From the Departments of Medicine and Medical
Biophysics, University of Toronto and Toronto General Research
Institute, University Health Network, Toronto, Ontario, Canada M5G 2MI;
and the ¶ Developmental Biology Program, Cancer and Blood Program,
Hospital for Sick Children and Department of Medical Genetics,
University of Toronto, Toronto, Ontario, Canada M5G
Received for publication, June 22, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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In human, germ line mutations in the
tumor suppressor retinoblastoma (Rb) predispose individuals to
retinoblastoma, whereas somatic inactivation of Rb contributes to the
progression of a large spectrum of cancers. In mice, Rb is highly
expressed in restricted cell lineages including the neurogenic,
myogenic, and hematopoietic systems, and disruption of the gene leads
to specific embryonic defects in these tissues. The symmetry between Rb
expression and the defects in mutant mice suggest that transcriptional
control of Rb during embryogenesis is pivotal for normal development. We have initiated studies to dissect the mechanisms of transcriptional regulation of Rb during development by promoter lacZ
transgenic analysis. DNA sequences up to 6 kilobase pairs upstream of
the mouse Rb promoter, isolated from two different genomic libraries, directed transgene expression exclusively to the developing nervous system, excluding skeletal muscles and liver. Expression of the transgene in the central and peripheral nervous systems, including the
retina, recapitulated the expression of endogenous Rb during embryogenesis. A promoter region spanning ~250 base pairs upstream of
the transcriptional starting site was sufficient to confer expression
in the central and peripheral nervous systems. To determine whether
this expression pattern was conserved, we isolated the human Rb 5'
genomic region and generated transgenic mice expressing lacZ under control of a 1.6-kilobase pair human Rb
promoter. The human Rb promoter lacZ mice also expressed
the transgene primarily in the nervous system in several independent
lines. Thus, transgene expression directed by both the human and mouse
Rb promoters is restricted to a subset of tissues in which Rb is
normally expressed during embryogenesis. Our findings demonstrate that
regulatory elements directing Rb expression to the nervous system are
delineated within a well defined core promoter and are regionally
separated from elements, yet to be identified, that are required for
expression of Rb in the developing hematopoietic and skeletal muscle systems.
The tumor suppressor Rb1
was originally identified as a gene that predisposes individuals to
retinoblastoma in infants and osteosarcoma and other malignancies in
later stages of life (1). Rb was subsequently found to be frequently
inactivated in human cancers either by mutations in the gene itself or
by alterations in upstream factors that control the activity of the
protein (2). During most of the G1 phase of the cell cycle,
hypo-phosphorylated Rb inhibits progression into S phase by forming
specific complexes with certain transcription factors. One of the main
partners of Rb, E2F1, regulates expression of genes required for the
transition into S phase and DNA synthesis (3). Mitogenic signals are
propagated through the G1 cyclins and
cyclin-dependent kinases, which phosphorylate and
inactivate Rb, thereby allowing progression into S phase (4). Differentiation signals inhibit the G1 cyclins and
associated cyclin-dependent kinases, thereby maintaining Rb
in the under-phosphorylated, active state. Active Rb is required for
permanent withdrawal from the cell cycle and suppression of cell death
during the onset of differentiation (5-7). There is also evidence that
under-phosphorylated Rb can interact with differentiation factors such
as MyoD and myogenin during myogenesis (8) and C/EBP Another major mechanism of regulation of Rb is at the transcriptional
level. Although initial analysis indicated that Rb is ubiquitously
expressed in adult mouse tissues (11), subsequent studies revealed that
Rb is up-regulated during differentiation of various cell types
in vitro (12, 13). By using in situ hybridization
analysis, we have shown (14) that levels of Rb transcripts are
temporally and spatially regulated during embryogenesis. High
expression of Rb is restricted to a subset of tissues during development including the nervous system, hematopoiesis, skeletal muscles, and lens. The pattern of Rb expression correlates very well
with the phenotype of Rb mutant mice. Rb knockout mice die at embryonic
day 13-14 with specific defects in cell cycle exit, cell survival, and
terminal differentiation during neurogenesis, hematopoiesis, and lens
development (15-17). A defect in terminal myogenesis was also observed
in Rb mutant embryos partially rescued to birth by a Rb minigene
composed of the mouse Rb promoter and Rb cDNA (7, 18).
