MXD/MIZ1 complexes activate transcription of MYC-repressed genes

MXD proteins are transcription repressors that antagonize the E-box dependent activation of genes by MYC. MYC together with MIZ1 acts also as a repressor of a subset of genes, including cell cycle inhibitor genes such as p15 and p21. A role of MXDs in regulation of MYC-repressed genes is not known. Here we report that MXDs are functionally expressed in U2OS cells and activate transcription of p15 and p21, and other MYC-repressed genes. Activation of transcription was dependent on the interaction of MXDs with MIZ1, and on an intact DNA binding domain. MIZ1-binding deficient MXD mutants interacted with MAX and were active as repressors of MYC-activated genes but failed to activate MYC-repressed genes. Mutant MXDs with reduced DNA binding affinity interacted with MAX and MIZ1 but neither repressed nor activated transcription. Overexpression of MXDs attenuated proliferation of U2OS cells predominantly via MIZ1-dependent induction of p21. Our data show that MXDs and MYC have a reciprocally antagonistic potential to regulate transcription of mutual target genes.


Introduction
MYC-associated factor X (MAX) and its binding partners comprise a family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors, which are implicated in the regulation of cell growth, proliferation, differentiation, apoptosis and tumorgenesis (Carroll et al., 2018;Conacci-Sorrell et al., 2014;Poole and van Riggelen, 2017).
Complexes of MAX with MYC and its homologs MYCN and MYCL bind to enhancer-box motifs (E-boxes) and promote expression of target genes (Conacci-Sorrell et al., 2014).
Under physiological conditions MYC is expressed in response to mitogens and promotes cell growth and proliferation (Armelin et al., 1984;Carroll et al., 2018;Hasmall et al., 1997;Lutterbach and Hann, 1994;Wang et al., 2011). Elevated expression or activation of MYC is associated with uncontrolled cellular growth and proliferation and supports the development of cancer, and MYC or its homologues are overexpressed, amplified or deregulated in many cancer types (Dang, 2012;Kalkat et al., 2017). MYC has been reported to function as a regulator of specific target genes (Kress et al., 2015;Muhar et al., 2018;Sabo et al., 2014;Walz et al., 2014) and/or as a general amplifier of transcription of active genes on a genome-wide scale (Baluapuri et al., 2019;Gerlach et al., 2017;Lin et al., 2012;Nie et al., 2012). MYC interacts with several coactivators and RNA-polymerase II (Pol II) associated factors including chromatin modifiers, transcription initiation and elongation factors that are implicated in transcription activation, but also with several complexes and assemblies involved in transcriptional repression (Baluapuri et al., 2019;Kress et al., 2015;Poole and van Riggelen, 2017).
MYC has been shown to facilitate the release of promoter-proximally paused Pol II (Rahl et al., 2010), enhance mRNA capping (Cowling and Cole, 2010), facilitate the transfers PAF1 to Pol II (Gerlach et al., 2017;Jaenicke et al., 2016), and enhance rate and processivity of transcription elongation by loading SPT5 onto Pol II (Baluapuri et al., 2019), and thereby support transcription of most active genes. In contrast, rapid depletion of MYC in leukemia and colon cancer cell lines affects transcription of a small subset of genes, suggesting that expression of a rather limited set of activated genes might depend on the presence of MYC (Muhar et al., 2018).

