Identification of Direct Thyroid Hormone Response Genes Reveals the Earliest Gene Regulation Programs during Frog Metamorphosis
2009; Elsevier BV; Volume: 284; Issue: 49 Linguagem: Inglês
10.1074/jbc.m109.066084
ISSN1083-351X
AutoresBiswajit Das, Rachel A. Heimeier, Daniel R. Buchholz, Yun‐Bo Shi,
Tópico(s)Physiological and biochemical adaptations
ResumoThyroid hormone (T3) is essential for normal development and organ function throughout vertebrates. Its effects are mainly mediated through transcriptional regulation by T3 receptor (TR). The identification and characterization of the immediate early, direct target genes are thus of critical importance in understanding the molecular pathways induced by T3. Unfortunately, this has been hampered by the difficulty to study gene regulation by T3 in uterus-enclosed mammalian embryos. Here we used Xenopus metamorphosis as a model for vertebrate postembryonic development to identify direct T3 response genes in vivo. We took advantage of the ability to easily induce metamorphosis with physiological levels of T3 and to carry out microarray analysis in Xenopus laevis and genome-wide sequence analysis in Xenopus tropicalis. This allowed us to identify 188 up-regulated and 249 down-regulated genes by T3 in the absence of new protein synthesis in whole animals. We further provide evidence to show that these genes contain functional TREs that are bound by TR in tadpoles and that their promoters are regulated by TR in vivo. More importantly, gene ontology analysis showed that the direct up-regulated genes are enriched in categories important for transcriptional regulation and protein degradation-dependent signaling processes but not DNA replication. Our findings thus revealed the existence of interesting pathways induced by T3 at the earliest step of metamorphosis. Thyroid hormone (T3) is essential for normal development and organ function throughout vertebrates. Its effects are mainly mediated through transcriptional regulation by T3 receptor (TR). The identification and characterization of the immediate early, direct target genes are thus of critical importance in understanding the molecular pathways induced by T3. Unfortunately, this has been hampered by the difficulty to study gene regulation by T3 in uterus-enclosed mammalian embryos. Here we used Xenopus metamorphosis as a model for vertebrate postembryonic development to identify direct T3 response genes in vivo. We took advantage of the ability to easily induce metamorphosis with physiological levels of T3 and to carry out microarray analysis in Xenopus laevis and genome-wide sequence analysis in Xenopus tropicalis. This allowed us to identify 188 up-regulated and 249 down-regulated genes by T3 in the absence of new protein synthesis in whole animals. We further provide evidence to show that these genes contain functional TREs that are bound by TR in tadpoles and that their promoters are regulated by TR in vivo. More importantly, gene ontology analysis showed that the direct up-regulated genes are enriched in categories important for transcriptional regulation and protein degradation-dependent signaling processes but not DNA replication. Our findings thus revealed the existence of interesting pathways induced by T3 at the earliest step of metamorphosis. IntroductionThyroid hormone (T3) 2The abbreviations used are: T3thyroid hormoneTRthyroid receptorTREthyroid response elementRXR9-cis-retinoic acid receptorGOGene ontologyCHXcycloheximideqRTquantitative reverse transcriptionChIPchromatin immunoprecipitation. is critical for adult organ homeostasis and function and also essential for vertebrate development (1.Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar, 2.Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1501) Google Scholar, 3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley & Sons, Inc., New York1999Google Scholar, 5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar, 6.Freake H.C. Oppenheimer J.H. Annu Rev. Nutr. 1995; 15: 263-291Crossref PubMed Scopus (155) Google Scholar, 7.Franklyn J.A. Gammage M.D. Trends Endocrinol. Metab. 1996; 7: 50-54Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 8.Silva J.E. Thyroid. 