Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

ABSTRACTATP-dependent chromatin remodelers regulate the DNA accessibility required of virtually all nuclear processes. Biochemical studies have provided insight into remodeler action at the nucleosome level, but how these findings translate to activity on chromatin fibers in vitro and in vivo remains poorly understood. Here, we present a massively multiplex single-molecule platform allowing high-resolution mapping of nucleosomes on fibers assembled on mammalian genomic sequences. We apply this method to distinguish between competing models for chromatin remodeling by the essential ISWI ATPase SNF2h: linker-length-dependent dynamic positioning versus fixed-linker-length static clamping. Our single-fiber data demonstrate that SNF2h operates as a density-dependent, length-sensing chromatin remodeler whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. In vivo, this activity manifests as different regulatory modes across epigenomic domains: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, SNF2h slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Overall, our generalizable approach provides molecularly-precise views of the processes that shape nuclear physiology. Concurrently, our data illustrate how a mammalian chromatin remodeling enzyme can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus.

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The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Biomolecules ◽

10.3390/biom11030360 ◽

2021 ◽

Vol 11 (3) ◽

pp. 360

Author(s):

Pieterjan Debie ◽

Noemi B. Declerck ◽

Danny van Willigen ◽

Celine M. Huygen ◽

Bieke De Sloovere ◽

...

Keyword(s):

Single Molecule ◽

Gamma Ray ◽

Nuclear Imaging ◽

Primary Amines ◽

Resection Margins ◽

Fluorescent Light ◽

Single Structure ◽

Fluorescence Images

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

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A Novel Human Ada2 Homologue Functions with Gcn5 or Brg1 To Coactivate Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.23.19.6944-6957.2003 ◽

2003 ◽

Vol 23 (19) ◽

pp. 6944-6957 ◽

Cited By ~ 46

Author(s):

Nickolai A. Barlev ◽

Alexander V. Emelyanov ◽

Paola Castagnino ◽

Philip Zegerman ◽

Andrew J. Bannister ◽

...

Keyword(s):

Histone Acetylation ◽

Chromatin Remodeling ◽

Adaptor Protein ◽

Saga Complex ◽

Yeast Two Hybrid ◽

Functional Assays ◽

Two Hybrid ◽

Histone Acetyltransferase Activity

ABSTRACT In yeast, the transcriptional adaptor yeast Ada2 (yAda2) is a part of the multicomponent SAGA complex, which possesses histone acetyltransferase activity through action of the yGcn5 catalytic enzyme. yAda2, among several SAGA proteins, serves to recruit SAGA to genes via interactions with promoter-bound transcription factors. Here we report identification of a new human Ada2 homologue, hAda2β. Ada2β differs both biochemically and functionally from the previously characterized hAda2α, which is a stable component of the human PCAF (human Gcn5 homologue) acetylase complex. Ada2β, relative to Ada2α, interacted selectively, although not stably, with the Gcn5-containing histone acetylation complex TFTC/STAGA. In addition, Ada2β interacted with Baf57 (a component of the human Swi/Snf complex) in a yeast two-hybrid screen and associated with human Swi/Snf in vitro. In functional assays, hAda2β (but not Ada2α), working in concert with Gcn5 (but not PCAF) or Brg1 (the catalytic component of hSwi/Snf complex), increased transcription via the B-cell-specific transcription factor Pax5/BSAP. These findings support the view that Gcn5 and PCAF have distinct roles in vivo and suggest a new mechanism of coactivator function, in which a single adaptor protein (Ada2β) can coordinate targeting of both histone acetylation and chromatin remodeling activities.

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Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01152.2007 ◽

2008 ◽

Vol 294 (2) ◽

pp. H699-H707 ◽

Cited By ~ 46

Author(s):

Ellen Steward Pentz ◽

Maria Luisa S. Sequeira Lopez ◽

Magali Cordaillat ◽

R. Ariel Gomez

Keyword(s):

Chromatin Remodeling ◽

In Vitro Model ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Histone H4 Acetylation ◽

Renin Gene ◽

Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

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Synthesis runs counter to directional folding of a nascent protein domain

Nature Communications ◽

10.1038/s41467-020-18921-8 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Xiuqi Chen ◽

Nandakumar Rajasekaran ◽

Kaixian Liu ◽

Christian M. Kaiser

Keyword(s):

Single Molecule ◽

Molten Globule ◽

Elongation Factor ◽

Protein Domain ◽

Folding Pathway ◽

Native Structure ◽

Folding Intermediates ◽

C Terminus

Abstract Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

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Sp1-Induced Upregulation of LBX2-AS1 Aggravates The Progression of Glioblastoma By Targeting The miR-491-5p/LIF Axis

