Kai, L. | Search bioRxiv

bioRxiv2023

The spreading of facultative H3K9me3-heterochromatin drives congenital disease

Heterochromatin marked by trimethylated histone 3 at lysine 9 (H3K9me3) plays fundamental roles in reprogramming to direct cell fate determination in higher eukaryotes. However, the upstream factors that guide the establishment and spreading of H3K9me3-heterochromatin, leading to human developmental malformations, remain elusive. In this study, we found that Cdk13, a member of RNA polymerase II (RNAPII) kinase, suppresses congenital heart syndrome by preventing global facultative H3K9me3-heterochromatin spreading. Additionally, Cdk13 directs the phosphorylation of a large set of heterochromatin proteins at specific sites, which are required for the interaction between HP1 and histone H3K9 methyltransferases. Furthermore, we identified a compound, an inhibitor of heterochromatin regulators, that can alleviate syndromic heart defects in Cdk13-mutant mice through the inhibition of H3K9me3-heterochromatin spreading. In summary, this study reveals a novel role and mechanism of Cdk13-triggered facultative H3K9me3-heterochromatin spreading in human genetic disease and paves the way for the treatment of congenital heart syndrome.

genetics↗

bioRxiv2022

One-pot synthesis of prenylated proteins utilizing E. coli cell-free expression

Bottom-up synthetic biology is a powerful tool for uncovering the mechanisms underlying vital biological processes, such as signaling and cell polarization. The core principle of reconstituting cellular functions in their minimal forms can be achieved through modular protein design. However, assembling multiple purified proteins into a functional and synchronized system remains a technical challenge. The fact that many regulatory proteins show direct or indirect membrane interactions further exacerbates the complications. Here, we introduce the Cell-Free prenylated Protein Synthesis (CFpPS) system which enables the production of prenylated proteins in a single reaction mix, through reconstituted prenylation machinery. Not only does the CFpPS system offer a fast and reliable method for producing solubilized prenylated proteins, but it can also produce the protein of interest directly in the vicinity of biomimetic membranes, thus enabling microscopy-based functional assessment. As proof of principle, we demonstrate synthesis and solubilization of various important signaling proteins from the Ras superfamily, as well as membrane binding and extraction of the key polarity regulator Cdc42. Furthermore, our method can be used to confer membrane affinity to any protein, simply by adding a 4-peptide motif to the C-terminus of the protein. In sum, the CFpPS system offers a versatile and effective platform for designing peripheral membrane proteins for synthetic biology applications.

synthetic biology↗