A two-edged role for the transposable element Kiddo in the rice ubiquitin2 promoter

Miniature inverted repeat transposable elements (MITEs) are thought to be a driving force for genome evolution. Although numerous MITEs are found associated with genes, little is known about their function in gene regulation. Whereas the rice ubiquitin2 (rubq2) promoter in rice (Oryza sativa) line IR24 contains two nested MITEs (Kiddo and MDM1), that in line T309 has lost Kiddo, providing an opportunity to understand the role of MITEs in promoter function. No difference in endogenous rubq2 transcript levels between T309 and IR24 was evident using RT-PCR. However, promoter analysis using both transient and stably transformed calli revealed that Kiddo contributed some 20% of the total expression. Bisulfite genomic sequencing of the rubq2 promoters revealed specific DNA methylation at both symmetric and asymmetric cytosine residues on the MITE sequences, possibly induced by low levels of homologous transcripts. When methylation of the MITEs was blocked by 5-azacytidine treatment, a threefold increase in the endogenous rubq2 transcript level was detected in IR24 compared with that in T309. Together with the observed MITE methylation pattern, the detection of low levels of transcripts, but not small RNAs, corresponding to Kiddo and MDM1 suggested that RNA-dependent DNA methylation is induced by MITE transcripts. We conclude that, although Kiddo enhances transcription from the rubq2 promoter, this effect is mitigated by sequence-specific epigenetic modification.