1995; 5: 481-492Crossref PubMed Scopus (308) Google Scholar, 9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar). T3 deficiency during development leads to severe developmental defects in mammals, including cretinism in human, which is characterized by severe short stature and mental retardation (5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar). During early mammalian development, there is little T3 in the fetus, although some maternal T3 reaches the embryo. High levels of T3 are present only during the so-called postembryonic period, which spans from several months prior to birth to several months after birth in human (3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 10.Howdeshell K.L. Environ. Health Perspect. 2002; 110: 337-348Crossref PubMed Scopus (306) Google Scholar). This is a critical period of organ growth and maturation, and thus, not surprisingly, T3 deficiency during this period causes severe developmental defects (5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar, 11.Porterfield S.P. Hendrich C.E. Endocr. Rev. 1993; 14: 94-106PubMed Google Scholar, 12.Hsu J.H. Brent G.A. TEM. 1998; 9: 103-112Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Interestingly, appropriate levels of maternal T3 are also important to ensure proper mammalian development (13.de Escobar G.M. Obregón M.J. del Rey F.E. Best Pract. Res. Clin. Endocrinol. Metab. 2004; 18: 225-248Crossref PubMed Scopus (431) Google Scholar, 14.de Escobar G.M. Obregón M.J. del Rey F.E. Public Health Nutrition. 2007; 10: 1554-1570Crossref PubMed Scopus (238) Google Scholar, 15.Anselmo J. Cao D. Karrison T. Weiss R.E. Refetoff S. JAMA. 2004; 292: 691-695Crossref PubMed Scopus (211) Google Scholar). These observations suggest that proper levels of T3 in both the mother and fetus are critical for mammalian development, which makes it difficult to separate the direct effects of T3 on the development of the fetus versus indirect effects through maternal influence. Furthermore, there are only a limited number of known direct T3 response genes in different model systems, and no systematic analysis has been carried out to isolate such genes in development. All these have hampered our understanding of how T3 regulates development in vivo.Amphibian metamorphosis is a postembryonic process that is also dependent on T3 (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley & Sons, Inc., New York1999Google Scholar, 16.Gilbert L.I. Tata J.R. Atkinson B.G. Metamorphosis: Post-embryonic Reprogramming of Gene Expression in Amphibian and Insect Cells. Academic Press, New York1996Google Scholar). Studies in anurans such as Xenopus laevis in the past century have shown that T3 controls every aspect of metamorphosis and is both necessary and sufficient (9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar, 18.Buchholz D.R. Tomita A. Fu L. Paul B.D. Shi Y.B. Mol. Cell. Biol. 2004; 24: 9026-9037Crossref PubMed Scopus (112) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). During metamorphosis, different tissues have different fates (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley & Sons, Inc., New York1999Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar). For example, the tail and gills undergo resorption, the brain, skin, intestine, and other visceral organs undergo remodeling while the limbs are generated de novo. All these changes are controlled by T3 in mostly organ-autonomous manner.T3 action is primarily mediated through thyroid receptors (TRs), which are transcription factors and are members of nuclear receptor superfamily (1.Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar, 2.Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1501) Google Scholar, 9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar, 20.Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 21.Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar). TRs bind to chromosomal sites in the promoter regions (known as thyroid response elements (TRE)) of direct response genes of T3. For T3-inducible genes, these binding sites are often composed of two direct repeats of the consensus sequence AGGTCA with a 4-bp spacer sequence. TR functions mainly as a heterodimer with 9-cis-retinoic acid receptor (RXR) at these TREs and represses and activates these genes by recruiting corepressor and coactivator complexes in the absence or presence of T3, respectively.We have previously proposed a dual function model for TR function during anuran metamorphosis (22.Shi Y.B. Wong J. Puzianowska-Kuznicka M. Stolow M.A. BioEssays. 1996; 18: 391-399Crossref PubMed Scopus (145) Google Scholar, 23.Sachs L.M. Damjanovski S. Jones P.L. Li Q. Amano T. Ueda S. Shi Y.B. Ishizuya-Oka A. