The Unique Transcriptional Activation Domain of Nuclear Factor-I-X3 Is Critical to Specifically Induce Marker Gene Expression in Astrocytes
2010; Elsevier BV; Volume: 286; Issue: 9 Linguagem: Inglês
10.1074/jbc.m110.152421
ISSN1083-351X
AutoresSandeep K. Singh, Katarzyna M. Wilczynska, Adrian T. Grzybowski, Jessie Yester, Bahiya Osrah, Lauren Bryan, Sarah Wright, Irene Griswold‐Prenner, Tomasz Kordula,
Tópico(s)RNA modifications and cancer
ResumoTranscription factors of the nuclear factor 1 (NFI) family regulate normal brain development in vertebrates. However, multiple splice variants of four NFI isoforms exist, and their biological functions have yet to be elucidated. Here, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including glial fibrillary acidic protein (GFAP) reporter, in astrocytes and glioma cells. The GAL4 fusion protein containing the TA domain of NFI-X3 strongly activates the GAL4 reporter, whereas the TA domain of NFI-X1 is ineffective. The expression of NFI-X3 is dramatically up-regulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers, such as GFAP and SPARCL1 (Secreted Protein, Acidic and Rich in Cysteines-like 1). Overexpression of NFI-X3 dramatically up-regulates GFAP and SPARCL1 expression in glioma cells, whereas the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II. Transcription factors of the nuclear factor 1 (NFI) family regulate normal brain development in vertebrates. However, multiple splice variants of four NFI isoforms exist, and their biological functions have yet to be elucidated. Here, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including glial fibrillary acidic protein (GFAP) reporter, in astrocytes and glioma cells. The GAL4 fusion protein containing the TA domain of NFI-X3 strongly activates the GAL4 reporter, whereas the TA domain of NFI-X1 is ineffective. The expression of NFI-X3 is dramatically up-regulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers, such as GFAP and SPARCL1 (Secreted Protein, Acidic and Rich in Cysteines-like 1). Overexpression of NFI-X3 dramatically up-regulates GFAP and SPARCL1 expression in glioma cells, whereas the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II. IntroductionAstrocytes are critical for the normal functions of the brain but also play a destructive role in many diseases of the central nervous system. Nevertheless, mechanisms controlling their differentiation and astrocyte-specific gene expression are only partially understood. Astrocyte differentiation is promoted by the activation of several signaling pathways, including the JAK-STAT pathway (1Miller F.D. Gauthier A.S. Neuron. 2007; 54: 357-369Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 2Barnabé-Heider F. Wasylnka J.A. Fernandes K.J. Porsche C. Sendtner M. Kaplan D.R. Miller F.D. Neuron. 2005; 48: 253-265Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 3Fukuda S. Abematsu M. Mori H. Yanagisawa M. Kagawa T. Nakashima K. Yoshimura A. Taga T. Mol. Cell Biol. 2007; 27: 4931-4937Crossref PubMed Scopus (106) Google Scholar), the activation of SMADs (Similar to Drosophila Mothers Against Decapentaplegic) (3Fukuda S. Abematsu M. Mori H. Yanagisawa M. Kagawa T. Nakashima K. Yoshimura A. Taga T. Mol. Cell Biol. 2007; 27: 4931-4937Crossref PubMed Scopus (106) Google Scholar, 4Gomes W.A. Mehler M.F. Kessler J.A. Dev. Biol. 2003; 255: 164-177Crossref PubMed Scopus (231) Google Scholar), the activation of Notch signaling (5Gaiano N. Nye J.S. Fishell G. Neuron. 2000; 26: 395-404Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 6Ge W. Martinowich K. Wu X. He F. Miyamoto A. Fan G. Weinmaster G. Sun Y.E. J. Neurosci. Res. 2002; 69: 848-860Crossref PubMed Scopus (158) Google Scholar), and the activation of genes encoding the nuclear factor-1 (NFI) 4The abbreviations used are: NFI, nuclear factor 1; CAT, chloramphenicol acetyltransferase; GFAP, glial fibrillary acidic protein; Luc, luciferase; TA, transcriptional activation; qPCR, quantitative PCR; MNase, micrococcal nuclease; PolII, polymerase II. family of transcription factors (7Bisgrove D.A. Monckton E.A. Packer M. Godbout R. J. Biol. Chem. 2000; 275: 30668-30676Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 8Cebolla B. Vallejo M. J. Neurochem. 