Interactive Fly, Drosophila

Chromatin mediated activation and silencing of retinoic acid responsive genes: Histone acetylation

Histone acetylation is thought to have a role in transcription. To gain insight into the role of histone acetylation in retinoid-dependent transcription, a study was performed of the effects of trichostatin A (TSA), a specific inhibitor of histone deacetylase, on P19 embryonal carcinoma cells. Coaddition of TSA and retinoic acid (RA) markedly enhances neuronal differentiation in these cells, although TSA alone does not induce differentiation but causes extensive apoptosis. Consistent with the cooperative effect of TSA and RA, coaddition of the two agents synergistically enhances transcription from stably integrated RA-responsive promoters. The transcriptional synergy by TSA and RA requires the RA-responsive element and a functional retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimer, both obligatory for RA-dependent transcription. TSA leads to promoter activation by an RXR-selective ligand, which otherwise is inactive in transcription. In addition, TSA enhances transcription from a minimum basal promoter, independently of the RA-responsive element. TSA alone or in combination with RA increases in vivo endonuclease sensitivity within the RA-responsive promoter, suggesting that TSA treatment might alter a local chromatin environment to enhance RXR/RAR heterodimer action. Thus, these results indicate that histone acetylation influences activity of the heterodimer, which is in line with the observed interaction between the RXR/RAR heterodimer and a histone acetylase (Minucci, 1997).

The binding of lipophilic hormones, retinoids and vitamins to members of the nuclear-receptor superfamily modifies the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300, which function as co-activators of transcription, and RIP-140, TIF-1 and TRIP-1/SUG-1 whose functions are unclear. A short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. The ability of SRC-1 to bind the estrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the estrogen receptor that are required for its ligand-induced activation function. It is proposed that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins (Heery, 1997).

The functionally conserved proteins CBP and p300 act in conjunction with other factors to activate transcription of DNA. A new factor, p/CIP, has been discovered that is present in the cell as a complex with CBP and is required for transcriptional activity of nuclear receptors and other CBP/p300-dependent transcription factors. The highly related nuclear-receptor co-activator protein NCoA-1 is also specifically required for ligand-dependent activation of genes by nuclear receptors. p/CIP, NCoA-1 and CBP all contain related leucine-rich charged helical interaction motifs that are required for receptor-specific mechanisms of gene activation, and that allow the selective inhibition of distinct signal-transduction pathways (Torchia, 1997).

Nuclear receptor corepressors - interaction with other nuclear receptors and other transcription factors

Cognate cDNAs are described for 2 of the 10 thyroid hormone receptor-associated proteins (TRAPs) that are immunopurified with thyroid hormone receptor alpha (TRalpha) from ligand-treated HeLa (alpha-2) cells. Both TRAP220 and TRAP100 contain LXXLL domains found in other nuclear receptor-interacting proteins, and both appear to reside in a single complex with other TRAPs (in the absence of TR). However, only TRAP220 shows a direct ligand-dependent interaction with TRalpha, and these interactions are mediated through the C terminus of TRalpha and (at least in part) the LXXLL domains of TRAP220. TRAP220 also interacts with other nuclear receptors (vitamin D receptor, retinoic acid receptor alpha, retinoid X receptor alpha, peroxisome proliferation-activated receptor [PPAR]alpha, PPARgamma and, to a lesser extent, estrogen receptor) in a ligand-dependent manner, whereas TRAP100 shows only marginal interactions with estrogen receptor, retinoid X receptor alpha, PPARalpha, and PPARgamma. Consistent with these results, TRAP220 moderately stimulates human TRalpha-mediated transcription in transfected cells, whereas a fragment containing the LXXLL motifs acts as a dominant negative inhibitor of nuclear receptor-mediated transcription both in transfected cells (TRalpha) and in cell free transcription systems (TRalpha and vitamin D receptor). These studies indicate that TRAP220 plays a major role in anchoring other TRAPs to TRalpha during the function of the TRalpha-TRAP complex and, further, that TRAP220 (possibly along with other TRAPs) may be a global coactivator for the nuclear receptor superfamily (Yuan, 1998).

CBF1/RBP-Jkappa, the mammalian homolog of Drosophila Suppressor of Hairless [Su(H)], switches from a transcriptional repressor to an activator upon Notch activation. The mechanism whereby Notch regulates this switch is not clear. Prior to induction, CBF1/RBP-Jkappa interacts with a corepressor complex containing SMRT (silencing mediator of retinoid and thyroid hormone receptors) and the histone deacetylase HDAC-1. This complex binds via the CBF1 repression domain, and mutants defective in repression fail to interact with the complex. Activation by Notch disrupts the formation of the repressor complex, thus establishing a molecular basis for the Notch switch. ESR-1, a Xenopus gene activated by Notch and X-Su(H), is induced in animal caps treated with TSA, an inhibitor of HDAC-1. The functional role for the SMRT/HDAC-1 complex in CBF1/RBP-Jkappa regulation reveals a novel genetic switch in which extracellular ligands control the status of critical nuclear cofactor complexes (Kao, 1998).

