Cloning of two human homologs of the Drosophila single-minded gene SIM1 on chromosome 6q and SIM2 on 21q within the Down syndrome chromosomal region

Abstract

As part of our effort to clone genes of human chromosome 21 that may contribute to Down syndrome, we have previously isolated four exons with homology to Drosophila single-minded (sim) gene, which encodes a transcription factor that is a master regulator of fruit fly neurogenesis. These exons were used to clone and characterize two human homologs of the Drosophila sim gene, SIM1 and SIM2, which map to chromosomes 6q16.3-q21 and 21q22.2, respectively; SIM2 maps within the so-called Down syndrome chromosomal region. Recently, two mouse homologs, Sim1 and Sim2, also have been identified. There is a high level of homology among human, mouse, and Drosophila sim genes in their amino-terminal half where the conserved bHLH, PAS1, PAS2, and HST domains are present. In contrast, the carboxy-terminal parts are only homologous between SIM1 and Sim1 and SIM2 and Sim2. Two isoforms (SIM2 and SIM2s) of human SIM2 have been detected that differ in their 3' ends. Northern blot analysis revealed one mRNA SIM1 species of approximately 9.5 kb and four different mRNA SIM2 species of 2.7, 3, 4.4, and 6 kb in human fetal kidney. The function of both human SIM1 and SIM2 is unknown. However, three copies of SIM2 may contribute to some specific Down syndrome phenotypes because of (1) mapping position, (2) potential function as transcriptional repressor, (3) likely dimerization with other transcription factors, (4) the temporal and spatial expression pattern of mouse Sim2, and (5) the potentially analogous role of human SIM2 to that of Drosophila sim during neurogenesis.

Figure 1

Comparison of human, mouse, and Drosophila SIM-predicted polypeptides. SIM1 (GenBank accession no. U70212) and SIM2 (U80456), human SIMs; Sim1 (D79209) and Sim2 (D63383), mouse SIMs; dsim (M19020), Drosophila single minded. SIM2s (U80457) represents the 570-amino-acid short isoform of SIM2. An asterisk (*) denotes identical and a period (.) denotes conserved amino acids, respectively. Amino acid color codes: (Red) Identical among all sequences; (blue) conserved among all sequences; (purple) identical between mouse and human SIM1; (turquoise) identical between mouse and human SIM2; (dark yellow) identical among all mammalian SIM proteins. SIM2s is shown from its point of divergence with SIM2.

Figure 2

The conserved domains bHLH, PAS1, PAS2, and HST of the SIMs are shown schematically together with their alignments to domains from additional proteins. (Red) Identical amino acids; (blue) conserved amino acids. The GenBank numbers of the different SIM sequences used are as in Fig. 1; trh (U33427), Drosophila trachealess; sima (U43090), Drosophila similar; hHIF-1a (U22431), human hypoxia-inducible factor-1α. SIM2s represents the 570-amino-acid short isoform of SIM2. An asterisk (*) denotes identical and a period (.) denotes conserved amino acids.

Figure 3

Chromosomal localization of the BAC clone KB153H1 containing the human SIM1 gene to 6q16 by FISH and Q-banding. (A) Metaphase chromosomes of human B-lymphoblastoid GM130B cell line hybridized with the BAC clone KB153H1. (B) Representative images of human chromosome 6. Hybridization signals appeared in the same position, 6q16, in all cases.

Figure 4

Mapping position of human and mouse SIM1 and SIM2 genes. The localization in mouse chromosomes was reported in Fan et al. (1996). Previously we have mapped SIM2 to chromosome 21 (Chen et al. 1995). SIM1 has been mapped to chromosome 6q16.3–q21 between markers WI-6516 and WI-6530 using the Genebridge 4 radiation hybrid panel. Diagrams are not to scale.

Figure 5

Northern blot analysis of SIM1 and SIM2 expression. The nonhomologous carboxy-terminal parts of SIM1 and SIM2 cDNAs were used as specific probes against filters containing poly(A)⁺ RNA from several human tissues (see Methods). The same filters were used sequentially for both hybridizations. Both genes are expressed in fetal kidney; SIM1 revealed an mRNA species of ∼9.5 kb (left), whereas SIM2 showed mRNA species of ∼2.7, 3, 4.4, and 6 kb (right).