Hepatic hydroxylation is an essential step in the metabolism and excretion of bile acids and is necessary to avoid pathologic conditions such as cholestasis and liver damage. In this report, we demonstrate that the human xenobiotic receptor SXR (steroid and xenobiotic receptor) and its rodent homolog PXR (pregnane X receptor) serve as functional bile acid receptors in both cultured cells and animals. In particular, the secondary bile acid derivative lithocholic acid (LCA) is highly hepatotoxic and, as we show here, a metabolic substrate for CYP3A hydroxylation. By using combinations of knockout and transgenic animals, we show that activation of SXR͞PXR is necessary and sufficient to both induce CYP3A enzymes and confer resistance to toxicity by LCA, as well as other xenotoxicants such as tribromoethanol and zoxazolamine. Therefore, we establish SXR and PXR as bile acid receptors and a role for the xenobiotic response in the detoxification of bile acids.

SummaryMicroRNAs are important players in stem cell biology. Among them, microRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain. Whether miR-9 plays a role in neural stem cell self-renewal and differentiation is unknown. We showed previously that nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector lacking the miR-9 recognition site rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses miR-9 pri-miRNA expression. MiR-9, by forming a negative regulatory loop with TLX, establishes a model for controlling the balance between neural stem cell proliferation and differentiation.

The rapid yet transient transcriptional activation of heat shock genes is mediated by the reversible conversion of HSF1 from an inert negatively regulated monomer to a transcriptionally active DNA-binding trimer. During attenuation of the heat shock response, transcription of heat shock genes returns to basal levels and HSF1 reverts to an inert monomer. These events coincide with elevated levels of Hsp70 and other heat shock proteins (molecular chaperones). Here, we show that the molecular chaperone Hsp70 and the cochaperone Hdj1 interact directly with the transactivation domain of HSF1 and repress heat shock gene transcription. Overexpression of either chaperone represses the transcriptional activity of a transfected GAL4-HSF1 activation domain fusion protein and endogenous HSF1. As neither the activation of HSF1 DNA binding nor inducible phosphorylation of HSF1 was affected, the primary autoregulatory role of Hsp70 is to negatively regulate HSF1 transcriptional activity. These results reveal that the repression of heat shock gene transcription, which occurs during attenuation, is due to the association of Hsp70 with the HSF1 transactivation domain, thus providing a plausible explanation for the role of molecular chaperones in at least one key step in the autoregulation of the heat shock response.

