Rapid Single-Pot Assembly of Modular Chromatin Proteins for Epigenetic Engineering

Haynes, Karmella A.; Priode, J. Harrison

doi:10.1007/978-1-0716-2847-8_14

Cited by 4 publications

(2 citation statements)

References 53 publications

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“…The multiple cloning site and the bridge fragments are invariable across the plasmid set and were obtained by gene synthesis. The multiple cloning site in each pUdO contains type IIP and type IIS restriction sites for both conventional and Golden Gate cloning, allowing single-pot digestion and ligation of multiple DNA fragments. − These restriction sites are flanked by strong unidirectional transcriptional terminators as well as two universal primer-binding sites for sequencing purposes (Figure S1). The bridge includes a bidirectional transcription terminator and two universal primer-binding sites to facilitate sequencing (Figure S1).…”

Section: Resultsmentioning

confidence: 99%

pUdOs: Concise Plasmids for Bacterial and Mammalian Cells

Manigat,

Connell,

Stewart

et al. 2024

ACS Synth. Biol.

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The plasmids from the Universitéd'Ottawa (pUdOs) are 28 small plasmids each comprising one of four origins of replication and one of seven selection markers, which together afford flexible use in Escherichia coli and several related gram-negative bacteria. The promoterless multicloning site is insulated from upstream spurious promoters by strong transcription terminators and contains type IIP or IIS restriction sites for conventional or Golden Gate cloning. pUdOs can be converted into efficient expression vectors through the insertion of a promoter at the user's discretion. For example, we demonstrate the utility of pUdOs as the backbone for an improved version of a Type III Secretion System reporter in Shigella. In addition, we derive a series of pUdO-based mammalian expression vectors, affording distinct levels of expression and transfection efficiency comparable to commonly used mammalian expression plasmids. Thus, pUdOs could advantageously replace traditional plasmids in a wide variety of cell types and applications.

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Section: Resultsmentioning

confidence: 99%

pUdOs: Concise Plasmids for Bacterial and Mammalian Cells

Manigat,

Connell,

Stewart

et al. 2024

ACS Synth. Biol.

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“…SRA constructs (SRA: SA0020_MV1, SRAmut1: SA0021_MV1, SRAmut2: SA0022_MV1) were generated via Golden Gate assembly 72 and cloned into the XbaI and SpeI sites of expression vector MV1. Annotated sequences are available at Benchling (https://benchling.com/hayneslab/f_/rmSYkAAU-synthetic-chromatin-actuators-2-0/).…”

Section: Annexin V Staining and Flow Cytometrymentioning

confidence: 99%

PIC recruitment by synthetic reader-actuators to polycomb-silenced genes blocks triple-negative breast cancer invasion

Williams

Hong

Jaffe

et al. 2023

Preprint

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Scientists have used small molecule inhibitors and genetic knockdown of gene-silencing polycomb repressive complexes (PRC1/2) to determine if restoring the expression of tumor suppressor genes can block proliferation and invasion of cancer cells. A major limitation of this approach is that inhibitors can not restore key transcriptional activators that are mutated in many cancers, such as p53 and members of the BRAF SWI/SNF complex. Furthermore, small molecule inhibitors can alter the activity of, rather than inhibit, the polycomb enzyme EZH2. While chromatin has been shown to play a major role in gene regulation in cancer, poor clinical results for polycomb chromatin-targeting therapies for diseases like triple-negative breast cancer (TNBC) could discourage further development of this emerging avenue for treatment. To overcome the limitations of inhibiting polycomb to study epigenetic regulation, we developed an engineered chromatin protein to manipulate transcription. The synthetic reader-actuator (SRA) is a fusion protein that directly binds a target chromatin modification and regulates gene expression. Here, we report the activity of an SRA built from polycomb chromodomain and VP64 modules that bind H3K27me3 and subunits of the Mediator complex, respectively. In SRA-expressing BT-549 cells, we identified 122 upregulated differentially expressed genes (UpDEGs, ≥ 2-fold activation, adjusted p < 0.05). On-target epigenetic regulation was determined by identifying UpDEGs at H3K27me3-enriched, closed chromatin. SRA activity induced activation of genes involved in cell death, cell cycle arrest, and the inhibition of migration and invasion. SRA-expressing BT-549 cells showed reduced spheroid size in Matrigel over time, loss of invasion, and activation of apoptosis. These results show that Mediator-recruiting regulators broadly targeted to silenced chromatin activate silenced tumor suppressor genes and stimulate anti-cancer phenotypes. Therefore further development of gene-activating epigenetic therapies might benefit TNBC patients.

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Synthetic biology and cell engineering—deriving new insights into cancer epigenetics

Franklin¹,

Haynes²

2023

Epigenetic Cancer Therapy

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Rapid Single-Pot Assembly of Modular Chromatin Proteins for Epigenetic Engineering

Cited by 4 publications

References 53 publications

pUdOs: Concise Plasmids for Bacterial and Mammalian Cells

pUdOs: Concise Plasmids for Bacterial and Mammalian Cells

PIC recruitment by synthetic reader-actuators to polycomb-silenced genes blocks triple-negative breast cancer invasion

Synthetic biology and cell engineering—deriving new insights into cancer epigenetics

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