From the Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294
Received for publication, September 15, 2000, and in revised form, October 23, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Erythroid Kruppel-like Factor (EKLF) is an
erythroid-specific transcription factor that plays a critical role in
The genes encoding Erythroid Kruppel-like Factor (EKLF) is one factor that is critical for
the developmental stage-specific switch from the Although a large amount of information suggests a critical role for
EKLF in globin gene switching, the mechanisms underlying EKLF
transactivation functions are not clearly understood. EKLF is
expressed, and is functional at all developmental stages, yet its
effects are apparent only during definitive hematopoiesis (19-21). The
molecular basis of this specificity is unknown. The dissection of EKLF
functional domains is an important step in elucidating these
mechanisms. Previous studies have localized a transactivation domain at
the amino-terminal region (aa 1-104) (22). In this report, we describe
the identification of a new transactivation domain. This domain,
consisting of amino acids 140-358, is sufficient for maximal
transactivation of the Plasmid Constructions--
The HA-EKLF plasmid was
constructed by subcloning a 63-base pair double-stranded oligomer (top:
5'-CATGGGTAGCAGCTACCCTTACGACGTGCCCGACTACGCCAGCCTGGGCGGCCCTAGCAGAGG-3'; bottom:
5'-CATGCCCCTGCTAGGGCCGCCCAGGCTGGCGTAGTCGGGCACGTCGTAAGGGTAGCTGCTACC-3') encompassing the HA epitope at the NcoI site of the
EKLF expression plasmid pSG5/EKLF (10). This HA-EKLF plasmid was used
as the parental plasmid for the generation of deletion mutants.
Restriction enzymes PvuII and Bpu10I cut in EKLF
cDNA at positions corresponding to codon 3. Restriction enzymes
BsaI, NarI, and AvaI cut at positions corresponding to codons 139, 225, and 254, respectively. Restriction enzyme SmaI cuts at positions corresponding to codons 155 and 255. Mutant HA- Cell Culture and Transfections--
K562 cells were grown and
electroporated as described previously (12). COS cells were
grown in Dulbecco's modified Eagle's medium, 10% fetal bovine
serum and transfected with different mutants along with
CMV-Lac-Z using the calcium chloride coprecipitation method.
Forty-eight hours after transfection, nuclear extracts were prepared
(23), and Western blots were performed using ~50 µg of nuclear
extracts that were normalized by Indirect Immunofluorescence--
Indirect immunofluorescence was
conducted as described previously (24), except that the cells
were blocked with 10% fetal bovine serum/phosphate-buffered saline for
10 min after the permeabilization step. Coverslips were mounted
in media containing propidium iodide (Vector Laboratories). 12CA5
anti-HA antibody (generous gift of Dr. Susan Ruppert) was used (1:500
dilution) as the primary antibody, and a FITC anti-mouse antibody
(Santa Cruz) was used as the secondary antibody (1:500 dilution).
Images were captured with a Hamamatsu 3CDC camera mounted on a Nikon
eclipse E800 microscope.
Gel Shift Assays--
COS cell nuclear extracts that were
normalized by We performed a mutational analysis of EKLF using a previously
established transient transfection assay (12) to map essential transactivation domains. The transactivation assay utilizes the human
erythroid cell line K562, which expresses little endogenous EKLF (12),
and an LCR - to
-globin gene switching during development. To identify
essential domains required for EKLF transactivation function, we
cotransfected a human erythroleukemia cell line (K562) with a locus
control region
/Luc-
/Cat reporter and an EKLF expression
vector. In this assay EKLF mediates a 500-fold induction of
/CAT
expression compared with controls. To map essential transactivation
domains, progressive NH2-terminal and internal
deletion mutants of EKLF were constructed. All EKLF mutants were
expressed at wild-type levels, localized to the nucleus, and bound DNA.
