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HEMATOPOIESIS
From the Programs of Cell Biology and Human
Genetics, Memorial Sloan-Kettering Cancer Center and
Sloan-Kettering Division, Joan and Sanford I. Weill Graduate School of
Medical Sciences of Cornell University; and the Department of Medicine
Biochemistry and Molecular Biology, Derald H. Ruttenberg Cancer Center,
Mount Sinai School of Medicine, New York, NY.
Monocyte differentiation induced by 1,25-dihydroxyvitamin
D3 (1,25(OH)2D3) is interrupted
during the course of acute promyelocytic leukemia (APL). One form of
APL is associated with the translocation t(11;17), which joins the
promyelocytic leukemia zinc finger (PLZF) and retinoic acid receptor
Steroid and vitamin hormones play an integral part
in the maturation and differentiation of hematopoietic cells. Members
of the steroid and nuclear receptor superfamily, including the retinoic acid receptor (RAR), have an important role in this process, presumably through the ligand-dependent activation of genes that initiate or
facilitate differentiation.1 The hormone
1,25-dihydroxyvitamin D3
(1,25(OH)2D3), a metabolite of vitamin D, can
induce differentiation of hematopoietic cell lines such as HL-60 and
U937 along a macrophage-monocyte lineage, including a decrease or
cessation in their proliferation.2-6 The effects of
1,25(OH)2D3 on differentiation are mediated
through its transcriptional modulation of key target genes by
heterodimers consisting of the vitamin D3 receptor (VDR)
and the retinoid X receptor.7-10 During differentiation of
hematopoietic cells, 1,25(OH)2D3 strongly
induces expression of macrophage-monocyte markers, such as CD14, the
lipopolysaccharide receptor, and the family of In a search for target genes that might mediate differentiation of
myelomonocytic cells by 1,25(OH)2D3, our
laboratory previously identified the cell-cycle inhibitor
p21WAF1/CIP1 and the homeobox gene
HoxA10 as direct transcriptional targets of
VDR.7,9 Furthermore, exogenous expression of these genes in U937 cells was sufficient to induce differentiation.8
Ligand-dependent activation of such target genes is thought to occur
through recruitment of coactivator complexes, including
p160-CREB-binding protein (CBP) and VDR interacting protein
(DRIP).13
Repression of VDR-dependent gene activation is not as well understood.
Clearly, proper control of ligand-dependent expression of genes
that mediate differentiation and cell-cycle arrest is an essential
component of the function of normal, noncancerous cells.
Aberrations in hormonal control of the differentiation process in
hematopoietic cells occur in such cancers as acute promyelocytic
leukemia (APL).
In APL, the M3 subtype of acute myelogenous leukemia, differentiation
of cells is blocked during an intermediate promyelocyte stage of
myeloid differentiation.14-18 These malignant
promyelocytes can be induced to differentiate into mature
polymorphonuclear leukocytes by providing pharmacologic doses of
all-trans retinoic acid (ATRA). In contrast, these cancers are
resistant to the effects of 1,25(OH)2D3. APL is
thought to be caused by one of several reciprocal translocations
involving RAR Several groups21-24 characterized the molecular mechanism
of PLZF-RAR The PLZF gene encodes a 673-amino acid protein that contains a broad
complex, tram-trak, bric-a-brac/pox virus zinc finger (BTB/POZ)
domain Because 1,25(OH)2D3 is a potent inducer of
monocytic differentiation32 and VDR is widely expressed in
hematopoietic cells, we wondered whether PLZF might oppose this effect.
We found that PLZF and VDR physically interact and that PLZF expression
both abrogates the ability of VDR to activate transcription and impairs its ability to mediate differentiation of U937 cells. This study provides the first characterization of a protein that may regulate 1,25(OH)2D3-dependent differentiation of
hematopoietic cells by blocking transcriptional activation by the receptor.
