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J Biol Chem, Vol. 273, Issue 37, 24223-24231, September 11, 1998


Identity of the beta -Globin Locus Control Region Binding Protein HS2NF5 as the Mammalian Homolog of the Notch-regulated Transcription Factor Suppressor of Hairless*

Lloyd T. LamDagger and Emery H. Bresnick§

From the University of Wisconsin Medical School, Department of Pharmacology, Madison, Wisconsin 53706

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Previously, we characterized a DNA-binding protein, HS2NF5, that bound tightly to a conserved region within hypersensitive site 2 (HS2) of the human beta -globin locus control region (LCR) (Lam, L. T., and Bresnick, E. H. (1996) J. Biol. Chem. 271, 32421-32429). The beta -globin LCR controls the chromatin structure, transcription, and replication of the beta -globin genes. We have now purified HS2NF5 to near-homogeneity from fetal bovine thymus. Two polypeptides of 56 and 61 kDa copurified with the DNA binding activity. The two proteins bound to the LCR recognition site with an affinity (3.1 nM) and specificity similar to mouse erythroleukemia cell HS2NF5. The amino acid sequences of tryptic peptides of purified HS2NF5 revealed it to be identical to the murine homolog of the suppressor of hairless transcription factor, also known as recombination signal binding protein Jkappa or C promoter binding factor 1 (CBF1). The CBF1 site within HS2 resides near sites for hematopoietic regulators such as GATA-1, NF-E2, and TAL1. An additional conserved, high affinity CBF1 site was localized within HS4 of the LCR. As CBF1 is a downstream target of the Notch signaling pathway, we propose that Notch may modulate LCR activity during hematopoiesis.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Transcription of the beta -globin genes is controlled by a powerful genetic element called the beta -globin locus control region (LCR).1 The human LCR consists of four erythroid-specific DNase I HSs, 10-50-kilobase pairs upstream of the beta -globin genes (1, 2). The LCR controls the chromatin structure, transcriptional activity, and replication timing of the beta -globin locus (3-5). A defining feature of the LCR is its ability to confer copy number-dependent and position-independent expression to a linked gene that is stably integrated into chromosomal DNA (3). The physiological importance of the LCR is highlighted by a chromosomal deletion associated with Hispanic beta -thalassemia, which removes part of the LCR and correlates with repression of the beta -globin genes (5).

An activity of the LCR distinct from traditional enhancers is that the activation property can be shared by multiple genes over long distances on a chromosome (6-8). We have proposed a mechanism of coordinate promoter activation involving the recruitment of chromatin remodeling enzymes, which mediate the decondensation of chromatin throughout the beta -globin domain (9, 10). The increase in DNA accessibility is manifested as general DNase I sensitivity (11). We hypothesize that this chromatin transition is necessary for the subsequent protein-DNA interactions occurring through promoters, enhancers, and silencers that determine the developmental expression pattern of the beta -globin genes. An alternative mechanism favors looping interactions between the LCR and individual beta -globin gene promoters (12-14), controlling the assembly of preinitiation complexes on the promoters. The chromatin disruption and looping models are not mutually exclusive, as the disruption may be necessary for subsequent promoter interactions. Both mechanisms share the requirement for transactivating proteins that function through the LCR.

Multiple hematopoietic and ubiquitous transcription factors interact with conserved sequences within the HSs of the LCR. HS2 is a strong erythroid-specific enhancer, which has been studied extensively (15-25). Factors interacting with HS2 include NF-E2 (26, 27), GATA-1 (28, 29), TAL-1 (30), USF (20, 31), SP1 (32), YY1 (33), and HS2NF5 (34). Mutational analysis suggests that many of these factors are required for optimal HS2-mediated transactivation in transgenic mice and stably transfected cell lines (20, 22, 34).

Previously, we had characterized protein-DNA interactions within a conserved region of HS2 containing an E box (34), which was a putative TAL1-binding site. TAL1 is a hematopoietic basic helix-loop-helix transcription factor that regulates hematopoiesis (35, 36). Although TAL1 binding was not detected using MEL and K562 cell nuclear extracts, a novel protein was identified, HS2NF5, that bound tightly to a site overlapping the E box. Recently, we performed a quantitative analysis of recombinant TAL1/E12 heterodimer DNA binding specificity (37). TAL1/E12 bound to oligonucleotides containing the HS2 E box with very low affinity, in contrast to the high affinity interaction with an optimal TAL1 site. As protein-protein interactions between TAL1 and LIM domain proteins alter the DNA binding specificity of TAL1 (38), we postulated that additional factors may allow TAL1 to associate stably with the E box. In contrast to our results with nuclear extracts, Etlinski et al. (30) detected TAL1 binding to the E box using K562 extracts.

