Functional Architecture of Atrophins

Abstract

Vertebrate genomes harbor two Atrophin genes, Atrophin-1 (Atn1) and Atrophin-2 (Atn2). The Atn1 locus produces a single polypeptide, whereas two different protein products are expressed from the Atn2 (also known as Rere) locus. A long, or full-length, form contains an amino-terminal MTA-2-homologous domain followed by an Atrophin-1-related domain. A short form, expressed via an internal promoter, consists solely of the Atrophin domain. Atrophin-1 can be co-immunoprecipitated along with Atrophin-2, suggesting that the Atrophins ordinarily function together. Mutations that disrupt the expression of the long form of Atrophin-2 disrupt early embryonic development. To determine the requirement for Atrophin-1 during development we generated a null allele. Somewhat surprisingly we found that Atrophin-1 function is dispensable. To gain a better understanding of the requirement for Atrophin function during development, an analysis of the functional domains of the three different gene products was carried out. Taken together, these data suggest that Atrophins function as bifunctional transcriptional regulators. The long form of Atrophin-2 has a transcriptional repression activity that is not found in the other Atrophin polypeptides and that is required for normal embryogenesis. Atrophin-1 and the short form of Atrophin-2, on the other hand, can act as potent and evolutionarily conserved transcriptional activators. Vertebrate genomes harbor two Atrophin genes, Atrophin-1 (Atn1) and Atrophin-2 (Atn2). The Atn1 locus produces a single polypeptide, whereas two different protein products are expressed from the Atn2 (also known as Rere) locus. A long, or full-length, form contains an amino-terminal MTA-2-homologous domain followed by an Atrophin-1-related domain. A short form, expressed via an internal promoter, consists solely of the Atrophin domain. Atrophin-1 can be co-immunoprecipitated along with Atrophin-2, suggesting that the Atrophins ordinarily function together. Mutations that disrupt the expression of the long form of Atrophin-2 disrupt early embryonic development. To determine the requirement for Atrophin-1 during development we generated a null allele. Somewhat surprisingly we found that Atrophin-1 function is dispensable. To gain a better understanding of the requirement for Atrophin function during development, an analysis of the functional domains of the three different gene products was carried out. Taken together, these data suggest that Atrophins function as bifunctional transcriptional regulators. The long form of Atrophin-2 has a transcriptional repression activity that is not found in the other Atrophin polypeptides and that is required for normal embryogenesis. Atrophin-1 and the short form of Atrophin-2, on the other hand, can act as potent and evolutionarily conserved transcriptional activators. The mouse and human genomes contain two Atrophin genes, the normal functions of which are not clear. Gain-of-function polyglutamine extension mutations in the Atrophin-1 gene (Atn1) cause dentato-rubro-pallidioluysial atrophy, a dominant neurodegenerative disease (1.Nagafuchi S. Yanagisawa H. Sato K. Shirayama T. Ohsaki E. Bundo M. Takeda T. Tadokoro K. Kondo I. Murayama N. et al.Nat. Genet. 1994; 6: 14-18Crossref PubMed Scopus (701) Google Scholar). The Atrophin-2 (Atn2) locus, also known as Arg-Glu repeat-encoding (Rere), was first described as encoding an Atrophin-1-related protein that could heterodimerize with Atn1 in vitro (2.Waerner T. Gardellin P. Pfizenmaier K. Weith A. Kraut N. Cell Growth & Differ. 2001; 12: 201-210PubMed Google Scholar, 3.Yanagisawa H. Bundo M. Miyashita T. Okamura-Oho Y. Tadokoro K. Tokunaga K. Yamada M. Hum. Mol. Genet. 