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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2658-2666
The Wsh, W57, and Ph
Kit Expression Mutations Define Tissue-Specific Control
Elements Located Between  23 and 154 kb Upstream of Kit
By
Georgina Berrozpe,
Inna Timokhina,
Steven Yukl,
Youichi Tajima,
Masao Ono,
Andrew D. Zelenetz, and
Peter Besmer
From the Molecular Biology Program Sloan-Kettering Institute, New
York, NY; and Cornell University Graduate School of Medical Sciences,
New York, NY.
 |
ABSTRACT |
The Kit and PDGFRa receptor tyrosine kinases are encoded in close
proximity at the murine white spotting (W) and patch
(Ph) loci. Whereas W mutations affect hematopoiesis,
melanogenesis, and gametogenesis, the Ph mutation affects
melanogenesis and causes early lethality in homozygotes. The
Wsh, W57, and Ph
mutations diminish Kit expression in certain cell types such as
mast cells and enhance it in others. The Wsh,
W57, and Ph mutations arose from deletions
and inversions affecting sequences in between the Kit and
PDGFRa genes. We have determined the precise location of the
breakpoint of the Wsh inversion and the endpoints
of the W57 deletion upstream of the Kit
transcription start site and examined the effect of these mutations on
Kit expression in mast cells and hematopoietic stem cells and lineage
progenitors. Our results indicate that positive elements controlling
Kit expression in mast cells mapping in between  23 and 154 kb
from the transcription start site can be dissociated from negative
elements controlling Kit misexpression during embryonic
development in the vicinity of the PDGFRa gene. In addition, we
have identified two clusters of hypersensitive sites in mast cells at
23 28 kb and 147 154 kb from the Kit gene
transcription start site. Analysis of these hypersensitive sites in
mutant mast cells indicates a role for HS4-6 in Kit expression
in mast cells. These findings provide a molecular basis for the
phenotype of these Kit expression mutations and they provide
insight into the complex mechanisms governing the regulation of
Kit expression.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE Kit RECEPTOR TYROSINE kinase (RTK) is
encoded at the white spotting (W) locus on mouse chromosome
5.1,2 W mutations affect hematopoiesis,
melanogenesis, and gametogenesis. In agreement with the pleiotropic
functions of the Kit receptor kinase in several cell systems during
embryonic development and in the adult animal, Kit expression
is restricted to distinct cell types in which the Kit receptor is known
to function, including: hematopoietic cell types, primordial germ
cells, spermatogonia, oocytes, melanoblasts, melanocytes, neuronal cell
populations, and interstitial cells of Cajal.3,4 Therefore,
the study of the mechanisms that control Kit expression in
various cell types is of great interest.
Many different W mutations have been identified and
characterized. Most of these mutations are structural Kit
mutations or null mutations that affect the different cellular
functions similarly.3,5,6 W mutations that affect
Kit expression have been characterized as well. The study of
such mutations provides the opportunity to gain insight into the
mechanism of tissue-specific Kit expression. Wsh,
Wbd, W57, and Ph are
Kit mutations that diminish Kit expression in certain cell types and enhance it in others. The Wsh and
Wbd mutations affect only melanogensis and tissue
mast cells and therefore these mice are fertile and not anemic, but
they are black-eyed whites and lack tissue mast cells.7,8
Whereas the mast-cell deficiency in Wsh mice
results from lack of Kit expression, enhanced Kit
expression in somitic dermatomes at the time of melanoblast
migration from the neural crest to the periphery may cause the
pigmentation defect.9,10 Ph/+ mice exhibit a
pigmentation deficiency similar to that of heterozygous
Wsh/+ mice. Our previous analysis showed that the
pigmentation deficiency in Ph/+ mice may also arise from an
effect of the Ph mutation on Kit expression during
embryogenesis.11 The W57 mutation
exerts a weak effect on melanogenesis and mast-cell numbers, but does
not affect erythropoiesis and gametogenesis. This mutation diminishes
Kit expression in mast cells as well as in some other cell
types.12,13 These observations suggested that the
Wsh, W57, and Ph
mutations affect some of the same distant 5' upstream elements
controlling Kit gene expression.9,10
The Kit RTK gene comprises 21 exons contained in 70 kb and maps
on mouse chromosome 5 in the vicinity of the receptor tyrosine kinases
PDGFRa and flk1.1,2,14 Long-range mapping
previously placed PDGFRa 200 to 400 kb 5' of Kit
and flk1 400 kb 3' of Kit. The molecular
basis of the Wsh, Wbd,
W57, and Ph mutations had been investigated