10.21203/rs.3.rs-242079/v1 ◽

2021 ◽

Author(s):

Wentao Li ◽

Ismatullah Soufiany ◽

Xiao Lyu ◽

Lin Zhao ◽

Chenfei Lu ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Cancer Progression ◽

Epithelial To Mesenchymal Transition ◽

Luciferase Reporter ◽

Mesenchymal Transition ◽

Stat3 Signaling ◽

Regulatory Effects

Abstract Background: Mounting evidences have shown the importance of lncRNAs in tumorigenesis and cancer progression. LBX2-AS1 is an oncogenic lncRNA that has been found abnormally expressed in gastric cancer and lung cancer samples. Nevertheless, the biological function of LBX2-AS1 in glioblastoma (GBM) and potential molecular mechanism are largely unclear. Methods: Relative levels of LBX2-AS1 in GBM samples and cell lines were detected by qRT-PCR and FISH. In vivo and in vitro regulatory effects of LBX2-AS1 on cell proliferation, epithelial-to-mesenchymal transition (EMT) and angiogenesis in GBM were examined through xenograft models and functional experiments, respectively. The interaction between Sp1 and LBX2-AS1 was assessed by ChIP. Through bioinformatic analyses, dual-luciferase reporter assay, RIP and Western blot, the regulation of LBX2-AS1 and miR-491-5p on the target gene leukemia Inhibitory factor (LIF) was identified. Results: LBX2-AS1 was upregulated in GBM samples and cell lines, and its transcription was promoted by binding to the transcription factor Sp1. As a lncRNA mainly distributed in the cytoplasm, LBX2-AS1 upregulated LIF, and activated the LIF/STAT3 signaling by exerting the miRNA sponge effect on miR-491-5p, thus promoting cell proliferation, EMT and angiogenesis in GBM. Besides, LBX2-AS1 was unfavorable to the progression of glioma and the survival. Conclusion: Upregulated by Sp1, LBX2-AS1 promotes the progression of GBM by targeting the miR-491-5p/LIF axis. It is suggested that LBX2-AS1 may be a novel diagnostic biomarker and therapeutic target of GBM.

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Rational design of protein-specific folding modifiers

10.1101/2020.04.28.064113 ◽

2020 ◽

Author(s):

Anirban Das ◽

Anju Yadav ◽

Mona Gupta ◽

R Purushotham ◽

Vishram L. Terse ◽

...

Keyword(s):

Single Molecule ◽

Rational Design ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Force Measurements ◽

Folding Nucleus ◽

Dynamics Simulations ◽

General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

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C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

10.1101/835082 ◽

2019 ◽

Cited By ~ 4

Author(s):

Laura Fumagalli ◽

Florence L. Young ◽

Steven Boeynaems ◽

Mathias De Decker ◽

Arpan R. Mehta ◽

...

Keyword(s):

Axonal Transport ◽

Motor Neurons ◽

Single Molecule ◽

Induced Pluripotent Stem Cell ◽

Cytoplasmic Dynein ◽

Inhibitory Interaction ◽

Hexanucleotide Repeat ◽

Repeat Expansions

ABSTRACTHexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies.

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Synthesis runs counter to directional folding of a nascent protein domain

10.1101/2020.04.29.068593 ◽

2020 ◽

Cited By ~ 1

Author(s):

Xiuqi Chen ◽

Nandakumar Rajasekaran ◽

Kaixian Liu ◽

Christian M. Kaiser

Keyword(s):

Single Molecule ◽

Molten Globule ◽

Elongation Factor ◽

Protein Domain ◽

Folding Pathway ◽

Native Structure ◽

Folding Intermediates ◽

C Terminus

AbstractFolding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

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The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

Frontiers in Oncology ◽

10.3389/fonc.2021.752573 ◽

2021 ◽

Vol 11 ◽

Author(s):

Dong Chen ◽

Yaqin Wang ◽

Feiya Yang ◽

Adili Keranmu ◽

Qingxin Zhao ◽

...

Keyword(s):

Prostate Cancer ◽

Real Time ◽

Expression Pattern ◽

Real Time Pcr ◽

Bioinformatic Analysis ◽

Biological Functions ◽

Quantitative Real Time Pcr ◽

Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

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Transcription factor clusters regulate genes in eukaryotic cells

eLife ◽

10.7554/elife.27451 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 38

Author(s):

Adam JM Wollman ◽

Sviatlana Shashkova ◽

Erik G Hedlund ◽

Rosmarie Friemann ◽

Stefan Hohmann ◽

...

Keyword(s):

Single Molecule ◽

Functional Unit ◽

Yeast Genome ◽

Gene Promoters ◽

Intrinsically Disordered ◽

Extracellular Signal ◽

Genome Model ◽

Nuclear Targets

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

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