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HEMATOPOIESIS
From the Department of Pathology and Laboratory of
Molecular Pathology, University of Texas Southwestern Medical Center,
Dallas; Division of Hematology/Oncology, Children's Hospital,
Department of Pediatrics, Harvard Medical School, Boston, MA; and
Department of Microbiology and Institute for Cellular and Molecular
Biology, University of Texas at Austin.
CCAAT displacement protein (cux/CDP) is an atypical homeodomain
protein that represses expression of several developmentally regulated
lymphoid and myeloid genes in vitro, including gp91-phox, immunoglobulin heavy chain, the T-cell receptor The development and function of the immune
system are controlled, in part, by the regulation of tissue-specific
gene expression. Orchestration of gene expression is modulated by a
combination of both positively and negatively acting transcription
factors. One such repressive factor is human CCAAT displacement protein (CDP), which belongs to a novel family of homeodomain proteins that
appear to bind DNA through repeated domains termed
"cut-repeats."1,2 CDP was initially identified as a
transcriptional repressor of the sea urchin sperm histone
H2B gene,3 and homologues include cux/CDP from
mouse,4 Clox from canine,5 cut from
Drosophila,6 and CDP from rat.7
Each of these 180- to 190-kd homologues contains 3 cut-repeats (CRs) in
addition to an atypical homeodomain (HD) located near the
carboxy-terminus. CDP requires cooperative interaction between either 2 CR domains or one CR domain and the homeodomain for DNA
binding.8,9 CDP preferentially recognizes AT-rich DNA
sequences9 that are often associated with nuclear matrix
association regions (MARs; reviewed by Scheuermann and Garrard10). CDP forms homomeric complexes in
coimmunoprecipitation experiments, presumably through a coiled-coil
domain located near the amino-terminus.11 A C-terminal
repression domain appears to associate with histone
deacetylases.12,13
CDP appears to play vital roles in regulating genes involved in
multilineage differentiation pathways. This has been exemplified in
Drosophila, where cell fate determination in multiple
lineages is altered by ectopic expression of
cut.14 The essential role of cut in
controlling normal development is demonstrated by the observation that
null mutations result in embryonic lethality.15,16 Within
mammalian systems CDP and its homologues function as repressors of a
wide variety of different genes within multiple systems (Wang and
colleagues17 and references therein). The binding of CDP homologues to promoters, enhancers, or MARs of tissue-specific genes
appears to be limited to developmental stages where target genes are
not expressed. Target sites for cux/CDP binding and repression have
been identified within myeloid- and lymphoid-specific genes and include
the gp91-phox promoter,18 neutrophil collagenase promoter,19 lactoferrin promoter,20
neutrophil gelatinase promoter,19 CD8a MAR,21
T-cell receptor cux/CDP appears to function through multiple mechanisms. In some cases,
in vitro studies indicate that cux/CDP is able to compete for
transcriptional activator binding, like BID/YY1, CP1, and IRF factors
in the gp91-phox promoter23,24 and Bright in the IgH
enhancer/MAR and promoter.17 Other experiments suggest cux/CDP might repress gene expression through higher-order changes in
chromatin structure including histone deacetylation.13
Many of the characterized cux/CDP binding sites also function as
nuclear matrix attachment sites.10,22,25 cux/CDP binding
can inhibit binding of MAR-containing DNA fragments to nuclear matrix
preparations in vitro,26 suggesting that cux/CDP may
influence gene expression by regulating nuclear localization.
To determine the roles of cux/CDP in controlling cell lineage
determination and function in vivo, a cux/CDP allele with a C-terminal deletion encompassing the homeodomain and repression domain
was generated using homologous gene targeting approaches in mice.
Mutation of the cux/CDP protein affects several organ systems,
including the hematolymphoid compartment, through cell-intrinsic and
environmental effects. Our data suggest that cux/CDP functions to
modulate the expression of lymphoid survival and/or death-inducing factors, which may include tumor necrosis factor (TNF).
Generation of the
To generate cux/CDP targeted embryonic stem cell
(ES) clones, the construct was linearized with NotI and
ligated to hairpin oligos to protect the ends from exonuclease
digestion. The construct was electroporated into the ES cell line CJ7
and G418 (200 µg/mL) and gancyclovir (2 µM) double-resistant clones
were isolated. Targeting was confirmed by a Southern blot of
BamHI digested DNA, probed with a 3-kb HindIII
probe, with a diagnostic band of 4.5 kb indicating the presence of a
targeted allele. Two clones were identified, which had homologously
recombined the targeting construct into the homeodomain and both had a
normal diploid karyotype (2n = 40 chromosomes). One clone was
selected for injection into C57BL/6J blastocysts, which were
transferred to 2.5-day pseudopregnant females. Chimeras were evaluated
for contribution of coat color by the ES cell agouti allele. Chimeras
with high ES cell contribution were backcrossed to C57BL/6J females.
The genotypes of offspring were determined by Southern blot and
polymerase chain reaction (PCR).
The ES clones homozygous for the targeted allele were generated from
heterozygous clones by increasing G418 concentrations to 2.8 mg/mL
(Figure 1B, lanes 2 and 3) and one was selected for injection into
RAG-2-deficient blastocysts to analyze the effect of the mutation on
lymphoid differentiation.
