scholarly journals In Vitro Reconstitution of PHO5 Promoter Chromatin Remodeling Points to a Role for Activator-Nucleosome Competition In Vivo

2010 ◽  
Vol 30 (16) ◽  
pp. 4060-4076 ◽  
Author(s):  
Franziska Ertel ◽  
A. Barbara Dirac-Svejstrup ◽  
Christina Bech Hertel ◽  
Dorothea Blaschke ◽  
Jesper Q. Svejstrup ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chromatin Remodeling ◽  
Classical Model ◽  
Nucleosome Formation ◽  
In Vitro Reconstitution ◽  
Dnase I Hypersensitive Site ◽  
Energy Dependent

ABSTRACT The yeast PHO5 promoter is a classical model for studying the role of chromatin in gene regulation. To enable biochemical dissection of the mechanism leading to PHO5 activation, we reconstituted the process in vitro. Positioned nucleosomes corresponding to the repressed PHO5 promoter state were assembled using a yeast extract-based in vitro system. Addition of the transactivator Pho4 yielded an extensive DNase I-hypersensitive site resembling induced PHO5 promoter chromatin. Importantly, this remodeling was energy dependent. In contrast, little or no chromatin remodeling was detected at the PHO8 or PHO84 promoter in this in vitro system. Only the PHO5 promoter harbors a high-affinity intranucleosomal Pho4 binding site (UASp) where Pho4 binding can compete with nucleosome formation, prompting us to test the importance of such competition for chromatin remodeling by analysis of UASp mutants in vivo. Indeed, the intranucleosomal location of the UASp element was critical, but not essential, for complete remodeling at the PHO5 promoter in vivo. Further, binding of just the Gal4 DNA binding domain to an intranucleosomal site could increase PHO5 promoter opening. These data establish an auxiliary role for DNA binding competition between Pho4 and histones in PHO5 promoter chromatin remodeling in vivo.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

scholarly journals Smarca5 mediated epigenetic programming facilitates fetal HSPC development in vertebrates

Blood ◽  
2020 ◽  
Author(s):  
Yanyan Ding ◽  
Wen Wang ◽  
Dongyuan Ma ◽  
Guixian Liang ◽  
Zhixin Kang ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Accessibility ◽  
Rna Seq ◽  
Chromatin Remodeler ◽  
Dynamic Changes ◽  
Functional Assays ◽  
Epigenetic Programming

Nascent HSPCs acquire definitive hematopoietic characteristics only when they develop into fetal HSPCs; however, the mechanisms underlying fetal HSPC development are poorly understood. Here, we profiled the chromatin accessibility and transcriptional features of zebrafish nascent- and fetal HSPCs using ATAC-seq and RNA-seq and revealed dynamic changes during HSPC transition. Functional assays demonstrated that chromatin remodeler-mediated epigenetic programming facilitates fetal HSPC development in vertebrates. Systematical screening of chromatin remodeler-related genes identified that smarca5 is responsible for the maintenance of chromatin accessibility at promoters of hematopoiesis-related genes in fetal HSPCs. Mechanistically, Smarca5 interacts with Nucleolin to promote chromatin remodeling, thereby facilitating genomic binding of transcription factors to regulate expression of hematopoietic regulators such as bcl11ab. Our results unravel a new role of epigenetic regulation and reveal that Smarca5-mediated epigenetic programming is responsible for fetal HSPC development, which will provide new insights into the generation of functional HSPCs both in vivo and in vitro.

scholarly journals Nuclear Ssr4 Is Required for the In Vitro and In Vivo Asexual Cycles and Global Gene Activity of Beauveria bassiana

mSystems ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Wei Shao ◽  
Qing Cai ◽  
Sen-Miao Tong ◽  
Sheng-Hua Ying ◽  
Ming-Guang Feng
Keyword(s):  
Chromatin Remodeling ◽  
Inorganic Ions ◽  
Global Gene Expression ◽  
Gene Activity ◽  
Fungal Genome ◽  
Cellular Events ◽  
First Time

Ssr4 is known to serve as a cosubunit of chromatin-remodeling SWI/SNF and RSC complexes in yeasts but has not been functionally characterized in fungi. This study unveils for the first time the pleiotropic effects caused by deletion of ssr4 and its role in mediating global gene expression in a fungal insect pathogen. Our findings confirm an essential role of Ssr4 in hydrophobin biosynthesis and assembly required for growth, differentiation, and development of aerial hyphae for conidiation and conidial adhesion to insect surface and its essentiality for insect pathogenicity and virulence-related cellular events. Importantly, Ssr4 can regulate nearly one-fourth of all genes in the fungal genome in direct and indirect manners, including dozens involved in gene activity and hundreds involved in metabolism and/or transport of carbohydrates, amino acids, lipids, and/or inorganic ions. These findings highlight a significance of Ssr4 for filamentous fungal lifestyle.

scholarly journals Generation of onco-enhancer enhances chromosomal remodeling and accelerates tumorigenesis