2000; 9: 1433-1442Crossref PubMed Scopus (55) Google Scholar). The Atn2 locus is more complex than the Atn1 locus, encoding two polypeptides from a larger palette of domains. In addition to the Atrophin domain, the Atn2 locus encodes the BAH, ELM2, SANT, and GATA domains, which are arranged in a configuration similar to that found in MTA-2, a protein that scaffolds the formation of a repressive chromatin remodeling complex, NuRD (4.Bowen N.J. Fujita N. Kajita M. Wade P.A. Biochim. Biophys. Acta. 2004; 1677: 52-57Crossref PubMed Scopus (251) Google Scholar). Atn2 and Atn1 gene products can be co-immunoprecipitated from embryo extracts, indicating that Atrophins probably carry out at least some of their cellular functions by acting together in a molecular complex (5.Zoltewicz J.S. Stewart N.J. Leung R. Peterson A.S. Development (Camb.). 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar). The single Atrophin gene products that are produced by the Drosophila melanogaster (Atro or grunge) and Caenorhabditis elegans (EGL-27) genomes have domain structures that are reminiscent of the long form of the vertebrate Atrophin-2 (6.Herman M.A. Ch'ng Q. Hettenbach S.M. Ratliff T.M. Kenyon C. Herman R.K. Development (Camb.). 1999; 126: 1055-1064Crossref PubMed Google Scholar, 7.Zhang S. Xu L. Lee J. Xu T. Cell. 2002; 108: 45-56Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), suggesting that this may be the ancestral function with Atn1 and the short form of Atn2 being a more recent evolutionary specialization. Genetic analysis of Atro in Drosophila indicates that it functions as a transcriptional co-repressor during development with important roles in segmentation and planar polarity (8.Wang L. Rajan H. Pitman J.L. McKeown M. Tsai C.C. Genes Dev. 2006; 20: 525-530Crossref PubMed Scopus (79) Google Scholar). Overexpression of a polyglutamine-expanded version of Atn1 in flies indicates that it too can function as a transcriptional repressor in this context (7.Zhang S. Xu L. Lee J. Xu T. Cell. 2002; 108: 45-56Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These studies are consistent with the general idea that Atrophins have an evolutionarily conserved function as transcriptional co-repressors. This is an attractive conclusion, but it cannot be used to explain why evolution has elaborated distinct gene products from an apparently single ancestral form. This evolutionary innovation appears to have been an early and functionally important one, because the teleost, avian, and mammalian lineages all carry two types of Atrophin genes. We have begun to explore the question of distinct functions of the two types of Atrophin gene products using a combination of genetic and biochemical analysis. We previously reported the phenotype of a mutation in Atn2 that selectively disrupts the function of the Atrophin-2 long-form gene product (5.Zoltewicz J.S. Stewart N.J. Leung R. Peterson A.S. Development (Camb.). 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar). Loss of Atr2L causes a diverse set of developmental defects, many of which are associated with a failure in the function of important signaling centers. For example, Shh is not expressed from the anterior notochord, Fgf8 expression in the anterior neural ridge is defective, and the Fgf8-expressing apical ectodermal ridge does not form properly in embryos unable to express Atr2L. Somitogenesis and heart development, processes that are not related in any obvious way to the signaling centers that are defective, also fail to proceed normally indicating that there are additional and diverse roles for Atr2L. To carry the analysis of Atrophin function further, we generated a null allele of Atn1 and carried out in vitro analyses of the Atrophin-2 and Atrophin-1 domains. These data support the idea that the Atrophin domain on its own has a distinct function that is supplied redundantly by the short form of Atrophin-2 (Atr2S) and Atrophin-1. Atrophin-1 Knock-out Mice−The targeting construct was produced by PCR amplification of fragments arms from ES 2The abbreviations used are: ES cell, embryonic stem cell; RERE, Arg-Glu repeat-encoding; CMV, cytomegalovirus; GFP, green fluorescent protein; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; PML, promyelocytic leukemia; POD, PML oncogenic domain; Atr2L, Atrophin-2 long form; Atr2S, Atrophin-2 short form; NLS, nuclear localization signal; HDAC, histone deacetylase; EGFR, epidermal