Fig 1: ASS1 is repressed in T cells(A) ATAC-seq at ASS1 in THP1 cells incubated in the indicated media for 72 h. ATF4 ChIP-seq from -Arg cells is shown for comparison. Bottom: overlay of ATAC-seq traces at the highlighted region of ASS1, mean of three replicates.(B) ATAC-seq at ASS1 in stimulated T cells, as in (A).(C) ChIP-qPCR for H3K9me3, H3K27me3, and H3K4me3 in stimulated T cells and THP1 cells incubated in the indicated media for 72 h. Data are represented as mean ± SEM; n = 4.(D) ChIP-qPCR for H3K9me3, H3K27me3, and ATF4 in stimulated T cells incubated for 72 h in complete medium (+Arg), medium containing 20 µM arginine, without (low Arg) or with (low Arg + 2HG) addition of 500 µM 2HG, or lacking arginine (-Arg). Data are represented as mean ± SEM; n = 4.(E) Representative western blot for ASS1 and ATF4 in stimulated T cells incubated in the indicated media for 72 h. Non-specific bands are indicated by an asterisk. Right: quantification, normalized to GAPDH, relative to +Arg. Data are represented as mean ± SEM; n = 5. **p < 0.01, ****p < 0.0001 (Dunnett’s multiple comparison test).(F) Model for ASS1 regulation in T cells and THP1 cells in response to arginine depletion. In THP1 cells accessibility at ASS1 allows ATF4 binding under low and -Arg conditions, inducing ASS1 expression. In T cells, ATF4 binding and ASS1 expression are regulated by two competing processes: ATF4 is active under low or -Arg conditions, but the ASS1 promoter is repressed. Under -Arg, elevated H3K9me3/H3K27me3 and reduced accessibility at ASS1 block ATF4 binding.See also Figure S4.
Fig 2: ASS1 upregulation is a common tumor response to arginine starvation(A) Growth of tumor cell lines in the indicated media. AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ALL, acute lymphoblastic leukemia. Data are represented as mean ± SD; n = 4.(B) qRT-PCR for ASS1 in the indicated cell lines, cultured in +Arg and -Arg media. Data are normalized to GAPDH, relative to +Arg in each cell line, represented as mean ± SD; n = 4 or 6 (HeLa). ***p < 0.001, ****p < 0.0001 (Šidák’s multiple comparison test).(C) Representative western blot for ASS1 and ATF4 in control (NT) HeLa cells or following KD of ASS1 or ATF4 in the indicated media for 72 h. (Right) Quantification, normalized to GAPDH, relative to +Arg NT cells. Data are represented as mean ± SEM; n = 3. *p < 0.05 (Dunnett’s multiple comparison test).(D) Growth of control (NT) HeLa cells or following KD of ASS1 or ATF4, incubated in the indicated media. Data are represented as mean ± SD; n = 3.(E) ChIP-qPCR for ATF4 and CEBPß levels in THP1 and HeLa cells incubated in the indicated media for 72 h. Data are represented as mean ± SEM; n = 3.(F) ChIP-qPCR for H3K9me3 and H3K27me3 in HeLa cells incubated in the indicated media for 72 h. Data are represented as mean ± SEM; n = 3.See also Figure S5.
