Although substantial progress has been made in defining transcription machinery components and the sequence elements they recognize, it remains difficult to define promoters and predict patterns of expression from their sequence. We show here that expression of Rin1 is restricted by cell type and, in neurons, is temporally regulated. We also show that a 945 bp Rin1 promoter fragment is sufficient to confer a similar pattern of expression on a reporter gene in cultured cells and transgenic animals. It remains possible, of course, that distal elements or 3′ UTR sequences not included in this fragment may contribute to regulated expression of Rin1 in vivo. Any such contribution would appear to be minor, but might, for instance, dictate the level of expression in neuron subtypes. Still, the promoter fragment described here should prove useful for moderate transgene expression restricted primarily to mature telencephalic neurons.
Localization of high level Rin1
expression to the telencephalic regions of the forebrain is unusual but not unique. A search of the Allen Brain Atlas (mouse.brain-map.org) for similar patterns of expression identified Icam5
(a.k.a. telencephalin) and Ptk2b
, both of which appear to have TATA-less promoters, as the highest matches (data not shown). Indeed, a 1.1 kb fragment of the Icam5 promoter was used successfully in conventionally constructed transgenic mice to direct expression to the telencephalon (Mitsui et al., 2007
). Comparison of the 945 bp Rin1
and 1.1 kb Icam5
promoter fragments revealed a partial, interrupted, alignment within the REST-like sequence (). Otherwise, these promoter fragments showed only modest similarity, highlighting the limitation of using sequence alignment for expression pattern prediction.
The presence of multiple E-box type Snail binding sites within the Rin1
promoter, and the detection of Snai1 binding to the promoter, strongly suggests a role for Snail family transcription factors in the repression of Rin1
transcription. This interpretation is also consistent with the observation that human SNAI1
is overexpressed and RIN1
is silenced in the breast tumor cell line ZR75-1, and that SNAI1
silencing restores RIN1
expression in these cells (Milstein et al., 2007
). We found, however, that deletion or mutation of individual Snail binding sites at best moderately increased expression from the Rin1
promoter. This may indicate that Snail family transcriptional repressors normally bind at multiple sites along the promoter, perhaps working cooperatively, and that any individual site contributes only modestly to repression.
The REST-like element described in this study extends over 26 base pairs and includes nucleotides that diverge from the consensus REST site. Although this element may act in part as a weak REST binding site for transcription silencing in non-neuronal cells (allowing low level expression in mammary epithelial cells), its striking conservation among mammalian Rin1
gene promoters suggests that it carries out another function. The reduced expression seen for both the RESTLm1 and RESTLm2 mutant promoters in differentiated P19 cells suggested that this element may positively regulate expression in neuronal cells. Indeed, other transcription factors have been shown to utilize subsets of distinct binding sites to achieve different outcomes (Blauwkamp et al., 2008
; So et al., 2007
). It should also be noted that REST functions in neuronal stem/progenitor cells (Johnson et al., 2008
; Su et al., 2006
) and is expressed in some mature neurons (Koenigsberger et al., 2000
; Palm et al., 1998
; Shimojo and Hersh, 2004
). Further, multiple REST isoforms (Palm et al., 1998
) and REST-containing protein complexes (Belyaev et al., 2004
) have been described. The Rin1
promoter REST-like element may work through recruitment of a specialized REST complex in neurons, perhaps without the CoREST factor needed for silencing (Andres et al., 1999
), and this may positively regulate expression. Alternatively, an unidentified neuronal transcription factor with binding site specificity similar to REST may function at this site.
The Rin1 gene product is an established Ras effector that stimulates Rab5-mediated endocytosis and Abl-mediated actin remodeling. The restriction of Rin1 protein to specific cell types and by stage of postnatal development should both confine the effects of these signaling pathways and prevent unwanted interference with Ras signaling through alternate effectors (e.g. Raf and PI3K). The combination of multiple Snail binding sites and a highly conserved REST-like element are likely to be instrumental in directing proper expression, but distal sequences and chromatin structure undoubtedly contribute additional regulation to Rin1 gene expression.