MYC directly transactivates CR2/CD21, the receptor of the Epstein-Barr virus, enhancing the viral infection of Burkitt lymphoma cells.

MYC is an oncogenic transcription factor dysregulated in about half of total human tumors. While transcriptomic studies reveal more than 1000 genes regulated by MYC, a much smaller fraction of genes is directly transactivated by MYC. Virtually all Burkitt lymphoma (BL) carry chromosomal translocations involving MYC oncogene. Most endemic BL and a fraction of sporadic BL are associated with Epstein-Barr virus (EBV) infection. The currently accepted mechanism is that EBV is the BL-causing agent inducing MYC translocation. Herein we show that the EBV receptor, CR2 (also called CD21), is a direct MYC target gene. This is based on several pieces of evidence: MYC induces CR2 expression in both proliferating and arrested cells and in the absence of protein synthesis, binds the CR2 promoter and transactivates CR2 in an E-box-dependent manner. Moreover, using mice with conditional MYC ablation we show that MYC induces CR2 in primary B cells. Importantly, modulation of MYC levels directly correlates with EBV's ability of infection in BL cells. Altogether, in contrast to the widely accepted hypothesis for the correlation between EBV and BL, we propose an alternative hypothesis in which MYC dysregulation could be the first event leading to the subsequent EBV infection.

A novel role of MNT as a negative regulator of REL and the NF-κB pathway.

MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix-loop-helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT-REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT-REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.

The MNT transcription factor autoregulates its expression and supports proliferation in MYC-associated factor X (MAX)-deficient cells.

The MAX network transcriptional repressor (MNT) is an MXD family transcription factor of the basic helix-loop-helix (bHLH) family. MNT dimerizes with another transcriptional regulator, MYC-associated factor X (MAX), and down-regulates genes by binding to E-boxes. MAX also dimerizes with MYC, an oncogenic bHLH transcription factor. Upon E-box binding, the MYC-MAX dimer activates gene expression. MNT also binds to the MAX dimerization protein MLX (MLX), and MNT-MLX and MNT-MAX dimers co-exist. However, all MNT functions have been attributed to MNT-MAX dimers, and no functions of the MNT-MLX dimer have been described. MNT's biological role has been linked to its function as a MYC oncogene modulator, but little is known about its regulation. We show here that MNT localizes to the nucleus of MAX-expressing cells and that MNT-MAX dimers bind and repress the MNT promoter, an effect that depends on one of the two E-boxes on this promoter. In MAX-deficient cells, MNT was overexpressed and redistributed to the cytoplasm. Interestingly, MNT was required for cell proliferation even in the absence of MAX. We show that in MAX-deficient cells, MNT binds to MLX, but also forms homodimers. RNA-sequencing experiments revealed that MNT regulates the expression of several genes even in the absence of MAX, with many of these genes being involved in cell cycle regulation and DNA repair. Of note, MNT-MNT homodimers regulated the transcription of some genes involved in cell proliferation. The tight regulation of MNT and its functionality even without MAX suggest a major role for MNT in cell proliferation.

Suppression of BCL6 function by HDAC inhibitor mediated acetylation and chromatin modification enhances BET inhibitor effects in B-cell lymphoma cells.

Multiple genetic aberrations in the regulation of BCL6, including in acetyltransferase genes, occur in clinically aggressive B-cell lymphomas and lead to higher expression levels and activity of this transcriptional repressor. BCL6 is, therefore, an attractive target for therapy in aggressive lymphomas. In this study romidepsin, a potent histone deacetylase inhibitor (HDACi), induced apoptosis and cell cycle arrest in Burkitt and diffuse large B-cell lymphoma cell lines, which are model cells for studying the mechanism of action of BCL6. Romidepsin caused BCL6 acetylation at early timepoints inhibiting its function, while at later timepoints BCL6 expression was reduced and target gene expression increased due to chromatin modification. MYC contributes to poor prognosis in aggressive lymphoma. MYC function is reduced by inhibition of chromatin readers of the bromodomain and extra-terminal repeat (BET) family, which includes BRD4. The novel combination of romidepsin and JQ1, a BRD4 inhibitor was investigated and showed synergy. Collectively we suggest that the combination of HDACi and BRD4i should be pursued in further pre-clinical testing.

Myc and cell cycle control.