Cell culture and transient transfections
Construction of PLZF-inducible cell lines
In vitro interaction assay Nuclear receptor proteins or domains were fused to glutathione-S-transferase (GST) and expressed in Escherichia coli. Putative interacting partners were translated in vitro (TNT kit; Promega) in the presence of sulfur 35 (35S)-methionine (Amersham, Piscataway, NJ). GST fusions and translated proteins were incubated in buffer A (170 mM potassium chloride [KCL], 20 mM Tris [pH 7.9], 20% glycerol, 0.2 mM EDTA, 4 mg/mL bovine serum albumin [BSA], 0.05% NP-40, 0.1 mM phenylmethylsulfonyl fluoride [PMSF], and 1 mM dithiothreitol [DTT]) for 3 hours before 3 washes in buffer B (same as buffer A but without BSA). Beads were resuspended in sodium dodecyl sulfate (SDS) sample buffer, separated by electrophoresis through 7.5% to 10% SDS polyacrylamide gels, dried, and exposed overnight at room temperature to Bio-Max film (Kodak, Rochester, NY).Immunoprecipitations Nuclear extracts were prepared from U937-PLZF45 cells as described previously.38 Then, 200 µg nuclear extract was resuspended in buffer A.1 (170 mM KCl, 20 mM Tris [pH 7.9], 10% glycerol, 0.2 mM EDTA, 0.05% NP-40, 0.1 mM PMSF, and 1 mM DTT) to a final volume of 445 µL. Extracts were precleared with normal rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA) and 50 µL protein A/G plus agarose beads (Santa Cruz Biotechnology) for 1 hour and then combined with 50 µL protein A/G plus beads, antibody, and either ethanol or 1,25(OH)2D3 (final concentration, 1 × 10 7 M) per reaction). Reactions were carried out
at 4°C for 2 hours and were followed by washing in buffer A.1 to 200 mM KCl. Washed pellets of beads were resuspended in SDS sample buffer,
separated by electrophoresis through 7.5% to 10% SDS polyacrylamide
gels, dried, and transferred to nitrocellulose filter paper. The
filters were subsequently immunoblotted for various proteins by using standard procedures. SKNO-1 cells were cultured in RPMI 1640 with 10%
FCS. Cells (1 × 107) were lysed for each
immunoprecipitation experiment. A PLZF mouse monoclonal antibody
(IgG2a isotype)30,39 and isotype control preimmune mouse monoclonal IgG2a (Jackson Immunoresearch,
West Grove, PA) were used at a concentration of 1 mg/mL. Subsequent steps in the immunoprecipitation were done as described
previously.40 The precipitated proteins were
electrophoresed through a 12% SDS-polyacrylamide gel and transferred
to an Immobilon P membrane (Millipore, Bedford, MA). Immunoblotting was
done by using standard methods.
Electrophoretic mobility shift assay Electrophoretic mobility shift assay (EMSA) was done as described previously41 by using 5 µg nuclear extract as described above. Cold specific and nonspecific competitor oligonucleotides were added to the reactions in 10 times the concentration of specific radioactive oligonucleotide. Antibody supershifts were done by adding anti-VDR monoclonal antibody or anti-PLZF polyclonal serum to the reactions. The vitamin D3 response element (VDRE) from the mouse osteopontin gene (OPN; also called Spp1) was generated as complementary oligonucleotides of the sequence 5'-GATCCACAAGGTTCACGAGGTTCACG-3' (top strand). The oligonucleotide used as the nonspecific control was a glucocorticoid response element of the top strand sequence 5'-GATCCGACCGAGAACAAGATGTTCTGTCGAG-3'.Differentiation assay and flow cytometry PLZF45 cells were washed in phosphate-buffered saline (PBS), transferred to medium without tetracycline, and allowed to grow for 16 hours before addition of 1,25(OH)2D3 (108 M), ATRA (106 M), or ethanol and
dimethyl sulfoxide. At various times, 1 × 106 cells were
obtained for flow cytometric analysis. Each sample was washed twice in
ice-cold PBS and 1% BSA and incubated in 150 µL cold PBS and 1% BSA
with 1.5 µL of either IgG1-fluorescein isothiocyanate conjugated
(FITC) and IgG1- R-phycoerythrin or IgG1-FITC anti-CD14 or
anti-CD11c (Caltag, Burlingame, CA) for 45 minutes. After incubation,
the cells were washed in PBS and 1% BSA 3 times; resuspended in 400 µL PBS, 1% BSA, and 0.5% paraformaldehyde; and stored at 4°C.
Flow cytometry was done on a fluorescence-activated cell-sorter scanner
(FACS Calibur; Becton Dickinson, Mountain View, CA).