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 2000; 126: 199-211Crossref PubMed Scopus (102) Google Scholar). According to this model, in the absence of T3, TR/RXR heterodimers repress direct target genes to ensure proper growth of the premetamorphic tadpoles and prevent premature metamorphic organ transformations. In the presence of T3, TR/RXR heterodimers activate these target genes to initiate metamorphosis. Over the years, studies by us and others have shown that TRs are both necessary and sufficient to mediate the metamorphic effects of T3 in X. laevis (9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). Thus all the remarkable morphological and developmental changes in the different tissues are effected through gene expression cascades initiated by TRs. Furthermore, TRs indeed have dual functions during metamorphosis. They recruit corepressor complexes in premetamorphic tadpoles to control metamorphic timing and recruit coactivator complexes to initiate metamorphosis (24.Paul B.D. Fu L. Buchholz D.R. Shi Y.B. Mol. Cell. Biol. 2005; 25: 5712-5724Crossref PubMed Scopus (56) Google Scholar, 25.Havis E. Sachs L.M. Demeneix B.A. EMBO Rep. 2003; 4: 883-888Crossref PubMed Scopus (53) Google Scholar, 26.Sato Y. Buchholz D.R. Paul B.D. Shi Y.B. Mech. Dev. 2007; 124: 476-488Crossref PubMed Scopus (53) Google Scholar, 27.Paul B.D. Buchholz D.R. Fu L. Shi Y.B. J. Biol. Chem. 2005; 280: 27165-27172Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28.Paul B.D. Buchholz D.R. Fu L. Shi Y.B. J. Biol. Chem. 2007; 282: 7472-7481Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In addition, the levels of coactivator complexes also regulate the rate of metamorphic progression (29.Matsuda H. Paul B.D. Choi C.Y. Hasebe T. Shi Y.B. Mol. Cell. Biol. 2009; 29: 745-757Crossref PubMed Scopus (41) Google Scholar).Using subtractive hybridization screening and microarrays, we and others have previously documented the differentially expressed genes in different organs during both T3-induced and natural metamorphosis (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley & Sons, Inc., New York1999Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar, 30.Denver R.J. Pavgi S. Shi Y.B. J. Biol. Chem. 1997; 272: 8179-8188Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 31.Buchholz D.R. Heimeier R.A. Das B. Washington T. Shi Y.B. Dev. Biol. 2007; 303: 576-590Crossref PubMed Scopus (74) Google Scholar, 32.Das B. Cai L. Carter M.G. Piao Y.L. Sharov A.A. Ko M.S. Brown D.D. Dev. Biol. 2006; 291: 342-355Crossref PubMed Scopus (100) Google Scholar, 33.Cai L. Das B. Brown D.D. Dev. Biol. 2007; 304: 260-271Crossref PubMed Scopus (11) Google Scholar). These studies have provided us with an understanding of the global gene expression changes and signaling pathways involved in metamorphosis. How these genes are regulated remains largely unknown, although a few have been shown to be direct target genes of TRs (34.Ranjan M. Wong J. Shi Y.B. J. Biol. Chem. 1994; 269: 24699-24705Abstract Full Text PDF PubMed Google Scholar, 35.Furlow J.D. Kanamori A. Endocrinology. 2002; 143: 3295-3305Crossref PubMed Scopus (46) Google Scholar, 36.Fu L. Tomita A. Wang H. Buchholz D.R. Shi Y.B. J. Biol. Chem. 2006; 281: 16870-16878Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 37.Puzianowska-Kuznicka M. Shi Y.B. J. Biol. Chem. 1996; 271: 6273-6282Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 38.Shi Y.B. Metamorphosis: Post-embryonic Reprogramming of Gene Expression in Amphibian and Insect Cells.in: Gilbert L.I. Tata J.R. Atkinson B.G. Academic Press, New York1996Google Scholar). Here we have made use of the recent advances in microarray analysis of gene expression in X. laevis and genomic sequencing in the highly related species Xenopus tropicalis to systematically identify direct target genes of T3, which are likely key players in propagating the effects of T3 in regulating metamorphosis. This is made possible in part by the fact that X. laevis and X. tropicalis undergo essentially identical T3-dependent metamorphosis. In addition, TR and RXR genes are regulated and function similarly in both species during development (39.Wang X. Matsuda H. Shi Y.B. Endocrinology. 