PCR analysis of mice genotypes
Mice containing a germline rearranged V(D)J segment of Ig heavy chain
specific for hapten 4-hydroxy-3-nitrophenyl28 were crossed
to the cux/CDP+/ Immunoblotting
Histochemistry and TUNEL assays Tissues for histochemistry were fixed in freshly prepared 4% paraformaldehyde in PBS overnight, embedded in paraffin wax, sectioned at 8 µm and counterstained with hematoxylin and eosin.Thymuses for TUNEL assays were cryopreserved and cryosectioned at 8 µm. TUNEL assays were performed according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN). Sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA) containing 1 µg/mL DAPI (Sigma) before fluorescence microscopy. Flow cytometry of hematopoietic organs Thymus and spleen single-cell suspensions from 2- to 5-week-old mice were obtained by grinding and filtration through nylon mesh into FACS buffer (PBS, 2% fetal bovine serum [FBS], 0.01% sodium azide, 2 mM EDTA). Marrows from femurs were flushed and cells suspended by pipetting. Cells were pelleted and resuspended in FACS buffer; then 1 to 2 × 106 cells were stained with the following antibodies: anti-B220 (RA3-6B2), anti-c-Kit (2B8), anti-CD25 (PC615.3 or 7D4), anti-IgM (polyclonal), anti-Mac-1 (M1/70), anti-CD43 (S7), anti-CD61 (2C9.G2), anti-CD4 (CT-CD4), anti-CD8 (53-6.7), anti-CD44 (IM7), and anti-CD3 (145-2C11). Cells not staining with
anti-Gr-1 (RB6-8C5), anti-Mac-1 (M1/70), anti-CD19 (1D3), anti-CD4
(H129.19), anti-CD8 (53-6.7), and anti-CD3 (145-2C11) were
defined as lineage negative in thymus.
In reconstitution experiments, antibodies anti-CD45.1 (A20) and
anti-CD45.2 (104) were used to distinguish between recipient Ly-5.1
(CD45.1) and donor Ly-5.2 (CD45.2) alloantigens, respectively. The
presence of the V Annexin V stain of apoptotic B cells was performed using an annexin V fluorescein isothiocyanate (FITC) kit (Oncogene, San Diego, CA) according to the manufacturer's instructions. Cells were stained with anti-B220-APC (RA3-6B2) antibody at the same time as annexin V in the supplied binding buffer. Most antibodies were directly conjugated with fluorochromes. In some instances biotin conjugates were used, which required a secondary layer of streptavidin-PerCP (Pharmingen, San Diego, CA). All antibodies were purchased from Pharmingen except anti-IgM (polyclonal), anti-CD25 (PC615.3), and anti-CD4 (CT-CD4), which were purchased from Caltag (Burlingame, CA). Flow cytometry was performed using the FACS Calibur (Becton Dickinson, San Jose, CA) and analysis restricted to viable cells defined by forward and side scatter. Colony-forming assays For myeloid/erythroid colony-forming assays, 2 × 105 bone marrow cells were seeded in methylcellulose containing erythropoietin (Epo, 2 U/mL) plus stem cell factor (SCF, 250 ng/mL) for erythroid burst-forming units (BFU-Es), or granulocyte-macrophage colony-stimulating factor (GM-CSF, 15 U/mL) plus interleukin-3 (IL-3) (15 U/mL) for granulocyte-macrophage colony-forming units (CFU-GMs). Cells were plated in triplicate and cultures were incubated in a fully humidified incubator with 5% CO2 before scoring by morphology after 14 days.Pre-BII cell colony-forming assays were performed by culturing 2 × 104 bone marrow cells in 0.9% base methylcellulose (Stem Cell Technologies, Vancouver, BC, Canada) containing 30% FBS (Stem Cell Technologies), 1:20 dilution of conditioned media containing IL-7 (kindly provided by Steven Bauer), 2 mM L-glutamine (Gibco BRL, Rockville, MD), and 0.1 mM 2-mercaptoethanol (Gibco BRL) in Iscoves modified Dulbecco medium (IMDM; Gibco BRL). Cultures were plated in duplicate and were incubated for 7 days (as above) prior to scoring of colonies by morphology. Frequency determination of pro-B/pre-BI colony-forming cells was performed by limiting dilution analysis. Stromal line ST231 was cultured at 3 × 104 cells/mL in 96-well dishes for 2 days in IMDM (Gibco BRL) with 10% FBS, 100 U penicillin, and 100 µg streptomycin antibiotics per milliliter and 50 µM 2-mercaptoethanol. Semiconfluent stroma plates were irradiated at 3000 rads and bone marrow cells added at limiting dilution (range, 150-0.62 cells/well) in media containing a 1:20 dilution of IL-7-conditioned media (equivalent to 100-200 U/mL). Cocultures were scored for the growth of lymphocyte pro-B/pre-BI colonies containing more than 10 cells after 7 days. The frequency of pro-B/pre-BI colony precursors was determined at the value of 37% negative wells when the fraction of negative wells was plotted against cells/well, according to the Poisson distribution.32 Bone marrow reconstitution Femurs and tibias were flushed with Hanks buffered saline solution (HBSS; Gibco BRL) containing 5% FBS. Viable cell concentration was adjusted to 5 × 106 cells/mL in HBSS with 0.5% FBS. Recipient 8- to 15-week-old C57BL/6-SJL mice, congenic for Ly-5.1 alloantigen, were irradiated with 950 rads (2 doses of 475 rads separated by 3 hours) from a 137Cs source and 1 × 106 bone marrow cells were injected into the lateral tail vein. Mice were housed in specific pathogen-free (SPF) facilities and were provided with neomycin sulfate (1.1 