growth factor receptor; E, embryonic day (e.g. E17). cell DNA. The fragments were assembled together with Lox sites made from synthetic oligonucleotides and a PGK-Neo cassette. The construct was sequence verified before being electroporated into the A14 ES cell lines, which were a kind gift from Dr. Bill Skarnes. Correct targeting events (fNEO allele) were identified using a PCR strategy and confirmed by Southern blotting. A correctly targeted ES cell line was electroporated with a Cre expression plasmid, and Cre-mediated deletion events that produced the Atn1Δ allele were identified using PCR. Both Atn1-fNEO and the Atn1Δ ES cell lines were used to generate chimeras and germ-line transmission. mRNA Expression−Radioactively labeled probes were synthesized from cDNA template using a Random Primed DNA labeling kit (Roche Applied Science) and [32P]dCTP (Amersham Biosciences), mouse multiple tissue Northern blots (Clontech) were hybridized and stripped according to manufacturer’s guidelines. For detection of Atrophin-2 transcript isoforms, two Atrophin-2 cDNA fragments (residues 1-1242 and 3162-4680) were used as template for 5′ and 3′ probe synthesis, respectively. To detect Atrophin-1, full-length cDNA was used as template. Human α-actin probe was synthesized from template supplied by Clontech. Constructs−Full-length Atrophin-1 and Atrophin-2 cDNAs were described in Zoltewicz et al. (5.Zoltewicz J.S. Stewart N.J. Leung R. Peterson A.S. Development (Camb.). 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar). Truncated versions of Atrophin-2 were constructed from the full-length clones by restriction fragment ligation or PCR amplification from the cloned cDNA using Expanded High Fidelity PCR (Roche Applied Science). All constructs were sequence verified. For transcription activity assays, full-length or fragments of Atrophin cDNA were cloned into pFA-CMV vector (Stratagene) using appropriate restriction enzyme sites to produce the amino-terminal Gal4 fusion protein. Constructs E through O of Fig. 3 encode the following Atrophin-2 amino acid residues: E, 1-616; F, 1-343; G, 344-570; H, 481-1559; I, 571-1140; J, 1055-1559; K, 1011-1360; L, 1011-1360 without “RE” repeat domain; M, 1148-1360; N, 1323-1559; O, 1055-1148. For protein localization studies, full-length or fragments of Atrophin-2 were cloned into pEGFP-N3 vector (Clontech) to generate carboxyl-terminal GFP fusion proteins. These truncated Atrophin-2-GFP constructs encode the following Atrophin-2 amino acid residues: 1, 1-1559; 2, 571-1559; 3, 1-1219; 4, 571-1140; 5, 571-1409. For Co-immunoprecipitation experiments, amino-terminal-(1-616) and carboxyl-terminal-(481-1559) Atrophin-2 fragments were constructed on pFLAG7 vector (Sigma) to produce amino-terminal FLAG-tagged fusion proteins. Cell Culture, Reporter Assay, and Immunofluorescence Microscopy−COS7, HeLa, and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum. For transcription assay, HEK293 cells were seeded in 12-well plates overnight; when they reached 80∼90% confluence, the cells were co-transfected with 100 ng of pFA-CMV constructs, 500 ng of hRL reporter vector and 50 ng of GL3 internal control plasmids using Lipofectamine 2000 (Invitrogen). Cells were harvested and lysed 24 h later. Renilla luciferase and firefly luciferase activity was measured using a dual luciferase assay kit (Promega) and a Lucy2 luminometer (Anthos Labtec Instruments). COS7 and HeLa cells were cultured and transfected with GFP constructs on 2-well slides. Cells were fixed 24 h later and subjected to a standard immunostaining protocol using antibodies recognizing HDAC1 (Affinity BioReagents), PML, or P300 (Santa Cruz Biotechnology), images were taken using a Zeiss LSM510 fluorescence confocal microscope. Western and Co-Immunoprecipitation−E17 mouse embryos were dissected and genotyped. The hearts were taken for protein extraction and Western blot using Atrophin-2 antibody 286-1 (Zoltewicz et al. (5.Zoltewicz J.S. Stewart N.J. Leung R. Peterson A.S. Development (Camb.). 