scholarly journals Multiple Interactions in Sir Protein Recruitment by Rap1p at Silencers and Telomeres in Yeast

2001 ◽  
Vol 21 (23) ◽  
pp. 8082-8094 ◽  
Author(s):  
Paolo Moretti ◽  
David Shore
Keyword(s):  
Complex Formation ◽  
Mating Type ◽  
Small Region ◽  
Carboxyl Terminus ◽  
Silent Chromatin ◽  
Histone Binding ◽  
Amino Terminal ◽  
Sir Complex ◽  
Two Hybrid

ABSTRACT Initiation of transcriptional silencing at mating type loci and telomeres in Saccharomyces cerevisiaerequires the recruitment of a Sir2/3/4 (silent information regulator) protein complex to the chromosome, which occurs at least in part through its association with the silencer- and telomere-binding protein Rap1p. Sir3p and Sir4p are structural components of silent chromatin that can self-associate, interact with each other, and bind to the amino-terminal tails of histones H3 and H4. We have identified a small region of Sir3p between amino acids 455 and 481 that is necessary and sufficient for association with the carboxyl terminus of Rap1p but not required for Sir complex formation or histone binding.SIR3 mutations that delete this region cause a silencing defect at HMR and telomeres. However, this impairment of repression is considerably less than that displayed by Rap1p carboxy-terminal truncations that are defective in Sir3p binding. This difference may be explained by the ability of the Rap1p carboxyl terminus to interact independently with Sir4p, which we demonstrate by in vitro binding and two-hybrid assays. Significantly, the Rap1p-Sir4p two-hybrid interaction does not require Sir3p and is abolished by mutation of the carboxyl terminus of Rap1p. We propose that both Sir3p and Sir4p can directly and independently bind to Rap1p at mating type silencers and telomeres and suggest that Rap1p-mediated recruitment of Sir proteins operates through multiple cooperative interactions, at least some of which are redundant. The physical separation of the Rap1p interaction region of Sir3p from parts of the protein required for Sir complex formation and histone binding raises the possibility that Rap1p can participate directly in the maintenance of silent chromatin through the stabilization of Sir complex-nucleosome interactions.

scholarly journals Sir3-Nucleosome Interactions in Spreading of Silent Chromatin in Saccharomyces cerevisiae

2008 ◽  
Vol 28 (22) ◽  
pp. 6903-6918 ◽  
Author(s):  
Johannes R. Buchberger ◽  
Megumi Onishi ◽  
Geng Li ◽  
Jan Seebacher ◽  
Adam D. Rudner ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Activity ◽  
Chromatin Fiber ◽  
Silent Chromatin ◽  
Histone H4 ◽  
Histone Binding ◽  
Aaa Atpase ◽  
Binding Domains ◽  
Sir Complex

ABSTRACT Silent chromatin in Saccharomyces cerevisiae is established in a stepwise process involving the SIR complex, comprised of the histone deacetylase Sir2 and the structural components Sir3 and Sir4. The Sir3 protein, which is the primary histone-binding component of the SIR complex, forms oligomers in vitro and has been proposed to mediate the spreading of the SIR complex along the chromatin fiber. In order to analyze the role of Sir3 in the spreading of the SIR complex, we performed a targeted genetic screen for alleles of SIR3 that dominantly disrupt silencing. Most mutations mapped to a single surface in the conserved N-terminal BAH domain, while one, L738P, localized to the AAA ATPase-like domain within the C-terminal half of Sir3. The BAH point mutants, but not the L738P mutant, disrupted the interaction between Sir3 and nucleosomes. In contrast, Sir3-L738P bound the N-terminal tail of histone H4 more strongly than wild-type Sir3, indicating that misregulation of the Sir3 C-terminal histone-binding activity also disrupted spreading. Our results underscore the importance of proper interactions between Sir3 and the nucleosome in silent chromatin assembly. We propose a model for the spreading of the SIR complex along the chromatin fiber through the two distinct histone-binding domains in Sir3.