by using pulsed-field gel electrophoresis (PFGE). Both the
Wsh and Wbd mutations were
shown to derive from the inversion of a 2 cmol/L segment of mouse
chromosome 5 with a proximal breakpoint between the Tec and the
Gabrb1 genes and a distal breakpoint between the PDGFRa
gene and Kit.9,11,13,15 The Ph mutation
was shown to arise from deletion of the entire PDGFRa
gene16 and the W57 mutation from
deletion of an 80 kb segment in between the PDGFRa and
Kit genes.13 Furthermore, the Kit coding
sequence is not altered in the Wsh,
Wbd, and W57
alleles.9,13
Enhancers and promoters of transcriptionally active genes are usually
associated with chromosomal regions that are in an open configuration
and are sensitive to nuclease digestion (DNase I hypersensitive
sites).17,18 Chromatin remodeling, the interaction of
transcription factors with cis-acting regulatory sequences contained in
DNase I hypersensitive sites and their interaction with the general
transcription machinery are essential steps in tissue-specific gene
expression.19,20 Therefore, the identification of DNase I
hypersensitive sites may serve as an important first step in unraveling
mechanisms governing this process. To gain insight into mechanisms
controlling Kit expression, we have investigated the molecular basis of
the Wsh, W57, and Ph
mutations and determined the precise location of the 3'
breakpoint of the Wsh inversion and the endpoints
of the W57 deletion. This information enabled us to
identify two clusters of hypersensitive sites near the
W57 deletion endpoints at 21 to 28kb
and at 147 to 154kb upstream of the c-kit transcription
start site in mast cells. In Wsh and
W57 mast cells the 3' hypersensitive sites,
HS4, HS5, and HS6, were found to be in a closed configuration implying
a critical role for these sites in mediating c-kit expression in mast
cells. These findings provide a molecular basis for the phenotype of
these Kit expression mutations and they provide insight into the
complex mechanisms involved in the regulation of Kit gene expression.
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MATERIALS AND METHODS |
Mice.
C57BL/6J and C57BL/6 Ph/+ mice were purchased from the
Jackson Laboratory (Bar Harbor, ME). C57BL/6
Wsh/Wsh were originally obtained from
Rudolf Jaenisch (Massachusetts Institute of Technology, Cambridge, MA)
and C57BL/6 Ph/Wsh mice were provided by Katia Manova.
Isolation and characterization of P1 clones.
Hybridization probes were derived by polymerase chain reaction (PCR)
amplification of various regions upstream of the Kit transcription
start site and used to screen a murine P1 library (Genome Systems, St
Louis, MO). PCR reactions were performed in a 50 mL reaction volume
using Perkin Elmer-Cetus (Norwalk, CT) recommendations. In
the initial screening of the library, one clone was identified. P1
recombinant plasmid DNA was prepared using a modified alkaline lysis
procedure (P1 manual; Genome Systems). The DNA was digested with
different enzymes and analyzed using PFGE or conventional
electrophoresis. For the isolation of P1 end sequences, we used the
plasmid rescue method. The P1 DNA was digested with a restriction
enzyme with a 6 base pair recognition sequence, that did not digest the
vector internally (Sac1, XbaI, BamH1). The vector was
recircularized, ligated, and used to transform Esherichia Coli.
The insert sequence was determined and used to derive hybridization
probes for rescreening of the P1 library. More than 5 P1 clones were
isolated to characterize more than 200 kb of upstream Kit sequences.
PFGE.
The preparation of samples for PFGE, enzyme digestions, and
electrophoresis was as described previously.9 After
electrophoresis gels were stained with EtBr for 30 minutes, exposed to ultraviolet light for 20 seconds, destained for 15 minutes, and soaked in 0.4 mol/L NaOH and 1.5 mol/L NaCl for 15 minutes. DNA was transferred to Zeta-Probe Nylon membranes from Bio-Rad
(Hercules, CA) in 0.4 mol/L NaOH and 1.5 mol/L NaCl
overnight. After blotting, the membranes were washed in 0.5 mol/L Tris
for 5 minutes and then in 2× SSC (1× SSC: 150 mmol/L NaCl, 15 mmol/L EDTA). Filters were prehybridized in 0.72 mol/L
sodium chloride, 40 mmol/L sodium phosphate pH 7.6, 4 mmol/L EDTA,
0.2% polyvinylpyrrolidone, 0.2% ficoll 400, 0.1% sodium dodecyl
sulfate (SDS), 2 mg denatured salmon sperm DNA at 65°C, and
hybridized in 0.72 mol/L sodium chloride, 40 mmol/L sodium phosphate pH
7.6, 4 mmol/L EDTA, 0.2% polyvinylpyrrolidone, 0.2% ficoll 400, 0.1%
SDS, 2 mg denatured salmon sperm DNA, and 10% dextran sulfate with
106 cts/min/mL at 65°C overnight. Filters were washed
15 minutes at room temperature in 2 × SSC , 0.1% SDS, and 3 × 30 minutes in 0.1 × SSC and 0.1% SDS at 65°C.
