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Regulating Intertwining Proteins
Previews 1061
tion cycle is a conserved feature of NMD. It is almost certainly a feature of C. elegans NMD (Page et al., 1999; Mango, 2001; Anders et al., 2003) and it appears to also be necessary in flies, as Drosophila melanogaster orthologs of SMG-1, -5, and -7 have been identified and shown to be essential for NMD (Gatfield et al., 2003). However, the situation in Saccharomyces cerevisiae is murky, as no yeast orthologs of SMG-1, -5, -6, and -7 have been found, yeast Upf1p has not been reported to be a phosphoprotein, and yeast Upf1p lacks the N-terminal region that mediates SMG-5 binding (Ohnishi et al., 2003). Nevertheless, yeast have protein kinases somewhat related to SMG-1 (called Tor1p and Tor2p) and an uncharacterized protein that has an extensive region of similarity to SMG-5 (called Tos5p) (Anders et al., 2003), indicating that the case for yeast is not closed. Another perplexing issue concerns the location of the proteins involved in NMD. Ohnishi et al. (2003) found that while SMG-5, -6, and -7 are primarily cytoplasmic, they also shuttle to the nucleus. This is reminiscent of UPF1, which resides mainly in the cytoplasm, but also shuttles to the nucleus (Mendell et al., 2002). What is the purpose of this shuttling? Do these proteins shuttle together to the nucleus to recycle UPF1 or do they go to the nucleus to engage in a pioneer round of translation, a controversial idea for which there is evidence both for and against (Dahlberg et al., 2003) (Figure 1). To address this question, it will be important to assess the phosphorylation status of nuclear UPF1 and to determine the nature of the UPF1-containing protein complexes in the nucleus (only cytoplasmic protein complexes were analyzed by Ohnishi et al. [2003] and Schell et al. [2003]). Now that many of the molecules involved in NMD have been identified and we are beginning to understand their
sequential interactions and subcellular locations, one hopes that we are arising from another night’s sleep, cleared of nonsense, and ready to solve the remaining riddles in this long-term puzzle.
Regulating Intertwining Proteins
existence of the bHLH domain (Ellenberger et al., 1994). The E47 homodimer is centered over its recognition site, with glutamate and arginine residues localized in the basic region interacting with the cytosine and adenine bases present in the cognate binding site. The HLH domain itself forms a parallel, four-helix bundle that is stabilized by van der Waals interactions between highly conserved hydrophobic residues that are present in both helices. The HLH family, now comprising more than 125 members in the human proteome, can be divided into distinct classes based on their expression patterns and biochemical properties (Massari and Murre, 2000). These classes include bHLH proteins, bHLH proteins that also contain a leucine zipper, and HLH proteins that lack a DNA binding domain and function as antagonists of the bHLH proteins. HLH proteins readily change dimerization partners during lymphocyte development. For example, human pre-B lineage cells express the bHLH proteins, E47 and E2-2, to form heterodimers, whereas in mature B lineage cells only E47 homodimers are present. In activated human mature B lineage cells E47 interacts with yet another bHLH protein, ABF-1. During the early stages
The basic helix-loop-helix (bHLH) proteins constitute a family of eukaryotic transcription factors that have the ability to dimerize through a coiled-coil region, characterized by a series of highly conserved hydrophobic residues that form a helix-loop-helix structure. In this issue of Molecular Cell, Firulli et al. demonstrate that dimerization of the bHLH proteins, HAND1 and HAND2, is regulated by phosphorylation of residues located within the HLH domain. Fifteen years ago, the Cabrera and Weintraub laboratories identified a domain, present in MyoD and members of the achaete-scute complex, that showed striking homology to the protooncogene c-myc (Davis et al., 1987; Villares and Cabrera, 1987). The myc homology region was also found in the immunoglobulin enhancer binding proteins, E12 and E47, and proposed to form a basic helix-loop-helix (bHLH) structure. Soon thereafter, the structure of the E47 DNA binding region, bound to its recognition site, was resolved and confirmed the
