Fig 2: USF2 represses lysosomal genes along with NuRD complex through H3K27 deacetylation.a Flowchart of the experiment to identify USF2-binding proteins. b Visualization of the binding network of USF2 binding partners obtained through LC-MS/MS using the STRING database. c Results of the local network cluster analysis in STRING using USF2 binding partners. d Heatmap depicting the enrichment of NURD complex components at USF2 binding sites. Each row indicates a 6 kb window centered on a USF2 binding site. e Binding between USF2 and NuRD complex subunits. Immunoprecipitation assay was performed by pulling down USF2, followed by immunoblotting with anti-HDAC1, anti-HDAC2, and anti-MTA1 antibodies to detect the endogenous protein expression levels. The representative images supported by the relevant statistics have been chosen from three independent preparations with similar outcomes. f Read density plots for ChIP-seq peaks of H3K27Ac in WT and Usf2−/− MEFs. g Visualization of USF2 and H3K27Ac ChIP-seq peaks, and ATAC-seq signals in USF2 target genes. h ChIP assays on USF2-dependent promoters in WT and Usf2−/− MEFs. n = 3 technical replicates. Statistics by two-tailed t-test using WT and Usf2−/− MEFs as a comparison. i Schematics of the repression mechanism of USF2-NuRD complex. Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Source data are provided as a Source Data file.

Fig 3: Phosphorylation of USF2 at S155 by GSK3β enhances DNA-binding activity.a Immunoblot analysis in the presence or absence of λ-phosphatase treatment in WT MEFs. b Schematic representation of well-characterized phosphorylation sites on USF2 and the associated kinases responsible for this modification. c Immunoblot analysis using phos-tagTM gel conducted after reconstituting WT, S155A, S222A, and T230A mutants in Usf2−/− MEFs. d ChIP assay on the promoters of lysosomal genes following the reconstitution of WT and S155A mutant in Usf2−/− MEFs. n = 3 technical replicates. Statistics by two-tailed t-test using USF2 WT and S155A mutant expressed Usf2−/− MEFs as comparison. e qRT-PCR assay of lysosomal genes conducted after reconstituting mock, WT, and S155A mutant in Usf2−/− MEFs. n = 3 technical replicates. Statistical analysis performed using a two-tailed t-test. Mock and USF2 S155A mutant rescued cells were individually compared to USF2 WT rescued cells. f Immunoblot analysis of lysosomal proteins conducted after reconstituting mock, WT, and S155A mutant in Usf2−/− MEFs. g Representative images of Lysotracker staining. Lysotracker assay was performed after reconstituting mock, WT, and S155A mutant in Usf2−/− MEFs. Lysotracker, red; Hoechst, blue. Scale bar, 20 μm. h Quantification of Lysotracker intensity per cell. Lysotracker assay was performed after reconstituting mock, WT, and S155A mutant in Usf2−/− MEFs. n = 18 biologically independent samples. Statistical analysis performed using a two-tailed t-test. Mock and USF2 S155A mutant rescued cells were individually compared to USF2 WT rescued cells. i Immunoblot analysis using phos-tagTM gel under normal and LiCl treated conditions at different time points. j Immunoblot analysis using phos-tagTM gel under normal, GSK3β-overexpressed, and LiCl treated conditions. k ChIP assay on the promoters of lysosomal genes in WT MEFs under normal and LiCl treated condition. n = 3 technical replicates. Statistics by two-tailed t-test using normal and LiCl treated WT as comparison. l Immunoblot analysis using phos-tagTM gel for USF2 immunoblotting under normal and GS conditions. m Immunoblot analysis under normal and GS conditions in WT MEFs. n Representative confocal microscopic images using GSK3β Ser9 phosphorylation antibody under normal and GS conditions. p-GSK3β (Ser9), green; DAPI, blue. Scale bar, 10 μm. o Schematics of the regulation of autophagy and lysosome genes by USF2 and TFEB under nutrient-rich or deficient condition. Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Figure 6/panel o Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Fig 4: Inhibition of USF2 stimulates clearance of α1-Antitrypsin Mutant Z.a Immunofluorescence staining to detect the expression of lysosomal proteins, such as Lamp1, in 16.5-day-old WT and Usf2−/− embryos. Lamp1, green; Hoechst, blue. Scale bar, 1 mm. b Immunoblot analysis of GFP-ATZ following the transfection of siNS or siUSF2 into HepG2 cell lines stably expressing GFP-ATZ (GFP-ATZ O/E HepG2). c Representative confocal microscopic images of GFP-ATZ in cells as in b. Scale bar, 10 μm. d Immunoblot analysis of GFP-ATZ O/E HepG2 cell lines with and without USF2 knockdown and in the presence or absence of Bafilomycin A1 (BafA1) treatment. e Quantification of GFP-ATZ protein levels relative to β-actin. n = 3 biologically independent samples. Statistical analysis performed using a two-tailed t-test. siNS and siUSF2+BafA1 cells were