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Previous Article | Next Article 
Molecular and Cellular Biology, January 2006, p. 117-130, Vol. 26, No. 1
0270-7306/06/$08.00+0 doi:10.1128/MCB.26.1.117-130.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Toshihiko Masui,1
Galvin H. Swift,1
Ling Shi,1,
R. Michael Henke,2 and
Raymond J. MacDonald1*
Department of Molecular Biology,1 Center for Neurosciences, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-91482
Received 25 July 2005/ Returned for modification 15 September 2005/ Accepted 8 October 2005
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ABSTRACT |
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INTRODUCTION |
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The pancreas is a multifunctional gland composed of both endocrine and exocrine tissues. The exocrine tissue comprises more than 90% of the adult pancreas and is composed of acini, which secrete digestive enzymes, and ducts, which secrete fluid and transport the acinar enzymes to the duodenum. Massive synthesis of the digestive enzymes is reflected in the pancreatic mRNA population: nearly 90% of the mRNA from the entire gland encodes a small number (about 20) of acinar secretory enzymes, such as amylases, elastases, chymotrypsinogens, and carboxypeptidases (12). The selective transcription of the acinar specific genes at such a high level is controlled largely by the PTF1 complex (6, 32). However, the mechanism of target-gene activation by PTF1 is unknown.
Functional binding sites for the PTF1 complex are present in the 5' promoter regions of all of the acinar digestive enzyme genes examined (6, 31). The binding site from the elastase 1 gene (Ela1) provides a model for the interaction of PTF1 with DNA and acinar cell-specific transcriptional activation. This site, known as the A element of the Ela1 enhancer, is located about 100 bp upstream of the 5' end of Ela1, is necessary and sufficient to direct acinar specific expression in transgenic mice (30), and in situ cooperates with two nearby elements (B and C) to direct the high level of transcriptional activation characteristic of Ela1 (18).
PTF1 is an unusual heterotrimeric bHLH transcription factor composed of PTF1a/P48 (a pancreas and neural specific bHLH protein), one of the common class A bHLH proteins, and a previously unidentified subunit (32, 33). (For clarity, we retain the use of p48/P48 [gene/protein], rather than Ptf1a/PTF1A, to distinguish the P48 subunit from the PTF1 complex.) PTF1 binding sites are bipartite with an E-box (preferably CACCTG) and a TC-box (TTTCCCA) spaced one or two helical turns apart, center to center (6, 31). Targeted deletion of the p48 gene causes pancreatic and cerebellar agenesis (14, 17, 36), so understanding the mechanism of transcriptional activation by PTF1 in differentiated acini will likely give insights into PTF1 action during pancreas and brain development as well.
We show that the previously unidentified third subunit of PTF1 from adult pancreas is RBP-L, an organ-specific mammalian variant of the CSL proteins. RBP-L provides the high activation potential of the complex, which is dependent on contact of all three subunits of the complex with DNA. A similar transcriptionally active complex can be reconstituted with RBP-J, the mediator of Notch signaling. The interaction of P48 with the RBP subunits requires two peptide motifs conserved in P48s from insects to mammals. One or both of these peptides are deleted in families with heritable permanent neonatal diabetes mellitus, in which infants are born without a pancreas and cerebellum (36). The similar developmental consequences for neonatal mice without P48 and infants with mutant P48 unable to bind RBP-J or -L suggest that most or all of the developmental functions of P48 require its ability to recruit an RBP into a PTF1 complex. Motifs similar to the RBP-interacting sites of P48 are present in other transcription factors; therefore, PTF1 may be one of a new family of complexes that use RBP-J in a Notch-independent manner.
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MATERIALS AND METHODS |
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Antibodies and immunofluorescence microscopy. The rabbit anti-P48/PTF1a was described previously (32). The rabbit anti-RBP-L was prepared by AnaSpec, Inc. (San Jose, CA), against PNAQEPAPDADTLLE, a sequence near the C terminus of mouse RBP-L, and affinity purified using the synthetic peptide. For electrophoretic mobility shift assay (EMSA), anti-RBP-J was from the Institute of Immunology Co., Ltd. (Tokyo, Japan); for chromatin immunoprecipitation (ChIP), anti-RBP-J was from Santa Cruz Biotechnology. The anti-RBP-L did not cross-detect RBP-J, and the anti-RBP-J did not cross-detect RBP-L in EMSA and Western blotting experiments with in vitro-synthesized RBP proteins. Anti-HEB serum (34) was a gift from S. Sawada. Anti-E2.2, anti-E12/E2-2, and anti-E47 were from BD Biosciences Pharmingen (San Diego, CA), and anti-c-Myc was from Santa Cruz Biotechnology (Santa Cruz, CA). Immunofluorescent localization of P48 and RBP-L was performed with 5-µm tissue sections from paraformaldehyde-fixed, paraffin-embedded adult mouse pancreas.
