Design, construction, and validation of histone-binding effectors in vitro and in cells

Stefan J. Tekel, Cassandra M. Barrett, Daniel A. Vargas, Karmella Haynes

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Chromatin is a system of nuclear proteins and nucleic acids that plays a pivotal role in gene expression and cell behavior, and is therefore the subject of intense study for cell development and cancer research. Biochemistry, crystallography, and reverse genetics have elucidated the macromolecular interactions that drive chromatin regulation. One of the central mechanisms is the recognition of post translational modifications (PTMs) on histone proteins by a family of nuclear proteins known as "readers." This knowledge has launched a wave of activity around the rational design of proteins that interact with histone PTMs. Useful molecular tools have emerged from this work, enabling researchers to to probe and manipulate chromatin states in live cells. Chromatin-based proteins represent a vast design space that remains under-explored. Therefore, we have developed a rapid prototyping platform to identify engineered fusion proteins that bind histone PTMs in vitro and regulate genes near the same histone PTMs in living cells. We have used our system to build gene activators with strong avidity for the gene silencing-associated histone PTM H3K27me3. Here, we describe procedures and data for cell-free production of fluorescently-tagged fusion proteins, ELISA-based measurement of histone PTM-binding, and a live cell assay to demonstrate that the fusion proteins modulate transcriptional activation at a site that carries the target histone PTM. This pipeline will be useful for synthetic biologists who are interested in designing novel histone-PTM-binding actuators and probes.

Original languageEnglish (US)
JournalBiochemistry
DOIs
StateAccepted/In press - Mar 17 2018

Fingerprint

Post Translational Protein Processing
Histones
Chromatin
Proteins
Fusion reactions
Genes
Nuclear Proteins
Reverse Genetics
Crystallography
Biochemistry
In Vitro Techniques
Rapid prototyping
Gene Silencing
Gene expression
Nucleic Acids
Transcriptional Activation
Assays
Actuators
Pipelines
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Design, construction, and validation of histone-binding effectors in vitro and in cells. / Tekel, Stefan J.; Barrett, Cassandra M.; Vargas, Daniel A.; Haynes, Karmella.

In: Biochemistry, 17.03.2018.

Research output: Contribution to journalArticle

Tekel, Stefan J. ; Barrett, Cassandra M. ; Vargas, Daniel A. ; Haynes, Karmella. / Design, construction, and validation of histone-binding effectors in vitro and in cells. In: Biochemistry. 2018.
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