Epigenetic Effects of Resveratrol on Oncogenic Signaling in Breast Cancer.

The crosstalk between oncogenic signaling pathways plays a crucial role in driving cancer development. We previously demonstrated that dietary polyphenols, specifically resveratrol (RSV) and other stilbenoids, epigenetically target oncogenes for silencing via DNA hypermethylation in breast cancer. In the present study, we identify signal transduction regulators among RSV-hypermethylated targets and investigate the functional role of RSV-mediated DNA hypermethylation in the regulation of Hedgehog and Wnt signaling. Non-invasive ER-positive MCF-7 and highly invasive triple-negative MCF10CA1a human breast cancer cell lines were used as experimental models. Upon 9-day exposure to 15 µM RSV, pyrosequencing and qRT-PCR were performed to assess DNA methylation and expression of GLI2 and WNT4, which are upstream regulators of the Hedgehog and Wnt pathways, respectively. Our results showed that RSV led to a DNA methylation increase within GLI2 and WNT4 enhancers, which was accompanied by decreases in gene expression. Consistently, we observed the downregulation of genes downstream of the Hedgehog and Wnt signaling, including common targets shared by both pathways, CCND1 and CYR61. Further analysis using chromatin immunoprecipitation identified increased H3K27 trimethylation and decreased H3K9 and H3K27 acetylation, along with abolishing OCT1 transcription factor binding. Those changes indicate a transcriptionally silent chromatin state at GLI2 and WNT4 enhancers. The inhibition of the Wnt signal transduction was confirmed using a phospho-antibody array that demonstrated suppression of positive and stimulation of negative Wnt regulators. In conclusion, our results provide scientific evidence for dietary polyphenols as epigenetics-modulating agents that act to re-methylate and silence oncogenes, reducing the oncogenic signal transduction. Targeting such an action could be an effective strategy in breast cancer prevention and/or adjuvant therapy.

A genome-wide association meta-analysis implicates Hedgehog and Notch signaling in Dupuytren's disease.

Dupuytren's disease (DD) is a highly heritable fibrotic disorder of the hand with incompletely understood etiology. A number of genetic loci, including Wnt signaling members, have been previously identified. Our overall aim was to identify novel genetic loci, to prioritize genes within the loci for functional studies, and to assess genetic correlation with associated disorders. We performed a meta-analysis of six DD genome-wide association studies from three European countries and extensive bioinformatic follow-up analyses. Leveraging 11,320 cases and 47,023 controls, we identified 85 genome-wide significant single nucleotide polymorphisms in 56 loci, of which 11 were novel, explaining 13.3-38.1% of disease variance. Gene prioritization implicated the Hedgehog and Notch signaling pathways. We also identified a significant genetic correlation with frozen shoulder. The pathways identified highlight the potential for new therapeutic targets and provide a basis for additional mechanistic studies for a common disorder that can severely impact hand function.

Unveiling the connection: Long-chain non-coding RNAs and critical signaling pathways in breast cancer.

Breast cancer is a complex and diverse condition that disrupts multiple signaling pathways essential for cell proliferation, survival, and differentiation. Recently, the significant involvement of long-chain non-coding RNAs (lncRNAs) in controlling key signaling pathways associated with breast cancer development has been discovered. This review aims to explore the interaction between lncRNAs and various pathways, including the AKT/PI3K/mTOR, Wnt/ß-catenin, Notch, DNA damage response, TGF-ß, Hedgehog, and NF-κB signaling pathways, to gain a comprehensive understanding of their roles in breast cancer. The AKT/PI3K/mTOR pathway regulates cell growth, survival, and metabolic function. Recent data suggests that specific lncRNAs can influence the functioning of this pathway, acting as either oncogenes or tumor suppressors. Dysregulation of this pathway is commonly observed in breast cancer cases. Moreover, breast cancer development has been associated with other pathways such as Wnt/ß-catenin, Notch, TGF-ß, Hedgehog, and NF-κB. Emerging studies have identified lncRNAs that modulate breast cancer's growth, progression, and metastasis by interacting with these pathways. To advance the development of innovative diagnostic tools and targeted treatment options, it is crucial to comprehend the intricate relationship between lncRNAs and vital signaling pathways in breast cancer. By fully harnessing the therapeutic potential of lncRNAs, there is a possibility of developing more effective and personalized therapy choices for breast cancer patients. Further investigation is necessary to comprehensively understand the role of lncRNAs within breast cancer signaling pathways and fully exploit their therapeutic potential.

