tested, did not produce a visible shift with each of the three probes
(lanes 1, 9, and 17). To
test whether this phenomenon could occur with a native Crx in the
retina, supershift EMSAs were carried out using a bovine retinal
extract and the Ret-4 probe in the presence of increasing amounts of
the recombinant Baf. Fig. 6C shows that, in the absence of
the recombinant Baf (lane 3), the retinal nuclear
extract produced two shifted bands with the Ret-4 probe as demonstrated
previously (5). The major band (labeled Crx) represents the
binding activity of Crx, whereas the minor band (labeled U)
represents the binding activity of a ubiquitously expressed protein.
Preincubation of the retinal nuclear extract with the increasing
amounts of the recombinant Baf (Baf-His) supershifted the Crx band to a
higher position(s) (labeled Baf/Crx) without affecting the U
band in a concentration-dependent manner (lanes
4-7 versus lane 3). This supershift was not observed with the His-negative control
(lane 8). Together, these results suggest that
interacting with Baf does not abolish the ability of Crx (or Crx-HD) to
bind to its DNA targets in vitro.
E80A, a Homeodomain Mutation of Crx Associated with Autosomal
Dominant Cone-Rod Dystrophy, Interferes with Both the Physical and
Functional Interaction of Crx and Baf--
Since Baf interacts with
the homeodomain of Crx, it is possible that mutations in the Crx
homeodomain could have some effect on Crx-Baf interaction. To test this
possibility, we carried out in vitro protein-protein
interaction assays using recombinant Baf and five mutant forms of human
CRX carrying homeodomain mutations identified from photoreceptor
degenerative diseases. Among the five CRX mutations, four (R41Q, R41W,
E80A, and R90W) are likely to be pathogenic, since they co-segregate
with either autosomal dominant cone-rod dystrophy (CORD2) or Leber
congenital amaurosis, whereas one (A56T) is of uncertain pathogenicity
because it has not been shown to co-segregate with a disease (18, 21,
23). Previous biochemical analysis of these mutants has demonstrated that A56T does not cause any detectable defects in CRX function and
therefore is likely to be a non-disease-causing variant (13). In
contrast, the four mutations co-segregating with disease cause abnormalities in the DNA binding and/or transactivation activity of CRX
(13). Interestingly, the E80A mutation leads to a "hyperactive" CRX
with a transactivation activity 2-fold greater than wild-type CRX (13).
To test whether these mutant forms of CRX could interact with Baf in a
similar manner as wild-type CRX, in vitro
co-immunoprecipitation assays were carried out using a Myc-tagged Baf,
anti-Myc antibody, and 35S-labeled CRX proteins carrying
each of the five homeodomain mutations. Fig.
7A demonstrates that the E80A
mutant had a lower binding affinity to Baf than the wild-type CRX and
the non-disease-causing variant A56T (lane 3 versus lanes 1 and 2).
Further analysis of three independent experiments using PhosphorImager
assays showed that the E80A mutation reduces the ability of CRX to bind
to Baf by 50% (data not shown). None of the other three CRX mutants
(R41Q, R41W, and R90W) had an affinity for Baf that was significantly different from wild-type CRX (data not shown). To confirm these results, supershift EMSAs with two different probes were carried out
using recombinant Baf and purified Crx-HD wild-type and E80A peptides.
As shown in Fig. 7B, the minimal amount of the Baf protein required for supershifting Crx-HD is higher for the E80A mutant than
that for the wild-type Crx (lanes 4-6
versus lanes 1-3 and lanes
10-12 versus lanes 7-9),
supporting a reduction in the affinity of Crx for Baf by the E80A
mutation. To see whether this reduction in the physical interaction of
Crx and Baf caused by the E80A mutation would affect the functional
interaction, we also tested whether the E80A mutation has any effect on
the sensitivity of CRX to Baf-mediated repression using transient
transfection assays in HEK293 cells. Consistent with a previous study
(13), in the absence of a recombinant Baf, E80A acted as a
"hyperactive" mutant, showing a 100% increase in transactivation
activity compared with the wild-type CRX (data not shown). A56T, in
contrast, showed the same activity as the wild-type CRX (data not
shown). An increasing amount of the Baf expression vector was then
added to these co-transfection assays. The transactivation activities
obtained from these experiments, presented as percentages of those
obtained in the absence of Baf (100%), are shown in Fig.
7C. The results indicated that the E80A mutant is less
sensitive to Baf-mediated repression than the wild-type CRX and the
A56T mutant. The sensitivity difference was significant when the
Baf/CRX DNA ratio was less than 2:1
(p = 0.0071 at the ratio of 0.5:1, and 0.004 at the
ratio of 1:1, respectively, for wild type versus E80A). As
an example, at a ratio of 0.5:1, Baf did not significantly repress the
trans-activating activity of E80A (p = 0.30;
comparing the samples with Baf versus without Baf), although
it did significantly repress both the wild-type CRX and A56T by about
34% under the same conditions. These results, combined with the
physical interaction data, suggest that the E80A mutation interferes
with both the physical and functional interaction of Crx and Baf.
