The region located between −13 and −9 kb of the mouse POMC
gene directs expression to hypothalamic neurons. Our previous results showed that the region between −13 and −2 kb upstream of the mouse POMC
transcriptional start site contains enhancers capable of directing proper cell-specific transgene expression to POMC neurons of the arcuate nucleus of the hypothalamus (66
). To facilitate the analysis of transgene expression, we inserted the coding region of EGFP into mouse Pomc
exon 2 immediately before the translational start codon of the POMC prohormone (Fig. , transgene 1). This 22-kb transgene containing the entire transcriptional unit of mouse Pomc
, together with 13 kb of 5′ flanking regions and 2 kb of 3′ flanking regions, drives accurate EGFP expression to nearly 100% of that of POMC-expressing neurons in the arcuate nucleus and has allowed for electrophysiological studies of living POMC neurons (3
). To further narrow the flanking regions necessary for Pomc
expression in the brain, we constructed two large deletions of transgene 1 by removing the region between −6.5 and −0.8 kb or between −9 and −0.8 kb (Fig. , transgenes 2 and 3). The expression pattern of EGFP was compared to that of endogenous Pomc
in the serial brain sections of transgenic mice carrying constructs 2 and 3 by using EGFP and ACTH polyclonal antibodies. Colocalization of EGFP in POMC neurons was assessed by red-fluorescent immunohistochemistry coupled to the ACTH antibody on Vibratome-sectioned brain slices of several independent transgenic mouse pedigrees. The analysis of deletion of transgenes 2 and 3 demonstrated that EGFP was coexpressed with Pomc
in neurons of the arcuate nucleus (Fig. ), as well as in pituitary corticotrophs and melanotrophs (data not shown), suggesting that the region between −9 and −0.8 kb is dispensable for neuron-specific expression of Pomc
in the hypothalamus. In addition, complete deletion of the fragment spanning from −13 to −9 (Fig. , transgene 4) prevented EGFP expression in the arcuate nucleus, although EGFP was properly expressed in pituitary corticotrophs and melanotrophs (data not shown). Transgenes 1 and 4 showed a consistent ectopic expression of EGFP in the subgranular layer of the dentate gyrus of the hippocampus probably due to the presence of a cryptic enhancer located in the −2- to −0.8-kb region of the Pomc
promoter, as recently reported (41
). These results suggest that the 4-kb region located between −13 and −9 kb upstream of the murine Pomc
gene contains one or more neuronal enhancers necessary to direct Pomc
expression to arcuate neurons.
FIG. 1. First set of transgenes used in this study. Transgenes 1 through 4 carry the whole transcriptional unit of mouse Pomc with the coding region of EGFP (E) inserted into exon 2 before the starting translational codon. Pomc exons are indicated by black boxes. (more ...)
FIG. 2. Reporter gene expression in the arcuate nuclei and pituitary glands of transgenic mice. (A and B) Coronal brain sections of a transgene 3 mouse immunostained for ACTH (panel A) and EGFP (panel B). The inset in panel B shows an example of colocalization (more ...)
We next sought to determine whether this 4-kb DNA region was sufficient to direct transgene expression to POMC neurons in the absence of the proximal Pomc promoter, which is known to be essential for expression in pituitary melanotrophs and corticotrophs. To this end, we constructed a transgene in which the region from kb −13 to −9 from the mouse Pomc gene was ligated upstream of a minimal heterologous promoter obtained from the Herpes simplex virus TK gene, followed by the hGH gene, which served as a reporter (Fig. , transgene 5). Transgenic mouse brains were analyzed for colocalization by in situ hybridization against endogenous Pomc mRNA by using a DIG-labeled riboprobe (developed with the brown chromogen diaminobenzidine) followed by immunohistochemistry against hGH (developed with the blue chromogen benzidine dihydrochloride). Interestingly, the fragment from positions −13 to −9 was able to target reporter gene expression to arcuate POMC neurons (Fig. ) in the total absence of sequences from the proximal promoter, exons, introns, or 3′ flanking regions of mouse Pomc, demonstrating that this distal 4-kb region contains neuronal-specific enhancer(s) sufficient to drive transcription in POMC arcuate neurons. This result was confirmed in three independent transgenic pedigrees that showed an average of 75% of POMC-positive arcuate neurons coexpressing the reporter hGH. Reporter gene expression was not detected in pituitary melanotrophs (Fig. ). The hGH riboprobe intensively labeled mouse somatotrophs in the pituitary anterior lobe due to cross-hybridization with mouse GH mRNA, precluding transgene expression analysis in corticotrophs. Variable patterns of ectopic transgene expression were also detected in the brains of these mouse lines in areas that included the supraoptic nuclei of the hypothalamus, amygdala, habenula, and cerebral cortex. Together, these results demonstrate that distal neuronal enhancers located within the −13- to −9-kb region of the mouse Pomc 5′ flanking region are sufficient to drive reporter expression to POMC neurons of the arcuate nucleus.
The Pomc neuron-specific region contains two highly conserved elements in mouse and human.
