ABSTRACT
Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2-H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for 'transcription therapy' of human disease.
Subject(s)
Genetic Therapy/methods , Genomic Imprinting , Insulin-Like Growth Factor II/genetics , Neoplasms/therapy , Zinc Fingers , Beckwith-Wiedemann Syndrome/therapy , Female , Gene Expression Regulation , Gene Targeting/methods , Genes, Tumor Suppressor , Genetic Engineering , Humans , Kidney Neoplasms/therapy , Male , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Wilms Tumor/therapyABSTRACT
BACKGROUND: During mouse prenatal development, the neighbouring insulin-like growth factor II (Igf2) and H19 loci are expressed monoallelically from the paternal and maternal alleles, respectively. Identical spatiotemporal expression patterns and enhancer deletion experiments show that the Igf2 and H19 genes share a common set of enhancers. Deletion of a differentially methylated region in the 5' flank of the H19 gene partially relieves the repression of the maternal Igf2 and paternal H19 alleles in the soma. The mechanisms underlying the function of the 5' flank of the H19 gene are, however, unknown. RESULTS: Chromatin analysis showed that the 5' flank of the mouse H19 gene contains maternal-specific, multiple nuclease hypersensitive sites that map to linker regions between positioned nucleosomes. These features could be recapitulated in an episomal-based H19 minigene, which was propagated in human somatic cells. Although the 5' flank of the H19 promoter has no intrinsic silencer activity under these conditions, it unidirectionally extinguished promoter-enhancer communications in a position-dependent manner, without directly affecting the enhancer function. CONCLUSIONS: The unmethylated 5' flank of the H19 gene adopts an unusual and maternal-specific chromatin conformation in somatic cells and regulates enhancer-promoter communications, thereby providing an explanation for its role in manifesting the repressed state of the maternally inherited Igf2 allele.
Subject(s)
Chromatin/chemistry , Enhancer Elements, Genetic , Insulin-Like Growth Factor II/genetics , Muscle Proteins/genetics , Promoter Regions, Genetic , RNA, Untranslated , Alleles , Animals , Blotting, Southern , Cell Line , Female , Fetus , Humans , Male , Mice , Muscle Proteins/metabolism , Plasmids , Polymerase Chain Reaction , Protein Conformation , RNA, Long Noncoding , Terminal Repeat Sequences , Transformation, GeneticABSTRACT
The major growth factors in bovine colostrum are transforming growth factor-beta s (TGF-beta 1 and TGF-beta 2) and insulin-like growth factors (IGF-1 and IGF-2). Recently, TGF-beta 2 content of bovine colostrum was measured using a TGF-beta 2 specific ELISA (1) and now we have validated ELISAs for for bovine TGF-beta 1 and IGF-1. The concentrations of IGF-1 and TGF-beta 1 in the first milking after calving were 248-1850 ng/ml and 12.4-42.6 ng/ml, respectively, and they declined in correlation with total protein concentration to 27.0-101 ng/ml (IGF-1) and 0.80-3.49 ng/ml(TGF-beta 1) by the fifth milkings. The amount of TGF-beta 1 was on average 5.3 +/- 1.4% of that of TGF-beta 2 and there is a high correlation (r = 0.966) between the concentrations of these growth factors in the same samples. No free TGF-beta 1 form of could be detected.
