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

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).


Introduction
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][2][3]). This network is comprised of basic helix-loophelix-leucine zipper (bHLHLZ) transcription factors (TFs) generally associated with activation (MYC) or repression (MXD) that compete for an obligate heterodimerization partner (MAX) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 exhibited abnormal and acellular seminiferous tubules (marked with blue asterisks in Fig 1G), and MLX KO epididymides contained decreased numbers of spermatozoa, which displayed highly abnormal morphologies relative to WT including malformed heads and abnormal midpiece and tail structures (S1C Fig), as well as populations of cells with an immature appearance compared with WT ( Fig 1G). As shown in Fig 1H, the GC identity of these immature appearing cells within the MLX KO epididymis was confirmed by staining with the pan-GC cytoplasmic marker DDX4 (also known as VASA). In WT mice, DDX4 is only detected at low levels in cells within the epididymis due to removal by phagocytosis of spermatid (St) cytoplasts or residual bodies [21] possessing this marker during the transition from round to elongated St (Fig 1H). These epididymal histological phenotypes of MLX KO were not present in Mlx heterozygous animals (S1B Fig) and were observed with varying severity from age P51 onward (S1D Fig). Note the combination of both abnormal spermatozoa and immature round cells in the epididymis of MLX KO animals even at this young age (S1E Fig).

Infertility of MLX KO male mice originates in the testes
To begin investigating a role for MLX in male fertility, we first determined the extent of MLX expression in the testes using immunofluorescent antibody staining. Fig 2A and 2B shows staining with anti-MLX throughout the testis, including GCs, Sertoli cells, interstitial cells, as well as in the lining of the epididymis, where spermatozoa mature and gain motility. The observed staining is specific, as no signal is detected from secondary antibody or from the same tissue derived from our MLX KO mice and stained with anti-MLX. We note that MLX expression in general appears to decrease with differentiation into the lumen of the seminiferous tubule (Fig 2B). Such widespread expression of MLX is consistent with other available data from human testes confirming the presence of this transcript in multiple testes cell types. The Human Protein Atlas [22] (http://www.proteinatlas.org) shows similar staining for MLX in human testis and epididymis, and single-cell RNA sequencing (scRNA-seq) of human testicular cell populations [23] shows MLX specifically enriched in Sertoli and primitive GCs (S2A Fig) and co-expressed with MLXIP (encoding MondoA) (S2B Fig). These data support a conserved role for MLX in human testes.
To understand the basis for the testicular phenotype in the MLX KO mice, we quantified aspects of the cellular biology of the testicular and epididymal GCs. Enumeration of daily sperm production (DSP) rate revealed both a significant decrease in DSP (Fig 3A), as well as diminished output of mature sperm to the epididymis in the MLX KO compared with WT mice (Fig 3B), consistent with decreased production in the testis. While the majority of WT cauda spermatozoa were motile and had a normal appearance, spermatozoa that reached the cauda epididymis in MLX KO animals exhibited both a lack of progressive motility ( Fig 3C) and abnormal morphology (Fig 3D), as shown previously in (S1C and S1E Fig). These features are consistent with the clinical symptoms of oligoasthenoteratozoospermia (OAT) as defined by decreased sperm number, lack of motility, and altered morphology of the sperm [24].
To determine whether a defect in androgen production correlated with the loss of fertility in MLX KO males, we quantified serum testosterone (T) between WT and MLX KO animals. Consistent with the lack of a change in the size of the seminal vesicle (Fig 3E), a T responsive tissue, we did not detect a change in serum T levels between genotypes (Fig 3F). By contrast, alterations in levels of important metabolites were detected upon liquid chromatography with tandem mass spectrometry (LAU : PleasenotethatLC À MS=MShasbeendefinedasliquidchromatograph C-MS/MS) metabolomic analysis of serum from WT versus MLX KO Table. Ribose-5-phosphate, pyruvate, and lactate were increased in MLX KO serum (S3B and S3C Fig), indicative of enhanced whole body glycolysis. MLX KO mice also exhibited alterations to metabolite levels associated with amino acid oxidation, such as decreased valine and a buildup of 2 downstream metabolites, 3-amino-isobutyrate and 2-hydroxy-isovaleric acid. Augmented glycolysis and alterations to oxidative substrates have also been reported in mice lacking MondoA or treated with a chemical inhibitor of MondoA [19] [10]. MondoA-MLX heterodimers are known to act through their downstream target, TXNIP, which, in turn, suppresses glycolysis [8]. Taken together, these results indicate that deletion of Mlx leads to a change, not in the production of T, but in whole body metabolism consistent with loss of MondoA-MLX activity, as well as alterations in normal testicular and epididymal tissue homeostasis. A model depicting changes in mitochondrial oxidative substrates upon MLX loss is shown in S3D Fig, suggesting a switch from glucose to amino acid oxidation.
As MLX functions as a TF in concert with its heterodimeric binding partners, MLX interacting proteins, we hypothesized that such a binding partner could also be required for male fertility. Since previous deletion of Mlxipl (encoding ChREBP) did not affect fertility [7], we gauged the requirement of Mlxip (encoding MondoA) for male fertility. As shown in Fig 3G, deletion of the gene encoding MondoA results in male-only infertility. Interestingly, in contrast to the MLX KO spermatozoa, which appeared abnormal, the spermatozoa from Mon-doA KO mice appear normal and are produced at normal number (S3E Fig) but are completely nonmotile (asthenospermic) (Fig 3H). This both supports a direct transcriptional requirement for MondoA-MLX activity in male fertility and suggests that MLX has functions independent of MondoA in the context of spermatogenesis.
Given the phenotypic differences between the MondoA and the MLX deleted mice, we sought to specifically determine the stage of the defect in spermatogenesis in the MLX KO mice. Staining for phospho-Histone H3ser10 (to detect mitotic and meiotic cells) indicated that GCs from both WT and MLX KO testes could undergo successful meiosis in the testis (Fig 3I). WT testis showed the expected stage-specific expression of γH2AX (marker of meiosis and DNA damage) decreasing with differentiation and present only in rare epididymal cells (most likely round St shed from the testes). By contrast, the MLX KO tissue display disrupted expression and the shedding of immature, γH2AX+ cells into the epididymis (Fig 3J). We also observed ectopic epididymal staining for the pan-GC marker DDX4 (see Fig 1H), which is normally only detected at low levels during the transition from round to elongated St.
As the cytoplasmic cell markers DDX4 and γH2AX are normally lost upon completion of meiosis, we tested whether testicular and epididymal cells from MLX KO mice were arresting in meiosis. All stages of meiosis (1, 2, and 4n DNA content) were observed in the testis and epididymis of both WT and MLX KO mice (Fig 3K), indicating that the cells transiting to the epididymis of MLX KO mice, although having significantly reduced total cell numbers compared with WT (Fig 3B), were postmeiotic (predominantly 1n). However, the MLX KO epididymal population displayed increased sub-1n DNA content, indicative of DNA fragmentation ( Fig  3K), consistent with the observed γH2AX expression. In conclusion, MLX KO males exhibit OAT, with decreased testicular St production accompanied by impaired transition from round to elongated St morphology. These immature postmeiotic cells maintain meiosis and stress markers and are shed to the epididymis where they appear to undergo apoptosis.

