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Molecular Endocrinology, doi:10.1210/me.2004-0006
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Molecular Endocrinology 18 (6): 1363-1375
Copyright © 2004 by The Endocrine Society

The Transcriptional Repressor Nkx6.1 Also Functions as a Deoxyribonucleic Acid Context-Dependent Transcriptional Activator during Pancreatic ß-Cell Differentiation: Evidence for Feedback Activation of the nkx6.1 Gene by Nkx6.1

Tessy Iype, David G. Taylor, Suzanne M. Ziesmann, James C. Garmey, Hirotaka Watada and Raghavendra G. Mirmira

Department of Internal Medicine and the Diabetes Center (T.I., S.M.Z., J.C.G., R.G.M.) and Department of Pharmacology (D.G.T., R.G.M.), University of Virginia, Charlottesville, Virginia 22903; and Department of Medicine (H.W.), Metabolism, and Endocrinology, Jutendo University School of Medicine, Tokyo 113-8421, Japan

Address all correspondence and requests for reprints to: Raghavendra Mirmira, University of Virginia Health System, 450 Ray C. Hunt Drive, Box 801407, Charlottesville, Virginia 22903. E-mail: mirmira{at}virginia.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the pancreas, the NK homeodomain transcription factor Nkx6.1 is required for the development of ß-cells and is believed to function as a potent repressor of transcription upon binding to A/T-rich sequences within the promoter region of target genes. Because the nkx6.1 promoter itself contains several such sequences, we considered the possibility that the expression level and restricted pattern of the nkx6.1 gene might be precisely regulated by one or more homeodomain transcription factors, including Nkx6.1 itself. In this report, we identify a novel ß-cell-specific enhancer element in the nkx6.1 gene between –157 and –30 bp (relative to the transcriptional start site) that harbors a conserved A/T-containing sequence flanked by G/C-rich stretches. Although the islet homeodomain-containing activator Pdx-1 was unable to stimulate transcription of a reporter gene through this enhancer element in mammalian cell lines, strikingly, Nkx6.1 robustly activated transcription through direct interaction with the A/T-rich sequence in this element. We demonstrate that this activation is indeed transcriptional in nature (and not secondary to translational effects) and is mediated by a modular acidic sequence within the COOH-terminal domain of Nkx6.1. We show by EMSAs that Nkx6.1 binds to the ß-cell-specific enhancer in vitro and by chromatin immunoprecipitation assays that Nkx6.1 natively occupies this region in vivo in ßTC3 cells. We therefore conclude that Nkx6.1 is a bifunctional transcription factor that serves to maintain the specific expression of its own gene during ß-cell differentiation while simultaneously effecting broader gene repression events.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MEMBERS OF THE homeodomain family of transcription factors are crucial for the development of multiple organ systems. Development of the mammalian pancreas relies on the actions of many such homeodomain proteins, including Hb9, Pdx1, Pax4, Nkx2.2, and Nkx6.1 (1). Targeted disruption of these and other factors has identified a pattern of transcription factor expression that controls the ordered differentiation of the various endocrine cell types within the pancreatic islets of Langerhans (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). The NK homeodomain factor Nkx6.1 directs the differentiation specifically of the insulin-secreting ß-cell. Mice deficient for Nkx6.1 display a dramatic reduction in ß-cell numbers (with insulin levels reduced to about 2% of wild type) but have near-normal development of all other endocrine and exocrine cell types (2). Of note, these mice also have significant defects involving V2 interneuron and motor neuron specification in the ventral spinal cord (11), thereby emphasizing an important parallel in developmental pathways leading to ß-cells and neurons.

To direct these specific developmental programs, Nkx6.1 is believed to control the expression of an array of critical target genes (2, 11, 12). Although these target genes have not been definitively identified, it has been reported that Nkx6.1 exerts its regulatory activity by binding preferentially to TAAT-containing DNA sequences within promoters (13, 14). Interestingly, this DNA binding activity is uniquely modulated by its acidic COOH-terminal domain, which is known to lower the intrinsic affinity of its homeodomain for DNA (14). Upon binding to DNA, Nkx6.1 acts as a potent repressor of gene transcription. This repression activity maps to the NH2-terminal domain of the protein, which consists of stretches of proline and alanine residues as well as the NK decapeptide (14, 15). The NK decapeptide, or TN domain, shares significant homology with the Groucho/transducin-like enhancer of split (TLE) corepressor interaction motif. Physical interactions between Nkx6.1 and Groucho/TLE have been demonstrated to produce a functional and potent repressor complex (15).

Nkx6.1 gene expression is subject to strict spatial and temporal control in both the central nervous system and pancreas (2, 11, 15, 16, 17). In the pancreas, the gene is initially expressed broadly in the developing mouse bud at embryonic d 10.5, but eventually becomes restricted exclusively to the ß-cells at the time of birth (embryonic d 18.5) (2). The nkx6.1 gene contains a TATA-less promoter with upstream putative recognition sequences for multiple ubiquitous and pancreas-specific transcription factors (including Pdx1, Nkx2.2, and Nkx6.1 itself). Studies using cell line models have identified that both transcriptional and translational mechanisms operate to regulate expression of the nkx6.1 gene (18). The regulatory region of the gene (between bp –1300 and –1 relative to a cluster of transcriptional start sites) contains sequences that enhance its transcription in ß-cells, whereas sequences within the 5' untranslated region (UTR) appear to be necessary for its efficient translation (18). However, no specific mechanisms have yet been identified to explain its restricted expression patterns during development.

