Fig 2: SAMD1 can polymerize in vitro and in vivo. (A) Representative immunofluorescence image of overexpressed SAMD1 in HEK293 cells. (B) Negative-staining EM image of full-length SAMD1. (C) Representative immunofluorescence image of overexpressed SAMD1 ΔSAM, SAMD1 ΔIDR, and SAMD1 WHmut (R45A/K46A) in HEK293 cells. (D) Schematic representation of deletion constructs used for experiments. FL = full length. (E, F) Representative immunofluorescence images of overexpressed SAMD1 IDR deletion constructs in HEK293 cells. (G) Quantification of fiber formation of panels (E) and (F). Shown is the relative number of transfected cells displaying fibers. Data represent mean ± SD of three biological replicates. Significance was evaluated using a two-way ANOVA with Dunnett’s multiple comparisons test, comparing SAMD1 mutant constructs to wild-type (WT) SAMD1. (***P < .001; **P < .01; *P < .05). (H) Negative-staining EM of SAMD1 ΔSAM, SAMD1-R498A/K514A, and SAMD1 ΔPA. Scale bar in panels (A), (C), (E), and (F) 10 μm. Cells in panels (A), (C), (E), and (F) were stained with an anti-HA antibody (green) and DAPI (blue).

Fig 3: Structural details of the interaction between L3MBTL3–SAM and SAMD1–SAM. (A) Detailed hydrogen-bonding interactions between L3MBTL3–SAM and SAMD1–SAM (PDB: 8Y77). (B) Electrostatic representation of the binding surfaces of SAMD1–SAM and L3MBTL3–SAM. (C) Hydrophobic interactions between SAMD1–SAM and L3MBTL3–SAM. (D) A potential model of the wild-type L3MBTL3–SAM/SAMD1–SAM complex that contains the self-associated L3MBTL3–SAM and SAMD1–SAM polymers. To generate the model, one molecule of the SAMD1–SAM pentamer and one molecule of the L3MBTL3–SAM polymer were superimposed with their corresponding molecules in the binary complex of SAMD1–SAM/L3MBTL3–SAM.

Fig 4: Role of PA-rich region for chromatin binding. (A) Reporter gene assay at CGI-driven luciferase genes (see Fig. 3A). (B) Western blot and (C) SAMD1 ChIP-qPCR of SAMD1 KO mouse ES cells [6] rescued with various SAMD1 constructs. (D) SAMD1 and L3MBTL3 ChIP-qPCR after SAMD1 rescue in SAMD1 KO mouse ES cells. (E) Western blot of SAMD1 KO mouse ES cells rescued with various SAMD1 constructs. (F) Quantification and (G) heatmap and profiles of SAMD1 ChIP-seq results at SAMD1-bound regions. See also Supplementary Fig. S9B. Statistical analysis in panel (F) was performed using a Kolmogorov–Smirnov test. Data in panels (A), (C), and (D) are represented as the mean of three biological replicates ± SD. The P-values were calculated using a two-way ANOVA with Dunnett’s test for multiple comparison (***P < .001; **P < .01; *P < .05).

Fig 5: Comparison of L3MBTL3 SAM and SAMD1 SAM domain polymer. (A) Mammalian-two-hybrid using SAM domains of SAMD1, L3MBTL3, and SFMBT1 [6]. (B) Top and side views of the SAMD1–SAM pentamer (PDB: 6LUJ) [6]. (C) Side and top views of the L3MBTL3–SAM polymer (PDB: 8Y76). (D) Details of the hydrophilic interactions between two self-associated L3MBTL3 SAM domains. Two molecules are colored in green and blue, respectively. (E) Electrostatic surfaces of the EH and ML surfaces of L3MBTL3–SAM. (F) Details of hydrophobic contacts between EH and ML surfaces of L3MBTL3–SAM. (G) Structural comparison of L3MBTL3 SAM and SAMD1 SAM domain polymers. Left shows the overlapped structures of one molecule each from L3MBTL3 SAM (green) and SAMD1 SAM (gray). Right shows the closely associated molecules of L3MBTL3 SAM (blue) and SAMD1 SAM (gray) from their polymers. (H) Overlapped gel-filtration profiles of the wild-type L3MBTL3–SAM and its E733A/D736A mutant. Profiles of standard proteins Conalbumin and Ribonuclease A are shown as references.