g/L) in drinking water. Contributions of donor cells to the reconstituted hematopoietic system were determined by flow cytometry. In most cases, reconstitution with donor cells was over 80%.TNF enzyme-linked immunosorbent assays Cells from bone marrow, spleen, and thymus were cultured at 5 × 106 cells/mL. Levels of secreted TNF in supernatants were measured using a mouse-specific TNF enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Biosource, Camarillo, CA). Bone marrow culture media was IMDM, 10% FBS, 100 U penicillin, and 100 µg streptomycin per milliliter, and 50 µM 2-mercaptoethanol. Thymocytes and splenocytes were cultured in Dulbecco modified Eagle medium (DMEM)-high glucose, 10% FBS, 55 µM 2-mercaptoethanol, 100 U penicillin and 100 µg streptomycin per milliliter, and 0.1 mM nonessential amino acids supplemented with concanavalin A (ConA; Sigma) at 6.25 mg/L. All media supplies were from Gibco BRL. Protein lysates were produced by homogenizing approximately 0.1 g tissue in 500 µL lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 µg/mL aprotinin, 10 µg/mL leupeptin, 50 mM NaF, 1 mM sodium orthovanadate, and 2% NP40) placed on ice for 30 minutes with vortexing every 10 minutes. Nuclei were pelleted at 14 000 rpm at 4°C for 10 minutes and supernatants were frozen in aliquots. Protein concentrations were determined as previously described. Equal amounts of protein per set of cux/CDP HD/HD and control tissues (range,
4.5-370 µg) were evaluated for TNF expression by ELISA.
Generation of the cux/CDP HD/HD) mutant mice were
present at 5.4% rather than the expected mendelian ratio of 25% (1219 mice analyzed). Genotype analysis of fetuses at 16 to 17 days found a
normal mendelian ratio (28%) of homozygous mutants, demonstrating that
death occurs at or shortly after birth.
Because the cux/CDP homeodomain was targeted for truncation, we
investigated whether a truncated protein was synthesized. An immunoblot
of thymic nuclear extracts from wild-type (+/+), heterozygous
(+/ cux/CDP HD/HD mice are
distinguishable from their heterozygous and wild-type (control)
littermates as early as 3 to 4 days postpartum due to their reduced
size and skin pallor. These mice have difficulty thriving, have a
reduced stature, gain little weight, have wavy whiskers, and lose fur
between 2 and 3 weeks of age (Figure
2A-B). A sexual dimorphism in mutant mice
viability was observed with 89.4% of the mutants surviving neonatal
lethality being phenotypically male. Few mice lived beyond 4 weeks of
age. Anatomic analysis of cux/CDPHD/HD
mice revealed that the size and cellularity of the thymus was dramatically reduced in comparison with control littermates (Figure 2C
and Table 1) with a preferential loss of
thymic cortex (Figure 2E). Furthermore, these mice suffered from
cachexia due to muscle wasting (Figure 2D) and loss of body fat,
including subcutaneous fat (Figure 2E). Bones appeared thin and flaky
(Figure 2E) and the hair follicles within skin appeared greater in
number and somewhat disorganized (Figure 2E). No obvious explanation
for the neonatal lethality was apparent by histologic examination of
vital organs.
cux/CDP  HD mutation on
lymphopoiesis and myelopoiesis by flow cytometry in mice that survived the neonatal period. Analysis of mutant bone marrow with pan-B lineage-specific marker B220 identified a 2- to 3-fold reduction in the
percentage and absolute numbers of B cells when compared to littermate
controls (Figure 3A and Table 1).
Analysis of c-Kit (Figure 3Ai-ii,), CD25 (Figure 3Aiii-iv),
and IgM (Figure 3Av-vi) within the B220 population to identify
pro-B/pre-BI, pre-BII and immature/mature B-lineage cells indicated
that the percentages of all B-lineage subpopulations were reduced in
mutant bone marrow (Table 1). This overall reduction was also observed
in pro-B/pre-BI and pre-BII colony-forming assays (Figure 3B). Although
all B-lineage subsets were reduced in number to some degree, the
CD25+ pre-BII population was preferentially affected. When
a B220+ gate was used, the CD25+ population was
reduced 2-fold in comparison with other B-lineage populations (Figure
3C). Decreases in these B-lineage populations could arise due to
suppressed differentiation or proliferation, or enhanced
apoptosis.
In the thymus, an average 5-fold reduction (1.2- to 64-fold range,
n = 17) in the number of thymocytes per gram body weight was observed
(Table 1). Although each of the CD4/CD8 populations were reduced in
number, the CD4+CD8+ population was
preferentially affected (Figure 3Axiii-xiv, and Table 1). Within the
lin In normal mice, the early expression of CD25 disappears in the
CD4 Reductions in lymphoid populations at the pre-BII and
CD4+CD8+ stages of development could arise if
cux/CDP In contrast to the decrease in lymphocytes in
cux/CDP Cell-intrinsic and environmental effects cause hematopoietic
defects in cux/CDP HD/HD mice could be due to
cell-intrinsic or microenvironmental influences. To distinguish between
these possibilities, bone marrow cells from
cux/CDPHD/HD mice and littermate controls
were transplanted into lethally irradiated C57BL/6-SJL recipients.