Many of the transcription factor genes involved in the ß-cell differentiation program are subject to either positive or negative transcriptional feedback by their respective encoded proteins (19, 20, 21, 22). Such feedback mechanisms may allow for either the rapid enhancement or attenuation of transcription in specific cell types, thereby permitting efficient spatial and temporal control of gene expression. Because the upstream regulatory region of the nkx6.1 gene contains potential recognition sequences for Nkx6.1 (18), we hypothesized that nkx6.1 transcription might be a target for feedback regulation. Although Nkx6.1 is a potent repressor of transcription, we also considered the possibility that this feedback could be positive in nature for two reasons: first, the nkx6.1 gene remains persistently expressed well into adulthood with little or no attenuation. Second, Nkx6.1 contains a negatively charged domain in the COOH terminus; such domains have been shown in some cases to activate transcription through the recruitment of basal transcriptional machinery (23, 24). In this report, we demonstrate for the first time that Nkx6.1 directly activates the expression of the nkx6.1 gene. By use of transcriptional and reporter gene assays, we show that this activation is transcriptional in nature and is indeed mediated by a discrete, acidic sequence within the COOH-terminal domain. This activation requires the interaction of Nkx6.1 with a specific A/T-containing sequence within a ß-cell-specific enhancer element in the nkx6.1 gene between –157 and –30 bp relative to the transcriptional start site. We therefore propose a model in which the ß-cell-specific expression of the nkx6.1 gene is maintained by a direct positive feedback mechanism involving a novel transcriptional activation function of Nkx6.1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of a Novel Proximal ß-Cell-Specific Enhancer (PBE) Element in the nkx6.1 Promoter
In prior studies, we have demonstrated the specific expression of nkx6.1 in pancreatic ß-cells and have identified a ß-cell-specific enhancer element in the nkx6.1 promoter between –840 and –771 bp (relative to a discrete cluster of transcriptional start sites) (18). However, the region of the promoter between –700 and +1 bp retains substantially greater activity in ß-cell lines compared with non-ß-cell lines (18), suggesting the existence of an additional ß-cell-specific enhancer element(s). To identify and characterize this enhancer element(s), we ligated various deletions of the nkx6.1 5'-flanking sequence (including the 5'-UTR) upstream of the luciferase reporter gene and transfected these constructs into mammalian cells. For these studies, we used a mouse ß-cell-derived line (ßTC3) that is reported to express the nkx6.1 gene and, as a control, a mouse fibroblast-derived line (NIH3T3) that does not. Western blot analysis (inset to Fig. 1AGo) confirmed the presence of Nkx6.1 in ßTC3 cells.



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  		Fig. 1. Identification of a Proximal ß-Cell-Specific Enhancer in the Mouse nkx6.1 Promoter

A, Reporter plasmids were constructed with the mouse nkx6.1 gene fragments indicated upstream of the luciferase coding sequence and were transfected into NIH3T3 cells (black bars) or ßTC3 cells (hatched bars). Percent luciferase activities were calculated with the activity from cells transfected with the backbone vector (pFoxLuc1) alone defined as 100%. Inset, Western blot demonstrating the expression of Nkx6.1 protein in ßTC3 cells but not in NIH3T3 cells. B, The PBE of the mouse promoter (–157 to –30 bp relative to the transcriptional start site) is aligned against sequences from the rat and human nkx6.1 genes from GenBank using the ClustalW algorithm in the MacVector software package. A region of A/T-rich sequence between –80 and –57 bp was identified that represents potential homeobox factor binding sites; the boxed sequence shows the homology of this region to the conserved rat I insulin promoter A3 element. The underlined sequence represents the probe used for EMSA studies in Fig. 6AGo. Asterisks identify the base pairs mutated by transversion in transfection and EMSA studies. C, Reporter plasmids containing either the –157- to –30-bp PBE fragment or the same fragment containing a mutation in the A/T-rich stretch upstream of the minimal rat prolactin promoter driving luciferase were transfected into NIH3T3 cells (black bars) or ßTC3 cells (hatched bars). Percent luciferase activities were calculated with the activity of cells transfected with the Prolactin-luciferase plasmid alone defined as 100%. *, P < 0.05 for the comparison shown as calculated by the paired Student’s t test. Experiments were performed in duplicate on at least three separate occasions. All data are shown as mean ± SE.

 
Figure 1AGo demonstrates that a reporter construct containing –1300 bp of the nkx6.1 promoter region directs expression of luciferase in ßTC3 cells but not in NIH3T3 cells. Deletion of the region between –380 and –157 bp revealed a 30% decrease in promoter activity in both ßTC3 and NIH3T3 cells, suggesting that a ubiquitous enhancer element resides within this region. Strikingly, however, nearly complete loss of residual promoter activity in ßTC3 cells was observed attending deletion of the region between –157 and –30 bp (no significant activity was observed for these constructs in NIH3T3 cells). These results suggest the existence of a discrete, PBE within the nkx6.1 promoter between –157 and –30 bp. Figure 1AGo shows that deletion of the PBE from the –1300 bp promoter results in an approximately 50% decrease in promoter activity in ßTC3 cells, thereby emphasizing the importance of this region within the context of the larger promoter.

Figure 1BGo shows that the PBE is a G/C-rich element that is highly conserved between the mouse, rat, and human nkx6.1 genes. Interestingly, the PBE contains an A/T-rich sequence (underlined in Fig. 1BGo) that shows distant homology to the well-characterized rat I insulin A3 promoter element, which is important in conferring ß-cell activity to the insulin gene (25). Figure 1CGo demonstrates that a single copy of the PBE can also function as a ß-cell-specific enhancer when linked to a heterologous (prolactin) promoter. Mutation of the A/T-rich sequence within the PBE significantly mitigates activity in ß-cells (Fig. 1CGo), suggesting that this sequence might serve as an important recognition sequence for ß-cell-specific transcription factors.