EMSAs and antibody supershifts. Nuclear extracts were prepared from rat pancreas, in vitro-translated (IVT) proteins were synthesized, and EMSAs including antibody supershifts were performed as previously described (30). Peptides for competition experiments were synthesized by the Protein Chemistry Technology Center (UT Southwestern, Dallas, TX). The double-stranded oligonucleotide for RBP-J binding had the sequence 5'-GTAGTAGTTGCTTTTCCCACG-3'.
RT-PCR analyses. The organ distribution of RNA transcripts for P48, RBP-L, and RBP-J was determined by RT-PCR analysis of RNA from 19 mouse organs. Pancreatic RNA was isolated by the guanidine thiocyanate technique (22), whereas RNA from other organs was isolated with TRIzol (Invitrogen, Carlsbad, CA). cDNA from each RNA was synthesized by Superscript II reverse transcriptase (Invitrogen) with oligo(dT) primer. Aliquots of each cDNA derived from the equivalent of 25 ng of total RNA template were amplified in 40-µl reactions with the following gene-specific primers: p48, 5'-CGCGTCTTTGTGCATATTGT-3' and 5'-CGGAGTTTCCTGGACAGAGT-3'; Rbp-L, 5'-GGAGCTGCACGGAGAAAA-3' and 5'-GTGTGAACTCGTGGTGGATG-3'; Rbp-J, 5'-GAATTTCCACGCCAGTTCAC-3' and 5'-ATACAGGGTCGTCTGCATCC-3'; and actin, 5'-AGCCATGTACGTAGCCATCC-3' and 5'-ACATCTGCTGGAAGGTGGAC-3'. The amplification conditions were 30 cycles for p48, Rbp-L, and Rbp-J and 25 cycles for actin of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C. The amplification products were analyzed by agarose gel electrophoresis and staining with ethidium bromide.
Coimmunoprecipitation and ChIP. Coimmunoprecipitation was performed with 75 µg of nuclear protein extract from rat pancreas, precleared and then incubated with 10 µg of anti-P48 or anti-cMyc immobilized on coupling beads.
Chromatin from rat pancreas and liver was prepared from formaldehyde cross-linked nuclei as described for rat liver chromatin (5) and sheared further by sonication. A total of 100 µl of purified chromatin in 900 µl of ChIP dilution buffer (Upstate, Lake Placid, NY) was incubated with 15 to 20 µg of antiserum. Blocked protein G-Sepharose beads (Upstate, Lake Placid, NY) were added to the chromatin. Bound chromatin was eluted from the beads, cross-linking was reversed, and the immunoprecipitated DNA was purified for PCR analysis. For sequential ChIP (9), half of the chromatin eluted from an initial immunoprecipitation was retained for the real-time PCR measurement of enrichment (below), and the remainder was used for a second immunoprecipitation. Ten percent of the immunoprecipitated DNA was amplified by 34 cycles of PCR for each PTF1 target gene. Quantification of ChIP enrichment of promoter regions was performed with SYBR Green Mastermix (Applied Biosystems, Foster City, CA) using the ABI prism 7700 (Applied Biosystems) and was calculated as the increase of Ela1 promoter DNA relative to that of the 28S rRNA gene.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A complex with the same electrophoretic mobility as authentic PTF1 can be reconstituted with IVT P48, RBP-L, or RBP-J, and one of the common bHLH proteins (e.g., HEB, E12, or E47) (Fig. 1C). Whereas P48 alone cannot bind the PTF1 binding site, heterodimers of P48 and any of the three common bHLH proteins can. The heterodimers bind the E-box, because the binding can be disrupted by a mutation in the E-box and is insensitive to changes in the TC-box (Fig. 2A). All three of the possible P48 heterodimers have faster electrophoretic mobilities than does PTF1. A complex with the mobility of authentic PTF1 forms with the addition of either RBP-J or RBP-L (Fig. 1C). The ability to form a trimeric complex with an RBP may not be unique to P48, but it is not a common property of bHLH proteins. For example, although heterodimers of ASCL1 or NEUROD1 with E12 can bind the E-box of the PTF1 site, we were unable to detect the formation of trimeric complexes with RBP-J or RBP-L (data not shown).
