The MammalianSingle-Minded(SIM) Gene: Mouse cDNA Structure and Diencephalic Expression Indicate a Candidate Gene for Down Syndrome

doi:10.1006/geno.1996.0332

Genomics

Volume 35, Issue 1, 1 July 1996, Pages 136-143

https://doi.org/10.1006/geno.1996.0332 Get rights and content

Abstract

We have recently isolated a human homolog (hSIM) of theDrosophila single-minded(sim) gene from the Down syndrome critical region of chromosome 21 using the exon trapping method. TheDrosophila simgene encodes a transcription factor that regulates the development of the central nervous system midline cell lineage. To elucidate the structure of the mammalian SIM protein, we have isolated cDNA clones from a mouse embryo cDNA library. The cDNA clones encode a polypeptide of 657 amino acids with a bHLH (basic–helix–loop–helix) domain, characteristic of a large family of transcription factors, and a PAS (Per-Arnt-Sim) domain in the amino-terminal half region. Both of these domains have striking sequence homology with human SIM andDrosophilaSIM proteins. In contrast, the carboxy-terminal half of the mouse SIM protein consists of a proline-rich region with no sequence homology to theDrosophilaSIM protein. A similar proline-rich domain is known for the activator domain of a number of transcription factors. Whole-mount embryoin situhybridization experiments revealed that the SIM mRNA is expressed prominently in the diencephalon of mouse embryos at 8–9.5 days postcoitum. The structural characteristics of the mouse SIM protein and its expression in the diencephalon during embryogenesis strongly suggest that the newly isolated mammalian SIM homolog may play a critical role in the development of the mammalian central nervous system. We propose that the human SIM gene may be one of the pathogenic genes of Down syndrome.

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Cited by (37)