When mutant EKLF proteins were tested for
/CAT activation, a novel
transactivation domain was identified. This novel domain, encompassing
amino acids (aa) 140-358, is sufficient for maximal
/CAT
activation. An 85-amino acid subdomain within this region (aa 140-225)
is essential for its activity. Interestingly, this central
transactivation subdomain is functionally redundant with the
amino-terminal domain (aa 1-139). Thus, EKLF possesses at least two
potent transactivation domains that appear to function in a redundant manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-like subunits of human hemoglobin are
expressed in a tissue- and developmental stage-specific pattern of
expression (1). Expression is exclusive to erythroid tissues, and
-,
-, and
-globin gene expression is predominately restricted to the
yolk sac, fetal liver, and bone marrow, respectively. The genes are
present within a 100-kb1
locus that contains a potent 22-kb enhancer termed the locus control
region (LCR) (2-5). Several groups have proposed that precise tissue
and developmental regulation is accomplished by complex protein-protein
interactions between factors that bind the LCR and those that bind the
promoters of individual genes (6, 7). The competition model suggests
that a number of erythroid-specific and ubiquitous factors bind the LCR
and enable this region to function as a potent enhancer. Downstream
genes then compete for productive interactions with the LCR (2-5). Presumably, yolk sac-, fetal liver-, and bone marrow-specific factors
bind to globin gene promoters and proximal enhancers and provide
individual genes with a competitive advantage for interaction with the
LCR at the appropriate developmental stage.
- to
-globin
expression (reviewed in Ref. 8). This erythroid-specific transcription
factor binds the
-globin promoter and activates high level
expression (9, 10). EKLF is a 358-amino acid protein containing an
amino-terminal, proline-rich transactivation region (aa 1-275) and a
COOH-terminal DNA binding domain (aa 276-358) (10). The DNA binding
domain consists of three C2H2 Kruppel-like zinc fingers that bind
specifically to the CCACACCCT motif at
90 of the
-globin promoter
(11). Although EKLF also binds to the CACCC box in the
promoter,
the binding affinity to
CACCC is 8-fold lower than the binding
affinity for
CACCC and EKLF preferentially activates the
gene
in transient transfection assays (12). Targeted deletion of EKLF in
mice results in a drastic reduction in the
-globin gene expression,
but
gene expression remains unaffected (13, 14). Finally,
persistence of
gene expression during development is observed in
EKLF knockout mice (15, 16). These observations are consistent with the view that EKLF plays a central role in
- to
-globin gene
switching by binding specifically to the
promoter and providing a
competitive advantage for interactions with the LCR in adult erythroid
tissue. Recent studies also suggest that EKLF exerts important
functions at sites other than the
-globin promoter (17, 18).
-globin promoter. Furthermore, an essential
region within this domain (aa 140-225) is completely redundant with
the amino-terminal transactivation domain (aa 1-139).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4-139 was generated by ligating three fragments as follows: a 0.7-kb AvrII-PvuII fragment, a
0.8-kb BsaI-BamHI fragment (BsaI end
was blunted by S1 nuclease), and a 3.3-kb
BamHI-AvrII fragment. Mutant HA-
4-225 was
generated by digesting HA-EKLF plasmid with Bpu10I and
NarI. The Bpu10I end was blunted with Klenow, and
the NarI end was blunted with S1. The 4.6-kb
Bpu10I(blunt)-NarI(blunt) fragment was
gel-purified and self-ligated. Mutant HA-
4-254 was generated by
digesting the HA-EKLF plasmid with Bpu10I and
AvaI. The Bpu10I end was blunted with Klenow, and
the AvaI end was blunted with S1. The 4.6-kb
Bpu10I(blunt)-AvaI(blunt) fragment was gel purified and self-ligated. Mutant HA-
140-226 was generated by ligating two fragments: a 1.2-kb AvrII-BsaI
fragment (the BsaI end was blunted with Klenow), and a
3.7-kb NarI-AvrII fragment (the Nar1
end was blunted with Klenow). Mutant HA-
255-358 was generated by
digesting the HA-EKLF plasmid with SmaI and
BamHI. The BamHI end was blunted with Klenow. The
4.6-kb SmaI-BamHI(blunt) fragment was
gel-purified and ligated with the 300-base pair SmaI fragment (codons 155-255) from EKLF cDNA. All constructs were sequenced and purified twice on cesium chloride gradients.
