ReviewVolume 6 | 2008
Cite this Article: Nuclear Receptor Signaling (2008) 6, e009.
Atrophin proteins: an overview of a new class of nuclear receptor corepressors
Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA (LW and CCT) and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA (LW), present address
The normal development and physiological functions of multicellular organisms are regulated by complex gene transcriptional networks that include myriad transcription factors, their associating coregulators, and multiple chromatin-modifying factors. Aberrant gene transcriptional regulation resulting from mutations among these elements often leads to developmental defects and diseases. This review article concentrates on the Atrophin family proteins, including vertebrate Atrophin-1 (ATN1), vertebrate arginine-glutamic acid dipeptide repeats protein (RERE), and Drosophila Atrophin (Atro), which we recently identified as nuclear receptor corepressors. Disruption of Atrophin-mediated pathways causes multiple developmental defects in mouse, zebrafish, and Drosophila, while an aberrant form of ATN1 and altered expression levels of RERE are associated with neurodegenerative disease and cancer in humans, respectively. We here provide an overview of current knowledge about these Atrophin proteins. We hope that this information on Atrophin proteins may help stimulate fresh ideas about how this newly identified class of nuclear receptor corepressors aids specific nuclear receptors and other transcriptional factors in regulating gene transcription, manifesting physiological effects, and causing diseases.
AIP: ATN1-interacting protein; AML: acute myeloid leukemia; ASH1: absent, small, or homeotic discs 1; ATN1: Atrophin-1; Atro: Atrophin; BAH: bromo adjacent homology; Bks: Brakeless; CHD4: chromodomain helicase DNA binding protein 4; CoREST: corepressor of REST; COUP-TF: chicken ovalbumin upstream promoter-transcription factor; DBD: DNA binding domain; DRPLA: dentatorubral-phallidolusian atrophy; EcR: ecdysone receptor; EGFR: epidermal growth factor receptor; ELM2: EGL-27 and MTA1 homology 2; Eve: Even-skipped; fgf8: fibroblast growth factor 8; ftz: fushi-tarazu; HDAC: histone deacetylase; HEF1: human enhancer of filamentation 1; HMTase: histone methyltransferase; IRSp53: insulin receptor tyrosine kinase substrate protein of 53kD; KD: lysine-aspartic acid; KE: lysine-glutamic acid; kni: knirps; LBD: ligand binding domain; LSD1: lysine specific demethylase 1; MBD: methyl CpG binding protein; MIER1: mesoderm induction early response 1; MTA: metastasis-associated protein; N-CoR: nuclear receptor corepressor; NuRD: nucleosome remodeling and histone deacetylase; Orc1: origin recognition complex 1; PBAF: polybromo, BRG-1 associated factors; PcG: polycomb group; PPAR: peroxisome proliferator-activated receptor; RAR: retinoic acid receptor; RD: arginin-aspartic acid; RERE: arginine glutamic acid repeats encoded protein; RE-repeats: arginine-glutamic acid dipeptide repeats; REST: RE1-silencing transcriptional factor; RSC: remodeling the structure of chromatin; RXR: retinoid-X receptor; SANT: SWI3/ADA2/N-CoR/TFIII-B; Sbb: Scribbler; SH3: Src homology 3; Sir: silent information regulator; SMRT: silencing mediator of retinoid and thyroid hormone receptors; SMRTER: SMRT-related ecdysone receptor interacting factor; SVP: Seven-Up; TGFβ: transforming growth factor β; Tll: Tailless; Tlx: vertebrate Tailless; TR: thyroid hormone receptor; trxG: trithorax group; USP: Ultraspiracle; UTR: untranslated region; ZNF: zinc finger protein.
Atrophin proteins are conserved transcriptional corepressors that, as we recently demonstrated, are involved in nuclear receptor signaling. This review article provides an overview of our current understanding of this new class of nuclear receptor corepressors, which includes vertebrate Atrophin-1 (ATN1), vertebrate arginine glutamic acid repeats encoded protein (RERE), and Drosophila Atrophin (Atro). This review discusses their identification, expression and cellular patterns, connections with nuclear receptor and epidermal growth factor signaling pathways, functional domains, associations with histone modifying factors, roles in animal development, and implications in human diseases. Deepening our understanding of the Atrophin proteins will expand our insights about how these nuclear receptor corepressors help transcriptional factors, such as nuclear receptors, regulate gene transcription, exert biological effects, and cause diseases.