Among vertebrate genomes, regulatory elements are often more conserved than average intergenic regions (16
). Therefore, in order to identify candidate enhancers within the −13- to −9-kb mouse Pomc
fragment, we sequenced this 4-kb region and compared it to the publicly available human POMC
locus sequence. Figure shows a global visualization of local sequence alignments (DOTTER program) (55
) between the regions from kb −13 to −9 and kb −11 to −7 from the mouse and human POMC
genes, respectively. Two regions of approximately 600 and 150 bp with high identities between the two species were readily identifiable. We named these two putative neuronal POMC enhancers nPE1 and nPE2. In addition to the conservation of nucleotide sequence, the distances from each element to the transcriptional start were also similar, indicating that the overall genomic organization at the POMC
locus is conserved between these two species (Fig. ).
FIG. 3. Identification of nPE enhancers. (A) Global visualization of local sequence alignments between regions from kb −13 to −9 of the mouse (vertical) and kb −11 to −7 of the human (horizontal) POMC genes by using the DOTTER (more ...) Conservation of neuronal enhancer function in the human POMC gene.
To determine whether the homologous human POMC region containing nPE1 and nPE2 sequences also has the functional capability to target transgenic expression to arcuate POMC neurons, we constructed a transgene in which the region between −11 and −8 kb of the human POMC gene (containing both nPE regions [Fig. ]) was ligated upstream of the minimal promoter of the chicken β-globin gene followed by the lacZ reporter gene (transgene 13 [Fig. ]). β-Galactosidase (β-Gal) activity was visualized in situ followed by immunohistochemistry against ACTH in coronal brain sections of transgenic mice from three independent pedigrees. Interestingly, X-Gal blue staining was evident in POMC-positive arcuate neurons (Fig. ), although the percentage of colocalization was highly variable among the three pedigrees analyzed (10, 68, and 95%). No reporter expression was detected in pituitary melanotrophs or corticotrophs (Fig. ). Different levels of ectopic β-Gal expression were observed in the brain of each transgenic pedigree in regions that included the amygdala, the habenula, the hippocampus, the cerebral cortex; the lateral, ventromedial, and arcuate nuclei of the hypothalamus; and some cells of the neural lobe of the pituitary that do not express Pomc (Fig. ). These results, together with those obtained from transgene 5 (see above), indicate that neuronal POMC and pituitary POMC gene expression are controlled by topographically distinct and functionally independent regulatory regions. In addition, the ability of the human genomic fragment containing nPE1 and nPE2 to target reporter expression to mouse POMC arcuate neurons suggests that these conserved sequences play a role in the expression of POMC in the human hypothalamus.
FIG. 4. Conservation of human POMC enhancer function. (A) Scheme of transgene 13. The region between kb −11 and −8 of the human POMC gene was ligated upstream of the minimal promoter of chick β-globin and the lacZ transgene. nPE regions (more ...) nPE1 and nPE2 are necessary to direct transgene expression to POMC arcuate neurons.
Within the region spanning kb −13 to −9 of the mouse Pomc promoter, nPE1 and nPE2 are the only nonrepetitive sequences that have been highly conserved during mammalian evolution. This conservation suggests that these sites may carry the necessary information to assure neuronal-specific expression of the POMC gene. To challenge this hypothesis, we constructed transgenic mice carrying deleted versions of the original POMC-EGFP transgene from kb −13 to +8 in which nPE1, nPE2, or both enhancers were deleted (transgenes 7, 8, and 9) (Fig. ). EGFP expression and colocalization with POMC were determined in brain and pituitary slices of transgenic mice. Transgenic mice lacking either nPE1 (construct 7) or nPE2 (construct 8) reproducibly targeted EGFP expression to over 90% of POMC-expressing neurons in the arcuate nucleus (Fig. ), demonstrating that the absence of either one of these elements was not sufficient to disrupt neuronal-specific expression. A truncation from kb −13 to −10.5 that removed nPE1, together with neighboring sequences (transgene 6) (Fig. ), also directed eutopic expression of EGFP in POMC arcuate neurons (data not shown), confirming that nPE1 is not absolutely necessary to guarantee neuronal Pomc gene expression. Nevertheless, the simultaneous deletion of nPE1 and nPE2 completely eliminated EGFP expression in POMC neurons of the arcuate in five independent transgenic lines (Fig. ). All transgenic lines carrying constructs 6, 7, 8, or 9 displayed EGFP expression in pituitary melanotrophs and corticotrophs (Fig. ), confirming that Pomc expression in the brain and pituitary gland is controlled by independent enhancers. Together, these results indicate that nPE1 and nPE2 function as neuronal transcriptional enhancers that are necessary for Pomc expression in arcuate neurons. In the absence of either one of these enhancers, the remaining element is still able to assure transgene expression in POMC neurons of the arcuate nucleus, indicating a functional redundancy between nPE1 and nPE2.
Second set of transgenes used in this study. nPE enhancers (gray boxes) were deleted from parental transgenes 1 and 5. Details are as described for Fig. .