Interestingly, whereas the mouse Rb minigene only suppresses the
Rb The correlation between Rb expression and the phenotypes of Rb mutant
embryos suggests the existence of a mechanism that ensures that Rb is
expressed in the appropriate tissues when cells receive signals to exit
the cell cycle and terminally differentiate. Analysis of
transcriptional regulation of Rb during embryogenesis may unravel this
developmental program. Here, we initiated studies to dissect the
transcriptional regulation of Rb during embryogenesis. We show that two
independent DNA segments containing the mouse Rb promoter can direct
expression of a linked reporter gene, lacZ, to the nervous
system, excluding the myogenic and hematopoietic systems. A region of
~250 bp upstream of the transcription starting site is sufficient to
confer lacZ expression during neurogenesis in the central
and peripheral nervous systems including the retina. Transgene
expression under control of the human Rb promoter was also detected
primarily in the nervous system in most transgenic lines. These
findings delineate the promoter region required for expression of Rb
during neurogenesis and indicate that different mechanisms, yet to be
defined, govern Rb expression during hematopoiesis and skeletal myogenesis.
Isolation of Mouse and Human Rb Promoter Genomic Regions--
A
genomic phage library, generated in
A human placenta genomic library (purchased from
CLONTECH) was screened with a 455-bp non-GC region
from the human genomic Rb promoter as a probe (21, 22), and several
recombinant phages were isolated and purified. A 4.5-kb
BamHI fragment containing the human promoter region was
identified by Southern blot of BamHI-digested recombinant
phage and was subcloned into pBluescript (phRb.B-B). This
BamHI fragment includes 2-kb human Rb promoter and
approximately 2.5 kb of the first exon and part of the first intron.
Physical mapping and DNA sequencing verified that this DNA fragment
contained the human Rb promoter.
Generation and in Vitro Verification of Transgenic
Constructs--
To create lacZ transgenic constructs, we
generated a plasmid pEZ25/26, a derivative of pBluescript, in which the
SacI-KpnI linker of pBS was replaced by a linker
with following sites: SpeI, XhoI,
NotI, NcoI, BamHI, SpeI.
The NotI site, located at the N terminus of lacZ
in this construct, pEZ25/26, is in frame with the NotI and
EagI sites in the mouse and human Rb promoters,
respectively. A lacZ-SV40 poly(A) cassette was introduced
between the NcoI and BamHI sites yielding
pEZ25/26.lacZ. To create mRbP(L).lacZ, a 4.5-kb Rb promoter region,
previously isolated from mouse L-cell genomic library (23), was
subcloned from the recombinant phage into pEZ25/26.lacZ. To create
pmRbP.(129).lacZ, the NotI fragment from pmRb(Not-Not) was
subcloned into a NotI-linearized pEZ25/26.lacZ. The
SfiI deletion was made by cutting with SfiI
(50 °C) followed by XhoI (at 37 °C), blunt ending with
Klenow, and re-ligation. To create hRbP.lacZ, an
XhoI-EagI fragment was purified from phRb.B-B and
subcloned into XhoI- plus NotI-digested
pEZ25/26.lacZ. Transgene plasmid DNA was purified on Qiagen
midi-columns. COS7 cells cultured in Dulbecco's modified Eagle's
medium plus 10% serum in 60-mm dishes were transfected with 8 µg of
plasmid DNA using the calcium-phosphate method, as described (24).
Twenty four hours after transfection, the medium was aspirated, and the
cells were fixed for 5 min in 4% paraformaldehyde (PFA) and
treated with X-gal solution as described (25). Intensive X-gal staining
confined primarily to the cytoplasm was observed microscopically within
1-2 h.
Generation and Analysis of Transgenic Mice--
To release the
insert DNA fragments, pmRbP(L).lacZ, pmRbP(129).lacZ, and
pmRbP. Analysis of Embryos and X-Gal Staining--
For timed pregnancy
and embryo staging, the mornings of vaginal plug observations were
considered as E0.5. Pregnant females were sacrificed by cervical
dislocation, the embryos were retrieved and fixed in 4% PFA for
30 min to 2 h depending on the developmental stage as described
(25). The embryos were washed several times in phosphate-buffered
saline and treated overnight with X-gal solution as in Ref. 25. Stained
embryos were photographed under a stereomicroscope (Leica). To identify
internal organs, the embryos were dissected sagittally and examined
microscopically. Some embryos were dehydrated, embedded, and sectioned
at 20 µM. Sections were deparaffinized with
xylenes, rehydrated with ethanol, counter-stained with eosin,
dehydrated again, and mounted with Permount (Fisher).
In Situ Hybridization--
In situ hybridization
analysis for Rb was performed as described (14). The Rb plasmid DNA was
linearized with BglII, and 35S-labeled antisense
riboprobe was prepared with T7 polymerase and used at 2 × 105 cpm/µl.
