Abstract
Transposable elements, including short interspersed repetitive elements (SINEs), comprise nearly half the mammalian genome. Moreover, they are a major source of conserved non-coding elements (CNEs), which play important functional roles in regulating development-related genes, such as enhancing and silencing, serving for the diversification of morphological and physiological features among species. We previously reported a novel SINE family, AmnSINE1, as part of mammalian-specific CNEs. One AmnSINE1 locus, named AS071, showed an enhancer property in the developing mouse diencephalon. Indeed, AS071 appears to recapitulate the expression of diencephalic fibroblast growth factor 8 (Fgf8). Here we established three independent lines of AS071-transgenic mice and performed detailed expression profiling of AS071-enhanced lacZ in comparison with that of Fgf8 across embryonic stages. We demonstrate that AS071 is a distal enhancer that directs Fgf8 expression in the developing diencephalon. Furthermore, enhancer assays with constructs encoding partially deleted AS071 sequence revealed a unique modular organization in which AS071 contains at least three functionally distinct sub-elements that cooperatively direct the enhancer activity in three diencephalic domains, namely the dorsal midline and the lateral wall of the diencephalon, and the ventral midline of the hypothalamus. Interestingly, the AmnSINE1-derived sub-element was found to specify the enhancer activity to the ventral midline of the hypothalamus. To our knowledge, this is the first discovery of an enhancer element that could be separated into respective sub-elements that determine regional specificity and/or the core enhancing activity. These results potentiate our understanding of the evolution of retroposon-derived cis-regulatory elements as well as the basis for future studies of the molecular mechanism underlying the determination of domain-specificity of an enhancer.
Highlights
Retroposons, including short interspersed repetitive elements (SINEs) and long interspersed repetitive elements (LINEs), can proliferate in a genome via reverse-transcription of an RNA intermediate and subsequent integration into random sites in the genome
We discovered a unique aspect of AS071 in gene regulation, in which the SINE-derived element contributes to conferring enhancer activity to the hypothalamus, providing a new clue to understand the importance of retroposons in the acquisition of cis-regulatory elements during evolution
Consistent lacZ expression was observed between E9.5 and E15.5 in three distinct diencephalic domains among the three independent stable lines (Figure 2A, Figure S2): the dorsal midline of the diencephalon extending from the caudal telencephalon (DD, green arrowhead), a narrow band stretch the lateral wall of the diencephalon from dorsal midline (LD, blue arrowhead), and the ventral midline of the hypothalamus (VMH, red arrowhead)
Summary
Retroposons, including short interspersed repetitive elements (SINEs) and long interspersed repetitive elements (LINEs), can proliferate in a genome via reverse-transcription of an RNA intermediate and subsequent integration into random sites in the genome This ‘copy and paste’ mechanism of amplification is called retrotransposition. It is widely accepted that the number and type of coding genes do not vary greatly among vertebrate species from fish to mammals and that protein-coding regions constitute only a small faction of each genome (1.2% [1]) These facts underscore the potentially important role of non-coding regions in gene regulation. This concept has become firmly established with the discovery of a large number of non-coding elements that are highly conserved across species [5,6,7,8] These conserved noncoding elements (CNEs) are assumed to be involved in generating different morphological and physiological features among species. Their evolutionary origins and functional role(s) leading to morphological differences among species have not been studied in detail
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