FIG. 6. nPE enhancers are necessary for POMC expression in the hypothalamus. (A through C) EGFP immunostainings performed on coronal hypothalamic sections of transgene 7 (panel A), 8 (panel B), and 9 (panel C) mice. Insets in panels A and B show colocalization (more ...)
To confirm the importance of these enhancers in directing reporter expression to POMC arcuate neurons, we introduced deletions of nPE1, nPE2, or both elements simultaneously into the original transgene 5 in which a minimal viral TK promoter was fused to the reporter gene hGH (transgenes 10, 11, and 12 [Fig. ]). As observed with the previous set of larger transgenes, deletion of either nPE1 or nPE2 alone did not prevent expression of reporter hGH in the arcuate POMC neurons (results not shown). However, the simultaneous deletion of both nPE1 and nPE2 completely abolished hGH expression in the POMC arcuate neurons of five independent lines of transgenic mice as assessed by anti-hGH immunohistochemistry (Fig. ). Transgenic mice carrying constructs 10, 11, and 12 did not show reporter gene expression in pituitary melanotrophs as predicted, but there was variable ectopic expression in the habenula, cerebral cortex, and hippocampus in all lines, showing that the transgenes were not inserted into transcriptionally silent regions (Fig. , inset). These results further demonstrate that nPE1 and nPE2 are responsible for the neuronal enhancer activity that we identified in the fragment from kb −13 to −9 of the mouse Pomc 5′ flanking region.
nPE1 and nPE2 are conserved in mammals but not in other vertebrates.
Since the POMC
gene is expressed in the ventromedial hypothalamic neurons of all vertebrate species studied to date, we investigated whether the neuronal regulatory elements nPE1 and nPE2 are conserved in different vertebrate orders. We searched the sequenced POMC
loci from the completed genome projects of rat, chimpanzee, and chicken and the teleost fishes pufferfish (T. rubripes
) and zebrafish (Danio rerio
), as well as the ongoing genome projects of the dog and the frog X. tropicalis
(see Materials and Methods). Figure shows a multiple percentage-identity plot (PIP) (53
) comparing the human POMC
locus to the complete mouse, rat, chimpanzee, chicken, frog, pufferfish and zebra fish POMC
loci, as well as to a partial dog sequence. All mammalian sequences showed extensive identity to the human POMC
locus, including several conserved noncoding blocks. Among these, nPE1 and nPE2 displayed a particularly greater degree of conservation, evidenced by long stretches and a high percentage of sequence identity relative to those of other less conserved intergenic or intronic regions. The chimpanzee POMC
locus was almost 100% identical to the human locus in coding and noncoding regions, as expected given the close evolutionary distance between these two primate species. In contrast, nonmammalian POMC
genes did not show significant blocks of sequence identity to the human POMC
locus, except for exons 2 and 3 (chicken and Xenopus
) and exon 3 (fishes). These data show that both nPE1 and nPE2 are strongly conserved among mammals but cannot be recognized in birds, amphibians, or teleost fishes. Figure shows a multiple alignment of nPE1 and nPE2 of four different mammals, including partial nPE sequences that we amplified by PCR from a bovine genomic phage clone containing the POMC
gene. The alignment shows several blocks of high sequence identity, which might indicate possible binding sites for transcription factors. nPE1 had an overall mouse and human similarity of 76%, with a 40-bp core of 100% identity, whereas mouse and human nPE2 had 90% similarity (138 bp out of 153 bp are identical). Interestingly, the similarities between mouse and human exons 1, 2, and 3 of 64, 87, and 82%, respectively, were not higher than the interspecies identity for nPE1 and nPE2.
FIG. 7. Conservation of POMC neuronal enhancers. (A) Comparison of the human POMC gene to several species by using the program PipMaker. The human POMC gene including 12 kb of the 5′ sequence was compared to equivalent regions of the mouse (Mus musculus (more ...)
A further comparison of human, mouse, dog, and cow nPE1 and nPE2 sequences by using the recently developed rVista version 2.0 bioinformatics tool (37
) allowed the detection of a limited number of conserved and aligned transcription factor binding sites that could determine the transcriptional regulation of Pomc
in arcuate neurons (Fig. ). Within nPE1, we identified one site that carried two adjacent consensus sequences for the POU domain transcription factors Brn 4.0 and OCT-1. Immediately downstream of this site was a consensus sequence for Elf-2, a member of the Ets transcription factor family. Interestingly, a sequence present at the 3′ end of nPE1 highly matched a consensus binding site for signal transducer and activator of transcription 3 (STAT3), a downstream effector of leptin known to upregulate Pomc
transcription in hypothalamic neurons. In addition, two conserved cAMP-responsive element binding (CREB-like) sites were present within nPE1.
Within nPE2, we identified a sequence that showed high similarity to the consensus binding site for the homeobox gene Nkx6.1 and the POU domain gene Brn2.0. Contiguous to this site, there is a sequence that carries overlapping matches for chicken ovoalbumin upstream promoter (COUP) and estrogen-related receptor alpha (ERRα), two transcription factors that belong to the orphan nuclear receptor family. Another canonical ERRα site was located further downstream.