Reconstitution was assayed at 4 and 8 weeks and 10 to 11 months after
transplantation. Donor cells were distinguished from recipient cells
using antibodies for the Ly-5 alloantigens.
At all time points, the total numbers and percentages of
B220+ B-cells in bone marrow were reduced in mice
reconstituted with cux/CDP
Thymuses from mice reconstituted with
cux/CDP Bone marrow myeloid cells positive for Mac-1+ (Figure 4 and
Table 2), CD43+ (not shown) or CD61+ (not
shown) but negative for B220, were again increased in percentage in
mice reconstituted with cux/CDP Levels of thymic and bone marrow B-cell apoptosis are elevated in
cux/CDP HD/HD mice,
we sought to determine whether these effects were due to enhanced cell
death. TUNEL assays on cux/CDPHD/HD thymus
sections indicated that the level of apoptosis was dramatically increased in the thymus (Figure 5A-C).
Furthermore, analysis of B220+ B cells in bone marrow by
annexin V staining demonstrated that B-cell apoptosis was elevated 2- to 3-fold in primary cux/CDPHD/HD mice
(Figure 5D-E). No difference was seen between the percentages of
annexin V+ cells in the B220 control and
cux/CDPHD/HD bone marrow populations
indicating that the enhanced apoptosis was confined to the B-cell
compartment (data not shown). Therefore, enhanced cell death is
responsible for thymic atrophy and loss of bone marrow B cells in
primary cux/CDPHD/HD mice.
TNF levels are elevated in
cux/CDP HD/HD bone marrow, our data
indicated that microenvironmental effects were responsible for the
thymic phenotype, suggesting that cux/CDP might be repressing
expression of a death-inducing factor or stimulating the expression of
a survival factor in normal mice. Many hematopoietic and
nonhematopoietic anomalies in the
cux/CDPHD/HD mice are similar to those
observed under specific conditions of TNF overexpression, particularly
the alopecia, cachexia, lymphopenia, and myeloid
hyperplasia.33,34 Therefore, we hypothesized that overexpression of TNF may be contributing to these abnormalities. To
determine whether hematopoietic organs were overexpressing TNF, bone
marrow cells, splenocytes, and thymocytes from
cux/CDPHD/HD and littermate controls were
cultured and levels of TNF secretion quantified by ELISA. Significantly
higher levels of TNF were observed in culture supernatants from
cux/CDPHD/HD bone marrow (1.5- to 2-fold
at 72 hours) and thymus (3- to 4-fold) (Figure
6A). TNF levels were also elevated in
protein extracts from hematopoietic and nonhematopoietic tissues.
Although variable, TNF protein levels were significantly higher in
cux/CDPHD/HD liver, lung, and thymus
relative to control tissues (Figure 6B). TNF levels in brain, muscle,
spleen, bone marrow, small intestine, heart, kidney, skin, and stomach
were not significantly different from control tissues (Figure 6B and
data not shown). Thymuses from
cux/CDPHD/HD mice expressed the highest
levels of TNF, approximately 3- to 7-fold higher than controls (Figure
6B). These data suggest that TNF could be a target gene for
cux/CDP-mediated repression and that TNF overexpression in
hematopoietic and nonhematopoietic tissues could be partly responsible
for the phenotypes observed in
cux/CDPHD/HD animals.
In this study we demonstrate that mice expressing low levels of a truncated cux/CDP protein have a complex phenotype, including partial neonatal lethality, runting, wavy fur and whiskers with latent alopecia, and muscle wasting with loss of body fat akin to cachexia. This complex phenotype is consistent with a role for cux/CDP in the transcriptional regulation of multiple target genes. Our findings also establish a pivotal role for cux/CDP in B and T
lymphopoiesis and myelopoiesis. B and T lymphoid demise could be
explained by several abnormalities Clonal expansion of developing lymphocytes occurs after productive
rearrangement of the IgH chain in B-lineage cells and TCR Our data strongly suggest that cell death accounts for lymphopenia in
cux/CDP Due to the pleiotropic abnormalities observed in
cux/CDP In contrast to the lymphopenia seen in
cux/CDP Due to the complex phenotype of the
cux/CDP Although the deletion of the homeodomain and C-terminal repression
domain has dramatic effects on protein function, the remaining cut
repeats in the cux/CDP We have shown that cux/CDP may play a role in repressing thymic TNF
expression and CD25 expression in thymocytes undergoing selection.
Indeed, CDP overexpression studies in a myeloid cell line has
identified that CDP can repress lipopolysaccharide-induced TNF
expression (unpublished observation, 2001). Consistent with the notion that CD25 may be a target gene for cux/CDP-mediated repression, the DNA binding activity of cux/CDP is up-regulated in
CD4+CD8+ thymocytes,22,52 a point
at which CD25 expression is down-modulated. However, it is unclear if
cux/CDP has a direct effect on CD25 transcription because CD25
expression can be induced by cytokines, including IL-1 and TNF, on
thymocyte precursors.53 It is possible that these or other
factors may directly or indirectly induce ectopic CD25 expression in
cux/CDP
We thank Fred Alt and Barry Sleckman for assistance with the
RAG-2
Submitted March 22, 2001; accepted August 10, 2001.