Nkx6.1 Activates the nkx6.1 Promoter through the PBE
Because A/T-containing sequences are potential homeodomain factor binding sites (26), we initially tested whether two ß-cell-restricted homeodomain transcription factors, Pdx-1 and Nkx6.1, might control transcription of the nkx6.1 gene through interaction with the PBE. Figure 2AGo demonstrates that overexpression of Pdx-1 in NIH3T3 cells causes activation of a reporter construct (–1300-bp reporter) that contains the previously identified distal ß-cell-specific enhancer of nkx6.1 (between –840 and –771 bp) (18), but not a construct containing only the PBE element (–380-bp reporter). By contrast, overexpression of Nkx6.1 leads to almost equivalent, 5- to 6-fold activation of both reporter constructs, suggesting that transcriptional activation by Nkx6.1 may be mediated through the PBE. Figure 2BGo confirms that promoter activation by Nkx6.1 in NIH3T3 cells is indeed mediated through the PBE, because deletion of the region between –157 and –30 bp within the context of the larger –1300-bp promoter results in complete loss of reporter activation. In addition, Nkx6.1 can modestly activate transcription (2-fold) through a single copy of the PBE linked to the heterologous rat prolactin promoter in NIH3T3 cells, but cannot significantly activate this reporter when a mutation in the A/T-rich sequence in the PBE is introduced (Fig. 2CGo). For the experiments shown in Fig. 2Go, A–C, a fixed quantity of Nkx6.1 expression plasmid (0.3 µg) was used, because a titration experiment with increasing amounts of plasmid revealed a plateau effect on luciferase activity at 0.3 µg of plasmid (data not shown).



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  		Fig. 2. Nkx6.1 Activates the nkx6.1 Promoter through the Proximal ß-Cell-Specific Enhancer

A, Reporter plasmids containing the indicated nkx6.1 gene fragments upstream of the luciferase coding sequence and CMV promoter-driven pBAT12 plasmids expressing Nkx6.1 or Pdx-1 cDNAs (or no cDNA) were cotransfected into NIH3T3 cells. Percent luciferase activities were calculated with the activity of cells transfected with the backbone reporter vector (pFoxLuc1) defined as 100%. The inset shows expression of Pdx-1 (top) and Nkx6.1 (bottom) by Western blot in NIH3T3 cells transfected with Pdx-1 and Nkx6.1 cDNAs, respectively. B, Reporter plasmids containing the indicated nkx6.1 gene fragments upstream of luciferase and a plasmid expressing Nkx6.1 cDNA (or no cDNA) were cotransfected into NIH3T3 cells. Percent luciferase activities were calculated with the activity of cells transfected with the backbone reporter vector (pFoxLuc1) defined as 100%. C, A reporter plasmid containing the –157 to –30-bp PBE fragment or the same fragment containing a mutation in the A/T-rich sequence upstream of the minimal rat prolactin promoter driving luciferase was cotransfected into NIH3T3 cells with a plasmid expressing Nkx6.1 cDNA (or no cDNA). Percent luciferase activities were calculated with the activity of cells transfected with the Prolactin-luciferase plasmid alone defined as 100%. *, P < 0.05 for the comparison shown, as calculated by the paired Student’s t test. All data are shown as mean ± SE.

 
Activation by Nkx6.1 Is at the Transcriptional (Not Translational) Level
Figure 2BGo also shows that activation of the –380-bp reporter by Nkx6.1 (as measured by luciferase enzyme activity) is abolished when the 5'-UTR of the nkx6.1 gene is deleted. This observation suggests that the 5'-UTR contains an important DNA element that is necessary for either transcriptional activation by Nkx6.1 or for proper translation of the nascent transcript. To distinguish between these two possibilities, we performed quantitation (by real-time RT-PCR) of luciferase mRNA levels (rather than quantitation of luciferase enzymatic activity) in NIH3T3 cells cotransfected with Nkx6.1 cDNA and the –380-bp reporter. Figure 3Go demonstrates that Nkx6.1 activates transcription of luciferase from the –380-bp reporter by about 5-fold, and that deletion of the 5'-UTR has no effect on this transcriptional capacity. These results are consistent with previous studies that identified an internal ribosomal entry site within the 5'-UTR that is necessary for efficient translation (18), and imply that transcriptional activation by Nkx6.1 does not require DNA sequence elements within the 5'-UTR. As a negative control for these studies, we observed that overexpression of just the Nkx6.1 homeodomain [amino acids (aa) 231–305, which contains no known transcriptional activity] did not activate transcription of either reporter construct (Fig. 3Go).



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  		Fig. 3. Nkx6.1 Activates nkx6.1 Gene Transcription Independently of the 5'UTR

Reporter plasmids containing the –380 bp nkx6.1 promoter with or without the 5'-UTR and pBAT12 plasmids expressing the Nkx6.1 and (231–305)Nkx6.1 cDNAs (or no cDNA) were cotransfected into NIH3T3 cells. To control for transfection efficiency and RNA recovery, a CMV promoter-driven LacZ plasmid was cotransfected in each experiment. Total RNA from cells was isolated and subject to real-time RT-PCR to quantitate luciferase and LacZ transcript levels as detailed in Materials and Methods. Relative luciferase mRNA levels were calculated with the levels of luciferase mRNA in cells transfected with pBAT12 alone defined as 1. Transcripts were quantitated in triplicate from transfection experiments performed on three separate occasions. Data represent the mean ± SE.

