scholarly journals Mapping and Functional Characterization of the TAF11 Interaction with TFIIA

2005 ◽  
Vol 25 (3) ◽  
pp. 945-957 ◽  
Author(s):  
M. M. Robinson ◽  
G. Yatherajam ◽  
R. T. Ranallo ◽  
A. Bric ◽  
M. R. Paule ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Regulation Of Gene Expression ◽  
Small Subunit ◽  
Molecular Organization ◽  
Functional Link ◽  
Essential Information ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
Promoter Dna

ABSTRACT TFIIA interacts with TFIID via association with TATA binding protein (TBP) and TBP-associated factor 11 (TAF11). We previously identified a mutation in the small subunit of TFIIA (toa2-I27K) that is defective for interaction with TAF11. To further explore the functional link between TFIIA and TAF11, the toa2-I27K allele was utilized in a genetic screen to isolate compensatory mutants in TAF11. Analysis of these compensatory mutants revealed that the interaction between TAF11 and TFIIA involves two distinct regions of TAF11: the highly conserved histone fold domain and the N-terminal region. Cells expressing a TAF11 allele defective for interaction with TFIIA exhibit conditional growth phenotypes and defects in transcription. Moreover, TAF11 imparts changes to both TFIIA-DNA and TBP-DNA contacts in the context of promoter DNA. These alterations appear to enhance the formation and stabilization of the TFIIA-TBP-DNA complex. Taken together, these studies provide essential information regarding the molecular organization of the TAF11-TFIIA interaction and define a mechanistic role for this association in the regulation of gene expression in vivo.

scholarly journals The TFIID Components Human TAFII140 andDrosophila BIP2 (TAFII155) Are Novel Metazoan Homologues of Yeast TAFII47 Containing a Histone Fold and a PHD Finger

2001 ◽  
Vol 21 (15) ◽  
pp. 5109-5121 ◽  
Author(s):  
Yann-Gaël Gangloff ◽  
Jean-Christophe Pointud ◽  
Sylvie Thuault ◽  
Lucie Carré ◽  
Christophe Romier ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Sequence Similarity ◽  
Protein A ◽  
Amino Acid Sequences ◽  
Molecular Organization ◽  
Phd Finger ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
High Degree

ABSTRACT The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAFIIs). TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAFIIs and overall molecular organization. In recent years, it has been assumed that all the metazoan TAFIIs have been identified, yet no metazoan homologues of yeast TAFII47 (yTAFII47) and yTAFII65 are known. Both of these yTAFIIs contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAFII25. We have cloned a novel mouse protein, TAFII140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component. TAFII140 shows extensive sequence similarity toDrosophila BIP2 (dBIP2) (dTAFII155), which we also show to be a component of DrosophilaTFIID. These proteins are metazoan homologues of yTAFII47 as their HFDs selectively heterodimerize with dTAFII24 and human TAFII30, metazoan homologues of yTAFII25. We further show that yTAFII65 shares two domains with theDrosophila Prodos protein, a recently described potential dTAFII. These conserved domains are critical for yTAFII65 function in vivo. Our results therefore identify metazoan homologues of yTAFII47 and yTAFII65.

scholarly journals Histone Folds Mediate Selective Heterodimerization of Yeast TAFII25 with TFIID Components yTAFII47 and yTAFII65 and with SAGA Component ySPT7

2001 ◽  
Vol 21 (5) ◽  
pp. 1841-1853 ◽  
Author(s):  
Yann-Gaël Gangloff ◽  
Steven L. Sanders ◽  
Christophe Romier ◽  
Doris Kirschner ◽  
P. Anthony Weil ◽  
...  
Keyword(s):  
Temperature Sensitive ◽  
Saga Complex ◽  
Yeast Two Hybrid ◽  
Conserved Residues ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
Necessary And Sufficient ◽  
Two Hybrid ◽  
Temperature Sensitive Phenotype