2006; 97: 1057-1070Crossref PubMed Scopus (62) Google Scholar, 9Krohn K. Rozovsky I. Wals P. Teter B. Anderson C.P. Finch C.E. J. Neurochem. 1999; 72: 1353-1361Crossref PubMed Scopus (71) Google Scholar, 10Amemiya K. Traub R. Durham L. Major E.O. J. Biol. Chem. 1992; 267: 14204-14211Abstract Full Text PDF PubMed Google Scholar, 11Steele-Perkins G. Plachez C. Butz K.G. Yang G. Bachurski C.J. Kinsman S.L. Litwack E.D. Richards L.J. Gronostajski R.M. Mol. Cell Biol. 2005; 25: 685-698Crossref PubMed Scopus (227) Google Scholar, 12das Neves L. Duchala C.S. Tolentino-Silva F. Haxhiu M.A. Colmenares C. Macklin W.B. Campbell C.E. Butz K.G. Gronostajski R.M. Godinho F. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 11946-11951Crossref PubMed Scopus (181) Google Scholar, 13Namihira M. Kohyama J. Semi K. Sanosaka T. Deneen B. Taga T. Nakashima K. Dev. Cell. 2009; 16: 245-255Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). During the vertebrate embryonic development, neurons are generated first, followed by glia. This neurogenic-to-gliogenic switch is induced by the activation of the JAK-STAT signaling pathway in neural precursors by neuron-derived cardiotrophin-1 (2Barnabé-Heider F. Wasylnka J.A. Fernandes K.J. Porsche C. Sendtner M. Kaplan D.R. Miller F.D. Neuron. 2005; 48: 253-265Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Specifically, STAT3 induces production of BMP-2, which subsequently activates SMAD1 (3Fukuda S. Abematsu M. Mori H. Yanagisawa M. Kagawa T. Nakashima K. Yoshimura A. Taga T. Mol. Cell Biol. 2007; 27: 4931-4937Crossref PubMed Scopus (106) Google Scholar). In turn, SMAD1 forms a complex with STAT3 and induces astrogliogenesis (14Nakashima K. Yanagisawa M. Arakawa H. Kimura N. Hisatsune T. Kawabata M. Miyazono K. Taga T. Science. 1999; 284: 479-482Crossref PubMed Scopus (720) Google Scholar). In addition to JAK-STAT and BMP-SMAD1 pathways, Notch signaling also affects gliogenesis by activation of RBP-Jκ transcriptional activity (6Ge W. Martinowich K. Wu X. He F. Miyamoto A. Fan G. Weinmaster G. Sun Y.E. J. Neurosci. Res. 2002; 69: 848-860Crossref PubMed Scopus (158) Google Scholar, 15Hermanson O. Jepsen K. Rosenfeld M.G. Nature. 2002; 419: 934-939Crossref PubMed Scopus (260) Google Scholar), induction of NFI expression (13Namihira M. Kohyama J. Semi K. Sanosaka T. Deneen B. Taga T. Nakashima K. Dev. Cell. 2009; 16: 245-255Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar), and concomitant demethylation of the astrocyte-specific regulatory elements.The NFI family transcription factors have recently emerged as important regulators of gliogenesis (13Namihira M. Kohyama J. Semi K. Sanosaka T. Deneen B. Taga T. Nakashima K. Dev. Cell. 2009; 16: 245-255Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 16Gronostajski R.M. Gene. 2000; 249: 31-45Crossref PubMed Scopus (423) Google Scholar, 17Mason S. Piper M. Gronostajski R.M. Richards L.J. Mol. Neurobiol. 2009; 39: 10-23Crossref PubMed Scopus (75) Google Scholar). These proteins, encoded by four genes highly conserved from chickens to humans (Nfia, Nfib, Nfic, and Nfix), regulate the transcription of various cellular and viral genes as well as viral DNA replication (16Gronostajski R.M. Gene. 2000; 249: 31-45Crossref PubMed Scopus (423) Google Scholar, 18Santoro C. Mermod N. Andrews P.C. Tjian R. Nature. 1988; 334: 218-224Crossref PubMed Scopus (491) Google Scholar). In vertebrates, products of these genes (NFI-A, -B, -C, and -X) share conserved N-terminal DNA binding and dimerization domains, followed by a subtype-specific domain and a variable C-terminal transactivation domain (19Roulet E. Armentero M.T. Krey G. Corthésy B. Dreyer C. Mermod N. Wahli W. Mol. Cell Biol. 1995; 15: 5552-5562Crossref PubMed Scopus (34) Google Scholar).In mammals, the NFI genes are expressed in overlapping patterns during embryogenesis, with high levels of expression of NFI-A, -B, and -X in the developing neocortex (20Chaudhry A.Z. Lyons