MLX deletion in Sertoli cells partially phenocopies the whole body deletion
Because Sertoli cells are known to play a critical role in the maintenance of the seminiferous epithelium to support GC development and prevent shedding of immature cells, we asked whether Sertoli cells were affected by MLX loss. We stained WT and MLX KO testes with anti-SOX9, which is present in Sertoli cell nuclei in a highly characteristic pattern at the periphery of seminal vesicle tubules. We found that SOX9 staining was essentially unaffected in seminal tubules from either WT or MLX KO testes with normal GC abundance ( Fig 4A). However, as noted above, a fraction of MLX KO seminiferous tubules appeared to be largely devoid of GCs yet maintained an outer ring of peripheral epithelial cells (Fig 1G). These acellular tubules in the MLX KO testes exhibited an increase in the density and frequency of peripheral SOX9 positive cells, possibly indicative of Sertoli cell dysfunction ( Fig 4A).
To determine whether Sertoli cell function was dependent on MLX, we compared our whole body deletion of Mlx with the effects of a targeted Sertoli cell Mlx deletion using Amh-Cre [26] in Mlx fl/fl mice. We found that, similar to MLX KO males, the Amh-Cre; Mlx fl/fl mice exhibited male infertility ( Fig 4B). However, unlike whole body knockout (KO) males with decreased testis and epididymis weight, there was no change in the weight of any of the tissues examined ( Fig  4C-4F). As observed in the whole body KO, Amh-Cre; Mlx fl/fl males exhibited defects in spermatogenesis, including decreased cauda epididymal sperm content resulting from diminished DSP, as well as loss of motility and abnormal morphology (Fig 4G-4J). Importantly, however, unlike whole body deletion of Mlx, the relative DSP rate was significantly higher in the Amh-Cre+ mice than in our whole body MLX KO (Fig 4K). Furthermore, mice bearing Sertoli-specific deletion of MLX did not possess acellular seminiferous tubules that are prevalent (accounting for about 10% of the total) in the whole body MLX KO mice ( Fig 4L, S4B Fig; quantified in S4C  Fig). These findings further confirm that the deleterious effects of MLX loss on fertility can originate within the testis and are consistent with the essential linkage between Sertoli cell function and spermatogenesis, including coupled glucose and lipid metabolism (reviewed in [27,28]). However, the differences in sperm production rate and lack of acellular tubules suggest that the MLX loss in Sertoli cells only partially phenocopies the constitutive loss of MLX.
In order to gauge where MondoA-MLX transcriptional activity is present, we stained WT and MLX KO testes for SOX9 and TXNIP, as a proxy. As shown in S4D Fig, TXNIP is widely expressed in the testes and greatly decreased with deletion of MLX. While TXNIP is highly expressed in human Sertoli cells (S4E Fig), the protein decreases in both GCs and Sertoli cells in the MLX KO , suggestive of MLX activity in both compartments. Consistent with this, another MondoA-MLX target, ARRDC4, is highly expressed in GC compared with Sertoli cells (S4F Fig), and Arrdc4 was recently demonstrated to support murine sperm maturation in vivo [29]. Given the widespread expression of MLX in the testis (Fig 2A and 2B), we reasoned that MLX loss is likely to have a cell autonomous effect on GC differentiation and spermiogenesis and decided to focus our further investigation on the whole body MLX KO .

