scholarly journals Advanced methods for the analysis of chromatin-associated proteins

2014 ◽  
Vol 46 (13) ◽  
pp. 441-447 ◽  
Author(s):  
Hector Guillen-Ahlers ◽  
Michael R. Shortreed ◽  
Lloyd M. Smith ◽  
Michael Olivier
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Affinity Purification ◽  
Interacting Proteins ◽  
Analytical Strategy ◽  
Multiple Loci ◽  
Associated Proteins ◽  
Shared Goal ◽  
Dna Protein Interactions

DNA-protein interactions are central to gene expression and chromatin regulation and have become one of the main focus areas of the ENCODE consortium. Advances in mass spectrometry and associated technologies have facilitated studies of these interactions, revealing many novel DNA-interacting proteins and histone posttranslational modifications. Proteins interacting at a single locus or at multiple loci have been targeted in these recent studies, each requiring a separate analytical strategy for isolation and analysis of DNA-protein interactions. The enrichment of target chromatin fractions occurs via a number of methods including immunoprecipitation, affinity purification, and hybridization, with the shared goal of using proteomics approaches as the final readout. The result of this is a number of exciting new tools, with distinct strengths and limitations that can enable highly robust and novel chromatin studies when applied appropriately. The present review compares and contrasts these methods to help the reader distinguish the advantages of each approach.

scholarly journals Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate

2006 ◽  
Vol 173 (4) ◽  
pp. 533-544 ◽  
Author(s):  
Chad D. Knights ◽  
Jason Catania ◽  
Simone Di Giovanni ◽  
Selen Muratoglu ◽  
Ricardo Perez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Biological Activities ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
P53 Gene ◽  
Protein Protein Interactions

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH2-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH2-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the “histone code” hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different “p53 cassettes,” each containing combination patterns of posttranslational modifications and protein–protein interactions.

scholarly journals The interactome of site-specifically acetylated linker histone H1

2021 ◽  
Author(s):  
Eva Hoellmueller ◽  
Katharina Greiner ◽  
Simon M Kienle ◽  
Martin Scheffner ◽  
Andreas Marx ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Affinity Purification ◽  
Histone H1 ◽  
Linker Histone ◽  
Translational Initiation ◽  
Site Specific ◽  
Linker Histone H1 ◽  
Initiation Factors ◽  
Genetic Code Expansion

Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by posttranslational modifications (PTMs) is rather limited. In this study, we report on the generation of site-specifically mono- and di-acetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification-mass spectrometry (AP-MS) and show that site-specific acetylation results in overlapping, but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD+-dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together our data suggests that site-specific acetylation of H1.2 plays a role in modulating protein-protein interactions.

scholarly journals Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

2014 ◽  
Vol 13 (6) ◽  
pp. 776-784 ◽  
Author(s):  
Alexander R. Paredez ◽  
Arash Nayeri ◽  
Jennifer W. Xu ◽  
Jana Krtková ◽  
W. Zacheus Cande
Keyword(s):  
Protein Identification ◽  
Binding Proteins ◽  
Affinity Purification ◽  
Actin Binding ◽  
Interacting Proteins ◽  
Actin Binding Proteins ◽  
Content Type ◽  
Cellular Processes ◽  
Multidimensional Protein Identification Technology ◽  
Associated Proteins

ABSTRACTConsistent with its proposed status as an early branching eukaryote,Giardiahas the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing fromGiardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins inGiardiausing affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that althoughGiardialacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton.

scholarly journals The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin

2020 ◽  
Vol 295 (14) ◽  
pp. 4748-4759 ◽  
Author(s):  
Hang Gyeong Chin ◽  
Pierre-Olivier Esteve ◽  
Cristian Ruse ◽  
Jiyoung Lee ◽  
Scott E. Schaus ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Organelle Transport ◽  
Histone H4 ◽  
Protein Protein Interactions ◽  
Molecule Inhibitor ◽  
Associated Proteins ◽  
Lysine Methyltransferase

Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein–protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311. Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro. In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.

scholarly journals The versatile interactome of chloroplast ribosomes revealed by affinity purification mass spectrometry

