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The glucose-sensing transcription factor MLX balances metabolism and stress to suppress apoptosis and maintain spermatogenesis

['Patrick A. Carroll', 'Basic Sciences Division', 'Fred Hutchinson Cancer Research Center', 'Seattle', 'Washington', 'United States Of America', 'Brian W. Freie', 'Pei Feng Cheng', 'Sivakanthan Kasinathan', 'Haiwei Gu']

Date: 2021-11

Male germ cell (GC) production is a metabolically driven and apoptosis-prone process. Here, we show that the glucose-sensing transcription factor (TF) MAX-Like protein X (MLX) and its binding partner MondoA are both required for male fertility in the mouse, as well as survival of human tumor cells derived from the male germ line. Loss of Mlx results in altered metabolism as well as activation of multiple stress pathways and GC apoptosis in the testes. This is concomitant with dysregulation of the expression of male-specific GC transcripts and proteins. Our genomic and functional analyses identify loci directly bound by MLX involved in these processes, including metabolic targets, obligate components of male-specific GC development, and apoptotic effectors. These in vivo and in vitro studies implicate MLX and other members of the proximal MYC network, such as MNT, in regulation of metabolism and differentiation, as well as in suppression of intrinsic and extrinsic death signaling pathways in both spermatogenesis and male germ cell tumors (MGCTs).

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: RNE is a member of the Scientific Advisory Boards of Kronos Bio Inc. and Shenogen Pharma, Beijing. There is no overlap between the research presented in this manuscript with the products or methods related to these companies.

Funding: PAC, PFC, BWF, TH, XW and RNE were supported by a grant from the National Cancer Institute at the National Institutes of Health USA ( https://www.cancer.gov/grants-training ): R35 CA231989 (to RNE). PAC was also supported by a postdoctoral fellowship T32 CA009657. Scientific Computing Infrastructure at the Fred Hutchinson Cancer Research Center was funded by an ORIP grant from the National Institutes of Health: S10OD028685 ( https://orip.nih.gov ). SK was supported by a fellowship from the National Institutes of Health USA F30CA186458 ( https://www.cancer.gov/grants-training ). HG and DR were supported by Cancer Center Support Grant, National Institutes of Health P30 CA015704 ( https://www.cancer.gov/grants-training ). JS is an Investigator of the Howard Hughes Medical Institute ( https://www.hhmi.org/programs ). DEA is supported by National Institutes of Health R01 GM055668 ( https://www.cancer.gov/grants-training ) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we report the phenotype associated with loss of MLX during normal murine development. As with deletion of either Mlxip (MondoA) or Mlxipl (Chrebp), loss of Mlx is not detrimental to normal embryonic development or organismal viability. However, all male homozygous null animals (MLX KO ) exhibit complete sterility with a dramatic increase in apoptosis of germ cells (GCs). Many of these phenomena are recapitulated by Sertoli cell–specific deletion of Mlx, directly implicating MLX in the normal function of this male-specific stromal cell. We link this phenotype to a broad integrated transcriptional program mediated by MLX within the MYC network that facilitates metabolism and directly suppresses apoptosis.

The MAX-centered network exists within a larger network, containing MAX-Like protein X (MLX), MondoA (also known as MLX-interacting protein, MLXIP), and carbohydrate response element binding protein (ChREBP, also known as MondoB and MLXIPL; reviewed in [ 5 , 6 ]). MLX heterodimerizes with a subset of MXD proteins as well as the glucose-sensing MondoA and ChREBP but is unable to heterodimerize with either MAX or MYC. ChREBP-MLX [ 7 ] and MondoA-MLX heterodimers are major regulators of glucose-responsive transcription in vitro and in vivo [ 8 – 11 ], and MLX function has been linked to the response to metabolic stress in multiple organisms [ 12 – 15 ]. Genetic ablation of MYC [ 16 ], MAX [ 17 ], and MNT [ 18 ] (the most ubiquitously expressed MXD family member) results in embryonic or perinatal lethality (in the case of MNT); however, loss of MondoA [ 19 ] or ChREBP does not interfere with overt development [ 7 ]. We previously demonstrated an obligate role for the MLX arm of the network in promoting survival of a wide range of tumor cells with deregulated MYC by facilitating metabolic reprogramming and suppressing apoptosis. However, cells expressing endogenously regulated MYC were found to tolerate MondoA or MLX loss [ 20 ].