scholarly journals Conservation of Histone Binding and Transcriptional Repressor Functions in a Schizosaccharomyces pombeTup1p Homolog

1999 ◽  
Vol 19 (12) ◽  
pp. 8461-8468 ◽  
Author(s):  
Yukio Mukai ◽  
Eri Matsuo ◽  
Sharon Y. Roth ◽  
Satoshi Harashima
Keyword(s):  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Chimeric Protein ◽  
Evolutionary Conservation ◽  
Histone Binding ◽  
Amino Terminal ◽  
Corepressor Complex ◽  
Related Proteins ◽  
Repressor Activity

ABSTRACT The Ssn6p-Tup1p corepressor complex is important to the regulation of several diverse genes in Saccharomyces cerevisiae and serves as a model for corepressor functions. To investigate the evolutionary conservation of these functions, sequences homologous to the S. cerevisiae TUP1 gene were cloned fromKluyveromyces lactis (TUP1) andSchizosaccharomyces pombe (tup11 +). Interestingly, while the K. lactis TUP1 gene complemented an S. cerevisiae tup1 null mutation, the S. pombe tup11 + gene did not, even when expressed under the control of the S. cerevisiae TUP1 promoter. However, anS. pombe Tup11p-LexA fusion protein repressed transcription of a corresponding reporter gene, indicating that this Tup1p homolog has intrinsic repressor activity. Moreover, a chimeric protein containing the amino-terminal Ssn6p-binding domain of S. cerevisiae Tup1p and 544 amino acids from the C-terminal region of S. pombe Tup11p complemented the S. cerevisiae tup1 mutation. The failure of native S. pombe Tup11p to complement loss of Tup1p functions in S. cerevisiaecorresponds to an inability to bind to S. cerevisiae Ssn6p in vitro. Disruption of tup11 + in combination with a disruption of tup12 +, anotherTUP1 homolog gene in S. pombe, causes a defect in glucose repression of fbp1 +, suggesting thatS. pombe Tup1p homologs function as repressors in S. pombe. Furthermore, Tup11p binds specifically to histones H3 and H4 in vitro, indicating that both the repression and histone binding functions of Tup1p-related proteins are conserved across species.

scholarly journals The CBP Bromodomain and Nucleosome Targeting Are Required for Zta-Directed Nucleosome Acetylation and Transcription Activation

2003 ◽  
Vol 23 (8) ◽  
pp. 2633-2644 ◽  
Author(s):  
Zhong Deng ◽  
Chi-Ju Chen ◽  
Michaela Chamberlin ◽  
Fang Lu ◽  
Gerd A. Blobel ◽  
...  
Keyword(s):  
Complex Formation ◽  
Histone Acetylation ◽  
Protein Sequencing ◽  
Creb Binding Protein ◽  
Barr Virus ◽  
Amino Terminal ◽  
Viral Promoters ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV)-encoded lytic activator Zta is a bZIP protein that can stimulate nucleosomal histone acetyltransferase (HAT) activity of the CREB binding protein (CBP) in vitro. We now show that deletion of the CBP bromo- and C/H3 domains eliminates stimulation of nucleosomal HAT activity in vitro and transcriptional coactivation by Zta in transfected cells. In contrast, acetylation of free histones was not affected by the addition of Zta or by deletions in the bromo or C/H3 domain of CBP. Zta stimulated acetylation of oligonucleosomes assembled on supercoiled DNA and dinucleosomes assembled on linear DNA, but Zta-stimulated acetylation was significantly reduced for mononucleosomes. Western blotting and amino-terminal protein sequencing indicated that all lysine residues in the H3 and H4 amino-terminal tails were acetylated by CBP and enhanced by the addition of Zta. Histone acetylation was also dependent upon the Zta basic DNA binding domain, which could not be substituted with the homologous basic region of c-Fos, indicating specificity in the bZIP domain nucleosome binding function. Finally, we show that Zta and CBP colocalize to viral immediate-early promoters in vivo and that overexpression of Zta leads to a robust increase in H3 and H4 acetylation at various regions of the EBV genome in vivo. Furthermore, deletion of the CBP bromodomain reduced stable CBP-Zta complex formation and histone acetylation at Zta-responsive viral promoters in vivo. These results suggest that activator- and bromodomain-dependent targeting to oligonucleosomal chromatin is required for stable promoter-bound complex formation and transcription activity.