MicroRNA­1 regulates the development of osteoarthritis in a Col2a1­Cre­ERT2/GFPfl/fl­RFP­miR­1 mouse model of osteoarthritis through the downregulation of Indian hedgehog expression.

The present study assessed the effects of microRNA­1 (miR­1) on the development of osteoarthritis using human tissues and a Col2a1­Cre­ERT2/GFPfl/fl­RFP­miR­1 mouse model of osteoarthritis. Human cartilage tissues (n=20) were collected for reverse transcription­quantitative polymerase chain reaction (RT­qPCR), histological analysis and immunohistochemistry experiments. A transgenic mouse model of osteoarthritis was established by subjecting Col2a1­Cre­ERT2/GFPfl/fl­RFP­miR­1 transgenic mice to anterior cruciate ligament transection (ACLT). Mice were subjected to radiography and in vivo fluorescence molecular tomography (FMT), while mouse tissues were collected for histological analysis, RT­qPCR and Safranin O staining. It was found that the miR­1 level was downregulated, whereas the levels of Indian hedgehog (Ihh), as well as those of its downstream genes were upregulated in human osteoarthritic cartilage. In the transgenic mice, treatment with tamoxifen induced miR­1, as well as collagen, type II (Col2a1) and Aggrecan (Acan) expression; however, it decreased Ihh, glioma­associated oncogene homolog (Gli)1, Gli2, Gli3, smoothened homolog (Smo), matrix metalloproteinase (MMP)­13 and collagen type X (Col10) expression. Safranin O staining revealed cartilage surface damage in the non­tamoxifen + ACLT group, compared with that in the tamoxifen + ACLT group. Histologically, an intact cartilage surface and less fibrosis were observed in the tamoxifen + ACLT group. Immunohistochemistry revealed that the protein expression of Ihh, Col10, and MMP­13 was significantly higher in the joint tissues of the non­tamoxifen + ACLT group than in those of the tamoxifen + ACLT group. However, Col2a1 expression was lower in the joint tissues of the non­tamoxifen + ACLT group than in those of the tamoxifen + ACLT group. The results of RT­qPCR and FMT further confirmed these findings. On the whole, the findings of the present study demonstrate that miR­1 expression protects against osteoarthritis­induced cartilage damage and gene expression by inhibiting Ihh signaling.

Epigenetic regulation of mammalian Hedgehog signaling to the stroma determines the molecular subtype of bladder cancer.

In bladder, loss of mammalian Sonic Hedgehog (Shh) accompanies progression to invasive urothelial carcinoma, but the molecular mechanisms underlying this cancer-initiating event are poorly defined. Here, we show that loss of Shh results from hypermethylation of the CpG shore of the Shh gene, and that inhibition of DNA methylation increases Shh expression to halt the initiation of murine urothelial carcinoma at the early stage of progression. In full-fledged tumors, pharmacologic augmentation of Hedgehog (Hh) pathway activity impedes tumor growth, and this cancer-restraining effect of Hh signaling is mediated by the stromal response to Shh signals, which stimulates subtype conversion of basal to luminal-like urothelial carcinoma. Our findings thus provide a basis to develop subtype-specific strategies for the management of human bladder cancer.

The role of Hedgehog-responsive fibroblasts in facial nerve regeneration.

