Differential Transcriptional Regulation by Mouse Single-minded 2s

Abstract

Single-minded 1 and 2 are unique members of the basic helixloop-helix Per-Arnt-Sim family as they are transcriptional repressors. Here we report the identification and transcriptional characterization of mouse Sim2s, a splice variant of Sim2, which is missing the carboxyl Pro/Ala-rich repressive domain. Sim2s is expressed at high levels in kidney and skeletal muscle; however, the ratio of Sim2 to Sim2s mRNA differs between these tissues. Similar to full-length Sim2, Sim2s interacts with Arnt and to a lesser extent, Arnt2. The effects of Sim2s on transcriptional regulation through hypoxia, dioxin, and central midline response elements are different than that of full-length Sim2. Specifically, Sim2s exerts a less repressive effect on hypoxia-induced gene expression than full-length Sim2, but is just as effective as Sim2 at repressing TCDD-induced gene expression from a dioxin response element. Interestingly, Sim2s bind to and activates expression from a central midline response element-controlled reporter through an Arnt transactivation domain-dependent mechanism. The differences in expression pattern, protein interactions, and transcriptional activities between Sim2 and Sim2s may reflect differential roles each isoform plays during development or in tissue-specific effects on other proteinmediated pathways.The basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) 2 proteins comprise a growing family of transcription factors that play key roles during development and in sensing and adapting to changes in the environment. Individual PAS proteins are known to control morphogenesis, circadian rhythmicity, responses to hypoxia and toxin metabolism. These proteins contain a bHLH motif, which mediates dimerization with other bHLH proteins and contributes to determining DNA binding specificity. The PAS domain, named after the founding members of this family (period-arylhydrocarbon nuclear translocator-single minded), is a multifunctional protein surface responsible for such diverse activities as ligand binding, PAS protein dimerization, and non-PAS protein interactions (1).In addition to environmental adaptation, some members of the bHLH-PAS family regulate development. In Drosophila, single-minded (sim) acts as the master regulator of central nervous system midline development by controlling expression of many genes required for differentiation. Similar to other bHLH-PAS proteins, sim functions as a heterodimer with arnt (2). This complex binds to central midline elements (CME) in the regulatory regions of target genes to activate expression of proteins required for proper central midline establishment (2, 3).Two mammalian homologs of sim, Sim1 and Sim2, have been identified (4 -6). These proteins share a high degree of similarity in their PAS domains, but little conservation is apparent in their carboxyl termini. Sim1 and Sim2 interact with Arnt, but differ from Drosophila sim, the aryl hydrocarbon receptor (AHR) and hypoxia inducible factor (HIF) by functioning as transcriptional repressors (7, 8). Sim1 and Sim2 are expresse...

The HIF1α-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element

“…Another repressive mode of SIM2 is by sequestration of ARNT from other bHLH/PAS transcription factors, a mechanism by which SIM2 can interfere with the function of the hypoxia-inducible factor 1a (HIF1a) (Woods and Whitelaw, 2002). Conversely, on some promoters, the SIM2/ARNT heterodimer can activate transcription via the transactivation domain of ARNT (Metz et al, 2006;Woods et al, 2008). The expression status of SIM2s is emerging as an interesting candidate for a solid tumour marker.…”

“…Targeted deletion of SIM2 in mice results in scoliosis, major disruptions in craniofacial structures and death shortly after birth owing to a breathing defect (Goshu et al, 2002;Shamblott et al, 2002), which shows that it has important roles during development. Alternative splicing produces a short (SIM2s) protein variant in mice and humans (Chrast et al, 1997;Metz et al, 2006). The human SIM2s (hSIM2s) splice variant arises from an alternate readthrough event into the last intron of SIM2, resulting in a 570-amino-acid protein that contains only one of the two SIM2 repression regions and a final 44 residues of the unique C-terminal sequence (Chrast et al, 1997).…”

Sim2 prevents entry into the myogenic program by repressing MyoD transcription during limb embryonic myogenesis

“…Sim2 contains a bHLH domain, two PAS (Per-Arnt-Sim) domains and one HST (Hif1-/SIM/TRH) domain. Although most bHLH-PAS proteins are transcriptional activators, Sim2 acts as a repressor of transcription in mammalian cells (Moffett et al, 1997;Moffett and Pelletier, 2000;Metz et al, 2006). The repressor activity has been localised into two independent repression domains in the C-terminal region of mouse Sim2 protein (Moffett et al, 1997;Moffett and Pelletier, 2000;Metz et al, 2006).…”

“…The antisense mRNA probes were used as previously described: chick (c) Fgfr4, cPax3, cMyoD and cDelta1 ; mouse (m) Myf5, mMyoD, mMyog and GFP (Bonnet et al, 2010); Xenopus MyoD (Li, H. Y. et al, 2010); cLmx1b (Michaud et al, 1997); and cSim2 and mSim2 (Coumailleau and Duprez, 2009). The mSim2 probe used for endogenous mSim2 expression in mouse limbs recognises exon 11 and consequently does not recognise the splice variant mSim2s (Metz et al, 2006). The mSim2 probe used after chick electroporation experiments was specially designed not to crossreact with endogenous cSim2 expression, by PCR amplification using the Sim2N2 primers (see Table S1 in the supplementary material) from the from mSim2(Myc)pEFIRESpuro (Woods et al, 2008).…”

“…Although most bHLH-PAS proteins are transcriptional activators, Sim2 acts as a repressor of transcription in mammalian cells (Moffett et al, 1997;Moffett and Pelletier, 2000;Metz et al, 2006). The repressor activity has been localised into two independent repression domains in the C-terminal region of mouse Sim2 protein (Moffett et al, 1997;Moffett and Pelletier, 2000;Metz et al, 2006). The location of the SIM2 gene on the human chromosome 21, its expression in brain and the behaviour of transgenic mice with three copies of the Sim2 gene, have led to the suggestion that SIM2 is important in the mental retardation associated with Down's syndrome (Ema et al, 1999;Chrast et al, 2000).…”