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

(A) IGV snapshot showing the 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 were previously published71. (B) RT-qPCR analysis of ZMAT3-WT and ZMAT3-KO HCT116 cells from 3 biological replicates. GAPDH served as the housekeeping gene control. (C) Colony formation assays performed from ZMAT3-WT and ZMAT3-KO HCT116 cells in 3 biological replicates. (D) Notched box plot of the log2fold change (FC) in RNA abundance of differentially expressed genes from RNA-Seq of ZMAT3-KO and ZMAT3-WT HCT116 cells. Median values for each group are indicated at the top of each box, and the number of RNAs for which data were obtained for each group is indicated at the bottom. (E) Volcano plot showing differentially expressed proteins (shown in red) identified by global quantitative proteomics from ZMAT3-WT and ZMAT3-KO HCT116 cells. (F) Most significantly enriched pathways identified by GSEA of genes significantly upregulated (p<0.05) in the ZMAT3-KO versus ZMAT3-WT based on quantitative proteomics data. (G) TMT mass spectrometry peptide abundance of HKDC1 in ZMAT3-WT and ZMAT3-KO HCT116 cells. Values represent the average of five biological replicates for ZMAT3-WT and four biological replicates for ZMAT3-KO cells. (H) IGV snapshot showing ZMAT3 and HKDC1 transcripts from RNA-seq of 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 in ZMAT3-WT and ZMAT3-KO HCT116 cells. GAPDH served as the housekeeping gene control. RT-qPCR was performed in biological triplicates. (C, D) RT-qPCR analysis from the indicated cell lines in biological triplicates following transfection with control (CTRL) siRNA or ZMAT3 siRNAs for 72 h. GAPDH served as the housekeeping gene control. (E) Immunoblotting of whole-cell lysates from HCT116 and HepG2 cells after siRNA-mediated knockdown of ZMAT3 or HKDC1 for 72 h. GAPDH served as the loading control. (F) Fold change in Zmat3, Trp53, Mdm4 and Hkdc1 mRNA levels from RNA-seq analysis of Zmat3 knockout and wild-type MEFs. (G) Analysis of HKDC1 mRNA levels in normal colon tissue and CRC samples from the TCGA COAD cohort. N indicates the number of samples in each group. (H) Fold change in Trp53, Zmat3, Cdkn1a and Hkdc1 mRNA levels from RNA-seq analysis of Trp53 knockout and wild-type MEFs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001

ZMAT3 inhibits mitochondrial respiration via HKDC1.

(A) Glucose uptake was measured using a 2-deoxyglucose analog and a luminescence-based enzymatic assay in ZMAT3-WT and ZMAT3-KO HCT116 cells in presence or absence of HKDC1. For SW122 and HEPG2 cells, relative glucose uptake was measured following siRNA-mediated knockdown of HKDC1 and/or ZMAT3. (B, C) Metabolic flux assays were performed to measure basal glycolysis rate and basal mitochondrial respiration rate in HCT116 cells after ZMAT3 and/or HKDC1 knockdown. (D, E) Incucyte live-cell proliferation assays and CCK8-based cell proliferation assays in ZMAT3-WT and ZMAT3-KO HCT116 cells in the presence or absence of siRNA-mediated HKDC1 knockdown. Data represent mean ± SEM of three independent experiments. ∗p < 0.05, (∗∗) p < 0.01, (∗∗∗) p<0.001.

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

(A) IGV snapshots from RNA-seq data following knockdown of p53 using p53 siRNAs in HCT116 cells. (B) Fold change for p53, p21, ZMAT3, and HKDC1 mRNA levels from RNA-seq of HCT116 cells transfected with siCTRL and sip53. (C, D) HCT116 cells were transfected with siCTRL or p53 siRNAs for 48 h. ZMAT3, p53 and HKDC1 mRNA or protein were measured by RT-qPCR (C) or immunoblotting of whole-cell lysates (D). GAPDH served as the housekeeping gene control. (E) Fold change in ZMAT3, p21, HKDC1 and p53 mRNA levels from RNA-seq of HCT116 cells treated with DMSO or Nutlin for 6 h. “ns” denotes not significant. (F) Immunoblotting of whole-cell lysates from ZMAT3-WT and ZMAT3-KO HCT116 with or without Nutlin treatment for 24 h. GAPDH served as the loading control. (G, H) Doxycycline (Doxy)- inducible ZMAT3-FLAG-HA HCT116 cells were treated with 2 µg/mL doxycycline for 48 h. ZMAT3 mRNA and ZMAT3-FLAG-HA protein induction were measured by RT-qPCR (G) and immunoblotting using an anti-HA antibody (H). GAPDH served as the housekeeping control. (I, J) Doxycycline-inducible ZMAT3-FLAG-HA HCT116 cells were transfected with CTRL siRNA or p53 siRNAs for 48 h, followed by 48 h of doxycycline treatment. ZMAT3, p53, and HKDC1 mRNA and protein levels were measured by RT-qPCR (I) or immunoblotting from whole-cell lysates (J). GAPDH served as the 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 of the workflow used to identify ZMAT3-FLAG-HA interacting proteins by IP-mass spectrometry in HCT116 cells expressing doxycycline-induced ZMAT3-FLAG-HA. (B) Volcano plot showing significantly enriched proteins (shown in red) identified by anti-FLAG IPs followed by mass spectrometry in the presence and absence of doxycycline in ZMAT3-FLAG-HA HCT116 cells. The vertical dotted line denotes a >10-fold enrichment cutoff. JUN was strongly enriched in the ZMAT3-FLAG IPs. (C) IGV snapshot showing JUN, POLR2A, H3K27Ac and H3K4Me3 peaks at the HKDC1 locus from ChIP-seq data from the ENCODE cell line datasets (accessions from top to bottom: ENCSR000FAH, ENCSR000EDG, ENCSR000EEK, ENCSR000EUU, ENCSR661KMA and ENCSR333OPW). The JUN binding motif (TGASTCA) is shown in blue (positive strand) and in red (negative strand). (D) IP followed by immunoblotting using anti-FLAG beads and whole-cell lysates from untreated (no doxy) or doxy-treated ZMAT3-FLAG-HA HCT116 cells. Ten percent of cell lysate was used as input. GAPDH served as the loading control. (E, F) ZMAT3- WT and ZMAT3-KO HCT116 cells were transfected with CTRL siRNA or JUN siRNAs for 48 h, followed by RT-qPCR (E) or immunoblotting of whole-cell lysates (F). GAPDH served as the housekeeping control. (G) JUN ChIP-qPCR was performed in biological triplicates from ZMAT3-WT and ZMAT3-KO HCT116 cells to determine the enrichment of JUN at HKDC1 intron 1. (H) Luciferase assays were performed in biological triplicates following JUN and/or ZMAT3 knockdown, and pGL4 or pGL4 construct containing the HKDC1 intron 1 region. ∗p < 0.05, ∗∗p < 0.01

Model of ZMAT3-mediated regulation of HKDC1 expression and mitochondrial respiration.

In ZMAT3-WT cells, p53 activates ZMAT3 transcription, leading to ZMAT3 protein binding to the transcription factor JUN. This interaction inhibits JUN binding to the HKDC1 locus, resulting in low HKDC1 expression and controlled mitochondrial respiration and cell proliferation. In ZMAT3-KO cells, JUN actively binds to the HKDC1 locus and upregulates its expression, leading to increased mitochondrial respiration and enhanced cell proliferation.