SMRT has been shown to associate with mSin3A (see Drosophila Sin3A) and the histone deacetylase HDAC1 as part of a large corepressor complex. It was reasoned that CBF1-mediated repression should be compromised in the presence of an inhibitor of histone deacetylase such as trichostatin A (TSA). This was tested in Xenopus animal caps, which are known to respond to the Notch signaling pathway. Specifically, the effects of TSA were assessed on the expression of ESR-1, an E(spl)-related gene from Xenopus. Expression of ESR-1, like similar genes in Drosophila, is induced in neural tissue by activated (cytoplasmic) forms of Xenopus Notch or by the Notch ligand, X-Delta-1. Because induction of ESR-1 expression by Notch appears to be mediated by Xenopus Su(H) [X-Su(H)], it was asked whether induction is enhanced by TSA. Such a result would support the idea that X-Su(H) might inhibit the expression of Notch target genes such as ESR-1 via the SMRT deacetylase complex. Expression of ESR-1 in neuralized animal caps is induced by X-Delta-1 in a dose-dependent manner, with the effects of X-Delta-1 on ESR-1 expression saturating at 1 ng of injected RNA. In the presence of TSA, the induction of ESR-1 transcripts in response to X-Delta-1 is enhanced two- to threefold at each dose. In the presence of TSA, the lowest dose of X-Delta-1 RNA (0.25 ng) induces levels of ESR-1 mRNA comparable to the saturating level induced in the absence of TSA. These results are consistent with the prediction that Notch target genes are derepressed by treatment with TSA, possibly through the inhibition of SMRT-associated histone deacetylases. To assay whether HDAC-1 associates with CBF1 in vivo, coimmunoprecipitation experiments were performed. Cells were transfected with Flag-CBF1 in the presence or absence of HDAC-1 and immunoprecipitated with Flag antibody. The HDAC-1 is coimmunoprecipiated only in the presence of CBF1. An interaction between CBF1 and HDAC-1 was also assessed through GST pull-down assays. HDAC-1 interacts with GST-CBF1. As with SMRT, HDAC-1 does not interact with a repression-defective mutant, CBF1. On the basis of these results, a model is proposed to explain this switch in which CBF1/RBP-Jkappa mediates repression of genes through the recruitment of a corepressor complex containing SMRT and histone deacetylase activity. In the absence of any positive acting factor, that is, TAN-1 (translocation-associated Notch, a truncated form of Notch1 that contains only the cytoplasmic domain), the CBF1/RBP-Jkappa exists as a corepressor complex. In the presence of activated Notch signaling, the intracellular domain of Notch would translocate to the nucleus and displace the corepressor complex. It remains to be tested whether a Notch/CBF1 complex recruits a coactivating complex (Kao, 1998).

Interaction of RAR with Polycomb protein complexes

Ectopic repression of retinoic acid (RA) receptor target genes by PML/RARA (promyelocytic locus gene/RA receptor alpha fusion protein) and PLZF/RARA fusion protein through aberrant recruitment of nuclear corepressor complexes drives cellular transformation and acute promyelocytic leukemia (APL) development. In the case of PML/RARA, this repression can be reversed through treatment with all-trans RA (ATRA), leading to leukemic remission. However, PLZF/RARA ectopic repression is insensitive to ATRA, resulting in persistence of the leukemic diseased state after treatment, a phenomenon that is still poorly understood. This study shows that, like PML/RARA, PLZF/RARA expression leads to recruitment of the Polycomb-repressive complex 2 (PRC2) Polycomb group (PcG) complex to RA response elements. However, unlike PML/RARA, PLZF/RARA directly interacts with the PcG protein Bmi-1 and forms a stable component of the PRC1 PcG complex, resulting in PLZF/RARA-dependent ectopic recruitment of PRC1 to RA response elements. Upon treatment with ATRA, ectopic recruitment of PRC2 by either PML/RARA or PLZF/RARA is lost, whereas PRC1 recruited by PLZF/RARA remains, resulting in persistent RA-insensitive gene repression. Bmi-1 is essential for the PLZF/RARA cellular transformation property and implicates a central role for PRC1 in PLZF/RARA-mediated myeloid leukemic development (Boukarabila, 2009).

RAR and long-term potentiation and depression

Hippocampal long-term potentiation (LTP) and long-term depression (LTD) are the most widely studied forms of synaptic plasticity thought to underlie spatial learning and memory. RARbeta deficiency in mice virtually eliminates hippocampal CA1 LTP and LTD. It also results in substantial performance deficits in spatial learning and memory tasks. Surprisingly, RXRgamma null mice exhibit a distinct phenotype in which LTD is lost whereas LTP is normal. Thus, while retinoid receptors contribute to both LTP and LTD, they do so in different ways. These findings not only genetically uncouple LTP and LTD but also reveal a novel and unexpected role for vitamin A in higher cognitive functions (Chiang, 1998).

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