Elsevier

Brain Research

Volume 810, Issues 1–2, 9 November 1998, Pages 1-8
Brain Research

Research report
Dissociation of IGF2 and H19 imprinting in human brain

https://doi.org/10.1016/S0006-8993(98)00783-5Get rights and content

Abstract

The human IGF2 and H19 genes are imprinted in most normal tissues. Alterations of genomic imprinting or loss of imprinting (LOI) have been observed in a number of malignant tumors. Although LOI has been linked to tumorigenesis, loss of IGF2 imprinting has also been observed in choroid plexus and leptomeninges in normal mouse brain. We have therefore analyzed the allelic expression of both IGF2 and H19 in human fetal brain and in different regions of human adult brain. In the brains of fetuses of 6–12 weeks gestation, both IGF2 and H19 were transcribed from both parental alleles. In contrast, strictly monoallelic expression of both IGF2 and H19 was observed in all other fetal tissues, suggesting a tissue-specific LOI in the central nervous system. In adult brain, LOI of IGF2 was region-specific. IGF2 was expressed from both parental alleles in the pons, but not in globus palludus, Raphe nucleus and hypothalamus. H19 expression was drastically reduced in adult brain compared to fetal brain, and was detectable only in the pons and globus palludus. In contrast to IGF2, the expression of H19 in adult pons was monoallelic. Examination of IGF2 promoter usage indicated predominant utilization of promoter P3 in all fetal and adult brain tissues. The LOI of IGF2 therefore reflects biallelic expression from the predominant promoter. IGF2 transcripts derived from the less abundant promoter P1, however, showed monoallelic expression in the adult pons. Our results suggest that IGF2 and H19 undergo ontogenetic changes in allelic expression and that there is dissociation of IGF2 and H19 imprinting in both fetal and adult human brain.

Introduction

Insulin-like growth factor-II (IGF-II) is a mitogenic peptide which is crucial for normal fetal growth [5]. IGF-II is synthesized in most tissues, and while its production declines rapidly in rodents soon after birth, in humans, it circulates in relatively high concentrations throughout life. Several species of IGF-II peptide have been described in human CSF and brain: an abundant 7.5 kDa peptide identical to the circulating form of IGF-II, and larger species which may represent pro-IGF-II and an IGF-II variant 11, 38. IGF-II is found throughout the human brain, with especially high levels found in the hypothalamus, the thalamus, mammillary bodies, fornix, and especially in the anterior and posterior pituitary [12]. Recent experiments have shown that IGF-II is an important neurotropic factor in the peripheral nervous system 22, 37and a neuroprotective factor in the CNS [4].

In the classic study demonstrating that Igf2 is an imprinted gene, transcribed only from the paternal allele, DeChiara et al. showed that Igf2 was expressed from both parental alleles in the leptomeninges [6]. Subsequent work has shown that Igf2 is also biallelically expressed in both rat [25]and mouse CNS [14]. This tissue-specific lack of imprinting suggests that Igf2 may be more extensively expressed in the CNS, since transcription can occur from both parental alleles. H19 is located downstream from IGF2, and it is paternally imprinted [36]. The gene contains no open reading frame, and it is believed that it codes for an RNA which has regulatory activity [10]. In many systems, IGF2 and H19 are reciprocally imprinted [23]. Since IGF-II may play an important role in CNS development and adult neurophysiology, we have examined the allelic expression of IGF2 and H19 in human fetal and adult brains.

Section snippets

Human tissues

Frozen adult brain specimens from a normal control group were obtained from the National Neurological Research Specimen Bank, VAMC, Los Angeles. Fresh frozen tissues from normal fetuses of 6–12 weeks of gestation were obtained from the Central Laboratory for Human Embryology Tissues, University of Washington, Seattle.

Nucleic acid extraction

Total nucleic acid (TNA), DNA and RNA were extracted from frozen specimens as described previously [32].

Polymerase chain reaction

PCR, reverse transcriptase PCR and multiplex PCR assays for promoter usage

Results

Although IGF2 and H19 are expressed abundantly in various fetal tissues, the level of expression of both genes is reduced in adult tissues, with the exception of persistent IGF2 expression in human adult liver. Northern blotting and ribonuclease protection assays (RPA) are established techniques to quantitate relative levels of mRNA expression in various tissues. However due to the limitation of the detection sensitivity of Northern blots and the limited amount of human CNS mRNA available for

Discussion

In this study, we have shown that IGF2 was transcribed predominantly from promoter P3 in both fetal and adult brain. This is in contrast to adult liver, the major site of IGF2 production, where IGF2 is transcribed from promoter P1 and is therefore not imprinted [32]. In all fetal tissues that we examined, IGF2 was expressed from a single parental allele, except for brain where IGF2 was always biallelically expressed, providing an exception to the rule that only P1 normally directs transcription

Acknowledgements

We thank the National Neurological Research Specimen Bank, VAMC, Los Angeles, CA 90073 for adult human CNS specimens and the Central Laboratory for Human Embryology Tissue, University of Washington, Seattle, for fetal tissues.

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