 
The Acidic COOH Terminal Domain of Nkx6.1 Directly Mediates Transcriptional Activation of the nkx6.1 Promoter
Whereas our prior studies identified a powerful transcriptional repression domain at the NH2 terminus of Nkx6.1 (14), a potential activation domain was not previously identified. To determine whether a discrete domain within Nkx6.1 is responsible for transcriptional activation, we cotransfected cDNAs encoding various deletions of Nkx6.1 with the –380-bp nkx6.1 reporter plasmid into NIH3T3 cells, and examined the ability of each deletion to activate the reporter (Fig. 4Go). Western blots and/or EMSA confirmed that the deletional constructs were expressed at comparable levels and capable of binding to DNA (Ref.14 and data not shown). Figure 4AGo demonstrates that constructs containing at least aa 306–338 within the acidic COOH terminus are capable of activating the reporter to the same magnitude as the full-length protein. Importantly, the COOH terminus requires the presence of the homebox (aa 229–305) for activation of this reporter because fusion of the COOH terminus (aa 306–364) to the heterologous yeast Gal4 DNA binding domain (DBD) does not lead to reporter activation (Fig. 4AGo). This result implies that the apparent transcriptional activation domain in the COOH terminus either overlaps with residues within the homeodomain or requires a specific function of the homeodomain (such as DNA binding or protein-protein interaction). To distinguish between these possibilities, we tested the ability of Gal4-DBD/Nkx6.1 fusion proteins to activate a reporter gene containing Gal4 recognition sequences upstream of a minimal promoter driving luciferase. As shown in Fig. 4BGo, whereas overexpression of a protein containing the Gal4-DBD fused to the COOH terminus (aa 306–364) of Nkx6.1 alone leads to a 2-fold activation of the reporter, this activation is enhanced (to 4-fold) when a portion of the homeodomain is included in the fusion (aa 261–364). By contrast, when similarly fused to the Gal4-DBD, the homeodomain alone (aa 231–305) had no significant effect on transcription (Fig. 4BGo). These findings confirm that Nkx6.1 contains a modular activation domain that includes a portion of the homeodomain and the COOH terminus.



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  		Fig. 4. Transcriptional Activation Maps to the COOH Terminus of Nkx6.1

A, The –380 bp nkx6.1 promoter reporter plasmid indicated at the top of the figure was cotransfected with pBAT12 plasmids expressing the Nkx6.1 cDNAs indicated schematically on the left into NIH3T3 cells. Percent luciferase activities were calculated with the activity of cells transfected with pBAT12 alone (backbone vector) defined as 100%. HD, Homeodomain; GAL4 DBD, Gal4 DNA binding domain. B, Reporter plasmid containing five tandem copies of the GAL4 upstream activating sequence (UAS) upstream of the adenovirus E1b promoter driving luciferase was cotransfected with expression plasmids encoding the individual Gal4 fusion constructs indicated schematically on the left into NIH3T3 cells. Percent luciferase activities were calculated with the activity of cells transfected with a plasmid encoding the isolated Gal4-DBD set as 100%.

 
To assess whether activation by Nkx6.1 is a result of a primary effect upon the nkx6.1 gene (as opposed to an indirect effect upon another gene, whose product subsequently activates the nkx6.1 gene), we performed in vitro transcription assays using bacterially purified proteins. Figure 5Go shows that in the presence of HeLa cell nuclear extract in vitro, (231–338)Nkx6.1 (a construct containing the homeodomain and minimal activation segment of the COOH terminus) causes almost 3-fold relative activation of the –380-bp reporter compared with the homeodomain alone. This activation is significantly attenuated when a mutation of the A/T-rich region of the PBE is introduced, and is completely abolished when the PBE is deleted (Fig. 5Go). The relatively modest activation observed in these experiments is consistent with the weak initiation of transcription in vitro observed for TATA-less promoters with multiple transcriptional start sites (27). Because transcription in vitro is not subject to effects secondary to transcription and translation of other genes, these results suggest a direct activation of the nkx6.1 gene by the COOH terminus of Nkx6.1.



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  		Fig. 5. Nkx6.1 Activates Transcription of the nkx6.1 Promoter in Vitro

Reporter plasmids containing the nkx6.1 gene promoter fragments indicated upstream of the luciferase coding sequence were incubated in the presence of HeLa nuclear extract, NTPs, 32P-UTP, and 250 ng of bacterially purified (231–305)Nkx6.1 or (231–338)Nkx6.1. 32P-UTP-labeled HeLaScribe control RNA was added at the conclusion of the 30 min incubation to serve as an RNA recovery standard. Reactions were subject to electrophoresis on a 5% polyacrylamide-urea gel, and luciferase transcripts were quantitated by phosphorimager analysis and normalized to the recovery standard. Relative luciferase transcript levels were calculated with the level of transcript in the reaction containing (231–305)Nkx6.1 defined as 1. Individual reactions were performed on at least three separate occasions, and data represent the mean ± SE. X in the middle construct, Mutation of the A/T-rich stretch of the PBE (see Fig. 1BGo).

