Regulation of the ERBB-2 Promoter by RBPJkappa and NOTCH*

(Received for publication, December 13, 1996)

Yanyun Chen Dagger , Wolfgang H. Fischer § and Gordon N. Gill Dagger

From the Dagger  Department of Medicine, University of California San Diego, La Jolla, California 92093-0650 and § The Salk Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Within the human ERBB-2 gene promoter, a 100-base pair region 5' to the TATA box enhances basal transcription 200-fold. Two palindromes present within this 100-base pair region are important for transcription. The palindrome binding protein was purified to homogeneity and found to be identical to RBPJkappa , the mammalian homolog of Drosophila Suppressor of Hairless (Su(H)). Recombinant RBPJkappa bound the ERBB-2 promoter with affinity comparable with that seen with well characterized RBPJkappa binding sites. RBPJkappa activated an ERBB-2 palindrome-containing promoter in 293 cells. Because in Drosophila Su(H) acts downstream of NOTCH and because NOTCH·Su(H)/RBPJkappa stimulates transcription from target promoters, NOTCH-IC, a constitutively active form of NOTCH, was tested for effects on the ERBB-2 palindrome. NOTCH-IC further increased RBPJkappa -mediated transcription on wild type but not mutant ERBB-2 palindrome. Thus, RBPJkappa can activate ERBB-2 transcription and serve as an anchor to mediate NOTCH function on the ERBB-2 gene.


INTRODUCTION

The human ERBB-2 gene, located on chromosome 17q21, encodes a 1255-amino acid protein-tyrosine kinase receptor (1, 2). This receptor is widely expressed at higher levels during fetal development than in the adult, where it is detected primarily in epithelial cells (3, 4). Amplification and overexpression is frequent in adenocarcinomas, especially in those arising in the breast and ovary where overexpression directly correlates with poorer patient outcomes (5, 6). Protein overexpression is frequently the result of gene amplification; however, tumors are reported to overexpress ERBB-2 mRNA and protein from single copy genes. Even with gene amplification, mRNA expression/gene is increased, indicating that transcriptional control mechanisms are important (6, 7).

To deduce transcriptional control mechanisms, the human ERBB-2 gene promoter has been sequenced and found to contain typical CAAT and TATA elements and four Alu sequences within 3.65 kilobases of the proximal promoter (8-10). A 100-bp region upstream of the TATA box increases promoter activity 200-fold. This 100-bp enhancer contains an Sp1 site near its 5' end and a CAAT box near its 3' end (11). It also contains two palindromic sequences that are conserved in the rat and mouse ERBB-2 promoters (12). Using reporter constructs, these palindromes were shown to exert both positive and negative regulation of the human ERBB-2 promoter (11).

We previously described a palindrome binding protein (PBP)1 that bound to the half-site of each ERBB-2 palindrome with the core recognition sequence TGGGAG (13). We now report that protein sequence analysis of purified PBP identifies it as RBPJkappa (recombination signal binding protein of immunoglobulin Jkappa gene). RBPJkappa , which was initially cloned by Matsunami et al. (14) based on recognition of the Jkappa recombination signal sequence, was subsequently isolated based on recognition of the Epstein-Barr virus (EBV) C promoter (designated as CBF1) (15-17) and of the adenovirus pIX gene promoter (18). RBPJkappa is the mammalian homolog of Drosophila Suppressor of Hairless (Su(H)) (19, 20). Further analysis indicated that although RBPJkappa contained a 40-amino acid region of homology to integrase, it lacked such activity (21) and recognized a composite sequence consisting of the heptamer recombination recognition site and a BamHI linker (15, 22). RBPJkappa is widely expressed with two mouse and three human splicing variants identified (23).

