Marathe, I. A. | Search bioRxiv

bioRxiv2024

Setdb1 and Atf7IP form a hetero-trimeric complex that blocks Setdb1 nuclear export

AbstractHistone H3K9 methylation (H3K9me) by Setdb1 silences retrotransposons (rTE) by sequestering them in constitutive heterochromatin. Atf7IP is a constitutive binding partner of Setdb1 and is responsible for Setdb1 nuclear localization, activation and chromatin recruitment. However, structural details of the Setdb1/Atf7IP interaction have not been evaluated. We used Alphafold2 predictions and biochemical reconstitutions to show that one copy of Setdb1 and two copies of Atf7IP form a hetero-trimeric complex in vitro and in cells. We also find that Atf7IP self-associates, forming multimeric complexes that are resolved upon Setdb1 binding. Setdb1 binds to Atf7IP through coiled coil interactions that include both Setdb1 nuclear export signals (NES). Atf7IP directly competes with CRM1 to bind the Setdb1 NES motifs, explaining how Atf7IP prevents CRM1-mediated nuclear export of Setdb1. Setdb1 also forms hetero-trimeric complexes with the Atf7IP paralog Atf7IP2 and we show that Setdb1 can form mixed heterotrimers comprising one copy of each Setdb1, Atf7IP and Atf7IP2. Atf7IP and Atf7IP2 are co-expressed in many tissues suggesting that heterotrimers with different compositions of Atf7IP and Atf7IP2 may differentially regulate H3K9me by fine-tuning Setdb1 localization and activity.

biochemistry↗

bioRxiv2024

Topological rearrangements activate the HerA-DUF anti-phage defense system

Leveraging the rich structural information provided by AlphaFold, we used integrated experimental approaches to characterize the HerA-DUF4297 (DUF) anti-phage defense system, in which DUF is of unknown function. To infer the function of DUF, we performed structure-guided genomic analysis and found that DUF homologs are universally present in bacterial immune defense systems. One notable homolog of DUF is Cap4, a universal effector with nuclease activity in CBASS, the most prevalent anti-phage system in bacteria. To test the inferred nuclease function of DUF, we performed biochemical experiments and discovered that the DUF only exhibits activity against DNA substrates when it is bound by HerA. To understand how HerA activates DUF, we determined the structures of DUF and the HerA-DUF complex. DUF forms large oligomeric assemblies with or without HerA, suggesting that oligomerization per se is not sufficient for DUF activation. Instead, DUF activation requires dramatic topological rearrangements that propagate from HerA to the entire HerA-DUF complex, leading to reorganization of DUF for effective DNA cleavage. We further validated these structural insights by structure- guided mutagenesis. Together, these findings reveal dramatic topological rearrangements throughout the HerA-DUF complex, challenge the long-standing dogma that protein oligomerization alone activates immune signaling, and may inform the activation mechanism of CBASS.

biochemistry↗