DNase I-hypersensitive Analysis--
Brains and carcasses were
recovered from staged embryos. Nuclear preparation, chromatin digestion
with DNase I, and DNA extraction were performed as described (26). DNA
samples were further digested with SacI or BglII
to detect the CP or 5'-CR, respectively, and analyzed by Southern blot
hybridization using dextran sulfate and GeneScreen Plus nylon membranes
as recommended by the supplier (Dupont) as described (23).
Mouse Rb Promoter Region from L-cell Genomic Library Directs
Transgene Expression Exclusively to the Nervous System--
We
previously described the isolation and in vitro
characterization of a mouse Rb promoter region isolated from a
mouse L-cell genomic library (23). To explore the expression pattern of
the Rb promoter during embryogenesis, the promoter (L) was placed upstream of the Escherichia coli
The expression pattern of the mRbP(L).lacZ transgenes is consistent
with the phenotypes of the partially rescued mgRb:Rb Mouse Rb Promoter Region from 129/sv Genomic Library Also Directs
Transgene Expression Exclusively to the Nervous System--
The
specific expression of the mouse Rb promoter (L) in the nervous system
could reflect the absence of essential regulatory elements for
expression in other tissues. Alternatively, this promoter DNA from a
mouse L-cell genomic library could harbor mutations, deletions, or
rearrangements that might have disrupted important regulatory elements
required for faithful expression of the transgene. To address these
issues, we re-isolated the mouse Rb promoter region from a 129/sv-mouse
tissue genomic library (Stratagene) (Fig.
2a). A 6-kb Rb promoter
fragment from the recombinant phage was subcloned into a
lacZ cassette, yielding mRbP(129).lacZ (Fig. 2b),
and three transgenic founders mRbP(129).lacZ 1, 2, and 10 were created
(Table I). Whole mount X-gal staining revealed that the three
independent transgenic lines expressed lacZ exclusively in
the nervous system, excluding the liver and skeletal muscles, in a
manner that was indistinguishable from the mRbP(L).lacZ lines (Fig. 2,
c versus d and e; Table
I).
The initial X-gal analysis was performed at E13.5 when endogenous Rb
expression is maximal (14). To determine the temporal expression
pattern of the mRbP(129).lacZ transgene during embryogenesis, embryos
were collected from E9.5 to E17.5 and subjected to whole mount X-gal
staining followed by cross-section analysis. The patterns of X-gal
staining were then compared with endogenous Rb expression at similar
developmental stages as detected by in situ hybridization analysis (Fig. 3). This comparison
revealed that independent mRbP(129).lacZ lines expressed
lacZ in the nervous system in a pattern that recapitulated the expression of endogenous Rb during neurogenesis. Specifically, both
endogenous Rb and the mRbP.lacZ transgenes were expressed in the
developing neural tube and brain folds, the spinal cord, dorsal root
ganglia, other cranial nerves, and the ganglion layer of the retina
(Fig. 3 and Table I). Even in younger or older embryos, there was no
evidence of X-gal staining in the liver and skeletal muscles where Rb
is normally expressed, suggesting that regulatory regions required for
expression of Rb in these tissues are located outside the first 6 kb of
the mouse Rb promoter.
Identification of Homologous DNA Sequences and DNase
I-hypersensitive Sites around the Rb Promoter--
We next sought to
identify regulatory DNA sequences that might be involved in controlling
Rb expression during neurogenesis. The mouse Rb core promoter (CP) is
located about 200 bp upstream of the translation initiation codon and
overlaps the major transcription starting site (Fig.
4a) (23). The human and mouse
Rb core promoter sequences are virtually identical (21, 23). They both
lack characteristic TATA and CAAT elements and contain
consensus-binding sites for Sp1, ETS, ATF, and E2F (23, 28, 29).
Analysis of natural mutations in low penetrant retinoblastoma patients (30) and mutagenesis in vitro (23) revealed that the Sp1, ETS, and ATF consensus sites are positively required for expression, whereas the E2F site functions as a negative element in some cellular contexts (23). Since many regulated genes contain upstream enhancers that control spatial expression, we searched the human and mouse upstream DNA sequences for the presence of conserved homologous regions. We identified a 5'-conserved region (5'-CR) at nucleotides
The binding of transcription factors to promoters and enhancer elements
is accompanied by destabilization of the chromatin, and this can
experimentally be visualized as increased sensitivity to certain
enzymes such as DNase I (26, 31). To determine whether the chromatin
around the conserved regions in the Rb promoter is in open
conformation, we examined the presence of DNase I hypersensitive (HS)
sites around the mouse Rb promoter during embryogenesis. A strong HS
region was found in the CP in brain and carcass tissues from E13.5
(Fig. 4, b-d) and E17.5 (not shown) embryos. This region consisted of a major HS band corresponding to the CP as well as two
smaller HS fragments, indicating the presence of destabilized nucleosomal regions within and immediately upstream of the CP (Fig.
