Maintenance of genomic imprinting by G9a/GLP complex of histone methyltransferases in embryonic stem (ES) cells
Abstract
DNA methylation refers to an addition of a methyl group to the 5 position of
the cytosine pyrimidine ring. As the best characterized epigenetic mark, DNA
methylation plays an important role in a plethora of biological functions, including
gene repression, genomic imprinting, silencing of retro-transposons and X
chromosome inactivation. Genomic imprinting refers to the mono-allelic expression
of certain genes according to their parent-of-origin. In mammals, the expression of
imprinted genes is controlled by the cis-acting regulatory elements, termed imprinted
control regions (ICRs). ICRs are marked by parent-of-origin-specific DNA
methylation and loss of DNA methylation at ICRs also causes aberrant expression of
imprinted genes. Therefore it is believed that the genomic imprinting is a DNA
methylation-associated epigenetic phenomenon. As accurate expression of imprinted
genes is essential for normal embryonic growth, energy homeostasis, development
of the brain and behaviour and abnormal expression of imprinted genes leads to
numerous clinical phenotype and human disorders, it is important to investigate how
the imprinted DNA methylation is stably maintained in mammals.
DNA methyltransferases (DNMTs) are the main enzymes that play a in the
establishment and maintenance of imprinted DNA methylation. In primordial germ
cells (PGCs), DNMT3A and DNMT3L are involved in the establishment of
imprinted DNA methylation. Whereas once established, the imprinted DNA
methylation is maintained by DNMT1, DNMT3A and DNMT3B, but mainly by
DNMT1. In addition, some other enzymes and DNA binding proteins also play a
role in this process. One of the best examples is ZFP57, which forms a complex with
KAP1 and SETDB1. ZFP57 maintains imprinted DNA methylation by recognizing a
methylated hexa-nucleotide and recruits DNMTs to the ICRs in mammalian
embryonic stem (ES) cells. Interestingly, DNA methylation analysis combined with
promoter microarrays carried out in our lab suggested that imprinted DNA
methylation is absent from some of the maternal ICRs in ES cells genetically null for
G9a, a histone H3 lysine 9 methylase. This indicates that G9a might also play a role
in the maintenance of imprinted DNA methylation.
In my work, I found that the repressive H3K9me2 and imprinted DNA
methylation are absent from several analysed ICRs in embryonic stem (ES) cells
genetically null for either G9a or its partner histone methyltransferase GLP. A
knockdown of G9a in ES cells reproduced these observations suggesting that
G9a/GLP complex is required for the maintenance of imprinted DNA methylation. I
also found that neither wild type nor catalytically inactive G9a can restore the loss of
imprinted DNA methylation in G9a-/- ES cells. Chromatin immunoprecipitation
(ChIP) combined with bisulfite DNA sequencing showed that imprinted DNA
methylation was present on the H3K9me2-marked allele indicating a direct role for
G9a in maintenance of genomic imprinting. Using a pharmacological inhibitor of
G9a and mutagenesis analyses, I found that G9a maintains the imprinted DNA
methylation independently of its catalytic activity and recruits DNMTs to the ICRs
via its ankyrin repeat domain. Dimerization of G9a with GLP is also essential for the
maintenance of genomic imprinting in ES cells. In summary, in addition to establish
H3K9me2, histone methyltransferases G9a and GLP also play an essential role in the
maintenance of genomic methylation imprints in ES cells.