scholarly journals Two related ARID family proteins are alternative subunits of human SWI/SNF complexes

2004 ◽  
Vol 383 (2) ◽  
pp. 319-325 ◽  
Author(s):  
Xiaomei WANG ◽  
Norman G. NAGL ◽  
Deborah WILSKER ◽  
Michael VAN SCOY ◽  
Stephen PACCHIONE ◽  
...  
Keyword(s):  
Dna Binding ◽  
Direct Evidence ◽  
Potential Interaction ◽  
Atpase Subunit ◽  
Reading Frame ◽  
Atpase Subunits ◽  
A Subunit ◽  
Distinct Subunit ◽  
Binding Behaviour

p270 (ARID1A) is a member of the ARID family of DNA-binding proteins and a subunit of human SWI/SNF-related complexes, which use the energy generated by an integral ATPase subunit to remodel chromatin. ARID1B is an independent gene product with an open reading frame that is more than 60% identical with p270. We have generated monoclonal antibodies specific for either p270 or ARID1B to facilitate the investigation of ARID1B and its potential interaction with human SWI/SNF complexes in vivo. Immunocomplex analysis provides direct evidence that endogenous ARID1B is associated with SWI/SNF-related complexes and indicates that p270 and ARID1B, similar to the ATPase subunits BRG1 and hBRM, are alternative, mutually exclusive subunits of the complexes. The ARID-containing subunits are not specific to the ATPases. Each associates with both BRG1 and hBRM, thus increasing the number of distinct subunit combinations known to be present in cells. Analysis of the panels of cell lines indicates that ARID1B, similar to p270, has a broad tissue distribution. The ratio of p270/ARID1B in typical cells is approx. 3.5:1, and BRG1 is distributed proportionally between the two ARID subunits. Analysis of DNA-binding behaviour indicates that ARID1B binds DNA in a non-sequence-specific manner similar to p270.

scholarly journals Communication between distinct subunit interfaces of the cohesin complex promotes its topological entrapment of DNA

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Vincent Guacci ◽  
Fiona Chatterjee ◽  
Brett Robison ◽  
Douglas E Koshland
Keyword(s):  
Dna Binding ◽  
Chromosome Structure ◽  
Biological Activities ◽  
Conformation Change ◽  
Entry And Exit ◽  
Reversible Dissociation ◽  
Independent Functions ◽  
Opening And Closing ◽  
Distinct Subunit

Cohesin mediates higher order chromosome structure. Its biological activities require topological entrapment of DNA within a lumen(s) formed by cohesin subunits. The reversible dissociation of cohesin’s Smc3p and Mcd1p subunits is postulated to form a regulated gate that allows DNA entry and exit into the lumen. We assessed gate-independent functions of this interface in yeast using a fusion protein that joins Smc3p to Mcd1p. We show that in vivo all the regulators of cohesin promote DNA binding of cohesin by mechanisms independent of opening this gate. Furthermore, we show that this interface has a gate-independent activity essential for cohesin to bind chromosomes. We propose that this interface regulates DNA entrapment by controlling the opening and closing of one or more distal interfaces formed by cohesin subunits, likely by inducing a conformation change in cohesin. Furthermore, cohesin regulators modulate the interface to control both DNA entrapment and cohesin functions after DNA binding.

scholarly journals Inhibition of p53 DNA Binding Function by the MDM2 Protein Acidic Domain

2011 ◽  
Vol 286 (18) ◽  
pp. 16018-16029 ◽  
Author(s):  
Brittany Cross ◽  
Lihong Chen ◽  
Qian Cheng ◽  
Baozong Li ◽  
Zhi-Min Yuan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Wild Type ◽  
Methyl Transferase ◽  
Reading Frame ◽  
Acidic Domain ◽  
Dna Binding Activity ◽  
Binding Function

MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. Alternate reading frame binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild type conformation and allows p53-MDM2-SUV39H1 complex to bind DNA. These results provide further evidence for an ubiquitination-independent mechanism of p53 regulation by MDM2 and reveal how MDM2-interacting repressors gain access to p53 target promoters and repress transcription. Furthermore, we show that the MDM2 inhibitor Nutlin cooperates with the proteasome inhibitor Bortezomib by stimulating p53 DNA binding and transcriptional activity, providing a rationale for combination therapy using proteasome and MDM2 inhibitors.