G.E. Gronostajski R.M. Dev. Dyn. 1997; 208: 313-325Crossref PubMed Scopus (176) Google Scholar). The Nfia and Nfib knock-out mice are characterized by neuroanatomical defects, including agenesis of the corpus callosum, loss of specific midline glial populations, and a 5–10-fold decrease in the expression of glial fibrillary acidic protein (GFAP), which is an astrocyte marker (11Steele-Perkins G. Plachez C. Butz K.G. Yang G. Bachurski C.J. Kinsman S.L. Litwack E.D. Richards L.J. Gronostajski R.M. Mol. Cell Biol. 2005; 25: 685-698Crossref PubMed Scopus (227) Google Scholar, 12das Neves L. Duchala C.S. Tolentino-Silva F. Haxhiu M.A. Colmenares C. Macklin W.B. Campbell C.E. Butz K.G. Gronostajski R.M. Godinho F. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 11946-11951Crossref PubMed Scopus (181) Google Scholar, 21Shu T. Butz K.G. Plachez C. Gronostajski R.M. Richards L.J. J. Neurosci. 2003; 23: 203-212Crossref PubMed Google Scholar). In addition, Nfib-deficient mice present aberrant hippocampus and pons formation and die due to the defects in lung development (11Steele-Perkins G. Plachez C. Butz K.G. Yang G. Bachurski C.J. Kinsman S.L. Litwack E.D. Richards L.J. Gronostajski R.M. Mol. Cell Biol. 2005; 25: 685-698Crossref PubMed Scopus (227) Google Scholar). The immature nestin-positive glia populate the hippocampus of Nfib-deficient mice but fail to mature into GFAP-positive astrocytes (22Barry G. Piper M. Lindwall C. Moldrich R. Mason S. Little E. Sarkar A. Tole S. Gronostajski R.M. Richards L.J. J. Neurosci. 2008; 28: 12328-12340Crossref PubMed Scopus (67) Google Scholar). In contrast, disruption of the Nfic gene results in early postnatal defects in tooth formation, including the loss of molar roots and aberrant incisor development (23Steele-Perkins G. Butz K.G. Lyons G.E. Zeichner-David M. Kim H.J. Cho M.I. Gronostajski R.M. Mol. Cell Biol. 2003; 23: 1075-1084Crossref PubMed Scopus (156) Google Scholar). The Nfix knock-out mice have recently been generated by two independent groups (24Driller K. Pagenstecher A. Uhl M. Omran H. Berlis A. Gründer A. Sippel A.E. Mol. Cell Biol. 2007; 27: 3855-3867Crossref PubMed Scopus (101) Google Scholar, 25Campbell C.E. Piper M. Plachez C. Yeh Y.T. Baizer J.S. Osinski J.M. Litwack E.D. Richards L.J. Gronostajski R.M. BMC Dev. Biol. 2008; 8: 52Crossref PubMed Scopus (113) Google Scholar), which reported multiple effects (26Pekarik V. Belmonte J.C. J. Biol. 2008; 7: 29Crossref PubMed Scopus (8) Google Scholar). The Nfix gene knock-out causes postnatal lethality in most of the animals and leads to hydrocephalus and partial agenesis of the corpus callosum (24Driller K. Pagenstecher A. Uhl M. Omran H. Berlis A. Gründer A. Sippel A.E. Mol. Cell Biol. 2007; 27: 3855-3867Crossref PubMed Scopus (101) Google Scholar). These mice also develop a deformation of the spine with kyphosis, due to a delay in ossification of vertebral bodies and a progressive degeneration of intervertebral disks. However, Nfix knock-out mice survive on a soft chow diet but are characterized by increased brain weight, expansion of the brain along the dorsal ventrical axis, and aberrant formation of the hippocampus (25Campbell C.E. Piper M. Plachez C. Yeh Y.T. Baizer J.S. Osinski J.M. Litwack E.D. Richards L.J. Gronostajski R.M. BMC Dev. Biol. 2008; 8: 52Crossref PubMed Scopus (113) Google Scholar). In summary, the knock-out phenotypes suggest that NFI-A, -B, and -X are important for normal brain development; however, the identity of the affected cell type(s) is not clear. Recently, both NFI-A and -B were shown to regulate gliogenesis in the chick embryo, with NFI-A also controlling the maintenance of neural precursors (27Deneen B. Ho R. Lukaszewicz A. Hochstim C.J. Gronostajski R.M. Anderson D.J. Neuron. 2006; 52: 953-968Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). More recently, NFI-C and -X were shown to regulate the expression of late astrocyte markers during the differentiation of neural precursors into astrocytes in vitro (28Wilczynska K.M. Singh S.K. Adams B. Bryan L. Rao R.R. Valerie K. Wright S. Griswold-Prenner I. Kordula T. Stem Cells. 