Gautam, G. | bioRxiv Science

bioRxiv2023

ATAC and SAGA histone acetyltransferase modules facilitate transcription factor binding to nucleosomes in an acetylation independent manner

Transcription initiation involves the coordination of multiple events, starting with activators binding specific DNA target sequences, which recruits transcription co-activators to open chromatin and enable binding of general transcription factors and RNA polymerase II to promoters. Two key human transcriptional coactivator complexes, ATAC (ADA-Two-A-Containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), target genomic loci to increase promoter accessibility. To better understand the function of ATAC and SAGA histone acetyltransferase (HAT) complexes, we used in vitro biochemical and biophysical assays to characterize human ATAC and SAGA HAT module interactions with nucleosomes and how a transcription factor (TF) coordinates these interactions. We found that ATAC and SAGA HAT modules bind nucleosomes with high affinity, independent of post-translational modifications (PTMs) and TFs. ATAC and SAGA HAT modules directly interact with the VP16 activator domain and a TF containing this domain enhances HAT module acetylation activity. Surprisingly, ATAC and SAGA HAT modules increase TF binding to its DNA target site within the nucleosome by an order of magnitude independent of histone acetylation. Altogether, our results reveal synergistic coordination between HAT modules and a TF, where ATAC and SAGA HAT modules: (i) acetylate histones to open chromatin, and (ii) facilitate TF targeting within nucleosomes independently of their acetylation activity.

molecular biology↗

bioRxiv2023

Engineering the ADDobody protein scaffold for generation of high-avidity ADDomer super-binders

Adenovirus-derived dodecamer (ADDomer) nanoparticles comprise 60 copies of Adenovirus penton base protein (PBP). ADDomer is thermostable, rendering the storage, transport and deployment of ADDomer-based therapeutics independent of a cold-chain. To expand the scope of ADDomer nanoparticles for new applications, we engineered ADDobodies. ADDobodies represent the crown domain of the PBP, genetically separated from its multimerization domain. We inserted heterologous sequences into hyper-variable loops in the crown domain. The resulting ADDobodies were expressed at high yields in Escherichia coli, are monomeric and maintain thermostability. We solved the X-ray structure of an ADDobody prototype validating our design. We demonstrated that ADDobodies can be used to select a specific binder against a target in in vitro selection experiments using ribosome display, with an enrichment factor of [~]104-fold in one selection round. We show that ADDobodies can be converted back into ADDomers by genetically reconnecting the selected ADDobody with the PBP multimerization domain from a different species, giving rise to a multivalent nanoparticle, called Chimera, confirmed by a 2.2 [A] structure determined by cryogenic electron microscopy (cryo-EM). Chimera comprises 60 binding sites, resulting in ultra-high, picomolar avidity to the target.

synthetic biology↗