Soon after the discovery of the Myc gene (c-Myc), it became clear that Myc expression levels tightly correlate to cell proliferation. The entry in cell cycle of quiescent cells upon Myc enforced expression has been described in many models. Also, the downregulation or inactivation of Myc results in the impairment of cell cycle progression. Given the frequent deregulation of Myc oncogene in human cancer it is important to dissect out the mechanisms underlying the role of Myc on cell cycle control. Several parallel mechanisms account for Myc-mediated stimulation of the cell cycle. First, most of the critical positive cell cycle regulators are encoded by genes induced by Myc. These Myc target genes include Cdks, cyclins and E2F transcription factors. Apart from its direct effects on the transcription, Myc is able to hyperactivate cyclin/Cdk complexes through the induction of Cdk activating kinase (CAK) and Cdc25 phosphatases. Moreover, Myc antagonizes the activity of cell cycle inhibitors as p21 and p27 through different mechanisms. Thus, Myc is able to block p21 transcription or to induce Skp2, a protein involved in p27 degradation. Finally, Myc induces DNA replication by binding to replication origins and by upregulating genes encoding proteins required for replication initiation. Myc also regulates genes involved in the mitotic control. A promising approach to treat tumors with deregulated Myc is the synthetic lethality based on the inhibition of Cdks. Thus, the knowledge of the Myc-dependent cell cycle regulatory mechanisms will help to discover new therapeutic approaches directed against malignancies with deregulated Myc. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

MYC oncogene in myeloid neoplasias.

MYC is a transcription factor that regulates many critical genes for cell proliferation, differentiation, and biomass accumulation. MYC is one of the most prevalent oncogenes found to be altered in human cancer, being deregulated in about 50 % of tumors. Although MYC deregulation has been more frequently associated to lymphoma and lymphoblastic leukemia than to myeloid malignancies, a body of evidence has been gathered showing that MYC plays a relevant role in malignancies derived from the myeloid compartment. The myeloid leukemogenic activity of MYC has been demonstrated in different murine models. Not surprisingly, MYC has been found to be amplified or/and deregulated in the three major types of myeloid neoplasms: acute myeloid leukemia, myelodysplastic syndromes, and myeloproliferative neoplasms, including chronic myeloid leukemia. Here, we review the recent literature describing the involvement of MYC in myeloid tumors.

p21 as a transcriptional co-repressor of S-phase and mitotic control genes.

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.

The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner.

BACKGROUND: CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear. RESULTS: Using immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF. CONCLUSIONS: Our data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes.

A cell cycle role for the epigenetic factor CTCF-L/BORIS.

CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis.

Transcription factors Sp1 and p73 control the expression of the proapoptotic protein NOXA in the response of testicular embryonal carcinoma cells to cisplatin.

Testicular germ cell tumors (TGCTs) are highly responsive to and curable by cisplatin-based chemotherapy even in advanced stages. We have studied the molecular mechanisms involved in the induction of apoptosis in response to cisplatin, and found that proapoptotic Noxa is transcriptionally up-regulated following cisplatin exposure, even in the absence of p53, in NTERA2 cisplatin-sensitive cells but not in 1411HP-resistant cells. Blockade of Noxa reduced the apoptotic response of embryonal carcinoma (EC) NTERA2 cells to cisplatin. A detailed analysis of the Noxa promoter revealed that p73 and Sp1-like factors, Sp1 and KLF6, played key roles in the transcriptional control of this gene. Overexpression of TAp73 induced Noxa whereas the dominant negative isoform ΔNp73, reduced the levels of Noxa after cisplatin exposure in NTERA2 and 2102EP. Interestingly, down-regulation of Sp1 increased Noxa expression in response to cisplatin. However, blockade of KLF6 decreased cisplatin-induced up-regulation of Noxa in EC cell lines. In addition, tissue microarray analyses of TGCTs revealed that expression of Noxa correlates with good clinical prognosis in patients with embryonal carcinoma. Thus, our data show the transcriptional network that regulates Noxa in EC cells, which is key for their apoptotic response to cisplatin-based chemotherapy, and propose Noxa as a predictive factor of therapeutic response.

MYC in chronic myeloid leukemia: induction of aberrant DNA synthesis and association with poor response to imatinib.

Untreated chronic myeloid leukemia (CML) progresses from chronic phase to blastic crisis (BC). Increased genomic instability, deregulated proliferation, and loss of differentiation appear associated to BC, but the molecular alterations underlying the progression of CML are poorly characterized. MYC oncogene is frequently deregulated in human cancer, often associated with tumor progression. Genomic instability and induction of aberrant DNA replication are described as effects of MYC. In this report, we studied MYC activities in CML cell lines with conditional MYC expression with and without exposure to imatinib, the front-line drug in CML therapy. In cells with conditional MYC expression, MYC did not rescue the proliferation arrest mediated by imatinib but provoked aberrant DNA synthesis and accumulation of cells with 4C content. We studied MYC mRNA expression in 66 CML patients at different phases of the disease, and we found that MYC expression was higher in CML patients at diagnosis than control bone marrows or in patients responding to imatinib. Further, high MYC levels at diagnosis correlated with a poor response to imatinib. MYC expression did not directly correlate with BCR-ABL levels in patients treated with imatinib. Overall our study suggests that, as in other tumor models, MYC-induced aberrant DNA synthesis in CML cells is consistent with MYC overexpression in untreated CML patients and nonresponding patients and supports a role for MYC in CML progression, possibly through promotion of genomic instability.

