DNA Methylation Precedes Chromatin Modifications under the Influence of the Strain-Specific Modifier Ssm1

Abstract

Ssm1 is responsible for the mouse strain-specific DNA methylation of the transgene HRD. In adult mice of the C57BL/6 (B6) strain, the transgene is methylated at essentially all CpGs. However, when the transgene is bred into the DBA/2 (D2) strain, it is almost completely unmethylated. Strain-specific methylation arises during differentiation of embryonic stem (ES) cells. Here we show that Ssm1 causes striking chromatin changes during the development of the early embryo in both strains. In undifferentiated ES cells of both strains, the transgene is in a chromatin state between active and inactive. These states are still observed 1 week after beginning ES cell differentiation. However, 4 weeks after initiating differentiation, in B6, the transgene has become heterochromatic, and in D2, the transgene has become euchromatic. HRD is always expressed in D2, but in B6, it is expressed only in early embryos. The transgene is already more methylated in B6 ES cells than in D2 ES cells and becomes increasingly methylated during development in B6, until essentially all CpGs in the critical guanosine phosphoribosyl transferase core are methylated. Clearly, DNA methylation of HRD precedes chromatin compaction and loss of expression, suggesting that the B6 form of Ssm1 interacts with DNA to cause strain-specific methylation that ultimately results in inactive chromatin.

Identification of Ssm1b, a novel modifier of DNA methylation, and its expression during mouse embryogenesis

“…The HRD transgene is in active chromatin in Ssm1d adults, but heterochromatic in adult Ssm1b mice (Padjen et al, 2005). In undifferentiated ESCs of both strains, the transgene is in a chromatin state intermediate between active and inactive.…”

“…This intermediate state is still observed in both B6 and D2 ESCs 1 week after removal of leukemia inhibitory factor (LIF) and feeder fibroblasts, except that in B6 the HRD transgene becomes associated with the methylated DNA-binding protein Mecp2. After differentiation in culture, in B6 ESCs HRD is heterochromatic, whereas in D2 the HRD transgene assumes an active chromatin state (Padjen et al, 2005). HRD transgenic RNA is expressed in D2 in all stages, but in B6 only in undifferentiated ESCs and during the first 3.5 days after differentiation in culture.…”

“…When HRD is carried in certain inbred strains of mice (Mus musculus), such as C57BL/6 (B6), it is highly methylated at CpG nucleotides. HRD is, however, unmethylated in other strains, such as DBA/2 (D2) (Engler et al, 1991;Weng et al, 1995;Padjen et al, 2005). Unmethylated HRD transgenes are transcribed and undergo V(D)J recombination (Engler and Storb, 1999).…”

“…However, for de novo methylation, a targeting mechanism must exist. The strain-specific modifier Ssm1 is a candidate for a novel targeting factor as it causes specific gene silencing via DNA methylation and chromatin compaction (Padjen et al, 2005). …”

Gene-Environment Interactions and Epigenetic Basis of Human Diseases

“…In addition, direct binding of DNMT3a and DNMT1 to HDAC1 was observed (Fuks et al, 2000;Fuks et al, 2001), and histone methylation is also coupled with methylated human Alu elements (Kondo and Issa, 2003). Although many studies have suggested that DNA methylation may act as a primary player in silencing target genes preceding other epigenetic pathways (Hashimshony et al, 2003;Padjen et al, 2005;Schubeler et al, 2000), there are also scenarios where DNA methylation seems to act as a secondary event to stabilize gene silencing status initiated by existing chromatin modifications (Bachman et al, 2003;Mutskov and Felsenfeld, 2004;Tamaru and Selker, 2001). Studies on the kinetics of silencing of transgenes show that loss of histone acetylation and H3-K4 methylation is the first step leading to reversible transcriptional repression, which is followed by H3-K9 methylation and promoter DNA methylation to stabilize the silenced chromatin state (Mutskov and Felsenfeld, 2004).…”