The mouse homologue of the human receptor-interacting protein 140 (RIP140) was isolated from a mouse embryonic cDNA library in yeast two-hybrid screening experiments by using the ligand binding domain (LBD) of nuclear orphan receptor TR2 as the bait. The receptor-interacting domains of mouse RIP140 were mapped to the regions containing the LXXLL motif (where L is leucine and X is any amino acid), and the RIP140-interacting domain of TR2 was mapped to its C-terminal 10-to 20-amino-acid sequence, a putative activation function 2 (AF-2) region. In a GAL4 reporter system and a reporter driven by the proximal region of the TR2 promoter, RIP140 functioned as a corepressor for both a GAL4 DNA binding domain (BD)-TR2 fusion and the wild-type receptor. When tethered to the BD of GAL4, RIP140 exerted a trans-repressive effect on the GAL4 reporter. In addition, RIP140 suppressed the retinoic acid (RA) receptor-mediated RA induction in a dosedependent manner. Finally, it was demonstrated that in the presence of RIP140, a cytosolic, green fluorescent protein-tagged TR2 LBD translocated into the nucleus, and TR2 and RIP140 were coimmunoprecipitated from the cell extract, indicating that the interaction between RIP140 and the LBD of TR2 occurred in vivo. The potential biological role of RIP140 in TR2-modulated transcriptional activity is discussed.Nuclear receptors regulate target gene expression by binding to their cognate DNA response elements and recruiting associate proteins to the transcription machinery (19, 45). Recently, different coactivators and corepressors for several nuclear receptors have been identified (16). For instance, the nuclear receptor corepressor (N-CoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT) have been shown to function as corepressors for retinoic acid (RA) receptor ␣ (RAR␣), thyroid hormone receptors (T 3 Rs), and orphan receptor COUP-TFI (4, 15, 36). The newly identified nuclear protein Nab1 is a corepressor for the orphan receptor NGFI-A family (38), and SUN-CoR is able to potentiate transcriptional repression by T 3 Rs and RevErb (44). In the coactivator category, the transcriptional mediator/intermediary factor 2 and steroid receptor coactivator 1 (SRC-1) are known to mediate transcriptional activation of RAR, estrogen receptor (ER), retinoid X receptor (RXR), and T 3 Rs (8,14,33,39,42). With respect to the working mechanism of corepressors and coactivators, it has been demonstrated that their actions involve the alteration of chromatin structure, such as the acetylation status of histone proteins (13,34,37).The orphan receptor TR2 belongs to the superfamily of nuclear receptors (21,31). This receptor gene is expressed most abundantly in the testes of adult animals, particularly the developing germ cell populations (22,24,25). During embryonic stages, TR2 expression is highest between embryonic day 8 (E8) and E12 (22). It is also known that this gene encodes two isoform receptors, one retaining the entire ligand binding domain (LBD) and the other truncated at ...

A unique heterodimerization pathway involving orphan receptors TR2 and TR4 is demonstrated. TR2 and TR4 preferentially form heterodimers in solution as well as on DNA elements containing a direct repeat-5 (DR5). The in vitro interaction between TR2 and TR4 is demonstrated by the yeast and the mammalian two-hybrid interaction assays, the pull-down assay, and the gel mobility shift assay. The in vivo interaction is demonstrated by following the intracellular localization of fusion receptors tagged with a green fluorescent protein. The dimerization is mediated by the ligand binding domains, and the three leucine residues on helix 10 of TR2 are critical for this interaction. In addition, coexpression of these two receptors exerts a much stronger repressive activity on a DR5-containing reporter than expressing either receptor alone. In the developing testis, TR2 and TR4 are coexpressed in the same testicular cell populations and exhibit a parallel pattern of expression along development. The preferential heterodimerization between TR2 and TR4 and their coexistence in specific germ cell populations suggest a physiological role of TR2/TR4 heterodimers in germ cell development.Nuclear receptors constitute a super family of transcription factors that regulate gene expression in a wide variety of biological processes such as growth, differentiation, and development. These transcription regulators modulate the transcription efficiency of their target genes by binding to specific DNA sequences in the promoters of these target genes, thereby recruiting corepressors or coactivators to the transcription machinery (1). The most common form of receptor-DNA interaction is the binding of repeated DNA sequences, either in a direct or an inverted orientation, by dimeric receptors. In most cases, nuclear receptors preferentially form heterodimeric pairs with a common partner, one of the retinoid receptor X (RXR) 1 family members (2). It is widely accepted that RXR family provides the common partners for all the nuclear receptors that are able to form heterodimers. The orphan receptors belong to the super family of nuclear receptors; however, the biological significance of these orphan members has been debated because of the lack of specific ligands for these receptors. Recently, the biological functions of several orphan receptors have been revealed in gene-targeted mice and by linkage analysis. For example, mice deficient in COUP-TFII or hepatocyte nuclear factor-4 displayed embryonic lethality (3), and chromosomal deletion of the ROR ␣ gene resulted in a staggered phenotype (4). In addition, it has been suggested that the signaling pathways of some orphan receptors can be coupled to certain established biological pathways, such as the orphan receptor COUP-TFII in the bone morphogenetic protein-4 (BMP-4) pathway (5).We have previously isolated and characterized a mouse orphan receptor TR2-11 gene that is expressed most abundantly in the developing germ cells (6). Later, we have identified two isoforms of this receptor, one encoding ...

