ZMAT3 depletion results in increased expression of genes related to glucose metabolism in colorectal cancer cells.

(A) IGV snapshot shows location of the two sgRNAs used to generate ZMAT3-KO HCT116 cells, the observed 57 bp deletion near sgRNA#2 and the p53 ChIP-seq peak in the ZMAT3 locus in response to p53 activation upon Nutlin treatment. The p53 ChIP-seq data was previously published66. (B) RT-qPCR was performed from 3 biological replicates of ZMAT3-WT and ZMAT3-KO HCT116 cells. GAPDH served as a housekeeping gene control. (C) Colony formation assays were performed from 3 biological replicates of ZMAT3-WT and ZMAT3-KO HCT116 cells. (D) Notched box plot of the log2FC in RNA abundance of differentially expressed genes after RNA-Seq analysis of ZMAT3-KO versus ZMAT3-WT HCT116 cells. Median values for each group are indicated at the top, and the number of RNAs for which data were obtained for each group is indicated at the bottom. (E) Volcano plots showing differentially expressed proteins (shown in red) identified by performing quantitative proteomics from ZMAT3-WT and ZMAT3-KO HCT116 cells. (F) Most significantly enriched pathways in the gene set enrichment analysis of significantly upregulated genes (p<0.05) from the ZMAT3-KO/ZMAT3-WT quantitative proteomics analysis. (G) TMT mass spectrometry peptide abundance of HKDC1 protein in ZMAT3-WT and ZMAT3-KO HCT116 cells. The values are the average of five biological replicates for ZMAT3-WT and four biological replicates for ZMAT3-KO cells. (H) IGV snapshot for ZMAT3 and HKDC1 from RNA-seq from ZMAT3-WT and ZMAT3-KO HCT116 cells. ∗p < 0.05, ∗∗∗∗p < 0.0001.

ZMAT3 negatively regulates HKDC1 expression in diverse cell types.

(A,B) RT-qPCR and immunoblotting for HKDC1 from ZMAT3-WT and ZMAT3-KO HCT116 cells. GAPDH served as a housekeeping gene control. RT-qPCR are values are the average of three biological replicates. (C, D) RT-qPCR was performed in biological triplicates for ZMAT3 and HKDC1 mRNAs from HCT116, SW1222, HCEC-1CT and HEPG2 cells after transfection with a control (CTRL) siRNA or ZMAT3 siRNAs for 72 hr. GAPDH served as a housekeeping gene control. (E) Immunoblotting was performed for endogenous ZMAT3 and HKDC1 from HCT116 and HepG2 whole cell lysates after siRNA-mediated knockdown of ZMAT3 or HKDC1 for 72 hr. GAPDH served as housekeeping gene control. (F) Fold change for Zmat3, Trp53, Mdm4 and Hkdc1 mRNAs is shown from the RNA-seq from Zmat3 knock-out and wild-type MEFs. (G) Analysis of HKDC1 mRNA levels from normal colon tissues or CRC patient samples from the TCGA COAD cohort. N refers to the number of samples in each group. (H) Fold change for Trp53, Zmat3, Cdkn1a and Hkdc1 mRNAs is shown from the RNA-seq from Trp53 knock-out and wild-type MEFs. N refers to the number of samples in each group. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001

ZMAT3 inhibits mitochondrial respiration and proliferation via HKDC1.

(A) 2-Deoxyglucose analog of glucose together with luminescence-based enzymatic assay was used to assess relative glucose uptake in ZMAT3-WT and ZMAT3-KO HCT116 cells in presence and absence of HKDC1. For SW122 and HEPG2 cells relative glucose was measured in the presence and absence of siRNA-mediated knockdown of HKDC1 and ZMAT3 alone or in combination. (B, C) Metabolic flux assays were performed for basal glycolysis rate and basal mitochondrial respiration rate in ZMAT3 and/or HKDC1 knockdown in HCT116 cells. (D, E) Incucyte live cell proliferation assays and CCK8-based cell proliferation in HCT116 ZMAT3-WT and KO cells in the presence and absence of siRNA-mediated knockdown HKDC1. ∗p < 0.05, (∗∗) p < 0.01, (∗∗∗) P<0.001. The results are the average of three independent experiments.

p53 negatively regulates HKDC1 expression in a ZMAT3-dependent manner.

(A) IGV snapshots from the RNA-seq data following knockdown of p53 with p53 siRNAs. Data shows increased HKDC1 mRNA levels and decreased ZMAT3 mRNA levels upon p53 knockdown in HCT116 cells. (B) Fold change is shown for p53, p21, ZMAT3, and HKDC1 mRNAs from the RNA-seq performed from siCTRL and sip53 transfected HCT116 cells. (C, D) HCT116 cells were transfected with CTRL siRNA or p53 siRNAs for 48 hr. The levels of ZMAT3, p53, and HKDC1 mRNA or protein were measured by RT-qPCR (C) or immunoblotting from whole cell lysates (D). GAPDH was used as housekeeping gene control. (E) Fold change for ZMAT3, p21, HKDC1 and p53 mRNAs is shown from the RNA-seq from HCT116 cells treated with DMSO or Nutlin for 6 hr. (F) Immunoblotting was performed for HKDC1, ZMAT3 and p21 from ZMAT3-WT and ZMAT3-KO HCT116 cells with or without Nutlin treatment for 24 hr. GAPDH served as the loading control. ns refers to not significant. (G,H) Doxycycline inducible ZMAT3-FLAG-HA HCT116 cells treated with 2ug/ml doxycycline for 48 hr. The levels of ZMAT3 mRNA or protein induction were measured by RT-qPCR (G) or immunoblotting from whole cell lysates against HA antibody(H). GAPDH was used as housekeeping gene control. (I, J) Doxycycline inducible ZMAT3-FLAG-HA HCT116 cells were transfected with CTRL siRNA or p53 siRNAs for 48 hr followed by 48hr doxycycline treatment. The levels of ZMAT3, p53, and HKDC1 mRNA or protein were measured by RT-qPCR (I) or immunoblotting from whole cell lysates (J). GAPDH was used as housekeeping gene control. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001

ZMAT3 inhibits HKDC1 transcription by interacting with the transcription factor JUN.