scholarly journals Communication between distinct subunit interfaces of the cohesin complex promotes its topological entrapment of DNA

2019 ◽  
Author(s):  
Vincent Guacci ◽  
Fiona Chatterjee ◽  
Brett Robison ◽  
Douglas Koshland
Keyword(s):  
Dna Binding ◽  
Chromosome Structure ◽  
Biological Activities ◽  
Conformation Change ◽  
Entry And Exit ◽  
Reversible Dissociation ◽  
Independent Functions ◽  
Opening And Closing ◽  
Distinct Subunit

ABSTRACTCohesin mediates higher-order chromosome structure. Its biological activities require topological entrapment of DNA within a lumen(s) formed by cohesin subunits. The reversible dissociation of cohesin’s Smc3p and Mcd1p subunits are postulated to form a regulated gate that allows DNA entry and exit into the lumen. We assessed gate-independent functions of this interface in yeast using a fusion protein that joins Smc3p to Mcd1p. We show that in vivo all the regulators of cohesin promote DNA binding of cohesion by mechanisms independent of opening this gate. Furthermore, we show that this interface has a gate-independent activity essential for cohesin to bind chromosomes. We propose this interface regulates DNA entrapment by controlling the opening and closing of one or more distal interfaces formed by cohesin subunits, likely by inducing a conformation change in cohesin. Furthermore, cohesin regulators modulate the interface to control both DNA entrapment and cohesin functions after DNA binding.

scholarly journals Characterization of devH, a Gene Encoding a Putative DNA Binding Protein Required for Heterocyst Function inAnabaena sp. Strain PCC 7120

2000 ◽  
Vol 182 (12) ◽  
pp. 3572-3581 ◽  
Author(s):  
Pratibha B. Hebbar ◽  
Stephanie E. Curtis
Keyword(s):  
Dna Binding ◽  
Time Course ◽  
Nitrogen Starvation ◽  
Receptor Protein ◽  
Fixed Nitrogen ◽  
Gene Encoding ◽  
Reading Frame ◽  
Pcc 7120

ABSTRACT The devH gene was identified in a screen forAnabaena sp. strain PCC 7120 sequences whose transcripts increase in abundance during a heterocyst development time course. The product of devH contains a helix-turn-helix motif similar to the DNA binding domain of members of the cyclic AMP receptor protein family, and the protein is most closely related to the cyanobacterial transcriptional activator NtcA. devH transcripts are barely detectable in vegetative cells and are induced approximately fivefold after nitrogen starvation. This induction is absent in the two developmental mutants hetR and ntcA. The gene is expressed as monocistronic transcripts with multiple 5′ termini, and the ∼500-bp region 5′ to devH was shown to have promoter activity in vivo. The devH gene was insertionally inactivated by the integration of plasmid sequences within the open reading frame. Nitrogen starvation of the devH mutant induces heterocysts of wild-type morphology, but the mutant is inviable in the absence of fixed nitrogen and unable to reduce acetylene aerobically.

scholarly journals Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

2014 ◽  
Vol 28 (6) ◽  
pp. 899-911 ◽  
Author(s):  
Sylvia C. Hewitt ◽  
Leping Li ◽  
Sara A. Grimm ◽  
Wipawee Winuthayanon ◽  
Katherine J. Hamilton ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Gene ◽  
Mutant Mouse ◽  
Estrogen Receptor Α ◽  
Binding Activity ◽  
Estrogen Response Element ◽  
Dna Binding Domain ◽  
Estrogen Response ◽  
Uterine Growth

Abstract Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

scholarly journals Ubiquitylome Analysis Reveals a Central Role for the Ubiquitin-Proteasome System in Plant Innate Immunity

2021 ◽  
Author(s):  
Xiyu Ma ◽  
Chao Zhang ◽  
Do Young Kim ◽  
Yanyan Huang ◽  
Elizabeth Chatt ◽  
...  
Keyword(s):  
Plant Immunity ◽  
High Sensitivity ◽  
Ubiquitin Proteasome System ◽  
Immune Recognition ◽  
Atpase Subunit ◽  
Ample Evidence ◽  
Network Analyses ◽  
Affinity Enrichment ◽  
Regulatory Loop

Abstract Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 minutes with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.

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