Advances in clinical and molecular understanding of the FMR1 premutation and fragile X-associated tremor/ataxia syndrome

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5'-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5'-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5'-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5'-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5'-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75(exonIII-/-) mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase(-/-) mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.

Adverse events of upper GI endoscopy

Quality Measures for Colonoscopy: A Critical Evaluation

Nucleosome packaging regulates many aspects of DNA metabolism and is thought to mediate genetic instability and transcription of expanded trinucleotide repeats. Both instability and transcription are sensitive to repeat length, tract purity, and CpG methylation. CAT or AGG interruptions within the (CAG)n or (CGG)n tracts of spinocerebellar ataxia, type 1 or fragile X syndrome, respectively, confer increased genetic stability to the repeats. We report the formation of nucleosomes on sequences containing pure and interrupted (CAG)n and (CGG)n repeats having lengths above and below the genetic stability thresholds. Increased lengths of pure repeats led to increased and decreased propensities for nucleosome assembly on the (CAG)n and (CGG)n repeats, respectively. CpG methylation of the CGG repeat further reduced assembly. CAT interruptions in (CAG)n tracts decreased nucleosome assembly. In contrast, AGG interruptions in (CGG)n tracts did not affect assembly by hypoacetylated histones. The latter observation was unaltered by CpG methylation of the repeats. However, nucleosome assembly by hyperacetylated histones on interrupted CGG tracts was increased relative to pure tracts and this effect was abolished by CpG methylation. Thus, CAT or AGG interruptions can modulate the ability of (CAG)n and (CGG) tracts to assemble into chromatin and the effect of the AGG interruptions is dependent upon both the methylation status of the DNA and the acetylation status of the histones. Compared with the genetically unstable pure repeats, both interruptions permit a propensity of nucleosome assembly closer to that of random (genetically stable) sequences, suggesting an association of nucleosome assembly of trinucleotide repeats and genetic instability.

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Adverse events associated with EUS and EUS-guided procedures

Cerebral function monitors during pediatric cardiac surgery: Can they make a difference?

During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.

Electrocardiographic features of patients with COVID-19: One year of unexpected manifestations