The importance of HS2NF5 in LCR function was suggested by a mutational analysis of the HS2NF5-binding site of HS2 (34). This site was necessary for optimal enhancer activity in stable and transient transfection assays. Since HS2NF5 had an apparently unique DNA binding specificity, a variable distribution in cell lines, and was the major activity that bound with high affinity to a functionally important region of HS2, we chose to purify HS2NF5. Here, we describe the identification of two forms of HS2NF5 as the previously cloned transcription factor known as Su(H) in Drosophila (39, 40), and RBPJkappa or CBF12 in mammals (41, 42). As Su(H) and CBF1 are critical developmental regulators and downstream targets of the Notch signaling pathway, we discuss a potential role for Notch signaling in LCR activity and hematopoiesis.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Buffers

Buffer A contained 20 mM HEPES (pH 7.2), 0.1 mM EDTA, 5% glycerol, 50 mM NaCl. Buffer B contained 20 mM Tris (pH 7.8), 0.1 mM EDTA, 5% glycerol, 50 mM NaCl. Buffer C contained 25 mM HEPES (pH 7.6), 20% glycerol. Buffer D contained 20 mM Tris (pH 7.5), 150 mM NaCl, 0.2 mM EDTA. Buffer E contained 20 mM HEPES (pH 7.5), 250 mM sucrose, 500 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamide. Buffer H contained 20 mM HEPES (pH 7.5), 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamide. All buffers contained 5 mM dithiothreitol.

Cell Culture

The mouse erythroleukemia cell line MEL was propagated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 4 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. The cell line was grown in a humidified incubator at 37 °C, in the presence of 5% carbon dioxide. MEL cells were treated with 1.5% dimethyl sulfoxide for 72 h prior to isolating nuclear extract.

Preparation of Fetal Calf Thymus Nuclear Extract

Fetal calf thymus (from approximately 4-month-old calves) was obtained from Peck Slaughterhouse (Milwaukee, WI). The fresh thymus was washed with cold phosphate-buffered saline and then mixed with 2 volumes of buffer H at 4 °C. The mixture was homogenized in a Waring blender with two pulses of 40 s each. The blended material was filtered through four sheets of cheesecloth and then centrifuged at 545 × g for 15 min. The pellet was washed with 2 volumes of buffer H by resuspension and centrifugation at 545 × g for 5 min. The pellet was then resuspended by vigorous shaking in 2 volumes of buffer E, and the mixture was centrifuged at 142,413 × g for 40 min. The supernatant was adjusted to 25% (NH4)2SO4 by gradual addition of solid (NH4)2SO4 with stirring. The mixture was stirred for 30 min, and the precipitate was collected by centrifugation at 21,000 × g for 15 min. The supernatant was adjusted to 50% (NH4)2SO4 as described above, and the precipitated material was collected by centrifugation at 21,000 × g for 15 min. The pellet was dissolved in a minimal volume of buffer A for Resource-S chromatography. The solution was dialyzed against the same buffer overnight. The insoluble material was removed by centrifugation for 15 min at 21,000 × g, and the resulting nuclear extract was used for the chromatography. All procedures were performed at 4 °C.

Electrophoretic Mobility Shift Assay

DNA binding reactions were performed as described previously (34). DNA binding activity was quantitated by analyzing gels with a PhosphorImager (Molecular Dynamics).

Chromatography

All chromatographic steps were carried out with an Amersham Pharmacia Biotech fast pressure liquid chromatography system at 4 °C.

Resource-S Chromatography-- The crude extract was chromatographed in three consecutive runs on a 20-ml Resource-S cation exchange column (Amersham Pharmacia Biotech) equilibrated in buffer A. Proteins were resolved with a 50-450 mM NaCl gradient in buffer A at a flow rate of 2.5 ml/min. HS2NF5 DNA binding was measured by EMSA in the presence of 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech). HS2NF5 activity eluted as a homogeneous peak between 200 and 380 mM NaCl. The fractions containing maximal HS2NF5 activity were pooled and dialyzed overnight against 2 liters of buffer B.

Q-Sepharose Chromatography-- The dialyzed material from the Resource-S column was centrifuged for 15 min at 21,000 × g at 4 °C. The supernatant was applied to a 12-ml Q-Sepharose anion-exchange column (Amersham Pharmacia Biotech) equilibrated in buffer B. Proteins were resolved with a 100-400 mM NaCl gradient in buffer B at a flow rate of 2.5 ml/min. HS2NF5 DNA binding was measured by EMSA in the presence of 0.5 µg of poly(dI-dC). HS2NF5 activity eluted as a major peak between 160 and 260 mM NaCl. A minor peak eluted at a slightly lower salt concentration but was not analyzed further, as it was present in fractions containing a high concentration of protein, which would have reduced considerably the fold purification. The fractions containing maximal HS2NF5 activity were pooled.

