Suino-Powell, K. | bioRxiv Science

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↗

bioRxiv2021

Generation of a New Frizzled 2 Flox Mouse Model to Clarify Its Role in Development

It is currently accepted that Wnt receptors, Frizzleds (Fzd), have high functional redundancy, making individual receptors challenging to target therapeutically. Specifically, Fzd2 is believed to be functionally redundant with Fzd1 and Fzd7, findings which were based largely on previously published global knockout mouse studies. Conversely, a Fzd2 global knockout mouse model developed by the International Mouse Phenotype Consortium (IMPC) is early embryonic lethal, suggesting Fzd2 is critical for early embryonic development. If global deletion of Fzd2 leads to early lethality, floxed models are necessary to identify tissue-specific phenotypes. We found that a previously published Fzd2 flox model does not fully delete Fzd2 function. To reconcile the contradictory findings in Fzd2 mouse models and allow for tissue-specific studies of Fzd2, we have generated a new flox model using a modified two-cell homologous recombination CRISPR approach. We demonstrated successful simultaneous insertion of two loxP sites fully surrounding the Fzd2 gene and confirmed cre-mediated recombination deletes the sequence between the loxP sites leading to a Fzd2 null allele. Preliminary studies suggest global knockouts are early embryonic lethal and full characterization of the tissue-specific effects of Fzd2 deletion is currently underway. This work suggests Fzd2 uniquely regulates development and emphasizes the importance of thorough validation of newly generated mouse models.

cell biology↗