Expression profiling reveals decreased spermatogenesis, altered metabolism, and increased stress in MLX KO testes
To gauge the altered transcriptome of MLX KO tissue, we used RNA sequencing (RNA-seq) to profile the RNA of whole testes from age-matched littermates of WT verified fertile breeders  (J) IF analysis of  testis and epididymis stained for the indicated protein (200×, scale bar = 200 uM). (K) Flow cytometry analysis of single cell suspension from testis and epididymis of WT versus MLX KO mice stained for DNA content. Shown for all is the mean with SEM with p-values shown from paired t test for groups of 2 and ANOVA for groups of 3 or more ( � p < 0.05, �� p < 0.01, ��� p < 0.001, ��� p < 0.0001). The underlying data for Fig 3A-3H can be found in S1 Data, and the underlying data for Fig 3K can Table). Gene set enrichment analysis (GSEA) indicated enrichment for only the Spermatogenesis Hallmark Gene Set in the WT testes, which correlates with normal spermatogenesis in these mice ( Fig 5C). However, compared with WT, the MLX KO tissue is enriched for gene expression signatures related to multiple metabolic pathways including fatty acid metabolism, glycolysis, and oxidative phosphorylation. We also noted enrichment for stress pathways in the MLX KO tissue, including inflammatory and interferon responses, tumor necrosis factor alpha (TAU : PleasenotethatTNFahasbeendefinedastumornecrosisfactoralphainthesentenceWealsonote NFα), nuclear factor kappa B (NAU : PleasenotethatNFkBhasbeendefinedasnuclearfactorkappa FκB), cytokine-activated Janus kinase (JAU : PleasenotethatJA AK)-signal transducer and activator of transcription (SAU : PleasenotethatSTAThasbeendefinedassignaltransducer TAT) and WNT signaling, and apoptosis (Fig 5C, S3 Table).
Spermatogenesis is a highly choreographed developmental program that can be separated into 3 broad categories enriched in 3 GC types (spermatogonia (Spg), spermatocyte (Sc), and spermatid (St)), which undergo, respectively, self-renewal/mitosis, meiosis, and spermiogenesis (see schematic in Fig 5D). These cellular states are lineage specified through the activities of specific TFs and their targets, several of which are listed in Fig 5D. Similar to GSEA, Enrichr analysis [33,34] for ChIP set enrichment analysis (CHEA) was employed to identify TFs associated with DEGs from WT compared with MLX KO testes. Most noteworthy is that genes downregulated in MLX KO cells are significantly associated with loss of CREM and MYBL1 (required for spermiogenesis [35] and meiosis [36], respectively), while up-regulated genes were associated with the more primitive spermatogonial TFs DMRT1 [37], OCT4 [38], and MYC [39] (S5A Fig). This suggests an incomplete block in normal spermatogenesis in MLX KO GCs with a loss of late markers and an accumulation of more primitive markers of GC differentiation.
We next asked whether genes whose expression is modulated by MLX deletion overlap with those controlled by key transcriptional regulators of spermatogenesis. We prepared volcano plots using previously reported DMRT1-bound [30] (Fig 5G) and CREM-bound [31] ( Fig 5H) genes from mouse testis that overlap with DEGs determined by our RNA-seq data. DMRT1 is known to balance mitosis and meiosis induction [40], and the trend toward up-regulation of these targets (217 up versus 117 down) is consistent with the entry of these cells into meiosis, as further evidenced by the presence of cells with 1N DNA content (see Fig 3K). The decreased expression of both MYBL1 and CREM targets is likely to be associated with stress during meiosis and spermiogenesis, respectively. Indeed, while the majority of CREM-bound targets are down-regulated in our MLX KO RNA-seq analysis (1031 down versus 534 up), we find that a large fraction of DEGs in MLX KO testes correspond to up-regulated genes previously shown to be linked to deletion of Crem, but not all of which are directly bound by CREM (622/652 up-regulated genes, as opposed to 640/898 down-regulated genes) [32] (CREM-LFC, Fig 5I). Our data implicate MLX in DMRT1-and CREM-regulated pathways critical for mammalian spermatogenesis.
In order to highlight significantly altered transcripts from the GSEA/CHEA categories identified, we generated heat maps for the top 100 down-regulated and up-regulated DEGs (Fig 5E and 5F). These include loss of both primitive SSC and Spg markers such as Eomes, Lhx1, and Prom1 (purple tabs), as well as the more differentiated St markers Tnp1, Tnp2, Prm1, and Prm2 (green tabs). We observe a concerted gain of apoptosis markers and effectors (red tabs), which include Timp1, Casp4, Atf3, Igfbp3, Casp12, and Fas up-regulation ( Fig 5F) as well as loss of Txnip, a proapoptotic protein that is MLX dependent for expression [8] (S4D Fig). Intriguingly, we also see the up-regulation of many genes associated with Sertoli dysfunction and stress, including the induction of the feminizing signaling molecule Wnt4 and stress markers known to be Sertoli enriched [41].
Immunoblotting of whole testes confirmed that a subset of these differentially expressed transcripts are also altered at the protein level. As shown in S5B Fig We employed immunofluorescence on WT and MLX KO testes to ascertain in situ, which populations of cells are altered upon loss of MLX. While both WT and MLX KO testes stain positive for the pan-GC marker DDX4, the mature St marker PGK2 is significantly decreased in MLX KO tissue ( Fig 5J). The residual PGK signal detected in Fig 5J is due to PGK1, present in interstitial and somatic cells. DDX4 expression confirms the GC fate of a large fraction of the testis cells. This, and the enrichment for DMRT1 targets among up-regulated DEGs (Fig 5G), suggests that the proliferative Spg population is still present in MLX KO testes. Consistent with this, the proliferation mark Ki67 is relatively unchanged in MLX KO

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 MLX KO 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 (sAU : PleasenotethatsiRNAhasbeendefinedassmallinterf iRNA) 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. 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 (WAU : Pleasenotethatt B) analysis of isolated cells from WT and MLX KO 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, MLX KO 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 MLX KO 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 MLX KO 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 acetylcarnitine (C2-carnitine), consistent with diminished expression of CRAT (another CREM target down in the MLX KO 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 MLX KO testes exhibited increased Fas mRNA and protein, we asked whether MLX plays a broader and cell autonomous role in regulating the FAS death unnett 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 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 MLX KO mice. As shown in Fig 6G, FASL selectively kills both MLX KO 3T3 cells and primary B cells while minimally affecting WT cells under normal culture conditions. We note that the MLX KO and WT 3T3 cells are equally viable under standard culture conditions. However, as we previously reported, MLX KO 3T3 cells undergo rapid apoptosis following enforced MYC expression [20]. Importantly, the expression of FAS protein is also elevated in MLX KO  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].

MGCTs require MLX for survival
As male GC progenitors (Spg) are the target of transformation in MGCT development, we extended our observations on the requirement for MLX in spermatogenesis and cellular survival by silencing MLX, and its dimerization partners MondoA or ChREBP, in a MGCT cell line (NTera2). Knockdown of MLX or MondoA in NTera2 cells resulted in a significant reduction in both the expression of SSC markers (Fig 6B, S6 Fig) and in viability (Fig 6H), while siChREBP has no effect on NTera2 cells, as expected, since it is not expressed. These data are consistent with both cell type-specific effects of MondoA, as well as a cell autonomous requirement for MondoA-MLX in GC tumors.
As shown by immunoblot, NTera2 cells treated with siMLX exhibited loss of MLX-dependent metabolic targets, similar to the MLX KO

