Publications by authors named "James Chuang"

9 Publications

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Essential histone chaperones collaborate to regulate transcription and chromatin integrity.

Genes Dev 2021 May 22;35(9-10):698-712. Epub 2021 Apr 22.

Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.
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http://dx.doi.org/10.1101/gad.348431.121DOI Listing
May 2021

The conserved elongation factor Spn1 is required for normal transcription, histone modifications, and splicing in Saccharomyces cerevisiae.

Nucleic Acids Res 2020 10;48(18):10241-10258

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

Spn1/Iws1 is a conserved protein involved in transcription and chromatin dynamics, yet its general in vivo requirement for these functions is unknown. Using a Spn1 depletion system in Saccharomyces cerevisiae, we demonstrate that Spn1 broadly influences several aspects of gene expression on a genome-wide scale. We show that Spn1 is globally required for normal mRNA levels and for normal splicing of ribosomal protein transcripts. Furthermore, Spn1 maintains the localization of H3K36 and H3K4 methylation across the genome and is required for normal histone levels at highly expressed genes. Finally, we show that the association of Spn1 with the transcription machinery is strongly dependent on its binding partner, Spt6, while the association of Spt6 and Set2 with transcribed regions is partially dependent on Spn1. Taken together, our results show that Spn1 affects multiple aspects of gene expression and provide additional evidence that it functions as a histone chaperone in vivo.
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http://dx.doi.org/10.1093/nar/gkaa745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544207PMC
October 2020

Spt6 Is Required for the Fidelity of Promoter Selection.

Mol Cell 2018 11 11;72(4):687-699.e6. Epub 2018 Oct 11.

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.
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http://dx.doi.org/10.1016/j.molcel.2018.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239972PMC
November 2018

Coupling Yeast Golden Gate and VEGAS for Efficient Assembly of the Violacein Pathway in Saccharomyces cerevisiae.

Methods Mol Biol 2018 ;1671:211-225

Institute for Systems Genetics, New York University Langone Medical Center, New York, NY, 10016, USA.

The ability to express non-native pathways in genetically tractable model systems is important for fields such as synthetic biology, genetics, and metabolic engineering. Here we describe a modular and hierarchical strategy to assemble multigene pathways for expression in S. cerevisiae. First, discrete promoter, coding sequence, and terminator parts are assembled in vitro into Transcription Units (TUs) flanked by adapter sequences using "yeast Golden Gate" (yGG), a type IIS restriction enzyme-dependent cloning strategy. Next, harnessing the natural capacity of S. cerevisiae for homologous recombination, TUs are assembled into pathways and expressed using the "Versatile Genetic Assembly System" (VEGAS) in yeast. Coupling transcription units constructed by yGG with VEGAS assembly is a generic and flexible workflow to achieve pathway expression in S. cerevisiae. This protocol describes assembly of a five TU pathway for yeast production of violacein, a pigment derived from Chromobacterium violaceum.
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http://dx.doi.org/10.1007/978-1-4939-7295-1_14DOI Listing
July 2018

Coordinated regulation of acid resistance in Escherichia coli.

BMC Syst Biol 2017 01 6;11(1). Epub 2017 Jan 6.

BE605 Course, Biomedical Engineering, Boston University, Boston, USA.

Background: Enteric Escherichia coli survives the highly acidic environment of the stomach through multiple acid resistance (AR) mechanisms. The most effective system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic protons and excrete GABA. The first described system, AR1, does not require an external amino acid. Its mechanism has not been determined. The regulation of the multiple AR systems and their coordination with broader cellular metabolism has not been fully explored.

Results: We utilized a combination of ChIP-Seq and gene expression analysis to experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and csiR. Our data identified all previously in vivo confirmed direct interactions and revealed several others previously inferred from gene expression data. Our data demonstrate that nac and csiR directly modulate AR, and leads to a regulatory network model in which all four TFs participate in coordinating acid resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with internally derived glutamate. This hypothesis makes several testable predictions that we confirmed experimentally.

Conclusions: Our data suggest that the regulatory network underlying AR is complex and deeply interconnected with the regulation of GABA and glutamate metabolism, nitrogen metabolism. These connections underlie and experimentally validated model of AR1 in which the decarboxylation enzymes of AR2 are used with internally derived glutamate.
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http://dx.doi.org/10.1186/s12918-016-0376-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217608PMC
January 2017

Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae.

Nucleic Acids Res 2015 Jul 8;43(13):6620-30. Epub 2015 May 8.