Phenyl-Sepharose Chromatography-- Ammonium sulfate was added to the pooled material from the Q-Sepharose column to a final concentration of 1 M. The mixture was applied to a 10-ml phenyl-Sepharose hydrophobic column (Amersham Pharmacia Biotech) equilibrated in buffer B containing 1 M ammonium sulfate. Proteins were resolved with a 1 to 0 M ammonium sulfate gradient in buffer B at a flow rate of 2 ml/min. HS2NF5 DNA binding was measured in the presence of 0.25 µg of poly(dI-dC). HS2NF5 activity eluted as a homogeneous peak between 150 and 300 mM ammonium sulfate. The fractions containing maximal HS2NF5 activity were pooled and dialyzed overnight against 1 liter of buffer C.

DNA-Cellulose Chromatography-- The pooled material from the phenyl-Sepharose column was applied to a 5-ml double-stranded DNA-cellulose (Sigma) column equilibrated in buffer C. Proteins were resolved with a 150-450 mM KCl gradient in buffer C at a flow rate of 1 ml/min. HS2NF5 DNA binding was assayed in the presence of 0.1 µg of poly(dI-dC). HS2NF5 activity eluted as a homogenous peak between 270 and 390 mM KCl. Protein concentrations of the pooled samples from each of the first three columns were determined by the Bradford assay. Proteins were analyzed by resolving on 9% SDS-PAGE, followed by silver staining. In place of the DNA-cellulose step, we also used sequence-specific DNA affinity chromatography with the concatamerized HS2NF5 site coupled to Sepharose. Although HS2NF5 could also be isolated with this column, the yield of HS2NF5 was lower than with the DNA-cellulose column (data not shown).

Gel Filtration Chromatography-- The pooled material from the DNA-cellulose column was dialyzed against buffer D and concentrated by lyophilization. The mixture was applied to a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated in buffer D, and proteins were eluted at a flow rate of 0.5 ml/min. HS2NF5 DNA binding was measured by EMSA in the presence of 0.1 µg of poly(dI-dC). After dialysis against Milli-Q filtered water and lyophilization, proteins were analyzed by 9% SDS-PAGE and silver staining.

Tryptic Proteolysis and Amino Acid Sequence Determination

Fractions of DNA-cellulose-purified material were dialyzed against Milli-Q water and concentrated by lyophilization. The proteins were resolved by 9% SDS-PAGE and detected by Coomassie Blue staining. After thorough destaining, gel pieces containing the 56- and 61-kDa forms of HS2NF5 were excised and subjected to carboxyamidomethylation proteolysis with trypsin as described previously (43). Mass spectroscopy sequencing was performed by Bill Lane of the Harvard Microchemistry Facility. The eluted peptides were separated by microcapillary reverse-phase HPLC coupled to the electrospray ionization source of a Finnigan LCQ quadrupole ion trap mass spectrometer. Peptides were eluted from the column with a linear gradient of 0-50% acetonitrile in 0.05% acetic acid at a flow rate of 700 nl/min. Ionization was assisted with a coaxial sheath liquid of 70% methanol, 0.05% acetic acid. Spectra were acquired as successive sets of three scan modes as follows: full scan MS over the m/z range 395-1118 amu, followed by two data-dependent scans on the most abundant ion in that full scan. These data-dependent scans allowed the automatic acquisition of a high resolution (zoom) scan to determine charge state and exact mass and MS/MS spectra for peptide sequence information. Base peak relative ion abundance corresponded to a load of 5-35 fmol by comparison with a standard peptide mixture analyzed under identical conditions. Interpretation of the resulting MS/MS spectra of the peptides was facilitated by searching NCBI non-redundant protein and EST data bases with the algorithm Sequest (44).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Comparison of MEL Cell and Fetal Calf Thymus HS2NF5-- Quantitative DNA binding studies suggested that the amount of HS2NF5 in nuclear extracts of MEL and K562 cells was low, which would make purification of ample amounts for sequencing difficult. Instead, we found that HS2NF5 DNA binding activity was present in nuclear extracts of fetal calf thymus, and it resembled HS2NF5 from MEL and K562 cells. To compare the DNA sequence specificity of MEL and thymus HS2NF5, we performed EMSAs using nuclear extracts fractionated on a Resource-S column, followed by a Q-Sepharose column.

As shown in Fig. 1, titrations were performed with increasing concentrations of radiolabeled oligonucleotides containing a wild-type or mutated HS2NF5 site (Fig. 1A) and a constant amount of fractionated extract. The HS2NF5-DNA complexes formed with MEL cell and thymus extracts had an identical mobility on the nondenaturing gel. A mutant oligonucleotide, Mut-1, was tested, which had been shown previously not to bind HS2NF5 (34). No HS2NF5 binding to Mut-1 was detected with either extract. In contrast, another mutated oligonucleotide, Mut-2, which had been shown previously not to affect HS2NF5 binding (34) was bound by both MEL and thymus HS2NF5. Thus, the DNA binding activity of MEL and thymus HS2NF5 appeared to be indistinguishable, justifying the use of thymus extract for purifying HS2NF5.