-gal activity. The anti-HA antibody
12CA5-HRP (Roche Molecular Biochemicals) was used at a 1:2500 dilution,
and gels were developed using the ECL system (Amersham Pharmacia
Biotech). Transactivation assays were performed as described previously
(12). Each construct was tested in at least three independent
experiments. Within each experiment, each construct was electroporated
in duplicate. CAT assays were performed in duplicate on each
electroporated sample, and
-gal assays were performed in triplicate
on each electroporated sample. The CAT assay values were normalized by
-gal values.
-gal activity (~20 µg) as described above were
used in gel shift assays using a double-stranded oligomer encompassing
the mammalian
promoter CACCC box (8). The binding reactions were
carried out in a buffer containing 5 mM Tris, 0.5 mM dithiothreitol, 0.5 mM EDTA, 25 mM NaCl, and 1% Ficoll. Before loading the sample on the
gel, a 0.1 volume of 20% Ficoll was added to each reaction. For
supershift/ablation assays, HA antibody was included in the binding reaction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
reporter (HS2-
/Luc-
/Cat) that contains wild-type human
- and
-globin promoters (Fig.
1). In this assay, full-length EKLF
activates
/CAT expression 500-fold compared with vector alone;
therefore, the system provides a sensitive, quantitative assay for
transactivation domains. We first tagged EKLF at the NH2
terminus with the influenza HA epitope (HA-EKLF) to allow detection of
the protein in transfected cells. HA-EKLF transactivated the
-globin
promoter in the HS2-
/Luc-
/Cat reporter at the same level
(500-fold) as wild-type EKLF. Using this parental plasmid, we
constructed three progressive amino-terminal truncations of HA-EKLF:
HA-
4-139 that deletes amino acids 4 through 139, HA-
4-225 that
deletes amino acids 4 through 225, and HA-
4-254 that deletes amino
acids 4 through 254 (Fig. 2A).
The same methionine start site was retained for all mutants to avoid
differences in the site of translation initiation. As an added control,
untagged versions of each mutant were also produced and tested in the
same assay; no differences between HA-tagged and untagged proteins were
observed (data not shown).
View larger version (10K):
[in a new window]
Fig. 1.
Constructs used in the transactivation
assay. The reporter plasmid HS2 /Luc-
/Cat has been
described previously (12). This construct contains a human 1.5-kb
KpnI-BglII HS2 fragment linked to a human
-globin gene promoter (
299 to +37) driving the firefly luciferase
(Luc) gene and a human
-globin promoter (
265 to +48)
driving the chloramphenicol acetyltransferase (Cat) gene.
The activator plasmid (pSG5/HA-EKLF) contains an SV40 promoter driving
HA-EKLF (wild-type or mutant). A CMV/Lac-Z gene was
cotransfected with the activator and the reporter into K562 cells to
serve as a control for transfection efficiency.
View larger version (14K):
[in a new window]
Fig. 2.
Deletion mapping of the transactivation
domain. A, schematic of HA-EKLF deletion constructs.
The DNA-binding domain (DBD) is shown as a solid
box, and the HA tag is shown as a stripped box.
B, histogram of transactivation results. The HS2
/Luc-
/Cat reporter was cotransfected with either vector
alone, full-length, or deletion mutants of HA-EKLF, and
CMV-Lac-Z into K562 cells. CAT activities were normalized to
-galactosidase levels. Full-length HA-EKLF activated
/CAT levels
500-fold relative to the vector alone and is depicted as 100%. The
transactivation capacities of the mutants are shown as percentage of
wild-type HA-EKLF activity. The error bars denote S.E. of at
least three independent experiments. The values for each construct are
as follows: vector, 0.2 ± 0.07%; HA-
4-139, 99 ± 21%;
HA-
4-225, 2.9 ± 2%; HA-
4-254, 1 ± 0.6%;
HA-
140-226, 95 ± 23%.