The Atn1 gene was initially cloned by Christopher Ross’s group at Johns Hopkins University as CTG-B37 in their screens for human genes containing CTG/CAG or CCG/CGG triplet repeats [Li et al., 1993]. In 1994, the Atn1 gene was assigned to the short arm of chromosome 12 and was simultaneously reported by Masao Yamada (National Children’s Medical Research Center, Japan) and Shoji Tsuji (Niigata University, Japan) to be a causative factor for the neurodegenerative disease DRPLA (dentatorubral-pallidoluysian atrophy) when the CAG repeat within the coding region of the Atn1 gene is expanded [Koide et al., 1994; Nagafuchi et al., 1994]. In addition to the glutamine-repeat (coded by the CAG repeat), ATN1 also contains two arginine-glutamic acid dipeptide-like repeats (RE-repeats) that reside at its C-terminus. In search of potential ATN1 homologues, Masao Yamada’s group identified RERE [Yanagisawa et al., 2000], named after the RE-like repeats present at the C-terminus of the protein. Owing to its resemblance with ATN1, RERE has also been referred to as Atrophin-2.
In 2002, Drosophila Atro was reported by two groups independently: Tian Xu’s group at Yale University, using a dominant female sterile-FRT/FLP approach, identified Atro during their search for lethal mutations that affect growth and patterning [Zhang et al., 2002]; and Steve Kerridge and Laurent Fansano’s group at the IBDML (Marseille, France), isolated Atro through an EMS mutagenesis in their screening for mutations that affect the expression of the region-specific pattern gene teashirt [Erkner et al., 2002]. In the latter study, Atro was also referred to as Grunge. Drawing on their characterization of Atro mutants, both groups reported that Atro is an essential gene involved in multiple developmental pathways.
Northern blot analysis reveals that Atn1 transcripts are ubiquitously expressed in a variety of neuronal and non-neuronal tissues, including brain, heart, lung, kidney, placenta, skeletal muscle and liver [Kanazawa, 1998; Onodera et al., 1995]. At the cellular level, ATN1 is predominantly localized to the cytoplasm of neuronal cells [Knight et al., 1997], although in cultured HeLa cells, transiently expressed ATN1 was found to be present within the nucleus [Okamura-Oho et al., 1999; Yanagisawa et al., 2000].
The Rere gene codes for two transcripts with estimated sizes of 7.4 and 9.4 kb. Their expression varies considerably in different tissues, being particularly abundant in the cerebellum, testis, uterus, prostate, skeletal muscle and kidney, and relatively lower in lung, colon and leukocytes [Waerner et al., 2001; Yanagisawa et al., 2000]. The cellular localization of RERE protein was reported based on transient overexpression conditions. In transfected HeLa cells, although a low level of RERE can be detected in the cytoplasm, RERE localizes predominantly to the nucleus, where it forms a speckle-like pattern resembling that of promyelocytic leukemia bodies [Waerner et al., 2001; Yanagisawa et al., 2000].
In Drosophila, Atro is at first ubiquitously distributed throughout the embryo during the early stage [Erkner et al., 2002; Zhang et al., 2002]. During later stages of embryogenesis, a higher level of Atro becomes more apparent at the ventral nerve cord region. At the third instar larvae stage, Atro resumes its ubiquitously expressed pattern in various examined imaginal tissues. Throughout different developmental stages, Atro mainly localizes to the nucleus of cells, within which endogenous Atro forms a speckle-like pattern [Zhang et al., 2002] . When overexpressed in human cells, consistently, Atro also forms a nuclear speckle pattern [Wang et al., 2008; Wang et al., 2006], indicating that formation of a nuclear speckle pattern is a shared property of Atro and RERE. Interestingly, this unique cellular feature does not apply to ATN1, probably because it lacks the SANT (SWI3/ADA2/N-CoR/TFIII-B) domain (more information about this domain will be provided below), which is essential for RERE to form nuclear foci [Wang et al., 2006].
