Research report
The melanin-concentrating hormone gene in human: flanking region analysis, fine chromosome mapping, and tissue-specific expression

https://doi.org/10.1016/S0169-328X(97)00018-1Get rights and content

Abstract

Genomic sequences encoding the human melanin-concentrating hormone (MCH) were isolated from a YAC library and subcloned in pUC vector using a novel E. coli transformation method. A 4.1-kb fragment encompassing ≈1.0 kb of the 5′-end-flanking region, the three exons–two introns of the coding region and ≈1.7 kb of the 3′-end-flanking region, was sequenced. Comparison with the rat MCH gene indicated strong conservation in the 5′-flanking region, in particular over the putative TATA box, CAAT box, GRE and AP-1 elements that could potentially regulate MCH gene expression. FISH with a fluorescent MCH genomic probe on human chromosomes and PCR analysis of a YAC panel mapped MCH to chromosome 12q23.1 in a region flanked by D12S1074 and D12S1030 markers. Expression of the MCH RNA species and pro-MCH-derived peptides (MCH and NEI) was investigated in human tissues by combining Northern blotting, RT-PCR, in situ hybridization, immunohistochemistry and RIA. In the human brain, MCH mRNA and MCH/NEI peptides were predominantely expressed in the lateral hypothalamus in agreement with the known distribution of MCH expression in rat. In addition, MCH gene products were detected in extra-hypothalamic sites, such as the pallidum, neocortex and cerebellum. In peripheral tissues, MCH mRNA was identified in several organs, including the thymus, brown adipose tissue, duodenum and testis. An additional shorter MCH gene transcript, likely the result of alternate splicing, was revealed in several brain areas and peripheral tissues. While only fully processed MCH and NEI were found in hypothalamus, a different peptide form, bearing MCH and NEI epitopes, was detected in peripheral organs. This represents the first evidence for differential processing of pro-MCH in mammals. © 1997 Elsevier Science B.V. All rights reserved.

Introduction

Melanin-concentrating hormone (MCH) is a cyclic peptide originally isolated from salmon pituitary gland [25]and later from rat and human hypothalami 32, 52. Mammalian MCH is encoded within a precursor of 165 amino acids which also gives rise to two other peptides, neuropeptide-glutamic acid-isoleucine (NEI) and neuropeptide-glycine-glutamic acid (NGE) 33, 34. Of these two peptides, only NEI has so far been isolated and characterized from rat hypothalamic cells [36]and peripheral tissues 22, 23.

The main sites of synthesis of MCH mRNA and derived peptides in rat brain are the lateral hypothalamus area (LHA) and neighboring zona incerta (ZI) [4]. The same locations for MCH perikarya were found in other mammalian species, including man 1, 33. The restricted localization of neurons expressing pro-MCH-derived peptides in the hypothalamus together with a widespread distribution of fibers throughout the brain strongly suggests that the MCH networks represents an interneuronal system possibly involved in many neuronal functions. I.c.v. injections of MCH inhibit or activate food-intake behavior depending on the concentration of peptide used and/or rat strains studied 41, 42. In addition, MCH and NEI act as functional antagonists in the activation of the hypothalamus-pituitary-adrenal gland axis after an acute stress [5]. Both MCH and NEI induce secretion of oxytocin in the neurohypophysis [36]. Centrally, MCH was shown to antagonize α-MSH on grooming behavior [14], on sensorimotor integration [30], on learning acquisition [29]or on aggression and anxiety [17]. All of these data are consistent with the down-regulation of MCH gene activity observed after chronic stress [38]or osmotic stress [39]. Peripherally, MCH and associated peptides predominate in the gastrointestinal and genital tracts where they appear to act as paracrine or autocrine factors 21, 22, 23.

The exon–intron structure of the MCH genes was established after PCR amplification of genomic DNA from the rat, mouse and human [7]. The rat MCH gene has been also cloned following screening of a genomic library but only 380 nucleotides upstream of the transcriptional initiation site were published and no regulatory element was mapped [50]. Strikingly, a single MCH gene was identified in rodents while two distinct gene systems were discovered in human, one corresponding to the authentic gene and highly homologous to its rat and mouse counterparts [7]and the other defined as the variant gene on the basis of partial sequence homology with the authentic gene [8]. The human authentic gene (PMCH locus) was mapped to 12q23–q24 and two copies of the variant gene, designed PMCHL1 and PMCHL2, were located on chromosome 5p14 and 5q12–q13, respectively [37]. The authentic MCH gene might be involved in human neural disorders, such as Darier's disease [12]or the spinocerebellar ataxia type 2 locus (SCA 2) [16].

In order to initiate genetic linkage studies and to further investigate the role and regulation of MCH and associated peptides in human complex behaviors, such as food-intake behavior, it is necessary to isolate the 5′- and 3′-flanking regions of the MCH gene and to identify the products of MCH gene transcription and MCH mRNA translation. In this article, we have established the genomic organization of the human authentic MCH gene and identified several regulatory elements which might be involved in its regulation. In addition, MCH RNA species were identified in unexpected sites of the human brain and in several peripheral organs as well. Finally, a particular processing of pro-MCH was found in peripheral tissues of human neonates and adults.

Section snippets

YAC library screening

YAC libraries from the CEPH (Paris, France) were screened by PCR using 30 pmol of two MCH-specific primers: HMCH2 (5′ CCA GCA AGA TTG GTA GAC TCT TCC CAG CAT 3′) and HMCH6 (5′ TTC TTA AAT CAT GGT CTG CCA CTG AAT CTG 3′) in a final volume of 30 μl containing 0.2 U of Taq polymerase (Appligene) and 0.8 mM dNTPs. Amplification conditions were as follows: denaturation at 94°C, annealing at 52°C and extention at 72°C for 1 min each during 30 cycles. The PCR products were separated on a 0.8% agarose

Cloning of the coding and flanking parts of the human MCH gene

Isolation of the MCH gene was achieved by screening a YAC library. Two YAC clones, 908f7 and 886a6, were clearly positive after DNA amplification with MCH gene-specific primers, viz. HPCR3 and HPCR4 (Fig. 1B). A BamHI/XhoI fragment of ≈8.9 kb in length and a XbaI fragment of ≈3.3 kb in length were then subcloned in pUC19 vector by using a modified procedure, including ethidium bromide treatment before transformation (Zhixing and Nahon, submitted). Several bacterial colonies were selected and

Discussion

In this paper, we report the isolation and sequencing of a clone containing ≈4.0 kb of genomic DNA encompassing the human MCH gene. This was a long-standing task which was only solved by screening YAC contigs from the CEPH (Paris, France) and by using a novel subcloning technique characterized by incubating ethidium bromide with recombinant plasmid before bacteria transformation. When phage libraries were grown on standard bacterial host strains or with the host strain MB408, designed to allow

Acknowledgements

We are grateful to Drs. A. Beaudet (McGill University, Montreal, Quebec, Canada), P. Kitabgi and F. Presse (IPMC, Valbonne, France) for critical reading of the manuscript and to Dr. J. Grosgeorge (LGMCH, Nice, France) for technical assistance. We thank very much Drs. D. Le Paslier (CEPH, Paris, France), P. Vernier (Institut A. Fessard, Gif-sur-Yvette, France) and F. Agid (Hôpital Pitié-Salpétrière, Paris, France) for the generous gift of the YAC library, human RNA and tissues samples,

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    1

    Present address: Department of Biology, Beijing Normal University, Beijing 100875, P.R. China.

    2

    Present address: Centre CNRS-INSERM de Pharmacologie-Endocrinologie, 141 rue de la Cardonille, 34094 Montpellier, France.

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