MLX directly regulates male GC development in coordination with MAX
To identify genomic binding sites for MLX and MAX, we carried out chromatin immunoprecipitation and sequencing (ChIP-Seq) on WT and MLX KO  in the WT tissue and 627 gene loci in the MLX KO tissue (association defined as within 5 Kb of the transcription start site (TAU : PleasenotethatTSShasbeendefinedastranscriptionstartsiteinthesentenc SS) of a locus) (Fig 7A). Among the subset of gene promoters that exhibited binding by both MAX and MLX (Fig 7A) are promoters of previously reported targets such as Txnip and Arrdc4 as well as loci encoding protamine and transition proteins (Prm and Tnp, respectively), key genes involved in spermatogenesis (Fig 7B). Differentially expressed transcripts in WT versus MLX KO testes, as detected by RNA-seq, showed significant correlation with MLX and MAX binding (Fig 7C-7D'), suggesting that these factors contribute directly to the regulation of genes whose expression is altered upon MLX loss. While genes occupied by MAX or MLX in general tend to display decreased expression in MLX deleted testes, many MAX or MLX bound genes were also found to be up-regulated subsequent to MLX loss (Fig 7C and 7D).
Because we had observed a correlation between MLX loss and altered expression of genes regulated by male-specific TFs CREM and DMRT1 (Fig 5G and 5H), we assessed overlap between MLX and CREM and DMRT1 binding sites, revealing highly significant binding by MLX to a subset of CREM-regulated genes (Fig 7E). MAX binding is also apparent at CREM targets, although decreased in the MLX KO ( Fig 7E' and 7G). While the CREM TF binds to a significant number of DMRT1 target genes, few DMRT1 regulated genes are also bound by MLX or MAX (Fig 7E and 7E'). This is consistent with our data indicating that relative to WT, MLX KO testes exhibit significantly decreased expression of CREM-bound genes but not DMRT1-bound genes ( Fig 7F and 7F'). The CREM and DMRT1 TFs are considered to be essential mediators of spermatogenesis and spermiogenesis, and the overlap between MLX and CREM genomic occupancy is consistent with similarities between MLX and CREM loss of function and a proapoptotic phenotype in male GCs [35]. We note that the testes-specific binding to key male GC-specific genes (e.g., Prm and Tnp) (Fig 7B) is also consistent with the observed male-specific fertility phenotype of MLX null mice, as female GCs do not express protamines or transition proteins known to be associated with sperm-specific genomic compaction.
We also note that in cells from MLX KO testes, MAX occupancy is altered: MAX binds only 19% (164/874) of its targeted gene loci detected in WT testes ( Fig 7A). However, In MLX KO testes, MAX occupies new sites that it did not occupy in WT testes (Fig 7A). We observed a similar shift in MAX occupancy in MLX KO 3T3 cells (see below). We find that 60% of genes bound by MAX in MLX KO testis cells are down-regulated ( Fig 7H and 7H'). Of the 53 loci in the MLX KO testis in which de novo MAX binding appears to "replace" MLX, 9 de novo MAXbound genes were differentially expressed in MLX KO that are functionally important in the testes. These include genes known to regulate MYC (Senp1 [46] and MYCN (Ptprd [47]) and/or OCT4 protein stability (Senp1 [48], WNT signaling (Chd11) [49], and Ctnna3 [50]), promote apoptosis (Prune2 [51]), and regulate DNA damage response, specifically in spermatogenic cells (Rnf138 [52,53]) (see S4 Table). These findings indicate that the cellular response to MLX loss involves a shift in the MAX dependent transcriptional machinery to genes involved in stress response.
In order to more broadly delineate the pathways in which MLX and MAX bound genes are involved, we utilized Enrichr analysis [34] of both the Molecular Signature Database (MSigDB) as well as CHEA database of our ChIP-Seq data to identify pathways altered, as well as transcriptional regulators with a high likelihood of associating with MAX and MLX target genes.  S4 Table).

MNT and MLX TFs bind metabolic and stress targets shared with MAX
Our data showing that the majority of loci bound by MAX in MLX KO testis cells are down-regulated ( Fig 7H and 7H') raise the possibility that loss of MLX promotes heterodimerization of MAX with a transcriptional repressor within the network. To better understand the role of other network members in modulating MLX activity, we extended our genomic occupancy analysis to WT and MLX KO 3T3 cells, a system that permits us to directly assess the effect of manipulating the expression of network members. One such network member is the transcriptional repressor MNT, which has been shown to independently dimerize with MAX as well as with MLX and also to form MNT-MNT homodimers [54]. MLX, MAX, and MNT exhibit a similar genomic distribution of occupancy proximal and downstream of the TSS within regions significantly enriched for the E-Box sequence motif (Fig 8A and 8B). MAX binds to the largest share of loci, while MLX and MNT occupy a subset of loci occupied by MAX ( Fig  8C). Moreover, data from individual tracks indicate that multiple MYC network members can occupy the same promoter regions and exhibit subtle changes in occupancy (in the case of the Txnip promoter) upon MLX deletion ( Fig 8D). Interestingly, as observed for MAX in testes (Fig 7A), the genes bound by both MAX and MNT shift between WT and MLX KO 3T3 cells (S8A Fig). CHEA analysis of the 69 de novo MAX binding sites in MLX KO 3T3 cells implies a functional conservation with de novo MAX pathways in the testis (S7D and S8C Figs). Both MLX and MNT deletions independently induce increased protein expression of BIM and the intrinsic stress response protein ATF4 (Fig 8E), suggesting that MNT-MLX heterodimers contribute to the response to stress. Notably, loss of MNT induces TXNIP (which is lost upon MLX deletion), indicating that it likely functions in concert with MAX and/or MLX to suppress TXNIP expression, thereby promoting glycolysis (see model in Fig 8F). To further explore the potential role of MNT in apoptosis suppression in GCs, we treated male GC-derived lines with siRNAs against MLX and MNT. While no effect was observed in GC-2 mouse Sc-like lines treated with siRNA against MLX, MondoA, or MNT (S9A Fig), siRNA against MNT in the GC-1-spg Spg-like mouse GC line decreased viability to a similar extent as siRNAs against MLX (Fig 9A). Moreover, siMNT treatment of GC-1-spg cells strongly induced both TXNIP and BIM (Fig 8C) as we earlier described observed in MNT KO 3T3 cells. These responses may be related to the elevated expression of MNT in GC-1-spg compared with GC-2 cells (S9B Fig). MNT expression, as well as expression of MLX and Mon-doA, is also required for the survival of human MGCT-derived NTera2 cells (Figs 6H and 9B). Furthermore, loss of either MLX or MNT triggers cell death in the NTera2 cell line by activating similar stress pathways (e.g., BIM, γH2AX, cleaved-PARP) as up-regulated in the tissues and cells derived from MLX KO mice (Figs 8E and 9E). Together, our data support roles for MLX and MNT in the growth and survival of multiple cell types.
We previously reported that MondoA knockdown in MYCN-driven neuroblastoma cells leads to induction of apoptosis due, at least in part, to decreased FASN (fatty acid synthase) expression and attenuated fatty acid biosynthesis. The MondoA deficient neuroblastoma cells were rescued by addition of oleic acid (OA; a monounsaturated C18:1 fatty acid) [20]. We confirmed that this was also the case in MondoA knockdown NTera2 cells (S9D Fig). Because