 
Nkx6.1 Interacts with the PBE in Vitro and in ßTC3 Cells in Vivo
To address whether Nkx6.1 can bind to the PBE, we performed EMSAs using bacterially purified Nkx6.1 homeodomain (aa 231–305) and a double-stranded oligonucleotide corresponding to the PBE as probe. The region of the PBE selected as a probe (–77 to –38 bp) contains the conserved A/T-rich sequence that might serve as a binding site for the Nkx6.1 homeodomain (see Fig. 1BGo). Figure 6AGo demonstrates that (231–305)Nkx6.1 can directly bind to the PBE probe in an EMSA (lane 1). Binding to this probe is competed by unlabeled oligonucleotide corresponding to the A/T-rich segment of the PBE (Fig. 6AGo, lanes 2 and 3), but not by an oligonucleotide containing a mutation in the A/T-rich segment (Fig. 6AGo, lanes 4 and 5). Importantly, however, the apparent binding affinity of (231–305)Nkx6.1 to the PBE probe in vitro is substantially lower (by about 10-fold) than that observed for binding to a probe containing an ideal homeodomain consensus sequence (ATTA, data not shown). This observation, therefore, leaves open the prospect that the interaction of Nkx6.1 with other proteins in the nucleus may enhance or facilitate binding to the PBE. Similar EMSAs using ßTC3 nuclear extract (which contains full-length Nkx6.1 protein) and Nkx6.1 antiserum resulted in no clearly identifiable complex corresponding to Nkx6.1 binding, as multiple bands were observed that might potentially obscure the expected complex (data not shown). Instead, to demonstrate that Nkx6.1 natively interacts with the PBE in ßTC3 cells, we performed chromatin immunoprecipitation (ChIP) assays using formaldehyde-cross-linked chromatin from ßTC3 cells. A proximal region of the nkx6.1 promoter overlapping the PBE (–324 to –141 bp) was amplified by real-time PCR from chromatin immunoprecipitated with Nkx6.1 antiserum and compared with results using chromatin immunoprecipitated with normal rabbit serum. Figure 6BGo demonstrates that the nkx6.1 promoter is enriched approximately 5-fold in extracts immunoprecipitated with Nkx6.1 antiserum. Because DNA is fragmented to an average of 800 bp before ChIP, our primers would detect association of Nkx6.1 with the A/T-rich segment of the PBE (even though they do not directly encompass this region). As a positive control in these studies, we observed that another potential Nkx6.1 target promoter, insulin (a region encompassing –126 to –229 bp relative to the transcriptional start site that contains the A3 element), is also enriched in extracts immunoprecipitated with Nkx6.1 antiserum. By contrast, however, Nkx6.1 antiserum did not enrich sequences from the albumin gene, which is not expressed in ß-cells (Fig. 6BGo). These ChIP results therefore demonstrate that Nkx6.1 is physically associated with a region of the nkx6.1 promoter harboring the PBE in ßTC3 cells.



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  		Fig. 6. Nkx6.1 Binds to the Proximal ß-Cell-Specific Enhancer of the nkx6.1 Gene

A, EMSA was performed using bacterially purified (231–305)Nkx6.1 and a 32P-labeled probe corresponding to the PBE. The PBE probe sequence (indicated in Fig. 1BGo) corresponds to bp –77 to –38 of the nkx6.1 promoter. The mutant competitor PBE oligonucleotide contains transversion mutations within the A/T-rich sequence as indicated in Fig. 1BGo. Protein (100 ng) was incubated with probe ± competitor for 15 min. at room temperature and subject to electrophoresis on a 5% polyacrylamide gel. Unlabeled wild-type (WT) and mutant (MUT) competitor oligonucleotides were added at approximately 100- and 800-fold molar excess as indicated. Arrow indicates position of the shifted complex. FP, Free probe. B, ChIP assays were performed using formaldehyde-cross-linked chromatin from ßTC3 cells and either Nkx6.1 antiserum or normal rabbit serum (No Ab), as described in Materials and Methods. Coimmunoprecipitated chromatin was quantitatively analyzed for recovery of the albumin, proximal nkx6.1, or insulin promoters by SYBR Green-I real-time PCR using specific primers, and compared with input chromatin before immunoprecipitation. Fold-Nkx6.1 association to each gene was calculated by comparing recovery of gene fragments from ChIP using Nkx6.1 antiserum to ChIP using normal rabbit serum (defined as 1). PCR quantitations were performed in triplicate from four independent ChIP samples, and data represent the mean ± SE. P values for the comparison shown were calculated by the paired Student’s t test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The proper development of the pancreatic ß-cell depends on the expression of several transcription factors in a precise spatial and temporal manner (1). Key among these factors is Nkx6.1, which is required for the final step of the major pathway leading to differentiation of ß-cells. Nkx6.1 gene expression is regulated by complex mechanisms involving transcriptional and translational control, the latter of which is dependent upon regulatory sequences within the 5'-UTR (18). In this report, we have characterized a novel transcriptional activation domain in the Nkx6.1 protein that appears to function through a ß-cell-specific enhancer in the proximal nkx6.1 gene. Our results provide the first evidence that Nkx6.1 directly functions in a positive feedback loop maintaining nkx6.1 gene transcription.

Nkx6.1 is expressed broadly in the early developing pancreatic bud but becomes more restricted to the ß-cell as development proceeds (2). Because the expression pattern of Nkx6.1 spatially and temporally follows that of Pdx-1 and Nkx2.2 in the developing mouse pancreas, it has been suggested that both these factors may initiate and subsequently maintain nkx6.1 gene expression through interaction with a distal ß-cell-specific enhancer (between –840 and –771 bp) (5, 18, 28). Our results confirm that Pdx-1 can activate nkx6.1 transcription through the distal region of the promoter, and other studies involving inactivation of the pdx-1 gene in developing and mature ß-cells confirm the role of Pdx-1 in the maintenance of nkx6.1 gene expression (29, 30). Once nkx6.1 transcription and translation are initiated, our data suggest that transcriptional feedback may mediate ongoing expression of the gene. Transcriptional feedback is an important phenomenon controlling the expression of several pancreatic transcription factors. For example, the paired-homeodomain factor Pax4 directly binds to the pax4 promoter and represses transcription of its own gene, thereby providing a negative feedback mechanism accounting for its transient expression in the pancreas during development (20). By contrast, Pdx-1 acts to activate its own gene promoter, thus allowing for a positive feedback loop that allows for maintenance of pdx1 expression throughout development and into adulthood (19). Our data demonstrate that Nkx6.1 can activate transcription of its own gene 6-fold when heterologously expressed in non-ß-cell types.