The activation of genes by MYC/MAX is antagonized by the MAX-Dimerization (MXD)
proteins, and MAX Network Transcriptional Repressor (MNT), henceforth collectively referred to as MXDs and MGA (Carroll et al., 2018). MXDs and MGA are bHLH transcription factors that form complexes with MAX and bind to the same E-boxes as MYC/MAX (Carroll et al., 2018;Conacci-Sorrell et al., 2014). MXDs recruit via their SID domain mSIN3-HDAC1/2 co-repressor complexes and repress transcription (Laherty et al., 1997;van Riggelen et al., 2010b).
Opposite to MYC, MXDs support cell cycle arrest and differentiation Lahoz et al., 1994;Yang and Hurlin, 2017). Genetic studies in mice confirmed the antagonism between MYC and MXDs. MXD1 -/mice show increased proliferation and detained differentiation of granulocyte precursors (Foley et al., 1998). Mice lacking MXD2(MXI1) display multiple histological abnormalities due to increased cell proliferation in several tissues, and are more susceptible to spontaneous and induced cancerogenesis (Schreiber-Agus et al., 1998). Depletion of MNT triggers increased cell proliferation (Hurlin et al., 2003;Nilsson et al., 2004). Mice bearing a deletion of Mnt in mammary glands develop spontaneous tumors with increased frequency, phenocopying transgenic overexpression of MYC (Toyo-oka et al., 2006). Finally, human MNT, MXD1 and MXD2 genes are located in regions that are frequently mutated in different cancer types (Cvekl et al., 2004;Edelmann et al., 2012;Schaub et al., 2018;Shapiro et al., 1994;Wechsler et al., 1994).
MYC, in particular in oncogenic or overexpressed conditions, has also the potential to repress transcription. The underlying mechanisms are not fully understood and a comprehensive set of bona-fide MYC-repressed genes is not known. This is in part due to the fact that MYC supports cell growth and proliferation, and thus, directly or indirectly promotes expression of all genes when compared to the transcription rates of resting cells. Hence, upon normalization lower than average activation of genes may appear as relative repression even though the genes are actually activated . However, MYC has been shown to interact with the zinc-finger transcription factor MIZ1 (ZBTB17) and the related transcription factors, SP1 and YY1 (Poole and van Riggelen, 2017). MIZ1 regulates embryonic development and differentiation (Adhikary et al., 2003;Walz et al., 2014;Wolf et al., 2013;Wolf et al., 2015). MYC in association with MIZ1 has been shown to repress genes, including the cyclin-dependent kinase (CDK) inhibitor genes p15 (CDKN2B), p21 (CDKN1A) and p27 (CDKN1B) and the circadian transcription factor genes BMAL1 (ARNTL), CLOCK and NPAS2 (Shostak et al., 2016;Staller et al., 2001;Walz et al., 2014;Wu et al., 2003;Yang et al., 2001). Mutations compromising the interaction of MYC with MIZ1 specifically affect the repressing but not the activating potential of MYC (Herold et al., 2002;Shostak et al., 2016;Si et al., 2010), indicating that MYC together with MIZ1 has the potential to directly repress transcription. In oncogenic conditions overexpressed MYC may recruit MIZ1 to a larger number of genes and attenuate their transcription (Lorenzin et al., 2016;Walz et al., 2014;Wolf et al., 2015). A low ratio of MYC versus MIZ1 occupancy (Lorenzin et al., 2016) and/or low relative affinity of promoters for MYC (de Pretis et al., 2017)  The development of lymphoma in mice is critically dependent on the interaction of MYC with MIZ1 (van Riggelen et al., 2010a). When the MYC/MIZ1 interaction is challenged by mutation, the repressive capacity of MYC is decreased, its pro-proliferative functions are reduced, and self-renewal of stem cells is compromised (Kerosuo and Bronner, 2016;Shostak et al., 2016).
In this study we addressed specifically the question whether and how MXDs have the potential to impact expression of genes that are repressed by MYC together with MIZ1.
Using U2OS cells, which endogenously express MXDs, we report the surprising observation that MNT, MXD1, and MXD2 activate transcription of specific MYCrepressed genes, in parallel to their known function as transcriptional repressors of MYC-activated genes. We show that activation of transcription by MXDs relies on their physical interaction with MIZ1, and requires functional DNA binding and corepressor recruitment domains. We show that MXDs inhibit U2OS cell growth and proliferation through activation of MYC-repressed genes, and MXD-dependent activation of p21 was particularly crucial.

Endogenous MXDs activate the p21 core promoter in U2OS cells
MXDs repress genes that are activated by MYC but a role of MXDs in the regulation of genes that are repressed by MYC has not been investigated. Since MXDs belong, like MYC, to the bHLH-ZIP family, we analyzed available ENCODE ChIP-seq data of MXD2 (GSM935498), MNT (GSE91968) and MYC (GSM822286, GSM822301). As expected, the analysis revealed that MXD2 and MNT binding overlaps with MYC binding (Fig. 1A).
MXD2 and MNT were also recruited to MIZ1 binding sites in genes that are repressed by MYC such as p21 and VAMP4 (Fig. 1A). Expression of MXDs is tightly regulated and cell-type specific (Hooker and Hurlin, 2006). Available RNA-seq data indicate that MIZ1, MYC as well as its antagonists MXD1, MXD2, and MNT are expressed in U2OS cells (Fig. 1B) (Elkon et al., 2015;Ibarra et al., 2016). To assess whether MXDs are functional and impact expression of MYC repressed genes we analyzed expression of a p21-luc reporter. This luciferase reporter contains only the core promoter of the p21 gene. Hence, it is highly likely that putative changes in p21-luc repression are directly due to regulation of transcription. p21-luc was previously shown to be repressed by MYC in MIZ1-dependent manner (Shostak et al., 2016;Wu et al., 2003). When U2OS cells were transfected with siRNAs against MYC the expression level of p21-luc was elevated (Fig. 1C), indicating that endogenous MYC had limited its expression. Surprisingly, expression of p21-luc was reduced when cells were treated with previously validated siRNAs against MXDs (Corn et al., 2005;Wu et al., 2012;Xu et al., 2007)  The above measurements were done with confluent U2OS cells, which are growing rather slowly. We chose these conditions to avoid or minimize potential indirect effects on gene expression that could be associated with MXD-dependent differences in cell number (due to growth or apoptosis). Colorimetric cell counting by WST-8 staining indicated that induction of MXDs had little impact on cell growth and viability under such conditions (Supplemental Fig. S2C), and quantification of GAPDH expression from the entire cultures confirmed the results of the WST-8 assay (Supplemental Fig. S2D).
Hence, the MXD-dependent increase in expression levels of p15, p21, and p27 (lucreporters and endogenous genes) is due to transcriptional regulation.

