Flanigan, S. F. | bioRxiv Science

bioRxiv2024

Modular cloning of multigene vectors for the baculovirus system and yeast

Recombinant macromolecular complexes are often produced by the baculovirus system, using multigene expression vectors. Yet, the construction of baculovirus-compatible multigene expression vectors is complicated and time-consuming. Furthermore, while the baculovirus and yeast are popular protein expression systems, no single method for multigene vector construction is compatible with both. Here we present the modular cloning (MoClo) Baculo toolkit for constructing multigene expression vectors for the baculovirus system and, through compatibility with the MoClo Yeast toolkit, also for yeast. Vector construction by MoClo Baculo utilises Golden Gate assembly, which does not require PCR, primers or the sequencing of intermediate products. As a proof of principle, MoClo Baculo was used to construct baculovirus and yeast multigene vectors expressing the four- and five-subunit human Polycomb Repressive Complex 2. We show that MoClo Baculo simplifies and expedites the construction of multigene expression vectors for the baculovirus system and provides compatibility with yeast as an alternative expression system.

biochemistry↗

bioRxiv2020

Convergent evolution between PALI1 and JARID2 for the allosteric activation of PRC2

The polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identities. JARID2 is the only accessory subunit of PRC2 that known to trigger an allosteric activation of methyltransferase. Yet, this mechanism cannot be generalised to all PRC2 variants as, in vertebrates, JARID2 is mutually exclusive with most of the accessory subunits of PRC2. Here we provide functional and structural evidence that the vertebrate-specific PRC2 accessory subunit PALI1 emerged through a convergent evolution to mimic JARID2 at the molecular level. Mechanistically, PRC2 methylates PALI1 K1241, which then binds to the PRC2-regulatory subunit EED to allosterically activate PRC2. PALI1 K1241 is methylated in mouse and human cell lines and is essential for PALI1-induced allosteric activation of PRC2. High-resolution crystal structures revealed that PALI1 mimics the regulatory interactions formed between JARID2 and EED. Independently, PALI1 also facilitates DNA and nucleosome binding by PRC2. In acute myelogenous leukemia cells, overexpression of PALI1 leads to cell differentiation, with the phenotype abrogated by a separation-of-function PALI1 mutation, defective in allosteric activation and active in DNA binding. Collectively, we show that PALI1 facilitates catalysis and substrate binding by PRC2 and provide evidence that subunit-induced allosteric activation is a general property of holo-PRC2 complexes.

biochemistry↗