Cell culture.
Mast cells were grown from bone marrow of adult mice in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 10% conditioned medium
from X63 interleukin (IL)-3 producing cells, nonessential amino acids,
sodium pyruvate. Nonadherent cells were harvested and fed weekly and
maintained at a cell density of 5 × 105
cells/mL.33 HCD57 cells were kindly provided by Harvey
Lodish (MIT) and they were grown in Iscoves's modified Dulbecco's
medium (IMDM) supplemented with 20% FCS and 1UmL1
eosinophil peroxidase (EPO). The Melan-a cell line was kindly provided
by Dot Bennett (St George Hospital Medical School, London,
UK) and they were grown in RPIM 1640 supplemented with 10% FCS and 200 nmol/L 12-o-tetradecanoylphorbol-13 acetate (TPA).
RNA and DNA isolation and blot analysis.
Total cellular RNA was extracted from each of the samples by treatment
with guanidine-HCl followed by phenol extraction and ethanol precipitation according with the method of Chirgwin et al.22 Total cellular RNA was fractionated in 1%
agarose-formaldehyde gels, transferred to nylon membranes (Nytran;
Schleicher and Schuell, Keene, NH), and prehybridization
and hybridization performed as previously described.23,24
The murine 3.7 kb Kit complementary DNA (cDNA) labeled with
32P-dCTP, prepared by the random primer method, was used as
a probe for hybridization.25
Genomic DNA was extracted from samples by treatment with SDS and
proteinase K followed by extraction with phenol/chloroform and ethanol
precipitation.26 Conditions for DNA transfer,
prehybridization, hybridization, and washing were the same as for PFGE.
Preparation of nuclei.
Nuclei were prepared as described by Siebenlist et al.27
Approximately 2 × 108 mast cells or HCD57 cells were
harvested, washed in cold phosphate-buffered saline (PBS), and
resuspended in 10 mL of ice-cold nuclear isolation buffer (60 mmol/L
KCL, 15 mmol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L ethyleneglycoltetracetic acid (EGTA), 15 mmol/L Tris-HCl (pH 7.4), 0.5 mmol/L dithiothreitol (DTT), and 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF). Cells were disrupted by mixing in 0.7 mL of a cold
10% Igepal CA-630. This solution was then layered onto 27 mL of
ice-cold nuclear isolation buffer containing 1.5 mol/L sucrose. Nuclei
were collected by sedimentation at 3,000 rpm. The procedure to isolate
nuclei from liver was essentially as described. One gram of liver was
homogenized using a Dounce homogenizer in 10 mL of a buffer containing
10 mmol/L hepes pH 7.6, 16 mmol/L KCl, 0.15 mmol/L spermine, 0.5 mmol/L
spermidine, 1 mmol/L EDTA, 1.5 mol/L sucrose, 10% glycerol, 0.5 mmol/L
DTT, 0.5 mmol/L PMSF, 1% Igepal CA-630. The homogenate was layered
onto 27 mL of the above solution and sedimented in an SW24 rotor at
24,000 rpm.
DNAse l digestion of nuclei.
The nuclear pellet was resuspended in 1 mL of a solution containing 10 mmol/L tris pH 7.5, 1.5 mmol/L MgCl2, 250 mmol/L sucrose, 0.5 mmol/L DTT, 1 mmol/L PMSF, and 5% glycerol. The nuclei were counted and aliquoted in a volume of 500 mL each and digested with
0.025 mg/mL of DNAse l (Worthington, Lakewood, NJ) for 2, 3, 4, 6, 8, and 10 minutes at 37°C and the reaction was terminated by addition of 100 mL of 250 mmol/L of EDTA, 300 mL of 1% of SDS and
0.1 mg/mL proteinase K. DNA was subsequently isolated by repeated organic extractions and ethanol precipitations. Restriction digests, electrophoresis, transfer of DNA, and hybridization were performed as
described above.
Antibodies.
Monoclonal antibodies against the following surface molecules were
used: biotin-labeled anti-CD3 (145-2C11), biotin-labeled anti-B220
(RA3-6B2), biotin-labeled anti-Mac-1 (M1/70), biotin-labeled anti-GR1
(8C5), biotin-labeled anti-erythroid antigen (TER119), phycoerythrin
(PE)-labeled anti-Sca-1 (E13), fluorescein isocyanate (FITC)-labeled
anti-Kit (2B8 and ACK2). Biotinylated lineage cocktail antibodies were
visualized with streptavidin Per CP (Becton Dickinson, Mountain View, CA).