A recessive variant in SIM2 in a child with complex craniofacial anomalies and global developmental delay
2022, European Journal of Medical Genetics
Citation Excerpt :
Human single-minded 2 homolog gene (SIM2) encodes a 667 amino acid protein that is part of a family of basic helix-loop-helix (bHLH) and the PER-ARNT-SIM (PAS) domains transcription factors that play an essential role in central nervous system midline cell development and gene expression (Nambu et al., 1990, 1991). Human SIM2 was initially identified by exon trapping from a Down syndrome critical region (DSCR) on chromosome 21 and was suggested to be a candidate gene for association with many of the pathophysiological features of Down syndrome, including brain development abnormalities, facial dysmorphology, and intellectual impairment, primarily due to its expression at fetal stages of brain development and within regions outside the brain consistent with sites of Down syndrome clinical hallmarks (Rahmani et al., 1989; Chen et al., 1995; Yamaki et al., 1996; Rachidi et al., 2005). However, to date no patients harboring SIM2 point-mutations had been reported, and thus the evidence for causality had been largely due to its location within the DSCR, in which contiguous gene deletions or duplications overlapping SIM2 (up to 70 genes in some reports) cause craniofacial anomalies and various developmental disorders.
Rare deletions and duplications on the long arm of Chromosome 21 have previously been reported in many patients with craniofacial and developmental phenotypes. However, this Down Syndrome Critical Region (DSCR) contains multiple genes, making identifying a single causative gene difficult. Here, we report a case of a boy with bicoronal craniosynostosis, facial dysmorphism, developmental delay, and intellectual impairment who was found by whole genome sequencing to have a homozygous missense mutation in the Single-Minded Homolog 2 (SIM2) gene (c.461 A > G, p.Tyr154Cys) within the DSCR. SIM2 encodes an essential bHLH and PAS domain transcription factor expressed during fetal brain development and acts as a master regulator of neurogenesis. This variant is globally very rare, segregates in the family, and is predicted to be highly deleterious by in silico analysis, 3D molecular modeling of protein structure, and functional analysis of zebrafish models. Zebrafish expressing the human SIM2^p.Y154C variant displayed a progressed microcephaly-like phenotype and head shape abnormalities. When combined with careful phenotyping of the patient vis-à-vis previously reported cases harboring structural variants in this critical 21q22 region, the data support a pathogenic role of SIM2 in this complex syndrome and demonstrates the utility of next-generation sequencing in prioritizing genes in contiguous deletions/duplications syndromes and diagnosing microarray-negative patients in the craniofacial clinic.
Transcription factor single-minded 2 (SIM2) is ubiquitinated by the RING-IBR-RING-type E3 ubiquitin ligases
2005, Experimental Cell Research
Human single-minded 2 (SIM2) is a member of the basic helix–loop–helix/Per–Arnt–Sim (bHLH/PAS) family of transcription factors and is associated with the etiology of Down syndrome phenotype. Here, we examined a possibility of the post-translational modification of SIM2 protein by transfecting various expression constructs followed by the analysis with immunoprecipitation and Western blotting. In fact, transient expression of SIM2 cDNA in HEK293 cells revealed poly-ubiquitination of SIM2 protein. In the stable transfectants, a proteasome inhibitor MG132 protected the poly-ubiquitinated SIM2 protein from degradation. Furthermore, in the cells co-transfected with SIM2 and each of four different E3 ubiquitin ligases, SIM2 was immunoprecipitated with the RING–IBR–RING-type E3 ubiquitin ligases, Parkin and HHARI, but it was not immunoprecipitated with other E3 ligases, such as one RING-type Siah-1 and the PHD type AIRE. A series of deletion constructs revealed that Parkin actually binds to SIM2 with the IBR (294–377)–RING2 (378–465) domains and that the sites for poly-ubiquitination of SIM2 reside within the PAS1–PAS2 region (aa 141–289). We postulated that transcription factor SIM2 and E3 ubiquitin ligase Parkin may interact each other to play an important physiological role in the brain development which is controlled by ubiquitination.
Spatial and temporal localization during embryonic and fetal human development of the transcription factor SIM2 in brain regions altered in Down syndrome
2005, International Journal of Developmental Neuroscience
Citation Excerpt :
Sim protein, acting as a tissue-specific partner, is activated by heterodimerisation with Tango, a general dimerization partner, and then transferred to the nucleus where it can bind specific DNA sequences, called CNS-Midline Element (CME) and regulate the expression of target genes, such as hedgehog (hh), involved in the cell-lineage specific development of the CNS (Mellerick and Nirenberg, 1995; Sonnenfeld et al., 1997; Ohshiro and Saigo, 1997; Ward et al., 1998). Similarly to Drosophila sim, the mammalian Sim genes, Sim1 and Sim2, are characterized by restricted expression patterns, particularly in the developing CNS (Dahmane et al., 1995; Ema et al., 1996a,b; Yamaki et al., 1996; Fan et al., 1996). Sim1 and Sim2 proteins interact with the ubiquitous partners Arnt and Arnt2, Tango orthologs, and migrate in the nucleus (Swanson et al., 1995; Probst et al., 1997) where they can activate or repress target genes (Ema et al., 1996a,b; Moffett et al., 1997; Moffett and Pelletier, 2000; Epstein et al., 2000; Woods and Whitelaw, 2002; Liu et al., 2003).
Human SIM2 is the ortholog of Drosophila single-minded (sim), a master regulator of neurogenesis and transcriptional factor controlling midline cell fate determination. We previously localized SIM2 in a chromosome 21 critical region for Down syndrome (DS). Here, we studied SIM2 gene using a new approach to provide insights in understanding of its potential role in human development. For the first time, we showed SIM2 spatial and temporal expression pattern during human central nervous system (CNS) development, from embryonic to fetal stages. Additional investigations were performed using a new optic microscopy technology to compare signal intensity and cell density [M. Rachidi, C. Lopes, S. Gassanova, P.M. Sinet, M. Vekemans, T. Attie, A.L. Delezoide, J.M. Delabar, Regional and cellular specificity of the expression of TPRD, the tetratricopeptide Down syndrome gene, during human embryonic development, Mech. Dev. 93 (2000) 189–193]. In embryonic stages, SIM2 was identified predominantly in restricted regions of CNS, in ventral part of D1/D2 diencephalic neuroepithelium, along the neural tube and in a few cell subsets of dorsal root ganglia. In fetal stages, SIM2 showed differential expression in pyramidal and granular cell layers of hippocampal formation, in cortical cells and in cerebellar external granular and Purkinje cell layers. SIM2 expression in embryonic and fetal brain could suggest a potential role in human CNS development, in agreement with Drosophila and mouse Sim mutant phenotypes and with the conservation of the Sim function in CNS development from Drosophila to Human. SIM2 expression in human fetal brain regions, which correspond to key structures for cognitive processes, correlates well with the behavioral phenotypes of Drosophila Sim mutants and transgenic mice overexpressing Sim2. In addition, SIM2-expressing brain regions correspond to the altered structures in DS patients. All together, these findings suggest a potential role of SIM2 in CNS development and indicate that SIM2 overexpression could participate to the pathogenesis of mental retardation in Down syndrome patients.