Each deletion construct was cotransfected with the LCR -
reporter
and Lac-Z control and tested for the ability to transactivate the
-globin promoter. The results are depicted in Fig. 2B. In these experiments full-length HA-EKLF (WT) activated the
-globin promoter 500-fold relative to the vector alone and is depicted as
100%. The activities of different mutants are expressed as a
percentage of WT. Surprisingly, HA-
4-139 activated the
promoter at wild-type levels (99 ± 21%) even though this deletion removed the transactivation domain previously identified by Chen and Bieker (amino acids 1-104) (22). A further 85 amino acid deletion
(HA-
4-225) drastically reduced
/CAT activation to 2.9 ± 2%. Deletion to amino acid 255 (HA-
4-254) resulted in very low
levels of
/CAT activation (1.0 ± 0.6%). These data
demonstrate that amino acids 140-358 encompass a potent
transactivation domain that is sufficient for maximal transactivation
of the
-globin promoter. Furthermore, the results suggest that an
85-amino acid subdomain (amino acids 140-225) is critical for the
function of this domain.
To ensure proper integrity of the mutants, we first assessed EKLF
protein levels after transfection into COS cells. Mutant constructs
were cotransfected with a Lac-Z plasmid to control for
transfection efficiency, and Western blots were conducted on normalized
nuclear extracts using an anti-HA antibody. These results are
illustrated in Fig. 3. All mutant EKLFs
were detected at near wild-type levels, suggesting that the proteins
were stable.
|
We next assessed the subcellular localization of the mutants to
determine whether the inactivity of HA-4-225 and HA-
4-254 could
be explained by protein mislocalization. Mutant constructs were
transfected into COS cells, and HA-EKLFs were detected by indirect
immunofluorescence with a FITC-labeled (green) anti-HA antibody (Fig. 4, A-E).
Propidium iodide staining (red) was utilized to define the
nucleus (Fig. 4, H-L), and a two-color merge (Fig. 4,
O-S) was used to assess EKLF nuclear localization. As
illustrated in the figure, all mutant EKLFs localized to the nucleus.
These data suggest that the transcriptional inactivity of HA-
4-225 and HA-
4-254 does not result from protein mislocalization.
|
Finally, we examined all mutant EKLFs for the ability to bind DNA. Gel
shift assays were performed with nuclear extracts obtained from COS
cells following transfection of the HA constructs described above. The
binding sequence was a labeled oligomer (20 base pairs) encompassing
the mammalian -globin CACCC site (Fig.
5). Control extracts from cells
transfected with the vector alone are shown in lanes 2 and
3. The background bands result from endogenous proteins that
are known to bind the CACCC motif (8); as expected, HA antibody does
not ablate or supershift these bands. Lanes 4 and
5 represent extracts from cells transfected with full-length HA-EKLF. Unfortunately, the EKLF gel shift band is obscured by a
background band and, therefore, cannot be detected. However, unique gel
shift bands that are distinct from endogenous bands are detected for
all deletion mutants. Lanes 6, 8, and
10 represent extracts from cells transfected with EKLF
mutants HA-
4-139, HA-
4-225, and HA-
4-254, respectively.
Specific bands that migrate at the predicted size are observed in each
of these lanes. These data demonstrate that all mutant EKLFs bind DNA.