Supported by Public Health Service grants GM50329 (R.H.S.), HL49196 (E.N.J.), and CA31534 and GM31689 (P.W.T.). E.J.N. is a fellow of the Lucille P. Markey Foundation.
A.M.S., J.A.L., and A.G. contributed equally to the work in this article.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Richard H. Scheuermann, Department of Pathology and Laboratory of Molecular Pathology, 5323 Harry Hines Blvd, Dallas, TX 75390-9072; e-mail: scheuerm{at}utsw.swmed.edu.
1.
Aufiero B, Neufeld EJ, Orkin SH.
Sequence-specific DNA binding of individual cut repeats of the human CCAAT displacement/cut homeodomain protein.
Proc Natl Acad Sci U S A.
1994;91:7757-7761
2.
Harada R, Dufort D, Denis-Larose C, Nepveu A.
Conserved cut repeats in the human cut homeodomain protein function as DNA binding domains.
J Biol Chem.
1994;269:2062-2067 3. Barberis A, Superti-Furga G, Busslinger M. Mutually exclusive interaction of the CCAAT-binding factor and of a displacement protein with overlapping sequences of a histone gene promoter. Cell. 1987;50:347-359[CrossRef][Medline] [Order article via Infotrieve].
4.
Valarche I, Tissier-Seta JP, Hirsch MR, Martinez S, Goridis C, Brunet JF.
The mouse homeodomain protein Phox2 regulates Ncam promoter activity in concert with Cux/CDP and is a putative determinant of neurotransmitter phenotype.
Development.
1993;119:881-896 5. Andres V, Nadal-Ginard B, Mahdavi V. Clox, a mammalian homeobox gene related to Drosophila cut, encodes DNA-binding regulatory proteins differentially expressed during development. Development. 1992;116:321-334[Medline] [Order article via Infotrieve]. 6. Neufeld EJ, Skalnik DG, Lievens PM, Orkin SH. Human CCAAT displacement protein is homologous to the Drosophila homeoprotein, cut. Nat Gen. 1992;1:50-55[CrossRef][Medline] [Order article via Infotrieve].
7.
Yoon SO, Chikaraishi DM.
Isolation of two E-box binding factors that interact with the rat tyrosine hydroxylase enhancer.
J Biol Chem.
1994;269:18453-18462
8.
Moon NM, Bérubé G, Nepveu A.
CCAAT displacement activity involves CUT repeats 1 and 2, not the CUT homeodomain.
J Biol Chem.
2000;275:31325-31334 9. Harada R, Berube G, Tamplin OJ, Denis-Larose C, Nepveu A. DNA-binding specificity of the cut repeats from the human cut-like protein. Mol Cell Biol. 1995;15:129-140[Abstract]. 10. Scheuermann RH, Garrard WT. MARs of antigen receptor and co-receptor genes. Crit Rev Eukaryot Gene Expr. 1999;9:295-310[Medline] [Order article via Infotrieve]. 11. Webb C, Zong T-T, Lin D, et al. Differential regulation of immunoglobulin gene transcription via nuclear matrix-associated regions. Cold Spring Harb Symp Quant Biol. 1999;64:109-118[CrossRef][Medline] [Order article via Infotrieve]. 12. Mailly F, Berube G, Harada R, Mao PL, Phillips S, Nepveu A. The human cut homeodomain protein can repress gene expression by two distinct mechanisms: active repression and competition for binding site occupancy. Mol Cell Biol. 1996;16:5346-5357[Abstract].
13.
Li S, Moy L, Pittman N, et al.
Transcriptional repression of the cystic fibrosis transmembrane conductance regulator gene, mediated by CCAAT displacement protein/cut homolog, is associated with histone deacetylation.
J Biol Chem.
1999;274:7803-7815
14.
Blochlinger K, Jan LY, Jan YN.
Transformation of sensory organ identity by ectopic expression of cut in Drosophila.
Genes Dev.
1991;5:1124-1135 15. Bodmer R, Barbel S, Sheperd S, Jack JW, Jan LY, Jan YN. Transformation of sensory organs by mutations of the cut locus of D melanogaster. Cell. 1987;51:293-307[CrossRef][Medline] [Order article via Infotrieve]. 16. Jack J, Dorsett D, Delotto Y, Liu S. Expression of the cut locus in the Drosophila wing margin is required for cell type specification and is regulated by a distant enhancer. Development. 1991;113:735-747[Abstract].
17.
Wang Z, Goldstein A, Zong RT, et al.
Cux/CDP homeoprotein is a component of NF-muNR and represses the immunoglobulin heavy chain intronic enhancer by antagonizing the bright transcription activator.
Mol Cell Biol.
1999;19:284-295
18.
Skalnik DG, Strauss EC, Orkin SH.
CCAAT displacement protein as a repressor of the myelomonocytic-specific gp91-phox gene promoter.
J Biol Chem.
1991;266:16736-16744
19.
Lawson ND, Khanna-Gupta A, Berliner N.
Isolation and characterization of the cDNA for mouse neutrophil collagenase: demonstration of shared negative regulatory pathways for neutrophil secondary granule protein gene expression.