PLOS BIOLOGY
The MLX transcription factor is required for male fertility in mice MLX is a functional heterodimeric partner for MondoA, we next tested whether OA treatment of MLX or MNT knockdown cells affected their growth and survival. As shown in Fig 9D, OA rescued growth of NTera2 cells arrested by knockdown of MLX or MNT. Moreover, OA reversed the decreased levels of MYCN, MAX, OCT4, and FASN expression in siMLX treated NTera2 cells (Fig 9E and 9F). Furthermore, MNT knockdown increased FASN expression ( Fig  9G, S9C Fig), while having no effect on MYCN, MAX, or OCT4 levels (Fig 9G). These findings are consistent with TCGA data from MGCTs where higher MLX transcript levels positively correlate with FASN expression, while MNT expression is inversely correlated with FASN ( Fig  9H). Taken together, these findings suggest that MNT represses a subset of MondoA-MLX targets (modeled in Fig 9I). Moreover, as fatty acid synthesis is associated with MGCTs in vivo [55], we hypothesize that MLX regulates lipid homeostasis in normal and transformed GCs.
Lastly, to extend our findings on the relevance of network activity to human male fertility, we investigated MNT expression in the Human Protein Atlas scRNA-seq database, which showed high GC expression, enriched in the Spg and Sc (S9E Fig). We next examined a dataset comparing normospermic to teratozoospermic men [56] that indicated a significant correlation of male sterility with decreased levels of mRNA encoding MLX, MNT, as well as CREM and its targets (including TNP and PRM transcripts) and increased levels of BCL2L11, FASN, and XBP1 (S9F Fig). These findings match many of the key changes observed in the MLX KO testes compared with WT and are consistent with a role for MNT-MLX interactions and their shared transcriptional targets in aspects of human male fertility by acting as regulators of mammalian spermatogenesis.

Discussion
Here, we describe a previously unexplored function of the MLX-based arm of the extended MYC network, namely an absolute requirement for MLX (and MondoA) in normal testis development and function. Inactivation of the MLX arm of the network leads to male-specific sterility, altered metabolism, and increased stress, accompanied by widespread activation of apoptosis in spermatogenesis. MYC, MYCN, MGA, and MAX have all been shown to play distinct and critical roles in male GC development. MYC/MYCN (and MAX) regulate SSC function, metabolism, and proliferation through PDPK1 [39], whereas MAX (heterodimerized with MGA) represses meiosis-associated transcription [57]. All 4 of these genes are also essential for normal embryonic development [16,17,[58][59][60]. By contrast, MLX (this report) and MondoA [19] are both dispensable for embryogenesis (Fig 1). Our characterization of spermatogenesis in MLX KO testes, as well as our genetic and genomic analyses, suggests that infertility is due to accumulation of cell autonomous defects in developing sperm as well as to a loss of Sertoli cell function in supporting survival and differentiation at multiple stages of spermatogenesis. The model depicted in Fig 10 posits that MLX loss in Spg and primary Scs leads to an inability to properly complete meiosis and a failure to produce normally differentiated round and elongated St and mature spermatozoa.
Human idiopathic OAT, which shares several characteristics with the MLX KO phenotype, is typically associated with a variety of metabolism-related pathologies, including metabolic syndrome, diabetes, obesity, and inflammation [61], all of which have been reported to be linked with dysregulation of MLX's dimerization partners MondoA and/or ChREBP (reviewed in [62,63]). While Mlx is not essential for normal embryonic development, previous studies have shown that MLX loss does lead to markedly diminished viability in a subset of cell types in vivo and ex vivo in the context of intrinsic and extrinsic metabolic stress (e.g., splenocytes, 3T3 cells, and tumors (e.g., MYCN-amplified neuroblastomas and MGCT)). This is consistent with the key role of nutrient sensing in male spermatogenesis and suggests possible routes of dysregulation associated with idiopathic OAT in humans.
Relevant to a critical role for MLX in metabolism during spermatogenesis, we observed that the serum and testes of MLX KO mice exhibit altered abundance of metabolites, consistent with a shift in glucose metabolism from oxidative phosphorylation, via the tricarboxylic acid (TAU : Pleaseno CA) cycle, to the production of lactate and to oxidation of alternative TCA substrates, such as branched-chain amino acids and fatty acids. Deletion of MondoA in mice leads to a similar change in serum metabolites, associated with enhanced glycolysis and activation of beta-oxidation [10,19]. Moreover, loss of the MondoA-MLX target TXNIP is sufficient to induce a similar metabolic profile, including augmented CPT1A expression and activity [42]. This suggests that the choice of oxidative substrate for the TCA cycle in testis is controlled at least in part by MondoA-MLX through its regulation of TXNIP. These metabolic alterations, observed in the MLX KO testes, are associated with GC apoptosis, concomitant with aseptic inflammation and immune cell activation, symptoms also associated with male infertility (reviewed in [64]). While MLX and TXNIP have been linked to inflammation [15], loss of TXNIP alone does not result in male sterility [65]. This suggests that critical MLX transcriptional targets in addition to TXNIP are responsible for the majority of the male-specific sterility phenotypes associated with loss of either MLX or MondoA. An example of such is Arrdc4, a bona fide MondoA-MLX target, bound by MLX in our dataset, and it has recently been shown to be required for normal male fertility in the mouse [29].
Our earlier work in MYCN amplified neuroblastomas following MondoA or MLX knockdown linked growth arrest and apoptosis with attenuated lipid biosynthesis that could be rescued by OA. Here, we find that MLX KO testes produce significantly increased levels of multiple acyl-carnitine species and increased levels of carnitine palmitoyltransferase (CPT1A) relative to WT, indicative of altered lipid metabolism and increased stress. Indeed, many DEGs directly bound by MLX have been shown to be involved in lipid metabolism and in spermatogenesis (see S4 Table and references therein). Therefore, altered expression of MLXdependent target genes controlling lipid metabolism may be at least 1 contributor to the stress accompanying MLX loss of function. Moreover, our data showing that MGCTs such as NTera2 are dependent on MLX, and whose growth arrest upon MLX knockdown is reversible by OA treatment, underscore the importance of lipid synthesis in MLX dependence.
Our transcriptional profiling and genomic occupancy analyses using ChIP-Seq has identified many other direct targets (i.e., genes bound and regulated) of MLX in the testes, including metabolic and stress effectors as well as many genes relevant to male-specific GC development (see S4 Table). Among the latter are a small subset of approximately 5,800 genes previously shown to be bound by the essential transcriptional regulator of spermiogenesis, CREM [31]. These MLX/CREM shared targets include genes encoding factors such as protamines and transition proteins as well as enzymes critical for apoptosis, mitochondrial activity, glucose, and lipid metabolism. All of these have been shown to be present and functional in spermatogenesis (S4 Table and references therein). The MYC/MYCN and CREM target PDPK1 is similarly bound by MLX and MAX and down-regulated in the MLX KO testes (S4 Table). PDPK1 is required for SSC function and also stabilizes MYC protein [39,66]. Important for metabolic Fig 8A-8D can be found in S1 Data. ChIP-Seq, chromatin immunoprecipitation and sequencing; E-box, Enhancer box; KO, knockout; MLX, MAX-Like protein X; TF, transcription factor; TSS, transcription start site; WB, western blot; WT, wild-type.

