- Email: [email protected]
The nano-scale architecture of the nucleus
HEADLINES
Where did you get that HAT? HATs are very much in fashion these days. Over the past few years, there has been a flurry of activity in identifying proteins with histone acetyl transferase (HAT) properties. Several coactivators of transcription, including the GCN5, p300/CBP and PCAF proteins, catalyse the acetylation of histones. It is proposed that the HAT activity of these coactivators is involved in the remodelling of nucleosomes and spurring changes in chromatin structure that regulate gene expression. Kawasaki et al.1 have added an interesting new member to this HAT collection. While studying the interaction between the transcription factor ATF-2 and the coactivator p300, Kawasaki et al. fortuitously discovered that ATF-2 by itself has intrinsic HAT activity. Purified ATF-2 was able to acetylate histones H2B and H4 (but not H3 or H2A) as free histones or when bound in mononucleosomes. The authors localized the HAT domain to the middle portion of ATF-2 (residues
112–350) between the activation domain and the basic leucine zipper (bZip) region. The ATF-2 sequence (residues 289–311) shares some similarity with a motif found in PCAF and GCN5 HAT proteins. Mutations within this sequence of ATF-2 abolished HAT activity and transactivation. Ultraviolet irradiation induces ATF-2 phosphorylation by the JNK/SAPK kinases on two crucial threonine residues (T69 and T71) in the transactivation domain. This phosphorylation event controls HAT activity. Mutation of T69 and T71 residues diminished interaction with p300 (the UV-induced intrinsic HAT activity) and transactivation of a reporter gene. The related bZip protein c-Jun had no HAT activity. ATF-2 appears to be an extremely versatile protein. Like many transcription factors, ATF-2 has a modular architecture: it contains a dimerization domain, a sequence-specific DNA-binding portion, a HAT domain and an N-terminal transactivation
domain. Phosphorylation of residues in the transactivation domain regulates interaction with coactivators and recruitment of HATs, as well as modulating the catalytic activity of its own HAT domain (perhaps by conformational changes). Future work will address the questions of when and why ATF-2 uses its HAT tricks and what are its natural cellular acetylation substrates. The observation that ATF-2 sits on the promoter of certain ‘immediate early genes’ suggests that it might be uniquely poised to open up chromatin and drive gene expression as part of a rapid response to cellular stress signals. ATF-2’s HAT skills will be the envy of many less-talented transcription factors.
1
Kawasaki, H. et al. (2000) ATF-2 has intrinsic histone acetyltransferase activity which is modulated by phosphorylation. Nature 405, 195–200
The nano-scale architecture of the nucleus
This month’s headlines were contributed by Søren Andersen, Paul Ko Ferrigno, Volker Haucke, Michael Mishkind, Jonathan Weitzman and Cezary Wojcik.
366
The post-reductionist era has been with us for some time, and cell biologists are now accomplished reconstructionists, building pictures of cellular structures from proteins identified through biochemistry and genetics. Understanding the beauty of cellular structures requires a knowledge of their inner architecture and engineering. A paper by Moir et al. addresses the process of DNA metabolism1. Although we know much about the enzymes involved, many, many questions remain. The first of these is: on what scaffolding does DNA replication take place? DNA replication localizes to apparently immobile structures, so-called factories, that lie on the inner aspect of the nuclear membrane. This suggests that the synthesis of new DNA would require the feeding of chromatin through the factory and raises interesting questions of mechanical engineering. Is chromatin supported on either side of
the factory, or does its weight drag it into the nuclear interior? Is the factory anchored in the nuclear membrane or to some other nuclear support? What engines drive chromatin through the factory – and how does the nucleus brace itself to withstand these forces? The foundation of nuclear envelope integrity has long been thought to be the nuclear lamina, and Moir et al. show clearly that, at least in Xenopus egg extracts, lamin structure is required for the elongation of replication forks. They find that the expression of a mutant human A-type lamin in the extracts leads to the formation of protein aggregates that include Xenopus lamins. These aggregates recruit at least two of the proteins (PCNA and RFC) involved in DNA replication. Because the initiation of DNA replication was not affected by lamina disruption, the authors suggest that normal lamin structure is required for the correct organization of elongation factors.
This raises another question: why would the ubiquitous lamin proteins recruit elongation factors? One alternative explanation for the authors’ observations is that the aggregates are serving as a physical brake to the movement of the replication fork. Another idea is that the role of the lamina could simply be to withstand the mechanical forces generated by elongation rather than to serve as the bedrock foundation of the factory. The complexity of Millennium domes, Eiffel towers and ‘Ferris wheels’ are likely just pale reflections of life at the heart of the cell.