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The binding of the three subunits to DNA is highly cooperative. For example, the PTF1 site of the rCPA-90 promoter does not bind a P48-E12 heterodimer, but the presence of a weak RBP site compensates and allows trimer binding via RBP-J/liter recognition of the TC-box (Fig. 2B). Conversely, the TC-boxes of the Ela1, Cpa-142, and Trpd promoter sites do not bind RBP-J/liter and yet are sufficient to recruit the PTF1 complex through collaborative binding to an E-box. Of the six PTF1-binding sites examined, only the Ctrb site could be bound independently by either a P48-AbHLH heterodimer or an RBP.
Cooperative binding may be facilitated by preformed trimeric PTF1 complexes. Anti-P48 coprecipitates RBP-L from nuclear extracts (data not shown), indicating that the trimeric PTF1 complex forms in vivo. Moreover, the addition of excess RBP-L or -J to an EMSA reaction prevents the binding of a P48-E12 heterodimer to an E-box in the absence of a paired TC-box (data not shown). However, RBP-J cannot inhibit the binding of a heterodimer containing a P48 with a mutation (W298A) that eliminates the interaction between P48 and RBP-J (see below, Fig. 4C). Thus, the ability of an RBP to inhibit the DNA binding of a P48-heterodimer requires its interaction with P48, and this interaction can occur in the absence of DNA binding.
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RBP-L is essential for the high transcriptional activity of PTF1. Forced expression of P48 and a common AbHLH (HEB, E47, or both) did not activate to high levels a cotransfected reporter driven by tandem repeats of a PTF1 binding site (26, 32). We tested whether RBP-L could supply the missing transcriptional activation. The human embryonic kidney cell line 293 has endogenous RBP-J and the common AbHLH proteins, but not P48 and RBP-L (data not shown). Coexpression of P48 and HEB in 293 cells activated a luciferase reporter driven by six copies of the Ela1 PTF1 binding site 18-fold higher than without the exogenous transcription factors (Fig. 3A). Overexpression of RBP-J did not change the extent of activation by P48 and HEB. Obata et al. showed previously that RBP-J could enhance activation by P48 on the PTF1-binding site of the Ctrb promoter threefold (26). In contrast, the addition of RBP-L boosted activation another 25-fold, to a total of 450-fold (Fig. 3A). P48 and RBP-L without the addition of exogenous HEB were nearly as effective (320-fold activation; Fig. 3A), due to the presence of ample endogenous AbHLH proteins. The supplemental activation provided by RBP-L depended on the incorporation of RBP-L into a PTF1 complex: a five-amino-acid deletion in P48 that prevents recruitment of RBP-L also eliminated the supplemental activation by RBP-L (see Fig. 4). Hence, RBP-L provides the high transcriptional activation of the PTF1 complex.
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Two motifs in the C-terminal domain of P48 are essential for the interaction with RBPJ/L. Comparison of the amino acid sequences for mouse, zebra fish, and fruit fly P48's revealed the conservation of two short peptide motifs, C1 (HSLSW) and C2 (WTPEDPR), in addition to the bHLH domain (Fig. 4A). To determine the function implied by this phylogenetic conservation, we tested the effects of mutations in C1 and C2 on the ability of P48 to form a trimeric DNA-binding complex (Fig. 4B). The deletion of either peptide or substitution of alanine for tryptophan in either peptide had no effect on the formation of DNA-binding heterodimers (Fig. 4C). In contrast, any of these changes did have pronounced and differential effects on the inclusion of RBP-J and RBP-L in a trimeric complex. The alterations of the C2 region prevented the association of RBP-J but had little or no effect with RBP-L. Conversely, deletion of the C1 peptide affected the recruitment of RBP-L more severely than that of RBP-J. Because tryptophan residues play key roles in the formation of other transcription factor complexes (15, 27), we tested whether the tryptophan residues in each of the peptides were necessary for P48 recruitment of the RBP-J or -L. Whereas substitution of alanine for either tryptophan 280 in C1 or 298 in C2 inhibited the recruitment of RBP-J, neither substitution detectably affected the recruitment of RBP-L. Deletion of amino acids 246 to 273, which are conserved among vertebrates but not to insects (Fig. 4A), severely affected the recruitment of RBP-L but much less so RBP-J (data not shown). These contrasting effects suggest that the binding requirements of the two RBPs emphasize different features in the C terminus of P48. The region for RBP-L binding is more extended than that for RBP-J and may correspond to the broad C-terminal region conserved among vertebrate P48 proteins.