2020 ◽  
Vol 48 (21) ◽  
pp. 12135-12150
Author(s):  
Peiwei Chai ◽  
Jie Yu ◽  
Ruobing Jia ◽  
Xuyang Wen ◽  
Tianyi Ding ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Tumor Formation ◽  
Epigenetic Mechanism ◽  
Therapeutic Concept ◽  
In Vivo Experiments ◽  
Disease Therapy ◽  
First Time

Abstract Chromatin remodeling impacts the structural neighborhoods and regulates gene expression. However, the role of enhancer-guided chromatin remodeling in the gene regulation remains unclear. Here, using RNA-seq and ChIP-seq, we identified for the first time that neurotensin (NTS) serves as a key oncogene in uveal melanoma and that CTCF interacts with the upstream enhancer of NTS and orchestrates an 800 kb chromosomal loop between the promoter and enhancer. Intriguingly, this novel CTCF-guided chromatin loop was ubiquitous in a cohort of tumor patients. In addition, a disruption in this chromosomal interaction prevented the histone acetyltransferase EP300 from embedding in the promoter of NTS and resulted in NTS silencing. Most importantly, in vitro and in vivo experiments showed that the ability of tumor formation was significantly suppressed via deletion of the enhancer by CRISPR-Cas9. These studies delineate a novel onco-enhancer guided epigenetic mechanism and provide a promising therapeutic concept for disease therapy.

scholarly journals The Role of Non-Catalytic Domains of Hrp3 in Nucleosome Remodeling

2021 ◽  
Vol 22 (4) ◽  
pp. 1793
Author(s):  
Wenbo Dong ◽  
Punit Prasad ◽  
Andreas Lennartsson ◽  
Karl Ekwall
Keyword(s):  
Chromatin Remodeling ◽  
Nucleosome Occupancy ◽  
Strong Association ◽  
Atpase Domain ◽  
Coupling Region ◽  
Nucleosome Remodeling ◽  
Catalytic Domains

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.

scholarly journals Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

2019 ◽  
Author(s):  
Adam S. B. Jalal ◽  
César L. Pastrana ◽  
Ngat T. Tran ◽  
Clare. E. Stevenson ◽  
David M. Lawson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Binding Activity ◽  
Terminal Domain ◽  
Dna Binding Activity

ABSTRACTThe tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on a centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the co-crystal structure of a C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain adopts alternate conformations. The multiple conformations of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIP-seq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain is a determinant of this variability. Finally, we show that the C-terminal domain of Caulobacter ParB possesses no or weak non-specific DNA-binding activity. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than wild-type in vivo. Taken all together, our results emphasize the role of the N-terminal domain in ParB spreading and faithful chromosome segregation in Caulobacter crescentus.

scholarly journals Circ-GALNT16 restrains colorectal cancer progression by enhancing the SUMOylation of hnRNPK

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Chaofan Peng ◽  
Yuqian Tan ◽  
Peng Yang ◽  
Kangpeng Jin ◽  
Chuan Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Binding ◽  
Rna Sequencing ◽  
Cancer Progression ◽  
Transcriptional Level ◽  
Binding Ability ◽  
Transcriptional Complex

Abstract Background Recent studies have investigated the role of circular RNAs (circRNAs) as significant regulatory factors in multiple cancer progression. Nevertheless, the biological functions of circRNAs and the underlying mechanisms by which they regulate colorectal cancer (CRC) progression remain unclear. Methods A novel circRNA (circ-GALNT16) was identified by microarray and qRT-PCR. A series of in vitro and in vivo phenotype experiments were performed to investigate the role of circ-GALNT16 in CRC. The FISH, RNA pulldown assay, RIP assay, RNA sequencing, coimmunoprecipitation, and ChIP were performed to investigate the molecular mechanisms of circ-GALNT16 in CRC progression. Results Circ-GALNT16 was downregulated in CRC and was negatively correlated with poor prognosis. Circ-GALNT16 suppressed the proliferation and metastatic ability of CRC cells in vitro and in vivo. Mechanistically, circ-GALNT16 could bind to the KH3 domain of heterogeneous nuclear ribonucleoprotein K (hnRNPK), which promoted the SUMOylation of hnRNPK. Additionally, circ-GALNT16 could enhance the formation of the hnRNPK-p53 complex by facilitating the SUMOylation of hnRNPK. RNA sequencing assay identified serpin family E member 1 as the target gene of circ-GALNT16 at the transcriptional level. Rescue assays revealed that circ-GALNT16 regulated the expression of Serpine1 by inhibiting the deSUMOylation of hnRNPK mediated by SUMO-specific peptidase 2 and then regulating the sequence-specific DNA binding ability of the hnRNPK-p53 transcriptional complex. Conclusions Circ-GALNT16 suppressed CRC progression by inhibiting Serpine1 expression through regulating the sequence-specific DNA binding ability of the SENP2-mediated hnRNPK-p53 transcriptional complex and might function as a biomarker and therapeutic target for CRC.