Morphometric analysis At various times during the PLZF-1,25(OH)2D3-ATRA induction experiment, 2 × 105 cells were removed from each growth condition, cytospun onto microscope slides at 600 rpm for 10 minutes, air dried, and fixed in 100% methanol for 5 minutes. They were then stained with Giemsa staining (Hema-tek 2000; Bayer Corp, Pittsburgh, PA). Several fields on each slide were examined, and the cell dimensions were measured from digitized images by using National Institutes of Health Image 1.62 software. Total-cell and nuclear areas were determined, and the percentage of each cell occupied by the nucleus was derived for each cell. Ten cells were measured, and the average nuclear-area percentage was calculated for each condition.
PLZF blocks 1,25(OH)2D3-activated transcription PLZF-RAR expression can block
1,25(OH)2D3-mediated monocyte
differentiation.20 To determine the possible molecular
mechanism for this activity, we used PLZF-RAR, PLZF, and VDR to
transfect U937 cells, along with a reporter gene containing a consensus VDRE, a 6-base pair direct repeat with a spacing of 3 nucleotides between the half sites. As shown in Figure
1A, exogenous expression of either PLZF
or PLZF-RAR inhibited VDR-mediated activation of a luciferase
reporter (lane 1 versus lanes 2 and 3), regardless of the presence of
the histone deacetylase (HDAC) inhibitor, TSA (lane 4 versus lanes 5 and 6). PLZF-RAR and PLZF were expressed at similar levels (data not
shown).
Because PLZF alone was capable of significantly blocking VDR-dependent
transactivation, we subsequently focused on PLZF. To test whether the
repression of VDR activity by PLZF was a result of general
transcriptional repression, PLZF was cotransfected with GAL-VP16 and a
GAL operator-containing responsive reporter in U937 cells. As shown in
Figure 1B, activation of the GAL reporter by VP16 was not affected by
exogenous expression of PLZF, indicating that PLZF does not generally
squelch transcription. Additionally, coexpression of PLZF with VDR did
not affect VDR expression levels (data not shown). For comparison, PLZF
was also cotransfected with RAR We addressed the question of whether the PLZF-mediated repression of
VDR function was due to a physical interaction between the proteins by
conducting in vitro pull-down assays to determine whether
35S-labeled PLZF could bind to GST-VDR or GST-RAR PLZF and VDR interact in cellular extracts The results of the in vitro interaction assay were confirmed by coimmunoprecipitation studies. Stable U937 cells capable of induced expression of PLZF on removal of tetracycline were used to assess an interaction between endogenous VDR and PLZF in a nuclear extract. Figure 2A shows the induction of PLZF expression after 36 hours of culture in medium without tetracycline. Nuclear extracts were prepared from cells in the presence and absence of tetracycline (the absence and presence of PLZF, respectively) and used in a coimmunoprecipitation assay with antibodies to both VDR and PLZF. The extracts (with and without PLZF) were precipitated with either anti-VDR or anti-PLZF antibodies and immunoblotted with the opposite antibody. As shown in Figure 2B, in the absence of PLZF, neither antibody precipitated the reciprocal protein. However, in the presence of PLZF, anti-VDR and anti-PLZF coprecipitated PLZF and VDR, respectively. As in the in vitro pull-down assays, the interaction between PLZF and VDR occurred independently of 1,25(OH)2D3.