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  		Fig. 1.   DNA binding activity of MEL cell and fetal calf thymus HS2NF5. A, sequences of the coding strands of wild-type and mutated oligonucleotides used in the EMSA. B, EMSA analysis of protein-DNA interactions. Increasing amounts (10, 20, and 40 fmol) of radiolabeled oligonucleotide duplex were incubated with a constant amount of fractionated nuclear extract from MEL cell and fetal calf thymus. The nuclear extracts were fractionated over Resource-S and Q-Sepharose columns as described under "Experimental Procedures." The positions of the unbound probe and the HS2NF5-DNA complex are indicated.

Purification of HS2NF5 from Fetal Calf Thymus-- The HS2NF5 purification scheme is outlined in Fig. 2. Fetal calf thymus nuclear extract was fractionated by ammonium sulfate precipitation, followed by chromatography on Resource-S, Q-Sepharose, phenyl-Sepharose, and double-stranded DNA-cellulose columns (data not shown). Protein samples from each stage of the purification were resolved by SDS-PAGE and analyzed by silver staining to evaluate the purity of HS2NF5. As shown in the extensively silver-stained gel of Fig. 3A, after four chromatographic steps, two major protein bands of 56 and 61 kDa were present in fractions 22-28. A smaller amount of protein from another purification is shown on the right to illustrate the copurification and purity of the 56- and 61-kDa proteins (Fig. 3B). Both the 56- and 61-kDa bands were characterized by microheterogeneity.


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  		Fig. 2.   HS2NF5 purification scheme.


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  		Fig. 3.   SDS-PAGE analysis of purified HS2NF5. A, fetal calf thymus nuclear extract (10 µg), the Resource-S pool (10 µg), the Q-Sepharose pool (10 µg), the phenyl-Sepharose pool (10 µg), and fractions from the DNA-cellulose column were analyzed by SDS-PAGE and visualized by silver staining. The positions of the two forms of HS2NF5 are indicated. B, purified HS2NF5 from another purification (fraction 24) was analyzed by SDS-PAGE and silver staining. C, comparison of the relative amount of 56- and 61-kDa polypeptides detected by silver staining and the DNA binding activity of HS2NF5 quantitated from EMSA (data not shown). D, densitometric scan of stained proteins in fraction 24 of panel A.

To show further that the 56- and 61-kDa bands represent HS2NF5, we compared the relative amount of DNA binding activity measured by EMSA (data not shown) with the relative amount of the 56- and 61-kDa polypeptides detected by densitometric analysis of the silver-stained bands (Fig. 3A). As the binding activity and protein correlated well, this supports the identity of the 56- and 61-kDa bands as HS2NF5 (Fig. 3C). The purity of HS2NF5 in fraction 24 from the DNA-cellulose column was estimated to be 94% by densitometric analysis (Fig. 3D) and reverse-phase chromatography on a microcapillary C18 HPLC column (data not shown). The purification results are summarized in Table I. A 52,450-fold purification of HS2NF5 was achieved, with a yield of 27%. These results were representative of 10 independent purifications with different samples of fetal calf thymus.

                              
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Table I
Purification of HS2NF5 from fetal calf thymus
Three hundred and fifteen grams of fetal calf thymus was used in this purification. The amount of 32P-oligonucleotide bound was measured with a PhosphorImager. The binding activity of HS2NF5 in the nuclear extract could not be accurately determined, since the crude extract contained substances that inhibited binding. The protein concentrations of fractions 22 and 24 of the DNA-cellulose column were estimated by SDS-PAGE and silver staining relative to standard proteins. The protein concentrations of fractions 26 and 28 were too low to be measured.

The DNA binding specificity of purified HS2NF5 was assessed by EMSA with a labeled wild-type HS2NF5 oligonucleotide and unlabeled wild-type and mutant competitors (Fig. 4A). Two mutations were shown previously (34) to reduce HS2 enhancer activity (E box mut, CAGATG changed to GTCGAC; HS2NF5 mut, TTCTCA changed to CTGCAG). In addition, as a control, we tested a third mutant (TAA mut, GCC changed to TAA), which we had shown previously to have no effect on HS2NF5 DNA binding (34). The wild-type and TAA mut oligonucleotides strongly reduced binding (wild type, 94.8 and 97.9% decrease at 50- and 200-fold excess, respectively; TAA mut, 79 and 93.2% decrease at 50- and 200-fold excess, respectively) (Fig. 4C). In contrast, the E box mut and HS2NF5 mut oligonucleotides only weakly competed for binding (E box mut, 48 and 61% decrease at 50- and 200-fold excess, respectively; HS2NF5 mut, 8.0 and 43% decrease at 50- and 200-fold excess, respectively), which agrees with our previous analysis of HS2NF5 DNA binding specificity in MEL cell nuclear extracts (34). Since the mutations of the E box mut and HS2NF5 mut oligonucleotides prevent HS2NF5 DNA binding and were shown previously to decrease HS2 enhancer activity (34), these results suggest that impaired HS2NF5 binding may be responsible for the inhibition.