ABSTRACT We show that the yeast TFIID (yTFIID) component yTAFII47 contains a histone fold domain (HFD) with homology to that previously described for hTAFII135. Complementation in vivo indicates that the yTAFII47 HFD is necessary and sufficient for vegetative growth. Mutation of highly conserved residues in the α1 helix of the yTAFII47 HFD results in a temperature-sensitive phenotype which can be suppressed by overexpression of yTAFII25, as well as by yTAFII40, yTAFII19, and yTAFII60. In yeast two-hybrid and bacterial coexpression assays, the yTAFII47 HFD selectively heterodimerizes with yTAFII25, which we show contains an HFD with homology to the hTAFII28 family We additionally demonstrate that yTAFII65 contains a functional HFD which also selectively heterodimerizes with yTAFII25. These results reveal the existence of two novel histone-like pairs in yTFIID. The physical and genetic interactions described here show that the histone-like yTAFIIs are organized in at least two substructures within TFIID rather than in a single octamer-like structure as previously suggested. Furthermore, our results indicate that ySPT7 has an HFD homologous to that of yTAFII47 which selectively heterodimerizes with yTAFII25, defining a novel histone-like pair in the SAGA complex.

scholarly journals Saccharomyces cerevisiae BUR6 encodes a DRAP1/NC2alpha homolog that has both positive and negative roles in transcription in vivo.

1997 ◽  
Vol 17 (4) ◽  
pp. 2057-2065 ◽  
Author(s):  
G Prelich
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Nucleotide Sequence Analysis ◽  
Detectable Effect ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
Tata Box Binding Protein ◽  
Mutant Phenotypes ◽  
Carboxy Terminal

BUR3 and BUR6 were identified previously by selecting for mutations that increase transcription from an upstream activating sequence (UAS)-less promoter in Saccharomyces cerevisiae. The bur3-1 and bur6-1 mutations are recessive, increase transcription from a suc2 delta uas allele, and cause other mutant phenotypes, suggesting that Bur3p and Bur6p function as general repressors of the basal transcriptional machinery. The molecular cloning and characterization of BUR3 and BUR6 are presented here. BUR3 is identical to MOT1, a previously characterized essential gene that encodes an ATP-dependent inhibitor of the TATA box-binding protein. Cloning and nucleotide sequence analysis reveals that BUR6 encodes a homolog of DRAP1 (also called NC2alpha), a mammalian repressor of basal transcription. Strains that contain a bur6 null allele are viable but grow extremely poorly, demonstrating that BUR6 is critical for normal cell growth in yeast. The Bur6p histone fold domain is required for function; an extensive nonoverlapping set of deletion alleles throughout the histone fold domain impairs BUR6 function in vivo, whereas mutations in the amino- and carboxy-terminal tails have no detectable effect. BUR6 and BUR3/MOT1 have different functions depending on promoter context: although the bur3-1 and bur6-1 mutations increase transcription from delta uas promoters, they result in reduced transcription from the wild-type GAL1 and GAL10 promoters. This transcriptional defect is due to the inability of the GAL10 UAS to function in bur6-1 strains. The similar phenotypes of bur6 and bur3 (mot1) mutations suggest that Bur6p and Mot1p have related, but not identical, functions in modulating the activity of the general transcription machinery in vivo.

scholarly journals Interaction of the Vaccinia Virus RNA Polymerase-Associated 94-Kilodalton Protein with the Early Transcription Factor

2009 ◽  
Vol 83 (23) ◽  
pp. 12018-12026 ◽  
Author(s):  
Zhilong Yang ◽  
Bernard Moss
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Transcription Initiation ◽  
Elongation Factor ◽  
Small Subunit ◽  
Functional Link ◽  
Early Transcription