To confirm that unique bands are EKLF-specific gel shifts, HA antibody
was included in the binding reactions. Lanes 7,
9, and 11 demonstrate that HA antibody
specifically ablates HA-
4-139, HA-
4-225, and HA-
4-254 gel
shifts. We conclude that all EKLF mutants bind DNA and, therefore, that
the transcriptional inactivity of HA-
4-225 and HA-
4-254 results
from deletion of a novel transactivation domain.
|
The results described above demonstrate that amino acids 4 to 139 of
EKLF are dispensable for maximal activation of the -globin promoter,
although this region encompasses the minimal activation domain
described by Chen and Bieker (22). One possible explanation for this
apparent discrepancy is that different systems were utilized to define
transactivation domains. Chen and Bieker (22) used a GAL4 DNA binding
domain fused to EKLF and tested the ability of fusion proteins to
activate a heterologous target promoter (GAL4 binding
sites-TATA-CAT) in 32DEpo1 cells. On the other hand, we utilized native
EKLF and the natural
-globin promoter in K562 cells. To determine
whether the amino-terminal domain of EKLF would transactivate in our
assay, we constructed a new mutant, HA-
140-226 (Fig.
2A), that essentially fuses the amino-terminal domain (amino
acids 1-139) to the transcriptionally inactive mutant HA-
4-225. We
then tested the ability of this protein to transactivate the
-globin
promoter in our HS2-
/Luc-
/Cat reporter (Fig.
2B). Interestingly, this mutant activated the
promoter
at WT levels (95 ± 23%). This result confirms that the
amino-terminal region of EKLF contains a potent transactivation domain
as originally described by Chen and Bieker (22). Furthermore, our data
demonstrate that this amino-terminal domain is functionally redundant
with the domain at aa 140-225. As expected, the HA-
140-226 EKLF
protein is stable (Fig. 3), localizes to the nucleus (Fig. 4
panels F, M, and T), and binds DNA
(Fig. 5, lanes 12 and 13).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results described above identify a novel transactivation
domain in the erythroid-specific transcription factor EKLF. This domain, which is contained within amino acids 140-358 of the protein, is sufficient for maximal transactivation of the -globin promoter (see HA-
4-139, Fig. 2B). Furthermore, an 85-amino acid
subdomain of this region (aa 140-225) is essential for activity;
deletion of this subdomain dramatically reduces transactivation
(HA-
4-225, Fig. 2B). Interestingly, this subdomain can
be functionally replaced by amino acids 1-139 (HA-
140-226, Fig.
2B), which contains a potent transactivation domain
described by Chen and Bieker (22). These studies demonstrate that EKLF
possesses at least two transactivation domains that function in a
redundant manner. In this respect, EKLF is similar to another
Kruppel-like family member, Sp1, which also contains two potent
transactivation domains that are functionally redundant (25). Mutant
constructs designed to delimit subregions within the 85-amino acid
central domain (aa 140-225) produced unstable proteins (data not
shown), precluding further definition of this domain.
Our results also demonstrate that amino acids 255-358 encompass
sequences sufficient for nuclear localization; the HA-4-254 EKLF mutant efficiently localized to the nucleus (Fig. 4,
panels E, L, and S), although the
protein did not activate transcription. To confirm this result we
generated a new construct HA-
255-358 that deletes amino acids
255-358. This mutant was expressed at WT levels in whole cell extracts
(data not shown). When tested for subcellular compartmentalization, the
mutant localized exclusively to the cytoplasm (Fig. 4, panels
G, N, and U). This result demonstrates that
the first 254 aa of EKLF do not contain a nuclear localization sequence. The best candidate for the nuclear localization sequence is
PKRSRR at position 260-265. This sequence is highly homologous to the
nuclear localization sequence (PKRGRR) identified in other Kruppel-like
family members (26).