Blood.
1998;91:2517-2524
20.
Khanna-Gupta A, Zibello T, Kolla S, Neufeld EJ, Berliner N.
CCAAT displacement protein (CDP/cut) recognizes a silencer element within the lactoferrin gene promoter.
Blood.
1997;90:2784-2795
21.
Banan M, Rojas IC, Lee WH, et al.
Interaction of the nuclear matrix-associated region (MAR)-binding proteins, SATB1 and CDP/Cux, with a MAR element (L2a) in an upstream regulatory region of the mouse CD8a gene.
J Biol Chem.
1997;272:18440-18452
22.
Chattopadhyay S, Whitehurst CE, Chen J.
A nuclear matrix attachment region upstream of the T cell receptor beta gene enhancer binds Cux/CDP and SATB1 and modulates enhancer-dependent reporter gene expression but not endogenous gene expression.
J Biol Chem.
1998;273:29838-29846
23.
Luo W, Skalnik DG.
CCAAT displacement protein competes with multiple transcriptional activators for binding to four sites in the proximal gp91phox promoter.
J Biol Chem.
1996;271:18203-18210
24.
Jacobsen BM, Skalnik DG.
YY1 binds five cis-elements and trans-activates the myeloid cell-restricted gp91(phox) promoter.
J Biol Chem.
1999;274:29984-29993
25.
Scheuermann RH, Chen U.
A developmental-specific factor binds to suppressor sites flanking the immunoglobulin heavy-chain enhancer.
Genes Dev.
1989;3:1255-1266
26.
Zong RT, Scheuermann RH.
Mutually exclusive interaction of a novel matrix attachment region binding protein and the NF-muNR enhancer repressor: implications for regulation of immunoglobulin heavy chain expression.
J Biol Chem.
1995;270:24010-24018 27. Tufarelli C, Fujiwara Y, Zappulla DC, Neufeld EJ. Hair defects and pup loss in mice with targeted deletion of the first cut repeat domain of the Cux/CDP homeoprotein gene. Dev Biol. 1998;200:69-81[CrossRef][Medline] [Order article via Infotrieve]. 28. Cascalho M, Ma A, Lee S, Masat L, Wabl M. A quasi-monoclonal mouse. Science. 1996;272:1649-1652[Abstract]. 29. Loh DY, Bothwell AL, White-Scharf ME, Imanishi-Kari T, Baltimore D. Molecular basis of a mouse strain-specific anti-hapten response. Cell. 1983;33:85-93[CrossRef][Medline] [Order article via Infotrieve].
30.
Yui K, Komori S, Katsumata M, Siegel RM, Greene MI.
Self-reactive T cells can escape clonal deletion in T-cell receptor V beta 8.1 transgenic mice.
Proc Natl Acad Sci U S A.
1990;87:7135-7139 31. Ogawa M, Nishikawa S, Ikuta K, Yamamura F, Naito M, Takahashi K. B cell ontogeny in murine embryo studied by a culture system with the monolayer of a stromal cell clone, ST2: B cell progenitor develops first in the embryonal body rather than in the yolk sac. EMBO J. 1988;7:1337-1343[Medline] [Order article via Infotrieve]. 32. Henry C, Marbrook J, Vann DC, Kodlin D, Wofsy C. Limiting dilution analysis. In: Lefkovits I, ed. Limiting Dilution of Cells in the Immune System. Cambridge United Kingdom: Cambridge University Press; 1979:138-152.
33.
Zhang X, Ren R.
Bcr-Abl efficiently induces a myeloproliferative disease and production of excess interleukin-3 and granulocyte-macrophage colony-stimulating factor in mice: a novel model for chronic myelogenous leukemia.
Blood.
1998;92:3829-3840 34. Glosli H, Veiby OP, Fjerdingstad H, et al. Effects of hTNFalpha expression in T cells on haematopoiesis in transgenic mice. Eur J Haematol. 1999;63:50-60[Medline] [Order article via Infotrieve]. 35. Shinkai Y, Rathbun G, Lam KP, et al. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell. 1992;68:855-867[CrossRef][Medline] [Order article via Infotrieve]. 36. Urbanek P, Wang ZQ, Fetka I, Wagner EF, Busslinger M. Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell. 1994;79:901-912[CrossRef][Medline] [Order article via Infotrieve]. 37. Vassalli P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol. 1992;10:411-452[CrossRef][Medline] [Order article via Infotrieve]. 38. Ware CF, VanArsdale S, VanArsdale TL. Apoptosis mediated by the TNF-related cytokine and receptor families. J Cell Biochem. 1996;60:47-55[CrossRef][Medline] [Order article via Infotrieve]. 39. Lewis S, Abrahamson G, Boultwood J, Fidler C, Potter A, Wainscoat JS. Molecular characterization of the 7q deletion in myeloid disorders. Br J Haematol. 1996;93:75-80[CrossRef][Medline] [Order article via Infotrieve].
40.
Johnson EJ, Scherer SW, Osborne L, et al.
Molecular definition of a narrow interval at 7q22.1 associated with myelodysplasia.
Blood.
1996;87:3579-3586
41.
Fischer K, Frohling S, Scherer SW, et al.