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  		Fig. 4.   DNA binding specificity of purified HS2NF5. A, sequences of the coding strands of wild-type and mutated oligonucleotides used in the EMSA. B, EMSA analysis. Fetal calf thymus HS2NF5 purified through the DNA-cellulose step (0.5 µl of dialyzed protein) was preincubated with 50- or 200-fold excess of unlabeled oligonucleotides and then incubated with the 32P-labeled wild-type HS2NF5 oligonucleotide (10 fmol). DNA binding activity was measured by EMSA. The positions of the unbound probe and the HS2NF5-DNA complex are indicated. C, quantitative analysis of DNA binding. The amount of 32P-oligonucleotide present in the HS2NF5-DNA complex was measured with a PhosphorImager, and the relative amount of complex formed was plotted. The plot shows averaged results from two independent experiments.

Chromatographic Separation of Two Forms of HS2NF5-- To determine whether the 56- and 61-kDa forms of HS2NF5 have distinct DNA binding properties or bind to DNA as a heteromer, we resolved the purified material on an analytical gel filtration column. As shown in Fig. 5, A and B, the 56- and 61-kDa proteins were partially separated by the column. The gel mobility shift formed by HS2NF5 segregated into two species, reflecting the partial separation of the two forms. The fractions containing predominantly either the 56- and 61-kDa forms of HS2NF5 retained DNA binding activity, suggesting that DNA binding does not require the formation of a heteromeric complex between the two forms. Furthermore, the copurification of HS2NF5 DNA binding activity with the 56- and 61-kDa polypeptides provided additional evidence for the identity of these proteins as HS2NF5.


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  		Fig. 5.   Resolution of 56- and 61-kDa forms of HS2NF5 on a Superdex 200 column. A, fractions containing HS2NF5 purified through the DNA-cellulose step were dialyzed and fractionated on a Superdex 200 column. Aliquots (5 µl) were assayed for DNA binding activity by EMSA with the 30-bp 32P-labeled wild-type oligonucleotide (30 fmol) of Fig. 1A. B, the fractions were dialyzed, dried, and analyzed by 9% SDS-PAGE and visualized by silver staining. The positions of the two forms of HS2NF5 are indicated.

We tested whether the two forms of HS2NF5 have distinct DNA binding activities. Even though both forms copurified on the DNA-cellulose column, their binding affinity could differ, as an affinity difference might not be apparent due to the high DNA concentration on the column. Quantitative EMSAs were used as described previously (31, 34, 45) to estimate the DNA binding affinity of the two forms of HS2NF5. Titrations were performed with a constant amount of purified HS2NF5 and increasing concentrations of the wild-type HS2NF5 oligonucleotide (Fig. 6A). Under these conditions, saturation is achieved upon depletion of the binding factor from the reaction by a vast stoichiometric excess of DNA. The estimated KD values for the 56- and 61-kDa forms of HS2NF5 were indistinguishable (KD = 3.1 ± 0.9 and 3.1 ± 1.1 nM, respectively) (Fig. 6B). The high affinity binding of purified thymus HS2NF5 corresponded well with our previous measurements of HS2NF5 in MEL cell nuclear extracts (KD = 5.8 nM) (34).


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  		Fig. 6.   Comparable DNA binding affinity of the 56- and 61-kDa forms of HS2NF5. A, quantitative EMSA analysis. Increasing amounts of 32P-labeled wild-type HS2NF5 oligonucleotide were incubated with purified HS2NF5, and DNA binding activity was measured by EMSA. The positions of the upper and lower HS2NF5-DNA complexes are indicated. B, quantitation of DNA binding by the two forms of HS2NF5. The concentration of HS2NF5-DNA complexes formed (pM) was determined with a PhosphorImager and plotted as a function of the total DNA concentration (nM) in the binding assay. Nonlinear regression analysis was used to estimate the equilibrium binding constant (KD) (mean ± S.E., n = 6).

Identity of HS2NF5 as the Mammalian Homolog of Su(H), CBF1-- The fractions containing maximal levels of HS2NF5 from the DNA-cellulose column were dialyzed, concentrated, and resolved by SDS-PAGE to separate the 56- and 61-kDa polypeptides. Proteins were detected by Coomassie Blue staining and then excised from the gel. The gel slices were incubated with trypsin; eluted peptides were resolved by reverse-phase microcapillary HPLC, and amino acid sequencing was performed by mass spectroscopy analysis. The sequences of two peptides from each of the 56- and 61-kDa forms were obtained. All four sequences were identical to the previously cloned murine homolog of the Drosophila transcription factor Su(H) (39, 40), also known as CBF1 (41). The positions of the peptide sequences relative to the sequence of Drosophila Su(H) and murine CBF1 are shown in Fig. 7.


