scholarly journals A Specificity and Targeting Subunit of a Human SWI/SNF Family-Related Chromatin-Remodeling Complex

2000 ◽  
Vol 20 (23) ◽  
pp. 8879-8888 ◽  
Author(s):  
Zuqin Nie ◽  
Yutong Xue ◽  
Dafeng Yang ◽  
Sharleen Zhou ◽  
Bonnie J. Deroo ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Gene Activation ◽  
Dna Interaction ◽  
Protein Protein Interactions ◽  
Baf Complex ◽  
Chromatin Remodeling Complexes

ABSTRACT The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc fromSaccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the β-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals Anti-inflammatory functions of the glucocorticoid receptor require DNA binding

2020 ◽  
Vol 48 (15) ◽  
pp. 8393-8407
Author(s):  
Laura Escoter-Torres ◽  
Franziska Greulich ◽  
Fabiana Quagliarini ◽  
Michael Wierer ◽  
Nina Henriette Uhlenhaut
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Drug Target ◽  
Immunosuppressive Drug ◽  
Rna Seq ◽  
Protein Protein Interactions ◽  
Inflammatory Conditions ◽  
Anti Inflammatory

Abstract The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR’s anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription. Using mice with a point mutation in GR’s zinc finger, that still tether via protein–protein interactions while being unable to recognize DNA, we demonstrate that DNA binding is essential for both transcriptional activation and repression. Performing ChIP-Seq, RNA-Seq and proteomics under inflammatory conditions, we show that DNA recognition is required for the assembly of a functional co-regulator complex to mediate glucocorticoid responses. Our findings may contribute to the development of safer immunomodulators with fewer side effects.

scholarly journals The HSA Domain of BRG1 Mediates Critical Interactions Required for Glucocorticoid Receptor-Dependent Transcriptional Activation In Vivo

2007 ◽  
Vol 28 (4) ◽  
pp. 1413-1426 ◽  
Author(s):  
Kevin W. Trotter ◽  
Hua-Ying Fan ◽  
Melissa L. Ivey ◽  
Robert E. Kingston ◽  
Trevor K. Archer
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Critical Factor ◽  
Receptor Complexes ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Eukaryotic Dna

ABSTRACT The packaging of eukaryotic DNA into chromatin can create an impediment to transcription by hindering binding of essential factors required for transcription. The mammalian SWI/SNF remodeling complex has been shown to alter local chromatin structure and facilitate recruitment of transcription factors. BRG1 (or hBrm), the central ATPase of the human SWI/SNF complex, is a critical factor for the functional activity of nuclear receptor complexes. Analysis using BRG1/SNF2h chimeras suggests BRG1 may contain previously uncharacterized functional motifs important for SWI/SNF. To identify these regions, BRG1 truncation and deletion mutants were designed, characterized, and utilized in a series of assays to evaluate transcriptional activation and chromatin remodeling by the glucocorticoid receptor. We identified a domain within the N terminus of BRG1 that mediates critical protein interactions within SWI/SNF. We find the HSA domain of BRG1 is required to mediate the interaction with BAF250a/ARID1A and show this association is necessary for transcriptional activation from chromatin mouse mammary tumor virus or endogenous promoters in vivo. These studies suggest BAF250a is a necessary facilitator of BRG1-mediated chromatin remodeling required for SWI/SNF-dependent transcriptional activation.

scholarly journals In Vivo Requirement of Activator-Specific Binding Targets of Mediator

2000 ◽  
Vol 20 (23) ◽  
pp. 8709-8719 ◽  
Author(s):  
Jin Mo Park ◽  
Hye-Suk Kim ◽  
Sang Jun Han ◽  
Moon-Sun Hwang ◽  
Young Chul Lee ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Specific Binding ◽  
Gene Activation ◽  
Dna Interaction ◽  
Activation Process ◽  
Binding Region ◽  
Physiological Conditions ◽  
Functional Analyses

ABSTRACT There has been no unequivocal demonstration that the activator binding targets identified in vitro play a key role in transcriptional activation in vivo. To examine whether activator-Mediator interactions are required for gene transcription under physiological conditions, we performed functional analyses with Mediator components that interact specifically with natural yeast activators. Different activators interact with Mediator via distinct binding targets. Deletion of a distinct activator binding region of Mediator completely compromised gene activation in vivo by some, but not all, transcriptional activators. These demonstrate that the activator-specific targets in Mediator are essential for transcriptional activation in living cells, but their requirement was affected by the nature of the activator-DNA interaction and the existence of a postrecruitment activation process.