View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7.
E80A, a homeodomain mutation of CRX, reduces
the strength of CRX-Baf interaction. A, E80A interferes
with the physical interaction of CRX and Baf: co-immunoprecipitation
assays. In vitro translated, 35S-labeled human
CRX (wild type (WT); lane 1) or its
mutants E80A (lane 3) and A56T (lane 2) were
incubated with a Myc-tagged Baf and immunoprecipitated using an
anti-Myc antibody (Santa Cruz Biotechnology, Inc.). Lanes
4-6 are input controls (one-twentieth volume used for
co-immunoprecipitation) for in vitro translated CRX
proteins. B, E80A interferes with the physical interaction
of Crx and Baf: supershift EMSAs. Equal amounts of the wild-type and
mutant (E80A) Crx-HD peptide were incubated with the Ret-4 and BAT-1
probe in the presence of the indicated amount of the Baf-His protein.
C, E80A interferes with the functional interaction of CRX
and Baf: co-transfection assays. The potency of Baf to repress the
wild-type and two mutant CRXs (E80A and A56T) was measured by transient
co-transfection assays in HEK293 cells. 100 ng of the hCRX
expression vector and an increasing amount of the Baf
expression vector (presented as a ratio of
Baf/CRX) were included in each co-transfection
assay with the rhodopsin promoter-luciferase construct pBR130-luc as
the reporter. The results were analyzed as described in the legend to
Fig. 6A and presented as percentage of transactivation
activity relative to those of samples without the Baf vector
(Baf/CRX = 0:1, 100%). The error
bars represent S.E. from four independent experiments
(n = 4).
|
|
The Human BAF Gene Tentatively Maps to the Chromosomal Region
Containing the Bardet-Biedl Syndrome-1 (BBS1) Locus--
The finding
that Baf forms a physical and functional interaction with Crx and that
a Crx mutation reduces this interaction raised the possibility that
mutations of Baf itself might cause a disease of the retina. We
therefore determined the genomic structure and chromosomal location of
the human BAF gene. A BLAST search of the human genome
databases from the NCBI and Celera Corp. revealed the presence of six
human sequences that were similar to the sequence of the bovine
cDNA fragment obtained from the yeast two-hybrid experiments (Fig.
8B). Of these six sequences,
two were possibly ancient pseudogenes with truncated reading frames and
a low level of homology with the bovine probe (sequences B4 and B5 in
Fig. 8B), and three carried longer, intronless reading
frames producing higher homology scores with the bovine open reading
frame and were considered more recent pseudogenes (B1, B2, and B6). One sequence (B3) corresponded to a putative three-exon gene encoding a
predicted protein that is 100% identical to bovine Baf
(Fig. 8A); we interpreted this to be the human
BAF homolog. The chromosomal structure of the human
BAF gene was determined by comparison between its mRNA
(GenBankTM accession number NM_003860) and a clone
containing a human DNA region from chromosome 11q13
(GenBankTM accession number AP001191). In particular, the
human BAF locus appeared to lie within chromosome 11q13, in
a region containing the gene responsible for BBS1 (OMIM: 209901) (46,
47). However, the precise location of sequence AP001191 differs among
the different human genome databases. Besides 11q13 (Celera; available on the World Wide Web at www.celera.com), some assemblies of the human
genome show, for example, that this sequence may be found in contigs
that are thought to be derived from chromosome 2q (Celera; Ensembl;
available on the World Wide Web at www.ensembl.org) or 14q (NCBI;
available on the World Wide Web at www.ncbi.nlm.nih.gov).
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 8.
Analysis of the human BAF
gene(s). A, chromosomal structure of the human
BAF gene. The coding region is hatched.
B, human paralogues of the bovine Baf protein. B
(i), The actual human BAF, encoded by the gene depicted in
A, corresponds to sequence B3 and is 100% identical to the
predicted bovine protein. All other sequences are from intronless open
reading frames, possibly belonging to retropseudogenes. The chromosomal
location of such sequences is indicated in brackets. Stop
codons are indicated by X. B (ii),
phylogenetic tree of the sequences depicted in B
(i) (see "Experimental Procedures").
