Roger D. Kornberg

Twenty-Five Years of the Nucleosome, Fundamental Particle of the Eukaryote Chromosome

The chromatin field needs much more information about structure beyond the nucleosome. Even the trajectory of the DNA entering and exiting the nucleosome, immediately beyond the core particle, is unclear (reviewed by Prunell 1998xA topological approach to nucleosome structure and dynamics (the linking number paradox and other issues) . Prunell, A. Biophys. J. 1998; 74: 2531–2544Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesPrunell 1998). The pattern of coiling a chain of nucleosomes in a thicker fiber remains uncertain (reviewed by Ramakrishnan 1997xHistone H1 and chromatin higher-order structure. Ramakrishnan, V. Crit. Rev. Eukaryot. Gene Expr. 1997; 7: 215–230Crossref | PubMedSee all ReferencesRamakrishnan 1997). Whereas additional coiling in a succession of higher helices would be a most plausible mechanism of further condensation, alternative hypotheses have been advanced. Many of the difficulties of analyzing chromatin problems stem from the variability inherent in higher order chromatin structures. Existing methods of structure determination require averaging and thus are of limited use in the face of variability.Solution of the higher order structure problem is crucial for understanding chromatin function. Histone tail modifications and interactions with other proteins, important for regulation, seem likely to influence higher order structure more than core particle structure. There is, however, insufficient evidence that acetylation actually causes chromatin unfolding, and only a suggestion of the interplay between acetylation and the chromatin remodeling events that affect core particle structure (14xOrdered recruitment of transcription and chromatin-remodeling factors to a cell cycle– and developmentally regulated promoter. Cosma, M.P, Tanaka, T, and Nasmyth, K. Cell. 1999; 97: 299–311Abstract | Full Text | Full Text PDF | PubMed | Scopus (545)See all References, 81xThe nucleosome remodeling complex, Snf/Swi, is required for the maintenance of transcription in vivo and is partially redundant with the histone acetyltransferase, gcn5. Sudarsanam, P, Cao, Y, Wu, L, Laurent, B.C, and Winston, F. EMBO J. 1999; 18: 3101–3106Crossref | PubMed | Scopus (86)See all References). Histone phosphorylation, long correlated with chromosome condensation, has recently been linked to transcriptional activity (reviewed by Bjorklund et al. 1999xGlobal transcription regulators of eukaryotes. Bjorklund, S, Almouzni, G, Davidson, I, Nightingale, K.P, and Weiss, K. Cell. 1999; 96: 759–767Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesBjorklund et al. 1999). There is no information as to the structural or functional consequences of other modifications, such as ubiquitination and glycosylation. Histone H1 is thought to promote condensation, but an understanding of its structural role and the connection with gene activity is lacking. Elucidation of gene silencing by heterochromatin similarly depends on determination of its structure.Finally, functional analysis in cell-free systems must be extended beyond the nucleosome to the chromosomal context. Histone–DNA complexes assembled in vitro reveal effects of HAT and chromatin-remodeling complexes on transcription (56xRole of nucleosome remodeling factor NURF in transcriptional activation of chromatin. Mizuguchi, G, Tsukiyama, T, Wisniewski, J, and Wu, C. Mol. Cell. 1997; 1: 141–150Abstract | Full Text | Full Text PDF | PubMedSee all References, 49xRequirement of RSF and FACT for transcription of chromatin templates in vitro. LeRoy, G, Orphanides, G, Lane, W.S, and Reinberg, D. Science. 1998; 282: 1900–1904Crossref | PubMedSee all References, 88xTranscriptional activators direct histone acetyltransferase complexes to nucleosomes. Utley, R.T, Ikeda, K, Grant, P.A, Cote, J, Steger, D.J, Eberharter, A, John, S, and Workman, J.L. Nature. 1998; 394: 498–502Crossref | PubMed | Scopus (406)See all References), but in vivo, promoters are associated with additional, nonhistone proteins, which influence the locations of nucleosomes and undoubtedly the higher order configuration of chromatin as well. These associations extend, in the broadest sense, to such DNA elements as locus control regions, which regulate the structure and activity of entire chromosomal domains. Only when transcription, replication, recombination, and other DNA transactions have been reconstituted in vitro with naturally assembled chromatin templates will a full understanding of the nucleosome be achieved.*To whom correspondence should be addressed (e-mail: kornberg@ stanford.edu).

A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II

A mediator was isolated from yeast that enabled a response to the activator proteins GAL4-VP16 and GCN4 in a transcription system reconstituted with essentially homogeneous basal factors and RNA polymerase II. The mediator comprised some 20 polypeptides, including the three subunits of TFIIF and other polypeptides cross-reactive with antisera against GAL11, SUG1, SRB2, SRB4, SRB5, and SRB6 proteins. Mediator not only enabled activated transcription but also conferred 8-fold greater activity in basal transcription and 12-fold greater efficiency of phosphorylation of RNA polymerase II by the TFIIH-associated C-terminal repeat domain (CTD) kinase, indicative of mediator-CTD interaction. A holoenzyme form of RNA polymerase II was independently isolated that supported a response to activator proteins with purified basal factors. The holoenzyme proved to consist of mediator associated with core 12-subunit RNA polymerase II.