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar)) and antiglyceraldehyde-3-phosphate dehydrogenase (Chemicon). HEK293 cells were cultured in 60-mm dishes overnight to 70-80% confluence and then transfected with either FLAG7 empty vector or amino- or carboxyl-terminal truncated constructs using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 h and lysed with supplemented with and (Roche Applied Science). extracts, together with either mouse or were with protein (Amersham overnight at were with in and then protein was using and at 100 for were on and to were with antibody and (Santa Cruz Biotechnology), according to a standard Western A of the requirement for Atn1 during development we generated an allele in which of are with sites We then generated a germ-line allele in which Cre-mediated the which the and of the Both PCR and Southern blot analysis were used to the of the deletion not produced and at that Atn1 is To that the mutation been we also carried out Northern blot analysis using The truncated was produced by the allele the of two genes, a is that the Atn2 gene a of Atrophin We additional with which to Atn1 and analysis expression of Atn1 and Atn2 but not the expression of the two Atn2 gene products (2.Waerner T. Gardellin P. Pfizenmaier K. Weith A. Kraut N. Cell Growth & Differ. 2001; 12: 201-210PubMed Google Scholar, 3.Yanagisawa H. Bundo M. Miyashita T. Okamura-Oho Y. Tadokoro K. Tokunaga K. Yamada M. Hum. Mol. Genet. 2000; 9: 1433-1442Crossref PubMed Scopus (55) Google Scholar, J.S. Stewart N.J. Leung R. Peterson A.S. Development (Camb.). 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar). To the expression of the Atrophins during we Northern blots with probes recognizing the Atr2L and All three of the gene products are expressed at and data not The Atr2L has expression through of but is as embryos whereas the has two early and in embryogenesis. this expression it that or of the Atrophin-2 gene products could for the of Atrophin-1. To determine of Atn2 expression to the of Atn2 to for of we measured the protein of Atn2 in Atn1 and in Fig. the expression of protein is by of Mutations that disrupt expression were not but previously we described two different that the expression of Atr2L (5.Zoltewicz J.S. Stewart N.J. Leung R. Peterson A.S. Development (Camb.). 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar). To the of Atr2L could a requirement for Atrophin-1, we the two were at and were and We were also to that were and without that are for Atr2L mutations are not which to a for Atr2L in the of an this analysis some it is consistent with the idea that either Atn1 is that is of a similar function that for the of A of and functional domains have been described for Atrophin-1 to a Atrophin-2 Y. Miyashita T. K. Yamada M. Hum. Mol. Genet. 1999; PubMed Scopus Google Scholar, K. C. J. Y. N. J. Cell 2000; PubMed Scopus Google Scholar, J. K. J. Mol. Cell. PubMed Scopus Google Scholar). To we could functions that were Atrophin-1 and we carried out an in vitro analysis of their functional domains. Atr2L has functions that cannot be by Atrophin-1 or Atr2S, of its functional domains was carried out as of a for studies have that the version of human Atrophin-1 can function as a transcriptional co-repressor when expressed in The Drosophila Atrophin gene can also function as a co-repressor in this assay the idea that this is an of Atrophins than a function produced by the dominant The domain of the Drosophila gene product is similar to that of Atr2L, not that of and Atrophins can it to that is the or the function of Atrophin-1. To this question more we carried out a of Atrophin-1 and Atrophin-2 functions in a transcriptional reporter A dual luciferase reporter in mammalian cells was used in which Atrophin-1, Atr2L, and were with the Gal4 domain and co-transfected with a luciferase reporter gene of Gal4 sites of a we found that the human Atrophin-1, the mouse Atrophin-1 has potent transcriptional can also function as an transcriptional in this assay transcription Atr2L was in this assay it transcriptional suggesting that the Atrophin-2 amino-terminal domain may have as by its domain and with HDAC1 (5.Zoltewicz J.S. Stewart N.J. Leung R. Peterson A.S. Development (Camb.). 