Department of Biochemistry and Molecular Pharmacology, New York University Langone School of Medicine, New York City, NY 10016, USA Institute for Systems Genetics, New York University Langone School of Medicine, New York City, NY 10016, USA High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.
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http://dx.doi.org/10.1093/nar/gkv466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513848PMC
July 2015

Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units.

ACS Synth Biol 2015 Jul 23;4(7):853-9. Epub 2015 Mar 23.

†Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 550 First Avenue, New York, New York 10016, United States.

We have adapted the Golden Gate DNA assembly method to the assembly of transcription units (TUs) for the yeast Saccharomyces cerevisiae, in a method we call yeast Golden Gate (yGG). yGG allows for the easy assembly of TUs consisting of promoters (PRO), coding sequences (CDS), and terminators (TER). Carefully designed overhangs exposed by digestion with a type IIS restriction enzyme enable virtually seamless assembly of TUs that, in principle, contain all of the information necessary to express a gene of interest in yeast. We also describe a versatile set of yGG acceptor vectors to be used for TU assembly. These vectors can be used for low or high copy expression of assembled TUs or integration into carefully selected innocuous genomic loci. yGG provides synthetic biologists and yeast geneticists with an efficient new means by which to engineer S. cerevisiae.
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http://dx.doi.org/10.1021/sb500372zDOI Listing
July 2015

Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA.

ACS Synth Biol 2013 Aug 11;2(8):473-7. Epub 2013 Mar 11.

Department of Molecular Biology and Genetics and High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Multichange ISOthermal (MISO) mutagenesis is a new technique allowing simultaneous introduction of multiple site-directed mutations into plasmid DNA by leveraging two existing ideas: QuikChange-style primers and one-step isothermal (ISO) assembly. Inversely partnering pairs of QuikChange primers results in robust, exponential amplification of linear fragments of DNA encoding mutagenic yet homologous ends. These products are amenable to ISO assembly, which efficiently assembles them into a circular, mutagenized plasmid. Because the technique relies on ISO assembly, MISO mutagenesis is additionally amenable to other relevant DNA modifications such as insertions and deletions. Here we provide a detailed description of the MISO mutagenesis concept and highlight its versatility by applying it to three experiments currently intractable with standard site-directed mutagenesis approaches. MISO mutagenesis has the potential to become widely used for site-directed mutagenesis.
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http://dx.doi.org/10.1021/sb300131wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040258PMC
August 2013

Endoscopic reconstruction of anterior and middle cranial fossa defects using acellular dermal allograft.

Laryngoscope 2003 Mar;113(3):496-501

Department of Otolaryngology and Communicative Disorders, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

Objective: To report our experience in reconstructing defects of the anterior and middle cranial fossa skull base using endoscopic placement of acellular dermal allograft (AlloDerm, LifeCell Corp., The Woodlands, TX).

Study Design: Retrospective chart review.

Methods: In all cases, the skull base repair was completed with a similar technique. After identification of the defect boundaries, endoscopic transnasal repair was performed through placement of a layered reconstruction of acellular dermal allograft, septal bone/cartilage, and acellular dermal allograft, which were all placed on the intracranial side of the defect. A mucosal free graft was draped over the reconstruction. Fibrin glue was used to hold the mucosal graft in place, and the reconstruction was supported by both absorbable and nonabsorbable nasal packing.

Results: Eight patients with nine skull base defects underwent the procedure for repair of cerebrospinal fluid rhinorrhea. All defects were successfully repaired. One patient underwent successful reconstruction of bilateral ethmoid roof defects that resulted from endoscopic resection of ethmoid adenocarcinoma. Twenty-four patients underwent primary resection of hypophyseal adenomas. Twenty-three patients had macroadenomas, and intraoperative cerebrospinal fluid leaks were noted in 11 patients. Sellar repairs after trans-sphenoidal hypophysectomy were successful in 22 of 24 patients. One patient with hypophysectomy required reoperation (1 of 24 [4%]) for secondary closure of a cerebrospinal fluid leak. Serious complications were avoided in all patients. Patients were followed for a period ranging from 5 to 57 months (mean period, 34 mo).

Conclusions: Acellular dermal allograft can be successfully used for the reconstruction of anterior and middle cranial fossa skull base defects. This allograft, which is easy to manipulate endoscopically, provides an effective seal and barrier in skull base reconstruction and avoids the need for a donor site.
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http://dx.doi.org/10.1097/00005537-200303000-00019DOI Listing
March 2003