Identification of high-confidence α-arrestin PPIs

(A) Phylogenetic tree of α-arrestins from human (6, top) and Drosophila (12, bottom) based on protein sequences. The numbers in parentheses indicate the length of each protein. aa, amino acids; Arr_N: Arrestin N domain; Arr_C: Arrestin C domain; PPxY: PPxY motif. (B) Shown is a schematic flow of AP/MS experiments and computational analysis. (C) ROC curves of SAINTexpress scores along with mean AUC values. The arrows point to the cutoff scores used in subsequent studies in human (left) and Drosophila (right). (D) (Top) The fraction of “high-confid” (high-confidence) and raw (unfiltered) PPIs that are supported by known affinities between short linear motifs and protein domains in human (left) and Drosophila (right). One-sided, Fisher’s exact test was performed to test the significance. (Bottom) The sum of log2 spectral counts (“log2 spec”) of proteins with WW domains that were reported to interact with each α-arrestin in the high-confidence or raw PPI sets are depicted as heatmap. (E) The α-arrestins and their interactomes were hierarchically clustered based on the log2 mean spectral counts and summarized for human (top) and Drosophila (bottom) in the heatmaps. The functionally enriched protein class in the clustered interactomes are indicated on the top. Proteins that were reported to interact with α-arrestins in literatures and databases are selectively labeled on the bottom. On the right, the functional composition of the clustered α-arrestin interactomes are summarized as the sum of log2 mean spectral counts, which are colored to correspond with the labels on the left.

Network of α-arrestins and their associated protein complexes

Network of α-arrestins and the functional protein complexes that significantly interact with them in human (A) and Drosophila (B). α-arrestins are colored yellow and prey proteins in protein complexes are colored according to the SAINTexpress scores (averaged if the protein interacts with multiple α-arrestins). The thickness of the green arrows indicates the strength of the interaction between α-arrestins and the indicated protein complexes, which was estimated with FDR of complex association scores (see “Materials and Methods”). UB, ubiquitination; HDAC, histone deacetylase; COMPASS, complex proteins associated with Set1; SMN, survivor of motor neurons; TFIIIC, transcription factor III C; RNA polII, RNA polymerase II; MCM, minichromosome maintenance protein complex; SAC, spindle assembly checkpoint; NSL, non-specific lethal; WASH, Wiskott-Aldrich syndrome protein and scar homolog; Arp2/3, actin related protein 2/3; TEF, transcription elongation factor.

A substantial fraction of α-arrestin-PPIs are conserved across species

Human and Drosophila α-arrestins and their orthologous interactomes are hierarchically clustered based on log2-transformed mean spectral counts. They are then manually grouped based on their shared biological functions and assigned distinct colors. The names of orthologous proteins that interact with α-arrestins are displayed on the right side of the heatmap.

TXNIP knockdown induces a global decrease in chromatin accessibility and gene expression

(A-B) HeLa cells were treated with either siRNA against TXNIP (siTXNIP) or negative control (siCon) for 48 hours (hr) and analyzed of changes in the mRNA (A) and protein levels (B) of TXNIP. Gray dots depict actual values of each experiment and bar plots indicate mean ± standard deviation (sd). ***FDR < 0.001 (see “Materials and Methods”) for RNA-seq. *P < 0.05, *** P < 0.001 (two-sided paired Student‘s t-test) for RT-qPCR and western blots. (A) Expression levels of RNAs were quantified by RNA-seq (left, log2 counts per million mapped reads (CPM), see “Materials and Methods”) and RT-qPCR. (B) Protein levels were first visualized by western blot analysis of lysates from HeLa cells and band intensities of three independent experiments were quantified using image J software (right). (C) A schematic workflow for detecting dACRs and DEGs using ATAC- and RNA-seq analyses, respectively. (D) Volcano plots of differential chromatin accessibility for all ACRs (left) and those associated with promoters (right). (E) Volcano plots of differential gene expression. (D-E) Blue dots denote “dACRs(-)” of significantly decreased chromatin accessibility (D) and “Down” genes of significantly down-regulated genes (E) in siTXNIP-treated cells compared to control (FDR ≤ 0.05, log2(siTXNIP / siCon) ≤ −1); red dots denote “dACRs(+)” of significantly increased chromatin accessibility (D) and “Up” genes of significantly up-regulated genes (E) in siTXNIP-treated cells compared to control (FDR ≤ 0.05, log2(siTXNIP / siCon) ≥ 1). Black dots denote data points with no significant changes. (F) Changes in chromatin accessibility of ACRs located in the promoter region of genes were plotted as CDFs. Genes were categorized into three groups based on changes in RNA levels (“Up”, “Down” as in (E) and “None” indicating genes with −0.5 ≤ log2(siTXNIP / siCon) ≤ 0.5). The number of genes in each group are shown in parentheses and P values in the left upper corner were calculated by one-sided KS test. (G) Top 10 GO terms (biological process and molecular function) enriched in genes that exhibited decreased chromatin accessibility at their promoter and decreased RNA expression upon TXNIP knockdown (Table S11).

