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

Abstract

Histone H3K9 methylation (H3K9me) by Setdb1 silences retrotransposons (rTEs) by sequestering them in 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 elucidated. 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.

Keywords: gene silencin; heterochromatin; histone methylation; transposable element (TE).

Figure 1

Setdb1 and Atf7IP interact through their coiled coil domains.A, domain architecture of Setdb1 and Atf7IP with the corresponding pcoils prediction for each protein. TTD, Triple Tudor Domain; MBD, methyl binding domain; pre, pre-set domain; Set, set domain; ps, post set domain; FN3, fibronectin type 3 domain. B, Alphascreen binding assay using Ni²⁺ and streptavidin beads showing interaction between his-sumo-Setdb1(2-115) and biotin-MBP-Atf7IP(574-666). Standard deviation of the data is shown as error bars. C, Alphascreen assay as shown in (B), but with increasing amounts of his-sumo-Setdb1 (closed circles) or increasing amounts of biotin-MBP-Atf7IP(574-666) (open circles). D, Superose 6 size exclusion chromatogram (top) showing elution of his-sumo-Setdb1(2-115), MBP-Atf7IP(574-666) and the his-sumo-Setdb1(2-115)/Atf7IP(574-666) complex. SDS-PAGE gels (bottom) of the elution fractions from the size exclusion run. E, SDS-page gel bands of purified his-Setdb1(11-104)/MBP-Atf7IP(585-666) and MBP-Setdb1(2-115)/his-sumo-Atf7IP(574-666) complexes.

Figure 2

Two molecules of Atf7IP bind to a single Setdb1.A, Alphafold2 prediction of the 1:1 Setdb1:Atf7IP complex. B, Alphafold2 prediction of the 1:2 Setbd1:Atf7IP complex. C and D, close up views of Atf7IP C631 and Setdb1 in the 1:1 and 1:2 Alphafold2 predictions. E, SDS-PAGE gel showing the results of cysteine crosslinking experiments with Setdb1 and Atf7IP Cys mutants. F, native Mass spectra of the intact MBP-Setdb1(2-115)/his-sumo-Atf7IP(574-666) complex. G, SEC-MALS trace of the intact MBP-Setdb1(2-115)/His-Sumo-Atf7IP(574-666) complex. H, SDS-PAGE analysis of fractions within peak one and peak two from the SEC-MALS experiment shown in (G).

Figure 3

Setdb1 and Atf7IP form a multimeric complex in cells.A, pulldown design that allows discrimination between dimer and multimer Setdb1/Atf7IP complexes. B and C, flag pulldowns from HEK293 cells expressing the indicated proteins. D, characteristic Alphafold2 prediction of the Atf7IP(574-666)/Atf7IP(574-666) complex showing poor accuracy (left), cysteine-cysteine crosslinking assay of a cysteine-free mutant of MBP-Atf7IP(574-666) containing only C631 with and without beta-mercaptoethanol (BME). E, gel filtration assay of MBP-Atf7IP (574-666) with and without Tween20. F, flag pulldowns from HEK293 cells expressing the indicated proteins.

Figure 4

ATF7IP binds to Setdb1 NES motifs and blocksCrm1binding.A, Alphafold prediction of the 1:2 Setdb1:Atf7IP heterotrimer. The location of the Setdb1 NES motifs are indicated and colored orange. B and C, top: close up views of the Setdb1 NES motifs showing detailed interactions between each NES and each copy of Atf7IP. Critical NES residues of Setdb1 and interacting residues of Atf7IP are shown as sticks. Bottom: Sequence of each Setdb1 NES motif with the critical hydrophobic NES residues indicated with ɸ. D, Alphascreen binding assay between MBP-Atf7IP(574-666) and his-sumo-Setdb1(2-114) with the indicates truncations. Standard deviation of the data is shown as error bars. E, GST pulldown assays showing competition for Setdb1 binding between Atf7IP and Crm1.

Figure 5

Atf7IP2 mimics Atf7IP heterotrimeric interaction with Setdb1.A, domain architecture of Atf7IP2 (top) with the corresponding pcoils prediction (bottom). FN3 = fibronectin type 3 domain. B, sequence alignment showing the similarity of coiled-coiled regions between Atf7IP and Atf7IP2. Interfacial residues in Atf7IP are highlighted in yellow. C and D, Alphafold2 predictions of the 1:1 Setdb1(2-114):Atf7IP2(297-388) complex and the 1:2 Setdb1(2-114):Atf7IP2(297-388) complex. E, sequential his and strep pulldown of the MBP-Setdb1(2-114)/his-sumo-Atf7IP2(297-388)/Strep-Atf7IP2(297-388) heterotrimer. F, Alphafold2 prediction of the Setdb1(2-114)/Atf7IP(574-666)/Atf7IP2(297-388) mixed trimer. G, sequential his and strep pulldown of the MBP-Setdb1(2-114)/his-sumo-Atf7IP(574-666)/Strep-Atf7IP2(297-388) mixed trimer. H and I, flag pulldowns from HEK293 cells expressing the indicated proteins.