scholarly journals Reconstitution of Glucocorticoid Receptor-Dependent Transcription In Vivo

2004 ◽  
Vol 24 (8) ◽  
pp. 3347-3358 ◽  
Author(s):  
Kevin W. Trotter ◽  
Trevor K. Archer
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Factor Loading ◽  
Baf Complex ◽  
Functional Remodeling ◽  
Chromatin Template ◽  
Necessary And Sufficient

ABSTRACT We developed a model system to study glucocorticoid receptor (GR)-mediated chromatin remodeling by the BRG1 complex. Introduction of the BRG1 ATPase into the SW-13 cell line initiates the formation of a functional remodeling complex. This complex is able to induce transcriptional activation from a transiently transfected promoter with wild-type and chromatin-remodeling-deficient BRG1 mutants, suggesting that the complex possesses a coactivator function independent from remodeling. Transactivation from a chromatin template requires the BRG1 remodeling function, which induces regions of hypersensitivity and transcription factor loading onto the integrated MMTV promoter. We report that BRG1 remodeling activity is required for GR-mediated transactivation and that this activity cannot be replaced by other ATP-dependent remodeling proteins. Further characterization of the BRG1-associated factors (BAFs) present in these cells (for example, the expression of BAF250 but not BAF180) reveals that the BAF complex rather than the polybromo-associated BAF complex is the necessary and sufficient chromatin-remodeling component with which the receptor functions in vivo. These results in conjunction with previous findings demonstrate that the GR functions with multiple forms of the SWI/SNF complex in vivo.

scholarly journals The Significance of Tetramerization in Promoter Recruitment by Stat5

1999 ◽  
Vol 19 (3) ◽  
pp. 1910-1918 ◽  
Author(s):  
Susan John ◽  
Uwe Vinkemeier ◽  
Elisabetta Soldaini ◽  
James E. Darnell ◽  
Warren J. Leonard
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Gene Activation ◽  
Interleukin 2 ◽  
Regulatory Region ◽  
Tryptophan Residue ◽  
Protein Protein Interactions ◽  
Tetramer Formation ◽  
Wide Range ◽  
The Individual

ABSTRACT Stat5a and Stat5b are rapidly activated by a wide range of cytokines and growth factors, including interleukin-2 (IL-2). We have previously shown that these signal transducers and activators of transcription (STAT proteins) are key regulatory proteins that bind to two tandem gamma interferon-activated site (GAS) motifs within an IL-2 response element (positive regulatory region III [PRRIII]) in the human IL-2Rα promoter. In this study, we demonstrate cooperative binding of Stat5 to PRRIII and explore the molecular basis underlying this cooperativity. We demonstrate that formation of a tetrameric Stat5 complex is essential for the IL-2-inducible activation of PRRIII. Stable tetramer formation of Stat5 is mediated through protein-protein interactions involving a tryptophan residue conserved in all STATs and a lysine residue in the Stat5 N-terminal domain (N domain). The functional importance of tetramer formation is shown by the decreased levels of transcriptional activation associated with mutations in these residues. Moreover, the requirement for STAT protein-protein interactions for gene activation from a promoter with tandemly linked GAS motifs can be relieved by strengthening the avidity of protein-DNA interactions for the individual binding sites. Taken together, these studies demonstrate that a dimeric but tetramerization-deficient Stat5 protein can activate only a subset of target sites. For functional activity on a wider range of potential recognition sites, N-domain-mediated oligomerization is essential.

scholarly journals c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein.

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029 ◽  
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

2009 ◽  
Vol 29 (15) ◽  
pp. 4103-4115 ◽  
Author(s):  
Hui Huang ◽  
Ming Yu ◽  
Thomas E. Akie ◽  
Tyler B. Moran ◽  
Andrew J. Woo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Fetal Liver ◽  
Gel Filtration ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Megakaryocyte Differentiation

ABSTRACT The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

scholarly journals Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling

2004 ◽  
Vol 3 (2) ◽  
pp. 264-276 ◽  
Author(s):  
Qi Fan ◽  
Lijia An ◽  
Liwang Cui
Keyword(s):  
Plasmodium Falciparum ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Northern Analysis ◽  
Binding Assays ◽  
Core Histones ◽  
Chromatin Remodeling Complexes

ABSTRACT The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain. The Plasmodium falciparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in P. falciparum, PfADA2. Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P. falciparum.

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