Molecular cytogenetic delineation of deletions and translocations involving chromosome band 7q22 in myeloid leukemias.
Blood.
1997;89:2036-2041
42.
Liang H, Fairman J, Claxton DF, Nowell PC, Green ED, Nagarajan L.
Molecular anatomy of chromosome 7q deletions in myeloid neoplasms: evidence for multiple critical loci.
Proc Natl Acad Sci U S A.
1998;95:3781-3785 43. Tosi S, Scherer SW, Giudici G, Czepulkowski B, Biondi A, Kearney L. Delineation of multiple deleted regions in 7q in myeloid disorders. Genes Chromosomes Cancer. 1999;25:384-392[CrossRef][Medline] [Order article via Infotrieve]. 44. Zeng WR, Watson P, Lin J, et al. Refined mapping of the region of loss of heterozygosity on the long arm of chromosome 7 in human breast cancer defines the location of a second tumor suppressor gene at 7q22 in the region of the CUTL1 gene. Oncogene. 1999;18:2015-2021[CrossRef][Medline] [Order article via Infotrieve]. 45. Ishwad CS, Ferrell RE, Hanley K, et al. Two discrete regions of deletion at 7q in uterine leiomyomas. Genes Chromosomes Cancer. 1997;19:156-160[CrossRef][Medline] [Order article via Infotrieve]. 46. Zeng WR, Scherer SW, Kkoutsilieris M, et al. Loss of heterozygosity and reduced expression of the CUTL1 gene in uterine leiomyomas. Oncogene. 1997;14:2355-2365[CrossRef][Medline] [Order article via Infotrieve].
47.
He LZ, Tribioli C, Rivi R, et al.
Acute leukemia with promyelocytic features in PML/RARalpha transgenic mice.
Proc Natl Acad Sci U S A.
1997;94:5302-5307
48.
Fernex C, Caillol D, Capone M, Krippl B, Ferrier P.
Sequences affecting the V(D)J recombinational activity of the IgH intronic enhancer in a transgenic substrate.
Nucleic Acids Res.
1994;22:792-798
49.
Angelin-Duclos C, Calame K.
Evidence that immunoglobulin VH-DJ recombination does not require germ line transcription of the recombining variable gene segment.
Mol Cell Biol.
1998;18:6253-6264
50.
Bories JC, Demengeot J, Davidson L, Alt FW.
Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.
Proc Natl Acad Sci U S A.
1996;93:7871-7876
51.
Bouvier G, Watrin F, Naspetti M, Verthuy C, Naquet P, Ferrier P.
Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development.
Proc Natl Acad Sci U S A.
1996;93:7877-7881
52.
Lauzurica P, Krangel MS.
Temporal and lineage-specific control of T cell receptor alpha/delta gene rearrangement by T cell receptor alpha and delta enhancers.
J Exp Med.
1994;179:1913-1921
53.
Zuniga-Pflucker JC, Di J, Lenardo MJ.
Requirement for TNF-alpha and IL-1 alpha in fetal thymocyte commitment and differentiation.
Science.
1995;268:1906-1909
© 2001 by The American Society of Hematology.
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  B. J. Wilson, R. Harada, L. LeDuy, M. D. Hollenberg, and A. Nepveu CUX1 Transcription Factor Is a Downstream Effector of the Proteinase-activated Receptor 2 (PAR2) J. Biol. Chem., January 2, 2009; 284(1): 36 - 45. [Abstract] [Full Text] [PDF]
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|
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 M. Truscott, R. Harada, C. Vadnais, F. Robert, and A. Nepveu p110 CUX1 Cooperates with E2F Transcription Factors in the Transcriptional Activation of Cell Cycle-Regulated Genes Mol. Cell. Biol., May 15, 2008; 28(10): 3127 - 3138. [Abstract] [Full Text] [PDF]
|
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 G. Stratigopoulos, S. L. Padilla, C. A. LeDuc, E. Watson, A. T. Hattersley, M. I. McCarthy, L. M. Zeltser, W. K. Chung, and R. L. Leibel Regulation of Fto/Ftm gene expression in mice and humans Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2008; 294(4): R1185 - R1196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. O'Connell, D. S. Rao, A. A. Chaudhuri, M. P. Boldin, K. D. Taganov, J. Nicoll, R. L. Paquette, and D. Baltimore Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder J. Exp. Med., March 17, 2008; 205(3): 585 - 594. [Abstract] [Full Text] [PDF]
|
|
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 |
|
 A. Iulianella, M. Sharma, M. Durnin, G. B. Vanden Heuvel, and P. A. Trainor Cux2 (Cutl2) integrates neural progenitor development with cell-cycle progression during spinal cord neurogenesis Development, February 15, 2008; 135(4): 729 - 741. [Abstract] [Full Text] [PDF]
|
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|
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 |
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 R. Harada, C. Vadnais, L. Sansregret, L. Leduy, G. Berube, F. Robert, and A. Nepveu Genome-wide location analysis and expression studies reveal a role for p110 CUX1 in the activation of DNA replication genes Nucleic Acids Res., January 17, 2008; 36(1): 189 - 202. [Abstract] [Full Text] [PDF]
|
|
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|
|
|
|
 |
|