2008; 149: 5610-5618Crossref PubMed Scopus (71) Google Scholar). As the regulation of immediate early, direct TR target genes by T3 should be independent of new protein synthesis, to identify these direct targets, we treated premetamorphic X. laevis tadpoles with T3 to induce metamorphosis and included protein synthesis inhibitors to block the synthesis of new proteins. We identified the resulting T3 response genes by using an X. laevis microarray. Gene ontology (GO) analysis revealed that these genes are enriched in categories important for the earliest steps of cellular transformations during metamorphosis. To determine whether these genes are regulated by TR directly at the transcriptional level, we then carried out bioinformatics analysis of the homologous genes in X. tropicalis followed by in vitro DNA binding studies. These results showed that essentially all of the genes had one or more functional TREs in and around their promoters. More importantly, we demonstrated that TR was bound to these TREs in tadpoles and regulated their promoters in vivo. Thus, our studies not only identified many direct TR target genes in vivo but also revealed a number of signaling transduction pathways that are regulated by T3 as the first step toward inducing metamorphosis.DISCUSSIONT3 regulates diverse developmental processes and organ function and metabolisms in different vertebrate species. Although non-genomic actions of T3 undoubtedly contribute to some of the biological effects of T3 (58.Davis P.J. Davis F.B. Thyroid. 1996; 6: 497-504Crossref PubMed Scopus (215) Google Scholar), TR is generally believed to be the main mediator of T3 action through transcriptional regulation. The immediate early, direct target genes of TR likely play critical roles to transduce the biological effects of T3. While a number of microarrays have been carried out to identify T3 response genes in different species, the T3-resposne genes identified from such studies include both immediate early, direct target genes of TR as well as downstream, late T3 response genes. Amphibian metamorphosis is an ideal model system to study T3 action in development because of its total dependence on T3 and the ability to easily manipulate this process without any potential complications of maternal effects as in mammals (3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley & Sons, Inc., New York1999Google Scholar). Furthermore, TR has been shown to be both necessary and sufficient for the metamorphic effects of T3, suggesting an essential role of direct TR target genes in this process (9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). Here, we have combined the advantages of two highly related frog species, microarray analysis in X. laevis and genome sequence information in X. tropicalis, to not only identify immediate early, direct target genes of T3 but also provide evidence to support their regulation by TR in developing tadpoles in vivo. Furthermore, our gene ontology analysis has revealed interesting pathways induced by T3 at the earliest stages of the metamorphic process.Our microarray analysis in X. laevis identified 188 genes up-regulated and 249 genes down-regulated by T3 in the absence of new protein synthesis. Interestingly, most of these genes were not found as T3 response genes in the absence of CHX during the 15-h treatment. Although the exact reasons remain to be determined, some possibilities include the following. First, CHX can stabilize some mRNAs (Fig. 3). This might have altered the magnitude of the changes for some genes. Some genes with the extent of T3 regulation near the 1.3-fold cut-off might change from being a regulated gene to being a non-regulated gene and vice versa in the presence of CHX. In addition, some of the T3-regulated genes might be expressed at very low levels and thus not detectable or with more variable signals on the microarray in the absence of CHX by the microarray. Upon stabilization by CHX, they were now detectable with more consistent signals as T3-regulated genes. Second, some of the genes regulated by T3 treatment alone were late response genes and were thus not regulated by T3 in the presence of CHX. Finally, undoubtedly some genes with a fold of regulation by T3 around the 1.3-fold cut-off might be missed in the T3 alone group or T3 plus CHX group due to some expected experimental variations. Thus, it is quite likely that most of the genes regulated by T3 in the presence of CHX are true T3 response genes during metamorphosis.The above conclusion is also supported by our studies in X. tropicalis, a closely related diploid species of X. laevis within the Xenopodinae subfamily of anurans. We have shown previously that TR and RXR genes are regulated and function similarly in X. tropicalis and X. laevis (39.Wang X. Matsuda H. Shi Y.B. Endocrinology. 