Preparation and staining of bone marrow and bone marrow mast cells
(BMMC).
Bone marrow was obtained by flushing the femurs and tibias with PBS
containing 2% calf serum. Erythrocytes were eliminated by lysis with
NH4Cl. The remaining cells stained with the indicated combinations of antibodies. Cell were stained in PBS with 2% calf serum for 15 minutes on ice, washed in PBS with 2% calf serum, and
centrifuged through a serum layer. The labeled cells were analyzed with
single-laser fluorescence-activated cell sorter (FACS; Becton
Dickinson). Kit expression of BMMC was determined using FITC-labeled
anti-Kit ACK2 antibodies.
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RESULTS |
Molecular cloning of the Kit upstream region and
characterization of the breakpoint and deletion endpoints of the
Wsh and W57 mutations.
We had previously identified the Wsh breakpoint in
the upstream region of the Kit gene by using PFGE.11 In
addition, an 80 kb deletion in the Kit upstream region was recently
reported in the W57 allele.13 To define
the nature of these two mutations more precisely, 200 kb of the Kit
upstream region was molecularly cloned by identifying overlapping P1
clones obtained from Genome Systems and a map was established with
several restriction enzymes (Fig 1A). DNA
from control and Wsh/Wsh mice was
digested with the enzymes Pmel and Pmel+Notl, resolved by PFGE and then
transferred to a blotting membrane. Blots were hybridized with various
probes corresponding to Kit upstream sequences (Fig 1A). Probe
2 consisting of a Kpnl-Alul fragment located at 24 Kb
upstream from the Kit transcription start site, detected a Pmel
fragment of 160 Kb and a Pmel+Notl fragment of 90 kb in control DNA, in
contrast in Wsh/Wsh DNA smaller
fragments were detected (Fig 2A).
Hybridization of the same blot with probe 3 consisting of a
Xbal-Bglll fragment located at 106 kb from the Kit
transcription start site, identified the same restriction fragments in
control DNA as with probe 2, whereas in
Wsh/Wsh DNA two larger fragments were
detected (Fig 2B). These results suggested that the 3'
Wsh/Wsh breakpoint is located between
the two probes. To characterize the breakpoint more precisely, blots
containing control and Wsh/Wsh DNA
digested with several restriction enzymes and resolved by agarose gel
electrophoresis were hybridized with different probes derived from
sequences between the Alul-Kpnl and the XbaI-BglII probes. A 0.9 kb Hindlll-Accl fragment corresponding to
sequences at 72 kb from the Kit transcription start site
identified distinct Wsh/Wsh
EcoR1, HindIII, and AccI fragments defining the
Wsh breakpoint at 72 kb (Fig 1B, 2C). In
HindIII and AccI digested Wsh/Wsh DNA (Fig 2 C), two fragments were
detected that we presume to represent breakpoint fragments from both
the two endpoints of the inversion.
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| Fig 1.
Schematic representation of W/Kit upstream
region: (A) Restriction map of Kit upstream region. Sites for
the restriction enzymes Not1 (N), EcoR1 (E), XbaI (X),
SalI (S), MluI (M), ClaI (C ),
PmeI (P), HindIII (H), AccI (A),
Sac1 (Sc), BamH1 (B), PstI (Ps) are indicated.
Sites for restriction enzymes determined for the entire region are
shown without brackets. Sites for enzymes for which only a partial map
exists are indicated with brackets. Scale in kilobases is shown. The
relative position and size of overlapping P1 clones used to establish
this restriction map is shown below. (B) Scheme used for the
determination of the Wsh breakpoint. The relative
position of probes used is indicated. A 0.9 kb
HindIII-AccI fragment located at 72 kb was
used to identify Wsh breakpoint. (C)
Schematic of the identification of the W57
deletion endpoints located 34 to 38 kb and 146 to 147
kb upstream of the Kit transcription start site. The 0.6 kb
BamH1-PstI and the 0.8 kb Xbal-Xhol
probes used for the identification of the 5' and the 3',
respectively, deletion endpoints are indicated.
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| Fig 2.