RSC, an Essential, Abundant Chromatin-Remodeling Complex

A novel 15-subunit complex with the capacity to remodel the structure of chromatin, termed RSC, has been isolated from S. cerevisiae on the basis of homology to the SWI/SNF complex. At least three RSC subunits are related to SWI/SNF polypeptides: Sth1p, Rsc6p, and Rsc8p are significantly similar to Swi2/Snf2p, Swp73p, and Swi3p, respectively, and were identified by mass spectrometric and sequence analysis of peptide fragments. Like SWI/SNF, RSC exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structure. RSC is, however, at least 10-fold more abundant than SWI/SNF complex and is essential for mitotic growth. Contrary to a report for SWII/SNF complex, no association of RSC (nor of SWI/SNF complex) with RNA polymerase II holoenzyme was detected.

Nucleosomes Unfold Completely at a Transcriptionally Active Promoter

It has long been known that promoter DNA is converted to a nuclease-sensitive state upon transcriptional activation. Recent findings have raised the possibility that this conversion reflects only a partial unfolding or other perturbation of nucleosomal structure, rather than the loss of nucleosomes. We report topological, sedimentation, nuclease digestion, and ChIP analyses, which demonstrate the complete unfolding of nucleosomes at the transcriptionally active PHO5 promoter of the yeast Saccharomyces cerevisiae. Although nucleosome loss occurs at all promoter sites, it is not complete at any of them, suggesting the existence of an equilibrium between the removal of nucleosomes and their reformation.

Nucleosomes inhibit the initiation of transcription but allow chain elongation with the displacement of histones

Promoters were assembled in nucleosomes or ligated to nucleosomes and transcribed with SP6 RNA polymerase or with mammalian RNA polymerase II and accessory factors. Neither polymerase would initiate transcription at a promoter in a nucleosome, but once engaged in transcription, both polymerases were capable of reading through a nucleosome. In the course of readthrough transcription, the histones were displaced from the DNA, as shown by the exposure of restriction sites and by a shift of the template to the position of naked DNA in a gel. It may be true, in general, that processive enzymes will traverse regions of DNA organized in nucleosomes and displace histones.

RELATIONSHIP OF CDK-ACTIVATING KINASE AND RNA POLYMERASE II CTD KINASE TFIIH/TFIIK

KIN28, a member of the p34cdc2/CDC28 family of protein kinases, is identified as a subunit of yeast RNA polymerase transcription factor IIH (TFIIH) on the basis of sequence determination, immunological reactivity, and copurification. KIN28 is, moreover, one of three subunits of TFIIK, a subassembly of TFIIH with protein kinase activity directed toward the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II. Itself a phosphoprotein, KIN28 interacts specifically with the two largest subunits of RNA polymerase II. Previous work of others points to two further associations: KIN28 interacts in vivo with the cyclin CCL1, and KIN28 and CCL1 are homologous to human MO15 and cyclin H, which form the cyclin-dependent kinase-activating kinase (CAK). We show that human CAK possesses the CTD kinase activity characteristic of TFIIH.

A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

A trithorax-group complex purified from Saccharomyces cerevisiae is required for methylation of histone H3.

Histone methylation has emerged as an important mechanism for regulating the transcriptional accessibility of chromatin. Several methyltransferases have been shown to target histone amino-terminal tails and mark nucleosomes associated with either euchromatic or heterochromatic states. However, the biochemical machinery responsible for regulating histone methylation and integrating it with other cellular events has not been well characterized. We report here the purification, molecular identification, and genetic and biochemical characterization of the Set1 protein complex that is necessary for methylation of histone H3 at lysine residue 4 in Saccharomyces cerevisiae. The seven-member 363-kDa complex contains homologs of Drosophila melanogaster proteins Ash2 and Trithorax and Caenorhabditis elegans protein DPY-30, which are implicated in the maintenance of Hox gene expression and regulation of X chromosome dosage compensation, respectively. Mutations of Set1 protein comparable to those that disrupt developmental function of its Drosophila homolog Trithorax abrogate histone methylation in yeast. These studies suggest that epigenetic regulation of developmental and sex-specific gene expression are species-specific readouts for a common chromatin remodeling machinery associated mechanistically with histone methylation.

A novel mediator between activator proteins and the RNA polymerase II transcription apparatus

One gene activator protein may interfere with the effects of another in eukaryotic cells. We report here that a hybrid yeast-herpes gene activator protein inhibits transcriptional activation by a thymidine-rich DNA element in yeast. This example of activator interference can be faithfully reproduced in vitro. Interference is reversed by a partially purified yeast component, but not by RNA polymerase II or various polymerase II transcription factors. We conclude that the partially purified yeast component is a novel factor, and we suggest this factor mediates the transcriptional activation process.

Dual roles of a multiprotein complex from S. cerevisiae in transcription and DNA repair

Yeast RNA polymerase II initiation factor b, homolog of human TFIIH, is a protein kinase capable of phosphorylating the C-terminal repeat domain of the polymerase; it possesses a DNA-dependent ATPase activity as well. The 85 kd and 50 kd subunits of factor b are now identified as RAD3 and SSL1 proteins, respectively; both are known to be involved in DNA repair. Factor b interacts specifically with another DNA repair protein, SSL2. The ATPase activity of factor b may be due entirely to that associated with a helicase function of RAD3. Factor b transcriptional activity was unaffected, however, by amino acid substitution at a conserved residue in the RAD3 nucleotide-binding domain, suggesting that the ATPase/helicase function is not required for transcription. These results identify factor b as a core repairosome, which may be responsible for the preferential repair of actively transcribed genes in eukaryotes.