 E. M. Sloand, A. S. M. Yong, S. Ramkissoon, E. Solomou, T. C. Bruno, S. Kim, M. Fuhrer, S. Kajigaya, A. J. Barrett, and N. S. Young Granulocyte colony-stimulating factor preferentially stimulates proliferation of monosomy 7 cells bearing the isoform IV receptor PNAS, September 26, 2006; 103(39): 14483 - 14488. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Krupp, L. E. Yaich, R. J. Wessells, and R. Bodmer Identification of Genetic Loci That Interact With cut During Drosophila Wing-Margin Development Genetics, August 1, 2005; 170(4): 1775 - 1795. [Abstract] [Full Text] [PDF]
|
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S.-H. Jung, C. J. Evans, C. Uemura, and U. Banerjee The Drosophila lymph gland as a developmental model of hematopoiesis Development, June 1, 2005; 132(11): 2521 - 2533. [Abstract] [Full Text] [PDF]
|
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Q. Zhu, U. Maitra, D. Johnston, M. Lozano, and J. P. Dudley The Homeodomain Protein CDP Regulates Mammary-Specific Gene Transcription and Tumorigenesis Mol. Cell. Biol., June 1, 2004; 24(11): 4810 - 4823. [Abstract] [Full Text] [PDF]
|
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P. Mitra, R.-L. Xie, R. Medina, H. Hovhannisyan, S. K. Zaidi, Y. Wei, J. W. Harper, J. L. Stein, A. J. van Wijnen, and G. S. Stein Identification of HiNF-P, a Key Activator of Cell Cycle-Controlled Histone H4 Genes at the Onset of S Phase Mol. Cell. Biol., November 15, 2003; 23(22): 8110 - 8123. [Abstract] [Full Text] [PDF]
|
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|
 S. Tsutsumi, T. Taketani, K. Nishimura, X. Ge, T. Taki, K. Sugita, E. Ishii, R. Hanada, M. Ohki, H. Aburatani, et al. Two Distinct Gene Expression Signatures in Pediatric Acute Lymphoblastic Leukemia with MLL Rearrangements Cancer Res., August 15, 2003; 63(16): 4882 - 4887. [Abstract] [Full Text] [PDF]
|
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|
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 J. A. Martinez-Climent, A. A. Alizadeh, R. Segraves, D. Blesa, F. Rubio-Moscardo, D. G. Albertson, J. Garcia-Conde, M. J. S. Dyer, R. Levy, D. Pinkel, et al. Transformation of follicular lymphoma to diffuse large cell lymphoma is associated with a heterogeneous set of DNA copy number and gene expression alterations Blood, April 15, 2003; 101(8): 3109 - 3117. [Abstract] [Full Text] [PDF]
|
|
|
|
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B. Goulet, P. Watson, M. Poirier, L. Leduy, G. Berube, S. Meterissian, P. Jolicoeur, and A. Nepveu Characterization of a Tissue-specific CDP/Cux Isoform, p75, Activated in Breast Tumor Cells Cancer Res., November 15, 2002; 62(22): 6625 - 6633. [Abstract] [Full Text] [PDF]
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 A. K. Gillingham, A. C. Pfeifer, and S. Munro CASP, the Alternatively Spliced Product of the Gene Encoding the CCAAT-Displacement Protein Transcription Factor, Is a Golgi Membrane Protein Related to Giantin Mol. Biol. Cell, November 1, 2002; 13(11): 3761 - 3774. [Abstract] [Full Text] [PDF]
|
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 P. Goebel, A. Montalbano, N. Ayers, E. Kompfner, L. Dickinson, C. F. Webb, and A. J. Feeney High Frequency of Matrix Attachment Regions and Cut-Like Protein x/CCAAT-Displacement Protein and B Cell Regulator of IgH Transcription Binding Sites Flanking Ig V Region Genes J. Immunol., September 1, 2002; 169(5): 2477 - 2487. [Abstract] [Full Text] [PDF]
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F. Boudreau, E. H. H. M. Rings, G. P. Swain, A. M. Sinclair, E. R. Suh, D. G. Silberg, R. H. Scheuermann, and P. G. Traber A Novel Colonic Repressor Element Regulates Intestinal Gene Expression by Interacting with Cux/CDP Mol. Cell. Biol., August 1, 2002; 22(15): 5467 - 5478. [Abstract] [Full Text] [PDF]
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M. X. Luong, C. M. van der Meijden, D. Xing, R. Hesselton, E. S. Monuki, S. N. Jones, J. B. Lian, J. L. Stein, G. S. Stein, E. J. Neufeld, et al. Genetic Ablation of the CDP/Cux Protein C Terminus Results in Hair Cycle Defects and Reduced Male Fertility Mol. Cell. Biol., March 1, 2002; 22(5): 1424 - 1437. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
and 
chains, and
CD8. To determine how this activity affects cell development in vivo, a
hypomorphic allele of cux/CDP was created by gene targeting. Homozygous
mutant mice (cux/CDP
enhancer (M. Krangel, written communication, July 1999) and the
immunoglobulin heavy chain (IgH) intronic enhancer/MAR.
Fix III library.
a block in differentiation, a block
in proliferative expansion, or enhanced cell death. A block in
differentiation is unlikely due to the fact that mature B and T cells
are present in normal proportions and numbers in peripheral lymphoid
organs and there is no accumulation of undifferentiated cells, as seen
in mice deficient in RAG-2
transgene.