Activation of the nkx6.1 gene by Nkx6.1 requires a PBE element located between –157 and –30 bp relative to the nkx6.1 transcriptional start site. Although this region contains predominantly G/C sequences, it includes a conserved A/T-rich stretch that is reminiscent of a ß-cell enhancer in the proximal rat insulin promoter (see Fig. 1BGo). Using a stringent in vitro oligonucleotide site selection strategy, we and others previously demonstrated that the Nkx6.1 homeodomain preferentially recognizes 5'-TAAT-3'- (or 5'-ATTA-3') DNA sequences (13, 14). A similar, though not identical, sequence (5'-ATTT-3') is present in the A/T stretch of the PBE; we found by EMSA that purified (231–305)Nkx6.1 can bind to this A/T-rich region of the promoter. In this regard, the highly homologous transcription factor Nkx6.2/Gtx has been shown to bind to a variety of A/T-rich sequences in vitro and mediate transcriptional activity in vivo through interaction with these sites (31). These EMSA studies taken together with our ChIP studies in ßTC3 cells strongly point to a direct interaction of Nkx6.1 with the PBE in vitro and in vivo, respectively. Importantly, however, our EMSA studies also suggest that the interaction of Nkx6.1 with the PBE in vitro is weaker than its interaction with a DNA fragment containing a TAAT sequence (data not shown). This observation raises at least two intriguing possibilities for the interaction of Nkx6.1 with the PBE in vivo: first, DNA binding by Nkx6.1 in vivo might be a facilitated phenomenon, such that it is enhanced by specific posttranslational modifications or by interactions with neighboring transcription factors; similar mechanisms have been observed to significantly enhance the binding in vivo of several DNA binding proteins, including HMG proteins, Ets-1, and PEA3 (32, 33, 34, 35). Second, it is possible that in vivo Nkx6.1 may not directly bind DNA at all, but might instead be tethered to DNA through interaction with another transcription factor. The occurrence of G/C-rich sequences in the PBE suggests the potential for interaction of Nkx6.1 with G/C-box binding proteins such as the SP1-like and/or Krüppel-like factors (36). The interaction of homeodomain factors with this family of proteins has been recently described (37); however, in initial studies we have not observed any functional or physical interactions of SP1 with Nkx6.1 (data not shown).

By use of luciferase reporter gene analysis (with measurement of both luciferase transcript levels and enzymatic activity), we demonstrate for the first time that Nkx6.1 has the capacity to activate transcription. Previously, only the transcriptional repressor activity of Nkx6.1 has been described in both the pancreas and central nervous system. The repressor activity localizes to sequences in the NH2 terminus and appears to involve at least two mechanisms: one dependent upon Groucho/TLE corepressor interactions (15) and the other mediated through stretches of Ser, Ala, and Pro residues in a manner similar to that of the Engrailed protein of Drosophila (14). Importantly, our data point to the coexistence of a transactivation domain located between aa 306–338 within the COOH terminus (with maximal activation requiring a portion of the homeodomain). Interestingly, the sequences within this region have been shown to autoinhibit DNA binding activity (14). Of the 33 amino acids within this region, 15 (45%) are either Asp or Glu. Similar acidic transcriptional activation domains have been described for several transcription factors, and are believed to function by recruiting components of the basal transcriptional machinery, such as transcription factor (TF) IIB and TFIID, to the promoter (23, 24). This mechanism provides an explanation for how basal transcriptional components could be assembled on the TATA-less promoter of the nkx6.1 gene.

The potential for both transcriptional activation and repression by the same transcription factor suggests that specific regulation must occur for one activity or the other to predominate. In the case of Nkx6.1, the nature of the DNA sequence in the enhancer regions of genes may provide some clue as to how this regulation might occur, and we therefore propose the model shown in Fig. 7Go. We have demonstrated previously that when the enhancer region of target genes (e.g. the insulin gene) contains isolated or tandem classic homedomain binding sites (TAAT or ATTA), Nkx6.1 directly binds to DNA and causes repression of gene transcription through interactions involving its NH2-terminal domain (see top half of Fig. 7Go) (14). By contrast, we demonstrate here that when a noncanonical homeodomain binding site (ATTT) is in the proximity of a G/C-rich region (as in the nkx6.1 gene), Nkx6.1 causes activation of transcription through interactions involving its COOH-terminal domain (see bottom half of Fig. 7Go). A similar dependence upon DNA sequence has been observed for the activity of other bifunctional transcription factors, including SP3, glucocorticoid receptor, and Ets-1 (38, 39, 40). Allosteric modulation of Nkx6.1 structure, possibly through contacts with neighboring transcription factors, may account for its DNA context-dependent activities. Implicit in this hypothesis is the possibility that during transcriptional activation Nkx6.1 may not directly bind to DNA itself but becomes associated with it through interactions with other proteins. Although the identity of the putative interacting protein(s) remains elusive, we anticipate that it will likely represent a ubiquitously expressed transcription factor because activation by Nkx6.1 through the PBE can occur in a very divergent cell type (NIH3T3 cells) and in vitro using HeLa nuclear extract.