4d). Two additional bands of ~1.5-2.5 kb were identified, suggesting the existence of HS sites within the first intron of Rb. In
contrast, analysis of the 5'-CR and upstream DNA sequences revealed no
obvious HS regions in chromatin extracted from E13.5-E17.5 brain
tissues (data not shown). A very faint signal in the predicted location
of the 5'-CR was detected in nuclei isolated from carcasses of E13.5
embryos (data not shown). Thus, the main HS region in the Rb promoter
appears to surround the CP; the 5'-CR may be important for certain cell
lineages in carcasses or other embryonic or adult tissues.
Sequences Required for Expression of Rb in the Nervous System Are
Independent of the 5'-CR and Confined to the First 250 bp Upstream of
the Transcriptional Starting Site--
The presence of multiple HS
sites around the Rb core promoter but not at the 5'-CR, suggested that
the core promoter alone may direct transgene expression to the nervous
system, independently of the 5'-CR. To examine this possibility
directly, we generated a deletion construct, mRb
We note that the relative levels of transgene expression within the
nervous system varied among and within several lines. For example,
mRb The Human Rb Promoter Region Directs Transgene Expression Primarily
to the Nervous System--
The exclusive expression pattern of the
mRbP.lacZ transgenes in the nervous system (Figs. 1-3 and 5) is
consistent with our observation that the mouse Rb minigene can only
rescue the neurogenic defect of Rb
Five transgenic hRbP.lacZ lines were established, and the expression
patterns of lacZ were determined during embryogenesis (Fig.
6, c Although Rb is expressed in all adult tissues (11), the gene is
highly expressed only in a subset of cell lineages during development
(Fig. 3, e, g, and i) (14). The correlation
between Rb expression and the spectrum of developmental defects in Rb null mice suggests the existence of a mechanism that coordinates the
expression of Rb during embryogenesis with the onset of terminal differentiation in certain tissues. We initiated a transgenic analysis,
described herein, with the goal of defining the regulatory regions that
control Rb expression during embryogenesis and elucidating this
developmental program. We found that mouse Rb promoter DNA sequences up
to 6 kb in length directed transgene expression exclusively to the
central and peripheral nervous systems, including the developing retina. Our results suggest that regulatory regions required for expression of Rb in the liver and skeletal muscles where Rb is normally
expressed may be physically separated from regulatory elements that
control Rb expression in the nervous system. This regulatory
arrangement, also found in other genes (33), is consistent with the
notion that an ancestor Rb might have initially evolved to exert
neurogenic functions and was later recruited by other tissues that
acquired appropriate regulatory elements elsewhere in the Rb locus. The
regulatory DNA sequences required for transcription of Rb in the liver,
lens, and skeletal muscles may be located in other genomic regions
surrounding the Rb gene. Alternatively, the 3'-UTR of Rb may control
the stability of the Rb message in the liver, lens, and skeletal muscles.
Arguably, the Rb promoter contains all the necessary regulatory
elements, but the lacZ cassette suppresses the Rb promoter specifically in the liver, lens, and skeletal muscles but not in the
nervous system. We consider this possibility unlikely because mouse Rb
promoter minigenes, containing the Rb cDNA rather than lacZ, are also expressed exclusively in the nervous system
and specifically rescue the neurogenic but not hematopoietic skeletal muscle and lens defects in Rb In addition to the 4.5-kb Rb minigene (Fig. 1a), another Rb
minigene capable of partially suppressing the Rb In contrast to the mouse minigenes, which only partially rescue the
Rb It is somehow puzzling that the highly conserved 5' region, 5'-CR, is
not involved in transcriptional activation as determined by transgenic
and DNase I-hypersensitive analyses. The 5'-CR may, however,
communicate with upstream or downstream elements and assist in
expression of Rb in some specific cell lineages or adult tissues. We
delineated the region required for mouse Rb transgene expression during
neurogenesis to 250 bp upstream of the transcription starting site.
This region contains several consensus-binding sites including an E2F
site that functions as a negative element in vitro (23).
This E2F site may allow autoregulation by Rb or cross-regulation by
p107 (and/or p130). The latter possibility is attractive considering
the almost mutually exclusive expression patterns of Rb and p107 during
embryogenesis (14). Interestingly, the core promoter (RbP
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
during
adipogenesis (9) and promote transcriptional activation of responsive
genes (10).