1
Moir, R.D. et al. (2000) Disruption of nuclear lamin organization blocks the elongation phase of DNA replication. J. Cell Biol. 149, 1179–1191
trends in CELL BIOLOGY (Vol. 10) September 2000
Where did you get that HAT? HATs are very much in fashion these days. Over the past few years, there has been a flurry of activity in identifying proteins with histone acetyl transferase (HAT) properties. Several coactivators of transcription, including the GCN5, p300/CBP and PCAF proteins, catalyse the acetylation of histones. It is proposed that the HAT activity of these coactivators is involved in the remodelling of nucleosomes and spurring changes in chromatin structure that regulate gene expression. Kawasaki et al.1 have added an interesting new member to this HAT collection. While studying the interaction between the transcription factor ATF-2 and the coactivator p300, Kawasaki et al. fortuitously discovered that ATF-2 by itself has intrinsic HAT activity. Purified ATF-2 was able to acetylate histones H2B and H4 (but not H3 or H2A) as free histones or when bound in mononucleosomes. The authors localized the HAT domain to the middle portion of ATF-2 (residues
112–350) between the activation domain and the basic leucine zipper (bZip) region. The ATF-2 sequence (residues 289–311) shares some similarity with a motif found in PCAF and GCN5 HAT proteins. Mutations within this sequence of ATF-2 abolished HAT activity and transactivation. Ultraviolet irradiation induces ATF-2 phosphorylation by the JNK/SAPK kinases on two crucial threonine residues (T69 and T71) in the transactivation domain. This phosphorylation event controls HAT activity. Mutation of T69 and T71 residues diminished interaction with p300 (the UV-induced intrinsic HAT activity) and transactivation of a reporter gene. The related bZip protein c-Jun had no HAT activity. ATF-2 appears to be an extremely versatile protein. Like many transcription factors, ATF-2 has a modular architecture: it contains a dimerization domain, a sequence-specific DNA-binding portion, a HAT domain and an N-terminal transactivation
domain. Phosphorylation of residues in the transactivation domain regulates interaction with coactivators and recruitment of HATs, as well as modulating the catalytic activity of its own HAT domain (perhaps by conformational changes). Future work will address the questions of when and why ATF-2 uses its HAT tricks and what are its natural cellular acetylation substrates. The observation that ATF-2 sits on the promoter of certain ‘immediate early genes’ suggests that it might be uniquely poised to open up chromatin and drive gene expression as part of a rapid response to cellular stress signals. ATF-2’s HAT skills will be the envy of many less-talented transcription factors.
1
Kawasaki, H. et al. (2000) ATF-2 has intrinsic histone acetyltransferase activity which is modulated by phosphorylation. Nature 405, 195–200
The nano-scale architecture of the nucleus
This month’s headlines were contributed by Søren Andersen, Paul Ko Ferrigno, Volker Haucke, Michael Mishkind, Jonathan Weitzman and Cezary Wojcik.
366
The post-reductionist era has been with us for some time, and cell biologists are now accomplished reconstructionists, building pictures of cellular structures from proteins identified through biochemistry and genetics. Understanding the beauty of cellular structures requires a knowledge of their inner architecture and engineering. A paper by Moir et al. addresses the process of DNA metabolism1. Although we know much about the enzymes involved, many, many questions remain. The first of these is: on what scaffolding does DNA replication take place? DNA replication localizes to apparently immobile structures, so-called factories, that lie on the inner aspect of the nuclear membrane. This suggests that the synthesis of new DNA would require the feeding of chromatin through the factory and raises interesting questions of mechanical engineering. Is chromatin supported on either side of
the factory, or does its weight drag it into the nuclear interior? Is the factory anchored in the nuclear membrane or to some other nuclear support? What engines drive chromatin through the factory – and how does the nucleus brace itself to withstand these forces? The foundation of nuclear envelope integrity has long been thought to be the nuclear lamina, and Moir et al. show clearly that, at least in Xenopus egg extracts, lamin structure is required for the elongation of replication forks. They find that the expression of a mutant human A-type lamin in the extracts leads to the formation of protein aggregates that include Xenopus lamins. These aggregates recruit at least two of the proteins (PCNA and RFC) involved in DNA replication. Because the initiation of DNA replication was not affected by lamina disruption, the authors suggest that normal lamin structure is required for the correct organization of elongation factors.
This raises another question: why would the ubiquitous lamin proteins recruit elongation factors? One alternative explanation for the authors’ observations is that the aggregates are serving as a physical brake to the movement of the replication fork. Another idea is that the role of the lamina could simply be to withstand the mechanical forces generated by elongation rather than to serve as the bedrock foundation of the factory. The complexity of Millennium domes, Eiffel towers and ‘Ferris wheels’ are likely just pale reflections of life at the heart of the cell.
1
Moir, R.D. et al. (2000) Disruption of nuclear lamin organization blocks the elongation phase of DNA replication. J. Cell Biol. 149, 1179–1191
trends in CELL BIOLOGY (Vol. 10) September 2000