PTF1 activity requires the interaction between P48 and RBP-L. To verify that the transcriptional activity of P48 and RBP-L is dependent on their presence in the PTF1 complex, we tested whether the mutations of the C1 and C2 peptides also inhibited transcriptional activation in transfected cells. To derive an effective transfection assay for RBP-J with its weak activation potential, we created a strong, constitutively active form by fusing the VP16 activation domain to the N terminus of RBP-J, similar to that described by (19, 26). P48W298A, which could recruit RBP-L but not RBP-J (Fig. 4C), retained much of its transcriptional activity in combination with RBP-L but lost nearly all activity with VP16RBP-J (Fig. 4D). The effects of the W280A substitution were similar, though more modest. P48 lacking the C1 peptide region had little activity in combination with VP16RBP-J and was completely inactive with RBP-L (Fig. 4D), a finding consistent with its relative ability to interact with the RBPs (Fig. 4C). The congruence between the ability of the RBPs to interact with P48 and their ability to stimulate activation of a PTF1-reporter gene confirms that their activity on PTF1 sites derives from the trimeric complex.
The PTF1 complex is conserved from flies to mammals. To determine whether a PTF1-like complex might be ancient, we tested whether the Drosophila orthologs of P48, E12, and RBP-J [FER1, DA and SU(H), respectively] could form a trimeric complex on the Ela1 PTF1-binding site. Indeed, the Drosophila proteins formed a DNA-binding complex that required all three proteins (Fig. 5A). Moreover, any combination containing the three types of subunits from Drosophila and mammals could form a trimeric DNA-binding complex. Like the mammalian PTF1, the Drosophila FER1-DA-SU(H) complex did not bind versions of the PTF1-site containing the diagnostic nucleotide substitutions in the E- and TC-boxes (Fig. 5B). Furthermore, coexpression of SU(H) with P48 and HEB in 293 cells activated the PTF1-responsive reporter to a level (70-fold over the basal) intermediate between PTF1-J and PTF1-L (Fig. 5C). The striking phylogenetic conservation of form and function indicates that the trimeric complex is an important regulator.
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Human p48 mutations disrupt the association of RBPs with the complex.
Two naturally occurring mutations in human p48 are associated with permanent neonatal diabetes mellitus, a genetic disorder characterized by the loss of pancreatic and cerebellar development (36). One of the mutations causes the deletion of the C terminus of P48, including peptide C2; the other deletes the region containing both C1 and C2 (Fig. 8A). We tested whether these deletions affect the ability of P48 to bind an RBP, as would be predicted. Normal human P48 readily formed PTF1 complexes with E12 and either RBP-J or RBP-L (Fig. 8C) and supported the characteristic high transcriptional activation in cell transfection assays (Fig. 8D). Although the mutant forms (which have more extensive alterations than the mouse mutants tested in Fig. 4) could form heterodimers with E12, they were unable to recruit RBP-J or -L into a trimeric complex (Fig. 8C). The small amount of the trimer formed with P48-
C2 (Fig. 8C, asterisk) provided less than 3% of the normal transcriptional activity (Fig. 8D). The developmental defects correlate with the inability of P48 to bind an RBP while retaining the ability to bind an AbHLH (Fig. 8C) and verify the importance of P48-RBP interactions in vivo.