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  		Fig. 7. Model for Nkx6.1 Action

Nkx6.1 is schematically represented as consisting of an NH2-terminal repression domain (R), a DNA-binding homedomain (HD), and a COOH-terminal activation domain (A). In the formation of a transcriptional repression complex (top half of figure), Nkx6.1 recognizes isolated or tandem ATTA-containing DNA sequences and thereby interacts with Gro/TLE via the NH2-terminal domain (14 15 ). In the formation of a transcriptional activation complex (bottom half of figure), Nkx6.1 recognizes an ATTT-containing DNA sequence that is flanked by G/C-rich sequences and thereby interacts with components of the basal transcriptional machinery (e.g. TFIIB, TFIID). Our data do not exclude the possibility that binding to DNA in the formation of an activation complex might be facilitated by interactions with other ubiquitous transcription factors (represented by the circle with ?). Helical lines represent DNA.

 
Taken together, our data suggest a positive feedback mechanism for the expression of the nkx6.1 gene. We hypothesize that during early pancreatic development transcription of the nkx6.1 gene is initiated by cell type-specific activators, which may include factors such as Pdx-1 and Nkx2.2. Within the cell types that can translate the gene product, subsequent amplification of nkx6.1 expression occurs through a positive feedback loop mediated by Nkx6.1 protein. Cell types with the lowest initial transcriptional and translational levels cannot maintain a strong feedback loop and therefore diminish in their capacity to express the gene. This hypothesis offers one plausible mechanism for how Nkx6.1, an otherwise powerful transcriptional repressor, remains robustly produced in the face of broader gene suppression events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Recombinant Plasmids and Mutagenesis
All plasmids were constructed using standard recombinant DNA techniques, and all constructs generated by PCR were confirmed by sequencing. The Escherichia coli expression vectors pET(231–305)Nkx6.1 and pET(231–338)Nkx6.1 were made by amplifying the appropriate coding fragment of hamster Nkx6.1 by PCR, then ligating it to the NcoI and XhoI sites of the COOH-terminal 6X-His vector, pET21d (Novagen, Madison, WI). The cytomegalovirus (CMV) promoter plasmids for the expression of intact Nkx6.1 and its fragments in mammalian cells were described previously (14). To generate reporter plasmids, fragments of the 5' region of the mouse nkx6.1 gene (obtained by PCR amplification) were ligated to the SalI and BamHI sites upstream of the luciferase gene in the plasmids pFoxLuc1 or pFoxLucPrl (41). The Gal4-Adenovirus E1b reporter plasmid (pG5FOXLucE1b) and Gal4-DBD fusion plasmids were described previously (14, 18).

Mutagenesis of the mouse nkx6.1 gene was performed using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) according to manufacturer’s instructions. The following oligonucleotide (and its complementary strand) was used in making the mutation (mutation underlined): 5'-CGTCCTCGAAAGTTCGACGGGGGGCCCCCCACCTCCC.

Cell Culture and Transient Transfections
The mouse ß-cell line ßTC3 was maintained in DMEM supplemented with 15% horse serum, 2.5% fetal bovine serum, and 1% penicillin/streptomycin. The mouse fibroblast cell line, NIH3T3, was maintained in DMEM supplemented with 10% newborn calf serum and 1% penicillin/streptomycin. For transient mammalian cell transfections, 5 x 104 NIH3T3 or 5 x 105 ßTC3 cells were plated in six-well tissue culture plates 24 h before the transfection. A total of 2 µg of plasmid DNA were mixed with 6 µl of Transfast reagent (Promega, Madison, WI), and transfections were performed according to the manufacturer’s protocol. Cells were harvested 48 h after transfection and luciferase activities were measured using a commercially available assay kit (Promega) and an FB15 luminometer (Zylux, Oak Ridge, TN). A total of 0.3 µg of cDNA expression plasmid, 0.8 µg of luciferase reporter plasmid, and 0.05 µg of a CMV promoter-driven LacZ control plasmid were used in each transfection. The balance of the 2 µg of DNA per transfection was made up by adding pBluescript. Data were normalized to ß-galactosidase activity and represent the average of at least three independent transfections ± SE.

Real-time RT-PCR
For measurement of luciferase transcript levels by real-time RT-PCR, NIH3T3 cells were transfected as above in six-well tissue culture plates, and harvested 48 h later for isolation of total RNA using the RNeasy kit (QIAGEN, Valencia, CA) according to manufacturer’s protocol. Five micrograms of the total RNA were reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA) and primers specific to firefly luciferase (5'-AAGCAGTGGTATCAACGCAGAGTGCTTCTGCCAACCGAACGGAC) and LacZ (5'-TCCAGATAACTGCCGTCACTCCAAC) coding sequences. The reaction was purified and excess primers were removed by use of a PCR clean-up column (QIAGEN). Luciferase and LacZ transcripts were quantitated in the resulting cDNA by real-time PCR using SYBR Green I monitoring as previously described (42, 43). The forward and reverse primers to amplify firefly luciferase mRNA were, respectively: 5'-TCGCCAGAAAGTAGGGGTCG and 5'-AAGCAGTGGTATCAACGCAGAGT. The forward and reverse primers to amplify LacZ mRNA were, respectively: 5'-TCAATCCGCCGTTTGTTCCCAC and 5'-TCCAGATAACTGCCGTCACTCCAAC. Forty cycles of PCR were performed under the following conditions: 95 C for 15 sec, 55 C for 15 sec, and 72 C for 15 sec.

Protein Expression and Purification
Expression and purification of (231–305)Nkx6.1 and (231–338)Nkx6.1 proceeded as described previously (14) with some modifications. Briefly, E. coli strain BL21(DE3)pLysS was transformed with either the pET(231–305)Nkx6.1 or pET(231–338)Nkx6.1. E. coli cultures were grown to an OD600 of 0.6–1.0 in 1 liter Luria-Bertani broth before the induction of protein expression with 1 mM isopropyl ß-D-thiogalactopyranoside. Induced cultures were then pelleted, resuspended in imidazole lysis buffer [50 mM NaH2PO4 (pH 8.0), 30 mM imidazole, 300 mM NaCl, 1 mM dithiothreitol, 5% glycerol], and lysed by sonication. The resulting bacterial lysate was applied to a 2-ml bed volume column containing nickel-nitrolo-triacetic acid Agarose (QIAGEN), and protein was eluted from the column using a linear gradient of 30–250 mM imidazole prepared in lysis buffer. Protein was analyzed for purity (>95%) by SDS-PAGE, and protein concentrations were measured using the Bradford method.