BACKGROUND: Facial nerve paralysis is a significant cause of morbidity, affecting facial appearance, emotional expression, speech, oral competence, and vision. A more complete understanding of the complex cellular events required for successful nerve regeneration may reveal new therapeutic targets. The role of fibroblasts in regeneration, and the process by which the nerve reforms its three-dimensional structure after a transection injury, are not fully understood. The Hedgehog signaling pathway has been shown to mediate nerve sheath formation during development. We therefore sought to characterize the role of Hedgehog-responsive cells following transection of the facial nerve. METHODS: Two transgenic mouse lines with reporters for the downstream effector of Hedgehog signaling, Gli1, were used. The animals underwent a unilateral facial nerve transection injury, and the contralateral side served as a control. Facial nerves were analyzed via immunohistochemistry and immunofluorescence at predetermined time points as the facial nerve regenerated after the transection injury. RESULTS: There was a statistically significant increase in Gli1+ cells both at the site of injury and within the distal nerve segment over time. Gli1+ cells are fibroblasts within the nerve and appear to contribute to the reformation of the nerve sheath after injury. CONCLUSION: These findings describe a key signaling pathway by which fibroblasts participate in motor nerve regeneration. Fibroblasts that reside within the nerve respond to injury and may represent a novel therapeutic target in the context of facial nerve regeneration after transection injury.

Combined inhibition of atypical PKC and histone deacetylase 1 is cooperative in basal cell carcinoma treatment.

Advanced basal cell carcinomas (BCCs) circumvent Smoothened (SMO) inhibition by activating GLI transcription factors to sustain the high levels of Hedgehog (HH) signaling required for their survival. Unfortunately, there is a lack of efficacious therapies. We performed a gene expression-based drug repositioning screen in silico and identified the FDA-approved histone deacetylase (HDAC) inhibitor, vorinostat, as a top therapeutic candidate. We show that vorinostat only inhibits proliferation of BCC cells in vitro and BCC allografts in vivo at high dose, limiting its usefulness as a monotherapy. We leveraged this in silico approach to identify drug combinations that increase the therapeutic window of vorinostat and identified atypical PKC Ɩ/ʎ (aPKC) as a HDAC costimulator of HH signaling. We found that aPKC promotes GLI1-HDAC1 association in vitro, linking two positive feedback loops. Combination targeting of HDAC1 and aPKC robustly inhibited GLI1, lowering drug doses needed in vitro, in vivo, and ex vivo in patient-derived BCC explants. We identified a bioavailable and selective small-molecule aPKC inhibitor, bringing the pharmacological blockade of aPKC and HDAC1 into the realm of clinical possibility. Our findings provide a compelling rationale and candidate drugs for combined targeting of HDAC1 and aPKC in HH-dependent cancers.

Genetic evidence supporting the taxonomic separation of the Arabian and Northwest African subspecies of the desert hedgehog (Paraechinus aethiopicus).

Traditional subspecies call attention to differences between geographic populations with research potential, but their value is often in need of revision. Genetic data can be useful for evaluating the taxonomic validity of historical species and subspecies designations or for identifying morphologically cryptic divergent lineages worthy of further in-depth taxonomic study. The desert hedgehog (Paraechinus aethiopicus) has a wide but fragmented distribution in arid and semi-arid habitats from the northwest to the northeast of Africa and southwestern Asia, and its taxonomy is still unclear. We used mitochondrial (cytochrome b, Cyt b, and 12S ribosomal RNA, 12S) and nuclear (breast cancer type 1 susceptibility protein, BRCA1, and apolipoprotein B, Apob) DNA sequence data to assess the degree of genetic divergence between two of its three major proposed subspecies: Arabian (P. a. dorsalis) and Northwest African (P. a. deserti); this is the first molecular evaluation of the taxonomy of P. aethiopicus. Phylogenetic analyses, comparison of interspecific and intraspecific genetic distances observed across hedgehog species, and molecular species delimitation methods (distance-based clustering and tree-based), all indicate a level of genetic differentiation between dorsalis and deserti that is compatible with their taxonomic separation. Their divergence in the studied genes were consistently comparable to, or greater than, several intrageneric and a few intergeneric distances in hedgehogs. The Cyt b net Kimura 2-parameter distance between dorsalis and deserti was 10.8±1.3%, which is about the mean between congeneric species in reviews of Cyt b distances for mammals. This study, as a test of the genetic distinctiveness of dorsalis and deserti, suggests that they represent evolutionarily significant units and flags them for future phylogeographic and taxonomic investigations.