The role of endoscopy in the management of choledocholithiasis

Biochemical and genetic findings accumulated over the past decade have established that the condensation of eukaryotic DNA in chromatin functions not only to constrain the genome within the boundaries of the cell nucleus but also to suppress gene activity in a general manner. This genetic repression extends from the level of the nucleosome, the primary unit of chromatin organization, where coiling of DNA on the surface of the nucleosome core particle impedes access to the transcriptional apparatus, to the higher order folding of nucleosome arrays and the organization of silent regions of chromatin (for reviews see Refs. 1van Holde K. Zlatanova J. Arents G. Moudrianakis E. Elgin S.C.R. Chromatin Structure and Gene Expression. Oxford University Press, Oxford, UK1995: 1-26Google Scholar, 2Ramakrishnan V. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 83-112Crossref PubMed Scopus (132) Google Scholar, 3Pruss D. Hayes J.J. Wolffe A.P. Bioessays. 1995; 17: 161-170Crossref PubMed Google Scholar, 4Grunstein M. Annu. Rev. Cell Biol. 1990; 6: 643-678Crossref PubMed Google Scholar, 5Kornberg R.D. Lorch Y. Annu. Rev. Cell Biol. 1992; 8: 563-589Crossref PubMed Google Scholar, 6Fletcher T.M. Hansen J.C. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 149-188Crossref PubMed Google Scholar and 105Koshland D. Strunnikov A. Annu. Rev. Cell Biol. 1996; 12: 305-333Crossref Scopus (283) Google Scholar). Chromatin structure is inextricably linked to transcriptional regulation, and recent studies show how chromatin is perturbed so as to facilitate transcription (for reviews see Refs. 7Adams C.C. Workman J.L. Cell. 1993; 72: 305-308Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 8Paranjape S.M. Kamakaka R.T. Kadonaga J.T. Annu. Rev. Biochem. 1994; 63: 265-297Crossref PubMed Google Scholar, 9Kornberg R.D. Lorch Y. Curr. Opin. Cell Biol. 1995; 7: 371-375Crossref PubMed Scopus (95) Google Scholar, 10Felsenfeld G. Cell. 1996; 86: 13-19Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 12Krude T. Elgin S.C.R. Curr. Biol. 1996; 6: 511-515Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we review the substantial advances in the identification of histone acetyltransferases and histone deacetylases, whose opposing activities establish the steady-state level of histone acetylation, and progress in studies of multicomponent systems that require energy for the process of nucleosome disruption.Histone AcetylationSince the early discovery of histone acetylation by Allfrey and colleagues (13Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Google Scholar), this post-translation modification has been correlated with the processes of transcription and chromatin assembly. Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface (11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar). Hyperacetylation of histones is associated with transcriptional activity or the potential for activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin. Histone acetylation is also associated with the active deposition and maturation of newly assembled nucleosomes during DNA replication (for reviews see Refs. 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar and 15Loidl P. Chromosoma. 1994; 103: 441-449Crossref PubMed Google Scholar). Acetylation reduces the net positive charge of the histones and weakens interactions with DNA (16Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar), inhibits the higher order folding of nucleosome arrays (17Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Google Scholar, 18Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and disrupts specific interactions with nonhistone regulators, as shown for the yeast silencer and repressor proteins Sir3 and Sir4 (19Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (202) Google Scholar, 20Hecht A. Laroche T. Strahl-Bosinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (674) Google Scholar) and Tup-1 (21Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Google Scholar).Tetrahymena Histone Acetyltransferase A and Yeast Gcn5In a convergence of biochemical and genetic studies, cloning of the p55 catalytic subunit of Tetrahymena nuclear (A-type) histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; P/CAF, p300/CBP-associated factor; CBP, CREB-binding protein; CREB, cyclic AMP response element binding protein; SAS, something about silencing; MOZ, monocytic leukemia zinc finger; MOF, males absent on the first; TAF, TBP-associated factor; TBP, TATA-binding protein; HDAC, histone deacetylase. revealed substantial sequence identity with yeast Gcn5, previously defined genetically as a transcriptional coactivator (22Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1210) Google Scholar). The catalytic domain of the Gcn5 HAT is required for coactivator function in vivo, providing a genetic link between histone modification and transcriptional activation (23Candau R. Zhou J.X. Allis C.D. Berger S.L. EMBO J. 1997; 16: 555-565Crossref PubMed Scopus (174) Google Scholar). As a human GCN5 homolog has been identified, this HAT is likely to be widely conserved (24Candau R. Moore P.A. Wang L. Barlev N. Ying C.Y. Rosen C.A. Berger S.L. Mol. Cell. Biol. 1996; 16: 593-602Crossref PubMed Scopus (154) Google Scholar, 25Wang L. Mizzen C. Ying C. Candau R. Barlev N. Brownell J. Allis C.D. Berger S.L. Mol. Cell. Biol. 1997; 17: 519-527Crossref PubMed Google Scholar). Bacterially expressed yeast Gcn5 protein acetylates free histone H3 strongly at lysine 14 and histone H4 weakly at lysines 8 and 16 (26Kuo M.-H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (480) Google Scholar). However, unlike the native HAT A enzyme, recombinant Gcn5 cannot acetylate nucleosomal histones, implying that other subunits in the complex must influence its activity on chromatin. Genetic and biochemical studies reveal at least two interacting proteins, Ada2 and Ada3, that form a complex with Gcn5 (23Candau R. Zhou J.X. Allis C.D. Berger S.L. EMBO J. 1997; 16: 555-565Crossref PubMed Scopus (174) Google Scholar,27Marcus G.A. Silverman N. Berger S.L. Horiuchi J. Guarente L. EMBO J. 1994; 13: 4807-4815Crossref PubMed Scopus (234) Google Scholar, 28Horiuchi J. Silverman N. Marcus G.A. Guarente L. Mol. Cell. Biol. 1995; 15: 1203-1209Crossref PubMed Google Scholar, 29Georgakopoulos T. Gounalaki N. Thireos G. Mol. Gen. Genet. 