Genetic analyses indicate that Su(H) acts downstream of NOTCH in the signaling pathway of sensory peripheral nervous system development in Drosophila (19, 20). A similar pathway for lateral inhibition in neuronal development exists in mammalian species (24). The Drosophila Enhancer of split (E(spl)) and the mouse Hairy enhancer of split (Hes-1) complexes are downstream of NOTCH and Su(H)/RBPJkappa in this pathway (25, 26). In the best studied mechanism, the intracellular domain (IC) of the transmembrane protein NOTCH is translocated to the nucleus with RBPJkappa that serves as the site-specific DNA binding partner (26, 27). The NOTCH-IC·RBPJkappa complex activates transcription of basic helix loop proteins encoded by Hes-1. Events downstream of E(spl)/Hes-1 may be inhibitory (lateral inhibition in neuronal development (28) and inhibition of myogenesis (29)) or stimulatory. RBPJkappa also acts in a protein complex with the non-DNA-binding EBNA2 to regulate genes involved in EBV latency (15). The alteration of NOTCH that is necessary for nuclear translocation is reported to occur upon ligand binding (27) via proteolysis (30), via translocation that removes much of the ectodomain (31), and with retroviral insertion (32).

Because RBPJkappa could bind to the two palindromes in the ERBB-2 promoter, we examined its activity using reporter gene constructs. We present evidence that RBPJkappa stimulates promoter activity via wild type but not mutant ERBB-2 palindromic sequences; activity was markedly enhanced upon co-expression of the intracellular domain of NOTCH but was not affected by co-expression of EBNA2. These results suggest that ERBB-2 promoter activity is regulated by a protein complex that contains RBPJkappa .


MATERIALS AND METHODS

Cell Cultures, Transfections, and Reporter Assays

293 cells were grown in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% calf serum. F9 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

Plasmid DNA was transfected as a calcium phosphate precipitate (33). A beta -galactosidase expression vector under control of a cytomegalovirus gene promoter was co-transfected with the luciferase reporter gene constructs.

Luciferase activity was measured as described by de Wet et al. (34). Aliquots of cell extract were added to an assay reaction containing 100 mM potassium phosphate (pH 7.8), 5 mM ATP, and 15 mM MgSO4 in a volume of 0.35 ml. Reactions were initiated by the addition of 0.1 ml of 1 mM luciferin, and light readings were integrated over 10 s with a monolight 2110 luminometer. beta -Galactosidase activity was measured as described by Norton and Coffin (35). Promoter activity was expressed as light units of luciferase activity/A420 unit of beta -galactosidase activity.

Plasmid Constructs

The intracellular region of human NOTCH (NOTCH-IC) was a generous gift from Dr. David Baltimore, MIT. Viral EBNA2 and human RBPJkappa cDNAs were generous gifts from Dr. Diane Hayward, The Johns Hopkins University. The NOTCH-IC and RBPJkappa cDNAs were regenerated by polymerase chain reaction and subcloned into a modified pCDNA3 vector containing the luciferase gene translation initiation codon and the hemagglutinin (HA) epitope tag.

Eight copies of the wild type palindrome II (Pal II), four copies of palindrome II mutated in both sites (Mb), or four copies of the Hes-1 RBPJkappa binding site A were cloned in front of a herpes simplex virus thymidine kinase promoter-driven luciferase reporter construct to generate wtPal II-TK, mutPal II-TK, or Hes-TK, respectively.

Electrophoretic Mobility Shift Assay (EMSA)

Binding reactions contained 2 × 104 cpm of DNA probe and varied amounts of protein with or without competitors or antibody in a final volume of 20 µl containing 100 mM KCl, 20 mM Tris (pH 7.5), 1 mM EDTA, 5 mM dithiothreitol, 5 mM MgCl2, 1 µg of poly(dI-dC), and 4% glycerol. The mixtures were incubated for 20 min at room temperature, loaded on a 4 or 6% acrylamide gel that had been run for 30 min at 4 °C in 0.5 × TBE (25 mM Tris borate, 0.5 mM EDTA), and electrophoresed at 10 mA for 2 h. Gels were dried and autoradiographed overnight.

Anti-HA antiserum was purchased from BABCO (Berkeley Antibody Co., Berkeley, CA). Oligonucleotides were synthesized using an Applied Biosystems 380 DNA synthesizer. The oligonucleotides used in Figs. 1 and 3 have the following sequences, and the RBPJkappa consensus sequences are underlined (references are indicated for each sequence): Pal I, CTGGGAGTTGCCGACTCCCAG (13); Pal II, CTGGGAGCGCGCTTGCTCCCAA (13); Mb, CGAAACACGCGCTTGACAAAGA (13); M3, CTGGGAGCGCGCTTGACAAAGA (13); -596, GATGTGGTGGGAAAACCATTA (8); Cp, ACACGCCGTGGGAAAAAATTT (36); Hes, GTTACTGTGGGAAAGAAAGTC (26); LMP, CAAGCTGTGGGAATGCGGTGG (36); pIX, TTAAGGGTGGGAAAGAATATA (18); Sp1, GCTTGATCGGGGCGGGGCGATC (13).