ABSTRACT A multisubunit RNA polymerase (RPO) encoded by vaccinia virus (VACV), in conjunction with specific factors, transcribes early, intermediate, and late viral genes. However, an additional virus-encoded polypeptide referred to as the RPO-associated protein of 94 kDa (RAP94) is tightly bound to the RPO for the transcription of early genes. Unlike the eight RPO core subunits, RAP94 is synthesized exclusively at late times after infection. Furthermore, RAP94 is necessary for the packaging of RPO and other components needed for early transcription in assembling virus particles. The direct association of RAP94 with NPH I, a DNA-dependent ATPase required for transcription termination, and the multifunctional poly(A) polymerase small subunit/2′-O-methyltransferase/elongation factor was previously demonstrated. That RAP94 provides a structural and functional link between the core RPO and the VACV early transcription factor (VETF) has been suspected but not previously demonstrated. Using VACV recombinants that constitutively or inducibly express VETF subunits and RAP94 with affinity tags, we showed that (i) VETF associates only with RPO containing RAP94 in vivo and in vitro, (ii) the association of RAP94 with VETF requires both subunits of the latter, (iii) neither viral DNA nor other virus-encoded late proteins are required for the interaction of RAP94 with VETF and core RPO subunits, (iv) different domains of RAP94 bind VETF and core subunits of RPO, and (v) NPH I and VETF bind independently and possibly simultaneously to the N-terminal region of RAP94. Thus, RAP94 provides the bridge between the RPO and proteins needed for transcription initiation, elongation, and termination.

scholarly journals The Plant NF-Y DNA Matrix In Vitro and In Vivo

Plants ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 406 ◽  
Author(s):  
Nerina Gnesutta ◽  
Matteo Chiara ◽  
Andrea Bernardini ◽  
Matteo Balestra ◽  
David S. Horner ◽  
...  
Keyword(s):  
Saturation Mutagenesis ◽  
Sequence Specificity ◽  
Histone Fold ◽  
Nuclear Factor Y ◽  
Histone Fold Domain ◽  
Genes Encoding ◽  
Core Histones ◽  
Ccaat Box

Nuclear Factor Y (NF-Y) is an evolutionarily conserved trimer formed by a Histone-Fold Domain (HFD) heterodimeric module shared by core histones, and the sequence-specific NF-YA subunit. In plants, the genes encoding each of the three subunits have expanded in number, giving rise to hundreds of potential trimers. While in mammals NF-Y binds a well-characterized motif, with a defined matrix centered on the CCAAT box, the specificity of the plant trimers has yet to be determined. Here we report that Arabidopsis thaliana NF-Y trimeric complexes, containing two different NF-YA subunits, bind DNA in vitro with similar affinities. We assayed precisely sequence-specificity by saturation mutagenesis, and analyzed genomic DNA sites bound in vivo by selected HFDs. The plant NF-Y CCAAT matrix is different in nucleotides flanking CCAAT with respect to the mammalian matrix, in vitro and in vivo. Our data point to flexible DNA-binding rules by plant NF-Ys, serving the scope of adapting to a diverse audience of genomic motifs.

scholarly journals Functional Analysis of the SIN3-Histone Deacetylase RPD3-RbAp48-Histone H4 Connection in the XenopusOocyte

1999 ◽  
Vol 19 (9) ◽  
pp. 5847-5860 ◽  
Author(s):  
Danielle Vermaak ◽  
Paul A. Wade ◽  
Peter L. Jones ◽  
Yun-Bo Shi ◽  
Alan P. Wolffe
Keyword(s):  
Histone Deacetylase ◽  
Nuclear Import ◽  
Transcriptional Repression ◽  
Oocyte Nucleus ◽  
Histone H4 ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
Localization Signal ◽  
Carboxy Terminal

ABSTRACT We investigated the protein associations and enzymatic requirements for the Xenopus histone deacetylase catalytic subunit RPD3 to direct transcriptional repression in Xenopus oocytes. Endogenous Xenopus RPD3 is present in nuclear and cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nuclear. We cloned Xenopus RbAp48 and SIN3 and show that expression of RPD3, but not RbAp48 or SIN3, leads to an increase in nuclear and cytoplasmic histone deacetylase activity and transcriptional repression of the TRβA promoter. This repression requires deacetylase activity and nuclear import of RPD3 mediated by a carboxy-terminal nuclear localization signal. Exogenous RPD3 is not incorporated into previously described oocyte deacetylase and ATPase complexes but cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus. We show that RPD3 associates with RbAp48 through N- and C-terminal contacts and that RbAp48 also interacts with SIN3. XenopusRbAp48 selectively binds to the segment of the N-terminal tail immediately proximal to the histone fold domain of histone H4 in vivo. Exogenous RPD3 may be targeted to histones through interaction with endogenous RbAp48 to direct transcriptional repression of theXenopus TRβA promoter in the oocyte nucleus. However, the exogenous RPD3 deacetylase functions to repress transcription in the absence of a requirement for association with SIN3 or other targeted corepressors.