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar). To explore this we made truncated versions of Atr2L and in Fig. 3, this analysis that the amino-terminal has the to we not this in repression is potent with constructs the domain for constructs E and analysis of its to act as a transcriptional to the acid amino and Truncated in which this domain is are unable to whereas constructs this domain, in the of other Atrophin-2 that it is and for the transcriptional of This domain is conserved Atrophin-1 and with and The amino acid sequence is in and as has been in other transcriptional domains. The domain that we identified is to a of and acid studies the of this in the of Atrophins with To the of this on transcriptional we these using and found that they were not required for transcriptional Atrophin-2 to has three nuclear localization two in the domain and in the Atrophin domain. has sequence at its A that Atrophin-2 is to a domain, the PML oncogenic domain at the of that the Atr2L was known (2.Waerner T. Gardellin P. Pfizenmaier K. Weith A. Kraut N. Cell Growth & Differ. 2001; 12: 201-210PubMed Google Scholar). To the the functional domains of Atrophin-2 and its we GFP at the of Atr2L and and its localization in mammalian in Fig. 4, Atr2L and are nuclear with a The cellular localization of the two are suggesting that the this localization This also that the in the Atr2L amino-terminal domain is or To the of the domain, we made a of constructs with amino- or carboxyl-terminal of and Atr2L. that all three to to the not on the other hand, a domain required for of the carboxyl-terminal amino of either or Atr2L produced a protein with nuclear localization but without the localization of and Atr2L. This indicates that the acid carboxyl-terminal contains a localization A that Atr2L with the PML protein in a domain. in Fig. the nuclear of Atr2L with This that the acid at the carboxyl-terminal of and Atr2L is a required for the with HDAC1 and P300 in FLAG-tagged fragments of Atrophin-2 were expressed in HEK293 The Western blot detection of FLAG-tagged in control antibody or of HDAC1 with the amino-terminal transcriptional repressor was by Western blotting. The of the histone P300 with the carboxyl-terminal transcriptional of Atrophin-2 was also CBP, a related histone is not associated with either HEK293 cells were transfected with either empty vector three or FLAG-tagged fusion the of Atrophin-2 with HDAC1 and P300 can be in of these with Atrophin-2 and the PML protein. Cells were transfected with an Atrophin-2-GFP and GFP was To detect with the cells were with PML, and P300 with HDAC1 and P300 analysis of Atr2L functional domains that Atr2L can either or can act through of histone as P300 or on the other hand, is the of histone The found in are in histone P300 and in histone HDAC1 P. Cell. 2002; 108: Full Text Full Text PDF PubMed Scopus Google Scholar). This that Atrophin-2 may transcription by transcriptional in To this we and as and carried out to proteins. in Fig. when Atn2 amino- or carboxyl-terminal fragments were with HDAC1 was found to be associated with the amino-terminal consistent with the of this domain in transcriptional The carboxyl-terminal domain, on the other hand, is associated with was not in either of the To this the localization of the cells that transfected with the construct were with HDAC1 or P300 in Fig. HDAC1 was into the Atrophin-2 domain. In HDAC1 was the P300 a more nuclear in it also appears to have been into the Atrophin-2 domain. In there is Atrophin mutations produce segmentation cellular polarity defects, and embryonic The Drosophila Atrophin gene can function as a transcriptional co-repressor by with the and transcription to segmentation (7.Zhang S. Xu L. Lee J. Xu T. Cell. 2002; 108: 45-56Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, L. Rajan H. Pitman J.L. McKeown M. Tsai C.C. Genes Dev. 2006; 20: 525-530Crossref PubMed Scopus (79) Google Scholar). development of the with the domain of a cell to planar polarity M. L. J. K. S. H. Development (Camb.). PubMed Scopus Google Scholar). In this context it is not it is also acting as a transcriptional co-repressor in the development, genetic studies support the idea that as repressor of of the M. S. A. Dev. 2006; PubMed Scopus Google Scholar). In the of are have two encoding three different In a we described an Atn2 mutation that selectively expression of of the two isoforms, Atr2L, expressed from this locus (5.Zoltewicz J.S. Stewart N.J. Leung R. Peterson A.S. Development (Camb.). 