Miles F. Wilkinson Department of Immunology The University of Texas M.D. Anderson Cancer Center Houston, Texas 77030 Selected Reading Anders, K.R., Grimson, A., and Anderson, P. (2003). EMBO J. 22, 641–650. Chiu, S.-Y., Serin, G., Ohara, O., and Maquat, L.E. (2003). RNA 9, 77–87. Dahlberg, J.E., Lund, E., and Goodwin, E.B. (2003). RNA 9, 1–8. Gatfield, D., Unterholzner, L., Ciccarelli, F.D., Bork, P., and Izaurralde, E. (2003). EMBO J. 22, 3960–3970. Gehring, N.H., Neu-Yilik, G., Schell, T., Hentze, M.W., and Kulozik, A.E. (2003). Mol. Cell 11, 939–949. Gonza´lez, C.I., Bhattacharya, A., Wang, W., and Peltz, S.W. (2001). Gene 274, 15–25. Mango, S.E. (2001). Trends Genet. 17, 646–653. Mendell, J.T., ap Rhys, C.M.J., and Dietz, H.C. (2002). Science 298, 419–422. Ohnishi, T., Yamashita, A., Kashima, I., Schell, T., Anders, K.R., Grimson, A., Hachiya, T., Hentze, M.W., Anderson, P., and Ohno, S. (2003). Mol. Cell 12, this issue, 1187–1200. Page, M.F., Carr, B., Anders, K.R., Grimson, A., and Anderson, P. (1999). Mol. Cell. Biol. 19, 5943–5951. Schell, T., Ko¨cher, T., Wilm, M., Seraphin, B., Kulozik, A.E., and Hentze, M.W. (2003). Biochem. J. 373, 775–783.
Molecular Cell 1062
of thymocyte development, the E47 gene products form heterodimers with yet another bHLH protein, HEB. The DNA binding activity of this heterodimeric complex is further modulated at two developmental checkpoints (Engel and Murre, 2001). This has raised the question as to how HLH dimerization is regulated. One form of regulation is well established and involves members of the Id gene family. For example, the antagonist HLH protein, Id3, is induced during thymocyte maturation by pre-TCR and TCR-mediated signaling. High levels of Id3 readily form heterodimers with the E47 and HEB, to inactivate their DNA binding activity. Another level of regulation has been suggested by studies that showed that hypophosphorylation promotes the formation of E47 homodimers in mature B lineage cells (Sloan et al., 1996). An alternative model for the formation of E47 homodimers has been suggested by studies demonstrating an intermolecular disulphide bond in E47, which was detected only in B lineage cells (Benezra, 1994). Now Firulli et al. (2003) provide new data that go to the heart of HLH regulation. Their studies have focused on the activities of the bHLH proteins, HAND1 and HAND2. HAND proteins are expressed in the developing heart, neural crest cells that occupy the brachial arches, lateral mesoderm, and trophoblasts. Hand1 functions to promote the terminal differentiation of trophoblasts. Additionally, ectopic expression of HAND gene products in the limbs of both chickens and mice results in duplication of digits. These and other studies have suggested that the HAND proteins play a key role in the growth and patterning of the limb bud along the anteroposterior axis (Johnson and Tabin, 1997). Firulli et al. (2003) demonstrate that during trophoblast differentiation the activity of HAND proteins is modulated by phosphorylation. Specifically, they show that residues present in the HLH domain are diffentially phosphorylated during trophoblast differentiation. They show that both PKA and PKC have the ability to modify these residues and further they identify a phosphatase, PP2A, that specifically interacts with the N- and C-terminal portion of HAND1, to modulate phosphorylation. Finally, they show that mutations in amino acids within the HLH region that are differentially phosphorylated alter the ability of HAND proteins
to dimerize and severely affect the ability to induce polydactylily in the chick hindlimb. Together, these experiments are the first to demonstrate that HLH dimerization is regulated, at least in part, by phosphorylation. Since HAND1 and HAND2 are expressed in multiple tissues, it will be particularly interesting to mutate the relevant residues in the mouse germline. Analysis of such mutants should provide further insight into how HLH dimerization is controlled during distinct stages of embryonic development and how it affects developmental progression. Although some progress has been made with regard to the regulation of bHLH activity, little insight has been gained into how dimerization actually is regulated. The findings by Firulli et al. are an important step toward addressing this problem. Will other HLH proteins also utilize phosphorylation or other forms of modification as a means to regulate dimerization? Modulation of HLH dimerization by posttranslational modification provides an efficient mechanism to regulate transcription factor activity, and it is likely that it will be used in a wide variety of biological systems.