The physiologic nature of the PLZF-VDR interaction was explored further in the M2 leukemia cell line SKNO,42 in which both PLZF and VDR are expressed endogenously. VDR could not be detected by direct immunoblotting from SKNO lysates (Figure 2D, lane 2). However, coimmunoprecipitates with the PLZF monoclonal antibody reacted positively (6.1-fold increase over lysate alone with the IgG signal subtracted) when probed with an anti-VDR antibody (Figure 2D, lane 4). These results show that antibodies to PLZF and VDR can reciprocally immunoprecipitate both proteins and that this interaction occurs even at the low endogenous protein levels in SKNO cells. Because of the apparent ligand-independent nature of the interaction between PLZF and VDR, and given that PLZF can interact with the corepressors SMRT-N-CoR, HDAC-1, and Sin3A,21,28,43,44 we wondered whether VDR associates with components of a corepressor complex through its association with PLZF. As shown in Figure 2C, endogenous Sin3A was clearly immunoprecipitated by anti-PLZF antibodies in the presence of PLZF (lanes 4 and 5) but not by anti-VDR antibodies (lanes 1 and 2). Similar results were observed when VDR coimmunoprecipitations were blotted for SMRT and HDAC-1 (data not shown). Thus, although VDR interacted with PLZF and PLZF interacted with Sin3A and other repressors, a 3-way interaction was not detected. Delineation of interacting domains To determine how PLZF might block transcriptional activation by VDR, an in vitro protein affinity assay was used to identify the domains in PLZF and VDR responsible for the interaction. Figure 3A depicts a series of GST fusions containing all or part of VDR. The 35S-labeled, in vitro-translated PLZF was incubated with the individual VDR derivatives (Figure 3B). In vitro-translated PLZF interacted to a significant extent with full-length VDR (Figure 3B, lane 3) but also with the GST-VDR DBD (GST-VDRDBD; Figure 3B, lane 6). It did not interact with GST alone, the GST-VDR ligand-binding domain (GST-VDRLBD, or the hinge region (GST-VDRhinge) (lanes 2, 4, and 5). Consistent with the inability of PLZF to interact with GST-VDRLBD, a GAL-VDRLBD construct that strongly activated transcription from a GAL-UAS reporter in response to 1,25(OH)2D3 was not affected by coexpression of PLZF in U937 cells (data not shown).
An interaction with VDR by means of its DBD could explain why repression by PLZF is ligand independent. To address whether the interaction with the VDR DBD was specific, we conducted a pull-down study using the VDR DBD and another nuclear receptor DBD, that from the orphan receptor HNF4. PLZF interacted with GST VDRDBD but not with GST-HNF4DBD (Figure 3B, lanes 6 and 7), thereby indicating that the interaction is specific for VDR and not the result of promiscuous interaction with a hormone receptor zinc finger region. We used a similar analysis to determine the domains of PLZF protein
that facilitate VDR interaction. Figure 3C depicts a series of PLZF
mutants containing deletions covering the BTB/POZ domain (PLZF We correlated the ability of various PLZF mutants to interact with VDR
with their ability to repress VDR-dependent transactivation of
cotransfected reporter constructs in U937 cells. Deletion of the
BTB/POZ domain abrogated the ability of PLZF to repress VDR transactivation (Figure 4A, lanes 4 and
5) relative to full-length PLZF (lanes 2 and 3). Thus, we found that,
in vivo, the BTB/POZ domain plays an important role in the functional
repression of VDR, presumably through its ability to physically
associate with the receptor. Interestingly, an epitope-tagged version
of the BTB/POZ domain did not repress VDR-dependent transcription
(Figure 4B). Furthermore, deletion of the zinc finger region of PLZF
partly relieved the repression of VDR transactivation (Figure 4C),
whereas a deletion of the RD2 domain retained the ability to repress
VDR (Figure 4D). Therefore, we found that although the BTB/POZ domain is necessary and sufficient for the interaction of PLZF with VDR, it
cannot on its own repress transcription, a result that suggests the
existence of additional contributions from other moieties of PLZF.
PLZF is present in a VDR DNA-binding complex Our observation of a physical interaction between the VDR DBD and PLZF led us to consider that PLZF may affect the ability of VDR to bind its hormone response element or affect the nature of the DNA-bound complex so that it cannot activate transcription. To explore this issue, nuclear extracts prepared from U937-PLZF cells, grown in either the presence or absence of tetracycline to regulate expression of PLZF, were used in an EMSA (Figure 5). The probe used in this assay was a direct repeat with a spacing of 3 nucleotides (DR-3) derived from the VDRE of the mouse OPN gene promoter and was identical to the canonical DR-3 in the reporter construct used in the transfection experiments. As shown in Figure 5A, addition of extract yielded at least 4 shifted species (lane 2), which showed specific competition with the addition of cold excess VDRE but not a nonspecific competitor (lanes 3 and 4). In extract containing PLZF, there was a significant change in shift mobility (lane 5). This shift showed competition with the specific oligo (lane 6) and was not affected by the nonspecific oligo (lane 7). Addition of an anti-VDR antibody confirmed that these shifted species contained VDR. Anti-VDR antibodies could compromise binding (open arrow) or alter the mobility of several of the major species on the gel and induce a supershift (asterisk in lane 9). In the presence of PLZF, addition of anti-VDR antibody disrupted the large lower shift (solid arrow) and a high-molecular-weight shift appeared (2 asterisks in lane 11). The disappearance of a significant specific shift in the presence of the anti-VDR antibody in lanes 9 and 11 indicates that, in both cases, VDR was present in the complex but the complex was altered by the presence of PLZF. The most supershifted complexes in lanes 9 and 11 appear to have different mobilities, with lane 11 showing slightly more delay, a finding consistent with the presence of PLZF in the DNA-protein complex.