/
neurogenic defects, a human Rb minigene could completely rescue
the Rb
/
phenotype (16, 20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FixII from mouse 129/sv DNA
(purchased from Stratagene), was screened by plaque lift hybridization,
using nylon membranes (DuPont) as recommended by the manufacturer. A
BglII-SfiI fragment from the mouse Rb promoter was used as a probe. A recombinant phage,
-129-1, was
plaque-purified, and the phage DNA was extracted using Qiagen phage DNA
kit. The physical map of
-129-1 was determined by multiple
digestions with restriction enzymes. A 6-kb NotI fragment
was subcloned into pBluescript, yielding pmRb(Not-Not), and the
sequence of the core promoter and the 5'-CR was determined using
specific primers designed based on the sequence of the mouse Rb
promoter (7).
sfi.lacZ were digested with SpeI and phRbP.lacZ with BamHI. The DNA inserts were extracted from agarose gels
with a Qiagen kit and further purified on Elutip-D columns (Schleicher & Schuell) from Xymotech. Microinjection of DNA and generation of
chimeric mice were done in the transgenic mouse facilities at the
Hospital for Sick Children and the Ontario Cancer Institute. Transgenic
mice were initially identified by Southern blot hybridization and later
genotyped by polymerase chain reaction. lacZ was amplified with a forward primer EZ74 (5' GTT CCG TCA TAG CGA TAA CG) and reverse
primer EZ64 (5' TCA ATC CGG TAG GTT TTC CG), yielding a polymerase
chain reaction product of 649 bp.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase
(lacZ) reporter gene, and five
mRbP(L).lacZ transgenic founders were created (Fig. 1b; Table
I). F1 transgenic males were then used to
set up timed pregnancies with wild type females to obtain staged
embryos. X-gal staining of E13.5 embryos revealed that three
independent transgenic lines (mRbP(L).lacZ 19, 37, and 5) expressed the
transgene exclusively in the nervous system (Fig. 1 and Table I). Two
other lines, mRbP(L).lacZ 27 and 29 expressed the transgene in the
nervous system as well as the skin (not shown, Table I). Whole mount and cross-section analysis of the mRbP(L).lacZ 19, 37 and 5 transgenic embryos revealed exclusive X-gal staining in the central (brain, spinal
cord, Fig. 1e, top) and peripheral (dorsal root ganglia, Fig. 1e, bottom) nervous systems. X-gal staining was also
detected in the follicles of vibrissae (Fig. 1d) where
endogenous Rb is normally expressed (14). In contrast to endogenous Rb,
no X-gal staining was detected in the liver or any muscle mass
throughout embryogenesis (Fig. 1, c and e,
top; Table I).
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Fig. 1.
A mouse Rb promoter from L-cells directs
transgene expression exclusively to the nervous system.
a, a schematic structure of the mgRb minigene containing 4.5 kb of mouse Rb promoter isolated from mouse L-cells, the Rb cDNA,
and a human growth hormone sequence and polyadenylation site at the 3'
end. b, the mRbP(L).lacZ transgene contains the same Rb
promoter region fused to the bacterial -galactosidase gene
(lacZ) with an SV40 polyadenylation site at the 3' end.
c-e, mRbP(L).lacZ transgenic mice express lacZ
exclusively in the nervous system as visualized by staining with X-gal
(blue). c, expression of mRbP(L).lacZ in the
brain and spinal cord in a E12.5 embryo. d, expression of
mRbP(L).lacZ in vibrissae in an E13.5 embryo. e, sagittal
sections of a E12.5 mRbP(L).lacZ transgenic embryo reveal X-gal
staining in the spinal cord (top) and dorsal root ganglia
(DRG, bottom) but not in skeletal muscles such as
the tongue (T) muscles. Abbreviations: SC, spinal
cord; Vi, vibrissae; 4V, fourth ventricle;
T, tongue; DRG, dorsal root ganglia.
Differential expression of endogenous Rb versus mouse and human Rb
promoter transgenes
/
fetuses (7,
18, 27). In these fetuses, the neurogenic, but not hematopoietic,
skeletal muscle and lens defects in Rb
/
embryos are rescued by an
Rb minigene containing the same mouse Rb promoter (L) fused to the
mouse Rb cDNA (Fig. 1a). The mgRb:Rb
/
embryos
survive to birth and exhibit aberrant myogenesis and other developmental defects, whereas the neurogenic phenotype is specifically suppressed (7, 18, 27).
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Fig. 2.