Characterization of the 3' inversion breakpoint in
Wsh/Wsh DNA. (A, B) DNA from spleen
cells was prepared in agarose plugs, digested with the restriction
enzymes PmeI and PmeI+Not1 and resolved by PFGE. The blot was
hybridized sequentially with probes 2 and 3 (see Fig 1A), panels A and
B, respectively. In +/+ DNA both probes identified same-size
fragments. In contrast in Wsh DNA the two probes
identified two different fragments. (C) Hybridization of a blot
containing Wsh/Wsh and +/+ DNA
digested with EcoR1, HindIII, and AccI and
hybridized with the 0.9 kb HindIII-AccI probe (see
Fig1B) corresponding to sequences at 72 kb identified fragments of
distinct sizes in Wsh/Wsh. Size markers
are indicated.
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To identify the W57 deletion endpoints, PFGE and
regular Southern blots containing
W57/W57 and control DNA
digested with various enzymes were hybridized with a panel of probes
corresponding to upstream Kit sequences. A BamHl-Pstl
fragment corresponding to sequences at 148 kb identified distinct W57/W57 EcoR1 and
HindIII fragments defining the 5' deletion endpoint at
146147 kb (Fig 1C, 3A). An
Xbal-Xhol fragment corresponding to sequences at
34 kb identified the same polymorphic EcoRl fragment as
the 5' BamHl-Pstl probe (Figs 1C and 3B). Therefore, the
W57 3' deletion endpoint is located 34
to 38 kb from the Kit transcription start site. In
agreement with this conclusion, an XbaI-XbaI probe corresponding to sequences at 39 kb was unable to detect any fragments in W57 DNA.
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| Fig 3.
Characterization of deletion endpoints in
W57/W57 and Ph/+ DNA. (A)
Blot hybridization using a 0.6 kb BamH1-PstI probe
corresponding to sequences at 147 kb showed novel DNA fragments in
W57 DNA digested with the restriction enzymes
EcoR1 and HindIII (see Fig 1C). (B) Blot hybridization
using a 0.8 kb XbaI-Xhol probe corresponding to
sequences at 34 identified the same EcoRl fragment as the
BamHl-Pstl probe (see Fig1C). PFGE analysis of high molecular
weight DNA from +/+ and Ph/+ mice digested
with Not1. Sequential hybridization with probes 2 and 4 (see Fig 1) is
shown. Identical fragments were detected in Ph DNA with both
probes. Size markers are indicated.
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The Ph mutation arose as a result of a deletion of the PDGFRa chain
gene. The 3' endpoint of this deletion lies in between the PDGFRa
and the Kit genes, but its precise location is not known. To further
define the 3' Ph deletion endpoint, a PFGE blot containing Ph/+
and control DNA digested with Not I was hybridized with probe 2 and
then with probe 4, a Hindlll fragment corresponding to
sequences at 200 kb from the Kit transcription start (Fig 1A).
Both probes identified identical control and Ph restriction fragments, indicating that the 3' deletion endpoint in the
Ph allele is located more than 200 kb upstream of the
Kit transcription start site (Fig 3C).
Effect of the Wsh, W57, and
Ph mutations on Kit expression in mast cells and in
hematopoietic progenitors.
Although the Wsh and Ph mutations both
cause ectopic Kit expression in dermatomal cells during
embryonic development,9,11 the Wsh and
W57 mutations abolish9 or severely
diminish13 Kit expression in mast cells, and mutant mice
have a mast-cell deficit (Fig 4). It was
therefore of interest to determine whether the Ph mutation affects Kit expression in mast cells as well. Because homozygous Ph/Ph mutant mice die early during embryogenesis, we chose to analyze Kit RNA expression in BMMC derived from
Ph/Wsh mice. The levels of Kit RNA in
normal and mutant mast cells determined by Northern blot analysis are
shown in Fig 4. The Kit RNA levels in
Ph/Wsh mast cells are comparable to those
in +/+ and Ph/+ BMMC, implying that the Ph
mutation does not affect Kit expression in these cells.
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| Fig 4.
Expression of Kit RNA in mast cells from +/+,
Ph/+, Ph/Wsh, and
W57/W57 mice. Total RNA prepared from
bone marrow-derived mast cells was fractionated by gel electrophoresis
and analyzed by blot hybridization. Total cellular RNA (10 mg) was used
per lane and hybridization was performed with a Kit cDNA probe.
Hybridization with an actin probe is shown below to indicate equal
loading.