1995; 246: 723-728Crossref PubMed Scopus (53) Google Scholar). Binding of Ada2 to the activation domains of the transcriptional activators VP16 and Gcn4 in vitro suggests a mechanism by which promoter targeting of Gcn5 might be achieved (30Silverman N. Agapite J. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11665-11668Crossref PubMed Scopus (96) Google Scholar,31Barlev N.A. Candau R. Wang L. Darpino P. Silverman N. Berger S.L. J. Biol. Chem. 1995; 270: 19337-19344Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar).P/CAF, p300/CBP, SAS, MOZ, and MOFAn increasing number of putative or demonstrated histone acetyltransferases have emerged in the past year. P/CAF (p300/CBP-associated factor) is a novel histone acetyltransferase isolated on the basis of sequence similarity to human and yeast Gcn5 (32Yang X.-J. Ogryzko V.V. Nishizawa J. Howard B.H. Nakayani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1283) Google Scholar), which interacts with the highly related transcriptional coactivators p300/CBP. Like Gcn5, recombinant P/CAF has intrinsic HAT activity for free histones H3 and H4, but unlike Gcn5, P/CAF is also able to acetylate nucleosomal histone H3. p300/CBP (CREB-binding protein) is itself a histone acetyltransferase with no resemblance in sequence to the other acetyltransferases (33Ogryzko V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2274) Google Scholar, 106Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1480) Google Scholar). The bacterially expressed p300/CBP protein is unique among HAT polypeptides in that it can acetylate all four core histones free in solution or when complexed in the nucleosome; acetylation on histone H4 occurs at lysines 5, 8, 12, and 16, the same positions that are subject to acetylation in vivo (32Yang X.-J. Ogryzko V.V. Nishizawa J. Howard B.H. Nakayani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1283) Google Scholar). p300 and CBP are known to physically interact with numerous transcription factors activated by signaling cascades, including CREB, c-Jun/v-Jun, Fos, and nuclear hormone receptors, and are also targets for the E1A oncoprotein (for a review see Ref. 34Janknecht R. Hunter T. Nature. 1996; 383: 22-23Crossref PubMed Scopus (337) Google Scholar). Whether histones are substrates of p300 in vivo remains to be determined.The yeast SAS, human MOZ and Tip60, and fly MOF proteins constitute a different class of putative acetyltransferases, characterized by a ∼300-amino acid region of significant similarity that contains a C2CH zinc finger motif and a subregion similar to HATs and other acetyltransferases (35Reifsnyder C. Lowell J. Clarke A. Pillus L. Nat. Genet. 1996; 14: 42-49Crossref PubMed Scopus (234) Google Scholar, 36Borrow J. Stanton Jr., V.P. Andresen J.M. Becher R. Behm F.G. Chaganti R.S.K. Civin C.I. Disteche C. Dube I. Frischauf A.M. Horsman D. Mitelman F. Volinia S. Watmore A.E. Housman D.E. Nat. Genet. 1996; 14: 33-41Crossref PubMed Scopus (609) Google Scholar, 37Kamine J. Elangovan B. Subramanian T. Coleman D. Chjnnadurai G. Virology. 1996; 216: 357-366Crossref PubMed Scopus (237) Google Scholar, 38Hilfiker A. Hilfiker-Kleiner D. Pannuti A. Lucchesi J. EMBO J. 1997; 16: 2054-2060Crossref PubMed Scopus (353) Google Scholar). The biochemical properties or substrate specificities of these proteins involved in silencing (SAS), transcriptional activation (Tip60), leukemogenesis (MOZ), and dosage compensation (MOF) have not yet been described. Interestingly, recurrent translocation in a subtype of acute myeloid leukemia generates a novel fusion of MOZ with CBP, suggesting that the aberrant acetylation of histones or other chromosomal proteins could mediate leukemogenesis (36Borrow J. Stanton Jr., V.P. Andresen J.M. Becher R. Behm F.G. Chaganti R.S.K. Civin C.I. Disteche C. Dube I. Frischauf A.M. Horsman D. Mitelman F. Volinia S. Watmore A.E. Housman D.E. Nat. Genet. 1996; 14: 33-41Crossref PubMed Scopus (609) Google Scholar).TAF250TFIID, the general transcription factor complex of TBP (TATA-binding protein) and the associated TAF proteins, has been found to contain a HAT activity. Unique among the various TAFs, TAFII250 alone or in the TFIID complex has both a serine kinase activity selective for RAP74 (a subunit of TFIIF) and a HAT activity (39Dikstein R. Ruppert S. Tjian R. Cell. 1996; 84: 781-790Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 40Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J.L. Berger S.L. Kouzarides T. Nakatani Y. Aliis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Like Gcn5, TAFII250 preferentially acetylates free histone H3 over H4 and has little or no activity on nucleosomal histones (40Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J.L. Berger S.L. Kouzarides T. Nakatani Y. Aliis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). The domain of TAFII250 responsible for HAT activity maps to the central, conserved region and shows no obvious sequence similarity to other HATs. The finding of HAT activity in TAFII250 implies that TFIID could contribute toward destabilizing nucleosomes over core promoter elements, although the physiological substrates of the HAT activity remain to be determined. TFIID may possess yet an another capacity for assisting nucleosome disorder. Portions of several TAFs (DrosophilaTAFII42 and TAFII62) adopt a histone octamer-like substructure that might be employed as a histone octamer-like substructure, which could serve as a competitor for DNA binding with the core histones (41Hoffman A. Chiang C.-M. Oelgeschlager T. Xie X. Burley S.K. Nakatani Y. Roeder R. Nature. 1996; 380: 356-359Crossref PubMed Scopus (157) Google Scholar, 42Nakatani Y. Bagby S. Ikura M. J . Biol. Chem. 1996; 271: 6575-6578Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 43Xie X. Kokubo T. Cohen S.L. Mirza U.A. Hoffman A. Chait B.T. Roeder R. Nakatani Y. Burley S.K. Nature. 