Fig. 1. Nuclear protein binding to the palindromes of the ERBB-2 promoter. EMSA was carried out with 32P-labeled 100-bp ERBB-2 oligonucleotide probe (-329 to -230 relative to the translation start site at +1) incubated with F9 nuclear extract without (0) or with a 100-fold excess of unlabeled Sp1 or Pal II oligonucleotides. Positions of the Sp1 and PBP complexes are indicated by arrowheads. comp, competitor.
[View Larger Version of this Image (51K GIF file)]


Fig. 3. Binding of recombinant RBPJkappa to the ERBB-2 palindrome. A 22-bp 32P-labeled Pal II probe was incubated with nuclear extract made from 293 cells or from 293 cells transiently transfected with HA-RBPJkappa . Reactions were incubated without (0) or with a 100-fold excess of the indicated unlabeled competitors or with a monoclonal antibody against the HA tag. Ab, antibody; comp, competitor; WT, wild type Pal II; MT, mutant Pal II.
[View Larger Version of this Image (66K GIF file)]

PBP Purification and Peptide Sequencing

Purification of PBP was described previously (Ref. 13; see Fig. 2). Briefly, nuclear extract made from F9 cells was negatively absorbed to DE52 and to a DNA affinity column prepared using a mutant oligonucleotide (Mb) followed by two rounds of specific DNA affinity chromatography using palindrome II (CTGGGAGCGCGCTTGCTCCCAA). The final step was gel filtration chromatography. All active fractions eluting from the Superdex 200 fast protein liquid chromatography column were pooled and separated by SDS-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. After visualization by staining with Amido Black 10B, the protein band was excised and subjected to in situ digestion with trypsin. The excised band was treated with polyvinylpyrrolidone to prevent binding of the enzyme to the membrane. Digestion with trypsin (1 µg in 30 µl of 0.1 M TES, pH 8) was allowed to proceed overnight. The supernatants were then fractionated by reversed-phase HPLC. Fractions were collected manually, based on absorbance at 210 nm. Fractions corresponding to symmetrical peaks in the chromatogram were then subjected to chemical sequence analysis in an ABI470 (Applied Biosystems Inc.) protein sequencer (37) and analyzed by matrix-assisted laser desorption mass spectroscopy on a Bruker Reflex Time-of-Flight instrument. Two HPLC fractions of the protein digest were sequenced. One fraction yielded DGYIHYGQTVK (Fraction 29). The mass spectrum contained one strong signal at m/z = 1280. The other fraction was Q(TP)(AV)(QL)(VL)(DP)(AV)(DT)(DL)(PV)XSQL (Fraction 46) where residues given in parentheses were observed in the same sequencing cycle, indicating the presence of two peptides in that fraction. The mass spectrum contained a major signal at m/z = 1235 and a weak signal at m/z = 1750. A BLAST computer homology search (38) revealed that all these sequences were identical to tryptic fragments of mouse/Xenopus RBPJkappa . All molecular masses determined were within 0.05% of the masses calculated for the corresponding RBPJkappa tryptic fragments.


Fig. 2. Identification of PBP as RBPJkappa . A, purification scheme for PBP. MT, mutant Pal II; WT, wild type. B, purified PBP before trypsin digestion. C, diagram of mouse RBPJkappa and three peptides sequenced. The region of proposed integrase homology is filled.
[View Larger Version of this Image (15K GIF file)]


RESULTS

Palindromes of the ERBB-2 Gene Interact with a Specific Nuclear Protein, PBP

Previous studies showed that a 100-bp region upstream of the TATA box of the ERBB-2 gene promoter increased basal promoter activity 200-fold (11). Two palindromic sequences are a prominent feature of this 100-bp enhancer element in addition to a strong Sp1 site near the 5' end and a CAAT box near the 3' end. The distal palindrome (Pal I) overlaps the Sp1 site, and the proximal palindrome, Pal II, overlaps the CAAT box (8, 9). When this 100-bp region was used as a probe in EMSA, proteins in F9 nuclear extract formed several complexes (Fig. 1). The complexes that could be effectively competed by an Sp1 consensus sequence are represented by Sp1 and proteins with a recognition motif similar to Sp1. A complex designated as PBP could not be competed by the Sp1 consensus oligonucleotide but was effectively competed by the palindrome sequence. F9 cells thus contain a specific palindrome binding activity.