A mouse Rb promoter isolated from 129/sv
genomic library directs transgene expression exclusively to the nervous
system. a, schematic presentation of a recombinant
phage, -129-1, encompassing the Rb first exon, promoter region, and
first intron. The
-129-1 clone was isolated from a mouse 129/sv
tissue library using the indicated BglII-SfiI
genomic fragment from the mouse Rb promoter (L) as a probe.
b, schematic presentation of the mRbP(129).lacZ transgene.
c, whole mount X-gal staining of E13.5 mRbP(L).lacZ embryo.
d, whole mount X-gal staining of E13.5 mRbP(129).lacZ
embryo. Note the apparent identical X-gal staining in the mRbP(L).lacZ
and mRbP(129).lacZ lines. Positive staining was observed in the
ganglion layer of the retina (R), cranial nerves such as the
trigeminal ganglia (Tg), dorsal root ganglia
(DRG), the vibrissae (Vi), and throughout the
brain and spinal cord (SC). No expression was observed in
the liver (L) or skeletal muscles where endogenous Rb is
normally expressed. e, a mid-sagittal dissection of E13.5
mRbP(129).lacZ embryo revealing expression in the nervous system but
not in developing muscles or liver. 4V, fourth
ventricle.
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Fig. 3.
Temporal expression of mouse Rb promoter
transgenes recapitulates endogenous Rb expression during
neurogenesis. a-c, lateral view of whole mount
(a) and mid-sagittal dissections (b and
c) of mRbP.lacZ transgenic mice during the indicated stages
of development. Expression is restricted to the developing nervous
system. d-i, comparison of transgene expression of
mRbP.lacZ mice in sagittal (d and f) and
cross-sections (h) with endogenous Rb expression detected by
in situ hybridization (e, g, and i).
Expression of the mRbP.lacZ transgene is restricted to the nervous
system, whereas endogenous Rb is also highly expressed in the liver and
skeletal muscles. Abbreviations: FV, fourth ventricle;
DRG, dorsal root ganglia; SC, spinal cords;
SM, skeletal muscles; L, liver; Lu,
lung; LV, lateral ventricle; TG, trigeminal
ganglia.
1545 to
1465 in human and
1141 to
1065 in the mouse (Fig. 4a). The 5'-CR contains potential binding sites for bHLH and
MYB factors within two sequential regions that share 32/40 and 23/23 nucleotide identity in human and mice. In addition, there are clusters
of bHLH- and GATA-binding sites and an NF
B/REL site around both
5'-CRs, which are not embedded in conserved regions but are found
both in human and mouse (Fig. 4a).
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Fig. 4.
Conserved regions and DNase I-hypersensitive
sites in the Rb promoter. a, human and mouse Rb
promoters share two regions of high homology. The core promoter
(CP) contains consensus-binding sites for Sp1, ETS, ATF, and
E2F. Mutagenesis of these elements established their importance
in vitro (23, 29). Mutation in the Sp1 and ATF sites were
identified in low penetrance retinoblastoma patients (30). The
5'-conserved region (5'-CR) contains consensus sites for bHLH and MYB
factors. There are also bHLH, GATA, and NF B/REL sites in the two
promoters in non-conserved regions adjacent to the 5'-CR. As indicated,
sequence analysis of the 5'-CR identified an A to
G difference in the 129/sv sequence compared with the
published sequence of mouse L cells (23), which increases the homology
between the human and mouse 5'-CR. b-d, DNase
I-hypersensitive analysis of the mouse Rb core promoter in carcass and
brain tissues from E13.5 embryos. Chromatin was partially digested with
increasing amounts of DNase I, and the DNA was purified, digested with
SacI, and Southern-blotted onto nylon membranes. The blots
were hybridized with a 32P-labeled
SacI-BglII probe shown in d. Identical
results were obtained in other experiments in which a PCR
fragment of 364 bp upstream of the BglII site was used as a
probe (not shown). A major DNase I-hypersensitive region was found at
about 1.2 kb downstream of the SacI site corresponding to
the core promoter of Rb. Additional HS sites were detected upstream and
downstream (within the first intron) of the core promoter.
Sfi.lacZ, in which
the 5'-CR and most upstream DNA sequences were removed (Fig.
5a). Five mRb
Sfi.lacZ transgenic founders were generated, three of which expressed the transgene (numbers 3, 7, and 16). The mRb
Sfi.lacZ 16 transgene was
expressed in the nervous system as well as other tissues such as
isolated groups of muscles and the heart (Table I). In contrast, both
mRb
Sfi.lacZ 3 and 7 lines expressed lacZ only in the
nervous system in a pattern that appeared identical to mRbP(129).lacZ and mRbP(L).lacZ transgenic mice (Fig. 5, b-i). At various
developmental stages, the mRb
Sfi.lacZ 3 and 7 transgenes were
detected in the central and peripheral nervous systems, follicles of
vibrissae, and retina but not in liver, skeletal muscles, or other
tissues (Fig. 5, b-i). Thus, the Rb 5'-CR is dispensable
for expression in the nervous system, and a segment of 450 bp
encompassing the Rb core promoter contains all the regulatory elements
required to direct Rb expression to the central and peripheral nervous systems during embryogenesis.
