Fig 2: Crosstalk between Spindlin1-HBx and DDB1-HBx engagements.a AlphaFold model of free HBx (left) and structure model of HBx binding to Spindlin1 (light magenta-green), DDB1 (cyan-gray, PDB ID 3I7H), and Bcl-2 (yellow-white, PDB ID 5FCG). b Schematic model of HBx transitioning from an inactive state to an active one by binding to Spindlin1, DDB1, and Bcl-2. c Co-IP of HA-HBx with endogenous DDB1 using anti-HA antibodies in HepG2-NTCP cells, which were transfected with Ctrl siRNA or Spindlin1 siRNA 1. d WB analyzing the SMC6 level in Spindlin1 knockdown HepG2-NTCP cells expressing HBx. Cells were transfected with Ctrl siRNA or Spindlin1 siRNA 1 for 72 h and then transfected with HA-HBx or empty vector. After 96 h, cells were harvest for WB analyzation. (c–d) n = 3 independent experiments. e A schematic model of Spindlin1-HBx mediated transcriptional derepression of cccDNA minichromosome. Chromatinized HBV genome undergoes epigenetic reprogramming from a repressive state to an active state via a poised intermediate state. The transcriptionally inactive HBV genome is typically silenced by host restriction factors, such as PML, Sp100, SMC5/6, H3K9 methyltransferases (H3K9MT), and HP1, which form a repressive chromatin state marked by H3K9me3 (left). To overcome the heterochromatin barrier, histone H3K4 methyltransferases (H3K4MT) can target H3K9me3-containing cccDNA chromatin to create bivalent H3 “K4me3-K9me3” modifications49. Spindlin1-HBx complex then binds to H3 “K4me3-K9me3” and competes off HP1 from the poised state chromatin (middle). Finally, Spindlin1 and HBx cooperatively establish an active chromatin state of HBV cccDNA in concert with transcription factor (TF) and co-activators such as CBP/p300 to promote HBV transcription (right). Source data are provided as a Source Data file.

Fig 3: AMBRA1 regulates ubiquitin-dependent degradation of aurora kinases in a context-dependent manner. A Complex structure prediction using AlphaFold2_mmseqs2 for AMBRA1WD40 with DDB1, cyclin D1, AURKA or AURKB. Top, cartoon illustration of predicted heteromeric protein complexes. Interchain AlphaFold2 contacts (< 8 Å) are shown as straight lines colored by predicted alignment error (PAE). AMBRA1 residues implicated in DDB1 binding are highlighted in green. Predicted template modeling scores (pTM) are indicated. Bottom, pairwise PAE scores for all protein complexes. B Heat map showing log2 fold changes of label-free quantification values from FLAG affinity purification and mass spectrometry detection in AMBRA-FLAG versus FLAG expressing ATRT cells. Cells were investigated with and without treatment of the CRL inhibitor MLN4924. C Co-immunoprecipitation analyses followed by western blot for selected potential AMBRA1 interactors and substrates. Previously identified interactors and substrates of AMBRA1 (CUL4A, DDB1, and cyclin D1) were included as controls. D Top: Immunoassays of cyclin D1, cyclin D3, AURKA, and AURKB for BT12 and CHLA06 cells, both in wildtype and AMBRA1-knockout conditions. Cells were treated with DMSO, 0.4 μM Baf-A1, or 1 μM MLN4924 for 4 h (cyclin D) or 12 h (aurora kinases). Increase in LC3B-II levels was used as a validation of autophagy inhibition. Bottom: Quantification of protein expression levels relative the corresponding DMSO control conditions. E Left: Immunoassays from His pull-down experiments for BT12 and CHLA266 cells, both in wildtype and AMBRA1-knockout conditions, transfected either with 6xHis-empty or 6xHis-tagged ubiquitin 48 h prior to pull-down. Right: Quantification of AURKA and AURKB ubiquitylation relative to total protein levels. F Model of context-dependent, CRL4AMBRA1-associated blockade of cell cycle regulators via degradation by the ubiquitin-proteasome system. Data are shown as mean ± SD (D, E). Statistics are derived from two-way ANOVA tests with Dunnett’s (D) or Sidak’s correction (E). *P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001

Fig 4: The effect of ANKRD11 overexpression on SETD5 expression and translation activity in ANKRD11-insufficient Neuro-2a cells(A) Immunoblot analysis (IB) of ANKRD11, SETD5, puromycin-incorporated newly synthesized proteins and DDB1 (loading control) in control (−) and ANKRD11-overexpressing (+) Neuro-2a cells with asterisks indicating nonspecific signals. ANKRD11 overexpression was induced by treatment with (1 μg/mL) doxycycline for 7 days in Neuro-2a cells containing a doxycycline-inducible ANKRD11 expression cassette.(B) RT-qPCR analysis of Setd5 mRNA levels in Ankrd11-disrupted Neuro-2a cells with (+) or without (−) ANKRD11 overexpression. Data are means ± SEM (n = 4 for each genotype). ∗∗∗∗p < 0.001 (Student’s t test).(C) Relative SETD5 protein levels measured as in Figure 6A. Data are means ± SEM (n = 4 independent experiments). ∗p < 0.05 (Student’s t test).(D) RT-qPCR analysis of pre-rRNA levels in Ankrd11-disrupted Neuro-2a cells with (+) or without (−) ANKRD11 overexpression. Data are means ± SEM (n = 4 for each genotype). ∗∗p < 0.01 (Student’s t test).(E) Relative translational activity measured as in Figure 6A. Data are means ± SEM (n = 4 independent experiments). ∗p < 0.05 (Student’s t test).

Fig 5: The effect of SETD5 overexpression on translation activity in ANKRD11-insufficient Neuro-2a cells(A) Immunoblot analysis (IB) of SETD5, ANKRD11, puromycin-incorporated newly synthesized proteins and DDB1 (loading control) in control (−) and SETD5-overexpressing (+) Neuro-2a cells with asterisks indicating nonspecific signals. SETD5 overexpression was achieved by treatment with (50 ng/mL) doxycycline for 5 days in Neuro-2a cells containing a doxycycline-inducible SETD5 expression cassette.(B) Relative translational activity measured as in Figure 5A. Data are means ± SEM (n = 4 independent experiments). ∗p < 0.05 (Student’s t test).(C) RT-qPCR analysis of pre-rRNA levels in Ankrd11-disrupted Neuro-2a cells with (+) or without (−) SETD5 overexpression. Data are means ± SEM (n = 4 for each genotype). ∗∗p < 0.01 (Student’s t test).(D) RT-qPCR analysis of Ankrd11 mRNA levels in Ankrd11-disrupted Neuro-2a cells with (+) or without (−) SETD5 overexpression. Data are means ± SEM (n = 4 for each genotype).(E) Relative ANKRD11 protein levels measured as in Figure 5A. Data are means ± SEM (n = 4 independent experiments).