A recent study shows that Atn1 is not an essential gene during mouse development, because Atn1 knockout mouse is indistinguishable from wild-type mouse [Shen et al., 2007]. In contrast, Rere is required for both mouse and zebrafish to develop and to survive [Asai et al., 2006; Plaster et al., 2007; Zoltewicz et al., 2004]. In mouse, mutations of Rere cause a failure in closing the anterior neural tube and fusion of the telencephalic and optic vesicles during early embryogenesis [Zoltewicz et al., 2004]. Other defects include a smaller first branchial arch with a deficit in the mesenchymal component, a fail-to-loop heart tube and somites with irregular shapes and sizes. All homozygous mice die shortly after E9.5, possibly owing to cardiac failure. Accompanying most of these described phenotypes is the disruption of important signaling centers, including the loss of sonic hedgehog in the anterior notochord, and defective expression of fibroblast growth factor 8 (fgf8) in the anterior neural ridge. In zebrafish, modulations of Fgf8 signaling by RERE were also observed in the context of posterior mesoderm formation, midbrain-hindbrain boundary maintenance, and the development of pharyngeal cartilage and inner ear [Asai et al., 2006; Plaster et al., 2007]. The apparently divergent effects of ATN1 and RERE on animal development suggest that their functions are not equivalent. Since ATN1 resembles a truncated form of RERE (Figure 1), the portion of RERE that is missing from ATN1 could account for their functional differences.
As the Drosophila counterpart of RERE, Atro is also crucial for the development of Drosophila. Embryos deficient of maternal or both the maternal and zygotic component of Atro exhibit severe segmentation defects [Erkner et al., 2002; Kankel et al., 2004; Zhang et al., 2002]. Such Atro mutant embryos also lack some ventral patterning elements, and often show holes in the ventral cuticle, suggesting that in addition to its better characterized involvement in anterior-posterior patterning, Atro also participates in dorsal-ventral axis patterning. In embryos devoid of Atro protein (using a null Atro allele), the expression patterns of several segmentation gap genes, such as hunchback, Krüpple and knirps (kni), are altered [Erkner et al., 2002; Wang et al., 2006]. The altered expression of gap genes can partly explain the subsequent abnormal expression of pair-rule genes, such as fushi-tarazu (ftz) and hairy. However, evidence from experiments using hypomorphic Atro alleles reveals that Atro also functions downstream of the gap genes. For example, embryos derived from females carrying hypomorphic Atro alleles do not show loss of continuous segments (gap gene phenotype), but, instead, display loss of even-numbered (ftz-dependent) engrailed stripes [Kankel et al., 2004; Zhang et al., 2002]. Consistently, the cuticles of such Atro mutant embryos also display strong ftz-like segmentation defects. Although these ftz-phenotypes could result from the loss of the repressive activity of a pair-rule protein Even-skipped (Eve), as suggested in [Zhang et al., 2002], other regulatory pathways that control the expression of ftz may be affected by Atro mutations as well.
Atro is also involved in other developmental pathways. For example, mutations of Atro affect planar polarity in the eyes in a non-autonomous manner [Fanto et al., 2003; Zhang et al., 2002], and also cause patterning defects in the legs [Erkner et al., 2002; Wehn and Campbell, 2006]. Atro can antagonize the activity of EGFR (epidermal growth factor receptor) as well [Charroux et al., 2006], since in the Drosophila wing, mutation of Atro or reduced expression of Atro results in ectopic vein formation [Charroux et al., 2006; Kankel et al., 2004; Wang et al., 2008]. (Note that wing vein formation is known to be initiated by activated EGFR [Martin-Blanco et al., 1999]). The possibility that Atro negatively regulates EGFR signaling is further supported by the genetic interactions between Atro and several key genetic components in the EGFR signaling pathways, including argos, rolled, pointed, and yan (anterior open), in both Drosophila wing and eye [Charroux et al., 2006].
The function of Atro has also been connected with microRNA. Steve Cohen’s group at Temasek, Singapore, reported recently that Atro is a target of miR-8, after they identified four potential miR-8 binding sites at the 3’ untranslated region (3’UTR) of the Atro gene [Karres et al., 2007]. In support of their finding, the leg phenotype displayed by mir-8 mutants is similar to that caused by the overexpression of Atro [Charroux et al., 2006; Karres et al., 2007]. Additionally, reducing Atro activity can rescue the survival rate of these mir-8 mutant flies [Karres et al., 2007]. Regulation of Atro by miR-8 appears to be a conserved feature, because the same group found that 3’UTR of the Rere gene also contains three potential targets of miR-429 and miR-200b, which are the vertebrate counterparts of miR-8. Therefore, a key function of miR-8 and miR-429/miR-200b may be to fine-tune the expression levels of Atro and RERE in Drosophila and in vertebrates. Consequently, altered expression of miR-8, -200b, or -429 is expected to affect the developmental pathways that are regulated by Atro or RERE.
Our lab recently defined Atrophin proteins as corepressors of nuclear receptors through our work with Drosophila Tailless (Tll) and vertebrate Tlx [Wang et al., 2006], which are two closely related nuclear receptors belonging to the 2E subgroup of the nuclear receptor family (NURSA, http://www.nursa.org/). An interaction between Tlx and ATN1 was also reported by Ron Evans’ lab at the Salk Institute [Zhang et al., 2006].