Fig 3: ATF4-induced ASS1 upregulation facilitates citrulline-dependent growth of THP1 cells(A) Key proteins in arginine uptake and biosynthesis.(B) Forest plot showing changes in gene expression, based on microarray analysis (see Figure 1D). Horizontal bars show interquartile range.(C) ASS1 and ATF4 expression in primary AML blasts or non-transformed monocytic and myelocytic cells from healthy donors (Quek et al., 2016). Samples are colored by donor. Bars show mean ± SD. **p < 0.01, ***p < 0.001 (Mann-Whitney test).(D) Western blot for ASS1 and ATF4 in stimulated T cells and THP1 cells incubated for 72 h in complete medium (+), medium containing 20 µM arginine (low), or lacking arginine (-). Two exposures of the ASS1 blot are shown for clarity. Representative of five replicates.(E) Quantification of (D), normalized to GAPDH, relative to +Arg T cells. Expression in T cells is shown on a smaller scale for clarity. Data are represented as mean ± SEM; n = 5. *p < 0.05, **p < 0.01, ***p < 0.001 (Dunnett’s multiple comparison test). ns, not significant.(F) Growth of control (NT) THP1 cells or following KD of ASS1 or ATF4 in the indicated media. Data are represented as mean ± SD; n = 4.(G) Representative western blot for ASS1 and ATF4 in control (NT) THP1 cells or following KD of ASS1 or ATF4 in the presence (+) and absence (-) of arginine for 72 h. Right: quantification, normalized to GAPDH, relative to +Arg NT cells. Data are represented as mean ± SEM; n = 3. *p < 0.05 (Dunnett’s multiple comparison test).(H) CTV-labeled CD8+ T cells were transduced with a GFP-ASS1 coexpression plasmid. Cells were analyzed for GFP and CTV levels after 96 h in the indicated media. Three technical replicates are shown.(I) Proportion of GFP-positive cells from the analysis in (H), normalized to the -Arg ratio. Data are represented as mean ± SD; n = 5 from two donors. **p < 0.01 (paired t test).See also Figure S2.
Fig 4: IFNAR1 Contributes to Infection-Induced Metabolic Reprogramming of Hepatocytes(A) Depiction of the urea cycle, expression, and concentrations of the associated genes and serum metabolites in naive and LCMV-clone-13-infected Alb-Cre ERT2 Ifnar1fl/fl (Ifnar1?/?) and Ifnar1+/+ mice (n = 3).(B) Uniform Manifold Approximation and Projection (UMAP) clustering of single-cell RNA-seq data of primary hepatocytes sorted from naive or infected wild-type animals 2 days after LCMV infection.(C and D) Expression levels and correlation with Ifit1 levels (IFN-I-stimulated gene) of (C) Otc and (D) Ass1 in hepatocytes isolated from naive versus infected animals (n = 2; pooled for each condition). Each dot represents a single hepatocyte.(E) Representative immunohistochemical staining of OTC, ASS1, and STAT1 in liver sections of naive or LCMV-infected (2 and 8 days after infection) wild-type mice.(F) Quantification of immunohistochemical staining of OTC, ASS1, and STAT1 using HistoQuest software (n = 4 and 8 pictures per mouse were quantified).(G) Expression of Otc and Ass1 in sorted primary hepatocytes isolated from naive and LCMV-infected (2 and 8 days after infection) mice (n = 3).For metabolite data in (A), one of two representative experiments is shown. For (B)–(G), single-cell transcriptomic and histological data are derived from one experiment. Symbols represent the arithmetic mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t test). Log 2 fold changes and adjusted p values for single-cell RNA-seq data were computed as described in the STAR Methods. See also Figure S4.
Fig 5: ATF4 activates ASS1 transcription via an intronic enhancer(A) Reference-normalized ChIP-seq for ATF4 and CEBPß at ASS1 in stimulated T cells and THP1 cells incubated in the indicated media for 72 h, and ChIP-seq for H3K4me3, H3K27ac, and H3K4me1 in THP1 cells in +Arg medium (Godfrey et al., 2019). Gray bars show qPCR primer locations.(B) Reference-normalized ChIP-seq for ATF4 and CEBPß at SLC7A1, as in (A).(C) Sequences of the enhancer region in parental (wild type [WT]) and mutant THP1 cells. PAM sequences are underlined.(D) ChIP-qPCR for ATF4 and H3K27ac in WT and mutant THP1 cells, incubated in +Arg or -Arg medium for 72 h. Data are represented as mean ± SEM; n = 3.(E) Western blot for ASS1 and ATF4 in WT and mutant THP1 cells, incubated in +Arg or -Arg medium for 72 h. Representative of three replicates.(F) Growth of WT and mutant THP1 cells, incubated in the indicated media. Data are represented as mean ± SD; n = 3.See also Figure S3.
Supplier Page from Abcam for Anti-ASS1 antibody [EPR12398]