p53-induced RNA-binding protein ZMAT3 inhibits transcription of a hexokinase to suppress mitochondrial respiration

Reviewer #1 (Public review):

Summary:

ZMAT3 is a p53 target gene that the Lal group and others have shown is important for p53-mediated tumor suppression, and which plays a role in the control of RNA splicing. In this manuscript, Lal and colleagues perform quantitative proteomics of cells with ZMAT3 knockout and show that the enzyme hexokinase HKDC1 is the most upregulated protein. Mechanistically, the authors show that ZMAT3 does not appear to directly regulate the expression of HKDC1; rather, they show that the transcription factor c-JUN was strongly enriched in ZMAT3 pull-downs in IP-mass spec experiments, and they perform IP-western to demonstrate an interaction between c-JUN and ZMAT3. Importantly, the authors demonstrate, using ChIP-qPCR, that JUN is present at the HKDC1 gene (intron 1) in ZMAT3 WT cells and shows markedly enhanced binding in ZMAT3 KO cells. The data best fit a model whereby p53 transactivates ZMAT3, leading to decreased JUN binding to the HKDC1 promoter, and altered mitochondrial respiration.

Strengths:

The authors use multiple orthogonal approaches to test the majority of their findings.

The authors offer a potentially new activity of ZMAT3 in tumor suppression by p53: the control of mitochondrial respiration.

Weaknesses:

Some indication as to whether other c-JUN target genes are also regulated by ZMAT3 would improve the broad relevance of the authors' findings.

Reviewer #2 (Public review):

Summary:

The study elucidates the role of the recently discovered mediator of p53 tumor suppressive activity, ZMAT3. Specifically, the authors find that ZMAT3 negatively regulates HKDC1, a gene involved in the control of mitochondrial respiration and cell proliferation.

Strengths:

Mechanistically, ZMAT3 suppresses HKDC1 transcription by sequestering JUN and preventing its binding to the HKDC1 promoter, resulting in reduced HKDC1 expression. Conversely, p53 mutation leads to ZMAT3 downregulation and HKDC1 overexpression, thereby promoting increased mitochondrial respiration and proliferation. This mechanism is novel; however, the authors should address several points.

Weaknesses:

The authors conduct mechanistic experiments (e.g., transcript and protein quantification, luciferase assays) to demonstrate regulatory interactions between p53, ZMAT3, JUN, and HKDC1. These findings should be supported with functional assays, such as proliferation, apoptosis, or mitochondrial respiration analyses.

Reviewer #3 (Public review):

Summary:

In their manuscript, Kumar et al. investigate the mechanisms underlying the tumor suppressive function of the RNA binding protein ZMAT3, a previously described tumor suppressor in the p53 pathway. To this end, they use RNA-sequencing and proteomics to characterize changes in ZMAT3-deficient cells, leading them to identify the hexokinase HKDC1 as upregulated with ZMAT3 deficiency first in colorectal cancer cells, then in other cell types of both mouse and human origin. This increase in HKDC1 is associated with increased mitochondrial respiration. As ZMAT3 has been reported as an RNA-binding and DNA-binding protein, the authors investigated this via PAR-CLIP and ChIP-seq but did not observe ZMAT3 binding to HKDC1 pre-mRNA or DNA. Thus, to better understand how ZMAT3 regulates HKDC1, the authors used quantitative proteomics to identify ZMAT3-interacting proteins. They identified the transcription factor JUN as a ZMAT3-interacting protein and showed that JUN promotes the increased HKDC1 RNA expression seen with ZMAT3 inactivation. They propose that ZMAT3 inhibits JUN-mediated transcriptional induction of HKDC1 as a mechanism of tumor suppression. This work uncovers novel aspects of the p53 tumor suppressor pathway.

Strengths:

This novel work sheds light on one of the most well-established yet understudied p53 target genes, ZMAT3, and how it contributes to p53's tumor suppressive functions. Overall, this story establishes a p53-ZMAT3-HKDC1 tumor suppressive axis, which has been strongly substantiated using a variety of orthogonal approaches, in different cell lines and with different data sets.

Weaknesses:

While the role of p53 and ZMAT3 in repressing HKDC1 is well substantiated, there is a gap in understanding how ZMAT3 acts to repress JUN-driven activation of the HKDC1 locus. How does ZMAT3 inhibit JUN binding to HKDC1? Can targeted ChIP experiments or RIP experiments be used to make a more definitive model? Can ZMAT3 mutants help to understand the mechanisms? Future work can further establish the mechanisms underlying how ZMAT3 represses JUN activity.