2008; 149: 5610-5618Crossref PubMed Scopus (71) Google Scholar). In addition, of the a few direct TR target genes in X. laevis that have been analyzed in X. tropicalis, all are regulated by T3 similarly in X. tropicalis (39.Wang X. Matsuda H. Shi Y.B. Endocrinology. 2008; 149: 5610-5618Crossref PubMed Scopus (71) Google Scholar). 3B. Das, R. A. Heimeier, D. R. Buchholz, and Y.-B. Shi, unpublished observation. Our studies here also indicate that the corresponding genes of the newly identified CHX-resistant T3 response genes in X. laevis are similarly regulated in X. tropicalis, supporting the idea that the T3-dependent gene regulation program is conserved between X. tropicalis and X. laevis.Furthermore, our bioinformatic search showed that of the 46 CHX-resistant T3-induced genes with clear homologs between X. laevis and X. tropicalis, 45 have candidate TREs within 2000 bp of their putative transcription start site. In addition, biochemical studies showed that, of the genes analyzed, each has at least one TRE capable of binding to the TR/RXR heterodimer strongly in vitro. More importantly, our in vivo ChIP assay has shown that TR is bound to the TREs of newly identified target genes in premetamorphic X. tropicalis tadpoles and that the binding to some of the TREs increases upon T3 treatment, just like that in X. laevis. By using the oocyte transcription system, we have further demonstrated that TR regulates the promoters of the newly identified genes in vivo in the context of chromatin. Thus, most, if not all, of the genes discovered here are likely directly regulated by TR during Xenopus development. These findings allowed us for the first time to generate a Xenopus consensus TRE based on a large number of biochemically characterized, natural occurring TREs. The consensus TRE and the degeneracy identified here should help to refine future searches for TREs in other TR target genes by using position-specific probability matrix (MEME) (60.Bailey T.L. Elkan C. Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology. AAAI Press, Menlo Park, CA1994: 28-36Google Scholar). Of particular interest in this regard is the identification of direct target genes of T3 in different metamorphosing tissues, which are likely important to dictate tissue-specific transformations, an area that we are currently pursuing.Our microarray analysis showed that only a small fraction of genes was commonly regulated by T3 in the absence or presence of CHX. One reason is that the genes regulated by T3 alone likely include both immediate early, direct T3 response genes, which are CHX-resistant T3 response genes, and late, downstream T3 response genes. Thus, one would expect that the biological functions of the genes regulated by T3 in the absence of CHX and those in the presence of CHX would also only partially overlap. Indeed, our GO categories analysis revealed that there are a number of GO categories overlapping between the genes induced by T3 alone and those induced by T3 in the presence of CHX. On the other hand, there are few common GO categories between the down-regulated genes by T3 alone and those by T3 in the presence of CHX, consistent with the much fewer common down-regulated genes under the two conditions (Fig. 2).The direct T3 response genes (regulated by T3 in the presence of CHX) are enriched in GO categories related to transcriptional regulation, such as transcription factors (Table 3 and supplemental Table 5). Some of these transcription factors have been reported previously as direct targets of T3, such as TRβ and TH/bZIP (34.Ranjan M. Wong J. Shi Y.B. J. Biol. Chem. 1994; 269: 24699-24705Abstract Full Text PDF PubMed Google Scholar, 35.Furlow J.D. Kanamori A. Endocrinology. 2002; 143: 3295-3305Crossref PubMed Scopus (46) Google Scholar, 65.Ishizuya-Oka A. Ueda S. Shi Y.B. Dev. Genet. 