1996; 380: 316-322Crossref PubMed Scopus (223) Google Scholar).Histone DeacetylasesIn parallel with the identification of HAT enzymes, studies of histone deacetylase (HDAC) enzymes in human, yeast, andDrosophila have also advanced significantly. Affinity chromatography with the ligand trapoxin, a high affinity, irreversible inhibitor, resulted in the purification and cloning of a human deacetylase (44Tauton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1456) Google Scholar) composed of a catalytic subunit, HD-1, renamed HDAC1 (45Hassig C.A. Fleischer T.C. Billin A.N. Schreibert S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar), and a tightly associated WD repeat protein, RbAp48. The sequence of the HDAC1 showed very strong sequence identity to yeast Rpd3, previously identified genetically to be necessary for full repression and activation of a subset of genes (46Vidal M. Gaber R. Mol. Cell. Biol. 1991; 11: 6317-6327Crossref PubMed Google Scholar). There are five members of theRPD3 family in yeast, two of which (HDA1 andRPD3) are components of the major histone deacetylase activities, which fractionate as 350-kDa (HDA) and 600-kDa (HDB) complexes (47Carmen A.A. Rundlett S.E. Grunstein M. J. Biol. Chem. 1996; 271: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 48Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (495) Google Scholar).As anticipated from the catalytic properties of the encoded proteins, deletions of the HDA1 and RPD3 genes strongly reduce HDA and HDB activities, leading to hyperacetylation of histones H3 and H4 in vivo. However, the phenotypes of these deletions are somewhat surprising, as they increase repression rather than increase activation of telomeric loci (48Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (495) Google Scholar). Similarly, mutation of a Drosophila homolog of RPD3 displays an enhancer of position effect variegation phenotype, suggesting that loss of wild-type Drosophila RPD3 function as a histone deacetylase leads to increased gene silencing (49De Rubertis F. Kadosh D. Henchoz S. Pauli D. Reuter G. Struhl K. Spierer P. Nature. 1996; 384: 589-591Crossref PubMed Scopus (190) Google Scholar). These findings may perhaps be related to the acetylation of histone H4 at lysine 12, which is required for transcriptional silencing in yeast (50Braunstein M. Sorbel R.E. Allis C.D. Turner B.M. Broach J.R. Mol. Cell. Biol. 1996; 16: 4349-4356Crossref PubMed Google Scholar) and which is also associated with heterochromatin in Drosophila (51Turner B.M. Birley A.J. Lavender J. Cell. 1992; 69: 375-384Abstract Full Text PDF PubMed Google Scholar), despite net hypoacetylation.More in line with the anticipated involvement of histone deacetylases in transcriptional repression is the physical association of the heteromeric Mad (Mxi1)/Max DNA binding repressors involved in controlling cell proliferation and differentiation with mammalian RPD3 homologs and with Sin3, a conserved transcriptional co-repressor genetically linked to yeast Rpd3 (45Hassig C.A. Fleischer T.C. Billin A.N. Schreibert S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 52Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (815) Google Scholar). Immunopurification studies also reveal additional, novel polypeptides associated with Sin3 and histone deacetylase in human cell extracts (54Zhang Y. Iranti R. Erdjument-Bromage H. Tempst P. Reinberg D. Cell. 1997; 89: 357-364Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). YY1, a mammalian transcription factor that can serve as a repressor to histone deacetylase W.M. C. Y. D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar), and and for nuclear hormone receptors, interact with complexes mammalian Sin3 and histone deacetylase L. R. H. J. L. N. D. Nature. 1997; PubMed Google Scholar, T. T.M. M. 1997; PubMed Google Scholar, L. D. Hassig C.A. Ayer D.E. Schreiber S.L. Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar). of genes by the yeast protein also of a complex Sin3 and Rpd3 D. Struhl K. Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar). This of strong for a of repression by the of histone deacetylase complexes to the of so of chromatin structure a of that for is the of nucleosomal histones during the histones or from and how might a structure interactions between DNA binding S. M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google nucleosomes that are or to the structure of DNA have different for in chromatin are by proteins as and Burley S.K. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and by the complex itself L. A. D. M. Cell. 1997; 89: Full Text Full Text PDF PubMed Google chromatin are for a gene and at which of the activation the binding of regulators, of the or activities to specific chromosomal or they the chromatin of for other enzymes are by is the of the modification a nucleosome and how is the modification the activities of the histone acetyltransferases and histone deacetylases with the family of nucleosome on higher order chromatin or is it by higher order chromatin to these and other not only for the of gene but also for of Biochemical and genetic findings accumulated over the past decade have established that the condensation of eukaryotic DNA in chromatin functions not only to constrain the genome within the boundaries of the cell nucleus but also to suppress gene activity in a general manner. This genetic repression extends from the level of the nucleosome, the primary unit of chromatin organization, where coiling of DNA on the surface of the nucleosome core particle impedes access to the transcriptional apparatus, to the higher order folding of nucleosome arrays and the organization of silent regions of chromatin (for reviews see Refs. 1van Holde K. Zlatanova J. Arents G. Moudrianakis E. Elgin S.C.R. Chromatin Structure and Gene Expression. Oxford University Press, Oxford, UK1995: 1-26Google Scholar, 2Ramakrishnan V. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 83-112Crossref PubMed Scopus (132) Google Scholar, 3Pruss D. Hayes J.J. Wolffe A.P. Bioessays. 1995; 17: 161-170Crossref PubMed Google Scholar, 4Grunstein M. Annu. Rev. Cell Biol. 1990; 6: 643-678Crossref PubMed Google Scholar, 5Kornberg R.D. Lorch Y. Annu. Rev. Cell Biol. 1992; 8: 563-589Crossref PubMed Google Scholar, 6Fletcher T.M. Hansen J.C. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 149-188Crossref PubMed Google Scholar and 105Koshland D. Strunnikov A. Annu. Rev. Cell Biol. 1996; 12: 305-333Crossref Scopus (283) Google Scholar). Chromatin structure is inextricably linked to transcriptional regulation, and recent studies show how chromatin is perturbed so as to facilitate transcription (for reviews see Refs. 7Adams C.C. Workman J.L. Cell. 1993; 72: 305-308Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 8Paranjape S.M. Kamakaka R.T. Kadonaga J.T. Annu. Rev. Biochem. 1994; 63: 265-297Crossref PubMed Google Scholar, 9Kornberg R.D. Lorch Y. Curr. Opin. Cell Biol. 1995; 7: 371-375Crossref PubMed Scopus (95) Google Scholar, 10Felsenfeld G. Cell. 1996; 86: 13-19Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 12Krude T. Elgin S.C.R. Curr. Biol. 1996; 6: 511-515Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we review the substantial advances in the identification of histone acetyltransferases and histone deacetylases, whose opposing activities establish the steady-state level of histone acetylation, and progress in studies of multicomponent systems that require energy for the process of nucleosome Histone AcetylationSince the early discovery of histone acetylation by Allfrey and colleagues (13Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Google Scholar), this post-translation modification has been correlated with the processes of transcription and chromatin assembly. Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface (11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar). Hyperacetylation of histones is associated with transcriptional activity or the potential for activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin. Histone acetylation is also associated with the active deposition and maturation of newly assembled nucleosomes during DNA replication (for reviews see Refs. 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar and 15Loidl P. Chromosoma. 1994; 103: 441-449Crossref PubMed Google Scholar). Acetylation reduces the net positive charge of the histones and weakens interactions with DNA (16Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar), inhibits the higher order folding of nucleosome arrays (17Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Google Scholar, 18Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and disrupts specific interactions with nonhistone regulators, as shown for the yeast silencer and repressor proteins Sir3 and Sir4 (19Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (202) Google Scholar, 20Hecht A. Laroche T. Strahl-Bosinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (674) Google Scholar) and Tup-1 (21Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Google Scholar). the early discovery of histone acetylation by Allfrey and colleagues (13Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Google Scholar), this post-translation modification has been correlated with the processes of transcription and chromatin assembly. Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface (11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar). Hyperacetylation of histones is associated with transcriptional activity or the potential for activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin. Histone acetylation is also associated with the active deposition and maturation of newly assembled nucleosomes during DNA replication (for reviews see Refs. 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar and 15Loidl P. Chromosoma. 1994; 103: 441-449Crossref PubMed Google Scholar). Acetylation reduces the net positive charge of the histones and weakens interactions with DNA (16Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar), inhibits the higher order folding of nucleosome arrays (17Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Google Scholar, 18Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and disrupts specific interactions with nonhistone regulators, as shown for the yeast silencer and repressor proteins Sir3 and Sir4 (19Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (202) Google Scholar, 20Hecht A. Laroche T. Strahl-Bosinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (674) Google Scholar) and Tup-1 (21Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Google Scholar). Tetrahymena Histone Acetyltransferase A and Yeast Gcn5In a convergence of biochemical and genetic studies, cloning of the p55 catalytic subunit of Tetrahymena nuclear (A-type) histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; P/CAF, p300/CBP-associated factor; CBP, CREB-binding protein; CREB, cyclic AMP response element binding protein; SAS, something about silencing; MOZ, monocytic leukemia zinc finger; MOF, males absent on the first; TAF, TBP-associated factor; TBP, TATA-binding protein; HDAC, histone deacetylase. revealed substantial sequence identity with yeast Gcn5, previously defined genetically as a transcriptional coactivator (22Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1210) Google Scholar). The catalytic domain of the Gcn5 HAT is required for coactivator function in vivo, providing a genetic link between histone modification and transcriptional activation (23Candau R. Zhou J.X. Allis C.D. Berger S.L. EMBO J. 1997; 16: 555-565Crossref PubMed Scopus (174) Google Scholar). As a human GCN5 homolog has been identified, this HAT is likely to be widely conserved (24Candau R. Moore P.A. Wang L. Barlev N. Ying C.Y. Rosen C.A. Berger S.L. Mol. Cell. Biol. 1996; 16: 593-602Crossref PubMed Scopus (154) Google Scholar, 25Wang L. Mizzen C. Ying C. Candau R. Barlev N. Brownell J. Allis C.D. Berger S.L. Mol. Cell. Biol. 1997; 17: 519-527Crossref PubMed Google Scholar). Bacterially expressed yeast Gcn5 protein acetylates free histone H3 strongly at lysine 14 and histone H4 weakly at lysines 8 and 16 (26Kuo M.-H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (480) Google Scholar). However, unlike the native HAT A enzyme, recombinant Gcn5 cannot acetylate nucleosomal histones, implying that other subunits in the complex must influence its activity on chromatin. Genetic and biochemical studies reveal at least two interacting proteins, Ada2 and Ada3, that form a