Tll is a gap protein involved in specifying terminal cell fate during early embryogenesis [Mahoney and Lengyel, 1987; Pignoni et al., 1990], and it is also implicated in neurogenesis [Younossi-Hartenstein et al., 1997]. In parallel with the roles of Tll in Drosophila nervous system development, Tlx participates in regulating vertebrate retinal and forebrain development [Miyawaki et al., 2004; Roy et al., 2002; Zhang et al., 2006] and in maintaining adult neural stem cells [Shi et al., 2004; Sun et al., 2007; Zhang et al., 2008]. Both Tll and Tlx have long been known to exert potent transcriptional repression [Hoch et al., 1992; Pankratz et al., 1992; Pankratz et al., 1989; Yu et al., 1994] and have been defined as dedicated transcriptional repressors [Moran and Jimenez, 2006]. The underlying molecular mechanisms, however, remained unclear until our discovery of their association with Atrophin proteins.
Our results show that Tll/Tlx-Atrophin interactions are mediated through the ligand binding domain (LBD) of Tll/Tlx and the C-terminal regions of the three Atrophin proteins [Wang et al., 2006] (regions marked in blue, Figure 1). In keeping with these observed physical interactions between Tll/Tlx and Atrophin proteins, our in vivo studies in Drosophila demonstrated that Atro indeed assists Tll in repressing the expression of kni, a segmentation gap gene known to be a direct target of Tll [Pankratz et al., 1992]. The following data indicate that Atro is involved in the Tll-regulatory pathways: (1) depletion of Atro from Drosophila embryos leads to derepression of kni similar to that in tll mutant embryos; (2) mutation of Atro in tll mutant embryos enhances the kni derepression; and (3) Atro is naturally present in a region within the kni promoter containing a defined Tll-binding site, as demonstrated by chromatin immunoprecipitation assays.
A recent report reveals that regulation of kni by Atro also involves another nuclear protein, called Brakeless (Bks, also referred to as Scribbler, Sbb). Mattias Mannervik’s group (Stockholm University, Sweden) reported that Atro and Bks/Sbb physically and genetically interact with each other to regulate kni expression [Haecker et al., 2007]. This finding resonates with an earlier report from Gerard Campbell’s group at the University of Pittsburgh, which showed that Atro interacts genetically with sbb to repress the expression of several genes, including thickveins, runt, aristaless and Bar, in fly imaginal discs [Wehn and Campbell, 2006]. Mannervik’s group further demonstrated that a human homolog of Bks/Sbb, zinc finger protein 608 (ZNF608), can also interact with ATN1 directly [Haecker et al., 2007]. Based on this finding, it is possible that ZNF608 and another Bks/Sbb-related protein, ZNF609, may be involved in nuclear receptor signaling in vertebrates as well.
Aside from Tll/Tlx, our data also show that Atrophin proteins interact with additional nuclear receptors. For example, Atrophin proteins bind several members of the NR2F group, including human chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and its Drosophila homologue Seven-Up (SVP) [Wang et al., 2006]. This result was somewhat surprising, because NR2F proteins are also known to interact with SMRT family corepressor proteins [Bailey et al., 1997; Shibata et al., 1997], including SMRT (silencing mediator of retinoid and thyroid hormone receptors) [Chen and Evans, 1995], N-CoR (nuclear receptor corepressor) [Horlein et al., 1995], and Drosophila SMRTER (SMRT-related ecdysone receptor interacting factor) [Tsai et al., 1999]. Therefore, certain nuclear receptors, like COUP-TF/SVP, are able to bind two classes of nuclear receptor corepressors, perhaps by means of flexible structural configurations. Whether Atrophin- and SMRT-family proteins bind COUP-TF/SVP in a competitive or a collaborative manner, and whether Atrophin proteins can interact with additional nuclear receptors, are important questions that still wait to be addressed.
The strong transcriptional repressive activity associated with Atrophin proteins has been reported by numerous laboratories. For example, mutations of Atro cause derepression of several segmentation gap genes in Drosophila [Erkner et al., 2002]; Atro binds the transcriptional repressor Even-skipped (Eve) and mediates its transcriptional repressive effect [Zhang et al., 2002]; Atro and ATN1 can provide a heterologous Gal4 DNA-binding domain the ability to repress gene transcription in Drosophila [Zhang et al., 2002]; and the N-terminal regions of both RERE and Atro mediate st