Unique forms of human and mouse nuclear receptor corepressor SMRT

Abstract

Nuclear hormone receptors have been shown to repress transcription in the absence of ligand. This repression is mediated by a corepressor complex that contains the Sin3A protein and histone deacetylases (HDAC1 and 2). Studies by several groups demonstrate that this complex is recruited to nuclear receptors through the highly related corepressors SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor). We describe here the cloning, characterization, and chromosomal mapping of forms of human and mouse SMRT that includes a 1,000-aa extension, which reveals striking homology to the amino terminus of N-CoR. Structure and function studies of wild-type and natural splicing variants suggest the presence of 3-4 amino terminal domains that repress in a cooperative as well as mechanistically distinct fashion.

Figure 1

Alignment of h-SMRT and m-SMRT sequences. Proteins were aligned by using the clustal alignment program. The underlined region of m-SMRT corresponds to the region deleted from the β isoform (see text). The arrow indicates the start point of the human s-SMRT identified previously. The nucleotide sequences are available on request and have been submitted to GenBank (accession nos. h-SMRT: AF113003; m-SMRTα: AF113001; m-SMRTβ: AF113002).

Figure 2

Alignment of h-SMRT and human N-CoR (hN-Cor). (A) The protein sequences were aligned by using the clustal alignment program. Identical residues are white with a black background. The arrow indicates the start of s-SMRT. (B) The diagram highlights the regions of homology between the SMRT N-terminal 1,031 aa and the corresponding N-CoR sequence (GenBank accession no. AF044209). The amino acids 1–160 are colored black to indicate this region as the Siah2 interacting domain. Amino acids 255–312 represent the Sin3A interaction domain mapped in N-CoR. Within the SANT region (amino acids 312–668), the actual SANT repeats are highlighted in black. The black-shaded region in the RD2 homology region represents the further defined repression core between amino acids 845 and 986. Percent similarities were calculated for each region by using the blastp program with a bosum62 matrix.

Figure 3

Tissue distribution and chromosomal localization of SMRT. (A) Poly(A) RNA derived from 20 μg of total RNA from each indicated tissue was subjected to Northern blot analysis. The filter was hybridized with ³²P-labeled m-SMRT/PstI fragment (720 bp) (Upper). The relative position of RNA marker (kb) is labeled at the left. The filter was stripped and subsequently reprobed with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA for the purpose of loading control (Lower). A 10-kb major transcript was detected in all tissues examined, with relatively high expression in brain, lung, and spleen (lanes 1, 5, and 9). A minor transcript of 8.5 kb in size was present in most tissues. (B) Fluorescence in situ hybridization analysis of h-SMRT is shown with a SMRT-specific probe (green) and a chromosome 12-specific alpha satellite probe that hybridizes to the pericentromeric region (red). The arrows indicate the localization of the SMRT clone at band 12q24. The schematic diagram of chromosome 12 highlights the relative position of SMRT on the chromosome as being close to the 12q24 band. A more defined location is available from genemap 98 (see text).

Figure 4

Characterization of SMRT amino terminus. (A) The schematic illustrates the various GAL4-SMRT fusions constructed, their corresponding amino acid numbers, and repressing ability. The patterns of colored and striped regions are described in the Fig. 2B legend. The GAL4-DBD is depicted as a gray oval. The fold repression represents the ability of the GAL-4-SMRT constructs to repress the activity of a GAL4-TK-luc reporter in cotransfection experiments and is relative to GAL-DBD alone. (B) Representative data comparing the repressing abilities of various regions of the m-SMRT N terminus. The constructs are shown in A. Amino acids 1–301 correspond to RD1, 427–663 to the SANT region, 736-1031 to RD2, and 1–1031 to the full m-SMRT N terminus. (C) Representative data narrowing down the RD2 region to a 150-aa core from 845 to 986. (D) Representative data demonstrating the differing repression abilities of the two m-SMRT isoforms. GAL4-SMRTβ (1–85) corresponds to RD1 with the deletion shown in Figs. 1 and 4A. GAL4-SMRTβ (1–813) corresponds to the full N terminus of m-SMRT with the β isoform deletion.