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar). the of the Atr2L is the that is similar to that of Atr2L are embryonic with in heart and development, of Shh and Fgf8 expression from anterior signaling centers. have two types of Atrophin genes, Atn1 and two Atn2 the protein expressed by the Atn2 have not been the is consistent with or of the being of and is also suggesting that the function of Atn2 may be more conserved and as a for Atn2 in the of Fgf8 signaling has been described that is consistent with that in Y. R. S. A. 2006; Scopus Google Scholar). Gain-of-function extension mutations in Atn1 cause neurodegenerative The of Atrophin-1 is but that Atrophin-1 can with transcription repressor or and histone M. H. Yamada M. H. S. J. T.M. 2001; PubMed Scopus Google Scholar), have to the that Atrophin-1 functions as a transcriptional We that the functions of Atrophin are the two genes. Atn2 encodes two that to all Atrophin The function of the Atr2L gene product is and whereas that of Atrophin-1 is apparently with that of The two gene products may function as transcriptional in development and of normal function at different developmental and The functions are either similar that Atrophin-1 and can for or their functions are We not any developmental in Atn1 embryos obvious in This is consistent with the idea that can for of Atrophin-1 and additional to support the that extension mutations of Atn1 are than In cultured mammalian Atrophin-1 and a transcription and we the to a conserved acid the Atrophin domain. Atr2L contains an Atrophin domain and an amino-terminal domain. analysis of the transcriptional activity of Atrophin-2 domains potent transcriptional in its carboxyl-terminal domain and transcriptional repressor activity in its amino-terminal with the amino-terminal domain is associated with and the carboxyl-terminal domain, also found in Atrophin-1, is associated with histone These data suggest that and mammalian Atrophin-2 are transcriptional regulators. and function are conserved from Drosophila to with a similar domain and with early developmental in the as and other Atrophin-1 and are gene products and have the to function as transcriptional but they have the to have two of this of Atrophin for its in the of a in which and Atrophin-1 function are of their functions The of data for the of extension mutations in Atn1 are has been that Atrophin-1 as a transcriptional studies suggest that Atr2L is a transcription whereas Atrophin-1 and function as This is by the that Atr2L can with or Atrophin-1, suggesting that repressor function is at least in by the Atr2L is associated with This that extension in Atrophin-1 may the of Atrophin-1 to with complex may its with Atr2L. This the from and The that a to act as a repressor Atrophin-1, is also Atrophin-2 localization to is by a targeting a of which at least is at its carboxyl-terminal are a domain in and other important events in than as CBP, and have been found in P. Cell. 2002; 108: Full Text Full Text PDF PubMed Scopus Google Scholar). Atrophin-2 to and HDAC1 and This indicates that Atn2 may not with HDAC1 and P300 but also with other in extension have been to in domains reminiscent of J. H. K. T. A. A.S. Y. The H. J. C. A. PubMed Scopus Google Scholar, M. T. S. S. H. 2001; PubMed Scopus Google Scholar), suggesting that this localization may a in We found that localization to is not required for the transcriptional of Atrophin-2 in it that it is required in We are to of the Peterson for and to Dr. for and to Dr. for with