1997; 20: 329-337Crossref PubMed Scopus (25) Google Scholar). Interestingly, GO categories related to transcriptional regulation are not significantly enriched in the genes regulated by T3 alone, likely because of the presence of many late, downstream response genes in this case. These results support the idea that a transcription factor-mediated gene regulation cascade plays an important role in metamorphosis and suggest that the earliest members of the gene expression pathways leading to subsequent morphological changes involve a disproportionately larger fraction of transcription factors.Another GO category specifically enriched in the direct T3 target genes is metallopeptidases (Table 3 and supplemental Table 5). This suggests that the remodeling of the extracellular matrix and other protein degradation events are important early signaling events induced by T3 during metamorphosis. In fact, one of the first T3 direct response genes found during metamorphosis is stromelysin-3 (MMP-11) (36.Fu L. Tomita A. Wang H. Buchholz D.R. Shi Y.B. J. Biol. Chem. 2006; 281: 16870-16878Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 66.Shi Y.B. Brown D.D. J. Biol. Chem. 1993; 268: 20312-20317Abstract Full Text PDF PubMed Google Scholar, 67.Wang Z. Brown D.D. J. Biol. Chem. 1993; 268: 16270-16278Abstract Full Text PDF PubMed Google Scholar, 68.Patterton D. Hayes W.P. Shi Y.B. Dev. Biol. 1995; 167: 252-262Crossref PubMed Scopus (140) Google Scholar), which has been shown to be essential for T3-induced cell death during metamorphosis (43.Fu L. Ishizuya-Oka A. Buchholz D.R. Amano T. Matsuda H. Shi Y.B. J. Biol. Chem. 2005; 280: 27856-27865Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 69.Mathew S. Fu L. Fiorentino M. Matsuda H. Das B. Shi Y.B. J. Biol. Chem. 2009; 284: 18545-18556Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 70.Ishizuya-Oka A. Li Q. Amano T. Damjanovski S. Ueda S. Shi Y.B. J. Cell Biol. 2000; 150: 1177-1188Crossref PubMed Scopus (102) Google Scholar, 71.Fu L. Buchholz D. Shi Y.B. Mol. Reprod Dev. 2002; 62: 470-476Crossref PubMed Scopus (58) Google Scholar). Thus, signaling processes triggered through the action of metallopeptidases are another important early event toward regulating cell fate and behavior during metamorphosis.In addition, our GO analysis revealed the enrichment of categories related to cell cycle and DNA replication in the genes induced by T3 alone and/or by T3 in the presence of CHX. Interestingly, whereas cell cycle categories were enriched in the direct target genes, DNA replication categories were enriched in the genes induced by T3 alone. These suggest that, in the absence of protein synthesis, T3 may induce cell cycle changes. The direct target genes by themselves, however, are not sufficient for DNA replication but rather prepare the cells to enter the S-phase. This is strongly supported by the enrichment of G1/S phase transition genes but the absence of DNA replication genes, e.g. the MCM genes involved in replication fork (72.Ryu S. Driever W. Cell Cycle. 2006; 5: 1140-1142Crossref PubMed Scopus (26) Google Scholar), in the direct T3 target genes (Table 3 and supplemental Table 5). Likewise, the absence of the GO categories, related to ribosomal biogenesis and assembly in the CHX-resistant T3 response genes, and their presence in the genes induced by T3 alone, suggest the possibility that downstream genes are required to sustain cell proliferation. Undoubtedly, it would be of importance in the future to investigate the function of the direct target genes to test these predictions. IntroductionThyroid hormone (T3) 2The abbreviations used are: T3thyroid hormoneTRthyroid receptorTREthyroid response elementRXR9-cis-retinoic acid receptorGOGene ontologyCHXcycloheximideqRTquantitative reverse transcriptionChIPchromatin immunoprecipitation. is critical for adult organ homeostasis and function and also essential for vertebrate development (1.Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar, 2.Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1501) Google Scholar, 3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley & Sons, Inc., New York1999Google Scholar, 5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar, 6.Freake H.C. Oppenheimer J.H. Annu Rev. Nutr. 1995; 15: 263-291Crossref PubMed Scopus (155) Google Scholar, 7.Franklyn J.A. Gammage M.D. Trends Endocrinol. Metab. 1996; 7: 50-54Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 8.Silva J.E. Thyroid. 1995; 5: 481-492Crossref PubMed Scopus (308) Google Scholar, 9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar). T3 deficiency during development leads to severe developmental defects in mammals, including cretinism in human, which is characterized by severe short stature and mental retardation (5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar). During early mammalian development, there is little T3 in the fetus, although some maternal T3 reaches the embryo. High levels of T3 are present only during the so-called postembryonic period, which spans from several months prior to birth to several months after birth in human (3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 10.Howdeshell K.L. Environ. Health Perspect. 