To determine whether PLZF was present in the DNA-bound complex, we conducted similar antibody additions using a monoclonal antibody to PLZF (Figure 5B). Addition of the anti-PLZF antibody did not affect the shift pattern observed when PLZF was not present (lanes 12 and 13). In the presence of PLZF, increasing amounts of anti-PLZF antibody abrogated both the upper and lower prominent shifts observed in the absence of the antibody (lanes 16 and 17 versus lane 14). The data indicate that PLZF is present in the VDR-VDRE complexes. When coupled with the data on in vitro interaction and transfection described earlier, these findings suggest that PLZF and VDR from nuclear extracts can form complexes on a VDRE and that these complexes could be responsible for the inability of VDR to transactivate in the presence of PLZF. Monocytic differentiation induced by 1,25(OH)2D3 is inhibited by PLZF The stable, tetracycline-inducible U937 cell line PLZF45 was used to assay the effects of PLZF expression on monocytic differentiation induced by 1,25(OH)2D3 and, in comparison, ATRA. Both 1,25(OH)2D3 and ATRA induce differentiation of hematopoietic precursor cells as measured by surface-antigen expression and changes in cellular morphologic features. PLZF45 cells were cultured in medium without tetracycline, inducing PLZF expression; treated with 1,25(OH)2D3 or ATRA for 48 hours; and harvested for analysis of cell-surface antigens by flow cytometry (Figure 6). The fold change in mean fluorescence of the monocytic differentiation marker CD14 was measured as a function of PLZF expression and treatment with 1,25(OH)2D3 or ATRA (Figure 6A). PLZF alone minimally induced expression of CD14 over background levels (lane 2). ATRA did not induce expression of CD14 (lane 3). CD14 expression was induced by 1,25(OH)2D3, to 20 times the levels in uninduced cells, and this induction was substantially reduced by PLZF (Figure 6A, lane 5 versus lane 6). In contrast, PLZF expression did not block ATRA-induced differentiation, as measured by the monocyte-granulocyte marker CD11c after 48 hours of treatment with ATRA (Figure 6B, lane 3 versus lane 4) or alone (lane 1 versus lanes 2 and 6); instead, it appeared to contribute to induction with ATRA. PLZF also did not block induction of CD11c family members CD11b and CD11a or of CD18 (data not shown). Thus, PLZF appears, possibly through a specific block of VDR function, to alter CD14 but not CD11c surface-marker expression. These results suggest that PLZF may alter very specific 1,25(OH)2D3-induced monocytic differentiation pathways.
As multipotent precursor cells progress through monocytic
differentiation, they undergo morphologic changes. One morphologic indicator of differentiation is a reduction in the volume of the nucleus volume relative to that of the cytoplasm. We used the PLZF45
cell line to assess whether PLZF expression would alter 1,25(OH)2D3-dependent changes in cell
morphologic features (Figure 7A). For
U937T-PLZF45 cells without either 1,25(OH)2D3
treatment or PLZF expression, nuclei occupied 71.5% ± 2.0% of
total-cell area (Figure 7B, lane 1). Expression of PLZF (Figure 7A,
panel ii) did not cause a significant decrease in nuclear size.
In contrast, treatment with 1,25(OH)2D3
resulted in a striking decrease in nuclear area, to 45% ± 3.0%,
indicative of a differentiating cell (Figure 7A, panel iii). Expression
of PLZF during treatment with 1,25(OH)2D3
blocked the shrinking of nuclear volume (Figure 7A, panel iv, versus
Figure 7A panel iii, and Figure 7B; 61.0% ± 3.0% versus 45.0 ± 3.0%, respectively). Unlike results observed with 1,25(OH)2D3 treatment, PLZF expression had
little effect on the morphologic changes resulting from ATRA treatment
(Figure 8A). Treatment with ATRA resulted
in a 13% reduction in nucleus-to-cytoplasm area (Figure 8B,
lanes 2 and 3), a change that was not affected by the presence of PLZF
(Figure 8B, lanes 3 and 4). These results, along with the observed
effects of PLZF expression on
1,25(OH)2D3-induced CD14 surface expression,
strongly suggest that PLZF is a potent and specific regulator of
1,25(OH)2D3-dependent monocytic
differentiation.