A yeast two-hybrid screen using the conserved carboxyl terminus of the nuclear receptor corepressor SMRT as a bait led to the isolation of a novel human gene termed SHARP (SMRT/HDAC1 Associated Repressor Protein). SHARP is a potent transcriptional repressor whose repression domain (RD) interacts directly with SMRT and at least five members of the NuRD complex including HDAC1 and HDAC2. In addition, SHARP binds to the steroid receptor RNA coactivator SRA via an intrinsic RNA binding domain and suppresses SRA-potentiated steroid receptor transcription activity. Accordingly, SHARP has the capacity to modulate both liganded and nonliganded nuclear receptors. Surprisingly, the expression of SHARP is itself steroid inducible, suggesting a simple feedback mechanism for attenuation of the hormonal response. The transcription action of steroids, retinoids, and thyroid hormone and their cognate receptors (NRs) (Mangelsdorf and Evans 1995; are modulated by an extensive set of nuclear receptor cofactors Glass and Rosenfeld 2000;Westin et al. 2000). A great deal of effort has focused on the identification and characterization of the constituents of these complexes to understand the mechanistic basis of the regulated events. The recruitment of coactivator complexes is a critical step in hormone induction, whereas the recruitment of corepressor complexes mediates active repression of unliganded nuclear receptors. SMRT and N-CoR have been identified as nuclear receptor corepressors (Chen and Evans 1995;Horlein et al. 1995;Ordentlich et al. 1999). Various lines of evidence suggest that at least one mechanism underlying the repression activity of SMRT and N-CoR is through their recruitment of a histone deacetylase complex containing mSin3A and HDAC1 (Alland et al. 1997;Hassig et al. 1997;Heinzel et al. 1997;Laherty et al. 1997;Nagy et al. 1997;Zhang et al. 1997). Direct interaction of SMRT with the class II histone deacetylase (HDAC 4-7) independent of Sin3A provides yet another mechanism for SMRT-mediated transcriptional repression (Huang et al. 2000;Kao et al. 2000). Recruitment of histone deacetylase complexes by corepressors has been proposed to cause a local change in the chromatin structure, therefore resulting in transcriptional repression (Knoepfler and Eisenman 1999).A search for cofactors that mediate ligand-dependent transactivation by nuclear receptors led to the identification of coactivators such as CBP/p300, PCAF, and the p160 family members including SRC-1, GRIP1/TIF2, and ACTR/RAC3/p/CIP (Onate et al. 1995;Hong et al. 1996; Kamei et al. 1996;Yao et al. 1996;Chen et al. 1997;Torchia et al. 1997;Blanco et al. 1998). Among these factors, CBP, PCAF, SRC-1, and ACTR have been shown to possess intrinsic histone acetyltransferase activity, consistent with a role for induced histone acetylation in transcriptional activation (Bannister and Kouzarides 1996;Ogryzko et al. 1996;Yang et al. 1996;Chen et al. 1997;Spencer et al. 1997). Targeted deletion of SRC-1 or p/CIP causes partial hormone insensitivity, suggesting a criti...

SUMMARY DNA hydroxylation catalyzed by Tet dioxygenases occurs abundantly in embryonic stem cells and neurons in mammals. However, its biological function in vivo is largely unknown. Here we demonstrate that Tet1 plays an important role in regulating neural progenitor cell proliferation in adult mouse brain. Mice lacking Tet1 exhibit impaired hippocampal neurogenesis accompanied by poor learning and memory. In adult neural progenitor cells deficient in Tet1, a cohort of genes involved in progenitor proliferation were hypermethylated and down-regulated. Our results indicate that Tet1 is positively involved in the epigenetic regulation of neural progenitor cell proliferation in the adult brain.

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation.Neural stem cells are undifferentiated precursors that retain the ability to proliferate and self-renew, and they have the capacity to give rise to both neuronal and glial lineages (1). Although the functional properties of neural stem cells have been studied extensively, molecular mechanisms underlying neural stem cell self-renewal and differentiation are only beginning to be understood. One class of factors thought to be important in this process is microRNAs (miRNAs), which are short, 20-22 nucleotide RNA molecules that are expressed in a tissue-specific and developmentally regulated manner and function as negative regulators of gene expression in a variety of eukaryotes. MiRNAs are involved in numerous cellular processes including development, proliferation, and differentiation (2, 3). Studies based on expression patterns, predicted targets, and overexpression analyses suggest that miRNAs are key regulators in stem cell biology.The lethal-7 (let-7) gene is one of the first two miRNAs discovered in Caenorhabditis elegans (C. elegans), and the first known human miRNA (4, 5). Mature let-7 is highly conserved across species in both sequence and function. It plays an important role in development and cell maturation (6-9). Let-7 is expressed in both embryonic and adult brains (10-13). Recently, increased expression and maturation of let-7 has been observed during neural cell specification (8, 9). Let-7a, one of the members of the let-7 family, has been shown to play a role in neuronal differentiation of embryonic neural progenitors (14), while let-7b has been shown to reduce the self-renewal of aging neural stem cells through targeting the high-mobility group A (HMGA) family member Hmga2 expression (15). TLX (NR2E1) is an orphan nuclear receptor that i...