MXDs interact with MIZ1 and directly activate MYC-repressed genes
The bHLH domains of MYCs and MXDs are highly conserved (Fig. 3A) (Hurlin et al., 1997). In order to analyze whether MXDs physically interact with MIZ1, we expressed in

MYC and MXDs require DNA binding to modulate MIZ1 activity
To test whether MYC and MXDs require an intact bHLH domain in order to associate with MIZ1-target genes, we replaced in the basic region of the DNA binding domain two critical neighboring arginyl residues (RR) by aspartyls (DD) (see Fig. 3A). Recruitment of the corresponding mutants, MYC RR367DD , MNT RR232DD , MXD1 RR68DD , and MXD2 RR79DD , to MYC-activated and MYC-repressed genes was reduced ( Fig 4A). However, the RRto-DD substitutions in the DNA-binding domains of MYC and MXDs did not affect their ability to interact with MIZ1 in a pull-down assay (Supplemental Fig. S4A).
Similarly, RR-to-DD substitutions in the DNA binding domains of MXDs impaired their potential to repress 6xEbox-luc and their capacity to activate p21-luc (Fig. 4B). These data suggest that the DNA-binding domains of MXDs are required for repression and activation of genes. Since the RR-to-DD substitutions did not affect the interaction of MXDs with MIZ1, the data also indicate that MXDs did not indirectly activate p21-luc by squelching MIZ1 away from MYC/MIZ-repressed promoters.
MXDs harbor a SID domain by which they recruit co-repressor complexes (Laherty et al., 1997). The SID domains of MXDs are short N-terminal segments of about 20 amino acid residues (aa). We deleted the SID domains of MNT (aa 2-16), MXD1 (aa 2-20), and MXD2 (aa 2-20) (Supplemental Fig. S5). It has been previously shown that deletion of the SID domain does not affect the interaction of MXDs with MAX (Ayer et al., 1995). ChIP analysis revealed that binding of MXDSID mutants to the promoters of NCL and p21 was not compromised (Fig. 5A). Previous data (Hurlin et al., 1997) had shown that MXDSID mutants failed to repress MYC-activated genes. In agreement with these data MXDSID mutants failed to repress the 6Ebox-luc reporter in HEK293 cells (Fig. 5B). MNTSID even activated 6Ebox-luc. Surprisingly, however, the SID versions of MXDs were compromised in their ability to activate p21-luc (Fig. 5C), suggesting that the SID domain is also required for MIZ1-dependent transactivation of MYC-repressed genes.

MXDs inhibit cell growth in MIZ1-dependent manner
Expression of MXDs reduces cell growth and proliferation in various cellular models (Chin et al., 1995;Delpuech et al., 2007;Hurlin et al., 1997). Doxycycline-induced overexpression of MXDs also reduced proliferation of growing U2OS cells that were seeded at low density (Fig. 6A). To analyze whether MXDs inhibit growth in MIZ1dependent fashion we overexpressed the MIZ1-interaction mutants, MNT L258D , MXD1 L95D , and MXD2 L106D , which are functional repressors of MYC-activated genes (see Fig. 3E and Supplemental Fig. S3E, G). The capacity of MNT L258D , MXD1 L95D , and MXD2 L106D to inhibit cell growth and proliferation was severely blunted ( Finally, we addressed the role MXDs in regulating growth of U2OS cells via p21. When p21 was depleted in growing U2OS cells the growth rate did not further increase, suggesting that growth was limited by other factors. Overexpression of MXDs attenuated proliferation of U2OS cells but failed to do so when MXD-induced accumulation of p21 was prevented by siRNA treatment (Supplemental Fig. S7C, D).
Hence, the induction and/or repression of other genes by MXDs could obviously not compensate for the lack of p21. These data indicate that regulation of the CDK inhibitor gene, p21, is a major pathway by which MXDs and MIZ1 impact proliferation of U2OS cells.