MAX, and MNT in 3T3 cells with p-value calculated from a hypergeometric test. (D) ChIP-Seq tracks for MLX, MAX, and MNT on the indicated gene promoters from WT and MLX KO 3T3 cells. (E) WBs from WT and MLX KO and WT and MNT KO littermate 3T3 cell lines probed for the indicated proteins. (F) Diagram summarizing modes of MondoA, MNT, and MLX association and consequent transcriptional responses. The underlying data for
https://doi.org/10.1371/journal.pbio.3001085.g008 regulation of sperm motility are a number of sperm-specific glycolytic enzymes, such as PGK2 and GAPDHS. MLX targets Gapdhs, and loss of GAPDHS results in infertility and nonmotile spermatozoa [67]. Intriguingly, loss of either PGK2 or GAPDHS results in decreased motility, as well as elevated acyl-carnitine levels [68], similar to MLX KO . This is supportive of multiple levels of cross talk between glucose, glucose sensing, and lipid metabolism. MLX also shares target genes with CREM including a number of phospholipases required for male fertility including Ddhd1 [69] and Plcb1 [70]. These data strongly support the notion that MLX and its binding partners act as transcriptional mediators of critical events in mammalian spermatogenesis in concert with other transcriptional regulators such as CREM and MAX. Interestingly, neither MLX nor MAX occupy a significant number of loci that are targets of the DMRT1 testes-specific transcriptional regulator of differentiation. Thus, the direct activity of MLX and other MYC network members in spermatogenesis may, to some extent, be more focused on functions regulated through the CREM pathway.
Many of the MLX-linked metabolic and stress targets observed in testes are not cell type specific, and we find their expression altered in both seminiferous tubules and testicular interstitial cells, as well as in 3T3 cells derived from WT versus MLX KO embryos. MLX loss of function sensitizes these cells to apoptotic stimuli and effectors known to be dependent upon MYC (e.g., FASL-FAS interactions and BIM) [71,72]. Taken together, these data support not only a direct role for MLX in regulation of metabolic targets but also a role for transcriptional repression of apoptosis effectors (such as BIM), most likely mediated by MLX heterodimerization with repressors such as MNT.
Our genome-wide occupancy analyses demonstrate that genes bound by MLX are also bound by other members of the MYC network, such as MAX and MNT. Intriguingly, in the absence of MLX, we detect a shift in the occupancies of these other factors: For example, in MLX KO 3T3 cells, MAX and MNT bind to loci that they did not occupy in WT cells. Our analysis of loci newly occupied by MAX (de novo sites) following MLX deletion show that these comprise many genes implicated in the response to stress (S4 Table, blue highlighted section), suggesting a modulation in transcriptional programming upon MLX loss. Of particular note are loss of Rnf138 and induction of Prune2 whose altered expression would be expected to promote spermatogonial apoptosis [51,52]; Cdh11, a proapoptotic inhibitor of catenin signaling [49]; and Acadl, induced along with other beta-oxidation enzymes in Sertoli cells by phagocytosis of dead GCs [73]. Importantly, this phagocytosis by Sertoli cells is required for male fertility to recycle lipid and other metabolites to the developing primitive GCs, as blocking it results in infertility [68].
Our study further implicates the extended MYC network, and specifically its nutrient-sensing MLX arm, in the direct regulation of, and linkage between, differentiation, metabolism, and apoptosis. Importantly, metabolic programs change along with changes in cellular state and thus must be responsive to both extrinsic signals, such as mitogenic cues (via effectors  [75]. It is clear that functional interactions among MYC network members are relevant, not only in MYC-driven oncogenesis, but also during testes development (this work), and regeneration of both skeletal muscle and liver [76,77]). We anticipate that further genetic perturbation of the network in distinct biological contexts, coupled with high-resolution genomic analysis, will yield important insights into the molecular control of normal and abnormal cellular behavior in both tissue homeostasis and oncogenesis.

Animal use
All experiments involving mice were carried out under accordance with the guidelines of the following institutions: Ethics statement: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocol 50783 of the Fred Hutchinson Cancer Research Center. The animal experiments conducted at the University of Utah were performed under IACUC protocol 15-04012. Mice were euthanized by asphyxiation under carbon dioxide according to IACUC protocol. Every effort was made to minimize pain and suffering.

Generation of Mlx −/− mice
The Mlx KO allele was created in our lab by a homologous recombination targeted method in 129S4 AK7 murine ESCs. The targeting construct is in the PGKneoF2L2DTA backbone and is based upon the coding sequence of Mlx transcript variant 1 (encoding MLX-α protein). This construct includes in 5 0 to 3 0 order: (1) a 1,546 base pair (bp) 5 0 -homology arm including exons 1 and 2 of Mlx; (2) a 1,717 bp Loxp-flanked region encoding exons 3 to 6 (bHLHLZ domain) of Mlx; (3) a FRT-flanked PGKNEO-positive selection cassette; and (4) a 2,873 bp 3 0 homology arm spanning exon 7 of Mlx transcript variant 1 and a pGKDTA-negative selection cassette. Selected ESC clones were injected into blastocysts to generate chimeric animals. These chimeras were bred to ROSA26 FlpO/FlpO females [78] in the 129S4 co-isogenic background to remove the Frt-flanked NEO cassette to generate conditional KO mice or Meo2-Cre delete mice [79] to generate total KO mice. The FlpO allele or the Meox2-Cre allele was subsequently crossed out. Two independent mouse lines from independent ESC clones were found to be phenotypically indistinguishable. All the targeting mice were confirmed by PCR and Southern blotting with 5 0 external, 3 0 external, and NEO probes. To generate Sertoli cell-specific deletion of Mlx, floxed animals were bred with mice expression transgenic Cre-recombinase under the control of the Amh promoter [26]. derived residual bodies and apoptotic cells. These features are critical for male fertility, and there is a tight coordination between the Sertoli and GCs at every stage of development. (B) Constitutive deletion of Mlx leads to infertile male MLX KO mice. This is driven by loss of MLX targets, decreased MYCN expression, alterations to MAX function leading to perturbation of normal metabolism, GC apoptosis, and lack of proper spermiogenesis and maturation. There is also shedding of immature cells from the testis to the epididymis, suggestive of altered phagocytosis by Sertoli cells. Sertoli dysfunction is supported by the fact that, as shown in (C), Sertoli-specific Mlx deletion also results in male infertility. However, the block to DSP and the extent of apoptosis are significantly less severe than observed in the constitutive deletion (B) underscoring the cell autonomous requirement for MLX in GC differentiation. DSP, daily sperm production; E. St, elongating spermatid; GC, germ cell; KO, knockout; MLX, MAX-Like protein X; R. St, round spermatid; SSC, spermatogonial stem cell; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001085.g010