2020 ◽  
Author(s):  
Lisa Désirée Westrich ◽  
Vincent Leon Gotsmann ◽  
Claudia Herkt ◽  
Fabian Ries ◽  
Tanja Kazek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Environmental Changes ◽  
Affinity Purification ◽  
Small Subunit ◽  
Fine Tuning ◽  
Chloroplast Gene ◽  
Purification Method ◽  
Chloroplast Gene Expression ◽  
Associated Proteins

Abstract In plant cells, chloroplast gene expression is predominantly controlled through post-transcriptional regulation. Such fine-tuning is vital for precisely orchestrating protein complex assembly as for the photosynthesis machinery and for quickly responding to environmental changes. While regulation of chloroplast protein synthesis is of central importance, little is known about the degree and nature of the regulatory network, mainly due to challenges associated with the specific isolation of transient ribosome interactors. Here, we established a ribosome affinity purification method, which enabled us to broadly uncover putative ribosome-associated proteins in chloroplasts. Endogenously tagging of a protein of the large or small subunit revealed not only interactors of the holo complex, but also preferential interactors of the two subunits. This includes known canonical regulatory proteins as well as several new proteins belonging to the categories of protein and RNA regulation, photosystem biogenesis, redox control and metabolism. The sensitivity of the here applied screen was validated for various transiently interacting proteins. We further provided evidence for the existence of a ribosome-associated Nα-acetyltransferase in chloroplasts and its ability to acetylate substrate proteins at their N-terminus. The broad set of ribosome interactors underscores the potential to regulate chloroplast gene expression on the level of protein synthesis.

scholarly journals Single-molecule simultaneous profiling of DNA methylation and DNA-protein interactions with Nanopore-DamID

2021 ◽  
Author(s):  
Seth W Cheetham ◽  
Yohaann M.A Jafrani ◽  
Stacey B Andersen ◽  
Natasha Jansz ◽  
Adam D Ewing ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Single Molecule ◽  
Protein Interactions ◽  
Cytosine Methylation ◽  
Cpg Methylation ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Transcriptional Start Sites ◽  
Dna Protein Interactions

DNA-protein interactions and cytosine methylation control eukaryotic gene expression. Here, we present an approach to simultaneously detect cytosine methylation and DNA-protein interactions from single molecules, through selective sequencing of adenine-labelled DNA. Applying this approach to LaminB1-associated heterochromatin domains, we identify strict CpG methylation maintenance at transcriptional start sites amid a generalised relaxation of methylation, potentially to prevent ectopic aberrant heterochromatic gene expression.

scholarly journals Proximity Biotin Labeling Reveals KSHV Interferon Regulatory Factor Networks

2020 ◽  
Author(s):  
Ashish Kumar ◽  
Michelle Salemi ◽  
Resham Bhullar ◽  
Sara Guevara-Plunkett ◽  
Yuanzhi Lyu ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Complex ◽  
Viral Protein ◽  
De Novo ◽  
Affinity Purification ◽  
Innate Immune ◽  
Host Protein ◽  
Cellular Proteins ◽  
Interacting Proteins ◽  
Affinity Precipitation

AbstractStudies on “HIT&RUN” effects by viral protein are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled study of both static and dynamic protein-protein interactions. Here we applied PL method with recombinant Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV, a gamma-herpesvirus, uniquely encodes four interferon regulatory factors (IRFs 1-4) in the genome, and we identified KSHV vIRF-1 and vIRF-4 interacting proteins during reactivation. Fusion of mini-TurboID with vIRF-1 or vIRF-4 did not interfere with KSHV gene expression, DNA replication, or de novo infections. PL identified 213 and 70 proteins for vIRF-1 and vIRF-4 respectively, which possibly interact during KSHV reactivation, and 47 of those were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological functions such as mRNA processing and transcriptional regulation by TP53. Involvement of commonly interacting 44 cellular proteins in innate immune regulation were examined by siRNAs, and we identified that splicing factor 3B (SF3B) family proteins were clearly involved in interferons transcription and suppressed KSHV reactivation. We propose that recombinant TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication, and selective splicing factors may have a function beyond connecting two exon sequences to regulate innate immune responses.ImportanceViral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to use of affinity-precipitation approaches, the majority of our studies focused on how the virus takes advantage of the newly-formed protein interactions for its own replication. Proximity labeling (PL) however, can also highlight the transient and negative effects – those interactions which lead to dissociation from the existing protein complex. Here we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.

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