Galectin-3, histone deacetylases, and Hedgehog signaling: Possible convergent targets in schistosomiasis-induced liver fibrosis.

Schistosomiasis affects approximately 240 million people in the world. Schistosoma mansoni eggs in the liver induce periportal fibrosis and hepatic failure driven by monocyte recruitment and macrophage activation, resulting in robust Th2 response. Here, we suggested a possible involvement of Galectin-3 (Gal-3), histone deacetylases (HDACs), and Hedgehog (Hh) signaling with macrophage activation during Th1/Th2 immune responses, fibrogranuloma reaction, and tissue repair during schistosomiasis. Gal-3 is highly expressed by liver macrophages (Kupffer cells) around Schistosoma eggs. HDACs and Hh regulate macrophage polarization and hepatic stellate cell activation during schistosomiasis-associated fibrogenesis. Previously, we demonstrated an abnormal extracellular matrix distribution in the liver that correlated with atypical monocyte-macrophage differentiation in S. mansoni-infected, Gal-3-deficient (Lgals3-/-) mice. New findings explored in this review focus on the chronic phase, when wild-type (Lgals3+/+) and Lgals3-/- mice were analyzed 90 days after cercariae infection. In Lgals3-/- infected mice, there was significant inflammatory infiltration with myeloid cells associated with egg destruction (hematoxylin and eosin staining), phagocytes (specifically Kupffer cells), numerically reduced and diffuse matrix extracellular deposition in fibrotic areas (Gomori trichrome staining), and severe disorganization of collagen fibers surrounding the S. mansoni eggs (reticulin staining). Granuloma-derived stromal cells (GR cells) of Lgals3-/- infected mice expressed lower levels of alpha smooth muscle actin (α-SMA) and eotaxin and higher levels of IL-4 than Lgals3+/+ mice (real-time PCR). The relevant participation of macrophages in these events led us to suggest distinct mechanisms of activation that culminate in defective fibrosis in the liver of Lgals3-/- infected mice. These aspects were discussed in this review, as well as the possible interference between Gal-3, HDACs, and Hh signaling during progressive liver fibrosis in S. mansoni-infected mice. Further studies focused on macrophage roles could elucidate these questions and clear the potential utility of these molecules as antifibrotic targets.

Transcriptional Dynamics of Immortalized Human Mesenchymal Stem Cells during Transformation.

Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human fibroblasts and human mesenchymal stem cells (hMSC).

Daidzein suppresses tumor necrosis factor-α induced migration and invasion by inhibiting hedgehog/Gli1 signaling in human breast cancer cells.

In breast cancer, the cytokine tumor necrosis factor-α (TNF-α) induces cell invasion, although the molecular basis of it has not been clearly elucidated. In this study, we investigated the role of daidzein in regulating TNF-α induced cell invasion and the underlying molecular mechanisms. Daidzein inhibited TNF-α induced cellular migration and invasion in estrogen receptor (ER) negative MCF10DCIS.com human breast cancer cells. TNF-α activated Hedgehog (Hh) signaling by enhancing Gli1 nuclear translocation and transcriptional activity, which resulted in increased invasiveness; these effects were blocked by daidzein and the Hh signaling inhibitors, cyclopamine and vismodegib. Moreover, these compounds suppressed TNF-α induced matrix metalloproteinase (MMP)-9 mRNA expression and activity. Taken together, mammary tumor cell invasiveness was stimulated by TNF-α induced activation of Hh signaling; these effects were abrogated by daidzein, which suppressed Gli1 activation, thereby inhibiting migration and invasion.

BRCA1 and p53 regulate critical prostate cancer pathways.

