discovering-protein-interactions-characterizing-protein-function

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

high-throughput-cellular-profiling-targeted-protein-degradation

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

Promega Corporation

In the canonical Wnt signaling pathway, beta-catenin activates target genes through its interactions with Tcf/Lef-family transcription factors and additional transcriptional coactivators. The crystal structure of ICAT, an inhibitor of beta-catenin-mediated transcription, bound to the armadillo repeat domain of beta-catenin, has been determined. ICAT contains an N-terminal helilical domain that binds to repeats 11 and 12 of beta-catenin, and an extended C-terminal region that binds to repeats 5-10 in a manner similar to that of Tcfs and other beta-catenin ligands. Full-length ICAT dissociates complexes of beta-catenin, Lef-1, and the transcriptional coactivator p300, whereas the helical domain alone selectively blocks binding to p300. The C-terminal armadillo repeats of beta-catenin may be an attractive target for compounds designed to disrupt aberrant beta-catenin-mediated transcription associated with various cancers.

ICAT inhibits beta-catenin binding to Tcf/Lef-family transcription factors and the general coactivator p300 using independent structural modules.

Wnt growth factors mediate cell fate determination during embryogenesis and in the renewal of tissues in the adult. Wnts act by stabilizing cellular levels of the transcriptional coactivator beta-catenin, which forms complexes with sequence-specific DNA-binding Tcf/Lef transcription factors. In the absence of nuclear beta-catenin, Tcf/Lefs act as transcriptional repressors by binding to Groucho/TLE proteins. The molecular basis of the switch from transcriptional repression to activation during Wnt signaling has not been clear, in particular whether factors other than beta-catenin are required to disrupt the interaction between Groucho/TLE and Tcf/Lef. Using highly purified proteins, we demonstrate that beta-catenin displaces Groucho/TLE from Tcf/Lef by binding to a previously unidentified second, low-affinity binding site on Lef-1 that includes sequences just N-terminal to the DNA-binding domain, and that overlaps the Groucho/TLE-binding site.

Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation.

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.

Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3-OGT co-localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3-OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step-wise model, involving TET-OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation.

TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS.

Mapping of protein binding sites within the genome has been significantly advanced by microarray and sequencing technologies, yet the method traditionally used to isolate protein-DNA complexes, chromatin immunoprecipitation, has remained dependent of the use of antibodies. Furthermore, cross-linking is commonly used to trap protein-DNA complexes and the challenge of using antibodies has come in recognition of the cross-linked epitopes, sometimes limiting the success of the approach. Here we present a method, HaloCHIP, which utilizes a HaloTag protein fusion and corresponding interaction resin, HaloLink, for capture of cross-linked protein-DNA complexes directly from a cellular lysate. This process alleviates the need for using an antibody, yields the DNA fragments bound to a particular protein of interest, and allows for a variety of downstream analyses such as PCR, qPCR, microarrays, and sequencing.

Isolation of intracellular protein--DNA complexes using HaloCHIP, an antibody-free alternative to chromatin immunoprecipitation.

The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). We report here that HIF1A employs a specific variant of the Mediator complex to stimulate RNAPII elongation. The Mediator-associated kinase CDK8, but not the paralog CDK19, is required for induction of many HIF1A target genes. HIF1A induces binding of CDK8-Mediator and the super elongation complex (SEC), containing AFF4 and CDK9, to alleviate RNAPII pausing. CDK8 is dispensable for HIF1A chromatin binding and histone acetylation, but it is essential for binding of SEC and RNAPII elongation. Global analysis of active RNAPII reveals that hypoxia-inducible genes are paused and active prior to their induction. Our results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia.

HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia.

Wnts are secreted growth factors that have critical roles in cell fate determination and stem cell renewal. The Wnt/β-catenin pathway is initiated by binding of a Wnt protein to a Frizzled (Fzd) receptor and a coreceptor, LDL receptor-related protein 5 or 6 (LRP5/6). We report the 2.1 Å resolution crystal structure of a Drosophila WntD fragment encompassing the N-terminal domain and the linker that connects it to the C-terminal domain. Differences in the structures of WntD and Xenopus Wnt8, including the positions of a receptor-binding β hairpin and a large solvent-filled cavity in the helical core, indicate conformational plasticity in the N-terminal domain that may be important for Wnt-Frizzled specificity. Structure-based mutational analysis of mouse Wnt3a shows that the linker between the N- and C-terminal domains is required for LRP6 binding. These findings provide important insights into Wnt function and evolution.

structural Studies of Wnts and identification of an LRP6 binding site.

Acquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.

ArticleTotal : 2	Year
Publication title Cited by 11	2014
Publication title Cited by 27	2020

Article	Year
ICAT inhibits beta-catenin binding to Tcf/Lef-family transcription factors and the general coactivator p300 using independent structural modules. Molecular cell\| PubMed ID: 12408825	2002
Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation. Nature structural & molecular biology\| PubMed ID: 15768032	2005
Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics. Journal of proteome research\| PubMed ID: 22149079	2012
Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands. Current chemical genomics\| PubMed ID: 23248739	2012
TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS. The EMBO journal\| PubMed ID: 23353889	2013
Isolation of intracellular protein--DNA complexes using HaloCHIP, an antibody-free alternative to chromatin immunoprecipitation. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 23436357	2013
HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia. Cell\| PubMed ID: 23746844	2013
structural Studies of Wnts and identification of an LRP6 binding site. Structure (London, England : 1993)\| PubMed ID: 23791946	2013
KDM4A lysine demethylase induces site-specific copy gain and rereplication of regions amplified in tumors. Cell\| PubMed ID: 23871696	2013

Danette L. Daniels

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