PBP Is RBPJkappa

To determine the identity of PBP, it was purified to homogeneity from F9 cells using ion exchange, DNA affinity, and gel filtration chromatographies and SDS-polyacrylamide gel electrophoresis. Fig. 2A summarizes the purification steps. Fig. 2B shows purified PBP before trypsin digestion, and the peptides identified by protein sequence analysis and mass spectroscopy are shown in Fig. 2C. A computer homology search revealed that all three sequences were identical to tryptic fragments of RBPJkappa , a protein originally identified through binding to the Jkappa immunoglobulin recombination signal sequence (14).

To verify that RBPJkappa had palindrome binding activity, the protein was expressed as a HA-tagged fusion protein and transfected into 293 cells. Transient expression of HA-RBPJkappa resulted in a large increase of PBP activity that could be competed by wild type Pal II but not by Mb oligonucleotides. An antibody against the HA tag supershifted the complex, supporting the sequence identification of PBP as RBPJkappa (Fig. 3).

Binding of PBP Is Competed Effectively by Other RBPJkappa Binding Sites

RBPJkappa was originally isolated as a protein that bound to the Jkappa recombination signal sequence consisting of a heptamer and a nonamer with a 23-bp spacer (14). RBPJkappa protein is highly conserved from Caenorhabditis elegans to Homo sapiens (14, 15, 19, 20, 39). The consensus recognition sequence for the RBPJkappa family has subsequently been determined as GTGGGAA, which is present in all RBPJkappa binding sites characterized so far (22). The PBP core site CTGGGAGC is close but not identical to the consensus sequence (13). To compare the affinity of ERBB-2 palindromes with other RBPJkappa binding sites, palindromes as well as other characterized binding sites were used as unlabeled competitors in EMSA using partially purified F9 nuclear extract and Pal II as a probe. As shown in Fig. 4, the EBV Cp sequence is the optimal binding site. Pal I, Pal II, and half-site mutation M3 have affinity similar to that seen with the Hes-1 and pIX gene promoters. A perfect matching consensus site is located at -596 of the ERBB-2 gene promoter (8). It is, however, a poor competitor, indicating that flanking sequences significantly affect RBPJkappa binding affinity for the core consensus sequence.


Fig. 4. Comparison of palindrome binding affinity with other known binding sites. Increasing amounts of indicated competitors (5 and 20 ng) were included in the reaction mixtures that contained PBP purified from F9 cell nuclear extract and 32P-labeled ERBB-2 Pal II oligonucleotide. EMSA reactions were run as described under "Materials and Methods." comp, competitor.
[View Larger Version of this Image (65K GIF file)]

RBPJkappa -stimulated Transcription via the ERBB-2 Palindrome Is Augmented by NOTCH-IC but Not by EBNA2

To study the function of RBPJkappa on the ERBB-2 promoter palindrome, RBPJkappa was transfected into 293 cells along with reporter constructs containing wild type or mutant palindrome sequences (Fig. 5). The control reporter TK was induced 3-fold by RBPJkappa . RBPJkappa activated wtPal II-TK 16-fold, whereas mutPal II-TK was induced no more than the background activation seen with the vector TK. Constitutively active forms of mammalian NOTCH stimulated transcription of the Hes-1 gene presumably through RBPJkappa and its binding site on the Hes-1 promoter (26). The intracellular domain of NOTCH was transfected to determine whether an active form of human NOTCH (NOTCH-IC) had any effect on the ERBB-2 gene (Fig. 5). NOTCH-IC alone did not increase reporter activity above background. However, co-transfection of NOTCH-IC with RBPJkappa activated wtPal II-TK activity 82-fold. NOTCH-IC failed to affect the basal stimulation of RBPJkappa seen with TK and mutPal II-TK. The effect of NOTCH-IC was thus specific to the wild type ERBB-2 palindrome-containing gene and was dependent on RBPJkappa .