A Two-Tiered Transcription Regulation Mechanism that Protects Germ Cell Identity
Germ cells are specialized cells that have the potential to develop into any type of tissue in the body. Mechanisms that protect germ cells from inappropriate differentiation are therefore crucial to reproduction, and may serve as models for understanding how pluripotency and identity are also maintained in somatic stem cell lineages. In various organisms, early embryonic germ cell precursors are protected from somatic differentiation cues by mechanisms that transiently and globally silence mRNA transcription (Leatherman and Jongens, 2003; Tomioka et al., 2002). In these cell lineages, early differentiation steps are guided by translational control of stored mRNAs until a germline transcription program takes over. In the nematode C. elegans, the identity of early germline precursors is initially maintained through successive
In the November issue of Developmental Cell, Schaner and colleagues (2003) describe remarkable versatility in how the embryonic germ lineage blocks differentiation: in early C. elegans germ cell precursors transcription is silenced but chromatin remains open, and then after lineage restriction a conserved inhibitory chromatin architecture appears.
Cornelis Murre Division of Biological Sciences, 0377 University of California, San Diego La Jolla, California 92093 Selected Reading Benezra, R. (1994). Cell 79, 1057–1067. Davis, R.L., Weintraub, H., and Lassar, A.B. (1987). Cell 51, 987–1000. Ellenberger, T.D., Fass, D., Arnaud, M., and Harrison, S. (1994). Genes Dev. 10, 970–980. Engel, I., and Murre, C. (2001). Nat. Rev. Immunol. 1, 193–199. Firulli, B.A., Howard, M.J., McDaid, J.R., McIlreavey, L., Dionne, K.M., Centonze, V.E., Cserjesi, P., Virshup, D.M., and Firulli, A.B. (2003). Mol. Cell 12, this issue, 1225–1237. Johnson, R.L., and Tabin, C.J. (1997). Cell 90, 979–990. Massari, M.E., and Murre, C. (2000). Mol. Cell. Biol. 20, 429–440. Sloan, S.R., Shen, C., McCarrick-Walmsley, R., and Kadesch, T. (1996). Mol. Cell. Biol. 16, 6900–6908. Villares, R., and Cabrera, C.V. (1987). Cell 50, 415–424.
tion cycle is a conserved feature of NMD. It is almost certainly a feature of C. elegans NMD (Page et al., 1999; Mango, 2001; Anders et al., 2003) and it appears to also be necessary in flies, as Drosophila melanogaster orthologs of SMG-1, -5, and -7 have been identified and shown to be essential for NMD (Gatfield et al., 2003). However, the situation in Saccharomyces cerevisiae is murky, as no yeast orthologs of SMG-1, -5, -6, and -7 have been found, yeast Upf1p has not been reported to be a phosphoprotein, and yeast Upf1p lacks the N-terminal region that mediates SMG-5 binding (Ohnishi et al., 2003). Nevertheless, yeast have protein kinases somewhat related to SMG-1 (called Tor1p and Tor2p) and an uncharacterized protein that has an extensive region of similarity to SMG-5 (called Tos5p) (Anders et al., 2003), indicating that the case for yeast is not closed. Another perplexing issue concerns the location of the proteins involved in NMD. Ohnishi et al. (2003) found that while SMG-5, -6, and -7 are primarily cytoplasmic, they also shuttle to the nucleus. This is reminiscent of UPF1, which resides mainly in the cytoplasm, but also shuttles to the nucleus (Mendell et al., 2002). What is the purpose of this shuttling? Do these proteins shuttle together to the nucleus to recycle UPF1 or do they go to the nucleus to engage in a pioneer round of translation, a controversial idea for which there is evidence both for and against (Dahlberg et al., 2003) (Figure 1). To address this question, it will be important to assess the phosphorylation status of nuclear UPF1 and to determine the nature of the UPF1-containing protein complexes in the nucleus (only cytoplasmic protein complexes were analyzed by Ohnishi et al. [2003] and Schell et al. [2003]). Now that many of the molecules involved in NMD have been identified and we are beginning to understand their
sequential interactions and subcellular locations, one hopes that we are arising from another night’s sleep, cleared of nonsense, and ready to solve the remaining riddles in this long-term puzzle.
