Biochemical Society Transactions (1999) 27 89
Chromatin, mctivators and eorepressors: molecular mechanismsto establish and maintainstates of gene activity. Alan P. Wolffe, Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, NIH, Bldg. 18T, Rm. 106, Bethesda, MD 20892543 I . E-mail
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Molecular Models for Regulation of Transcription by RNA Polymerase 111 M.Meisterernst Lab. Fur Molekulare Biologie Genzentrum, der Ludwig-Maximilians Universitat, Munchen 8 1375, Germany
Chromatin remodeling enzymes have a major role in transcriptional control. We have focussed on histone deacetylase complexes because of the essential role that the histone acetylation cycle has in early vertebrate development (Almouzni d., 1994, Dev. Biol. 165, 654). Three histone deacetylase complexes have been characterized following fractionation using column chromatography and immunoprecipitation: 1) the M i 2 deacetylase complex: this is a multisubunit molecular machine containing a nucleosomedependent ATPase of the SW12SNF2 superfamily required for histone deacetylation. We have coupled the remodeling activity of the SW12/SNF2 ATPase to the covalent modification of histones in the nucleosome (Wade d., 1998, Curr. Biol. 8, 843). 2) the MeCP2 deacetylase complex: this is a multisubunit enzyme complex containing the methyl CpG binding transcriptional repressor MeCP2, histone deacetylase and numerous components associated with DNA replication (Jones 1998, Nature Genetics 19, 187). We have previously found that chromatin assembly in essential for the repression of transcription on methylated DNA in XenoDus oocytes. 3) the nuclear receptor targeted histone deacetylase complex: we find that ceriiin transcriptional coactivators and corepressors targeted to nuclear receptors exist in a common complex in XenoDus explaining the role of nucleosomal architecture in the reversible activation and repression of transcription by nuclear receptors in the XenoDus oocyte (Wong 1998, EMBO J. 17, 520; Li d., 1998, EMBO J. 17,6300).
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REGULATION OF TRANSCRIPTION BY CHROMATIN REMODELING MACHINES Craig L. Peterson, Colin Logie, Jocelyn Krebs, Igor Gavin, and Laurie Boyer. Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, 373 Plantation St., University of Massachusetts Medical Center, Worcester, MA 01605. W e have icccntly desciihed a novel nucleosomal array assay that couples the activity of a nucleosome icmodeling activity to restiiction endonuclease activity. In our initial studies this assay was used to determine the kinctic parameters of ATP-dependent nucleosome disruption by the yeast SWI/SNF complex. We have now extended these studies to encompass a quantitative comparison of four other ATP-dependent remodeling enzymes, yeast RSC, human SWIISNF, Xenopus Mi-2, and Drosophiln CHRAC complexes. I n addition, we have used this quantitative assay to investigatc thc mechanism of ATP-dcpendent remodeling. W e find that SWIlSNF is ahle to efliciently remodel arrays of H3/H4 tetramers, suggesting that remodeling does not require reanangement or loss of the H 2 N H 2 B dimers. Furthermore, w e have used IAEDANS-labelled histone H3 and meauremcnts of steady state fluorescence to investigate. whether ATP-dependent remodeling by SWYSNF involves confonnational changes in the histone octamer. Our results indicate that ATP-dependent icmodeling does not influence thc structure of thc histone octamer. hut is restricted to changes i n nucleosomal DNA-histone interactions. Consistent with this view, we have compared the kinetics of SWIlSNF remodeling o l small. circular minichromosomes vs short, linear nucleosomal arrays, and we find that SWI/SNF remodcling is controlled hy DNA topology. We will present a new model for SWYSNF action involving an ATP-driven change in the topology of nucleosomal DNA. 810
The P C A F Histone Acetylase Complex Yoshihiro Nakatani
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, M D 20892
The adenovirus E I A transforming factor disturbs host cell growth control by interacting with cellular factors that normally function to repress cell proliferation. E l A transforming activity resides in two distinct domains, the targets of which include p300/CBP and the retinoblastoma protein family. While the RB pathway has been well studies, the p300/CBP pathway had not been clarified until our lab cloned the novel histone acetylase PCAF. Binding of E I A to p300/CBP disturbs access of PCAF acetylase to p3OO/CBP. In support of relevance of this competition, PCAF and E t A compete in regulation of cell cycle progression, differentiation, and transcriptional activation. To investigate P C A F function at the molecular level in greater detail, we purified PCAF in its native state. PCAF is found in a complex with more than 20 associated polypeptides. Strikingly, some polypeptides associated with PCAF are identical to the TBP-associated factors (TAFs) which are subunits of TFIID. Furthermore, some polypeptides show significant sequence similarity to other TAFs. Taken together, we have concluded that a histone octamer-like domain could be present within the PCAF complex, as we previously demonstrated in the TFIID complex. A possible role of the histone octamer-like structure in the P C A F complex may be the replacement of the histone octamer after relaxation of nucleosomal structure by acetylation of the histone tails. An important feature of the histone-like domains in the P C A F complex is the lack of regions corresponding to histone N-terminal tails. In rhis regard, if it replaces the histone octamer, the histone-like structure in the PCAF complex may play an architectural role in the maintenance of a transcriptionally active chromatin state. The fact that PCAF is found in a complex with more than 20 associated polypeptides suggests that E I A disturbs access of the PCAF complex to promoters through p300/CBP. In this regard, subunits in the P C A F complex may be involved in cellular events mediated by PCAF, i.e. regulation of transcription, cell cycle progression, and differentiation. Further study of these PCAF-associated factors should provide insights into the molecular mechanisms of these events.