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The presence of similar tryptophan-containing peptides in other transcription factors (Fig. 9D) suggests that Notch-independent functions of RBP-J/Su(H) may be more widespread. To determine whether these related motifs have the potential to mediate binding to RBP-J, we tested whether C2-like peptides we identified in HAIRLESS, KYOT2, and NFATc4 could disrupt the formation of the PTF1-J complex in vitro (Fig. 9E). Although the HAIRLESS and NFATc4 peptides were no more effective than the reversed-NOTCH1 peptide, the KYOT2 peptide was even more effective at low concentrations than those from P48 and NOTCH1.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cooperative binding properties of the PTF1 complexes and their ability to form prior to binding DNA have important regulatory implications. The cooperativity allows the use of variant E- and TC-boxes that cannot be bound individually by bHLH dimers or RBPs and so remain unoccupied and inactive in the absence of the trimeric complex. In addition, the efficient formation of the trimeric complex may limit the amount of P48 heterodimers and free RBPs and thereby prevent their binding to isolated E-boxes and RBP-sites. For PTF1-sites with near consensus half-sites, transcriptional activity might vary with changes in the composition of the bound complexes. For example, with limiting amounts of RBPs, P48-AbHLH dimers might predominate; with limiting P48, free RBP-L (or RBP-J) would be available for binding RBP sites with or without a paired E-box. In the adult pancreas, a cellular process ensures the formation of the PTF1 complex with highest transcriptional activity by excluding RBP-J in preference for RBP-L and coincidentally ensures that most of the RBP-J is available for Notch signaling.
Neither PTF1 complex is dependent on Notch signaling. The RBP subunits provide the transcriptional activity of the PTF1 complex. RBP-J and RBP-L are homologous proteins encoded by separate genetic loci; the amino acid sequences of the mouse proteins are 48% identical, despite highly divergent N- and C-terminal regions. High sequence conservation between the core regions of RBP-J and -L (67% for the mouse proteins), the retention of identical residues in RBP-L that in RBP-J contact DNA (36), and the same consensus DNA binding sequence indicate that the two isoforms have very similar structures and bind DNA in the same manner. The differences in the transactivation properties of RBP-J and -L likely lie in the divergent N- or C-terminal regions. The N and C regions of RBP-J augment the binding to NotchIC (39). The RBP-L C terminus is conserved across species and likely provides the potent transactivation that RBP-J lacks.
RBP-J mediates the transcriptional effects of Notch signaling by receiving the processed NotchIC fragment while bound to target genes (2, 35). The NotchIC binds RBP-J through a tryptophan-containing peptide motif that has been proposed to fit into a hydrophobic pocket on the beta-trefoil domain of the RBP-J (16, 38). This interacting motif has the core consensus 
WP ( is a hydrophobic residue), is conserved in Notch from arthropods to mammals, and is present in two other known RBP-J binding proteins, EBNA2 (20) and KyoT2 (40). For RBP-J in the PTF1 complex to respond to Notch signaling, the hydrophobic pocket would have to remain accessible to the NotchIC. We showed that the C2-peptide of P48 is a variant of the core consensus and performs the same function. Because the P48 C2 peptide can displace NotchIC from its complex with RBP-J and the equivalent Notch peptide displaces P48, Notch and P48 must compete for the same site on RBP-J. Indeed, overexpression of P48 disrupts the transcriptional activity of the RBPJ-NotchIC complex. Consequently, the incorporation of RBP-J in PTF1 is incompatible with its activation by the NotchIC.
Unlike monomeric RBP-J, RBP-L is not bound and activated by the NotchIC (24). RBP-L is inherently indifferent to Notch signaling, within or without the PTF1 complex. Whereas the C2 peptide of P48 is most important for binding RBP-J, the C1 peptide is more important for binding RBP-L. The C1 peptide is near the end of an extended region conserved among vertebrate P48s but not in the fly FER1. The appearance of RBP-L in the vertebrate lineage suggests that the conservation may be driven by selection for maintaining the interaction with RBP-L, distinct from RBP-J. The restricted tissue distribution of RBP-L indicates that its function may be limited largely to the maintenance or development of pancreatic tissues, the tracheal glands of the lung, and regions of the forebrain.
Proof that RBP-L plays a nonredundant function awaits an effective inactivation of the gene. Mouse Rbp-L is a complex gene comprising 12 exons and two transcriptional start sites (23). The downstream start creates a translational initiation codon in exon 5 that makes a shorter protein missing the first 123 N-terminal amino acids of the full-length RBP-L. Insertion of an nlacZ neo cassette with the simian virus 40 transcriptional terminator into the first exon disrupted the production of the large transcript but not of the short transcript (23). Mice homozygous for the disruption showed no obvious defects, but pancreatic function was not investigated and the sufficiency of the short form of RBP-L remains untested.