2009; 27: 1173-1181Crossref PubMed Scopus (35) Google Scholar).Alternative splicing is a common mechanism of generating transcription factors with diverse functions in the brain (29Vihma H. Pruunsild P. Timmusk T. Genomics. 2008; 92: 279-291Crossref PubMed Scopus (51) Google Scholar, 30Arteaga M.F. Coric T. Straub C. Canessa C.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 4459-4464Crossref PubMed Scopus (49) Google Scholar, 31Chu H.Y. Ohtoshi A. Mol. Cell Biol. 2007; 27: 3743-3749Crossref PubMed Scopus (9) Google Scholar, 32Michelhaugh S.K. Vaitkevicius H. Wang J. Bouhamdan M. Krieg A.R. Walker J.L. Mendiratta V. Bannon M.J. J. Neurochem. 2005; 95: 1342-1350Crossref PubMed Scopus (26) Google Scholar). Accordingly, transcripts of all four NFI genes are alternatively spliced, yielding many different proteins from a single gene (33Kruse U. Sippel A.E. J. Mol. Biol. 1994; 238: 860-865Crossref PubMed Scopus (76) Google Scholar, 34Altmann H. Wendler W. Winnacker E.L. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 3901-3905Crossref PubMed Scopus (42) Google Scholar) with several different splice variants of NFI-X identified in human cells (35Gründer A. Qian F. Ebel T.T. Mincheva A. Lichter P. Kruse U. Sippel A.E. Gene. 2003; 304: 171-181Crossref PubMed Scopus (21) Google Scholar). Here, we cloned and characterized a novel human NFI-X splice variant X3 (NFI-X3), which regulates gene expression in primary human astrocytes. This splice variant contains a unique transcriptional activation (TA) domain that is remarkably conserved in mammals, including mice, rats, dogs, macaques, and humans. Mechanistically, the TA domain of NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II.DISCUSSIONIt is apparent that NFIs are critical for the proper development of the brain; nevertheless, the vital target genes regulated by NFIs and the molecular mechanisms of their regulation remain elusive. To date, multiple NFI-responsive genes have been identified in the brain, including the genes encoding GFAP, ACT, SPARCL1, and S100B in astrocytes (28Wilczynska K.M. Singh S.K. Adams B. Bryan L. Rao R.R. Valerie K. Wright S. Griswold-Prenner I. Kordula T. Stem Cells. 2009; 27: 1173-1181Crossref PubMed Scopus (35) Google Scholar, 37Gopalan S.M. Wilczynska K.M. Konik B.S. Bryan L. Kordula T. J. Biol. Chem. 2006; 281: 13126-13133Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 47Besnard F. Brenner M. Nakatani Y. Chao R. Purohit H.J. Freese E. J. Biol. 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Because regulatory sequences of these genes can bind all NFIs, their transcriptional regulation in a given cell type, in part, depends on the availability of a particular NFI isoform, the presence of the specific splice variant, and probably the acquired post-transcriptional modifications of the NFIs. In fact, NFIs are expressed in complicated patterns during embryogenesis (20Chaudhry A.Z. Lyons G.E. Gronostajski R.M. Dev. Dyn. 1997; 208: 313-325Crossref PubMed Scopus (176) Google Scholar, 51Plachez C. Lindwall C. Sunn N. Piper M. Moldrich R.X. Campbell C.E. Osinski J.M. Gronostajski R.M. Richards L.J. J. Comp. Neurol. 2008; 508: 385-401Crossref PubMed Scopus (62) Google Scholar), with NFI-A and NFI-B expressed during early gliogenesis (27Deneen B. Ho R. Lukaszewicz A. Hochstim C.J. Gronostajski R.M. Anderson D.J. Neuron. 2006; 52: 953-968Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), whereas NFI-X and NFI-C are expressed later in the differentiation of astrocytes and control the expression of late astrocyte markers (28Wilczynska K.M. Singh S.K. Adams B. Bryan L. Rao R.R. Valerie K. Wright S. Griswold-Prenner I. Kordula T. Stem Cells. 