individually compared to siUSF2 cells. f Representative confocal microscopic images of GFP-ATZ O/E HepG2 cell lines with and without USF2 knockdown and in the presence or absence of BafA1 treatment. ATZ, green; Lamp1, red; DAPI, blue. Scale bar, 10 μm. g Quantification of GFP-ATZ intensity. n = 7 biologically independent samples. Statistical analysis performed using a two-tailed t-test. siUSF2, siNS+BafA1 and siUSF2+BafA1 cells were individually compared to siNS cells. h Quantification of co-localization of Lamp1 and GFP-ATZ. n = 3 biologically independent samples. Statistical analysis performed using a two-tailed t-test. siUSF2 treated cells were compared to siUSF2+BafA1 treated cells. i Immunoblot analysis of GFP-ATZ O/E HepG2 cell lines after overexpression of TFEB and knockdown of USF2. j Quantification of GFP-ATZ protein levels relative to β-actin. k Representative confocal images of GFP-ATZ O/E HepG2 cell lines after overexpression of TFEB and knockdown of USF2. Scale bar, 10 μm. l Quantification of GFP-ATZ intensity. n = 5 biologically independent samples. Statistical analysis performed using a two-tailed t-test. siNS, siNS+HA-TFEB cells were individually compared to siUSF2+HA-TFEB cells. m Schematics of therapeutic strategies for α1-antitrypsin deficiency by enhancing autophagy and lysosomal function through the inhibition of USF2 and the activation of TFEB. Data are presented as mean ± SEM. **, p < 0.01; ***, p < 0.001. Figure 7/panel m Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Fig 5: USF2 represses the biogenesis of functionally mature lysosomes.a A schematic that illustrates a screening process to identify lysosome-associated transcription factors and the corresponding transcription factor (TF) enrichment ranks from the ENCODE TF ChIP-seq database. b TF enrichment rank plot in differentially expressed genes. c TF enrichment rank plot in the term of lysosomal biogenesis d Venn diagram illustrating the overlapping enriched TFs related to lysosomes. e Immunoblot analysis of USF2 and Lamp1 expression in shNS HepG2 and shUSF2 HepG2 cell lines. f Representative images depict Lysosensor staining in shNS HepG2 and shUSF2 HepG2 cell lines. These images were captured using a confocal microscope under identical settings. The white guidelines indicate the cell boundaries. Lysosensor, green; Hoechst, blue. Scale bar, 20 μm. g Quantification of lysosomal number per cell and diameter per lysosome in shNS HepG2 and shUSF2 HepG2 cell lines. n = 4 biologically independent samples. Statistical analysis was performed using a two-tailed t-test. shUSF2 HepG2#1 and shUSF2 HepG2#2 cell lines were individually compared to shNS HepG2. h Schematic drawing of the generation of Usf2 whole-body knockout mice. i Summary of genotyping results for the offspring of Usf2 heterozygous crosses. “Expected” represents the theoretical number of offspring expected based on the Mendelian ratio for an analysis of similar size. The graph on the right represents the “Observed” in the table. j Representative TEM images of WT and Usf2−/− MEFs. Scale bar, 2 μm. High magnification of the boxed areas is shown on the right. Lysosomes (red arrows). k Immunoblot analysis of USF2 and Lamp1 expression in WT and Usf2−/− MEFs. l Representative confocal images of Lysosensor staining in WT and Usf2−/− MEFs. m Quantification of lysosomal number per cell and diameter per lysosome in WT and Usf2−/− MEFs. n = 4 biologically independent samples. Statistics by two-tailed t-test using WT and Usf2−/− MEFs as a comparison. n Representative confocal images of DQ-BSA staining. WT and Usf2−/− MEFs were treated with DQ-BSA. DQ-BSA, red; Hoechst, blue. Scale bar, 20 μm. o Quantification of DQ-BSA intensity per cell in WT and Usf2−/− MEFs. n = 6 biologically independent samples. Statistics by two-tailed t-test using WT and Usf2−/− MEFs as a comparison. p Analysis of the activity of lysosomal cathepsin D in WT and Usf2−/− MEFs. n = 2 biologically independent samples. Statistics by two-tailed t-test using WT and Usf2−/− MEFs as a comparison. q Immunoblot analysis of shNS, shUSF2, and USF2 (GFP-USF2) reconstituted shUSF2 HepG2 cell lines (left), and WT, Usf2−/−, and USF2 reconstituted Usf2−/− MEFs (right). r Representative images of Lysotracker staining in shUSF2 HepG2 cell line and Usf2−/− MEFs reconstituted with GFP-USF2. Lysotracker, red; GFP, green; Hoechst, blue. Scale bar, 10 μm. s Quantification of Lysotracker intensity per cell in shUSF2 HepG2 cell line and in Usf2−/− MEFs. n = 9 biologically independent samples for HepG2 cells and n = 5 biologically independent samples for MEFs. Statistics by two-tailed t-test using shNS and shUSF2 HepG2 cell line or WT and Usf2−/− MEFs as each comparison. Data are presented as mean ± standard error of the mean (SEM). *, p < 0.05; **, p < 0.01; ***, p < 0.001. Source data are provided as a Source Data file.