Nuclear targeting of a bacterial integrase that mediates site-specific recombination between bacterial and human target sequences.

TrwC is a bacterial protein involved in conjugative transfer of plasmid R388. It is transferred together with the DNA strand into the recipient bacterial cell, where it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell. Considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. We have searched for possible TrwC target sequences in the human genome. Recombination assays showed that TrwC efficiently catalyzes recombination between its natural target sequence and a discrete number of sequences, located in noncoding sites of the human genome, which resemble this target. We have determined the cellular localization of TrwC and derivatives in human cells by immunofluorescence and also by an indirect yeast-based assay to detect both nuclear import and export signals. The results indicate that the recombinase domain of TrwC (N600) has nuclear localization, but full-length TrwC locates in the cytoplasm, apparently due to the presence of a nuclear export signal in its C-terminal domain. The recombinase domain of TrwC can be transported to recipient cells by conjugation in the presence of the helicase domain of TrwC, but with very low efficiency. We mutagenized the trwC gene and selected for mutants with nuclear localization. We obtained one such mutant with a point A904T mutation and an extra peptide at its C terminus, which maintained its functionality in conjugation and recombination. This TrwC mutant could be useful for future TrwC-mediated site-specific integration assays in mammalian cells.

CTCF regulates the local epigenetic state of ribosomal DNA repeats.

BACKGROUND: CCCTC binding factor (CTCF) is a highly conserved zinc finger protein, which is involved in chromatin organization, local histone modifications, and RNA polymerase II-mediated gene transcription. CTCF may act by binding tightly to DNA and recruiting other proteins to mediate its various functions in the nucleus. To further explore the role of this essential factor, we used a mass spectrometry-based approach to screen for novel CTCF-interacting partners. RESULTS: Using biotinylated CTCF as bait, we identified upstream binding factor (UBF) and multiple other components of the RNA polymerase I complex as potential CTCF-interacting partners. Interestingly, CTCFL, the testis-specific paralog of CTCF, also binds UBF. The interaction between CTCF(L) and UBF is direct, and requires the zinc finger domain of CTCF(L) and the high mobility group (HMG)-box 1 and dimerization domain of UBF. Because UBF is involved in RNA polymerase I-mediated ribosomal (r)RNA transcription, we analyzed CTCF binding to the rDNA repeat. We found that CTCF bound to a site upstream of the rDNA spacer promoter and preferred non-methylated over methylated rDNA. DNA binding by CTCF in turn stimulated binding of UBF. Absence of CTCF in cultured cells resulted in decreased association of UBF with rDNA and in nucleolar fusion. Furthermore, lack of CTCF led to reduced binding of RNA polymerase I and variant histone H2A.Z near the rDNA spacer promoter, a loss of specific histone modifications, and diminished transcription of non-coding RNA from the spacer promoter. CONCLUSIONS: UBF is the first common interaction partner of CTCF and CTCFL, suggesting a role for these proteins in chromatin organization of the rDNA repeats. We propose that CTCF affects RNA polymerase I-mediated events globally by controlling nucleolar number, and locally by regulating chromatin at the rDNA spacer promoter, similar to RNA polymerase II promoters. CTCF may load UBF onto rDNA, thereby forming part of a network that maintains rDNA genes poised for transcription.

p21(Cip1) confers resistance to imatinib in human chronic myeloid leukemia cells.

Imatinib is a Bcr-Abl inhibitor used as first-line therapy of chronic myeloid leukemia (CML). p21(Cip1), initially described as a cell cycle inhibitor, also protects from apoptosis in some models. We describe that imatinib down-regulates p21(Cip1) expression in CML cells. Using K562 cells with inducible p21 expression and transient transfections we found that p21 confers partial resistance to imatinib-induced apoptosis. This protection is not related to the G2-arrest provoked by p21, a decrease in the imatinib activity against Bcr-Abl or a cytoplasmic localization of p21. The results suggest an involvement of p21(Cip1) in the response to imatinib in CML.