PLOS BIOLOGY
The MLX transcription factor is required for male fertility in mice

Mating tests
Pairs of sexually mature mice (1 of each sex) were housed in the same cage, and mating was observed with the seminal plugs and number of pups born recorded. For mendelian frequency, heterozygotes were bred with heterozygotes, and the progeny (F1) were genotyped. For KO mating tests, homozygous null males or females were bred with WT mice.

Metabolomics
Reagents. Acetonitrile, ammonium acetate, and acetic acid (LC-MS grade) were all purchased from Fisher Scientific (Pittsburgh, Pennsylvania, United States of America). The standard compounds corresponding to the measured metabolites were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and Fisher Scientific.
Serum sample preparation and LC-MS/MS measurement. Male mice (3 of each genotype WT, MLX KO ) were bled by retro-orbital eye bleed, and serum was isolated, then flash-frozen on dry ice before subsequent use for LC-MS/MS. Frozen serum samples were first thawed overnight at 4˚C, and 50 μL of each sample was placed in a 2-mL Eppendorf vial. The initial step for protein precipitation and metabolite extraction was performed by adding 500 μL MeOH and 50 μL internal standard solution (containing 1,810.5 μM 13 C 3 -lactate and 142 μM 13 C 5 -glutamic acid). The mixture was then vortexed for 10 seconds and stored at −20˚C for 30 minutes, followed by centrifugation at 14,000 RPM for 10 minutes at 4˚C. The supernatants (450 μL) were was collected into a new Eppendorf vial and dried using a CentriVap Concentrator (Labconco, Fort Scott, Kansas, USA). The dried samples were reconstituted in 150 μL of 40% PBS/60% ACN. A pooled sample, which was a mixture of all serum samples, was used as the quality control (QC) sample.
The LC-MS/MS experimental procedures were well documented in our previous studies [20,[80][81][82][83][84][85]. Briefly, all LC-MS/MS experiments were performed on a Waters Aquity I-Class UPLC-XenoTQ-S micro (Waters, Milford, Massachusetts, USA) system. Each sample was injected twice, 10 μL for analysis using negative ionization mode and 2 μL for analysis using positive ionization mode. Both chromatographic separations were performed in hydrophilic interaction chromatography (HILIC) mode on a Waters XBridge BEH Amide column (150 × 2.1 mm, 2.5 μm particle size, Waters). The flow rate was 0.300 mL/min, auto-sampler temperature was kept at 4˚C, and the column compartment was set at 40˚C. The mobile phase was composed of Solvents A (5 mM ammonium acetate in 90%H 2 O/ 10% acetonitrile + 0.2% acetic acid) and B (5 mM ammonium acetate in 90%acetonitrile/ 10% H 2 O + 0.2% acetic acid). After the initial 2 minutes isocratic elution of 90% B, the percentage of Solvent B was linearly decreased to 50% at t = 5 minutes. The composition of Solvent B maintained at 50% for 4 minutes (t = 9 minutes), and then the percentage of B was gradually raised back to 90%, to prepare for the next injection. The mass spectrometer is equipped with an electrospray ionization (ESI) source. Targeted data acquisition was performed in multiple reaction monitoring (MRM) mode. We monitored 121 and 80 MRM transitions in negative and positive mode, respectively (201 transitions in total). The whole LC-MS system was controlled by MassLynx software (Waters). The extracted MRM peaks were integrated using TargetLynx software (Waters).
Cell sample preparation and LC-MS/MS measurement. Isolated cells from the seminiferous tubules of age-matched WT and MLX KO mice (N = 3) were separated into 4 technical replicates of 1 × 10^6 cells each and flash frozen on dry ice. Soluble metabolites were extracted into 1 ml of 20:80% water:methanol before clearing the insoluble and drying down on a Speed-Vac before subsequent LC-MS/MS.
The LC-MS/MS experiments were performed on an Agilent 1260 LC-6410 QQQ-MS (Agilent Technologies, Santa Clara, California, USA) system. Moreover, 5 μL of each sample was injected for analysis using positive ionization mode. Chromatographic separation was performed using a Waters XSelect HSS T3 column (2.5 μm, 2.1 × 150 mm). The flow rate was 0.3 mL/min. The mobile phase was composed of Solvents A (100% H 2 O with 0.2% formic acid) and B (100% ACN with 0.2% formic acid). After the initial 0.5 minutes isocratic elution of 100% A, the percentage of Solvent A was linearly decreased to 5% at t = 10 minutes. Then the percentage of A remained the same (5%) for 5 minutes (t = 15 minutes). The metabolite identities were confirmed by spiking with mixtures of standard compounds. The extracted MRM peaks were integrated using Agilent Masshunter Workstation software (Agilent Technologies).
Serum T quantification. Blood was collected by either retro-orbital eye bleed or cardiac stick and heparinized plasma was isolated. T levels were determined by ELISA at the University of Virginia Center for Research in Reproduction (NICHD Grant # U54-HD028934) Charlottesville, Virginia.
Cell isolation from testicular tissue. Testes and epididymides were dissected, defatted, and processed as follows for cellular isolation: For seminiferous tubule cell isolation, testes were dissected, decapsulated, and subjected to an enzymatic digestion to isolate seminiferous tubules from the interstitial stromal cells (including Ledig and immune cells). Isolated tubules were then digested to release a single cell suspension of the seminiferous epithelium including GCs and Sertoli cells. For epididymal GCs, epididymides were dissected, and the caudal portion was cut to release cellular content of sperm. Single cell suspension was filtered to remove tissue yielding predominately mature spermatozoa from WT tissue. Spermatogenesis analysis. Testes and epididymides were dissected, defatted, and processed for analysis as previously described [86]. For calculation of testicular DSP rate (St count), testes were weighed and homogenized in 0.1 M sodium phosphate buffer (pH 7.4) with 0.1% Triton X-100 via 8 strokes of a 15-ml Kontes homogenizer. Homogenization-resistant St were counted on a hemocytometer, and St per gram per testis were calculated [87].
