DNMT1-Interacting RNAs Block Gene-Specific DNA Methylation

5-methylcytosine is found in the context of paired symmetrical methylation of a CpG site. In normal somatic cells, most CpG sites are heavily methylated, while CpG islands (sites of CpG clusters) in germ-line tissues and located near promoters, remain unmethylated thus allowing gene expression to occur.  When a CpG island in the promoter region of a gene is methylated, expression of the gene is repressed. The addition of methyl groups is controlled at several different levels in cells and is carried out by a family of enzymes called DNA methyltransferases (DNMTs).

Three DNMTs are required for establishment and maintenance of DNA methylation patterns. DNMT1 is responsible for the maintenance of DNA methylation patterns, while DNMT3a and 3b mediate establishment of new or de novo DNA methylation patterns. Even with this knowledge about DNMT function, questions still remain about how DNA methylation is altered.

Researchers at Harvard Stem Cell Institute, Beth Israel Deaconess Medical Center and Universita Cattolica del Sacro Cuore-Institute of Hematology, along with other collaborators sought to address this question by examining how transcription may be involved in regulating DNA methylation. In particular, they investigated the dynamics between DNMT1 and non-coding RNAs using the methylation sensitive gene CEBPA.

Non-coding RNAs (ncRNAs) are functional RNA molecules that are transcribed from DNA but are not translated into proteins. In general ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level.  Many long non-coding RNAs (lncRNAs) can complex with chromatin-modifying proteins and recruit their catalytic activity to specific sites in the genome, thereby modifying chromatin states and influencing gene expression.

The authors identified a functional ncRNA transcribed from the CEBPA locus, ecCEBPA (extra coding CEBPA), which regulates CEBPA methylation by interacting with DNMT1 thus preventing CEBPA gene methylation.

Their findings are summarized below:

• ecCEBPA knockdown led to a decrease of CEBPA mRNA expression and to a significant increase in DNA methylation.
• Overexpression of the downstream region of ecCEBPA resulted in a greater-than-threefold increase in mRNA expression and coincided with a decrease of DNA methylation in three regions of the CEBPA gene. Using reduced representation bisulphite sequencing (RRBS), these modifications were found to be specific for the CEBPA locus (and not other gene loci).  Their results also suggest that this may be a cell cycle-specific action during S phase of CEBPA synthesis.
• ecCEBPA directly interacts with DNMT1. ecCEBPA is expressed as a ~4.5 kb non-polyadenylated transcript and is enriched in the nuclear fraction suggesting a functional role. RIP (RNA immunoprecipitation) analysis using a DNMT1 antibody showed an enrichment of CEBPA transcripts in non-polyadenylated fractions compared to polyadenylated fractions.
• DNMT1 has a stronger affinity for RNA than DNA as indicated by a lower dissociation constant (K_d) for RNA than for DNA. This association was found to be dependent on the formation of RNA secondary structures.
• DNMT1 interacting RNAs (DiRs) that may regulate DNA methylation are widespread and associated with a multitude of biological processes.

Source: Read more about their findings and get all of the details here: Di Ruscio A., Ebralidze AK., et al, DNMT1-interacting RNAs Block Gene-Specific DNA Methylation, Nature (2013, Oct 9).

References: [1] Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115, USA [3] Università Cattolica del Sacro Cuore, Institute of Hematology, L.go A. Gemelli 8, Rome 00168, Italy [4].