scholarly journals MDMX acidic domain inhibits p53 DNA binding in vivo and regulates tumorigenesis

2018 ◽  
Vol 115 (15) ◽  
pp. E3368-E3377 ◽  
Author(s):  
Qingling Huang ◽  
Lihong Chen ◽  
Leixiang Yang ◽  
Xiaoling Xie ◽  
Lin Gan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Gene ◽  
Core Domain ◽  
Acidic Domain ◽  
Physiological Relevance ◽  
Important Regulator ◽  
Knockin Mouse

The MDM2 homolog MDMX oncoprotein is indispensable for inhibition of p53 during normal embryonic development and malignant transformation, yet how MDMX harnesses p53 functions is unclear. In addition to a canonical N-terminal p53-binding domain, recent work suggests the central acidic domain of MDMX regulates p53 interaction through intramolecular mimicry and engages in second-site interaction with the p53 core domain in vitro. To test the physiological relevance of these interactions, we generated an MDMX knockin mouse having substitutions in a conserved WW motif necessary for these functions (W201S/W202G). Notably, MDMXSG cells have normal p53 level but increased p53 DNA binding and target gene expression, and rapidly senesce. In vivo, MDMXSG inhibits early-phase disease in Eµ-Myc transgenic mice but accelerates the onset of lethal lymphoma and shortens overall survival. Therefore, MDMX is an important regulator of p53 DNA binding, which complements the role of MDM2 in regulating p53 level. Furthermore, the results suggest that the WW motif has dual functions that regulate p53 and inhibit Myc-driven lymphomas independent of p53.

scholarly journals General Transcriptional Coactivator PC4 Activates p53 Function

2004 ◽  
Vol 24 (5) ◽  
pp. 2052-2062 ◽  
Author(s):  
Sourav Banerjee ◽  
B. R. Prashanth Kumar ◽  
Tapas K. Kundu
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Physiological Role ◽  
Transcriptional Coactivator ◽  
Suppressor Activity ◽  
Tumor Suppressor Activity ◽  
In Vitro Interaction

ABSTRACT The function of p53 is modulated by several transcriptional coactivators that regulate its tumor suppressor activity. Here we report that human transcriptional coactivator PC4 enhances the DNA binding of p53 to its cognate site in vitro and directly interacts with p53 in vivo. In vitro interaction studies demonstrated that the C-terminal 30 amino acids (364 to 393) of p53 strongly interact with PC4. Surprisingly, PC4 also stimulates the sequence-specific DNA binding of p53 with the C-terminal 30 amino acids deleted (p53Δ30), suggesting that PC4 mediates enhancement of p53 DNA binding by a unique mechanism. We also demonstrated that PC4 can stimulate p53- and p53Δ30-mediated transactivation from a p53-responsive promoter. Furthermore, PC4 enhances p53- and p53Δ30-dependent apoptosis by inducing bax (a p53-targeted proapoptotic gene) gene expression. These results establish the first physiological role of PC4 as a transcriptional coactivator.

scholarly journals Role of the PAS Domain in Regulation of Dimerization and DNA Binding Specificity of the Dioxin Receptor

1998 ◽  
Vol 18 (7) ◽  
pp. 4079-4088 ◽  
Author(s):  
Ingemar Pongratz ◽  
Camilla Antonsson ◽  
Murray L. Whitelaw ◽  
Lorenz Poellinger
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Binding Specificity ◽  
Pas Domain ◽  
Bhlh Domain ◽  
Dna Binding Specificity ◽  
Dioxin Receptor

ABSTRACT The dioxin receptor is a ligand-regulated transcription factor that mediates signal transduction by dioxin and related environmental pollutants. The receptor belongs to the basic helix-loop-helix (bHLH)–Per-Arnt-Sim (PAS) family of factors, which, in addition to the bHLH motif, contain a PAS region of homology. Upon activation, the dioxin receptor dimerizes with the bHLH-PAS factor Arnt, enabling the receptor to recognize xenobiotic response elements in the vicinity of target genes. We have studied the role of the PAS domain in dimerization and DNA binding specificity of the dioxin receptor and Arnt by monitoring the abilities of the individual bHLH domains and different bHLH-PAS fragments to dimerize and bind DNA in vitro and recognize target genes in vivo. The minimal bHLH domain of the dioxin receptor formed homodimeric complexes, heterodimerized with full-length Arnt, and together with Arnt was sufficient for recognition of target DNA in vitro and in vivo. In a similar fashion, only the bHLH domain of Arnt was necessary for DNA binding specificity in the presence of the dioxin receptor bHLH domain. Moreover, the bHLH domain of the dioxin receptor displayed a broad dimerization potential, as manifested by complex formation with, e.g., the unrelated bHLH-Zip transcription factor USF. In contrast, a construct spanning the dioxin receptor bHLH domain and an N-terminal portion of the PAS domain failed to form homodimers and was capable of dimerizing only with Arnt. Thus, the PAS domain is essential to confer dimerization specificity of the dioxin receptor.

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