|
|
BBS1 is a multisystem, autosomal recessive disorder that includes
retinitis pigmentosa as one of its manifestations. Because of the
possible location of BAF within 11q13, we considered
BAF a candidate for BBS1. However, an analysis of leukocyte
DNA from 43 unrelated patients with Bardet-Biedl syndrome did not
reveal any sequence variations in the coding exons or flanking intron sequences. Since ~40% of families with Bardet-Biedl syndrome show linkage to the BBS1 region (46, 48, 49), it is highly likely that we
had cases caused by BBS1 in our set of 43 unrelated patients from
families in which linkage analysis had not been performed. Thus, this
negative result strongly suggests that BAF is not the BBS1
gene. Because of the ambiguity in the chromosomal localization of
BAF and the possibility that BAF mutations might
cause some other form of retinal degeneration whose locus has not yet
been mapped to the BAF locus, we extended our mutation
survey and screened 51 unrelated patients with Leber congenital
am-aurosis and 37 with autosomal recessive retinitis pigmentosa. Again,
no sequence variations were found.
| |
DISCUSSION |
Baf Interacts with Crx and Four Other Paired-like Homeodomain
Transcription Factors--
Using a yeast two-hybrid screen, we
identified Baf as protein that possibly interacts with Crx. Further
evidence supporting a direct interaction of Baf and Crx was provided by
pull-down assays, in vitro co-immunoprecipitation, and
supershift electrophoretic mobility shift assays. We also showed that
recombinant Crx and Baf are co-localized in the nucleus of HEK293 cells
after co-transfection. Both Crx and Baf proteins are present in the
nuclei of the photoreceptors and the bipolar cells of the mature mouse
retina as well as in the pinealocytes of the pineal gland, and the two
proteins can be co-immunoprecipitated from a bovine retinal extract
using a Crx antibody. These results suggest that Baf and Crx interact in vivo. Baf was originally identified as a host cellular
factor that plays a role in preventing retroviral DNA from
autointegration (integrating into the virus' own genome) and in the
formation of preintegration complexes (PICs) during retroviral
infection (42, 50, 51). Baf is evolutionarily conserved from
Caenorhabditis elegans to mammals (52) (Fig. 1),
implicating Baf as an important protein for cellular function. Baf is
reported to interact directly with the LEM
domain3-containing proteins,
including the nuclear lamin-binding proteins LAP2 (53) and emerin (29,
54). Our study demonstrates for the first time that Baf is also able to
bind directly to a paired-like homeodomain transcription factor.
Consistent with this, four other paired-like homeodomain proteins
(Chx10, Pax-6, Otx1, and Otx2) also have the ability to interact with
Baf in vitro. NMR and x-ray crystallography studies
demonstrate that both Baf and the paired-like homeodomain independently
form a higher order -helical structure important for binding to DNA
and for dimerization (52, 55-57). This helical structure may provide a
structural base for Baf interaction with a homeodomain. As an example,
based on the crystal structure of a complex of a paired-like
homeodomain and DNA, it was predicted that residue Glu42,
corresponding to Glu80 of the Crx protein, is an essential
residue for protein-protein interaction (57). Our results support this
prediction, since the E80A mutation affects the ability of Crx-HD to
bind to Baf but does not affect its binding to DNA. It would be
interesting to test whether other classes of the HD and non-HD
transcription factors with helical structures, such as helix-loop-helix
proteins, also interact with Baf.
Baf is believed to serve as a bridging molecule that binds to DNA
randomly in vitro (42, 58) and that co-localizes with chromosomal DNA during interphase and mitosis (53). However, our EMSA
studies with a recombinant Baf did not detect a Baf-DNA complex with
oligomers harboring Crx binding sites. This could be due to our use of
EMSA conditions favoring Crx binding specifically to its targets,
including the use of a low concentration of probes and a relatively low
amount of recombinant Baf. Alternatively, the presence of a His tag at
the N terminus of recombinant Baf might reduce its ability to bind DNA.
Nevertheless, the interaction of Crx-HD and Baf in vitro
appears to occur in the absence or presence of double-stranded DNA,
since a positive interaction was detected by both pull-down assays
(without DNA, as confirmed by UV spectrum and agarose gel analysis of
the protein preparations) and in vitro
co-immunoprecipitation assays in the presence of double-stranded DNA
templates. Further experiments are needed to determine whether the
interaction of Baf and Crx actually occurs in vivo and also
to determine the precise location of Crx-Baf complexes in the
photoreceptor cells.
Baf Might Regulate Gene Expression in Photoreceptors and/or
Pinealocytes--
The previously reported interaction of Baf with
nuclear lamin-associated proteins raises the possibility that Baf might
be indirectly involved in transcriptional regulation, since nuclear lamins have a role in localizing transcription factors or other proteins to the nuclear rim and in repressing transcription (59-61). Our results showing a physical and functional interaction of Baf with
Crx suggest that Baf may indeed be involved in transcriptional regulation, at least in the retina and possibly in the pineal gland.