RNAi transfection. Flexitube Gene Solutions siRNA mixtures (QiAU : Pleaseprovidethemanufactu agen, Germantown, MD, USA) were utilized to knockdown the indicated target. RNAiMax (Qiagen) was used according to the manufacturer's conditions. Cells were counted 72 to 96 hours posttransfection, and viability was monitored by trypan blue exclusion. For OA treatment, Oleic Acid Water Soluble (Sigma-Aldrich) was resuspended in sterile water. B220+ cell purification. Spleens were smashed, and B220+ B cells were purified from the splenocytes using AutoMACS system according to the manufacturer's recommendations. Purity was routinely over 95% pure, as assessed by flow cytometry.
Flow cytometry. Testis tissue was prepared as in [88]. Isolated seminiferous tubule or epididymal cells were resuspended in staining buffer with HOECHST, and samples were run on a Canto-2 Flow Cytometer and analyzed by FACS-Diva Software.
IHC tissue staining. Testicular and epididymal tissue was fixed in Modified Davidson's Fluid (MDF) as described [89], then embedded in paraffin and sectioned onto slides at 5 micron thickness. Slides were deparaffiinized, rehydrated, and antigen retrieval was utilized. For immunofluorescent IHC, Dako reagents were used (Block, Primary Dilution Buffer, Antifade mounting media), and Alexa-Flour 488nm secondary antibodies were used in combination with DAPI for staining. For tissue staining and IHC, samples prepared as described above were submitted to the FHCRC Experimental Histopathology Core and either stained with hematoxylin and eison, or stained with the indicated antibodies, visualized with the cromophore DAB and counterstained with hematoxylin to mark nuclei.
The staining of previously published Xenograft tissue of the NTera2 cells [45] were carried out by the Institute of Pathology of the University Medical Center Göttingen. Briefly, 4 micron thick sections were mounted on slides, deparaffinnized, rehydrated, and antigen retrieval was utilized. The slides were stained with primary antibody then biotinylated secondary antibodies using a REAL Detection System (LSAB+ kit; Dako). The signals were visualized using a REAL Streptavidin Alkaline Phosphatase kit (Dako), while Ki-67 staining was visualized with DAB. All samples were counterstained with hematoxylin, mounted in super mount medium, and analyzed via light microscopy.
WBs. Tissues and cell pellets were lysed in RIPA buffer. For whole testes sample preps, tissue was homogenized mechanically to facilitate lysis. Lysates were quantified by BCA assay (PAU : PleaseprovidethemanufacturerlocationforPierceinthesentenceLysateswerequantifiedby:::: ierce Biotechnology, Waltham, MA, USA) or normalized to cell number for equal loading. Samples were resolved on NuPAGE 4-12% Bis-Tris gradient gel before transferring to Nitrocellulose (0.2 micron). Blots were blocked with 5% Milk in TBST, washed with TBST, and probed with primary and secondary antibody in 5% Milk in TBST. The secondary antibody was HRP conjugated, and chemiluminescent detection was employed. Blots were exposed to Pro-Signal Blotting Film (GAU : PleaseprovidethemanufacturerlocationforGeneseeScientificinthesenten enesee Scientific, San Diego, CA, USA). RNA extraction and sequencing. RNA was extracted with Trizol reagent, quantified on a TapeStation. A total of 500 ng of RNA was submitted for library preparation through FHCRC Genomics Core. Libraries were aligned to mm10 using TopHat then processed with EdgeR or DE-Seq. Data were analyzed with GSEA, and volcano and violin plots were generated using ggplot.
ChIP-Seq. We performed ChIP-seq as previously described [90] with modifications to improve solubility of TFs, which tended to vary depending on cell type. The chromatin preparations from the testes were from a pool of testes from 6 WT/KO animals, and this material was not treated with MNase. The chromatin preparations from the B cells and 3T3 cell lines were treated with MNase. Briefly, after formaldehyde cross-linking, cell lysis, and chromatin fragmentation with MNase, the final SDS concentration after dilution of total chromatin was increased to 0.25% with addition of 20% SDS stock solution. Sonication was performed in a Covaris M220 focused ultrasonicator for 12 minutes with the following settings: 10% duty cycle, 75W peak incident power, 200 cycles/burst, and 6to 7˚C bath temperature. The SDS concentration of the sonicated chromatin solution was readjusted to 0.1% with dilution buffer. Immunoprecipitation was performed on the clarified chromatin (input) fraction from 10 × 10^6 cellular equivalents, and DNA was purified was using standard phenol:chloroform extraction. . We used 10 μg of antibody for each immunoprecipitation. To purified ChIP DNA, we added 10 pg of spike-in DNA purified from MNase-digested chromatin from Drosophila melanogaster S2 cells or Saccharomyces cerevisiae [91] to permit comparison between samples. DNA was then subjected to library preparation as previously described [92,93], and 25 × 25 paired-end sequencing was performed on an Illumina HiSeq 2500 instrument at the Fred Hutchinson Cancer Research Center Genomics Shared Resource.
For the B cells samples, an alternative library preparation was employed [94]. Sequencing datasets were aligned to the mouse mm10 genome assembly using Bowtie2. Datasets were also aligned to the dmel_r5_51 (D. melanogaster) or sc3 (S. cerevisiae) assemblies using Bowtie2 depending on the source of the spike-in DNA. Counts per bp were normalized as previously described by multiplying the fraction of mapped reads spanning each position in the genome by genome size [95] or by scaling to spike-in DNA [91]. Peaks were called using MACS. Plots were generated with ngs plot [96], or the R package ggplot2 Motif enrichment was done using HOMER [97]. Statistical analysis. Graphpad Prism was used for all statistical data analysis unrelated to NGS datasets. p-Values were calculated by hypergeometric test Student t test or 1-way ANOVA with a Dunnett test, when appropriate. For metabolomics analysis, MetaboAnalyst [25] was used. Gene lists of differentially expressed and/or bound targets were analyzed by Enrichr [34] and visualized using Appyter [98]. For RNA-seq and ChIP-Seq analysis, R Studio and Python were used. SAU : AbbreviationlistshavebeencompiledforthoseusedthroughoutSupportinginformat upporting information S1 Fig. (goes with Fig 1).