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

Abstract

The strain-specific modifier Ssm1 is responsible for the straindependent methylation of particular E. coli gpt-containing transgenic sequences. Here, we identify Ssm1 as the KRAB-zinc finger (ZF) gene 2610305D13Rik located on distal chromosome 4. Ssm1b is a member of a gene family with an unusual array of three ZFs. Ssm1 family members in C57BL/6 (B6) and DBA/2 (D2) mice have various amino acid changes in their ZF domain and in the linker between the KRAB and ZF domains. Ssm1b is expressed up to E8.5; its target transgene gains partial methylation by this stage as well. At E9.5, Ssm1b mRNA is no longer expressed but by then its target has become completely methylated. By contrast, in D2 embryos the transgene is essentially unmethylated. Methylation during B6 embryonic development depends on Dnmt3b but not Mecp2. In differentiating B6 embryonic stem cells methylation spreads from gpt to a co-integrated neo gene that has a similarly high CpG content as gpt, but neo alone is not methylated. In adult B6 mice, Ssm1b is expressed in ovaries, but in other organs only other members of the Ssm1 family are expressed. Interestingly, the transgene becomes methylated when crossed into some, but not other, wild mice that were kept outbred in the laboratory. Thus, polymorphisms for the methylation patterns seen among laboratory inbred strains are also found in a free-living population. This may imply that mice that do not have the Ssm1b gene may use another member of the Ssm1 family to control the potentially harmful expression of certain endogenous or exogenous genes.

The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses

“…In conclusion, our findings add to a growing body of evidence that at least some KRAB-ZFPs, including ZFP809, Gm6871 (Castro-Diaz et al 2014), ZNF91, ZNF93 (Jacobs et al 2014), and Ssm1b (Ratnam et al 2014) recognize and repress foreign DNA in mammals. Our approach of using epitope-tagged KRAB-ZFPs for ChIP-seq combined with genetic deletions of KRAB-ZFPs will be a valuable tool to identify additional ERV targeting KRAB-ZFPs and in general lead to important insights into the function of this large, rapidly evolving, and enigmatic protein family.…”

Ssm1b expression and function in germ cells of adult mice and in early embryos

“…Ssm1b belongs to a family of Krüppel‐associated box domain (KRAB) zinc finger genes we call the Ssm1 family (Figure S3). The Ssm1 family of genes was originally identified by PCR amplification of cDNA from various stages of development (Ratnam et al, ). The primers that amplified Ssm1b mRNA also captured several related mRNAs containing single nucleotide polymorphisms (SNPs) that alter the amino acid sequence in the zing finger and linker regions, but not in the KRAB domain (see Figure in Ratnam et al, ).…”

“…The Ssm1 family of genes was originally identified by PCR amplification of cDNA from various stages of development (Ratnam et al, ). The primers that amplified Ssm1b mRNA also captured several related mRNAs containing single nucleotide polymorphisms (SNPs) that alter the amino acid sequence in the zing finger and linker regions, but not in the KRAB domain (see Figure in Ratnam et al, ). The zinc fingers in this family have an unusual organization: three functional zinc fingers interspersed with three non‐functional ones (Ratnam et al, ).…”

“…Ssm1b is expressed in the early mouse embryo, specifically at the blastocyst stage up to Embryonic Day 7.5 (E7.5) (Ratnam et al, ); HRD methylation concurrently increases at these stages. Thus, HRD methylation follows a similar methylation profile as the rest of the genome, where genome‐wide de novo methylation initiated at the blastocyst stage results in high and uniform methylation patterns on both alleles (Reik, Dean, & Walter, ).…”

“…Ssm1b‐dependent HRD methylation was originally identified in the C57BL6/J strain (Engler et al, ). A lack of HRD methylation also correlated with an absence of Ssm1b expression in the DBA/2J strain: when Ssm1b was introduced into a non‐methylating mouse strain (DBA/2J × C3H/HeJ) F1, HRD became methylated (Ratnam et al, ). Surprisingly, (C3H/HeJ × DBA/2J) F1 mice maintain HRD in an unmethylated state throughout development, yet embryonic stem (ES) cells derived from C3H/HeJ blastocysts gain HRD methylation upon differentiation.…”