2009; 27: 1173-1181Crossref PubMed Scopus (35) Google Scholar). NFIs are also phosphorylated, and this modification affects their function (7Bisgrove D.A. Monckton E.A. Packer M. Godbout R. J. Biol. Chem. 2000; 275: 30668-30676Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).To date, the functional importance of the multiple NFI splice variants as well as the mechanisms controlling their generation remain largely unknown. Significantly, the NFI splice variants are evolutionarily conserved in mammals, and their splicing mostly affects the C termini encoding their TA domains, thus suggesting that these variants probably fulfill very specific biological functions (35Gründer A. Qian F. Ebel T.T. Mincheva A. Lichter P. Kruse U. Sippel A.E. Gene. 2003; 304: 171-181Crossref PubMed Scopus (21) Google Scholar). In particular, the inclusion of the exon 9 found in the NFI-A, NFI-B, and NFI-C generates a subset of relatively active transcription factors (NFI-A1, NFI-B1, and CTF-1). In contrast to these isoforms, the importance of alternative splicing of the NFI-X transcript was controversial, because Xenopus and mouse splice variants containing exon 9 were reported not to be particularly active (19Roulet E. Armentero M.T. Krey G. Corthésy B. Dreyer C. Mermod N. Wahli W. Mol. Cell Biol. 1995; 15: 5552-5562Crossref PubMed Scopus (34) Google Scholar, 52Nebl G. Cato A.C. Cell Mol. Biol. Res. 1995; 41: 85-95PubMed Google Scholar). However, the TA domain of Xenopus NFI-X3 is drastically different from the TA of the X3 variants found in mammals (Fig. 1). Moreover, the repressive properties of the previously cloned mouse NFI-X3 can be attributed to the partial TA domain that lacks 14 C-terminal amino acids (52Nebl G. Cato A.C. Cell Mol. Biol. Res. 1995; 41: 85-95PubMed Google Scholar). In fact, the full-length TA domain of the NFI-X3 splice variant is remarkably conserved in mammals, with 100% identity in primates and dog, and contains 17 conserved proline residues (Fig. 1). These proline residues are probably responsible for the relatively strong activation of both the GFAP and NFI reporters by NFI-X3 in astrocytes and glioma cells (Fig. 3, A and B), activation of GFAP and SPARCL1 expression in differentiating astrocytes (Fig. 5), and activation of GFAP and SPARCL1 expression in cells overexpressing NFI-X3 (Fig. 7). In contrast, NFI-X3Δ, which lacks its proline-rich TA domain, did not activate GFAP and SPARCL1 expression when overexpressed (Fig. 7). The importance of the NFI-X3 TA domain is further supported by the findings that the Gal4-X3TA domain fusion protein efficiently activated Gal4 reporter (Fig. 4), whereas siRNA specific to exon 9 (present in NFI-X3) abolished the activation of GFAP and SPARCL1 expression (Fig. 6). In contrast, NFI-X1 did not activate the GFAP reporter in astrocytes and glioma cells (Fig. 3A), mildly activated the NFI reporter in astrocytes (with approximately 10 times lower efficiency than NFI-X3) (Fig. 3B), and relatively moderately activated SPARCL1 and GFAP expression when stably overexpressed in glioma cells (Fig. 7). Moreover, a fusion protein containing the NFI-X1 TA domain and the Gal4 DNA-binding domain did not activate the Gal4 reporter (Fig. 4). These data suggest that NFI-X3 serves as a strong transcriptional activator, whereas NFI-X1 is either a repressor or a relatively very weak activator. Surprisingly, both NFI-X1 and NFI-X3 repress some genes independently of their TA domains, as observed for the p21 reporter (Fig. 3C) and p21 mRNA in cells overexpressing NFI-X1 and NFI-X3 (data not shown); nevertheless, the mechanism of repression remains to be established. In contrast to the TA domain of NFI-X3, a deletion of 8 amino acids (exon 1A) did not affect NFI-X3 binding to the gfap enhancer (Fig. 8) or the repression of the p21 reporter (Fig. 3C).The question remains as to whether NFI-X3 plays an important biological role. Our data argue that NFI-X3 is mainly responsible for the NFI-X-mediated regulation of GFAP and SPARCL1 expression in primary human astrocytes because the down-regulation of NFI-X3 expression has an identical effect as the down-regulation of expression of the total NFI-X pool (Fig. 6). Although NFI-X3 mRNA accounts for only ∼10% of the NFI-X mRNAs (Fig. 6A), the 10 times stronger activation potential (Fig. 3B) may explain its critical role in gene regulation. Moreover, the expression of NFI-X3 is strongly up-regulated during the differentiation of neural progenitors toward