scholarly journals A “Tagless” Strategy for Identification of Stable Protein Complexes Genome-wide by Multidimensional Orthogonal Chromatographic Separation and iTRAQ Reagent Tracking

2008 ◽  
Vol 7 (5) ◽  
pp. 1836-1849 ◽  
Author(s):  
Ming Dong ◽  
Lee Lisheng Yang ◽  
Katherine Williams ◽  
Susan J. Fisher ◽  
Steven C. Hall ◽  
...  
Keyword(s):  
Chromatographic Separation ◽  
Protein Complexes ◽  
Genome Wide ◽  
Itraq Reagent

scholarly journals Distinct CoREST complexes act in a cell-type-specific manner

2019 ◽  
Author(s):  
Igor Mačinković ◽  
Ina Theofel ◽  
Tim Hundertmark ◽  
Kristina Kovač ◽  
Stephan Awe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Germ Line ◽  
Protein Complexes ◽  
Specific Gene ◽  
S2 Cells ◽  
Cell Type ◽  
Specific Gene Expression ◽  
Genome Wide ◽  
A Cell ◽  
Cell Type Specific

Abstract CoREST has been identified as a subunit of several protein complexes that generate transcriptionally repressive chromatin structures during development. However, a comprehensive analysis of the CoREST interactome has not been carried out. We use proteomic approaches to define the interactomes of two dCoREST isoforms, dCoREST-L and dCoREST-M, in Drosophila. We identify three distinct histone deacetylase complexes built around a common dCoREST/dRPD3 core: A dLSD1/dCoREST complex, the LINT complex and a dG9a/dCoREST complex. The latter two complexes can incorporate both dCoREST isoforms. By contrast, the dLSD1/dCoREST complex exclusively assembles with the dCoREST-L isoform. Genome-wide studies show that the three dCoREST complexes associate with chromatin predominantly at promoters. Transcriptome analyses in S2 cells and testes reveal that different cell lineages utilize distinct dCoREST complexes to maintain cell-type-specific gene expression programmes: In macrophage-like S2 cells, LINT represses germ line-related genes whereas other dCoREST complexes are largely dispensable. By contrast, in testes, the dLSD1/dCoREST complex prevents transcription of germ line-inappropriate genes and is essential for spermatogenesis and fertility, whereas depletion of other dCoREST complexes has no effect. Our study uncovers three distinct dCoREST complexes that function in a lineage-restricted fashion to repress specific sets of genes thereby maintaining cell-type-specific gene expression programmes.

scholarly journals Epstein–Barr virus nuclear antigen 3A partially coincides with EBNA3C genome-wide and is tethered to DNA through BATF complexes

2014 ◽  
Vol 112 (2) ◽  
pp. 554-559 ◽  
Author(s):  
Stefanie C. S. Schmidt ◽  
Sizun Jiang ◽  
Hufeng Zhou ◽  
Bradford Willox ◽  
Amy M. Holthaus ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Latent Infection ◽  
Protein Complexes ◽  
Nuclear Antigen ◽  
Lymphoblastoid Cell Lines ◽  
Barr Virus ◽  
Genome Wide ◽  
Epstein Barr