The molecular basis for transactivation through the newly identified, central domain of EKLF is not known. However, some insight can be obtained from studies of the amino-terminal domain. The activity of this domain is regulated by phosphorylation of a conserved threonine residue within the recognition site of casein kinase-II (27). EKLF is heavily phosphorylated on serine and threonine residues, and several consensus sites for phosphorylation are present within the central domain (27); therefore, the activity of this region may also be regulated by phosphorylation. Alternatively, transactivation may be a function of the proline-rich nature of this region. The activation domains of several transcription factors are rich in prolines (28), and previous studies have demonstrated that a proline stretch of 10 residues is sufficient to confer high transactivation capacity when fused to the GAL4 DNA binding domain (29). The entire EKLF protein is rich in prolines, except for the zinc finger domain (aa 275-358); therefore, both the amino-terminal and central domains may function through proline-rich sequences.
The redundancy of amino-terminal and central domains could be achieved in several ways. Both domains may interact independently with the same coactivator. This has been observed for the two redundant Sp1 activation domains, both of which interact with the same coactivator, TAFII110 (a component of the TFIID complex) (30). Alternatively, the two domains may interact with different coactivators that have redundant activities. Previous studies have demonstrated that EKLF interacts physically with CBP, P300, and P/CAF in vivo, and GATA-1 in vitro (31, 32). Furthermore, Armstrong et al. (33) recently purified the SWI/SNF-related complex E-RC 1 based on its ability to interact functionally with EKLF in vitro. Additional experiments will be required to define the coactivators that interact with each EKLF transactivation domain.
As mentioned above, EKLF is phosphorylated extensively, and Zhang and
Bieker (31) recently demonstrated that specific lysines are acetylated
(Lys261, Lys270). These post-translational
modifications may promote interactions with specific coactivators and
factors of the basal transcription machinery. Differential
post-translational modifications of the two transactivation domains
during development may provide a mechanism for the temporal-specific
transcriptional activity of EKLF on the -globin promoter, and
identification of additional EKLF interacting proteins should provide
insights into globin gene switching.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Susan Ruppert for the generous gift of 12CA5 and Dr. Andrew Perkins for helpful suggestions on the gel shift assay. We are especially grateful to Dr. Lee Wall for technical advice on the gel shift assay. We also thank Dr. Tom Ryan, Dr. Dominic Ciavatta, and other members of Townes laboratory for many helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the NHLBI.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: National Institutes of Health, NICHD, Laboratory
of Molecular Embryology, Bethesda, MD 20892-5431.
§ To whom correspondence should be addressed. Tel.: 205-934-5294; Fax: 205-934-2889; E-mail: ttownes@uab.edu.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M008457200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
kb, kilobase pair(s);
LCR, locus control region;
EKLF, erythroid Kruppel-like
factor;
aa, amino acids;
HA, hemagglutinin;
-gal,
-galactosidase;
CAT, chloramphenicol acetyltransferase;
WT, wild-type;
CMV, cytomegalovirus.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Stamatoyannopoulos, G., Niehuis, A. W., Majerus, P., and Varmus, H. (eds) (1994) The Molecular Basis of Blood Diseases , W. B. Saunders Company, Philadelphia |
2. | Townes, T. M., and Behringer, R. R. (1990) Trends Genet. 6, 219-223[CrossRef][Medline] [Order article via Infotrieve] |
3. | Orkin, S. H. (1990) Cell 63, 665-672[Medline] [Order article via Infotrieve] |
4. | Dillon, N., and Grosveld, F. (1993) Trends Genet. 9, 134-137[CrossRef][Medline] [Order article via Infotrieve] |
5. | Engel, J. D. (1993) Trends Genet. 9, 304-309[CrossRef][Medline] [Order article via Infotrieve] |
6. | Behringer, R. R., Ryan, T. M., Palmiter, R. D., Brinster, R. L., and Townes, T. M. (1990) Genes Dev. 4, 380-389[Abstract] |
7. | Enver, T., Raich, N., Ebens, A. J., Papayannopoulou, T., Costantini, F., and Stamatoyannopoulos, G. (1990) Nature 344, 309-313[CrossRef][Medline] [Order article via Infotrieve] |
8. | Perkins, A. (1999) Int. J. Biochem. Cell Biol. 31, 1175-1192[CrossRef][Medline] [Order article via Infotrieve] |
9. | Bieker, J. J., and Southwood, C. M. (1995) Mol. Cell. Biol. 15, 852-860[Abstract] |
10. | Miller, I. J., and Bieker, J. J. (1993) Mol. Cell. Biol. 13, 2776-2786[Abstract] |
11. |
Feng, W. C.,
Southwood, C. M.,
and Bieker, J. J.