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  		Fig. 7.   Alignment of amino acid sequences of tryptic peptides from two forms of bovine HS2NF5 with murine CBF1 and Drosophila suppressor of hairless. Two tryptic peptide sequences were obtained from each of the 56- and 61-kDa forms of bovine HS2NF5 by mass spectroscopy analysis. The bovine sequences are depicted above the sequences of murine CBF1 and Drosophila suppressor of hairless (SWISS-PROT data base numbers P31266 and P28159, respectively). Conserved residues are depicted by the shaded boxes.

Binding of HS2NF5 to CBF1 Sites within LCRs from Different Species-- Although the HS2NF5 site within HS2 is partially conserved between human, galago, mouse, rabbit and goat, certain nucleotide substitutions in galago and mouse could potentially inhibit HS2NF5 binding. We have localized an additional conserved CBF1 site within the 280-bp minimal core of the HS4 subregion of the LCR, which also has enhancer activity (46). Fig. 8 shows a comparison of the HS2 and HS4 sequences, which contain the CBF1 sites, from different species.


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  		Fig. 8.   Conserved sequences within HS2 and HS4 of the beta -globin LCR. A, alignment of sequences from human, goat, galago, rabbit, and mouse HS2. The bold sequence depicts a predicted CBF1 recognition site within the HS2 region previously shown to bind HS2NF5 with high affinity. B, alignment of sequences from the human, galago, rabbit, and mouse beta -globin LCR within the HS4 core. The bold sequence depicts a predicted CBF1 recognition site, which is conserved between human, galago, and rabbit; the goat sequence has not been reported.

To determine the relative binding of purified HS2NF5 to these sites, a competitive DNA binding assay was performed with the radiolabeled wild-type HS2NF5 oligonucleotide of Fig. 1A and a 50- or 200-fold stoichiometric excess of radioinert oligonucleotides representing sequences from the human, goat, galago, and mouse LCR (Fig. 9). In addition, we compared the binding of purified HS2NF5 to the LCR sites with sites from Su(H) and CBF1 target genes. We tested oligonucleotides containing either a CBF1-binding site from the C promoter of EBV (Cp) (47) or a Su(H)-binding site from the Drosophila melanogaster Hes promoter (48) (Fig. 9A). The gel was analyzed with a PhosphorImager to quantitate binding (Fig. 9C). A 200-fold molar excess of either Cp or Hes strongly inhibited binding (100 and 98.6% decrease, respectively). Similarly, oligonucleotides containing the core sequence TTCTCAG (from the human and goat LCR) strongly inhibited binding (89.0 and 93.8% decrease, respectively). In contrast, oligonucleotides containing the galago and mouse sequences only weakly inhibited binding (27.4 and 46.5% decrease, respectively), relative to a control oligonucleotide containing a site for the erythroid- and megakaryocytic-specific transcription factor NF-E2 (39.3% decrease). Thus, these results show that purified HS2NF5 binds to the human and goat LCR sites with an affinity similar to the Cp and Hes sites; in contrast, galago and mouse LCR sites bound HS2NF5 with lower affinity.


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  		Fig. 9.   Comparison of HS2NF5 binding to recognition sites within HS2 and known CBF1 target genes. A, sequences of the coding strands of oligonucleotides with potential CBF1-binding sites from different species. Radioinert oligonucleotides were used as competitors in an EMSA. The CBF1 and HS2NF5 sites are indicated in bold and italics. B, EMSA analysis. Fetal calf thymus HS2NF5 purified through the phenyl-Sepharose step was preincubated with 50- or 200-fold excess of unlabeled oligonucleotides and then incubated with the 32P-labeled wild-type HS2NF5 oligonucleotide of Fig. 1A (30 fmol). DNA binding activity was measured by EMSA. The positions of unbound probe and the HS2NF5-DNA complex are indicated. C, quantitative analysis of DNA binding. The amount of 32P-oligonucleotide present in the HS2NF5-DNA complex was measured with a PhosphorImager, and the relative amount of complex formed was plotted.   

We also tested oligonucleotides containing other potential CBF1 sites within HS2 (at positions 11228 and 11421); these oligonucleotides inhibited HS2NF5 binding by 58.1 and 72.2%, respectively (data not shown), suggesting that these sites have a moderate affinity for HS2NF5. The competition data quantitated above, using a 200-fold excess of oligonucleotide, was qualitatively similar to the results using a 50-fold excess of oligonucleotide.

As indicated above, HS4 also contains a conserved region with a putative CBF1 site (Fig. 8B). This site is well conserved between human, galago, and rabbit but, analogous to the HS2 site, is not conserved in mouse; the sequence of goat HS4 has not been reported. To determine if the HS4 sequence binds CBF1 with high affinity, the competitive EMSA was done using a 50- and 200-fold excess of radioinert oligonucleotides containing sequences from the human, galago, and mouse LCR (Fig. 10A). The rabbit sequence was not tested, since it was identical to galago. The oligonucleotides with CBF1 sites from human and galago HS4 strongly competed for HS2NF5 binding (72.3 and 80.3% inhibition, respectively), similar to the oligonucleotide containing the CBF1 site from human HS2 (87.6% decrease) (Fig. 10C). In contrast, the mouse sequence only mildly inhibited binding (24.8% decrease), relative to the NF-E2 control (12.8% decrease). Thus, the CBF1 site within HS4 is conserved between human, galago, and rabbit.


