[Show abstract][Hide abstract] ABSTRACT: Expression profiling of embryonic stem (ES) cell differentiation in the presence of serum has been performed previously. It remains unclear if transcriptional activation is dependent on complex growth factor mixtures in serum or whether this process is intrinsic to ES cells once the stem cell program has been inactivated. The aims of this study were to determine the transcriptional programs associated with the stem cell state and to characterize mesoderm differentiation between serum and serum-free culture.
ES cells were differentiated as embryoid bodies in 10% FBS or serum-free media containing BMP4 (2 ng/ml), and expression profiled using 47 K Illumina(R) Sentrix arrays. Statistical methods were employed to define gene sets characteristic of stem cell, epiblast and primitive streak programs. Although the initial differentiation profile was similar between the two culture conditions, cardiac gene expression was inhibited in serum whereas blood gene expression was enhanced. Also, expression of many members of the Kruppel-like factor (KLF) family of transcription factors changed dramatically during the first few days of differentiation. KLF2 and KLF4 co-localized with OCT4 in a sub-nuclear compartment of ES cells, dynamic changes in KLF-DNA binding activities occurred upon differentiation, and strong bio-informatic evidence for direct regulation of many stem cell genes by KLFs was found.
Down regulation of stem cell genes and activation of epiblast/primitive streak genes is similar in serum and defined media, but subsequent mesoderm differentiation is strongly influenced by the composition of the media. In addition, KLF family members are likely to be important regulators of many stem cell genes.
[Show abstract][Hide abstract] ABSTRACT: Rev-erbbeta is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-alpha (RORalpha). RORalpha has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Reverbbeta and RORalpha are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erbbeta has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erbbeta regulates lipid metabolism in skeletal muscle. This lean tissue accounts for >30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erbbeta in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erbbeta decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (>15-fold) in mRNA expression of interleukin-6, an "exercise-induced myokine" that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (>20-fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erbbeta in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erbbeta may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth.
[Show abstract][Hide abstract] ABSTRACT: Rev-erbβ is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan
receptor-α (RORα). RORα has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis,
and inflammation. Reverbβ and RORα are expressed in similar tissues, including skeletal muscle; however, the pathophysiological
function of Rev-erbβ has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping
cognate sequences of these nuclear receptors that Rev-erbβ regulates lipid metabolism in skeletal muscle. This lean tissue
accounts for >30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary
site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin
sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the
regulatory role of Rev-erbβ in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse
Rev-erbβ decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (>15-fold) in mRNA expression of interleukin-6, an “exercise-induced myokine”
that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (>20-fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation.
This study implicates Rev-erbβ in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate
that selective modulators of Rev-erbβ may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle
Journal of Biological Chemistry 03/2005; 280(10):8651-8659. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rev-erbA/RVR are closely related orphan nuclear receptors (NRs) functioning as dominant transcriptional silencers through an association with the nuclear receptor corepressor N-CoR. In contrast with ligand-regulated NRs, Rev-erbA/RVR lack the ligand-binding domain (LBD) C-terminal activation helix, H12. In the case of retinoid acid receptor and thyroid hormone receptor, ligand binding is thought to reposition H12, causing corepressor dissociation and coactivator recruitment, thus leading to transcriptional activation. Here we present homology models of the Rev-erbA/RVR LBDs, which show that the putative ligand cavity is occupied by side chains, suggesting the absence of endogenous ligands. Modeling also revealed a very hydrophobic surface due to the absence of H12, exposing residues from H3, loop 3-4, H4, and H11. Mutation of specific residues from this surface severely impaired the in vitro and in vivo interaction of the Rev-erbA/RVR LED with the receptor-interacting domain of the corepressors N-CoR or its splice variant RIP13 Delta 1, reinforcing the view of the physical association of N-CoR with a LED surface encompassing H3-H4 and H11. Furthermore, mutations in the LED surface significantly reduced the ability of Rev-erbA and RVR to function as repressors of transcription. Interestingly, a hydrophobic surface comprised of H3-H4 and H12 in liganded NRs mediates the interaction with coactivators. Hence, it appears that corepressors and coactivators bind to overlapping surfaces of NR LBDs, the conformational change associated with H12 upon ligand binding resulting in a switch from a corepressor- to a coactivator-binding surface.