2002; 110: 337-348Crossref PubMed Scopus (306) Google Scholar). This is a critical period of organ growth and maturation, and thus, not surprisingly, T3 deficiency during this period causes severe developmental defects (5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar, 11.Porterfield S.P. Hendrich C.E. Endocr. Rev. 1993; 14: 94-106PubMed Google Scholar, 12.Hsu J.H. Brent G.A. TEM. 1998; 9: 103-112Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Interestingly, appropriate levels of maternal T3 are also important to ensure proper mammalian development (13.de Escobar G.M. Obregón M.J. del Rey F.E. Best Pract. Res. Clin. Endocrinol. Metab. 2004; 18: 225-248Crossref PubMed Scopus (431) Google Scholar, 14.de Escobar G.M. Obregón M.J. del Rey F.E. Public Health Nutrition. 2007; 10: 1554-1570Crossref PubMed Scopus (238) Google Scholar, 15.Anselmo J. Cao D. Karrison T. Weiss R.E. Refetoff S. JAMA. 2004; 292: 691-695Crossref PubMed Scopus (211) Google Scholar). These observations suggest that proper levels of T3 in both the mother and fetus are critical for mammalian development, which makes it difficult to separate the direct effects of T3 on the development of the fetus versus indirect effects through maternal influence. Furthermore, there are only a limited number of known direct T3 response genes in different model systems, and no systematic analysis has been carried out to isolate such genes in development. All these have hampered our understanding of how T3 regulates development in vivo.Amphibian metamorphosis is a postembryonic process that is also dependent on T3 (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley & Sons, Inc., New York1999Google Scholar, 16.Gilbert L.I. Tata J.R. Atkinson B.G. Metamorphosis: Post-embryonic Reprogramming of Gene Expression in Amphibian and Insect Cells. Academic Press, New York1996Google Scholar). Studies in anurans such as Xenopus laevis in the past century have shown that T3 controls every aspect of metamorphosis and is both necessary and sufficient (9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar, 18.Buchholz D.R. Tomita A. Fu L. Paul B.D. Shi Y.B. Mol. Cell. Biol. 2004; 24: 9026-9037Crossref PubMed Scopus (112) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). During metamorphosis, different tissues have different fates (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley & Sons, Inc., New York1999Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar). For example, the tail and gills undergo resorption, the brain, skin, intestine, and other visceral organs undergo remodeling while the limbs are generated de novo. All these changes are controlled by T3 in mostly organ-autonomous manner.T3 action is primarily mediated through thyroid receptors (TRs), which are transcription factors and are members of nuclear receptor superfamily (1.Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar, 2.Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1501) Google Scholar, 9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar, 20.Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 21.Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar). TRs bind to chromosomal sites in the promoter regions (known as thyroid response elements (TRE)) of direct response genes of T3. For T3-inducible genes, these binding sites are often composed of two direct repeats of the consensus sequence AGGTCA with a 4-bp spacer sequence. TR functions mainly as a heterodimer with 9-cis-retinoic acid receptor (RXR) at these TREs and represses and activates these genes by recruiting corepressor and coactivator complexes in the absence or presence of T3, respectively.We have previously proposed a dual function model for TR function during anuran metamorphosis (22.Shi Y.B. Wong J. Puzianowska-Kuznicka M. Stolow M.A. BioEssays. 1996; 18: 391-399Crossref PubMed Scopus (145) Google Scholar, 23.Sachs L.M. Damjanovski S. Jones P.L. Li Q. Amano T. Ueda S. Shi Y.B. Ishizuya-Oka A. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 2000; 126: 199-211Crossref PubMed Scopus (102) Google Scholar). According to this model, in the absence of T3, TR/RXR heterodimers repress direct target genes to ensure proper growth of the premetamorphic tadpoles and prevent premature metamorphic organ transformations. In the presence of T3, TR/RXR heterodimers activate these target genes to initiate metamorphosis. Over the years, studies by us and others have shown that TRs are both necessary and sufficient to mediate the metamorphic effects of T3 in X. laevis (9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). Thus all the remarkable morphological and developmental changes in the different tissues are effected through gene expression cascades initiated by TRs. Furthermore, TRs indeed have dual functions during metamorphosis. They recruit corepressor complexes in premetamorphic tadpoles to control metamorphic timing and recruit coactivator complexes to initiate metamorphosis (24.Paul B.D. Fu L. Buchholz D.R. Shi Y.B. Mol. Cell. Biol. 2005; 25: 5712-5724Crossref PubMed Scopus (56) Google Scholar, 25.Havis E. Sachs L.M. Demeneix B.A. EMBO Rep. 2003; 4: 883-888Crossref PubMed Scopus (53) Google Scholar, 26.Sato Y. Buchholz D.R. Paul B.D. Shi Y.B. Mech. Dev. 2007; 124: 476-488Crossref PubMed Scopus (53) Google Scholar, 27.Paul B.D. Buchholz D.R. Fu L. Shi Y.B. J. Biol. Chem. 2005; 280: 27165-27172Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28.Paul B.D. Buchholz D.R. Fu L. Shi Y.B. J. Biol. Chem. 2007; 282: 7472-7481Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In addition, the levels of coactivator complexes also regulate the rate of metamorphic progression (29.Matsuda H. Paul B.D. Choi C.Y. Hasebe T. Shi Y.B. Mol. Cell. Biol. 2009; 29: 745-757Crossref PubMed Scopus (41) Google Scholar).Using subtractive hybridization screening and microarrays, we and others have previously documented the differentially expressed genes in different organs during both T3-induced and natural metamorphosis (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley & Sons, Inc., New York1999Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar, 30.Denver R.J. Pavgi S. Shi Y.B. J. Biol. Chem. 1997; 272: 8179-8188Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 31.Buchholz D.R. Heimeier R.A. Das B. Washington T. Shi Y.B. Dev. Biol. 2007; 303: 576-590Crossref PubMed Scopus (74) Google Scholar, 32.Das B. Cai L. Carter M.G. Piao Y.L. Sharov A.A. Ko M.S. Brown D.D. Dev. Biol. 2006; 291: 342-355Crossref PubMed Scopus (100) Google Scholar, 33.Cai L. Das B. Brown D.D. Dev. 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Academic Press, New York1996Google Scholar). Here we have made use of the recent advances in microarray analysis of gene expression in X. laevis and genomic sequencing in the highly related species Xenopus tropicalis to systematically identify direct target genes of T3, which are likely key players in propagating the effects of T3 in regulating metamorphosis. This is made possible in part by the fact that X. laevis and X. tropicalis undergo essentially identical T3-dependent metamorphosis. In addition, TR and RXR genes are regulated and function similarly in both species during development (39.Wang X. Matsuda H. Shi Y.B. Endocrinology. 2008; 149: 5610-5618Crossref PubMed Scopus (71) Google Scholar). As the regulation of immediate early, direct TR target genes by T3 should be independent of new protein synthesis, to identify these direct targets, we treated premetamorphic X. laevis tadpoles with T3 to induce metamorphosis and included protein synthesis inhibitors to block the synthesis of new proteins. We identified the resulting T3 response genes by using an X. laevis microarray. Gene ontology (GO) analysis revealed that these genes are enriched in categories important for the earliest steps of cellular transformations during metamorphosis. To determine whether these genes are regulated by TR directly at the transcriptional level, we then carried out bioinformatics analysis of the homologous genes in X. tropicalis followed by in vitro DNA binding studies. These results showed that essentially all of the genes had one or more functional TREs in and around their promoters. More importantly, we demonstrated that TR was bound to these TREs in tadpoles and regulated their promoters in vivo. Thus, our studies not only identified many direct TR target genes in vivo but also revealed a number of signaling transduction pathways that are regulated by T3 as the first step toward inducing metamorphosis.
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