scholarly journals Inhibition of Androgen Receptor-Mediated Transcription by Amino-Terminal Enhancer of split

2001 ◽  
Vol 21 (14) ◽  
pp. 4614-4625 ◽  
Author(s):  
Xin Yu ◽  
Peng Li ◽  
Robert G. Roeder ◽  
Zhengxin Wang
Keyword(s):  
Androgen Receptor ◽  
Nuclear Extract ◽  
Basal Transcription ◽  
Hela Nuclear Extract ◽  
Protein Protein Interaction ◽  
Transcription System ◽  
Amino Terminal ◽  
Two Hybrid ◽  
Enhancer Of Split

ABSTRACT A yeast two-hybrid assay has identified an androgen-dependent interaction of androgen receptor (AR) with amino-terminal enhancer of split (AES), a member of the highly conserved Groucho/TLE family of corepressors. Full-length AR, as well as the N-terminal fragment of AR, showed direct interactions with AES in in vitro protein-protein interaction assays. AES specifically inhibited AR-mediated transcription in a well-defined cell-free transcription system and interacted specifically with the basal transcription factor (TFIIE) in HeLa nuclear extract. These observations implicate AES as a selective repressor of ligand-dependent AR-mediated transcription that acts by directly interacting with AR and by targeting the basal transcription machinery.

scholarly journals The Yng1p Plant Homeodomain Finger Is a Methyl-Histone Binding Module That Recognizes Lysine 4-Methylated Histone H3

2006 ◽  
Vol 26 (21) ◽  
pp. 7871-7879 ◽  
Author(s):  
David G. E. Martin ◽  
Kristin Baetz ◽  
Xiaobing Shi ◽  
Kay L. Walter ◽  
Vicki E. MacDonald ◽  
...  
Keyword(s):  
Histone H3 ◽  
General Role ◽  
Histone Binding ◽  
Phd Finger ◽  
Amino Terminal ◽  
Plant Homeodomain Finger ◽  
Plant Homeodomain ◽  
Insight Into

ABSTRACT The ING (inhibitor of growth) protein family includes a group of homologous nuclear proteins that share a highly conserved plant homeodomain (PHD) finger domain at their carboxyl termini. Members of this family are found in multiprotein complexes that posttranslationally modify histones, suggesting that these proteins serve a general role in permitting various enzymatic activities to interact with nucleosomes. There are three members of the ING family in Saccharomyces cerevisiae: Yng1p, Yng2p, and Pho23p. Yng1p is a component of the NuA3 histone acetyltransferase complex and is required for the interaction of NuA3 with chromatin. To gain insight into the function of the ING proteins, we made use of a genetic strategy to identify genes required for the binding of Yng1p to histones. Using the toxicity of YNG1 overexpression as a tool, we showed that Yng1p interacts with the amino-terminal tail of histone H3 and that this interaction can be disrupted by loss of lysine 4 methylation within this tail. Additionally, we mapped the region of Yng1p required for overexpression of toxicity to the PHD finger, showed that this region capable of binding lysine 4-methylated histone H3 in vitro, and demonstrated that mutations of the PHD finger that abolish binding in vitro are no longer toxic in vivo. These results identify a novel function for the Yng1p PHD finger in promoting stabilization of the NuA3 complex at chromatin through recognition of histone H3 lysine 4 methylation.

