Abstract
Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to ...Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.
Regulation of gene expression is essential for the functioning of all eukaryotic organisms. Understanding gene expression regulation requires determining which proteins interact with regulatory ...elements in chromatin. MS-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation, as it allows studying protein function and protein complex formation in their in vivo chromatin-bound context. Total chromatin isolated from cells can be directly analyzed using MS or further fractionated into transcriptionally active and inactive chromatin prior to MS-based analysis. Newly formed chromatin that is assembled during DNA replication can also be specifically isolated and analyzed. Furthermore, capturing specific chromatin domains facilitates the identification of previously unknown transcription factors interacting with these domains. Finally, in recent years, advances have been made toward identifying proteins that interact with a single genomic locus of interest. In this review, we highlight the power of chromatin proteomics approaches and how these provide complementary alternatives compared with conventional affinity purification methods. Furthermore, we discuss the biochemical challenges that should be addressed to consolidate and expand the role of chromatin proteomics as a key technology in the context of gene expression regulation and epigenetics research in health and disease.
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•An overview of proteomics methods to study chromatin and gene regulation.•Strength and limitations of the different approaches are highlighted.•An outlook on the outstanding challenges for chromatin proteomics.•Future directions for chromatin proteomics are discussed.
MS-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation. Total chromatin isolated from cells can be directly analyzed using MS, further fractionated into transcriptionally active and inactive chromatin, enriched for specific compartment or regions, and potentially used for single-locus isolation. This review highlights recent advances and discusses current challenges that should be addressed to further advance the field of chromatin proteomics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Understanding how chromatin is regulated is essential to fully grasp genome biology, and establishing the locus-specific protein composition is a major step toward this goal. Here we explain why the ...isolation and analysis of a specific chromatin segment are technically challenging, independently of the method. We then describe the published strategies and discuss their advantages and limitations. We conclude by discussing why significant technology developments are required to unambiguously describe the composition of small single loci.
Despite all efforts made to develop predictive biomarkers for antiangiogenic therapies, no unambiguous markers have been identified so far. This is due to among others the lack of standardized tests. ...This study presents an improved microvessel density quantification method in tumor tissue based on stereological principles and using whole-slide images. Vessels in tissue sections of different cancer types were stained for CD31 by an automated and validated immunohistochemical staining method. The stained slides were digitized with a digital slide scanner. Systematic, uniform, random sampling of the regions of interest on the whole-slide images was performed semi-automatically with the previously published applications AutoTag and AutoSnap. Subsequently, an unbiased counting grid was combined with the images generated with these scripts. Up to six independent observers counted microvessels in up to four cancer types: colorectal carcinoma, glioblastoma multiforme, ovarian carcinoma and renal cell carcinoma. At first, inter-observer variability was found to be unacceptable. However, after a series of consensus training sessions and interim statistical analysis, counting rules were modified and inter-observer concordance improved considerably. Every CD31-positive object was counted, with exclusion of suspected CD31-positive monocytes, macrophages and tumor cells. Furthermore, if interconnected, stained objects were considered a single vessel. Ten regions of interest were sufficient for accurate microvessel density measurements. Intra-observer and inter-observer variability were low (intraclass correlation coefficient > 0.7) if the observers were adequately trained.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Synovial sarcoma tumours contain a characteristic fusion protein, SS18-SSX, which drives disease development. Targeting oncogenic fusion proteins presents an attractive therapeutic opportunity. ...However, SS18-SSX has proven intractable for therapeutic intervention. Using a domain-focused CRISPR screen we identified the bromodomain of BRD9 as a critical functional dependency in synovial sarcoma. BRD9 is a component of SS18-SSX containing BAF complexes in synovial sarcoma cells; and integration of BRD9 into these complexes is critical for cell growth. Moreover BRD9 and SS18-SSX co-localize extensively on the synovial sarcoma genome. Remarkably, synovial sarcoma cells are highly sensitive to a novel small molecule degrader of BRD9, while other sarcoma subtypes are unaffected. Degradation of BRD9 induces downregulation of oncogenic transcriptional programs and inhibits tumour progression in vivo. We demonstrate that BRD9 supports oncogenic mechanisms underlying the SS18-SSX fusion in synovial sarcoma and highlight targeted degradation of BRD9 as a potential therapeutic opportunity in this disease.