The MYC/MAX/MXD network plays a critical role in both development and tumorigenesis as major mediators of transcriptional regulation of growth, metabolism, proliferation, apoptosis, and differentiation (for reviews, see [ 1 – 3 ]). This network is comprised of basic helix–loop–helix–leucine zipper (bHLHLZ) transcription factors (TFs) generally associated with activation (MYC) or repression (MXD) that compete for an obligate heterodimerization partner (MAX) in order to bind DNA and influence expression of shared target genes [ 4 ]. Typically, MYC-MAX responds to mitogenic signals to activate Enhancer box (E-box)-containing promoters, whereas MXD-MAX responds to the loss of mitogenic signals or differentiation cues to repress the same targets. This allows the network to balance proliferative cues with cell cycle entry and exit.

Results

Gene expression in fractionated testes cell populations We next fractionated the testicular tissue to remove interstitial (stromal and immune) cells from the seminiferous tubules (comprised predominantly of Spg, Sc, and St as well as Sertoli cells) in order to assess protein expression. In comparison with WT, fractionated MLXKO tubule cells show complete loss of MLX and strongly decreased expression of both MLX dimerization partners, MondoA and ChREBP (Fig 6A). Surprisingly, these cells also exhibit moderately decreased expression of the immature spermatogonial stem cell (SSC) markers MYCN, MAX, and OCT4, with no change in the expression of the MYC-antagonist MNT (Fig 6A). We had also noted diminished expression of the SSC marker EOMES in whole testes (S5B Fig). Importantly, small interfering RNA (siRNA) against MLX resulted in similar changes in the male germ cell tumor (MGCT) cell line NTera2 (Fig 6B), supporting a cell autonomous role for MLX in regulating the expression of these SSC markers. This suggests that MLX may impact stem cell function in male GCs as well as during subsequent differentiation. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. Analysis of DEGs from WT and MLXKO mice. (A–D) WB analysis of (A) isolated seminiferous tubule cells from WT and MLXKO mice; (B) NTera2 MGCT cells treated with siCTRL or siMLX; (C) Seminiferous tubule and (D) epididymal cells from WT versus MLXKO mice probed for the indicated proteins. (E) LC–MS relative abundance data for the indicated metabolites from isolated seminiferous tubule cells (N = 3 paired littermates, shown is the mean +/− SD, paired t test). (F) Model explaining MLX regulation of metabolic targets. (G) sFASL treatment of the indicated cell lines (N = 3 biological replicates, shown is the percent dead cells with mean +/− SD). (H) Relative viable cell number of the NTera2 cells after siRNA transfection with the indicated siRNA. siDEATH included as control for siRNA transfection efficacy (N = 4 independent experiments, shown is the mean +/− SEM). One-way ANOVA with a Dunnett correction was used for 6G and 6H (*p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001). The underlying data for Fig 6E, 6G and 6H can be found in S1 Data. DEG, differentially expressed gene; KO, knockout; LC–MS, liquid chromatography–mass spectrometry; MGCT, male germ cell tumor; MLX, MAX-Like protein X; sFASL, soluble FAS ligand; siRNA, small interfering RNA; WB, western blot; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001085.g006 We also assessed the expression of metabolic and stress targets identified by RNA-seq, as well as markers of spermatogenesis, in the seminiferous tubules by western blot (WB) analysis of isolated cells from WT and MLXKO mice. As shown in Fig 6C, the known MLX target TXNIP is decreased, and the marker of fatty acid beta-oxidation CPT1A is increased along with stress-related proteins including FAS, BIM, IGFBP3, and γH2AX concomitant with PARP cleavage, all of which are consistent with increased apoptosis. We also confirmed decreased expression of the mature St/spermatozoa marker PGK2, while the pan-GC marker DDX4 is not significantly altered. This further confirms a disruption of normal differentiation associated with elevated stress. In contrast with our observations in seminiferous tubules, cells isolated from epididymides did not robustly express MYC network or stem cell markers. However, as shown in Fig 6D, MLXKO epididymal cells did exhibit alterations to the same metabolic targets TXNIP and CPT1A, as well as elevated stress markers FAS, BIM, IGFBP3, γH2AX, and PARP cleavage. Epididymal cells from MLXKO mice also maintained the immature GC marker DDX4 (which is normally absent from epididymal cells of WT mice) and, consistent with spermiogenic defects, they also lacked PGK2. Interestingly, MLX appears to regulate many of the same proteins in cells isolated from the interstitium of the testes, supportive of a broad role for MLX in both stabilizing its binding partners and regulating metabolic targets (S5A Fig). In summary, MLX appears to regulate male GC function at multiple stages: While MYCN-MAX are expressed in the primitive Spg population, loss of MLX destabilizes MYCN, thereby potentially affecting stem cell function. MLX also appears to suppress stress as early as the initiation of meiosis, as γH2AX is induced then, but should resolve after the completion of genome reduction. While the genomes of MLXKO St are indeed reduced to haploid, the stress markers associated with meiosis (and others) are maintained. This correlates with loss of many late St markers and apoptosis. As Sertoli-specific deletion does not result in widespread apoptosis of GCs, this supports a broad role for MLX in facilitating a cell autonomous survival pathway in the male germline.