astrocytes and precedes the activation of GFAP and SPARCL1 expression (Fig. 5). Importantly, the ratio of NFI-X3 mRNA/total NFI-X mRNA increases ∼2-fold during the differentiation (Fig. 5). Conversely, this ratio is decreased in various glioma cells, already expressing low levels of all NFI-X transcripts (Fig. 2C), which may be viewed as cells that exited astrocyte differentiation. However, NFI-X3 expression is not restricted to glia because we were also able to detect NFI-X3 mRNA in other non-glial cells (data not shown), thus indicating that NFI-X3 probably regulates transcription in various cell types.Although NFIs were discovered over 2 decades ago (53Nagata K. Guggenheimer R.A. Enomoto T. Lichy J.H. Hurwitz J. Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 6438-6442Crossref PubMed Scopus (216) Google Scholar), the molecular mechanism of NFI-mediated gene activation still remains elusive. Interestingly, NFIs not only bind to their specific binding elements within the regulatory regions of target genes but also interact with other transcription factors (54Ravichandran V. Sabath B.F. Jensen P.N. Houff S.A. Major E.O. J. Virol. 2006; 80: 10506-10513Crossref PubMed Scopus (31) Google Scholar) and histone H3 (55Müller K. Mermod N. J. Biol. Chem. 2000; 275: 1645-1650Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Importantly, NFI-C (CTF-1) binding to histone H3, in part, depends on its TA domain (55Müller K. Mermod N. J. Biol. Chem. 2000; 275: 1645-1650Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). NFI-C can also be immunoprecipitated with remodeling complexes containing Brg1 (56Zhao L.H. Ba X.Q. Wang X.G. Zhu X.J. Wang L. Zeng X.L. Acta Biochim. Biophys. Sin. 2005; 37: 440-446Crossref Scopus (18) Google Scholar), suggesting that chromatin remodeling may be the mechanism regulating NFI-dependent transcription. Recently, the induction of the c-fos gene by MAPK signaling has been shown to depend on the acetylation relay switch that induces a local change in the nucleosome architecture allowing for NFI binding (57O'Donnell A. Yang S.H. Sharrocks A.D. Mol. Cell. 2008; 29: 780-785Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Nevertheless, how NFI recruitment increases c-fos expression is not clear. Our data allow us to postulate a novel model of gene activation that depends on the TA domain of NFI-X3. Our data suggest that binding of NFI-X3 (and to a lesser extent NFI-X1) to the gfap enhancer induces changes in the structure/occupancy of the nucleosomes positioned at the promoter of the gfap gene (Fig. 9B). This localized alteration is probably an eviction of the +1 nucleosome from the gfap promoter, which subsequently allows for the increased binding of the general transcription factors and the recruitment of PolII. In summary, our data suggest that NFI-X3 is a critical splice variant of the nfix gene, which regulates expression of late markers, such as GFAP, in astrocytes via a mechanism that depends, in part, on the induction of local alteration in chromatin architecture. IntroductionAstrocytes are critical for the normal functions of the brain but also play a destructive role in many diseases of the central nervous system. Nevertheless, mechanisms controlling their differentiation and astrocyte-specific gene expression are only partially understood. Astrocyte differentiation is promoted by the activation of several signaling pathways, including the JAK-STAT pathway (1Miller F.D. Gauthier A.S. Neuron. 2007; 54: 357-369Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 2Barnabé-Heider F. Wasylnka J.A. Fernandes K.J. Porsche C. Sendtner M. Kaplan D.R. Miller F.D. Neuron. 2005; 48: 253-265Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 3Fukuda S. Abematsu M. Mori H. Yanagisawa M. Kagawa T. Nakashima K. Yoshimura A. Taga T. Mol. Cell Biol. 2007; 27: 4931-4937Crossref PubMed Scopus (106) Google Scholar), the activation of SMADs (Similar to Drosophila Mothers Against Decapentaplegic) (3Fukuda S. Abematsu M. Mori H. Yanagisawa M. Kagawa T. Nakashima K. Yoshimura A. Taga T. Mol. Cell Biol. 2007; 27: 4931-4937Crossref PubMed Scopus (106) Google Scholar, 4Gomes W.A. Mehler M.F. Kessler J.A. Dev. Biol. 2003; 255: 164-177Crossref PubMed Scopus (231) Google Scholar), the activation of Notch signaling (5Gaiano N. Nye J.S. Fishell G. Neuron. 