Significance of the phylogenetic conservation of PTF1. We showed that the Drosophila orthologues of P48 (FER1), E12/E47 (DA), and RBP-J [SU(H)] form a trimeric complex with identical DNA-binding characteristics and similar transcriptional activity as mammalian PTF1. Because Rbp-L appears in vertebrates, the original PTF1-like complex used the mediator of Notch signaling, although in a manner that excluded it from the Notch signaling pathway. Because arthropods do not have an organ homologous to the exocrine pancreas, the ancient function of the PTF1 complex cannot be pancreatic function or development. A Notch-independent function that may be analogous to the action of PTF1 has been ascribed for SU(H) during the development of the mechanosensory organs of the Drosophila peripheral nervous system (3). The requirement for a PTF1 complex for proper formation of the cerebellum (see below) suggests that the ancient role is nervous system development.
Role of RBP in cerebellar and pancreatic development. Two mutations of human P48/PTF1A have been associated with the absence of pancreatic and cerebellar development in newborn infants (36). The functional defects caused by the mutations are now clear. One deletes a C-terminal region containing the C2 peptide; the other deletes the C1 peptide as well (see Fig. 4A). Sellick et al. showed that mouse P48 with the C2 deletion and a class A bHLH (E47) could not activate a reporter gene driven by a PTF1 site and concluded that the absence of activity was due to the inability of the mutant P48 to form the bHLH heterodimer. We showed instead that both truncated forms of human P48 can form DNA-binding heterodimers with class A bHLH proteins and that the transcriptional defect was due to the inability to recruit RBP-J or RBP-L into a PTF1 complex. Although the mutations delete more than just the peptides that mediate the interactions with RBP-J and -L, it seems apparent that the developmental defects in infants homozygous for the p48-truncations are largely, if not exclusively, due to the inability to form complexes containing RBP-J or RBP-L (or both). Because RBP-J is present in the developing cerebellum but RBP-L is not (10, 23; our unpublished results), the PTF1 complex required for cerebellar development must contain RBP-J, equivalent to the Drosophila complex of FER1:DA:SU(H). Because the human p48 mutations that eliminate the binding of P48 to RBPs appear to phenocopy the p48-null mouse mutations (36), the functions of P48 during cerebellar and pancreatic development may occur predominantly or solely within the context of the PTF1 complex.
As for most programs of organogenesis, pancreatic development requires proper Notch signaling (1, 8, 11, 25). Leach and coworkers showed that enforced activation of the Notch pathway by constitutive NotchIC expression can interfere with acinar development, possibly through the disruption of PTF1 binding activity (8). Our results now suggest that the interference may be due to competition between NotchIC and P48 for RBP-J. It is possible that Notch signaling affects acinar cell differentiation in part through this mechanism during normal pancreatic development as well.
Broad potential for RBP-J functions independently of Notch signaling. RBP-J is ubiquitous and therefore available for recruitment by transcriptional regulatory schemes in a wide variety of developmental programs (see, for example, references 3 and 36). We have shown that other DNA-binding transcription factors, e.g., KYOT2 (FHL1/SLIM), contain peptides related to the RBP-J-interacting motifs of Notch and P48, suggesting that these may also bind RBP-J. Indeed, KYOT2 is known to bind RBP-J and, due to its ability (like P48) to interfere with NotchIC activation in cell transfection overexpression experiments, has been proposed to be a negative regulator of Notch signaling (40). Other LIM-only proteins similar to KYOT2 act as docking molecules that assemble transcription factor complexes via LIM domains (28). Rather than interfering with RBP-J function, we suggest that KYOT2 may be an LIM-only adapter that integrates RBP-J into select transcription factor complexes.
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ACKNOWLEDGMENTS |
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This study was supported by Public Health Service grants DK61220 and DK55266 from the National Institute of Diabetes and Digestive and Kidney Diseases.
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FOOTNOTES |
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Present address: Eccles Institute of Human Genetics, University of Utah, Salt Lake City, Utah.
Present address: Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.
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HAIRLESS. The P48:E12 dimer was not affected.