Research reportDissociation of IGF2 and H19 imprinting in 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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