A novel in vitro system for analyzing parental allele-specific histone acetylation in genomic imprinting

Abstract

One of the obstacles in studying human genomic imprinting is distinguishing the parental origin of alleles in diploid cells. To solve this problem, we have constructed a library of mouse A9 hybrids in which individual clones contain a single human chromosome of known parental origin. Here we extend this in vitro system to the analysis of the role of histone acetylation in the allelic expression of human imprinted genes. The levels of histone H4 acetylation of the imprinted human LIT1, H19, and SNRPN genes were examined by a chromatin immunoprecipitation (ChIP) assay in mouse A9 hybrids with a single human chromosome of known parental origin. We demonstrated that H4 histones associated with the actively expressed alleles of imprinted LIT1, H19, and SNRPN genes were highly acetylated, whereas they were hypoacetylated in the silent alleles. Furthermore, treatment of A9 hybrids with trichostatin A (TSA), an inhibitor of histone deacetylase, resulted in transcriptional reactivation of the silent alleles for LIT1 and SNRPN, suggesting that histone deacetylation is one of the key regulatory mechanisms in genomic imprinting. These results indicate that our monochromosomal hybrid system is a new technology for analyzing histone modifications between parental alleles in human imprinted genes.

Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation

“…In the analysis of human genomic imprinting, polymorphisms are usually required to distinguish the parental origin of alleles. In the present study, we utilized monochromosomal A9 hybrids (20,33,34) to analyze parental allele-specific histone H3 acetylation at lysine 9/18 and histone H3 methylation at lysine 9 in the imprinted ARHI gene. The expressed paternal allele showed much higher level of histone H3-K9/18 acetylation than the silenced maternal allele on all three CpG islands.…”

Suberoylanilide Hydroxamic Acid, a Histone Deacetylase Inhibitor: Effects on Gene Expression and Growth of Glioma Cells In vitro and In vivo

“…Cells used in these experiments were subcultured for no more than 10 additional passages. All cells were incubated at 37jC in 5% CO 2 .…”

“…It is essential for the normal development of mammals and plays an important physiologic role (1,2). Histone deacetylation is mediated by histone deacetylases (HDAC).…”

The manipulation of chromosomes by mankind: the uses of microcell-mediated chromosome transfer

“…Further, Mitsuya et al (1999) were able to define expression and methylation differences for specific imprinted loci and also assess the abnormalities in chromosomes derived from patients with disease associated with imprinted loci. These uniparental monochromosome libraries have also permitted the identification of previously unknown imprinted genes (Arima et al 2000;Okita et al 2003) and the analysis of the histone acetylation status of individual genes from a single chromosome (Yoshioka et al 2001). Indeed, construction of monochromosome hybrids has even helped in the diagnosis of specific syndromes (Coveler et al 2002).…”