Myc roles in hematopoiesis and leukemia.

Hematopoiesis is a process capable of generating millions of cells every second, as distributed in many cell types. The process is regulated by a number of transcription factors that regulate the differentiation along the distinct lineages and dictate the genetic program that defines each mature phenotype. Myc was first discovered as the oncogene of avian leukemogenic retroviruses; it was later found translocated in human lymphoma. From then on, evidence accumulated showing that c-Myc is one of the transcription factors playing a major role in hematopoiesis. The study of genetically modified mice with overexpression or deletion of Myc has shown that c-Myc is required for the correct balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Enforced Myc expression in mice leads to reduced HSC pools owing to loss of self-renewal activity at the expense of increased proliferation of progenitor cells and differentiation. c-Myc deficiency consistently results in the accumulation of HSCs. Other models with conditional Myc deletion have demonstrated that different lineages of hematopoietic cells differ in their requirement for c-Myc to regulate their proliferation and differentiation. When transgenic mice overexpress c-Myc or N-Myc in mature cells from the lymphoid or myeloid lineages, the result is lymphoma or leukemia. In agreement, enforced expression of c-Myc blocks the differentiation in several leukemia-derived cell lines capable of differentiating in culture. Not surprising, MYC deregulation is recurrently found in many types of human lymphoma and leukemia. Whereas MYC is deregulated by translocation in Burkitt lymphoma and, less frequently, other types of lymphoma, MYC is frequently overexpressed in acute lymphoblastic and myeloid leukemia, through mechanisms unrelated to chromosomal translocation, and is often associated with disease progression.

Inhibition of cell differentiation: a critical mechanism for MYC-mediated carcinogenesis?

Despite its early discovery and relevance in cancer, the mechanisms by which MYC brings about tumorigenic transformation have not been clarified. MYC elicits a variety of biological activities, the proliferation promotion being the best studied. However, inhibition of cell differentiation was one of the first MYC activities described. The importance of differentiation impairment in MYC-induced tumorigenesis is demonstrated in transgenic mice models with conditional MYC expression, where MYC inactivation leads to tumor regression associated to re-differentiation of tumor cells. To explain the anti-differentiation effects of MYC, it has been argued that MYC impairs differentiation by preventing exit from the cell cycle. However, we have described models where MYC blocks the erythroid or neuronal differentiation without reversing the proliferative arrest. In these and other models discussed here, MYC-mediated inhibition of differentiation occurs by blocking the upregulation of transcription factors that control the differentiation. The importance of MYC anti-differentiation function has recently gained importance after the discovery that MYC is one of the four transcription factors able to reprogram differentiated cells into pluripotent cells. We will discuss the hypothesis that MYC engages common pathways as a "stemness" keeper and as an oncogene.

HCT116 cells deficient in p21(Waf1) are hypersensitive to tyrosine kinase inhibitors and adriamycin through a mechanism unrelated to p21 and dependent on p53.

p21(Waf1) (p21) was described as a cyclin-dependent kinase inhibitor, but other p21 activities have subsequently been described, including its ability to inhibit apoptosis in some models. Comparative work on the human colon cancer isogenic cell lines HCT116 and HCT116p21(-/-) led to the proposal that p21 protects colon cancer cells against apoptosis by genotoxic drugs. We asked whether p21 also protected from cell death induced by non-genotoxic drugs, such as tyrosine kinase inhibitors. We found that p21-deficient cells were dramatically more sensitive towards imatinib and gefitinib than parental cells. Interestingly, HCT116p21(-/-) also showed higher basal activity of protein kinases as c-Abl, c-Src, and Akt. We generated HCT116p21(-/-) sublines with inducible p21 expression and found that p21 did not rescue the hypersensitivity to imatinib. Moreover, down-regulation of p21 by enforced c-Myc expression or by p21 siRNA did not sensitize parental HCT116 cells. We found that, in HCT116p21(-/-) cells, p53 showed higher stability, higher transcriptional activity and phosphorylation in serines associated with p53 activity. Furthermore, silencing of p53 with siRNA and inactivation of p53 with a dominant negative mutant rescued the hypersensitive response to kinases inhibitors, 5-fluorouracil and adriamycin in HCT116p21(-/-) cells. Consistently, HCT116p53(-/-) cells are more resistant to imatinib than parental cells, suggesting that imatinib activity is partly dependent on p53 in colon cancer cells. We conclude that high p53 activity, rather than p21 deficiency, is the mechanism responsible for hypersensitivity to drugs of HCT116p21(-/-) cells. Therefore the role of p21 on apoptosis of HCT116 colon cancer cells should be re-evaluated.