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Fig. 5.
A 450-bp fragment encompassing the Rb core
promoter is sufficient to direct faithful transgene expression to the
developing nervous system. a, the mRbP sfi.lacZ
transgene extends 250 bp upstream of the transcription starting site of
Rb (450 bp upstream of the translation initiation codon) excluding the
5'-CR. (b and c and f and
g) Whole mount X-gal staining and cross-sections of E13.5
(b and f) and E17.5 (c and
g) mRbP(129).lacZ 7 embryos. d and e
and h and i, whole mount X-gal staining (3 h and
overnight) and cross-sections of E13.5 mRbP(129).lacZ 3 embryos. Note
the different expression levels in vibrissae and retina in the two
embryos. Abbreviations are as in Figs. 1-3.
Sfi.lacZ 7 showed weak expression in the follicles of vibrissae,
whereas mRb
Sfi.lacZ 3 was highly expressed in this tissue (Fig. 5,
c and i); within the mRb(129).lacZ 10 line, some embryos exhibited intense staining in the vibrissae, whereas other embryos, in the same litter, showed little expression (Table I and data
not shown).
/
mutant embryos (7, 18, 27). In
contrast, a human Rb minigene consisting of a 1.6-kb human Rb promoter
region, linked to the human Rb cDNA, completely rescues the Rb
/
lethal defect (16) and induces dwarfism in transgenic mice (32). This
suggests that unlike the mouse counterpart, the human Rb promoter may
either direct expression to all the tissues where Rb is normally
expressed or that the human promoter transgene is dysregulated in the
mouse, directing ubiquitous expression to many tissues at levels
sufficient to rescue the Rb
/
lethal defect. To test these
possibilities directly, we isolated the human Rb promoter from a normal
human placental genomic library (Fig.
6a). A 1.6-kb
BamHI-EagI segment containing the human Rb promoter was transferred to a lacZ cassette yielding
hRbP.lacZ (Fig. 6b). This construct is similar to the human
Rb minigene that was used to rescue the Rb
/
defect (32). However,
the hRbP.lacZ minigene utilizes an SV40 polyadenylation signal (Fig. 6b), whereas the human Rb minigene contains a
-globin
cassette at the 3' end (32).
View larger version (45K):
[in a new window]
Fig. 6.
Transgenic analysis of the human Rb
promoter. a, schematic presentation of a recombinant
phage clone, -h6, encompassing the human Rb first exon and promoter
region.
-h6 was isolated from a human placenta genomic library using
the indicated 455-bp genomic fragment from the human Rb promoter as a
probe. b, schematic presentation of the hRbP.lacZ transgene.
c-i, expression of the hRbP.lacZ transgene in several
independent lines is confined to the nervous system like the mRbP.lacZ
lines. j-m, the hRbP.lacZ 5 transgenic line exhibited
expression in other tissues including isolated groups of skeletal
muscles, the palate shelf and intestines. Most hRbP.lacZ lines
expressed lacZ in the intervertebral disc (ID)
(f, k and m). n, endogenous
Rb, revealed by in situ hybridization, was also detected in
the intervertebral disks, but expression was severalfolds lower than in
adjacent skeletal muscles, spinal cord and liver. Abbreviations are as
in Figs. 1-3.
m, and Table I). Three of these transgenic lines
(numbers 7, 21, and 22) exhibited X-gal staining primarily in the
nervous system like the mRbP.lacZ lines (Fig. 6, c-i). The
21 and 7 lines also exhibited some expression in the kidney, and number
22 was expressed at low level in the heart. The hRbP.lacZ 15 line
exhibited high level of expression in the retina and low levels in the
central and peripheral nervous systems (Table I). The hRbP.lacZ line 5 was expressed in the spinal cord, the cartilage of palate shelf, intervertebral disks, intestines. and isolated groups of skeletal muscles (Fig. 6, j
m, and data not shown). The expression
pattern of hRbP.lacZ 5 may represent activation of cryptic elements in the human Rb promoter at some integration sites. In addition to the
hRbP.lacZ 5, three other lines, 7, 21, and 22, expressed
lacZ in the intervertebral disc (ID, Fig.
6f, and Table I). As shown in Fig. 6n, endogenous
Rb transcripts were also detected, although at relatively low levels,
in the intervertebral disks. The relax expression patterns of the
hRbP.lacZ mice compared with the tight expression of the mRbP.lacZ
lines in the nervous system may point to some differences in the levels
of expression of the human and mouse Rb genes in certain tissues.