We were concerned that the well-documented ability of PLZF to cause
cell-cycle arrest might affect the differentiation results shown in
Figures 6 to 8. Table 1 shows the
cell-cycle profile of PLZF45 cells during the
1,25(OH)2D3-dependent treatments illustrated in
those figures. At the times described, PLZF induced a G1
arrest similar to that induced by 1,25(OH)2D3
alone (71.7% versus 72.6%). However, PLZF did not induce CD14, nor
did it change the nucleus-to-cytoplasm ratio. PLZF expression also did
not inhibit ATRA induction of CD11c or ATRA-mediated morphologic
changes. These results strongly suggest that, although PLZF does induce
a G1 arrest, it neither blocks all hormone-induced
differentiation nor induces general differentiation on its own.
Our study demonstrated that PLZF blocks transcriptional activation of a
canonical VDRE reporter construct, revealed a physical interaction between VDR and PLZF, and showed physiologic effects on VDR-dependent processes by PLZF. To more clearly define the role of
PLZF in VDR-mediated differentiation, we examined its effect on a
direct VDR target gene. Our laboratory previously identified the
cyclin-dependent kinase
inhibitor p21WAF1/CIP1 gene as a direct
transcriptional target of VDR.7 Expression of p21 was also
shown to be sufficient to begin differentiation of U937 cells along a
macrophage-monocyte lineage. Using a 2.4-kb fragment of the p21
promoter cloned upstream of the luciferase gene (Figure
9A), we here observed that coexpression
of VDR and PLZF resulted in a reproducible attenuation of
1,25(OH)2D3-mediated activation of p21
transcription (Figure 9B). RAR
In this study, we found that PLZF is a potent repressor of VDR-dependent transcriptional activation. At the molecular level, this repression appears to be due to a selective physical interaction between VDR and PLZF. We showed that PLZF and VDR can be coimmunoprecipitated from a hematopoietic precursor cell line and that this interaction appears to occur through the DBD of VDR. Consistent with these results, PLZF was detected in a VDR-VDRE-bound complex in vitro, suggesting that the PLZF-mediated block of VDR transactivation might be due to a PLZF-induced alteration in the nature of the receptor-DNA complex that is unfavorable for VDR-dependent transcriptional activation. The BTB/POZ domain of PLZF plays an essential role in both the
interaction of PLZF and VDR and subsequent repression of
transcription.46 In studies in Drosophila,
proteins containing BTB/POZ were shown to regulate gene
expression through local control of nucleosome positioning and
remodeling.47 The BTB/POZ domain of PLZF-RAR The BTB/POZ domain is able to associate with several corepressors,
notably Sin3A, HDAC1, SMRT, and N-CoR.21-24,28 Many
researchers have found that constitutive association of these
repressors with PLZF-RAR The BTB/POZ domain of PLZF is an obligate homodimer,25,46 and we found that when bound to its cognate site, PLZF forms a high-molecular-weight complex that may contain as many as 4 PLZF molecules.48 This information suggests that several molecules of PLZF could form a complex with the VDR when the 2 proteins interact. Hence, PLZF may not block VDR action by recruitment of corepressors but may instead work by steric blockade of the VDR. PLZF, like many of members of its family, contains not only a BTB/POZ domain but also 9 Krüppel-like zinc fingers (Cys2His2). We showed previously that PLZF can bind a specific DNA site and repress transcription of reporter genes.48,49 Our data suggest that PLZF is present in a DNA-bound complex with VDR. Although PLZF did not bind the VDRE probe used in the assay (data not shown), the transfection data suggest that the zinc fingers are partly responsible for repression of VDR transactivation (Figure 4C). In a previous study,50</ |
Top
Abstract
Introduction
2 leukocyte integrins including CD11a, CD11b, and
CD11c.
regions thought to be critical for transcriptional repression
by PLZF
) of 1,25(OH)2D3 or ATRA (10
BTB/POZ (