2000; 26: 395-404Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 6Ge W. Martinowich K. Wu X. He F. Miyamoto A. Fan G. Weinmaster G. Sun Y.E. J. Neurosci. Res. 2002; 69: 848-860Crossref PubMed Scopus (158) Google Scholar), and the activation of genes encoding the nuclear factor-1 (NFI) 4The abbreviations used are: NFI, nuclear factor 1; CAT, chloramphenicol acetyltransferase; GFAP, glial fibrillary acidic protein; Luc, luciferase; TA, transcriptional activation; qPCR, quantitative PCR; MNase, micrococcal nuclease; PolII, polymerase II. family of transcription factors (7Bisgrove D.A. Monckton E.A. Packer M. Godbout R. J. Biol. Chem. 2000; 275: 30668-30676Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 8Cebolla B. Vallejo M. J. Neurochem. 2006; 97: 1057-1070Crossref PubMed Scopus (62) Google Scholar, 9Krohn K. Rozovsky I. Wals P. Teter B. Anderson C.P. Finch C.E. J. Neurochem. 1999; 72: 1353-1361Crossref PubMed Scopus (71) Google Scholar, 10Amemiya K. Traub R. Durham L. Major E.O. J. Biol. 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Sendtner M. Kaplan D.R. Miller F.D. Neuron. 2005; 48: 253-265Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Specifically, STAT3 induces production of BMP-2, which subsequently activates SMAD1 (3Fukuda S. Abematsu M. Mori H. Yanagisawa M. Kagawa T. Nakashima K. Yoshimura A. Taga T. Mol. Cell Biol. 2007; 27: 4931-4937Crossref PubMed Scopus (106) Google Scholar). In turn, SMAD1 forms a complex with STAT3 and induces astrogliogenesis (14Nakashima K. Yanagisawa M. Arakawa H. Kimura N. Hisatsune T. Kawabata M. Miyazono K. Taga T. Science. 1999; 284: 479-482Crossref PubMed Scopus (720) Google Scholar). In addition to JAK-STAT and BMP-SMAD1 pathways, Notch signaling also affects gliogenesis by activation of RBP-Jκ transcriptional activity (6Ge W. Martinowich K. Wu X. He F. Miyamoto A. Fan G. Weinmaster G. Sun Y.E. J. Neurosci. Res. 2002; 69: 848-860Crossref PubMed Scopus (158) Google Scholar, 15Hermanson O. Jepsen K. Rosenfeld M.G. 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These proteins, encoded by four genes highly conserved from chickens to humans (Nfia, Nfib, Nfic, and Nfix), regulate the transcription of various cellular and viral genes as well as viral DNA replication (16Gronostajski R.M. Gene. 2000; 249: 31-45Crossref PubMed Scopus (423) Google Scholar, 18Santoro C. Mermod N. Andrews P.C. Tjian R. Nature. 1988; 334: 218-224Crossref PubMed Scopus (491) Google Scholar). In vertebrates, products of these genes (NFI-A, -B, -C, and -X) share conserved N-terminal DNA binding and dimerization domains, followed by a subtype-specific domain and a variable C-terminal transactivation domain (19Roulet E. Armentero M.T. Krey G. Corthésy B. Dreyer C. Mermod N. Wahli W. Mol. Cell Biol. 1995; 15: 5552-5562Crossref PubMed Scopus (34) Google Scholar).In mammals, the NFI genes are expressed in overlapping patterns during embryogenesis, with high levels of expression of NFI-A, -B, and -X in the developing neocortex (20Chaudhry A.Z. Lyons G.E. Gronostajski R.M. Dev. Dyn. 1997; 208: 313-325Crossref PubMed Scopus (176) Google Scholar). The Nfia and Nfib knock-out mice are characterized by neuroanatomical defects, including agenesis of the corpus callosum, loss of specific midline glial populations, and a 5–10-fold decrease in the expression of glial fibrillary acidic protein (GFAP), which is an astrocyte marker (11Steele-Perkins G. Plachez C. Butz K.G. Yang G. Bachurski C.J. Kinsman S.L. Litwack E.D. Richards L.J. Gronostajski R.M. Mol. Cell Biol. 2005; 25: 685-698Crossref PubMed Scopus (227) Google Scholar, 12das Neves L. Duchala C.S. Tolentino-Silva F. Haxhiu M.A. Colmenares C. Macklin W.B. Campbell C.E. Butz K.G. Gronostajski R.M. Godinho F. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 11946-11951Crossref PubMed Scopus (181) Google Scholar, 21Shu T. Butz K.G. Plachez C. Gronostajski R.M. Richards L.J. J. Neurosci. 