Fig. 5. Stimulation of ERBB-2 promoter palindrome II by RBPJkappa and NOTCH-IC. 293 cells were transfected with 2 µg of the indicated reporter constructs, 3 µg of RBPJkappa , 1.5 µg of NOTCH-IC, and 0.1 µg of cytomegalovirus-beta -galactosidase expression plasmids. Relative activity is the ratio of normalized luciferase activity in the presence of the indicated expression vectors divided by the activity in the presence of the parental vector pCDNA3 and in the absence of any expression plasmid. Normalized luciferase activity equals light units of luciferase activity/A420 unit of beta -galactosidase activity. The numbers are the mean ± S.D. (n = 3), and the experiment was repeated 3 times with similar results. When not shown, error bars are within the column lines.
[View Larger Version of this Image (34K GIF file)]

The effects of RBPJkappa and NOTCH-IC on ERBB-2 palindromes were compared with effects on the Hes-1 promoter. As shown in Fig. 6, NOTCH-IC alone was sufficient to induce activation of the Hes-1 promoter, whereas RBPJkappa alone did not stimulate the Hes-1 reporter. Co-transfection of RBPJkappa and NOTCH-IC caused no change in activity beyond that observed with NOTCH-IC alone. EBNA2 is a well described activator of some RBPJkappa response genes (15, 36). It up-regulates Cp and CD23 genes utilizing RBPJkappa and its binding sites on extended promoter elements. When EBNA2 was transfected into 293 cells, it had no effect on ERBB-2 or Hes-1 promoter activity. EBNA2 also had no effect on activation mediated by RBPJkappa and/or NOTCH-IC on either the ERBB-2 palindromes or the Hes-1 site. Thus EBNA2 requires interactions additional to those mediated via RBPJkappa (40), whereas effects of NOTCH·RBPJkappa occur on promoters containing only RBPJkappa response elements.


Fig. 6. Lack of effect of EBNA2 on Hes-1 and ERBB-2 palindromes. 293 cells were transfected with 1.5 µg of the indicated reporter constructs, 2 µg of RBPJkappa , 1.5 µg of NOTCH-IC, 1.3 µg of EBNA2, and 0.2 µg of cytomegalovirus-beta -galactosidase expression plasmids. Data are presented as in Fig. 5, and the experiment was repeated 2 times with similar results.
[View Larger Version of this Image (28K GIF file)]


DISCUSSION

The present studies identify the protein (PBP) that binds to the two palindromes of the proximal enhancer of ERBB-2 as RBPJkappa . Peptide sequences of tryptic fragments of PBP were identical to those of RBPJkappa (14-16). Although the ERBB-2 palindrome half-sites do not perfectly match the reported RBPJkappa consensus binding sequence (22), PBP/RBPJkappa bound to the palindromic sites with an affinity similar to that observed for the consensus sites. NFkappa B/Rel binding sites, which also resemble the palindrome half-sites, do not compete for PBP binding (13). Additionally, nuclear extracts containing epitope-tagged RBPJkappa formed a specific complex with ERBB-2 palindromes. Previous studies identified two components of PBP (13). We suggest that these likely represent splicing variants of RBPJkappa with varying N termini (23).

RBPJkappa is reported to function as a site-specific DNA binding protein that recruits additional transcription factors to target genes (17, 36, 41). RBPJkappa represses transcription from its cognate site in the adenovirus pIX gene promoter (18) and from a Gal4 binding site when expressed as a Gal4 fusion (42). A repression domain in RBPJkappa was identified that coincided with that required for activation by EBNA2 (42). EBNA2 was thus deduced to activate transcription by both masking the repression domain of RBPJkappa and by bringing a strong transactivation domain to the RBPJkappa binding site.