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  		Fig. 10.   HS2NF5 binding to a conserved CBF1 recognition site within HS4. A, sequences of the coding strands of oligonucleotides with predicted CBF1-binding sites from different species. Radioinert oligonucleotides were used as competitors in the EMSA. The CBF1-binding sites are indicated in bold and italics. B, EMSA analysis. Fetal calf thymus HS2NF5 purified through the phenyl-Sepharose step was preincubated with 50- or 200-fold excess of unlabeled oligonucleotides and then incubated with the 32P-labeled wild-type HS2NF5 oligonucleotide of Fig. 1A (30 fmol). DNA binding activity was measured by EMSA. The positions of the unbound probe and the HS2NF5-DNA complex are indicated. C, quantitative analysis of DNA binding. The amount of 32P-oligonucleotide present in the HS2NF5-DNA complex was measured with a PhosphorImager, and the relative amount of complex formed was plotted.   

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Identity of HS2NF5 as CBF1-- Previously, we had identified HS2NF5 as a protein that bound to a functionally relevant region of the human beta -globin LCR (34). Here, we have described the chromatographic purification of two forms of HS2NF5 from fetal calf thymus. Amino acid sequencing revealed that tryptic peptides from the two forms of HS2NF5 are homologous to the Drosophila transcription factor Su(H) and identical to its murine homolog, CBF1. Su(H) and CBF1 are highly conserved transcription factors that have been cloned from Drosophila (49), Caenorhabditis elegans (50), Xenopus (51), mouse (41), and human (42).

The two forms of HS2NF5 may result from alternative RNA splicing, as two distinct cDNAs encoding CBF1 have been isolated from a 38B9 pre-B cell cDNA library (52). The corresponding RNA molecules generated by alternative splicing, RBP-2 and RBP-2N, encode proteins of 526 and 487 amino acids, respectively, differing in their amino termini. RBP-2N was expressed in the pre-B cell line at 10-20 times the level of RBP-2. Despite the presence of two distinct transcripts, purified RBP-Jkappa from the same cell line was detected as a single Coomassie Blue-stained band of 60 kDa (41). In addition, CBF1 purified from HeLa cells was detected as a major 60-kDa band and a minor component that migrated slightly slower by SDS-PAGE (42). The 56- and 61-kDa thymic forms copurified in 10 purifications and were routinely present in approximately equal amounts, suggesting that the 56-kDa form does not arise from proteolysis. As they have an indistinguishable affinity for the HS2 oligonucleotide, the functional differences between the two forms are unclear (Fig. 6).

The consensus DNA recognition sequence for CBF1 has been reported to be TTCCCAC (53, 54). The sequences of functional CBF1 sites within Cp and Hes closely resemble the high affinity sites of HS2 and HS4. In addition to high affinity binding and transactivation, another parameter used to assess the functional importance of a binding site is sequence conservation (55). The CBF1 site within HS4 is well conserved between human, galago, and rabbit but not in mouse. The CBF1 site within HS2 is conserved between human and goat, but not with rabbit, galago, and mouse. Although the HS2 site is not conserved in all species, we have shown that it was important for optimal enhancer activity of human HS2 in stable and transient transfection assays (34). Mutagenesis of essential nucleotides of the HS2NF5-binding site overlapping the E box or nucleotides distinct from the E box resulted in a similar inhibition of HS2 enhancer activity. The functional significance of the conserved CBF1 site within HS4 is unclear, although it falls within the 280-bp minimal enhancer fragment (46) but outside of the 101-bp fragment required for HS formation (56).

Developmental Roles of Su(H), CBF1, and Notch-- Su(H) regulates neurogenesis, myogenesis, and the development of various other organs in Drosophila (reviewed in Ref. 57). A common theme that has emerged from studies of Drosophila, C. elegans, and Xenopus development is Su(H) mediates cell fate decisions. Su(H) controls the expression of genes critical for cell differentiation. Homozygous mutant mice lacking CBF1 have a severe developmental delay by day 8.5 of gestation, multiple abnormalities at day 9.5, and die before day 10.5 of gestation (58). In addition to the important developmental role of CBF1, CBF1 is a critical mediator of transactivation by the EBV protein, EBNA2.