The single CH domain of calponin is neither sufficient nor necessary for F-actin binding

1998 ◽  
Vol 111 (13) ◽  
pp. 1813-1821 ◽  
Author(s):  
M. Gimona ◽  
R. Mital
Keyword(s):  
Binding Site ◽  
Tandem Repeats ◽  
Actin Binding ◽  
Carboxyl Terminus ◽  
Amino Terminus ◽  
Binding Motif ◽  
Amino Terminal ◽  
Nonmuscle Cells ◽  
Carboxy Terminal

Calponins have been implicated in the regulation of actomyosin interactions in smooth muscle cells, cytoskeletal organisation in nonmuscle cells, and the control of neurite outgrowth. Domains homologous to the amino-terminal region of calponin have been identified in a variety of actin cross-linking proteins and signal transduction molecules, and by inference these ‘calponin homology (CH) domains’ have been assumed to participate in actin binding. We here report on the actin binding activities of the subdomains of the calponin molecule. All three mammalian isoforms of calponin (basic h1, neutral h2 and acidic) possess a single CH domain at their amino terminus as well as three tandem repeats proximal to the carboxyl terminus. Calponin h2 differs, however, from h1 in lacking a consensus actin-binding motif in the region 142–163, between the CH domain and the tandem repeats, which in h1 calponin can be chemically cross-linked to actin. Despite the absence of this consensus actin-binding motif, recombinant full-length h2 calponin co-sediments in vitro with F-actin, suggesting the presence of another binding site in the molecule. It could be shown that this binding site resides in the C-terminal tandem repeats and not in the CH domain. Thus, constructs of h2 calponin bearing partial or complete deletions of the triple repeated sequences failed to co-localise with actin stress fibres despite the presence of a CH domain. Deletion of the acidic carboxyl terminus, beyond the repeats, increased actin binding, suggesting that the carboxy-terminal tail may modulate actin association. Results obtained from transient transfections of amino- and carboxy-terminal truncations in h1 calponin were consistent with the established location of the actin binding motif outside and carboxy-terminal to the CH domain, and confirm that the presence of a single CH domain alone is neither sufficient nor necessary to mediate actin binding. Instead, the carboxy-terminal tandem repeats of h1 and h2 calponin are shown to harbour a second, independent actin binding motif.

scholarly journals I kappaB alpha physically interacts with a cytoskeleton-associated protein through its signal response domain.

1997 ◽  
Vol 17 (12) ◽  
pp. 7375-7385 ◽  
Author(s):  
P Crépieux ◽  
H Kwon ◽  
N Leclerc ◽  
W Spencer ◽  
S Richard ◽  
...  
Keyword(s):  
26S Proteasome ◽  
Cdna Clones ◽  
Human Homolog ◽  
Microtubule Organizing Center ◽  
Protein Motifs ◽  
Amino Terminal ◽  
Signal Response ◽  
Nf Kappab ◽  
Two Hybrid

The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I kappaB alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappaB alpha as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.

scholarly journals Targeting of the Yeast Ty5 Retrotransposon to Silent Chromatin Is Mediated by Interactions between Integrase and Sir4p

2001 ◽  
Vol 21 (19) ◽  
pp. 6606-6614 ◽  
Author(s):  
Weiwu Xie ◽  
Xiaowu Gai ◽  
Yunxia Zhu ◽  
David C. Zappulla ◽  
Rolf Sternglanz ◽  
...  
Keyword(s):  
Silent Chromatin ◽  
Amino Acid Residues ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
In Vitro Binding ◽  
C Terminus ◽  
Vitro Binding ◽  
Primary Determinant ◽  
Two Hybrid

ABSTRACT The Ty5 retrotransposons of Saccharomyces cerevisiaeintegrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR andHML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.

scholarly journals Cellular Splicing Factor RAF-2p48/NPI-5/BAT1/UAP56 Interacts with the Influenza Virus Nucleoprotein and Enhances Viral RNA Synthesis