2003; 23: 203-212Crossref PubMed Google Scholar). In addition, Nfib-deficient mice present aberrant hippocampus and pons formation and die due to the defects in lung development (11Steele-Perkins G. Plachez C. Butz K.G. Yang G. Bachurski C.J. Kinsman S.L. Litwack E.D. Richards L.J. Gronostajski R.M. Mol. Cell Biol. 2005; 25: 685-698Crossref PubMed Scopus (227) Google Scholar). The immature nestin-positive glia populate the hippocampus of Nfib-deficient mice but fail to mature into GFAP-positive astrocytes (22Barry G. Piper M. Lindwall C. Moldrich R. Mason S. Little E. Sarkar A. Tole S. Gronostajski R.M. Richards L.J. J. Neurosci. 2008; 28: 12328-12340Crossref PubMed Scopus (67) Google Scholar). In contrast, disruption of the Nfic gene results in early postnatal defects in tooth formation, including the loss of molar roots and aberrant incisor development (23Steele-Perkins G. Butz K.G. Lyons G.E. Zeichner-David M. Kim H.J. Cho M.I. Gronostajski R.M. Mol. Cell Biol. 2003; 23: 1075-1084Crossref PubMed Scopus (156) Google Scholar). The Nfix knock-out mice have recently been generated by two independent groups (24Driller K. Pagenstecher A. Uhl M. Omran H. Berlis A. Gründer A. Sippel A.E. Mol. Cell Biol. 2007; 27: 3855-3867Crossref PubMed Scopus (101) Google Scholar, 25Campbell C.E. Piper M. Plachez C. Yeh Y.T. Baizer J.S. Osinski J.M. Litwack E.D. Richards L.J. Gronostajski R.M. BMC Dev. Biol. 2008; 8: 52Crossref PubMed Scopus (113) Google Scholar), which reported multiple effects (26Pekarik V. Belmonte J.C. J. Biol. 2008; 7: 29Crossref PubMed Scopus (8) Google Scholar). The Nfix gene knock-out causes postnatal lethality in most of the animals and leads to hydrocephalus and partial agenesis of the corpus callosum (24Driller K. Pagenstecher A. Uhl M. Omran H. Berlis A. Gründer A. Sippel A.E. Mol. Cell Biol. 2007; 27: 3855-3867Crossref PubMed Scopus (101) Google Scholar). These mice also develop a deformation of the spine with kyphosis, due to a delay in ossification of vertebral bodies and a progressive degeneration of intervertebral disks. However, Nfix knock-out mice survive on a soft chow diet but are characterized by increased brain weight, expansion of the brain along the dorsal ventrical axis, and aberrant formation of the hippocampus (25Campbell C.E. Piper M. Plachez C. Yeh Y.T. Baizer J.S. Osinski J.M. Litwack E.D. Richards L.J. Gronostajski R.M. BMC Dev. Biol. 2008; 8: 52Crossref PubMed Scopus (113) Google Scholar). In summary, the knock-out phenotypes suggest that NFI-A, -B, and -X are important for normal brain development; however, the identity of the affected cell type(s) is not clear. Recently, both NFI-A and -B were shown to regulate gliogenesis in the chick embryo, with NFI-A also controlling the maintenance of neural precursors (27Deneen B. Ho R. Lukaszewicz A. Hochstim C.J. Gronostajski R.M. Anderson D.J. Neuron. 2006; 52: 953-968Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). More recently, NFI-C and -X were shown to regulate the expression of late astrocyte markers during the differentiation of neural precursors into astrocytes in vitro (28Wilczynska K.M. Singh S.K. Adams B. Bryan L. Rao R.R. Valerie K. Wright S. Griswold-Prenner I. Kordula T. Stem Cells. 2009; 27: 1173-1181Crossref PubMed Scopus (35) Google Scholar).Alternative splicing is a common mechanism of generating transcription factors with diverse functions in the brain (29Vihma H. Pruunsild P. Timmusk T. Genomics. 2008; 92: 279-291Crossref PubMed Scopus (51) Google Scholar, 30Arteaga M.F. Coric T. Straub C. Canessa C.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 4459-4464Crossref PubMed Scopus (49) Google Scholar, 31Chu H.Y. Ohtoshi A. Mol. Cell Biol. 2007; 27: 3743-3749Crossref PubMed Scopus (9) Google Scholar, 32Michelhaugh S.K. Vaitkevicius H. Wang J. Bouhamdan M. Krieg A.R. Walker J.L. Mendiratta V. 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Mechanistically, the TA domain of NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II.
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