Alternatively, this discrepancy may reflect partial dysregulation of
the human promoter in mouse tissues. Importantly, four of five
hRbP.lacZ lines directed transgene expression primarily to the nervous
system but not the liver and skeletal muscles where endogenous Rb is
highly expressed. Thus, both the human and mouse Rb core promoters
evolved to direct Rb expression to the nervous system. Other, yet to be
defined, regulatory elements, located outside the first 6 kb upstream
of the Rb core promoter, may be required for expression of Rb during
hematopoiesis and skeletal myogenesis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
embryos (7, 18, 27). We previously
thought that the expression pattern of these Rb minigenes reflected the
differential stability in different tissues of the human growth hormone
cassette at the 3' end of the Rb minigene (Fig. 1a). The
present study indicates that the Rb promoter dictates this expression
pattern. Since the Rb promoter directs expression exclusively to the
nervous system, the mgRb:Rb
/
fetuses can be viewed as null for Rb
in tissues other than the nervous system. As the Rb minigene can rescue
the mid-gestation death of Rb
/
embryos, but is only expressed in
the nervous system but not the hematopoietic system, it is tempting to
speculate that Rb
/
embryos die at E13.5-E14.5 due to a neurogenic
rather than hematopoietic deficiency.
/
defect contains a
1.3-kb mouse Rb promoter region and the first intron of Rb (7). Since
this minigene could also only rescue the Rb
/
neurogenic defect, it
is unlikely that the regulatory elements required for expression of Rb
in liver, lens, and muscles are located in the first intron. Thus, the
HS sites detected in the first intron of Rb (Fig. 4, b and
c) may not be important for expression in these tissues. To
identify the regulatory elements required for expression of Rb during
hematopoiesis, skeletal myogenesis, and lens development, large DNA
segments 20-40 kb upstream of the Rb CP, the 3'-UTR, and downstream
sequences will have to be subject to DNase I HS and transgenic analysis.
/
embryonic defect, a human Rb minigene consisting of a similar
promoter DNA segment used in our study can completely rescue the Rb
defect (16, 20). The discrepancy between these observations is not
clear but may be attributed to different 3'-UTR used in the two studies
or to the widespread, deregulated, expression of the human Rb transgene
in some transgenic lines (Fig. 6, j-m), which might be
sufficient for phenotypic rescue of the Rb
/
defect. Recently,
transgenic mice expressing N-terminal deletions of Rb under control of
the human Rb promoter were found to rescue the neurogenic but not
skeletal or hematopoietic defects of Rb
/
mutant embryos (16, 20),
yielding a phenotype similar to the mgRb:Rb
/
fetuses. It would be
interesting to determine the expression of the N-terminal Rb mutants in
the liver and muscles relative to the nervous system.
Sfi) is
sufficient to direct transgene expression to the central and peripheral
nervous systems as well as the retina (Fig. 5). Inactivation of the Rb
promoter by methylation or specific mutations has been implicated in
the initiation of retinoblastoma (19, 30). Our results suggest that
perhaps similar alterations may also suppress transcription of the Rb gene in other neuronal tissues. Although several transcription factors
were shown to bind the Rb core promoter in certain tissues, the
neurogenic factors that control the expression of Rb during development
are not known. Future experiments should allow us to identify the
cis-elements and the corresponding factors that control Rb expression
in the retina, the central, and peripheral nervous systems and study
their effects on expression of Rb in the contexts of normal development
as well as neoplastic transformation.
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FOOTNOTES |
---|
* This work was supported by Grant MT-14314 from the Medical Research Council of Canada (to E. Z.).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.
§ Both authors made equal contributions to this work.
Recipient of a scholarship from the Cancer Research Society
Inc./Medical Research Council of Canada. To whom correspondence should
be addressed: Depts. of Medicine and Medical Biophysics, University of
Toronto, Toronto General Research Institute, University Health Network,
67 College St., Rm. 407, Toronto, Ontario M5G 2M1, Canada. Tel.:
416-340-5106; Fax: 416-340-3453; E-mail:
eldad.zacksenhaus@utoronto.ca.
Published, JBC Papers in Press, October 2, 2000, DOI 10.1074/jbc.M005474200
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ABBREVIATIONS |
---|
The abbreviations used are:
Rb, retinoblastoma;
X-gal, 5-bromo-4-chloro-3-indolyl- -galactopyranoside;
lacZ,
-galactosidase;
E, embryonic day;
kb, kilobase pair(s);
bp, base pair(s);
3'-UTR, 3'-untranslated region;
5'-CR, 5'
conserved region;
CP, core promoter;
HS, hypersensitive site.
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