MLX regulates glucose and lipid metabolism and suppresses apoptosis In order to gauge the functional consequences of alterations to metabolism associated with loss of MLX, targeted LC–MS/MS was utilized to monitor glycolytic and beta-oxidation metabolites in isolated seminiferous tubule cells. As shown in Fig 6E, we detected increased intracellular glucose, consistent with the diminished expression of TXNIP, which is known to suppress glucose uptake, while there was no significant change in pyruvate or lactate levels, perhaps due to decreased expression of glycolytic enzymes that are targets of CREM (e.g., LDHA and LDHC). We also detected a significant increase in a number of acyl-carnitine species (the product of CPT1A enzymatic activity) (Fig 6E), while there was no change in acetyl-carnitine (C2-carnitine), consistent with diminished expression of CRAT (another CREM target down in the MLXKO testes) as opposed to the general up-regulation of fatty acid gene set in general, many of which are regulated by CREM (e.g., XBP1 and SREBF1). These changes are consistent with a role for MLX as a transcriptional regulator of metabolism in the seminiferous epithelium predominantly comprised of Spg, Sc, and St. A hypothetical model for putative targets of MLX responsible for these changes is shown in Fig 6F, including the previously reported positive correlation between MondoA-MLX and TXNIP [8] and the inverse correlation between TXNIP and CPT1A [42]. Because Fas has been established as a developmental regulator of cell survival during spermatogenesis [43] and cells from MLXKO testes exhibited increased Fas mRNA and protein, we asked whether MLX plays a broader and cell autonomous role in regulating the FAS death receptor. To this end, we determined the effect of soluble FAS ligand (sFASL) on immortalized 3T3 cells and primary B cells derived from WT versus MLXKO mice. As shown in Fig 6G, FASL selectively kills both MLXKO 3T3 cells and primary B cells while minimally affecting WT cells under normal culture conditions. We note that the MLXKO and WT 3T3 cells are equally viable under standard culture conditions. However, as we previously reported, MLXKO 3T3 cells undergo rapid apoptosis following enforced MYC expression [20]. Importantly, the expression of FAS protein is also elevated in MLXKO 3T3 cells and is suppressed by the reintroduction of any one of the 3 isoforms of MLX into null cells (S6B Fig). Reintroduction of MLX also stabilizes MondoA and MYC-MAX protein levels. Consistent with the established role of MondoA-MLX [20], the MLX target genes TXNIP, TOMM20, and FASN are repressed in the MLXKO cells, but robustly reexpressed with reconstitution of MLX (S6B Fig). This indicates a direct role for MLX in both activation of metabolic targets and suppression of FAS levels and suggests that MLX loss sensitizes cells to context-dependent death, not only as a consequence of MYC activation, but also in response to environmental factors such as FASL and glucose levels. We surmise that MLX normally attenuates stress and apoptosis during spermatogenesis, a process involving high metabolic demand, dependent upon a glycolytic program driven by both MYC and MYCN [39], as well as directly modified by FASL-FAS signaling [43].

[END]

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