(A) Schematic for identification of ZMAT3-FLAG interacting proteins by IP mass spectrometry from HCT116 cells expressing doxycycline-induced ZMAT3-FLAG-HA. (B) Volcano plot showing significantly enriched proteins (shown in red) identified by ZMAT3-FLAG IP followed by mass spectrometry, in presence or absence of doxycycline from ZMAT3-FLAG-HA HCT116 cells. JUN was strongly enriched in the ZMAT3-FLAG IPs. (C) IGV snapshot showing JUN, POLR2A, H3K27Ac and H3K3Me3 peaks at the HKDC1 locus from the ENCODE project (accession from top to bottom: ENCSR000FAH, ENCSR000EDG, ENCSR000EEK, ENCSR000EUU, ENCSR661KMA and ENCSR333OPW). JUN binding motif (TGASTCA) is shown in blue (positive strand) and in red (negative strand). (D) Immunoblotting was performed using anti-FLAG beads and whole cell lysates from no doxy or doxycycline treated ZMAT3-FLAG-HA HCT116 cells. 10% of total cell lysate was used for input. GAPDH used as loading control. (E, F) ZMAT3-WT and KO HCT116 cells were transfected with CTRL siRNA or JUN siRNAs for 48 hr. The levels of ZMAT3, JUN, and HKDC1 mRNAs or the corresponding proteins were measured by RT-qPCR (E) or immunoblotting from whole cell lysates (F). GAPDH was used as housekeeping gene control. (G) JUN ChIP-qPCR was performed in biological triplicates from HCT116 ZMAT3-WT and KO cells to determine enrichment of JUN at the HKDC1 promoter. (H) Luciferase assays were performed in biological triplicates upon JUN or ZMAT3 knockdown alone or in combination using the HKDC1 promoter reporter constructs. ∗p < 0.05, ∗∗p < 0.01

Schematic for ZMAT3-mediated regulation of HKDC1 expression and inhibition of mitochondrial respiration.

In ZMAT3-WT cells p53 activates ZMAT3 transcription, resulting in ZMAT3 protein binding to the transcription factor JUN. This inhibits the ability of JUN to bind to the HKDC1 promoter, low HKDC1 expression leading to controlled mitochondrial respiration and controlled cell proliferation. In ZMAT3-knockout cells, JUN actively binds to the HKDC1 promoter and upregulates its expression resulting in increased mitochondrial respiration and increased cell proliferation.

Extended results for Figure 1.

(A) Immunoblotting for ZMAT3, p53, p21 and GAPDH from ZMAT3-WT and ZMAT3-KO HCT116 cells with or without Nutlin treatment (24 hr). GAPDH served as the loading control. (B) Incucyte live cell proliferation assays were performed from ZMAT3-WT and ZMAT3-KO HCT116 cells. (C) Volcano plot for the differentially expressed genes identified by RNA-Seq from ZMAT3-WT and ZMAT3-KO HCT116 cells. Significantly expressed genes are indicated in red (p<0.05). (D) Most significantly enriched pathways identified by GSEA from the top 500 significantly upregulated genes (p<0.05) from ZMAT3-KO vs ZMAT3-WT RNA-seq.

Extended results for Figure 2.

(A) Volcano plot for the differentially expressed genes identified by RNA-Seq performed after transfection of HCT116 cells with siCTRL or siZMAT3 for 72 hr. Significantly expressed genes are indicated in red (p<0.05). (B) GSEA analysis was performed for the top 500 significantly upregulated genes (p<0.05) in ZMAT3 knockdown HCT116 cells identified by RNA-seq. (C, D) Venn diagram of showing comparisons of the indicated RNA-Seq data sets. 1,023 significant upregulated (C) and 1,042 downregulated (D) differentially expressed genes were shared between the ZMAT3-KO/ZMAT3-WT and siZMAT3/siCTRL comparisons. (E) Most significantly enriched pathways in the GSEA for the top 500 genes commonly upregulated genes (p<0.05) in ZMAT3-KO vs ZMAT3-WT and siZMAT3 vs siCtrl comparisons from the RNA-seq data. (F, G) ZMAT3 and HKDC1 mRNA levels were determined in CRC patient samples in the TCGA COAD cohort from p53-WT (wild-type) and p53-Mutant CRC patient samples.

Extended results for Figure 3.

Non-mitochondrial oxygen consumption in ZMAT3 and/or HKDC1 knockdown in HCT116 cells. Values are the average of four independents experiments.

Extended results for Figure 4.

(A) Volcano plot shows the differentially expressed genes from the RNA-Seq from sip53 and siCTRL transfected groups in HCT116 cells. Significantly expressed genes are shown in red (p<0.05). (B, C) Venn diagram and GSEA analysis for the genes commonly upregulated in RNA-Seq upon p53 knockdown and ZMAT-KO HCT116. (D, E) Venn diagram and GSEA analysis for the genes commonly downregulated in RNA-Seq upon p53 knockdown and ZMAT-KO HCT116.

Extended results for Figure 5.

(A) Schematic of full-length ZMAT3 protein showing three zinc finger motifs. (B) Immunoblot from 10% input and FLAG immunoprecipitation from doxycycline inducible ZMAT3-FLAG-HA HCT116 cell lysates treated with/without doxycycline for 48 hr.