scholarly journals Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites

1997 ◽  
Vol 136 (3) ◽  
pp. 501-513 ◽  
Author(s):  
Richard D. Shelby ◽  
Omid Vafa ◽  
Kevin F. Sullivan
Keyword(s):  
Cell Cycle ◽  
Histone H3 ◽  
Structural Elements ◽  
Histone Fold ◽  
Contact Sites ◽  
Nucleosomal Dna ◽  
Histone Fold Domain ◽  
Cycle Dependent

We investigated the requirements for targeting the centromeric histone H3 homologue CENP-A for assembly at centromeres in human cells by transfection of epitope-tagged CENP-A derivatives into HeLa cells. Centromeric targeting is driven solely by the conserved histone fold domain of CENP-A. Using the crystal structure of histone H3 as a guide, a series of CENPA/histone H3 chimeras was constructed to test the role of discrete structural elements of the histone fold domain. Three elements were identified that are necessary for efficient targeting to centromeres. Two correspond to contact sites between histone H3 and nucleosomal DNA. The third maps to a homotypic H3–H3 interaction site important for assembly of the (H3/H4)2 heterotetramer. Immunoprecipitation confirms that CENP-A self-associates in vivo. In addition, targeting requires that CENP-A expression is uncoupled from histone H3 synthesis during S phase. CENP-A mRNA accumulates later in the cell cycle than histone H3, peaking in G2. Isolation of the gene for human CENP-A revealed a regulatory motif in the promoter region that directs the late S/G2 expression of other cell cycle–dependent transcripts such as cdc2, cdc25C, and cyclin A. Our data suggest a mechanism for molecular recognition of centromeric DNA at the nucleosomal level mediated by a cooperative series of differentiated CENP-A–DNA contact sites arrayed across the surface of a CENP-A nucleosome and a distinctive assembly pathway occurring late in the cell cycle.

scholarly journals The N Terminus of the Centromere H3-Like Protein Cse4p Performs an Essential Function Distinct from That of the Histone Fold Domain

2000 ◽  
Vol 20 (18) ◽  
pp. 7037-7048 ◽  
Author(s):  
Yinhuai Chen ◽  
Richard E. Baker ◽  
Kevin C. Keith ◽  
Kendra Harris ◽  
Sam Stoler ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mutant Proteins ◽  
Histone Fold ◽  
Human Centromere ◽  
Kinetochore Assembly ◽  
Evolutionarily Conserved ◽  
Histone Fold Domain ◽  
N Terminus ◽  
Acid Domain

ABSTRACT Cse4p is an evolutionarily conserved histone H3-like protein that is thought to replace H3 in a specialized nucleosome at the yeast (Saccharomyces cerevisiae) centromere. All known yeast, worm, fly, and human centromere H3-like proteins have highly conserved C-terminal histone fold domains (HFD) but very different N termini. We have carried out a comprehensive and systematic mutagenesis of the Cse4p N terminus to analyze its function. Surprisingly, only a 33-amino-acid domain within the 130-amino-acid-long N terminus is required for Cse4p N-terminal function. The spacing of the essential N-terminal domain (END) relative to the HFD can be changed significantly without an apparent effect on Cse4p function. The END appears to be important for interactions between Cse4p and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex. Genetic and biochemical evidence shows that Cse4p proteins interact with each other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form functional mixed complexes. These results support different roles for the Cse4p N terminus and the HFD in centromere function and are consistent with the proposed Cse4p nucleosome model. The structure-function characteristics of the Cse4p N terminus are relevant to understanding how other H3-like proteins, such as the human homolog CENP-A, function in kinetochore assembly and chromosome segregation.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

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