BACKGROUND: Loss or mutations of the BRCA1 gene are associated with increased risk of breast and ovarian cancers and with prostate cancer (PCa) aggressiveness. Previously, we identified GADD153 as a target of BRCA1 protein, which increases doxorubicin sensitivity in human p53 -/- PCa cells (PC3). Considering that p53 is a crucial target in cancer therapy, in this work we investigated p53 role in the regulation of transcription of GADD153. METHODS: We performed reverse transcription quantitative PCR (RT-qPCR), western blot and luciferase assays to analyze GADD153 and/or BRCA1 expression in response to ultraviolet or doxorubicin exposure in PC3 p53 stable-transfected cells and LNCaP (p53+/+) cells. BRCA1 protein recruitment to GADD153 promoter was studied by chromatin immunoprecipitation-qPCR. To assess expression of BRCA1 and/or p53 target genes, we used a panel of stable-transfected PCa cell lines. We finally analyzed these genes in vivo using BRCA1-depleted PCa xenograft models. RESULTS: We found that GADD153 was highly induced by doxorubicin in PC3 cells; however, this response was totally abolished in LNCaP (p53wt) and in p53-restituted PC3 cells. Furthermore, BRCA1 protein associates to GADD153 promoter after DNA damage in the presence of p53. Additionally, we demonstrated that BRCA1 and/or p53 modulate genes involved in DNA damage and cell cycle regulation (cyclin D1, BLM, BRCA2, DDB2, p21(WAF1/CIP1), H3F3B, GADD153, GADD45A, FEN1, CCNB2), EMT (E-cadherin, ß-catenin, vimentin, fibronectin, slug, snail) and Hedgehog pathways (SHH, IHH, DHH, Gli1, PATCH1). Furthermore, xenograft studies demonstrated that BRCA1 knockdown in PC3 cells increased tumor growth and modulated these genes in vivo. CONCLUSIONS: Although BRCA1 induces GADD153 in a p53 independent manner, p53 abolished GADD153 induction in response to DNA damage. In addition, several important PCa targets are modulated by BRCA1 and p53. Altogether, these data might be important to understand the therapy response of PCa patients.

Gene cloning, expression and functional characterization of a proliferation-inducing ligand (APRIL) from hedgehog (Erinaceus europaeus).

Here we describe the identification of the hedgehog Erinaceus europaeus homologue of a proliferation-inducing ligand (APRIL) of the TNF family (designated heAPRIL). Hedgehog APRIL contains two cysteine residues (Cys(196) and Cys(211)), a furin protease cleavage site and a conserved putative N-glycosylation site (Asn(124)). Real-time quantitative PCR (qPCR) analysis revealed that heAPRIL could be detected in various tissues. MTT assays and flow cytometric analysis revealed that Nus-hesAPRIL and hesAPRIL could promote the survival/proliferation of splenic B cells. Laser scanning confocal microscopy analysis showed GFP-hesAPRIL could successfully bind to the APRIL receptors of lymphocytes.

Cross-species chromosome painting unveils cytogenetic signatures for the Eulipotyphla and evidence for the polyphyly of Insectivora.

Insectivore-like animals are traditionally believed among the first eutherian mammals that have appeared on the earth. The modern insectivores are thus crucial for understanding the systematics and phylogeny of eutherian mammals as a whole. Here cross-species chromosome painting, with probes derived from flow-sorted chromosomes of human, was used to delimit the homologous chromosomal segments in two Soricidae species, the common shrew (Sorex araneus, 2n = 20/21), and Asiatic short-tailed shrew (Blarinella griselda, 2n = 44), and one Erinaceidae species, the shrew-hedgehog (Neotetracus sinensis, 2n = 32), and human. We report herewith the first comparative maps for the Asiatic short-tailed shrew and the shrew-hedgehog, in addition to a refined comparative map for the common shrew. In total, the 22 human autosomal paints detected 40, 51 and 58 evolutionarily conserved segments in the genomes of common shrew, Asiatic short-tailed shrew, and shrew-hedgehog, respectively, demonstrating that the common shrew has retained a conserved genome organization while the Asiatic short-tailed shrew and shrew-hedgehog have relatively rearranged genomes. In addition to confirming the existence of such ancestral human segmental combinations as HSA 3/21, 12/22, 14/15 and 7/16 that are shared by most eutherian mammals, our study reveals a shared human segmental combination, HSA 4/20, that could phylogenetically unite the Eulipotyphlan (i.e., the core insectivores) species. Our results provide cytogenetic evidence for the polyphyly of the order Insectivora and additional data for the eventual reconstruction of the ancestral eutherian karyotype.

