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 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-confidence and all 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 interacting proteins with WW domains observed in the high confidence and all raw PPIs are visualized in the heatmap. (E) The α-arrestins and interacting prey proteins 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 groups of preys are indicated at the top. Previously reported proteins interacting with α-arrestins are labeled at the bottom. On the right, the functional composition of prey groups is summarized with the sum of log2 mean spectral counts of each prey group, which are colored to correspond with the labels on the left.

Network of α-arrestins and their interacting 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 of the PPIs. The gray edges indicate that evidence supporting the complex was provided by COMPLEAT and/or GO cellular components. The thickness of the green arrows indicates the strength of the interaction between α-arrestins and the indicated protein complexes, which was estimated with -log10 FDR of complex association scores. 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 are hierarchically clustered based on log2-transformed mean spectral counts of their orthologous interactome. They are then manually grouped according to 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. (A) Expression levels of RNAs were quantified by RNA-seq (left, log2 counts per million mapped reads (CPM), see “Processing of RNA-seq data” in “Materials and Methods”) and RT-qPCR (right, relative levels of TXNIP in siTXNIP compared to siCon condition, see “Quantitative Reverse-transcription PCR” in Supplementary Information). (B) Protein levels were first visualized by western blot analysis of lysates from HeLa Cells and band intensities of three independent experiments were quantified (right). (A-B) Gray dots depict actual values of each experiment and bar plots indicate mean ± standard deviation (sd). ***FDR < 0.001 (test of differential expression by edgeR (Robinson et al., 2010), see “Processing of RNA-seq data” in “Materials and Methods”) for RNA-seq. *P < 0.05, *** P < 0.001 (two-sided paired Student T test) for RT-qPCR and western blots. (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 (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 (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) Analysis of co-IP between the 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, with IgG 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 T test).

Interaction of ARRDC5 with the V-type ATPase 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%. ** P < 0.01. (D) Localization of Arrdc5 and the V-type ATPase in osteoclasts. The V-type ATPase 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. Dashed lines were used to outline representative osteoclasts (scale bar = 100 μm).

Fluorescence images showing HEK293 and S2R+ cells stably expressing GFP tagged α-arrestins

Representative images of HEK293 (A) and S2R+ (B) cells stably expressing GFP-tagged α- arrestins.

Affinity purification / mass spectrometry (AP/MS) data are highly reproducible and recapitulate known PPIs

(A) Average Pearson correlation coefficients of log2 spectral counts between replicates of AP/MS of each α-arrestin at varying cutoffs are shown (mean ± standard deviation(sd)). The cutoff used in this study, 6, is shown as a dashed line. (B) PCA plots based on log2 spectral counts of high-confidence PPIs for human (upper) and Drosophila (lower) are shown. (C) SAINTexpress scores and average spectral counts (log2) of the positive PPIs (Table S2A and C) are shown and density plots for each axis are also plotted. The positive PPIs that are included in the filtered set are selectively labeled.

Protein domains and subcellular localization of α-arrestin interactomes

(A) Protein domains enriched in each α-arrestin interactome for human (top) and Drosophila (bottom) are shown. The significance of the enrichment test (-log10 FDR) is indicated in shades of green, as depicted in the legend. SPOC, spen paralogue and orthologue C-terminal; MCM, minichromosome maintenance protein complex; FDRM, F for 4.1 protein, E for ezrin, R for radixin and M for moesin; TBP, TATA binding protein; GEF, guanine nucleotide exchange factor; THRAP3, thyroid hormone receptor-associated protein 3; BCLAF1, Bcl-2-associated transcription factor1; RMMBL, RNA metabolising metallo beta lactamase; CaMKII, C-terminus of the Calcium/calmodulin dependent protein kinases II; CPSF, cleavage and polyadenylation specificity factor; DCB, dimerization and cyclophilin-binding domain; FRAP, FKBP12-rapamycin complex-associated protein; ATM, ataxia telangiectasia mutant; THRAP, transformation/transcription domain associated proteins; MIF4G, middle domain of eukaryotic initiation factor 4G; AAA, ATPase family associated with various cellular activities; C4, C terminal tandem repeated domain in type 4 procollagen; SMC, structural maintenance of chromosomes. (B) Subcellular localizations of prey proteins of each α-arrestin for human (left) and Drosophila (right).

Phylogenetic tree showing relationships between α-arrestins from human and Drosophila

Phylogenetic tree of α-arrestins from both species based on protein sequences were drawn as in Figure 1A.

ATAC- and RNA-seq results are highly reproducible and ATAC-seq results exhibit a pattern typical of strong enrichment around TSSs of expressed genes

(A and B) PCA plots of ATAC- (A) and RNA-seq (B) results based on batch-corrected log2 counts and CPM, respectively. Numbers in parentheses are percentages of explained variance for the corresponding PCs. (C) Heatmaps of ATAC-seq read counts (read counts have been transformed into a log2 function and corrected for batch effects) in regions surrounding TSSs along with log2 (RNA level in siTXNIP-treated cells/RNA level in siCon-treated cells) for genes having the corresponding TSS are plotted for each sample.

Genomic locations of ACRs and association between chromatin landscapes and transcriptional activity

(A) Genomic locations of 70,746 consensus ACRs identified from ATAC-seq analysis. (B) Composition of dACRs(-), dACRs(+), and other ACRs (“others”, not significantly changed) under the TXNIP knockdown condition compared to the control. (C) Genomic locations of 4,825 dACRs(-) and 394 dACRs(+) are depicted. Colors in the bar plot have the same symbolism as in (A). (D) Cumulative distribution function (CDF) of mean changes in accessibility of all ACRs located in gene promoters. The genes were categorized into three groups (“None”, “Down”, and “Up”) as explained in Figure 4F. P values on the left upper corner were calculated with the one sided Kolmogorov-Smirnov (KS) test comparing “Down” or “Up” groups to the “None” group. (E) CDF of changes in accessibility of ACRs located in gene bodies. Changes in accessibility of ACRs whose intensity is highest among all ACRs located in gene bodies are depicted on the left and mean changes in accessibility of all ACRs located in gene bodies are depicted on the right. P values on the upper left corners are calculated in the same manner as in (D).

TXNIP depletion does not affect the protein level or subcellular localization of HDAC2 but represses transcription of target genes

(A) Representative immunofluorescence images of TXNIP and HDAC2 after HeLa cells were transfected with either siCon or siTXNIP for 48 hr (magnification ×600); TXNIP (red), HDAC2 (green), and DAPI (blue). (B) RT-qPCR results of four target genes whose RNA expression and chromatin accessibility in their promoters, quantified using high-throughput sequencing data, were observed to be strongly repressed in HeLa cell. Data are presented as the mean ± sd, n=3). Gray dots depict actual values of each experiment. *P < 0.05, ns: not significant (two sided paired Student T test).

Summary of ATAC- and RNA-seq read counts before and after processing. For ATAC-seq, the number of properly paired reads, filtered/deduplicated reads, and identified narrow peaks are summarized. For RNA-seq, the number of filtered and alignable reads are summarized. *Filtered/dedup reads, filtered/deduplicated reads

List of primer sequences used in this study.