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Hematopoietic stem cells and progenitors of the lymphoid, myeloid, and
erythroid lineages, as well as mast cells, all express high levels of
the Kit gene products. Because Kit expression is abolished or
diminished in mast cells from Wsh and
W57 mutant mice, it was of interest to determine
whether Kit expression levels in hematopoietic stem cells and lineage
progenitors were affected in Wsh and
W57 mutant mice. For this purpose, Kit protein
expression levels in lineage minus Sca+ and
Sca bone marrow cells from Wsh,
W57, and C57Bl6/J mice were determined by FACS
(Fig 5). Although the size of the
Sca+, Kit+ and Sca,
Kit+ cell populations were comparable in the bone marrow
from Wsh, W57, and C57Bl6/J
mice, the mean fluorescent intensity of these cell populations was
diminished in both the samples from the Wsh and
W57 mutant mice. The relative levels of Kit
expression in the Wsh/Wsh
lin, Sca+, Kit+, and
lin, Sca, and Kit+
compared with normal C57Bl6 bone marrow cells was 76% and 58%, respectively, and in W57/W57
bone marrow cells, Kit expression levels were less reduced, 83% and
75%, respectively. These results imply that control elements affected
by these mutations are necessary to obtain normal levels of Kit
expression in hematopoietic stem cells and progenitor cell populations.
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| Fig 5.
Flow cytometric analysis of wild-type
(+/+), Wsh/Wsh, and
W57/W57 lin bone marrow
progenitor cells. Dot plots (top) displaying Sca-1 (PE) and Kit (FITC)
surface expression of lin bone marrow
progenitor cells from +/+ (left),
Wsh/Wsh (center), and
W57/W57 (right) mice. The number in
the top corner of quadrants indicates subpopulation percentages.
Histograms show Kit (FITC) surface expression in the Sca-1 fraction (middle) and the Sca-1+ fraction
(bottom) of lin bone marrow progenitor cells from
+/+ (left), Wsh/Wsh (center),
and W57/W57 (right) mice. The
number in the top right corner of histogram boxes indicates mean
fluorescence of the particular gate.
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Identification of DNase 1 hypersensitive sites in the vicinity of the
W57 deletion endpoints.
Because in W57/W57 mutant mice
Kit expression in mast cells is severely diminished (Fig
4),13 and in Wsh/Wsh mice
Kit expression is abolished completely,9 it seemed
reasonable to presume that mast cell control elements were located in
the vicinity of the W57 deletion endpoints. To
identify these elements, we performed a DNase1 hypersensitive site
analysis with nuclei isolated from normal BMMC, Kit-expressing
erythroid HCD57 cells, Kit-expressing melan-a cells (a melanocyte cell
line), and liver as a negative control for Kit expression.
Nuclei were digested with DNase 1 for increasing time periods and DNA
isolated from these nuclei was digested with different restriction
enzymes, and analyzed by blot hybridization using various probes. These
analyses showed one cluster of at least three hypersensitive sites
(HS1, HS2, HS3) upstream of the 5' W57
deletion endpoint, and a second cluster of three hypersensitive sites
(HS4, HS5, HS6) downstream of the 3' deletion endpoint in normal
BMMC (Figs 6 and 7) and
three hypersensitive sites (HSM1, HSM2, HSM3) in the melanocyte cell
line melan-a. To detect the 5' hypersensitive sites, we used
three different probes (Sal-Eco, Sal-Sac, and Eco-Eco) contained within
a 5.5 kb Sac fragment corresponding to sequences located at
147154 kb from the Kit transcription start site
(Fig 6). A Kpn-Alu probe corresponding to sequences located at
24 kb, identified the hypersensitive site HS4 in a Sacl
fragment, and the hypersensitive sites HS5 and HS6 in a BamHl fragment in DNA from BMMC (Fig 7). Analysis of nuclei from melan-a cells detected three new hypersensitive sites, HSM1-3, two sites, HSM2
and HSM3, in the 5' cluster at 150 kb, and one site, HSM1, in the 3' cluster at 24 kb (Fig 6 and 7). However, no
hypersensitive sites were detected when nuclei from Kit-expressing
erythroid HCD57 cells or Kit-negative liver cells were analyzed (Figs 6 and 7). Analysis of these upstream hypersensitive sites in mutant mast
cells enabled us to investigate their role in directing Kit expression in mast cells more precisely. Interestingly, DNase I
hypersensitive site analysis with nuclei from mutant
W57/W57 and
Wsh/Wsh BMMC indicated
that the 3' hypersensitive sites HS4, HS5, and HS6 were in a
closed configuration in both mutant BMMC and no hypersensitive site was
generated, suggesting that this hypersensitive site may be important
for mast cell-specific Kit expression (Fig 7). In contrast,
the 5' hypersensitive sites HS1, HS2, and HS3 in
W57/W57 BMMC remained in an
open configuration (Fig 6).
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| Fig 6.