In vertebrates, a paradigm for CBF1 function has been developed from studies by Hayward and co-workers (59) on EBNA2, a transactivating protein of EBV. EBNA2 mediates target gene activation in B-cells and is necessary for EBV-induced B-cell transformation. EBNA2 lacks DNA binding activity but acts as a coactivator by physically interacting with DNA-bound CBF1 (42). CBF1 has a transcriptional repressive domain (59, 60) and thus may inhibit transcription unless associated with a coactivator. CBF1 was shown recently to interact with a LIM domain protein, KyoT2 (61), which negatively regulated transactivation by CBF1. TAL1 heterodimers also interact with LIM domain proteins, which modulate their DNA binding activity (38) and ability to control hematopoiesis (62). Interestingly, the CBF1 site of HS2 overlaps a TAL1 site, and both factors engage in interactions with homologous LIM domain proteins. In addition to EBNA2 and KyoT2, the intracellular domain of the transmembrane receptor Notch interacts with CBF1.

The Notch transmembrane receptor is activated by the binding of a transmembrane ligand such as Serrate or Delta on the surface of a neighboring cell (reviewed in Refs. 57, 63, and 65). Although the exact mechanism of Notch activation is unknown, a favored model suggests that Su(H) physically interacts with the intracellular domain of Notch in the inactive state (64). Upon activation, Su(H) is displaced by the cytosolic protein Deltex and translocates to the nucleus to activate target genes that control cell fate (57). In addition, the intracellular domain of Notch is liberated by site-specific proteolysis, allowing it to enter the nucleus and to potentially serve as a coactivator for Su(H), analogous to EBNA2 (64, 65). The Notch system is quite complex, as there are multiple Notch receptors and ligands (65).

A General Role for Notch Signaling in Hematopoietic Cells?-- CBF1 was first purified from a pre-B-cell line, based on its binding to the Jkappa recombination signal (41). It was later revealed that a CBF1-binding site was created by the concatamerization of this sequence (42). We have purified CBF1 from fetal calf thymus and have shown previously (34) that this DNA binding activity is present in erythroleukemia cell lines. Thus, CBF1 is present in multiple hematopoietic lineages. The broad distribution of CBF1 is consistent with the wide distribution of Notch (57).

Several lines of evidence implicate Notch signaling as a regulatory mechanism in hematopoiesis. First, a human homolog of Drosophila Notch, TAN-1, is expressed in CD34+ hematopoietic precursor cells (66). Second, expression of a constitutively active Notch mutant, consisting of the intracellular domain, in 32D myeloid progenitor cells inhibited granulocytic differentiation (67). Finally, enforced expression of activated Notch in the developing T-cells of transgenic mice resulted in an increase in CD8 and a decrease in CD4 T-cells (68). These studies along with the presence of CBF1 in multiple hematopoietic lineages, and the fact that other LCR-binding proteins regulate hematopoiesis (35, 36, 69, 70), strongly suggest a role for Notch in cell fate decisions during hematopoiesis.

In summary, we have purified HS2NF5 and identified it as CBF1. According to the Notch paradigm, the nuclear translocation of CBF1 is controlled through cell-cell interactions. Interactions between stromal cells and hematopoietic progenitor cells, which express Notch ligands and receptors, respectively (65), are important for hematopoiesis (71, 72). As we have detected CBF1 in nuclear extracts from multiple cell lines and the thymus, there may be constitutive Notch activity in these systems, if the subcellular distribution of CBF1 is strictly regulated by Notch. It will be important to determine whether Notch controls the subcellular distribution of CBF1 in erythroid cells, the occupancy of CBF1 sites within the LCR, and LCR function.

    ACKNOWLEDGEMENTS

We thank William S. Lane at the Harvard Microchemistry Facility for mass spectrometry and peptide sequencing. We also thank Brian K. Kay for a critical review of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK50107, the Leukemia Society of America, the Milwaukee Foundation, and the Pharmaceutical Research and Manufacturers of America Foundation.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.

Dagger Predoctoral Trainee of the Pharmaceutical Research and Manufacturers of America Foundation.

§ Leukemia Society of America Scholar and a Shaw Scientist. To whom correspondence should be addressed: University of Wisconsin Medical School, Dept. of Pharmacology, 387 Medical Science Bldg., 1300 University Ave., Madison, WI 53706. Tel.: 608-265-6446; Fax: 608-262-1257; E-mail: ehbresni{at}facstaff.wisc.edu.

The abbreviations used are: LCR, locus control region; Cp, C promoter from Epstein-Barr virus; CBF1, C promoter binding factor 1; EMSA, electrophoretic mobility shift assay; EBV, Epstein-Barr virus; EBNA2, Epstein-Barr virus nuclear antigen 2; Hes, hairy enhancer of splitHPLC, high pressure liquid chromatographyHS, hypersensitive siteRBP-Jkappa , recombination signal binding protein immunoglobulin Jkappa Su(H), suppressor of hairlessPAGE, polyacrylamide gel electrophoresisbp, base pair(s)MS, mass spectroscopymut, mutant.

2 CBF1 and RBP-Jkappa refer to the same protein, which will be indicated as CBF1 throughout.

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Top
Abstract
Introduction
Procedures
Results
Discussion
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