Although several isoforms of protein kinase C (PKC) have been implicated in T lymphocyte activation events, little is known about their mode of action. To address the role of PKCzeta in T cell activation, we have generated Jurkat T cell transfectants expressing either the wild type (J-PKCzeta) or "kinase-dead" mutant (J-PKCzeta(mut)) versions of this protein. Expression of PKCzeta but not PKCzeta(mut) increased transcriptional activation mediated by the NF-kappaB or nuclear factor of activated T cells (NFAT). PKCzeta cooperates with calcium ionophore and with NFAT1 or NFAT2 proteins to enhance transcriptional activation of a NFAT reporter construct. However, neither NFAT nuclear translocation nor DNA binding were in J-PKCzeta cells. Our results show that PKCzeta enhanced transcriptional activity mediated by Gal4-NFAT1 fusion proteins containing the N-terminal transactivation domain of human NFAT1. Interestingly, PKCzeta synergizes with calcineurin to induce transcriptional activation driven by the NFAT1 transactivation domain. Co-precipitation experiments showed physical interaction between PKCzeta and NFAT1 or NFAT2 isoforms. Even more, PKCzeta was able to phosphorylate recombinant glutathione S-transferase-NFAT1 (1-385) protein. These data reveal a new role of PKCzeta in T cells through the control of NFAT function by modulating the activity of its transactivation domain.

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