Identification of DNase I hypersensitivity site cluster
at 147 154 kb in upstream Kit region. Nuclei were prepared from
+/+ BMMC, HCD57 cells, melan-a cells and liver, and digested with
DNaseI for increasing periods of time. Subsequently, DNA was restricted
with Sac1, fractionated by electrophoresis and analyzed by blot
hybridization using different probes. The Sal-Eco, Sal-Sac, and Eco-Eco
hybridization probes derived from a Sac1 restriction fragment at 147
154 kb detected a cluster of hypersensitive sites in mast cells
(BMMC) and two sites in melan-a cells in the vicinity of the 5'
W57 breakpoint. DNA from liver nuclei contained no
hypersensitive sites in this region. Size markers are indicated. A
schematic representation of the hypersensitive sites in the distal Kit
promoter region around 147 154 kb is shown below.
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| Fig 7.
Identification of DNase I hypersensitive sites at 23
28 kb in the upstream Kit region. Nuclei were prepared from +/+,
Wsh/Wsh and
W57/W57 BMMC and from Kit expressing
HCD57 and melan-a cells and liver, and digested with DnaseI. DNA was
restricted with the restriction enzymes Sac1 and BamH1 (as
indicated), fractionated by electrophoresis, and analyzed by blot
hybridization. A Kpnl-Alul probe detected one hypersensitive
site in the Sac1 fragment located at 23 28 kb in +/+ BMMC,
near the 3' W57 deletion endpoint. No
hypersensitive site was detected in DNA from W57
and Wsh BMMC and from liver nuclei. The same
Kpnl-Alul probe detected two hypersensitive sites in the
BamHl fragment located at 21 25 in +/+ that were
absent in W57 BMMC. Whereas in DNA from melan-a
cells HS4-6 were absent, a new hypersensitive site HSM1 was detected.
Horizontal arrows identify sub-bands generated by hypersensitive sites.
Size markers are indicated. A schematic representation of the
hypersensitive sites in the distal Kit promoter region around 21 to
28 kb is shown below.
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| |
DISCUSSION |
The Kit gene is expressed and functions in several
hematopoietic cell types, including lineage progenitors,
megakaryocytes, mast cells, gametogenesis, melanogenesis, neuronal cell
populations, and interstitial cells of Cajal. Thus, understanding of
the mechanisms controlling the temporal and spatial expression of
Kit both during embryogenesis and in the adult is of great
importance. In this study, we have determined the molecular basis of
three different W mutations known to affect Kit
expression differentially: Wsh,
W57, and Ph. Previous analysis of the
Wsh, W57, and Ph
mutations by PFGE showed that they were formed as a result of an
inversion of a 2 cmol/L segment of mouse chromosome 5 with one
breakpoint located between the Kit and the PDGFRa
genes, an 80-kb deletion located between the Kit and PDGFRa genes
and deletion of the entire PDGFRa receptor gene, respectively. Our
analysis localizes the Wsh breakpoint to 72
kb and the W57 deletion endpoints to
3438 and 146147 kb, respectively.
Furthermore, the Ph deletion endpoint was determined to be more
than 200 kb upstream of the kit transcription start site. In a
recent study, another pigmentation mutation Rw that is
associated with a 30 cmol/L inversion of mouse chromosome 5 was shown
to have a distal breakpoint located between 160 and 220
kb from the Kit transcription start site.36
Chromosomal deletions and/or rearrangements are known to cause position
effects and influence tissue-specific expression of genes over long
distances. Position effects may arise by several mechanisms. First,
juxtaposition of euchromatic (transcriptionally active) with
heterochromatic (transcriptionally silent) regions in chromosomes may
affect the expression of genes in the vicinity of breakpoints. Second,
position effects may arise as a result of a rearrangement whereby a
gene and its regulatory regions are placed next to regulatory elements
of another gene thus affecting its expression. Third, position effects
may interrupt cis-regulatory elements involved in the control of
tissue-specific gene expression.28 The phenotypes that
result from the chromosomal rearrangements and/or deletions found in
the Wsh, Wbd,
W57, Ph, and Rw. alleles are
consistent with cell-type-specific position-effect mutations.29 Thus, in these mutant mice, chromosomal
rearrangements or deletions may either remove part of the regulatory
region of the Kit gene or affect chromatin conformation
preventing the binding of trans-acting factors to cis-regulatory elements.