Baf also physically interacts with four other paired-like homeodomain
transcription factors in vitro. Among the four, Otx2 is
expressed in several types of neurons in the retina, including photoreceptor cells (62), and it can bind to and
trans-activate the promoter of the photoreceptor gene
IRBP in vitro (63, 64). In contrast, Chx10 is expressed in
the bipolar cells of the retina and is involved in the proliferation of
the retinal progenitor cells and in the determination of bipolar cell
fate (43). Chx10 is a repressor of transcription and has been
postulated to prevent presumptive bipolar cells from adapting a
photoreceptor cell fate during development by repressing
Crx-dependent
activity.4 However, since the
homeodomain proteins can interact with multiple functional domains, the
physiological relevance of Baf interaction with these homeodomain
proteins remains to be established.
Increasing evidence suggests that a homeostasis of transcription factor
network consisting of activators and repressors is important for
controlling expression of tissue- or cell type-specific genes. As for
the expression of photoreceptor genes, a high level of Crx expression
may be required for overcoming the repressive effect of Baf, Chx10, or
other repressors. This requirement may explain why photoreceptor genes
are not expressed by bipolar cells although Crx is present at a low
level in these neurons (Fig. 4C). Our studies also suggest
that Baf may participate in transcriptional regulation via its
interaction with cell type-specific proteins, although Baf itself is
ubiquitously expressed. Future studies with conditional knockouts or
overexpression of Baf in specific tissues or cell types are required to
decipher the role of Baf in vivo.
The mechanism by which Baf represses Crx transactivation activity
requires further study. Interaction of Baf with Crx appears not to
abolish the binding of Crx to its target DNA based on our EMSA studies.
However, the possibility cannot be ruled out that in vivo,
Baf may act as a general repressor to block transcription by
nonspecifically binding to chromosomal DNA or by modifying chromatin
structure. Another possibility is that Baf localizes Crx to a specific
cellular compartment that prevents Crx from accessing its DNA targets.
A recombinant Baf demonstrates both nuclear and cytoplasmic
localization in transiently transfected HEK293 cells, whereas Crx
localizes primarily to the nucleus regardless of the presence or
absence of the Baf expression vector. This would argue that Baf has no
effect on the nuclear localization of Crx. However, Crx appears to
co-localize with Baf inside the nucleus during interphase, as
determined by immunocytochemistry and confocal microscopy. Considering
that Baf is associated with nuclear laminae, it is possible that Baf
could repress Crx by localizing the Crx protein to a specific nuclear
compartment. Alternatively, Baf could lead to a change(s) in the
conformation or stability of the Crx protein, or it could antagonize a
co-activator or potentiate a repressor of Crx. It is obvious that our
understanding of the effect of Baf on Crx activity is limited by the
use of transiently transfected HEK293 cells. It would be interesting to
determine whether the same effect could be seen in a photoreceptor cell
line. Unfortunately, a well characterized and stable photoreceptor cell
line is currently unavailable, and only a limited transfection efficiency can be achieved with existing retinoblastoma cell lines or
primary retinal cell cultures.
Is BAF Linked to Retinal Diseases?--
Loss of Baf in C. elegans by using the RNA interference (RNAi) technique
leads to an embryonic lethal phenotype due to abnormal segregation of
chromosomes during mitosis (58). More recently, it has been found that
mutations of emerin and lamin A/C, the two components of nuclear matrix
complexes that Baf is associated with, cause muscle and heart diseases
such as Emery-Dreifuss muscular dystrophy and lipodystrophy (65).
Furthermore, a disease-causing mutant of emerin poorly binds to BAF
in vitro and fails to assemble into the nuclear envelope
in vivo (29), suggesting that Baf is required for nuclear
envelope reassembly during mitosis and may contribute to the phenotype
of tissue-specific diseases caused by mutations in emerin or lamin A/C.
Our finding that the interaction of Baf and Crx is affected by a CRX
mutant associated with a photoreceptor dystrophy raises the possibility
that Baf may also contribute to the phenotype of CRX-associated retinal
diseases. In addition to CRX, two other retinal homeodomain
transcription factors that interacted with Baf in our study are also
linked to human retinal diseases with great heterogeneity, including
CHX10 that is associated with microphthalmia (66) and PAX-6 associated
with aniridia (67-69). Thus, BAF may be a candidate for a genetic
modifier that contributes to these diseases as well. It would be
interesting to test whether mutations in either Baf or these Baf
interaction partners could affect their physical interaction.
The evidence that Baf interacts with several proteins involved in
tissue-specific diseases allows us to hypothesize that BAF itself could be a candidate gene that, when mutated, might cause a
degeneration or maldevelopment of the retina. As the first step to
address this speculation, we considered the fact that the human BAF gene may lie in a chromosom
TOP
ABSTRACT
INTRODUCTION