2001 ◽  
Vol 75 (4) ◽  
pp. 1899-1908 ◽  
Author(s):  
Fumitaka Momose ◽  
Christopher F. Basler ◽  
Robert E. O'Neill ◽  
Akihiro Iwamatsu ◽  
Peter Palese ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Splicing Factor ◽  
Yeast Two Hybrid ◽  
Amino Terminal ◽  
Virus Rna ◽  
Rna Interaction ◽  
Two Hybrid

ABSTRACT Previous biochemical data identified a host cell fraction, designated RAF-2, which stimulated influenza virus RNA synthesis. A 48-kDa polypeptide (RAF-2p48), a cellular splicing factor belonging to the DEAD-box family of RNA-dependent ATPases previously designated BAT1 (also UAP56), has now been identified as essential for RAF-2 stimulatory activity. Additionally, RAF-2p48 was independently identified as an influenza virus nucleoprotein (NP)-interacting protein, NPI-5, in a yeast two-hybrid screen of a mammalian cDNA library. In vitro, RAF-2p48 interacted with free NP but not with NP bound to RNA, and the RAF-2p48–NP complex was dissociated following addition of free RNA. Furthermore, RAF-2p48 facilitated formation of the NP-RNA complexes that likely serve as templates for the viral RNA polymerase. RAF-2p48 was shown, in both in vitro binding assays and the yeast two-hybrid system, to bind to the amino-terminal region of NP, a domain essential for RNA binding. Together, these observations suggest that RAF-2p48 facilitates NP-RNA interaction, thus leading to enhanced influenza virus RNA synthesis.

Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella

2000 ◽  
Vol 113 (1) ◽  
pp. 103-112 ◽  
Author(s):  
A. Fujita ◽  
K. Nakamura ◽  
T. Kato ◽  
N. Watanabe ◽  
T. Ishizaki ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Binding ◽  
Pdz Domain ◽  
Small Gtpase ◽  
Regulatory Subunit ◽  
Yeast Two Hybrid ◽  
Fibrous Sheath ◽  
Amino Terminal ◽  
Two Hybrid

The small GTPase Rho; functions as a molecular switch that regulates various cellular processes such as cell adhesion, motility, gene expression and cytokinesis. We previously isolated several putative Rho; targets including rhophilin which bound selectively to the GTP-bound form of Rho;. Rhophilin is expressed highly in testis and is localized specifically in sperm flagella. The presence of a PDZ domain at the carboxy terminus of rhophilin suggested that rhophilin works as an adaptor molecule. To test this hypothesis, we employed a yeast two hybrid system using the rhophilin PDZ domain as a bait, and screened a mouse testis cDNA library. We isolated several positive clones containing the same insert. The open reading frame of the cDNA encoded a novel protein of 212 amino acids designated as ropporin from a Japanese word ‘oppo’ (the tail). The amino-terminal 40 amino acid sequence of ropporin showed high homology to that of the regulatory subunit of type II cAMP-dependent protein kinase, which is involved in dimerization and binding to A-kinase anchoring proteins. Consistently, a yeast two hybrid assay and gel filtration of recombinant ropporin indicated that ropporin dimerizes through this domain. Deletion analysis indicated that the carboxy-terminal four amino acids are essential for binding of ropporin to rhophilin, and ropporin and RhoV14 coprecipitated in the presence of rhophilin in vitro. Northern blot analysis showed that ropporin is exclusively expressed in testis, and induced at the late stage of spermatogenesis. This induction paralleled that of rhophilin. Immunocytochemistry using anti-ropporin antibody showed that ropporin is localized in the principal piece and the end piece of sperm flagella. Electronmicroscopy revealed that ropporin is mostly localized in the inner surface of the fibrous sheath while rhophilin is present in the outer surface of the outer dense fiber. These results suggest that rhophilin and ropporin may form a complex in sperm flagella.

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