The 360 base-pair fragment in HVS-1 of the mitochondrial genome were determined from ancient human remains excavated at Noen U-loke and Ban Lum-Khao, two Bronze and Iron Age archaeological sites in Northeastern Thailand, radio-carbon dated to circa 3,500-1,500 years BP and 3,200-2,400 years BP, respectively. These two neighboring populations were parts of early agricultural communities prevailing in northeastern Thailand from the fourth millennium BP onwards. The nucleotide sequences of these ancient samples were compared with the sequences of modern samples from various ethnic populations of East and Southeast Asia, encompassing four major linguistic affiliations (Altaic, Sino-Tibetan, Tai-Kadai, and Austroasiatic), to investigate the genetic relationships and history among them. The two ancient samples were most closely related to each other, and next most closely related to the Chao-Bon, an Austroasiatic-speaking group living near the archaeological sites, suggesting that the genetic continuum may have persisted since prehistoric times in situ among the native, perhaps Austroasiatic-speaking population. Tai-Kadai groups formed close affinities among themselves, with a tendency to be more closely related to other Southeast Asian populations than to populations from further north. The Tai-Kadai groups were relatively distant from all groups that have presumably been in Southeast Asia for longer-that is, the two ancient groups and the Austroasiatic-speaking groups, with the exception of the Khmer group. This finding is compatible with the known history of the Thais: their late arrival in Southeast Asia from southern China after the 10th-11th century AD, followed by a period of subjugation under the Khmers.

The mouse orphan nuclear receptor TR2-11-f suppressed the expression of reporters fused to a hormone response element of the mouse cellular retinoic acid-binding protein I gene promoter. TR2-11-f was able to bind to a direct repeat with four nucleotides in the spacer (5'TGACCTTTGGGGACCT3') located within this hormone response element as homodimers. The specificity of protein-DNA interactions was demonstrated by competition in gel retardation and antibody-mediated supershift reactions. The residues critical for TR2-11-f binding were mapped to both repeated sequences, whereas the spacer and the flanking sequences were less important. The Kd and Bmax of TR2-11-f homodimer binding to this direct repeat were determined to be 2.6 nM and 0.012 nM, respectively. By using a yeast two-hybrid system, it was demonstrated that dimerization of TR2-11-f was mediated by its ligand-binding domain. The actions of TR2-11-f in regulating cellular retinoic acid-binding protein I gene will likely influence retinoic action and availability within the cells.

The mouse orphan nuclear receptor TR2-11 functions as a repressor for reporter genes containing a direct repeat-5 or direct repeat-4 hormone response element. The functional domains responsible for its suppressive activity are defined, including the DNA-binding domain and the ligand-binding domain. The C-terminal 30 amino acid residues can be deleted without compromising its suppressive activity, whereas a deletion for 40 amino acids completely abolishes the suppressive activity and receptor dimerization, and reduces the DNA-binding affinity. Point mutation at three conserved leucine residues located on the predicted dimer interface abolishes the suppressive activity, receptor dimerization and its DNA binding property. However, mutation at two consecutive glutamate residues located within the hinge between the last two helices of the ligand-binding domain (helix 10 and helix 11 according to the human retinoid receptor X alpha structure) drastically reduces its DNA-binding affinity and abrogates the suppressive activity without compromising its ability to dimerize, indicating that receptor dimerization property can be functionally uncoupled from its suppressive activity. A transferable, active silencing activity is encoded within the DEF segment of the receptor molecule, as evidenced by the suppression of a GAL4 reporter by a chimeric protein containing the DNA-binding domain of GAL4 and the DEF segment of TR2-11. Moreover, the C-terminal 49 amino acid sequence is required for this trans-suppressive activity. It is suggested that TR2-11 functions as a repressor, mediated by mechanisms requiring high affinity DNA binding, receptor dimerization, and active silencing.