The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/β-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/β-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active β-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a β-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active β-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active β-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active β-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/β-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode. 8 Correspondence should be addressed to Y.S. (yshi@coh.org). 6,7 These authors contributed equally to this work.Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONSY.S. conceived and designed the study. Y.S., Q.Q., G.S., W.L., S.Y., P.Y. and C.Z. performed the experiments and analysed the data. Y.S., Q.Q., G.S., F.H.G. and R.M.E. interpreted the data. Y.S. wrote the paper with comments from Q.Q., G.S., R.T.Y., F.H.G. and R.M.E. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. The finding of neurogenesis in the adult brain led to the discovery of adult neural stem cells. Neural stem cells are defined as a subset of undifferentiated precursors that retain the ability to proliferate and self-renew and have the capacity to give rise to both neuronal and glial lineages [1][2][3][4] . Under normal conditions, neurogenesis in the adult mammalian brain is restricted to two discrete germinal centres: the subgranular layer of the hippocampal dentate gyrus 3 and the subventricular zones of the lateral ventricles 5,6 . A complete understanding of adult neural stem cells requires the identification of molecules that determine the self-renewal and multipotent characteristics of these cells.TLX is an orphan nuclear receptor that is expressed in vertebrate forebrains 7,8 . We showed previously that TLX is an important regulator of neural stem cell maintenance and self-renewal in both embryo...

TLX is a transcription factor that is essential for neural stem cell proliferation and self-renewal. However, the molecular mechanism of TLX-mediated neural stem cell proliferation and self-renewal is largely unknown. We show here that TLX recruits histone deacetylases (HDACs) to its downstream target genes to repress their transcription, which in turn regulates neural stem cell proliferation. TLX interacts with HDAC3 and HDAC5 in neural stem cells. The HDAC5-interaction domain was mapped to TLX residues 359 -385, which contains a conserved nuclear receptor-coregulator interaction motif IXXLL. Both HDAC3 and HDAC5 have been shown to be recruited to the promoters of TLX target genes along with TLX in neural stem cells. Recruitment of HDACs led to transcriptional repression of TLX target genes, the cyclin-dependent kinase inhibitor, p21 CIP1/WAF1 (p21), and the tumor suppressor gene, pten. Either inhibition of HDAC activity or knockdown of HDAC expression led to marked induction of p21 and pten gene expression and dramatically reduced neural stem cell proliferation, suggesting that the TLX-interacting HDACs play an important role in neural stem cell proliferation. Moreover, expression of a TLX peptide containing the minimal HDAC5 interaction domain disrupted the TLX-HDAC5 interaction. Disruption of this interaction led to significant induction of p21 and pten gene expression and to dramatic inhibition of neural stem cell proliferation. Taken together, these findings demonstrate a mechanism for neural stem cell proliferation through transcriptional repression of p21 and pten gene expression by TLX-HDAC interactions.coregulators ͉ NR2E1 ͉ self-renewal N uclear receptors are ligand-dependent transcription factors that regulate the expression of genes critical for a variety of biological processes, including development, growth, and differentiation. TLX is an orphan nuclear receptor that plays an important role in vertebrate brain functions (1-6). We have shown that TLX is an essential regulator of neural stem cell proliferation and self-renewal in the adult brain (3). TLX could act by controlling the expression of a network of downstream target genes to establish the undifferentiated and self-renewable state of neural stem cells. Elucidating the network regulated by TLX in producing these outcomes will be a significant advance in understanding neural stem cell self-renewal and neurogenesis.Nuclear receptors carry out transcriptional functions through the recruitment of positive and negative regulatory proteins, referred to as coactivators and corepressors (7,8). At least one mechanism underlying the repression activity of nuclear receptors is through the recruitment of histone deacetylase (HDAC) complexes. For example, HDAC1 and HDAC2 have been found in complexes with Sin 3 (9), nucleosome remodeling and deacetylation (NuRD) (10), nuclear receptor corepressor (N-CoR) (11), silencing mediator of retinoic and thyroid hormone receptors (SMRT) (12), and SMRT and HDAC-associated repressor proteins (SHARP) (13). HDAC4, HDA...