[Show abstract][Hide abstract] ABSTRACT: Repression of transcription by the classical nuclear receptors (e.g. TR, RAR), the orphan nuclear receptors (e.g. Rev-erbAalpha/beta), Mxi-1 and Mad bHLH-zip proteins and the oncoproteins PLZF and LAZ3/BCL6 is mediated by the corepressors N-CoR and SMRT. The interaction of the corepressors with the components involved in chromatin remodelling, such as the recruiting proteins Sin3A/B and the histone deacteylases HDAc-1 and RPD3, has been analysed in detail. The N-CoR/Sin3/HDAc complexes have a key role in the regulation of cellular proliferation and differentiation. However, the interaction of these corepressors with the basal transcriptional machinery has remained obscure. In this study we demonstrated that the N-terminalrepression domains and the receptor interactiondomains (RID) of N-CoR and its splice variants, RIP13a and RIP13Delta1, directly interact with TAFII32 in vivo and in vitro . We show that interaction domain II within the N-CoR and RIP13a RID is required for the interaction with TAFII32. We also observed that N-CoR directly interacts with each of the basal factors, TFIIB and TAFII70, and can simultaneously interact with all three basal factors in a non-competitive manner. Furthermore, we provide evidence that suggests the RVR/Rev-erbbeta-corepressor complex also interacts with the general transcriptional machinery, and that the physicalassociation of TFIIB with N-CoR also occurs in the presence of Sin3B and HDAc-1. Interestingly, we observed that N-CoR expression ablated the functional interaction between TFIIB and TAFII32 that is critical to the initiation of transcription. In conclusion, this study demonstrates that the N-terminal repressor region and the C-terminal RIDs are part of the corepressor contact interface that mediates the interaction with the general transcription factors, and demonstrates that TAFs can also directly interact with corepressors to mediate signals from repressors to the basal machinery. We also suggest that N-CoR interacts with the central components of the transcriptional initiation process (TFIIB, TAFs) and locks them into a non-functional complex or conformation that is not conducive to transcription.
Nucleic Acids Research 07/1998; 26(12):2899-907. · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rev-erbA alpha and RVR are orphan nuclear receptors that function as dominant transcriptional silencers. Ligand-independent repression of transcription by Rev-erbA alpha and RVR is mediated by the nuclear receptor corepressors, N-CoR and its variants RIP (RXR interacting protein) 13a and RIP13 delta 1. The physical association between the corepressors and Rev-erbA alpha and RVR is dependent on the presence of a receptor interaction domain (RID) in the N-CoR family. Our previous study demonstrated that the E region of RVR and Rev-erbA alpha is necessary and sufficient for the in vivo interaction with the nuclear receptor corepressor, RIP13 delta 1. The present investigation demonstrates that two corepressor interaction regions, CIR-1 and CIR-2, separated by approximately 150 amino acids in the E region of RVR, are required for the interaction with N-CoR, RIP13a, and RIP13 delta A. The D region is not required for the physical interaction. In contrast, the D and E regions of Rev-erbA alpha were necessary for the interaction with the N-CoR and RIP13a-RIDs in vivo, suggesting that RIP13 delta 1 and N-CoR/RIP13a differentially interact with Rev-erbA alpha. Mutagenesis of CIR-1, a novel domain that is highly conserved between RVR and Rev-erbA alpha, demonstrated that the N-terminal portion of helix 3 plays a key role and is absolutely necessary for the interaction with RIP13 delta 1, RIP13a, and N-CoR. The phenylalanine residues, F402 and F441, in RVR and Rev-erbA alpha, respectively, were critical residues in supporting corepressor interaction. Cotransfection studies demonstrated that repression of a physiological target, the human Rev-erbA alpha promoter, by RVR was significantly impaired by mutation of CIR-1 or deletion of CIR-2. Furthermore, overexpression of either the N-CoR/RIP13a or RIP13 delta 1-RIDs alleviated RVR-mediated repression of the Rev-erbA alpha promoter, demonstrating that corepressor binding mediates the repression of a native target gene by RVR. A minimal region containing juxtapositioned CIR-1 and CIR-2 was sufficient for corepressor binding and transcriptional repression. In conclusion, our study has identified a new corepressor interaction region, CIR-1, in the N terminus of helix 3 in the E region of RVR and Rev-erbA alpha, that is required for transcriptional silencing. Furthermore, we provide evidence that CIR-1 and CIR-2 may form a single corepressor interaction interface.
