Publications by authors named "Isaac A Chaim"

13 Publications

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Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes.

Nat Methods 2021 May 7;18(5):507-519. Epub 2021 May 7.

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.

RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.
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http://dx.doi.org/10.1038/s41592-021-01128-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8148648PMC
May 2021

Reintroduction of the archaic variant of in cortical organoids alters neurodevelopment.

Science 2021 02;371(6530)

Department of Pediatrics and Department of Cellular & Molecular Medicine, School of Medicine, Center for Academic Research and Training in Anthropogeny (CARTA), Kavli Institute for Brain and Mind, University of California, San Diego, La Jolla, CA 92037, USA.

The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 () plays a key role in neural development and function. also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in , which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.
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http://dx.doi.org/10.1126/science.aax2537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006534PMC
February 2021

Longitudinal assessment of tumor development using cancer avatars derived from genetically engineered pluripotent stem cells.

Nat Commun 2020 Jan 28;11(1):550. Epub 2020 Jan 28.

Ludwig Cancer Research San Diego Branch, 9500 Gilman Dr., CMM-East Room 3055, La Jolla, CA, 92093, USA.

Many cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that captures authentic cancer pathobiology. Orthotopic engraftment of the neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) results in formation of high-grade gliomas. Similar to patient-derived GBM, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, intra-tumor heterogeneity, and extrachromosomal DNA amplification. Re-engraftment of these primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. These cancer avatar models provide a platform for comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation.
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http://dx.doi.org/10.1038/s41467-020-14312-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6987220PMC
January 2020

Complex Oscillatory Waves Emerging from Cortical Organoids Model Early Human Brain Network Development.

Cell Stem Cell 2019 10 29;25(4):558-569.e7. Epub 2019 Aug 29.

Department of Pediatrics/Rady Children's Hospital San Diego, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Kavli Institute for Brain and Mind and Halıcıoğlu Data Science Institute, University of California, San Diego, La Jolla, CA 92093, USA; Center for Academic Research and Training in Anthropogeny (CARTA), La Jolla, CA 92093, USA. Electronic address:

Structural and transcriptional changes during early brain maturation follow fixed developmental programs defined by genetics. However, whether this is true for functional network activity remains unknown, primarily due to experimental inaccessibility of the initial stages of the living human brain. Here, we developed human cortical organoids that dynamically change cellular populations during maturation and exhibited consistent increases in electrical activity over the span of several months. The spontaneous network formation displayed periodic and regular oscillatory events that were dependent on glutamatergic and GABAergic signaling. The oscillatory activity transitioned to more spatiotemporally irregular patterns, and synchronous network events resembled features similar to those observed in preterm human electroencephalography. These results show that the development of structured network activity in a human neocortex model may follow stable genetic programming. Our approach provides opportunities for investigating and manipulating the role of network activity in the developing human cortex.
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http://dx.doi.org/10.1016/j.stem.2019.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778040PMC
October 2019

Nitric oxide induced S-nitrosation causes base excision repair imbalance.

DNA Repair (Amst) 2018 08 5;68:25-33. Epub 2018 May 5.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. Electronic address:

It is well established that inflammation leads to the creation of potent DNA damaging chemicals, including reactive oxygen and nitrogen species. Nitric oxide can react with glutathione to create S-nitrosoglutathione (GSNO), which can in turn lead to S-nitrosated proteins. Of particular interest is the impact of GSNO on the function of DNA repair enzymes. The base excision repair (BER) pathway can be initiated by the alkyl-adenine DNA glycosylase (AAG), a monofunctional glycosylase that removes methylated bases. After base removal, an abasic site is formed, which then gets cleaved by AP endonuclease and processed by downstream BER enzymes. Interestingly, using the Fluorescence-based Multiplexed Host Cell Reactivation Assay (FM-HCR), we show that GSNO actually enhances AAG activity, which is consistent with the literature. This raised the possibility that there might be imbalanced BER when cells are challenged with a methylating agent. To further explore this possibility, we confirmed that GSNO can cause AP endonuclease to translocate from the nucleus to the cytoplasm, which might further exacerbate imbalanced BER by increasing the levels of AP sites. Analysis of abasic sites indeed shows GSNO induces an increase in the level of AP sites. Furthermore, analysis of DNA damage using the CometChip (a higher throughput version of the comet assay) shows an increase in the levels of BER intermediates. Finally, we found that GSNO exposure is associated with an increase in methylation-induced cytotoxicity. Taken together, these studies support a model wherein GSNO increases BER initiation while processing of AP sites is decreased, leading to a toxic increase in BER intermediates. This model is also supported by additional studies performed in our laboratory showing that inflammation in vivo leads to increased large-scale sequence rearrangements. Taken together, this work provides new evidence that inflammatory chemicals can drive cytotoxicity and mutagenesis via BER imbalance.
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http://dx.doi.org/10.1016/j.dnarep.2018.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436541PMC
August 2018

In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities.

Proc Natl Acad Sci U S A 2017 11 9;114(48):E10379-E10388. Epub 2017 Nov 9.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139;

The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual differences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes [four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1)] can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyl-adenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, HO, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals.
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http://dx.doi.org/10.1073/pnas.1712032114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715766PMC
November 2017

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.

Cell 2017 Aug 10;170(5):899-912.e10. Epub 2017 Aug 10.

Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Stem Cell Program, University of California at San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA; Molecular Engineering Laboratory, A(∗)STAR, Singapore, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. Electronic address:

Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
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http://dx.doi.org/10.1016/j.cell.2017.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873302PMC
August 2017

A novel role for transcription-coupled nucleotide excision repair for the in vivo repair of 3,N4-ethenocytosine.