Molecular phylogenetic evidence confirming the Eulipotyphla concept and in support of hedgehogs as the sister group to shrews.

For more than a century, living insectivore-like mammals have been viewed as little removed from the ancestral mammalian stock based on their retention of numerous primitive characteristics. This circumstance has made "insectivores" a group of special interest in the study of mammalian evolution. included hedgehogs, moles, shrews, solenodons, golden moles, tenrecs, flying lemurs, tree shrews, and elephant shrews in Insectivora. Subsequently, morphologists excluded flying lemurs, tree shrews, and elephant shrews from Insectivora and placed these taxa in the orders Dermoptera, Scandentia, and Macroscelidea, respectively. The remaining insectivores constitute Lipotyphla, which is monophyletic based on morphology. In contrast, molecular data suggest that lipotyphlans are polyphyletic, with golden moles and tenrecs placed in their own order (Afrosoricida) in the superordinal group Afrotheria. Studies based on nuclear genes support the monophyly of the remaining lipotyphlans (=Eulipotyphla) whereas mitochondrial genome studies dissociate hedgehogs from moles and place the former as the first offshoot on the placental tree. One shortcoming of previous molecular studies investigating lipotyphlan relationships is limited taxonomic sampling. Here, we evaluate lipotyphlan relationships using the largest and taxonomically most diverse data set yet assembled for Lipotyphla. Our results provide convincing support for both lipotyphlan diphyly and the monophyly of Eulipotyphla. More surprisingly, we find strong evidence for a sister-group relationship between shrews and hedgehogs to the exclusion of moles.

Maximum likelihood analysis of the complete mitochondrial genomes of eutherians and a reevaluation of the phylogeny of bats and insectivores.

The complete mitochondrial genomes of two microbats, the horseshoe bat Rhinolophus pumilus, and the Japanese pipistrelle Pipistrellus abramus, and that of an insectivore, the long-clawed shrew Sorex unguiculatus, were sequenced and analyzed phylogenetically by a maximum likelihood method in an effort to enhance our understanding of mammalian evolution. Our analysis suggested that (1) a sister relationship exists between moles and shrews, which form an eulipotyphlan clade; (2) chiropterans have a sister-relationship with eulipotyphlans; and (3) the Eulipotyphla/Chiroptera clade is closely related to fereuungulates (Cetartiodactyla, Perissodactyla and Carnivora). Divergence times on the mammalian tree were estimated from consideration of a relaxed molecular clock, the amino acid sequences of 12 concatenated mitochondrial proteins and multiple reference criteria. Moles and shrews were estimated to have diverged approximately 48 MyrBP, and bats and eulipotyphlans to have diverged 68 MyrBP. Recent phylogenetic controversy over the polyphyly of microbats, the monophyly of rodents, and the position of hedgehogs is also examined.

Short interspersed elements (SINEs) from insectivores. Two classes of mammalian SINEs distinguished by A-rich tail structure.

Four tRNA-related SINE families were isolated from the genome of the shrew Sorex araneus (SOR element), mole Mogera robusta (TAL element), and hedgehog Mesechinus dauuricus (ERI-1 and ERI-2 elements). Each of these SINEs families is specific for a single Insectivora family: SOR, for Soricidae (shrews); TAL, for Talpidae (moles and desmans); ERI-1 and ERI-2, for Erinaceidae (hedgehogs). There is a long polypyrimidine region (TC-motif) in TAL, ERI-1, and ERI-2 elements located immediately upstream of an A-rich tail with polyadenylation signals (AATAAA) and an RNA polymerase III terminator (T(4-6)) or TCT(3-4)). Ten out of 14 analyzed mammalian tRNA-related SINE families have an A-rich tail similar to that of TAL, ERI-1, and ERI-2 elements. These elements were assigned to class T+. The other four SINEs including SOR element have no polyadenylation signal and transcription terminator in their A-rich tail and were assigned to class T-. Class T+ SINEs occur only in mammals, and most of them have a long polypyrimidine region. Possible models of retroposition of class T+ and T- SINEs are discussed.

The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia.

Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.