Previously, we showed ectopic Kit expression at distinct sites
during embryogenesis in Wsh and Ph mutant
mice.9,11 These observations suggested that in
Wsh, Wbd, and Ph mice
tissue-specific silencers involved in the regulation of Kit
expression during embryonic development were lost or inactivated. Based
on our mapping of the 3' extent of the Ph deletion, these negative control elements that are active during embryogenesis probably
lie more than 200 kb upstream from Kit. The presence of
positive tissue-specific regulatory elements is supported by the
finding that Kit expression is abolished or greatly diminished in mast cells derived from of Wsh, Wbd,
and W57 mutant mice. The positive elements
controlling Kit expression in mast cells could be located
between 3438 kb and 146147 kb and may be
affected in the Wsh, Wbd, and
W57 mutations. These positive elements are not
affected by the Ph mutation, because mast cells from Ph/+ and
Ph/Wsh mutant mice exhibit normal levels of
Kit RNA expression. The observation of a slight but significant
reduction of Kit expression in hematopoietic stem cells and
progenitors in both Wsh and W57
mutant mice further suggests that the upstream elements, affected by both the Wsh and the W57
mutations, are necessary for optimal Kit expression in these cells.
The chromatin in the vicinity of actively transcribed genes may be
altered, giving rise to short regions that are hypersensitive to
nucleases. We have used endonuclease DNase l sensitivity as a means to
identify and correlate changes in chromatin conformation in the
Kit upstream region with tissue-specific transcription of the
Kit gene. A cluster of at least three hypersensitive sites, HS1, HS2, and HS3 was identified between 147 and 154 kb
and another cluster of three hypersensitive sites, HS4, HS5, and HS6 between 23 and 28 kb upstream of the Kit
transcription start site, in Kit-expressing normal BMMC, but
not in Kit-expressing erythroid HCD57 cells and
melanocyte-derived melan-a cells and Kit-negative liver cells.
Interestingly, hypersensitive sites HS4, HS5, and HS6 were found to be
in a closed configuration in BMMC isolated from Wsh
and W57 homozygous mutant mice. These results
suggest that we have identified control elements that may be critical
for Kit expression in mast cells. Furthermore, we identified
three additional hypersensitive sites, HSM1, HSM2, and HSM3, in the
melanocyte cell line, melan-a, within the 5' and the 3'
hypersensitive site clusters. Because of their respective
localizations within the 5' and 3' hypersensitive site
clusters it is tempting to speculate that HSM1-3 have a role for
Kit expression in melanocytes.
The finding of tissue-specific hypersensitivity sites in the upstream
Kit region is reminiscent of the locus-control region (LCR) of
the  -globin locus. The globin LCR contains 4 hypersensitive sites
and directs position-independent and copy-number-dependent expression
of -globin transgenes.30,31,32 The hypersensitive sites
within the LCR have been ascribed distinct functions. HS3 directs an
activity that opens and remodels chromatin structure. Other sites
display enhancer activity.33,34 Furthermore, the factors
binding to these hypersensitive sites in the LCR synergize with each
other to produce optimal levels of -globin
expression.33,35 By analogy, the hypersensitive sites we
have identified may be part of a LCR that controls Kit
expression in mast cells. In Wsh the 5'
hypersensitive site region is displaced, hypersensitive sites HS4, HS5,
and HS6 are in a closed configuration and Kit expression in
mast cells is abolished, wherease in W57 the
5' hypersensitive sites (HS1, HS2, and HS3) are in an open configuration, the 3' hypersensitive sites HS4, HS5, and HS6 are in a closed configuration in mast cells and Kit expression in mast cells is diminished. Hypersensitive site HS4, HS5, and HS6 may be
in a closed configuration, in both the Wsh and the
W57 mutations, because they require an interaction
with upstream elements located in the displaced chromosomal segments to
mediate Kit expression in mast cells. In addition, these
results imply that the 5' and the 3' hypersensitive sites
in the Kit upstream region may function in synergy to produce
optimal Kit expression in mast cells. Future studies will be
aimed at the elucidation of the nature of the hypersensitive sites
HS1-HS6 and the mechanism by which they control Kit expression.
| |
ACKNOWLEDGMENT |
We thank Drs Frederic Gilles and Andre Goy for helpful technical advice
and discussions and Dr Katia Manova for mutant mice and discussions, Dr
Jeffrey Ravetch for letting us use his FACS machine and discussions,
and Drs Dale Dorsett, Rosemary Bachvarova, and Elizabeth Lacy for
numerous stimulating discussions. We thank Dr Dot Bennett for the
melan-a cells, Dr Harvey Lodish for HCD57 cells, and Dr Shin-Ichi
Nishikawa for a gift of ACK2 MoAb. We also thank Rose Manziano for her
dedicated help in the preparation of many reagents.
| |
FOOTNOTES |
Submitted January 22, 1999; accepted June 9, 1999.
Supported by grants from the National Institutes of Health and the
National Cancer Institute, R37 CA32926 and RO1-HL/DK55748 (P.B.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Peter Besmer, PhD, Memorial Sloan-Kettering
Cancer Center, 1275 York Ave, New York, NY 10021; e-mail:
p-besmer{at}ski.mskcc.org.
| |
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