Nucleic Acids Res 2017 04;45(6):3242-3252

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Etheno (ε) DNA base adducts are highly mutagenic lesions produced endogenously via reactions with lipid peroxidation (LPO) products. Cancer-promoting conditions, such as inflammation, can induce persistent oxidative stress and increased LPO, resulting in the accumulation of ε-adducts in different tissues. Using a recently described fluorescence multiplexed host cell reactivation assay, we show that a plasmid reporter bearing a site-specific 3,N4-ethenocytosine (εC) causes transcriptional blockage. Notably, this blockage is exacerbated in Cockayne Syndrome and xeroderma pigmentosum patient-derived lymphoblastoid and fibroblast cells. Parallel RNA-Seq expression analysis of the plasmid reporter identifies novel transcriptional mutagenesis properties of εC. Our studies reveal that beyond the known pathways, such as base excision repair, the process of transcription-coupled nucleotide excision repair plays a role in the removal of εC from the genome, and thus in the protection of cells and tissues from collateral damage induced by inflammatory responses.
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http://dx.doi.org/10.1093/nar/gkx015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389632PMC
April 2017

DNA Repair Capacity in Multiple Pathways Predicts Chemoresistance in Glioblastoma Multiforme.

Cancer Res 2017 01 28;77(1):198-206. Epub 2016 Oct 28.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.

Cancer cells can resist the effects of DNA-damaging therapeutic agents via utilization of DNA repair pathways, suggesting that DNA repair capacity (DRC) measurements in cancer cells could be used to identify patients most likely to respond to treatment. However, the limitations of available technologies have so far precluded adoption of this approach in the clinic. We recently developed fluorescence-based multiplexed host cell reactivation (FM-HCR) assays to measure DRC in multiple pathways. Here we apply a mathematical model that uses DRC in multiple pathways to predict cellular resistance to killing by DNA-damaging agents. This model, developed using FM-HCR and drug sensitivity measurements in 24 human lymphoblastoid cell lines, was applied to a panel of 12 patient-derived xenograft (PDX) models of glioblastoma to predict glioblastoma response to treatment with the chemotherapeutic DNA-damaging agent temozolomide. This work showed that, in addition to changes in O-methylguanine DNA methyltransferase (MGMT) activity, small changes in mismatch repair (MMR), nucleotide excision repair (NER), and homologous recombination (HR) capacity contributed to acquired temozolomide resistance in PDX models and led to reduced relative survival prolongation following temozolomide treatment of orthotopic mouse models in vivo Our data indicate that measuring the combined status of MMR, HR, NER, and MGMT provided a more robust prediction of temozolomide resistance than assessments of MGMT activity alone. Cancer Res; 77(1); 198-206. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-1151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100738PMC
January 2017

Inter-individual variation in DNA repair capacity: a need for multi-pathway functional assays to promote translational DNA repair research.

DNA Repair (Amst) 2014 Jul 26;19:199-213. Epub 2014 Apr 26.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address:

Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA repair capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic.
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http://dx.doi.org/10.1016/j.dnarep.2014.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071454PMC
July 2014

Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis.

Proc Natl Acad Sci U S A 2014 May 22;111(18):E1823-32. Epub 2014 Apr 22.

Department of Biological Engineering, Center for Environmental Health Sciences, Department of Biology, and The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.

The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.
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http://dx.doi.org/10.1073/pnas.1401182111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020053PMC
May 2014

Evaluation of the potential of novel PCL-PPDX biodegradable scaffolds as support materials for cartilage tissue engineering.

J Tissue Eng Regen Med 2012 Apr 5;6(4):272-9. Epub 2011 May 5.

Departamento de Biología Celular, Departamento de Ciencia de los Materiales, Universidad Simón Bolívar, Caracas, Venezuela.

Cartilage is a specialized tissue represented by a group of particular cells (the chondrocytes) and an abundant extracellular matrix. Because of the reduced regenerative capacity of this tissue, cartilage injuries are often difficult to handle. Nowadays tissue engineering has emerged as a very promising discipline, and biodegradable polymeric scaffolds are widely used as tissue supports. In cartilage injuries, the use of autologous chondrocyte implantation from non-affected cartilage zones has emerged as a very interesting technique, where chondrocytes are expanded in order to obtain a greater number of cells. Nevertheless, it has been reported that chondrocytes in bidimensional cultures suffer a dedifferentiation process. The present study sought, in the first place, to standardize a novel protocol in order to obtain primary cultures of chondrocytes from newborn rabbit hyaline cartilage from the xiphoid process. Second, the potential of porous three-dimensional (3D) biodegradable polymeric matrices as support materials for chondrocytes was evaluated: a novel poly(ε-caprolactone)-poly(p-dioxanone) (PCL-PPDX) blend in a 90:10 w:w ratio and poly(ε-caprolactone) (PCL). After achieving the standardization, a typical round-shaped chondrocyte morphology and the expression of collagen type II and aggrecan, evaluated by RT-PCR, were observed. Second-passage chondrocytes adhered effectively to these scaffolds, although cell growth at 7 days in culture was significantly less in the PCL-PPDX blend. After 3 weeks of culture on PCL-PPDX or PCL, the cells expressed collagen type II. The present study demonstrates the potential, unknown until now, of PCL-PPDX blend scaffolds in the field of cartilage tissue engineering.
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http://dx.doi.org/10.1002/term.430DOI Listing
April 2012