Regulating Intertwining Proteins
existence of the bHLH domain (Ellenberger et al., 1994). The E47 homodimer is centered over its recognition site, with glutamate and arginine residues localized in the basic region interacting with the cytosine and adenine bases present in the cognate binding site. The HLH domain itself forms a parallel, four-helix bundle that is stabilized by van der Waals interactions between highly conserved hydrophobic residues that are present in both helices. The HLH family, now comprising more than 125 members in the human proteome, can be divided into distinct classes based on their expression patterns and biochemical properties (Massari and Murre, 2000). These classes include bHLH proteins, bHLH proteins that also contain a leucine zipper, and HLH proteins that lack a DNA binding domain and function as antagonists of the bHLH proteins. HLH proteins readily change dimerization partners during lymphocyte development. For example, human pre-B lineage cells express the bHLH proteins, E47 and E2-2, to form heterodimers, whereas in mature B lineage cells only E47 homodimers are present. In activated human mature B lineage cells E47 interacts with yet another bHLH protein, ABF-1. During the early stages
The basic helix-loop-helix (bHLH) proteins constitute a family of eukaryotic transcription factors that have the ability to dimerize through a coiled-coil region, characterized by a series of highly conserved hydrophobic residues that form a helix-loop-helix structure. In this issue of Molecular Cell, Firulli et al. demonstrate that dimerization of the bHLH proteins, HAND1 and HAND2, is regulated by phosphorylation of residues located within the HLH domain. Fifteen years ago, the Cabrera and Weintraub laboratories identified a domain, present in MyoD and members of the achaete-scute complex, that showed striking homology to the protooncogene c-myc (Davis et al., 1987; Villares and Cabrera, 1987). The myc homology region was also found in the immunoglobulin enhancer binding proteins, E12 and E47, and proposed to form a basic helix-loop-helix (bHLH) structure. Soon thereafter, the structure of the E47 DNA binding region, bound to its recognition site, was resolved and confirmed the
Miles F. Wilkinson Department of Immunology The University of Texas M.D. Anderson Cancer Center Houston, Texas 77030 Selected Reading Anders, K.R., Grimson, A., and Anderson, P. (2003). EMBO J. 22, 641–650. Chiu, S.-Y., Serin, G., Ohara, O., and Maquat, L.E. (2003). RNA 9, 77–87. Dahlberg, J.E., Lund, E., and Goodwin, E.B. (2003). RNA 9, 1–8. Gatfield, D., Unterholzner, L., Ciccarelli, F.D., Bork, P., and Izaurralde, E. (2003). EMBO J. 22, 3960–3970. Gehring, N.H., Neu-Yilik, G., Schell, T., Hentze, M.W., and Kulozik, A.E. (2003). Mol. Cell 11, 939–949. Gonza´lez, C.I., Bhattacharya, A., Wang, W., and Peltz, S.W. (2001). Gene 274, 15–25. Mango, S.E. (2001). Trends Genet. 17, 646–653. Mendell, J.T., ap Rhys, C.M.J., and Dietz, H.C. (2002). Science 298, 419–422. Ohnishi, T., Yamashita, A., Kashima, I., Schell, T., Anders, K.R., Grimson, A., Hachiya, T., Hentze, M.W., Anderson, P., and Ohno, S. (2003). Mol. Cell 12, this issue, 1187–1200. Page, M.F., Carr, B., Anders, K.R., Grimson, A., and Anderson, P. (1999). Mol. Cell. Biol. 19, 5943–5951. Schell, T., Ko¨cher, T., Wilm, M., Seraphin, B., Kulozik, A.E., and Hentze, M.W. (2003). Biochem. J. 373, 775–783.