Discussion
MXD proteins encompass a group of transcriptional repressors that antagonize the activation of genes by MYC (Conacci-Sorrell et al., 2014). The aim of this work was to investigate whether and how MXDs impact on such genes that are not activated but repressed by MYC. We therefore focused specifically on the regulation of a selected group of gene promoters (mainly p15, p21, and p27) that were previously shown by various means to be directly repressed by MYC in MIZ1-dependent manner (Shostak et al., 2016;Si et al., 2010;Staller et al., 2001;Walz et al., 2014;Wu et al., 2003;Yang et al., 2001), and we restricted our analyses on U2OS cells, which express MXDs as well as MYC at functional levels. We report the unexpected and surprising finding that MXDs activated transcription of this validated group of MYC-repressed genes. Thus, the antagonizing role of MXDs appears to extends to the limb of genes that are repressed by MYC, i.e., gene that are activated MYC are repressed by MXDs, and vice versa, genes that are repressed MYC are activated by MXDs. Since the activating function of MYC is antagonized by MXDs is seems conceivable that also the repressing function of MYC requires regulatory counterbalance, in particular since MYC-repressed genes include crucial inhibitors of the cell cycle.
Several lines of evidence support that transcription of genes such as p15, p21, and p27 was directly activated by MXDs. Most importantly, MXDs are functionally expressed in U2OS cells and MXD downregulation resulted in elevated expression of the selected MYC-repressed genes. We controlled WST-8 staining that the apparent induction of these MYC-repressed genes was not artificially due to differences in number and size of living cells (biomass) in MXD-induced versus control-treated cultures (e.g. by less apoptosis of MXD-induced cells).
In addition to the analysis of endogenous gene expression we measured the impact of MXDs on luciferase reporter genes to assess the transcriptional regulation of the core promoters. The p21-luc reporter contains a short, truncated core promoter with binding sites for SP1, MIZ1, and MYC (Wu et al., 2003), and also for MXDs. Thus, analysis of Together, our data suggest that MXDs activated MYC-repressed genes in direct manner in addition to their function as repressors of MYC-activated genes. The dual function of MXDs is not unprecedented as MYC itself also acts as activator and repressor, and interacts via its MYC-boxes with factors associated with transcription activation and repression (Baluapuri et al., 2019;Kress et al., 2015;Poole and van Riggelen, 2017;Tu et al., 2018). The conditions and mechanisms specifying MYC as either (general) activator or repressor of particular genes are only partly understood.
MXDs repress MYC-activated genes by recruiting via their short (~20 aa) N-terminal SID domains mSIN3-HDAC co-repressor complexes (Laherty et al., 1997) but MXDs do not contain known domains for the recruitment of co-activators. SID deletions do not affect the recruitment of MXDs to their target genes but compromised repression of 6xEbox-luc, and, surprisingly, also activation of p21-luc. The underlying mechanism remains obscure and awaits further investigation.
Together, these data indicate that MXDs (at physiological and overexpressed levels in were compromised in their capacity to inhibit cell proliferation, just as a corresponding MIZ1-interaction mutant of MYC was recently shown to be compromised in its capacity to stimulate cell proliferation (Kerosuo and Bronner, 2016;Shostak et al., 2016;van Riggelen et al., 2010a). Hence, at least in U2OS cells MXDs antagonized MYC's proproliferative function predominantly in MIZ1-dependent manner via the regulation of MYC-repressed genes. Overexpression of MXDs failed to attenuate cell growth when expression of p21 was suppressed by siRNA, indicating that regulation of p21 was of particular importance in U2OS cells.
In summary, our results reveal that transcription factors of the MXD family, which are characterized repressors of MYC-activated genes, show transcription activating properties at genes that are repressed by MYC together with MIZ1. Thus, our findings suggest that activation and repression of genes by MYC could be coordinated by a reciprocal antagonism of MXD proteins.