(1994)
J. Biol. Chem.
269,
1493-1500 |
12. |
Donze, D.,
Townes, T. M.,
and Bieker, J. J.
(1995)
J. Biol. Chem.
270,
1955-1959 |
13. | Perkins, A. C., Sharpe, A. H., and Orkin, S. H. (1995) Nature 375, 318-322[CrossRef][Medline] [Order article via Infotrieve] |
14. | Nuez, B., Michalovich, D., Bygrave, A., Ploemacher, R., and Grosveld, F. (1995) Nature 375, 316-318[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Perkins, A. C.,
Gaensler, K. M.,
and Orkin, S. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12267-12271 |
16. | Wijgerde, M., Gribnau, J., Trimborn, T., Nuez, B., Philipsen, S., Grosveld, F., and Fraser, P. (1996) Genes Dev. 10, 2894-2902[Abstract] |
17. |
Perkins, A. C.,
Peterson, K. R.,
Stamatoyannopoulos, G.,
Witkowska, H. E.,
and Orkin, S. H.
(2000)
Blood
95,
1827-1833 |
18. |
Guy, L. G.,
Delvoye, N.,
and Wall, L.
(2000)
J. Biol. Chem.
275,
3675-3680 |
19. |
Guy, L. G.,
Mei, Q.,
Perkins, A. C.,
Orkin, S. H.,
and Wall, L.
(1998)
Blood
91,
2259-2263 |
20. | Southwood, C. M., Downs, K. M., and Bieker, J. J. (1996) Dev. Dyn. 206, 248-259[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Tewari, R.,
Gillemans, N.,
Wijgerde, M.,
Nuez, B.,
von Lindern, M.,
Grosveld, F.,
and Philipsen, S.
(1998)
EMBO J.
17,
2334-2341 |
22. | Chen, X., and Bieker, J. J. (1996) EMBO J. 15, 5888-5896[Abstract] |
23. | Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499[Medline] [Order article via Infotrieve] |
24. | Shiio, Y., Itoh, M., and Inoue, J. (1995) Methods Enzymol. 254, 497-502[Medline] [Order article via Infotrieve] |
25. | Courey, A. J., and Tjian, R. (1988) Cell 55, 887-898[Medline] [Order article via Infotrieve] |
26. |
Shields, J. M.,
and Yang, V. W.
(1997)
J. Biol. Chem.
272,
18504-18507 |
27. |
Ouyang, L.,
Chen, X.,
and Bieker, J. J.
(1998)
J. Biol. Chem.
273,
23019-23025 |
28. | Mitchell, P. J., and Tjian, R. (1989) Science 245, 371-378[Medline] [Order article via Infotrieve] |
29. | Gerber, H. P., Seipel, K., Georgiev, O., Hofferer, M., Hug, M., Rusconi, S., and Schaffner, W. (1994) Science 263, 808-811[Medline] [Order article via Infotrieve] |
30. | Hoey, T., Weinzierl, R. O., Gill, G., Chen, J. L., Dynlacht, B. D., and Tjian, R. (1993) Cell 72, 247-260[Medline] [Order article via Infotrieve] |
31. |
Zhang, W.,
and Bieker, J. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9855-9860 |
32. | Merika, M., and Orkin, S. H. (1995) Mol. Cell. Biol. 15, 2437-2447[Abstract] |
33. | Armstrong, J. A., Bieker, J. J., and Emerson, B. M. (1998) Cell 95, 93-104[Medline] [Order article via Infotrieve] |