Epstein–Barr Virus (EBV) conversion of B-lymphocytes to Lymphoblastoid Cell Lines (LCLs) requires four EBV nuclear antigen (EBNA) oncoproteins: EBNA2, EBNALP, EBNA3A, and EBNA3C. EBNA2 and EBNALP associate with EBV and cell enhancers, up-regulate the EBNA promoter, MYC, and EBV Latent infection Membrane Proteins (LMPs), which up-regulate BCL2 to protect EBV-infected B-cells from MYC proliferation-induced cell death. LCL proliferation induces p16INK4A and p14ARF-mediated cell senescence. EBNA3A and EBNA3C jointly suppress p16INK4A and p14ARF, enabling continuous cell proliferation. Analyses of the EBNA3A human genome-wide ChIP-seq landscape revealed 37% of 10,000 EBNA3A sites to be at strong enhancers; 28% to be at weak enhancers; 4.4% to be at active promoters; and 6.9% to be at weak and poised promoters. EBNA3A colocalized with BATF-IRF4, ETS-IRF4, RUNX3, and other B-cell Transcription Factors (TFs). EBNA3A sites clustered into seven unique groups, with differing B-cell TFs and epigenetic marks. EBNA3A coincidence with BATF-IRF4 or RUNX3 was associated with stronger EBNA3A ChIP-Seq signals. EBNA3A was at MYC, CDKN2A/B, CCND2, CXCL9/10, and BCL2, together with RUNX3, BATF, IRF4, and SPI1. ChIP-re-ChIP revealed complexes of EBNA3A on DNA with BATF. These data strongly support a model in which EBNA3A is tethered to DNA through a BATF-containing protein complexes to enable continuous cell proliferation.

scholarly journals Multiplex enCas12a screens show functional buffering by paralogs is systematically absent from genome-wide CRISPR/Cas9 knockout screens

2020 ◽  
Author(s):  
Merve Dede ◽  
Megan McLaughlin ◽  
Eiru Kim ◽  
Traver Hart
Keyword(s):  
Cell Lines ◽  
Protein Complexes ◽  
Essential Genes ◽  
Systematic Analysis ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
Synthetic Lethal Interactions ◽  
Crispr Screen ◽  
A Cell ◽  
Genetic Buffering

AbstractMajor efforts on pooled library CRISPR knockout screening across hundreds of cell lines have identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens are very low compared to the number of constitutively expressed genes in a cell, raising the question of why there are so few essential genes. Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observed that half of all constitutively-expressed genes are never hits in any CRISPR screen, and that these never-essentials are highly enriched for paralogs. We investigated paralog buffering through systematic dual-gene CRISPR knockout screening by testing algorithmically defined ~400 candidate paralog pairs with the enCas12a multiplex knockout system in three cell lines. We observed 24 synthetic lethal paralog pairs which have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions were present in at least two out of three cell lines and 14 of 24 (58%) were present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Together these observations strongly suggest that paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes due to genetic buffering in monogenic CRISPR-based mammalian functional genomics approaches.

scholarly journals Chromatin Architectural Factors as Safeguards against Excessive Supercoiling during DNA Replication

2020 ◽  
Vol 21 (12) ◽  
pp. 4504
Author(s):  
Syed Moiz Ahmed ◽  
Peter Dröge
Keyword(s):  
Dna Replication ◽  
Protein Complexes ◽  
Genome Replication ◽  
Relaxation Rates ◽  
Architectural Features ◽  
Genome Wide ◽  
New Findings ◽  
Accessory Factors ◽  
Novel Strategy ◽  
Supercoil Relaxation

Key DNA transactions, such as genome replication and transcription, rely on the speedy translocation of specialized protein complexes along a double-stranded, right-handed helical template. Physical tethering of these molecular machines during translocation, in conjunction with their internal architectural features, generates DNA topological strain in the form of template supercoiling. It is known that the build-up of transient excessive supercoiling poses severe threats to genome function and stability and that highly specialized enzymes—the topoisomerases (TOP)—have evolved to mitigate these threats. Furthermore, due to their intracellular abundance and fast supercoil relaxation rates, it is generally assumed that these enzymes are sufficient in coping with genome-wide bursts of excessive supercoiling. However, the recent discoveries of chromatin architectural factors that play important accessory functions have cast reasonable doubts on this concept. Here, we reviewed the background of these new findings and described emerging models of how these accessory factors contribute to supercoil homeostasis. We focused on DNA replication and the generation of positive (+) supercoiling in front of replisomes, where two accessory factors—GapR and HMGA2—from pro- and eukaryotic cells, respectively, appear to play important roles as sinks for excessive (+) supercoiling by employing a combination of supercoil constrainment and activation of topoisomerases. Looking forward, we expect that additional factors will be identified in the future as part of an expanding cellular repertoire to cope with bursts of topological strain. Furthermore, identifying antagonists that target these accessory factors and work synergistically with clinically relevant topoisomerase inhibitors could become an interesting novel strategy, leading to improved treatment outcomes.