Cell culture and transfections
U2OStx and HEK293 cells (Shostak et al., 2016) were maintained in DMEM with 10% FBS and 1x PenStrep. Cell culture reagents were obtained from Life Technologies unless indicated differently. Inducible U2OStx cells overexpressing different transgenes were obtained by stable transfection with AhdI-linearized pcDNA4/TO vector using Xfect (Clontech). Resistant clones were selected with 50 µg/ml hygromycin and 100 µg/ml zeocin (Invivogen) for 2 weeks and pooled together. For siRNA transfections, U2OS cells were seeded on 24 or 96 well plates and next day transfected with the siRNAs (sequences are given in Supplemental Table 1) using Lipofectamine RNAiMAX reagent.
Cells were kept in the siRNA transfection mix minimum 24 hours before further applications. For luciferase reporter assays, HEK293 cells were transfected with the indicated plasmids using Lipofectamine2000. Next day, luciferase expression was measured using Dual-luciferase Reporter Assay (Promega) and an EnSpire Reader (Perkin Elmer).
Vectors containing the ORFs of MNT and MXD1 were kindly provided by Prof. Bernhard Lüscher. The MXD2 ORF was amplified from U2OS cDNA. To produce inducible constructs V5-tagged ORFs of MXDs were cloned in pcDNA4/TO vector. Subsequent L-D, RRDD, and ∆SID mutagenesis, respectively, was performed using DF-Pfu polymerase (Bioron). Cloning and mutagenesis primers are available upon request.

Gene expression analysis
Total RNA from U2OStx cells was extracted with TriFaster (GeneON) and cDNA synthesis was performed with Maxima First Strand cDNA Synthesis Kit (Thermo). qPCR was performed on LightCycler 480 (Roche) using Maxima SYBR Green/ROX qPCR Master Mix (Thermo) and gene expression was quantified using a ∆∆Ct method relative to GAPDH. Primer sequences are listed in Supplemental Table 1.

ChIP
U2OStx cells overexpressing different transgenes were incubated with doxycycline for 24 h and then cross-linked in 1% formaldehyde for 10 min. Chromatin was prepared as described previously (Shostak et al., 2016). Sheared chromatin was incubated overnight at 4°C with 40 µl of salmon sperm DNA-blocked anti-FLAG (A2220, Sigma-Aldrich) and anti-V5 (A7345, Sigma-Aldrich) beads. Subsequently, precipitated chromatin was washed and recovered as previously described (Shostak et al., 2016). Samples were then analyzed by qPCR, and values were normalized to percentage of input. Primer sequences are listed in Supplemental Table 1.

Cell confluence and fluorescent microscopy
For proliferation assays, siRNA-transfected or untreated transgenic U2OStx cells were seeded on transparent 96 well plates (~3000 cells per well) and next day induced with standard growth medium containing 10 ng/ml doxycycline or PBS for control. To quantify apoptosis, growth medium was supplemented with Caspase-3/7 Green Reagent (4440, Essen Bioscience). Cell confluency and apoptosis were measured with an IncuCyte ZOOM reader (Essen Bioscience) using in-built software.

Co-immunoprecipitation and Western blotting
Protein lysates of U2OS cells were prepared by incubation with ice cold lysis buffer (Shostak et al., 2016) and subsequent sonication in the ultrasonic bath (Merck) for 10 min. Pre-cleared lysates (centrifugation at 16000xg for 10 min at 4°C) were boiled with 4x Laemmli buffer, separated using 12% SDS-PAGE and transferred on nitrocellulose membranes. Membranes were decorated with anti-FLAG (1:5000, M2, Sigma-Aldrich), anti-V5 (1:5000, 46-0705, Life Technologies), and anti-Tubulin (1:1000, WA3) antibodies in TBS 5% milk at 4°C overnight. Next day, membranes were incubated with respective HRP-conjugated secondary antibodies and exposed to X-ray films. For co-IP experiments, transfected HEK293 cells were collected and prepared as described above. Then cell lysates (500 µg total protein) were incubated with 40 µl of PBSwashed anti-FLAG M2 beads (Sigma-Aldrich) at 4°C overnight. Next day beads were washed 3 times with PBS supplemented with 500 mM NaCl and 1% Triton X100 and precipitated proteins were eluted by boiling in 4x Laemmli buffer and loaded on 12% SDS-PAGE gels.
AS performed the experiments and GS did the bioinformatics analysis. AS, AD and MB planned and designed the experiments and wrote the manuscript.  (Elkon et al., 2015;Ibarra et al., 2016). It should be noted that MAX protein is about an order of magnitude more stable than MYC, and hence believed to be present in excess over its binding partners MYC and MXDs (Blackwood et al., 1992).  Cells pre-treated with siRNA were transfected with reporter plasmid one day before DOX induction (n=3). Data are presented as mean ± SEM. * P < 0.05; Student's t-test.