[Show abstract][Hide abstract] ABSTRACT: COUP-TF II/ARP-1 is an 'orphan' steroid receptor that inhibits basal transcription, and represses trans-activation by the vitamin D, thyroid hormone and retinoid receptors. The molecular basis of repression by COUP-TF II remains obscure. In this study we utilized the GAL4 hybrid system to demonstrate that COUP-TF II contains sequences within the C-terminal region that encode a dominant transcriptional repressor that inhibits the ability of the potent chimeric transactivator GAL4VP16 to induce transcription. Mammalian two hybrid analysis demonstrated that COUP-TF II did not efficiently interact with either interaction domains I or II from N-CoR and RIP13. However, COUP-TF II efficiently interacts with a region comprised of interaction domains I + II from the corepressor, RIP13delta1. Efficient interaction of the orphan receptor with the corepressor was critically dependent on a large region comprised of the C, D and E domains of COUP-TF II, which correlated with the domain that maximally represses transcription. This investigation suggested that the N-CoR variant, RIP13delta1 interacts with a region of COUP-TF II that functions as a dominant transcriptional repressor.
The Journal of Steroid Biochemistry and Molecular Biology 11/1997; 63(4-6):165-74. DOI:10.1016/S0960-0760(97)00079-4 · 3.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rev-erbA alpha and RVR/Rev-erb beta/BD73 are orphan steroid receptors that have no known ligands in the 'classical sense'. These 'orphans' do not activate transcription, but function as dominant transcriptional silencers. The thyroid hormone receptor (TR) and the retinoic acid receptor (RAR) act as transcriptional silencers by binding corepressors (e.g. N-CoR/RIP13 and SMRT/TRAC-2) in the absence of ligands. The molecular basis of repression by orphan receptors, however, remains obscure, and it is unclear whether these corepressors mediate transcriptional silencing by Rev-erbA alpha and RVR. Recently, two new variants of N-CoR have been described, RIP13a and RIP13delta1. The characterisation of these splice variants has identified a second receptor interaction domain (ID-II), in addition to the previously characterised interaction domain (ID-I). This investigation utilised the mammalian two hybrid system and transfection analysis to demonstrate that Rev-erbA alpha and RVR will not efficiently interact with either ID-I or ID-II separately from RIP13a or RIP13delta1. However, they interact efficiently with a domain composed of ID-I and ID-II from RIP13a. Interestingly, the interaction of Rev-erbA alpha and RVR is strongest with ID-I and ID-II from RIP13delta1. Detailed deletion analysis of the orphan receptor interaction with RIP13/N-CoR rigorously demonstrated that the physical association was critically dependent on an intact E region of Rev-erbA alpha and RVR. Over-expression of the corepressor interaction domains (i.e. dominant negative forms of N-CoR/RIP13) could alleviate orphan receptor-mediated repression of transactivation by GALVP16. This demonstrated that these regions could function as anti-repressors. In conclusion, these data from two independent approaches demonstrate that repression by Rev-erbA alpha and RVR is mediated by an interaction of ID-I and ID-II of N-CoR, RIP13a and delta1 with the putative ligand binding domain of the orphan receptors.
