Histone Modification Analysis at Imprinted Genes in Mice

Posted May 12th, 2010 at 3:13 pm.

Nelly Khaselev
Mentor: Dr. Tamara Davis

Imprinted genes are genes whose expression is determined by parent of origin. Cells contain two alleles (different copies of a single gene), one from each parent, and for the majority of genes, both alleles are equally expressed. On the contrary, a small number of imprinted genes only express one allele: either the paternally or maternally inherited copy. This research aims to progress towards understanding the mechanism of this type of gene regulation.

In this laboratory, we are focusing on the study of histones, which are essential proteins associated with DNA in chromatin. Histones act as spools which wrap DNA in order to condense it and help regulate gene expression. We believe that histone modification, such as acetylation and methylation, is a key process in epigenetic regulation. The main question we are investigating is: are there different histone modifications associated with the expressed allele versus the silent allele of imprinted genes. To address this question we will be looking at Rasgrf1 on mouse chromosome 9, which is only paternally expressed in some tissues and biallelically expressed in other tissues.

Our research is delving into a new method of analysis which will focus on two types of histone modification: acetylation and methylation. Acetylation is the addition of an acetyl group to lysine, an amino acid, which removes positive charge and loosens the histones affinity for DNA. This type of histone modification is usually associated with active DNA transcription. Methylation has the opposite effect on histones. A methyl group is added to certain amino acids and increases the histones affinity for DNA and is usually associated with transcriptionally silent DNA. We will be using a new technique called chromatin immunoprecipitation (ChIP) to analyze the histone modification on the Rasgrfl gene in various tissues. This method will hopefully allow us to see that repressive modifications are present on silent alleles whereas active modifications are present on expressed alleles.

The ChIP method uses antibodies that are targeted to specific modified histones to precipitate the protein-DNA complex that occur naturally in cells. The histones are cross linked to the DNA with the use of formaldehyde. Following cross linking of the DNA/histone complex, the DNA is fragmented apart via sonication - the use of high frequency sound to break up the DNA into small fragments. The antibodies are then used to precipitate the targeted DNA-protein complex. The process of linkage is then reversed and the DNA fragments are separated from the protein. The DNA fragments are than quantified and identified with the use of PCR. This complicated process ultimately results with a way to see a difference between the DNA-protein binding complexes in imprinted genes.

Filed under: 2009,Davis, Dr. Tamara,Khaselev, Nelly by Ann Dixon

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