Sumoylation and desumoylation assays for a chromatin-remodelling complex in vivo and in vitro

2006 ◽  
Author(s):  
Jung Hwa Kim ◽  
Keun Il Kim ◽  
Sung Hee Baek
Keyword(s):  
Chromatin Remodelling ◽  
Chromatin Remodelling Complex

scholarly journals Interaction of RSC chromatin remodelling complex with nucleosomes is modulated by H3 K14 acetylation and H2B SUMOylation in vivo

2020 ◽  
Author(s):  
Neha Jain ◽  
Davide Tamborrini ◽  
Brian Evans ◽  
Shereen Chaudhry ◽  
Bryan J. Wilkins ◽  
...  
Keyword(s):  
Histone Variants ◽  
Chromatin Remodelling ◽  
List Type ◽  
Atpase Subunit ◽  
Chromatin Remodelling Complex ◽  
Key Points ◽  
Target Sites ◽  
Nucleosome Binding

AbstractChromatin remodelling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair and transcription. A key question is how these complexes find their target sites on chromatin. Here, we use genetically encoded photo-crosslinker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC (remodels the structure of chromatin) complex, on the nucleosome in living yeast. We find that the interaction of the Sth1 bromodomain with the H3 tail depends on K14 acetylation by Gcn5. This modification does not recruit RSC to chromatin but mediates its interaction with neighbouring nucleosomes. We observe a preference of RSC for H2B SUMOylated nucleosomes in vivo and show that this modification moderately enhances RSC binding to nucleosomes in vitro. Furthermore, RSC is not ejected from chromatin in mitosis, but its mode of nucleosome binding differs between interphase and mitosis. In sum, our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications most likely in combination with other components such as histone variants and transcription factors.Key PointsIn vivo photo-crosslinking reveals the footprint of the ATPase subunit of RSC on the nucleosome.RSC binds to H3 K14ac nucleosomes via the C-terminal bromodomain of its ATPase-subunit Sth1.RSC preferentially localizes to H2B-SUMOylated nucleosomes.

scholarly journals H3K36 methylation and DNA-binding are critical for Ioc4 recruitment and Isw1b remodeller function

2021 ◽  
Author(s):  
Jian Li ◽  
Lena Bergmann ◽  
Kimberly M Webb ◽  
Madelaine M Gogol ◽  
Philipp Voigt ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Functional Characterization ◽  
Full Length ◽  
Pwwp Domain ◽  
H3k36 Methylation ◽  
Chromatin Remodelling Complex

The Isw1b chromatin-remodelling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeller. Ioc4-PWWP preferentially binds H3K36me3-containing nucleosomes. The ability of the PWWP domain to bind DNA is required for this interaction. It is also promoted by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 as well as DNA are also critical for full-length Ioc4 binding to nucleosomes in vitro as well as its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain is necessary for efficient remodelling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing intragenic transcription initiation and the production of non-coding RNAs.

scholarly journals Kaposi's sarcoma-associated herpesvirus K8 protein interacts with hSNF5

2003 ◽  
Vol 84 (3) ◽  
pp. 665-676 ◽  
Author(s):  
Seungmin Hwang ◽  
Daeyoup Lee ◽  
Yousang Gwack ◽  
Hyesun Min ◽  
Joonho Choe
Keyword(s):  
Kaposi's Sarcoma ◽  
Expression System ◽  
Kaposi’S Sarcoma ◽  
Chromatin Remodelling ◽  
Significant Similarity ◽  
Early Protein ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus related to Epstein–Barr virus (EBV) and herpesvirus saimiri. KSHV open reading frame K8 encodes a basic region-leucine zipper protein of 237 aa that homodimerizes. K8 shows significant similarity to the EBV immediate-early protein Zta, a key regulator of EBV reactivation and replication. In this study, a carboxyl-terminal deletion mutant of K8, K8(1–115), that had strong transactivating properties was found. Screening using transcriptionally inactive K8(1–75) showed that K8 interacts and co-localizes with hSNF5, a cellular chromatin-remodelling factor, both in vivo and in vitro. This interaction requires aa 48–183 of hSNF5 and 1–75 of K8. In a yeast expression system, the ability of K8 and K8(1–115) to activate transcription requires the presence of SNF5, the yeast homologue of hSNF5. These data suggest a mechanism by which the SWI–SNF complex is recruited to specific genes. They also suggest that K8 functions as a transcriptional activator under specific conditions and that its transactivation activity requires its interaction with the cellular chromatin remodelling factor hSNF5.