TXNIP directly represses the recruitment of HDAC2 to target loci

(A) Co-IP assay showing the interaction between TXNIP and HDAC2 proteins. Lysates from HeLa cells that had been treated with either siCon or siTXNIP for 48 hr were subjected to IP and immunoblotting with antibodies recognizing TXNIP and HDAC2. IgG was used as the negative control. (B) Nuclear and cytoplasmic fractions of HeLa cells were analyzed with western blots following transfection with siCon or siTXNIP for 48 hr (left). Lamin B1 and GAPDH were used as nuclear and cytoplasmic markers, respectively. Western blot results from three independent experiments for TXNIP and HDAC2 were quantified as in Figure 4B. C, cytoplasm; N, nucleus. (C) Genomic regions showing RNA expression and chromatin accessibility at CD22 and L1CAM gene loci (top). Through the ChIP-qPCR analysis, the fold enrichment of HDAC2 and histone H3 acetylation (H3ac) at the CD22 and L1CAM promoter regions in HeLa cells treated with either siCon or siTXNIP for 48 hr were quantified (bottom). Data are presented as the mean ± sd (n=3, biological replicates). Gray dots depict actual values of each experiment. *P < 0.05, **P < 0.01, ns: not significant (two-sided paired Student’s t-test).

Interaction of ARRDC5 with the V-type ATPases in osteoclasts

(A) The human ARRDC5-centric PPI network. V-type and P-type ATPases, their related components, and extracellular exosomes are labeled and colored. Other interacting proteins are indicated with gray circles. (B) TRAP staining of osteoclasts. Cell differentiation was visualized with TRAP staining of GFP-GFP or GFP-ARRDC5 overexpressing osteoclasts (scale bar = 500 μm). TRAP-positive multinucleated cells (TRAP+ MNC) were quantified as the total number of cells and the number of cells whose diameters were greater than 200 μm. * P < 0.05. (C) Resorption pit formation on dentin slices. Cell activity was determined by measuring the level of resorption pit formation in GFP-GFP or GFP-ARRDC5 overexpressing osteoclasts (scale bar = 200 μm). Resorption pits were quantified as the percentage of resorbed bone area per the total dentin disc area using ImageJ software. The resorption area is relative to that in dentin discs seeded with GFP-GFP overexpressing osteoclasts, which was set to 100%. The colors of the bar plots are same as in (B). ** P < 0.01. (D) Relative mRNA levels of ARRDC5 in non-target control (Control) or shARRDC5-expressing osteoclasts (shARRDC5) measured by qPCR. * P < 0.05 (Student’s t-test, one-sided). (E) TRAP staining of osteoclasts. Cell differentiation was visualized with TRAP staining of “Control” or “shARRDC5” expressing osteoclasts (scale bar = 500 μm, left). TRAP-positive multinucleated cells (TRAP+ MNC) were quantified as the total number of cells (right). Colors of the bar plots are same as in (D). *** P < 0.001 (Student’s t-test). (F) The protein level of ATP6V1 in GFP-GFP or GFP-ARRDC5 overexpressing osteoclasts. The numbers represent independent samples for western blot analysis (left) and band intensities of three independent experiments were quantified (right). Colors of the bar plots are same as in (B) and (C). ns, not significant. (G) Localization of ARRDC5 and V-type ATPase V1 domain subunit (ATP6V1) in osteoclasts. ATP6V1 was visualized with immunofluorescence (red), GFP-GFP and GFP-ARRDC5 were visualized with GFP fluorescence (green), and nuclei were visualized with DAPI (blue). Representative fluorescence images are shown (scale bar = 100 μm). The region of interest, marked by the red boxes, was high-magnified and presented below. The integrated density of fluorescence was quantified using Image J software and expressed as relative fluorescence (right). The integrated density of fluorescence in GFP-GFP osteoclasts was established as the reference value, which was set to 1. * P < 0.05, ** P< 0.01 (Student’s t-test).