A novel nuclear localization signal in the human single-minded proteins SIM1 and SIM2
2004, Biochemical and Biophysical Research Communications
Citation Excerpt :
These results exclude Gln389 from the consensus sequence in various SIM proteins. To date, the SIM gene homologs have been isolated from a variety of species including human, mouse, rat, Xenopus, fugu, and zebrafish [4–8]. In these SIM proteins, we found the well-conserved amino acid sequence consisting of a cluster of basic amino acids with Pro and Tyr at the C-terminal end (RKxxKxK/RxxxxKxKxRxxPY) (Fig. 4).
Human Single-minded 1 (SIM1) and SIM2 genes were found as homologs of Drosophila sim gene which plays a key role in the midline cell lineage of the central nervous system. SIM proteins belong to a family of transcription factors, called bHLH/PAS. Here we examined the intracellular localization of SIM proteins using the expression constructs of whole SIM2 or SIM1 protein fused with enhanced green fluorescent protein (EGFP). The transient expression analysis revealed the nuclear localization of SIM proteins in the cultured cells. To identify the nuclear localization signal, we made expression constructs of EGFP-fusion protein consisting of various portions of SIM proteins. Transfection assay showed the presence of NLS activity in the small region of 23 and 21 amino acid residues at the central part of SIM2 and SIM1 proteins, respectively. Further analysis with amino acid substitution of this small region of SIM2 protein revealed the critical role of five amino acid residues (Arg367, Lys373, Pro385, Tyr386, and Gln389) in NLS activity. The consensus sequence of RKxxKx[K/R]xxxxKxKxRxxPY was estimated as a presumptive NLS in SIM proteins from various species. Thus, the NLS consisting of a cluster of basic amino acids with Pro and Tyr at the C-terminal end is novel and well conserved in the SIM proteins during evolution.
Differential activities of murine Single Minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop-helix/Per-Arnt-Sim homology transcription factors
2002, Journal of Biological Chemistry
The basic helix-loop-helix/Per-Arnt-Sim homology (bHLH/PAS) protein family comprises a group of transcriptional regulators that often respond to a variety of developmental and environmental stimuli. Two murine members of this family, Single Minded 1 (SIM1) and Single Minded 2 (SIM2), are essential for postnatal survival but differ from other prototypical family members such as the dioxin receptor (DR) and hypoxia-inducible factors, in that they behave as transcriptional repressors in mammalian one-hybrid experiments and have yet to be ascribed a regulating signal. In cell lines engineered to stably express SIM1 and SIM2, we show that both are nuclear proteins that constitutively complex with the general bHLH/PAS partner factor, ARNT. We report that the murine SIM factors, in combination with ARNT, attenuate transcription from the hypoxia-inducible erythropoietin (EPO) enhancer during hypoxia. Such cross-talk between coexpressed bHLH/PAS factors can occur through competition for ARNT, which we find evident in SIM repression of DR-induced transcription from a xenobiotic response element reporter gene. However, SIM1/ARNT, but not SIM2/ARNT, can activate transcription from the EPOenhancer at normoxia, implying that the SIM proteins have the ability to bind hypoxia response elements and affect either activation or repression of transcription. This notion is supported by co-immunoprecipitation of EPO enhancer sequences with the SIM2 protein. SIM protein levels decrease with hypoxia treatment in our stable cell lines, although levels of the transcripts encoding SIM1 and SIM2 and the approximately 2-h half-lives of each protein are unchanged during hypoxia. Inhibition of protein synthesis, known to occur in cells during hypoxic stress in order to decrease ATP utilization, appears to account for the fall in SIM levels. Our data suggest the existence of a hypoxic switch mechanism in cells that coexpress hypoxia-inducible factor and SIM proteins, where up-regulation and activation of hypoxia-inducible factor-1α is concomitant with attenuation of SIM activities.
The evolution of chordate neural segmentation
2002, Developmental Biology
Amphioxus is the closest relative to vertebrates but lacks key vertebrate characters, like rhombomeres, neural crest cells, and the cartilaginous endoskeleton. This reflects major differences in the developmental patterning of neural and mesodermal structures between basal chordates and vertebrates. Here, we analyse the expression pattern of an amphioxus FoxB ortholog and an amphioxus single-minded ortholog to gain insight into the evolution of vertebrate neural segmentation. AmphiFoxB expression shows cryptic segmentation of the cerebral vesicle and hindbrain, suggesting that neuromeric segmentation of the chordate neural tube arose before the origin of the vertebrates. In the forebrain, AmphiFoxB expression combined with AmphiSim and other amphioxus gene expression patterns shows that the cerebral vesicle is divided into several distinct domains: we propose homology between these domains and the subdivided diencephalon and midbrain of vertebrates. In the Hox-expressing region of the amphioxus neural tube that is homologous to the vertebrate hindbrain, AmphiFoxB shows the presence of repeated blocks of cells along the anterior–posterior axis, each aligned with a somite. This and other data lead us to propose a model for the evolution of vertebrate rhombomeric segmentation, in which rhombomere evolution involved the transfer of mechanisms regulating neural segmentation from vertical induction by underlying segmented mesoderm to horizontal induction by graded retinoic acid signalling. A consequence of this would have been that segmentation of vertebrate head mesoderm would no longer have been required, paving the way for the evolution of the unsegmented head mesoderm seen in living vertebrates.

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^☆: Sequence data from this article have been deposited with the DDBJ/GenBank/EMBL Data Libraries under Accession Nos. D64135 and D70838.

¹: To whom correspondence should be addressed. Telephone and Fax: 81-3-3351-2370. E-mail: [email protected].

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Regular ArticleThe MammalianSingle-Minded(SIM) Gene: Mouse cDNA Structure and Diencephalic Expression Indicate a Candidate Gene for Down Syndrome☆

Abstract

Regular Article
The MammalianSingle-Minded(SIM) Gene: Mouse cDNA Structure and Diencephalic Expression Indicate a Candidate Gene for Down Syndrome☆