EMSAs
Single-stranded oligonucleotide probes were 5' end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase. Labeled oligonucleotides were column-purified and annealed to an excess of complementary strand. EMSA reactions consisted of 100 ng purified (231–305)Nkx6.1 or 5 µg nuclear extract, 1 µg poly deoxyinosine/deoxycytosine, 10 µg BSA, and varying concentrations (100- to 800-fold molar excess) of cold competitor oligonucleotide. EMSA buffers and electrophoresis conditions were described previously (44). The following oligonucleotides were used in these experiments (top strands shown): PBE: 5'-CGAAAGTTCTCATTTTGGCCCCCCACCTCCCCTCCCTTGC; competitor PBE: 5'-GATCTCTCATTTTGGCAGTC; competitor mutant PBE: 5'-GATCTCTCCGGGTGGCAGTC.

In Vitro Transcription Assays
Direct transcriptional activation of the Nkx6.1 promoter was analyzed using the HeLaScribe in vitro Transcription System (Promega). A 25-µl reaction mixture consisting of 5 µl HeLa nuclear extract, 1x transcription buffer, 3 mM MgCl2, 15 U ribonuclease inhibitor, and 300 ng DNA template (the –380-bp luciferase reporter, the –380-bp mutant luciferase reporter, or the –30-bp luciferase reporter) was preincubated at 30 C for 30 min. The transcription reaction was then initiated with the addition of nucleotide triphosphate [10 mM CTP, GTP, and ATP, and 0.4 mM UTP (uridine triphosphate], 10 µCi {alpha}32P-UTP, and 250 ng of (231–305)Nkx6.1 or (231–338)Nkx6.1. The reactions were incubated at 30 C for 1 h then terminated with stop buffer and supplemented with 25 µl of in vitro-transcribed, 32P-labeled HeLaScribe control RNA to serve as an internal control for recovery. RNA products were isolated and purified using the RNeasy kit (QIAGEN) and subject to electrophoresis on a 5% polyacrylamide gel containing 7 M urea. Luciferase transcript was visualized and quantitated using a Typhoon phosphorimager (Amersham Pharmacia Biotech, Piscataway, NJ) and normalized to the internal recovery control.

Quantitative ChIP Assay
Protein-DNA cross-linking in ßTC3 cells, subsequent ChIP, and quantitation of coimmunoprecipitated promoter fragments by real-time PCR proceeded as we have detailed previously (42, 45). Antisera used in the immunoprecipitations were either anti-Nkx6.1 (a gift from Dr. Michael German, University of California, San Francisco, CA) or normal rabbit serum. ChIPs were performed on four independent occasions, and each ChIP was quantitated in triplicate on two separate occasions by real-time PCR for recovery of either the nkx6.1 or albumin promoters (to give a total of eight determinations for each promoter). Forward and reverse primers used to amplify the nkx6.1 gene (–324 to –141 bp relative to the transcriptional start site) were, respectively: 5'-GGAACCACTCTTTTCGCCAG and 5'-TAAACACCGCCTCCAATAGC. Forward and reverse primers used to amplify the insulin gene (–126 to –296 bp relative to the ins1 transcriptional start site) were, respectively: 5'-TCAGCCAAAGATGAAGAAGGTCTC and 5'-TCCAAACACTTGCCTGGTGC. Forward and reverse primers used to amplify the albumin gene were, respectively: 5'-TGGGAAAACTGGGAAAACCATC and 5'-CACTCTCACACATACACTCCTGCTG.

Western Blot Analysis
Expression of Pdx-1 and full-length and deleted fragments of Nkx6.1 in nuclear extracts (5 µg) from ßTC3 and NIH3T3 cells was measured by performing Western blot analysis using antisera to Pdx-1 or Nkx6.1 (both from Dr. Michael German) as described previously (14, 42). Western blots were visualized by using the ECL-Plus system (Amersham Pharmacia Biotech).


   ACKNOWLEDGMENTS
 
We thank Dr. S. Chakrabarti and Kyu Won Kim for assistance in these studies. We are grateful to Dr. M. German for providing Pdx-1 and Nkx6.1 antisera.


   FOOTNOTES
 
This work was supported by National Institutes of Health (NIH) Grants K08 DK02683 and R01 DK60581 and a grant from the Diabetes Action Research and Education Foundation (to R.G.M.), and NIH Training Grants T32 GM08136 (to D.G.T.) and T32 DK0732025 (to S.M.Z.).

Abbreviations: aa, Amino acids; ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus; DBD, DNA binding domain; PBE, proximal ß-cell-specific enhancer; TF, transcription factor; TLE, transducin-like enhancer of split; UTP, uridine triphosphate; UTR, untranslated region.

Received for publication January 7, 2004. Accepted for publication March 23, 2004.


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 DISCUSSION
 MATERIALS AND METHODS
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F. S. J. de Souza, A. M. Santangelo, V. Bumaschny, M. E. Avale, J. L. Smart, M. J. Low, and M. Rubinstein
Identification of Neuronal Enhancers of the Proopiomelanocortin Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting
Mol. Cell. Biol., April 15, 2005; 25(8): 3076 - 3086.
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