scholarly journals Pocket Protein Complexes Are Recruited to Distinct Targets in Quiescent and Proliferating Cells

2005 ◽  
Vol 25 (18) ◽  
pp. 8166-8178 ◽  
Author(s):  
Egle Balciunaite ◽  
Alexander Spektor ◽  
Nathan H. Lents ◽  
Hugh Cam ◽  
Hein te Riele ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Regulatory Networks ◽  
Protein Complexes ◽  
Proliferating Cells ◽  
Quiescent Cells ◽  
Pocket Protein ◽  
Genome Wide ◽  
E2f Transcription Factor ◽  
A Cell ◽  
Cycle Dependent

ABSTRACT Biochemical and genetic studies have determined that retinoblastoma protein (pRB) tumor suppressor family members have overlapping functions. However, these studies have largely failed to distinguish functional differences between the highly related p107 and p130 proteins. Moreover, most studies pertaining to the pRB family and its principal target, the E2F transcription factor, have focused on cells that have reinitiated a cell cycle from quiescence, although recent studies suggest that cycling cells exhibit layers of regulation distinct from mitogenically stimulated cells. Using genome-wide chromatin immunoprecipitation, we show that there are distinct classes of genes directly regulated by unique combinations of E2F4, p107, and p130, including a group of genes specifically regulated in cycling cells. These groups exhibit both distinct histone acetylation signatures and patterns of mammalian Sin3B corepressor recruitment. Our findings suggest that cell cycle-dependent repression results from recruitment of an unexpected array of diverse complexes and reveals specific differences between transcriptional regulation in cycling and quiescent cells. In addition, factor location analyses have, for the first time, allowed the identification of novel and specific targets of the highly related transcriptional regulators p107 and p130, suggesting new and distinct regulatory networks engaged by each protein in continuously cycling cells.

The Hbo1-Brd1/Brpf2 complex is responsible for global acetylation of H3K14 and required for fetal liver erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (9) ◽  
pp. 2443-2453 ◽  
Author(s):  
Yuta Mishima ◽  
Satoru Miyagi ◽  
Atsunori Saraya ◽  
Masamitsu Negishi ◽  
Mitsuhiro Endoh ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Protein Complexes ◽  
Fetal Liver ◽  
Genome Wide ◽  
Physiologic Function ◽  
A Subunit ◽  
H3k14 Acetylation ◽  
Regulator Genes ◽  
Biochemical Analyses ◽  
And Function

Abstract The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.

Quantitative Control for Stoichiometric Protein Synthesis

2021 ◽  
Vol 75 (1) ◽  
Author(s):  
James C. Taggart ◽  
Jean-Benoît Lalanne ◽  
Gene-Wei Li
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Protein Complexes ◽  
Annual Review ◽  
Publication Date ◽  
Rna Turnover ◽  
Regulatory Strategies ◽  
Open Questions ◽  
Genome Wide ◽  
Bacterial Protein Synthesis

Bacterial protein synthesis rates have evolved to maintain preferred stoichiometries at striking precision, from the components of protein complexes to constituents of entire pathways. Setting relative protein production rates to be well within a factor of two requires concerted tuning of transcription, RNA turnover, and translation, allowing many potential regulatory strategies to achieve the preferred output. The last decade has seen a greatly expanded capacity for precise interrogation of each step of the central dogma genome-wide. Here, we summarize how these technologies have shaped the current understanding of diverse bacterial regulatory architectures underpinning stoichiometric protein synthesis. We focus on the emerging expanded view of bacterial operons, which encode diverse primary and secondary mRNA structures for tuning protein stoichiometry. Emphasis is placed on how quantitative tuning is achieved. We discuss the challenges and open questions in the application of quantitative, genome-wide methodologies to the problem of precise protein production. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Sign in / Sign up

Export Citation Format

Share Document