RBPJkappa is the mammalian homolog of Drosophila Su(H), which functions in the NOTCH pathway and regulates development of the peripheral nervous system (19, 20). Su(H) interacts positively with NOTCH (43, 44) and negatively with Hairless (45). Fortini and Artavanis-Tsakonas (27) observed that Su(H) was translocated to the nucleus when the transmembrane receptor NOTCH was activated by binding the ligand Delta. Interestingly, an oncogene from a human T lymphoblastic leukemia (TAN-1) was identified as a truncated NOTCH lacking much of the extracellular domain (31). Kopan et al. (30) presented evidence that proteolytic processing of NOTCH could generate NOTCH-IC sufficient to act via a complex with RBPJkappa in the nucleus. RBPJkappa binds to the intracellular domain of NOTCH, and expression of RBPJkappa with ectodomain-deleted forms of NOTCH (delta EC-NOTCH), both with and without retention of the transmembrane domain, activates transcription from Hes-1 and EBV C promoters (26, 46). delta EC-NOTCH·RBPJkappa and EBNA2·RBPJkappa complexes exhibited similar transcriptional enhancement on EBV C promoter sites (46).

RBPJkappa stimulated transcription from a reporter gene containing wild type but not mutant ERBB-2 palindrome sequences. This activity was greatly enhanced by NOTCH-IC. Using immunofluorescence, we found that NOTCH-IC and RBPJkappa localize in the nuclei of 293 cells (data not shown). These observations support models of RBPJkappa ·NOTCH-IC heterodimers that act to enhance transcription from RBPJkappa response elements. Comparison of ERBB-2 palindrome and Hes-1 elements in reporter gene assays indicates a stronger effect of transfected RBPJkappa alone on ERBB-2 and a stronger effect of transfected NOTCH-IC alone on Hes-1. This may reflect a higher affinity in vivo of endogenous RBPJkappa for the Hes-1 site. RBPJkappa was not inhibitory on either site. On these sites, EBNA2 had no effect. This suggests that stimulatory effects of EBNA2 require interaction with additional proteins such as Spi-1/Spi-B, which bind to a more extended EBV C element (40).

Several recognition elements have been identified within the extended ERBB-2 promoter. Two AP2 and two Sp1 consensus binding sequences are found at -397, -359, -369, and -314, respectively, relative to the translation start site (8). Several breast cancer cell lines are reported to have a strong AP2 activity that increases expression via the response element located at -397 bp (47). A stimulatory factor that binds to the promoter region 3' of the TATA box has also been described (48). Analyses of the rat ERBB-2 promoter have identified a transacting factor that binds at -466 to -456 bp (49) and inhibition by co-expression of Rb (50), adenovirus E1A (51), and c-Myc (52). However, deletional analysis of the human ERBB-2 gene identified the region between -329 and -230 bp as the major strong enhancer region; it contains two functionally important palindromes (11). The activity of RBPJkappa , which binds to each palindrome half-site (13), is strongly augmented by NOTCH-IC. Interestingly, mice that contain homozygous deletions of ERBB-2 exhibit defects in development of cardiac trabeculae (53), but development of cranial neural crest-derived sensory ganglia and motor neurons was also impaired, implying important roles for ERBB-2 in both cardiac and neural development. Control of ERBB-2 expression by NOTCH signaling pathways that are known to be important in neuronal development in transgenic mice thus appears plausible.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant DK13149 and by a grant from the Markey Charitable Trust.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.
   To whom correspondence should be addressed: University of California San Diego, 9500 Gilman Dr. 0650, La Jolla, CA 92093-0650. Tel.: 619-534-4310; Fax: 619-534-0871; E-mail: ggill{at}ucsd.edu.
1   The abbreviations used are: PBP, palindrome binding protein; bp, base pair(s); EBV, Epstein-Barr virus; Su(H), Drosophila Suppressor of Hairless; Hes-1, Hairy enhancer of split; E(spl), Drosophila Enhancer of split; IC, intracellular domain; HA, hemagglutinin; Pal I, distal palindrome; Pal II, proximal palindrome; Mb, Pal II mutated in both sites; EMSA, electrophoretic mobility shift assay; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; HPLC, high pressure liquid chromatography; wtPal II-TK, wild type Pal II thymidine kinase; mutPal II-TK; mutated Pal II thymidine kinase; Hes-TK, Hes-1 thymidine kinase.

ACKNOWLEDGEMENTS

We thank Dr. Diane Hayward, The Johns Hopkins University, for human RBPJkappa and EBNA2 expression plasmids and Dr. David Baltimore, MIT, for the intracellular region of human NOTCH cDNA.


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