The neuregulin-1 (NRG-1)/receptor tyrosine-protein kinase erbB (ErbB) system is an endothelium-controlled paracrine system modulating cardiac performance and adaptation. Recent studies have indicated ...that NRG-1 has antifibrotic effects in the left ventricle, which were explained by direct actions on cardiac fibroblasts. However, the NRG-1/ErbB system also regulates the function of macrophages. In this study, we hypothesized that the antifibrotic effect of NRG-1 in the heart is at least partially mediated through inhibitory effects on macrophages. We also hypothesized that the antifibrotic effect of NRG-1 may be active in other organs, such as the skin and lung. First, in a mouse model of angiotensin II (ANG II)-induced myocardial hypertrophy and fibrosis, NRG-1 treatment (20 µg·kg
·day
ip) significantly attenuated myocardial hypertrophy and fibrosis and improved passive ventricular stiffness (4 wk). Interestingly, 1 wk after exposure to ANG II, NRG-1 already attenuated myocardial macrophage infiltration and cytokine expression. Furthermore, mice with myeloid-specific deletion of the
gene (
) showed an intensified myocardial fibrotic response to ANG II. Consistently, NRG-1 activated the ErbB4 receptor in isolated macrophages, inhibited phosphatidylinositide 3-kinase/Akt and STAT3 signaling pathways, and reduced the release of inflammatory cytokines. Further experiments showed that the antifibrotic and anti-inflammatory effects of NRG-1 were reproducible in mouse models of bleomycin-induced dermal and pulmonary fibrosis. Overall, this study demonstrates that the antifibrotic effect of NRG-1 in the heart is linked to anti-inflammatory activity NRG-1/ErbB4 signaling in macrophages. Second, this study shows that NRG-1 has antifibrotic and anti-inflammatory effects in organs other than the heart, such as the skin and lung.
Our study contributes to the understanding of the antifibrotic effect of neuregulin-1 during myocardial remodeling. Here, we show that the antifibrotic effect of neuregulin-1 is at least partially mediated through anti-inflammatory activity, linked to receptor tyrosine-protein kinase erbB-4 activation in macrophages. Furthermore, we show that this effect is also present outside the heart.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
10.
The Complexity of PRC2 Subcomplexes van Mierlo, Guido; Veenstra, Gert Jan C.; Vermeulen, Michiel ...
Trends in cell biology,
August 2019, 2019-08-00, 20190801, Volume:
29, Issue:
8
Journal Article
Peer reviewed
Open access
Polycomb repressive complex 2 (PRC2) is a multisubunit protein complex essential for the development of multicellular organisms. Recruitment of PRC2 to target genes, followed by deposition and ...propagation of its catalytic product histone H3 lysine 27 trimethylation (H3K27me3), are key to the spatiotemporal control of developmental gene expression. Recent breakthrough studies have uncovered unexpected roles for substoichiometric PRC2 subunits in these processes. Here, we elaborate on how the facultative PRC2 subunits regulate catalytic activity, locus-specific PRC2 binding, and propagation of H3K27me3, and how this affects chromatin structure, gene expression, and cell fate.
Polycomb repressive complex 2 (PRC2) is a multisubunit epigenetic protein complex that regulates gene expression by catalyzing trimethylation of histone H3 on lysine 27 (H3K27me3), which is essential for embryonic development.PRC2 can associate with a multitude of facultative subunits resulting in at least two different multimeric configurations called PRC2.1 and PRC2.2. However, the exact interplay between the two subcomplexes requires further study.PRC2.1 and PRC2.2 are recruited to target genes via distinct mechanisms and have divergent roles in gene silencing, but their combined action together with PRC1 is required for Polycomb function.Changes of cellular state, such as during the differentiation of embryonic stem cells, can evoke architectural and functional changes in the PRC2 subcomplexes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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