Nucleic Acids Research 12/1996; 24(22):4379-86. DOI:10.1093/nar/24.22.4379 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rev-erbA alpha is an orphan nuclear receptor that functions as a dominant transcriptional repressor. Tissue culture and in situ hybridisation studies indicated that Rev-erbA alpha plays an important role in mammalian differentiation and development. Previous studies have localised the silencing domain of Rev-erbA alpha to the D/E region of the orphan receptor. This study utilised the GAL4 hybrid system to demonstrate that efficient repression is mediated by 34 amino acids (aa) between aa 455 and 488 in the E region of the receptor. This domain contains the ligand binding domain (LBD)-signature motif [(F/W)AKxxxxFxxLxxxDQxxLL] and a region that, according to the recently published crystal structures of steroid receptors, would be predicted to form helix 5 of the canonical LBD structure. Fine deletions and site-specific mutagenesis indicated that both the LBD signature motif and helix 5 were necessary for efficient silencing. Utilising mammalian two hybrid technology, we have also demonstrated that Rev-erbA alpha does not associate with the interaction domain (aa 2218-2451) of the nuclear receptor corepressor, N-CoR, that is known to interact with the thyroid hormone and retinoic acid receptors. This suggested that transcriptional repression by Rev-erbA alpha is not mediated through an interaction with N-CoR. In conclusion, we have identified and characterised the minimal domain of Rev-erbA alpha, that mediates transcriptional repression by this orphan receptor.
Nucleic Acids Research 10/1996; 24(18):3490-8. · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RVR/Rev-erbβ/BD73 is an orphan steroid receptor that has no known ligand in the 'classical' sense. RVR binds as a monomer to an element which consists of an A/T-rich sequence upstream of the consensus hexameric half-site. However, RVR does not activate transcription and blocks transactivation of this element by ROR/RZR. The mechanism of RVR action remains obscure, hence we used the GAL4 hybrid system to identify and characterize an active transcrip- tional silencer in the ligand binding domain (LBD) of RVR. Rigorous deletion and mutational analysis demonstrated that this repressor domain is encoded by amino acids 416-449 of RVR. Furthermore, we demonstrated that efficient repression is dependent on the so-called LBD-specific signature motif, (F/W)AKXXXXFXXLXXXDQXXLL (which spans loop3-4 and helix 4) and helix 5 (H5; identified in the crystal structures of the steroid receptor LBDs). Although RVR is expressed in many adult tissues, including skeletal muscle, and during embryogenesis, its physiological function in differentiation and mammalian development remains unknown. Since other 'orphans4, e.g. COUP-TF II and Rev-erbAα, have been demon- strated to regulate muscle and adipocyte differenti- ation, we investigated the expression and functional role of RVR during mouse myogenesis. In C2C12 myogenic cells, RVR mRNA was detected in proliferat- ing myoblasts and was suppressed when the cells were induced to differentiate into post-mitotic, multinucleated myotubes by serum withdrawal. This decrease in RVR mRNA correlated with the appearance of muscle-specific markers (e.g. myogenin mRNA). RVR 'loss of function' studies by constitutive over-ex- pression of a dominant negative RVRΔE resulted in increased levels of p21Cip1/Waf1 and myogenin mRNAs after serum withdrawal. Time course studies indicated that expression of RVRΔE mRNA results in the precocious induction and accumulation of myogenin and p21 mRNAs after serum withdrawal. In addition, we demonstrated that over-expression of the COUP-TF II and Rev-erbAα receptors in C2C12 cells completely blocked induction of p21 mRNA after serum with- drawal. In conclusion, our studies identified a potent transcriptional repression domain in RVR, character- ized critical amino acids within the silencing region and provide evidence for the physiological role of RVR during myogenesis.
Nucleic Acids Research 10/1996; 24(18). · 9.11 Impact Factor