Molecular Cell 1062
of thymocyte development, the E47 gene products form heterodimers with yet another bHLH protein, HEB. The DNA binding activity of this heterodimeric complex is further modulated at two developmental checkpoints (Engel and Murre, 2001). This has raised the question as to how HLH dimerization is regulated. One form of regulation is well established and involves members of the Id gene family. For example, the antagonist HLH protein, Id3, is induced during thymocyte maturation by pre-TCR and TCR-mediated signaling. High levels of Id3 readily form heterodimers with the E47 and HEB, to inactivate their DNA binding activity. Another level of regulation has been suggested by studies that showed that hypophosphorylation promotes the formation of E47 homodimers in mature B lineage cells (Sloan et al., 1996). An alternative model for the formation of E47 homodimers has been suggested by studies demonstrating an intermolecular disulphide bond in E47, which was detected only in B lineage cells (Benezra, 1994). Now Firulli et al. (2003) provide new data that go to the heart of HLH regulation. Their studies have focused on the activities of the bHLH proteins, HAND1 and HAND2. HAND proteins are expressed in the developing heart, neural crest cells that occupy the brachial arches, lateral mesoderm, and trophoblasts. Hand1 functions to promote the terminal differentiation of trophoblasts. Additionally, ectopic expression of HAND gene products in the limbs of both chickens and mice results in duplication of digits. These and other studies have suggested that the HAND proteins play a key role in the growth and patterning of the limb bud along the anteroposterior axis (Johnson and Tabin, 1997). Firulli et al. (2003) demonstrate that during trophoblast differentiation the activity of HAND proteins is modulated by phosphorylation. Specifically, they show that residues present in the HLH domain are diffentially phosphorylated during trophoblast differentiation. They show that both PKA and PKC have the ability to modify these residues and further they identify a phosphatase, PP2A, that specifically interacts with the N- and C-terminal portion of HAND1, to modulate phosphorylation. Finally, they show that mutations in amino acids within the HLH region that are differentially phosphorylated alter the ability of HAND proteins
to dimerize and severely affect the ability to induce polydactylily in the chick hindlimb. Together, these experiments are the first to demonstrate that HLH dimerization is regulated, at least in part, by phosphorylation. Since HAND1 and HAND2 are expressed in multiple tissues, it will be particularly interesting to mutate the relevant residues in the mouse germline. Analysis of such mutants should provide further insight into how HLH dimerization is controlled during distinct stages of embryonic development and how it affects developmental progression. Although some progress has been made with regard to the regulation of bHLH activity, little insight has been gained into how dimerization actually is regulated. The findings by Firulli et al. are an important step toward addressing this problem. Will other HLH proteins also utilize phosphorylation or other forms of modification as a means to regulate dimerization? Modulation of HLH dimerization by posttranslational modification provides an efficient mechanism to regulate transcription factor activity, and it is likely that it will be used in a wide variety of biological systems.
A Two-Tiered Transcription Regulation Mechanism that Protects Germ Cell Identity
Germ cells are specialized cells that have the potential to develop into any type of tissue in the body. Mechanisms that protect germ cells from inappropriate differentiation are therefore crucial to reproduction, and may serve as models for understanding how pluripotency and identity are also maintained in somatic stem cell lineages. In various organisms, early embryonic germ cell precursors are protected from somatic differentiation cues by mechanisms that transiently and globally silence mRNA transcription (Leatherman and Jongens, 2003; Tomioka et al., 2002). In these cell lineages, early differentiation steps are guided by translational control of stored mRNAs until a germline transcription program takes over. In the nematode C. elegans, the identity of early germline precursors is initially maintained through successive
In the November issue of Developmental Cell, Schaner and colleagues (2003) describe remarkable versatility in how the embryonic germ lineage blocks differentiation: in early C. elegans germ cell precursors transcription is silenced but chromatin remains open, and then after lineage restriction a conserved inhibitory chromatin architecture appears.
Cornelis Murre Division of Biological Sciences, 0377 University of California, San Diego La Jolla, California 92093 Selected Reading Benezra, R. (1994). Cell 79, 1057–1067. Davis, R.L., Weintraub, H., and Lassar, A.B. (1987). Cell 51, 987–1000. Ellenberger, T.D., Fass, D., Arnaud, M., and Harrison, S. (1994). Genes Dev. 10, 970–980. Engel, I., and Murre, C. (2001). Nat. Rev. Immunol. 1, 193–199. Firulli, B.A., Howard, M.J., McDaid, J.R., McIlreavey, L., Dionne, K.M., Centonze, V.E., Cserjesi, P., Virshup, D.M., and Firulli, A.B. (2003). Mol. Cell 12, this issue, 1225–1237. Johnson, R.L., and Tabin, C.J. (1997). Cell 90, 979–990. Massari, M.E., and Murre, C. (2000). Mol. Cell. Biol. 20, 429–440. Sloan, S.R., Shen, C., McCarrick-Walmsley, R., and Kadesch, T. (1996). Mol. Cell. Biol. 16, 6900–6908. Villares, R., and Cabrera, C.V. (1987). Cell 50, 415–424.