scholarly journals Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Patrick Laurette ◽  
Thomas Strub ◽  
Dana Koludrovic ◽  
Céline Keime ◽  
Stéphanie Le Gras ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Affinity Purification ◽  
Regulatory Elements ◽  
Chromatin Remodelling Complex ◽  
Melanoma Cell Proliferation ◽  
Dna Replication And Repair ◽  
Chromatin Organisation ◽  
Proliferation In Vitro

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

Dynamics of gene targeting and chromatin remodelling by nuclear receptors

2000 ◽  
Vol 28 (4) ◽  
pp. 405-410 ◽  
Author(s):  
G. L. Hager ◽  
T. M. Fletcher ◽  
N. Xiao ◽  
C. T. Baumann ◽  
W. G. Müller ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Extract ◽  
Chromatin Remodelling ◽  
Drosophila Embryo ◽  
Multistep Process ◽  
Chromatin Structural ◽  
Mmtv Promoter ◽  
Green Fluorescent

Activation of the murine-mammary-tumour virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). We have reconstituted this nucleoprotein transition with chromatin assembled on MMTV LTR DNA with Drosophila embryo extracts, purified GR, and HeLa nuclear extract. Chromatin remodelling in vitro is ATP-dependent and maps to a region identical with that found in vivo. We demonstrate specific, glucocorticoid response element dependent, binding of purified GR to a large, multi-nucleosome MMTV chromatin array and show that GR-dependent chromatin remodelling is a multistep process. In the absence of ATP, GR binds to multiple sites on the chromatin array and inhibits nuclease access to GR recognition sites. On the addition of ATP, GR induces remodelling resulting in a large increase in access of enzymes to their sites within the transition region. These findings are complemented by studies in living cells; using a tandem array of MMTV-Ras reporter elements and a form of GR labelled with the green fluorescent protein, we have observed direct targeting of the receptor to response elements in live mouse cells. Whereas the ligand-activated receptor is associated with the MMTV promoter for observable periods, photobleaching experiments provide direct evidence that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment. The results both in vitro and in vivo are consistent with a dynamic model ('hit and run') in which GR first binds to chromatin after ligand activation, recruits a remodelling activity and is then lost from the template.

Interaction of RSC Chromatin Remodelling Complex with Nucleosomes is Modulated by H3 K14 Acetylation and H2B SUMOylation  In vivo

2020 ◽  
Author(s):  
Neha Jain ◽  
Davide Tamborrini ◽  
Brian Evans ◽  
Shereen Chaudhry ◽  
Bryan J. Wilkins ◽  
...  
Keyword(s):  
Chromatin Remodelling ◽  
Chromatin Remodelling Complex

scholarly journals 252 Follicle dermal papilla cells undergo chromatin remodelling processes in vivo and in vitro

2017 ◽  
Vol 137 (10) ◽  
pp. S236
Author(s):  
E. Carrasco ◽  
A. Gilmore ◽  
V. Botchkarev ◽  
C.A. Jahoda
Keyword(s):  
Dermal Papilla ◽  
Chromatin Remodelling ◽  
Dermal Papilla Cells

Induction and Differentiation of Dental Epithelium in Vivo and in Vitro

1982 ◽  
Vol 40 ◽  
pp. 142-145
Author(s):  
E. J. Kollar
Keyword(s):  
Connective Tissue ◽  
Oral Mucosa ◽  
Basal Lamina ◽  
Functional Derivatives ◽  
Specific Source ◽  
Dental Epithelium ◽  
Connective Tissue Stroma

The differentiation and maintenance of many specialized epithelial structures are dependent on the underlying connective tissue stroma and on an intact basal lamina. These requirements are especially stringent in the development and maintenance of the skin and oral mucosa. The keratinization patterns of thin or thick cornified layers as well as the appearance of specialized functional derivatives such as hair and teeth can be correlated with the specific source of stroma which supports these differentiated expressions.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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