Publications by authors named "Avi J Samelson"

9 Publications

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Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.

Cell 2021 Apr 9;184(9):2503-2519.e17. Epub 2021 Apr 9.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA. Electronic address:

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
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http://dx.doi.org/10.1016/j.cell.2021.03.025DOI Listing
April 2021

BRD2 inhibition blocks SARS-CoV-2 infection in vitro by reducing transcription of the host cell receptor ACE2.

bioRxiv 2021 Jan 19. Epub 2021 Jan 19.

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted an unbiased CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is an essential node in the cellular response to SARS-CoV-2 infection. BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells. BRD2 also controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates ACE2 levels. It is possible that the previously reported interaction between the viral E protein and BRD2 evolved to manipulate the transcriptional host response during SARS-CoV-2 infection. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.
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http://dx.doi.org/10.1101/2021.01.19.427194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836110PMC
January 2021

The folding and unfolding behavior of ribonuclease H on the ribosome.

J Biol Chem 2020 08 11;295(33):11410-11417. Epub 2020 Jun 11.

Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.
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http://dx.doi.org/10.1074/jbc.RA120.013909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450101PMC
August 2020

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.

IUCrJ 2020 Mar 26;7(Pt 2):306-323. Epub 2020 Feb 26.

Structural Biology Research Center, Institute of Materials Structure Science, KEK/High Energy Accelerator Research Organization, Tsukuba, Ibaraki 305-0034, Japan.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.
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http://dx.doi.org/10.1107/S205225252000072XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055375PMC
March 2020

Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex.

Science 2019 11 31;366(6468):971-977. Epub 2019 Oct 31.

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.

The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagA:RagC GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo-electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.
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http://dx.doi.org/10.1126/science.aax0364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945816PMC
November 2019

Kinetic and structural comparison of a protein's cotranslational folding and refolding pathways.

Sci Adv 2018 05 30;4(5):eaas9098. Epub 2018 May 30.

Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA 94720-3220, USA.

Precise protein folding is essential for the survival of all cells, and protein misfolding causes a number of diseases that lack effective therapies, yet the general principles governing protein folding in the cell remain poorly understood. In vivo, folding can begin cotranslationally and protein quality control at the ribosome is essential for cellular proteostasis. We directly characterize and compare the refolding and cotranslational folding trajectories of the protein HaloTag. We introduce new techniques for both measuring folding kinetics and detecting the conformations of partially folded intermediates during translation in real time. We find that, although translation does not affect the rate-limiting step of HaloTag folding, a key aggregation-prone intermediate observed during in vitro refolding experiments is no longer detectable. This rerouting of the folding pathway increases HaloTag's folding efficiency and may serve as a general chaperone-independent mechanism of quality control by the ribosome.
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http://dx.doi.org/10.1126/sciadv.aas9098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976279PMC
May 2018

Quantitative determination of ribosome nascent chain stability.

Proc Natl Acad Sci U S A 2016 11 7;113(47):13402-13407. Epub 2016 Nov 7.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;

Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein's energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.
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http://dx.doi.org/10.1073/pnas.1610272113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127326PMC
November 2016

HIV-1 Tat recruits transcription elongation factors dispersed along a flexible AFF4 scaffold.

Proc Natl Acad Sci U S A 2013 Jan 18;110(2):E123-31. Epub 2012 Dec 18.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

The HIV-1 Tat protein stimulates viral gene expression by recruiting human transcription elongation complexes containing P-TEFb, AFF4, ELL2, and ENL or AF9 to the viral promoter, but the molecular organization of these complexes remains unknown. To establish the overall architecture of the HIV-1 Tat elongation complex, we mapped the binding sites that mediate complex assembly in vitro and in vivo. The AFF4 protein emerges as the central scaffold that recruits other factors through direct interactions with short hydrophobic regions along its structurally disordered axis. Direct binding partners CycT1, ELL2, and ENL or AF9 act as bridging components that link this complex to two major elongation factors, P-TEFb and the PAF complex. The unique scaffolding properties of AFF4 allow dynamic and flexible assembly of multiple elongation factors and connect the components not only to each other but also to a larger network of transcriptional regulators.
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http://dx.doi.org/10.1073/pnas.1216971110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545800PMC
January 2013

Accessing protein conformational ensembles using room-temperature X-ray crystallography.

Proc Natl Acad Sci U S A 2011 Sep 14;108(39):16247-52. Epub 2011 Sep 14.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3220, USA.

Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation.
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http://dx.doi.org/10.1073/pnas.1111325108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182744PMC
September 2011