Carla Grandori

Direct activation of TERT transcription by c-MYC

The MYC proto-oncogene encodes a ubiquitous transcription factor (c–MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c–MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c–MYC and its role in tumorigenesis are poorly understood because few c–MYC targets have been identified. Here we show that c–MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c–MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c–MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated cells, and can both induce immortalization when constitutively expressed in transfected cells. Consistent with the recently reported association between MYC overexpression and induction of telomerase activity, we find here that the TERT promoter contains numerous c–MYC–binding sites that mediate TERT transcriptional activation. c–MYC–induced TERT expression is rapid and independent of cell proliferation and additional protein synthesis, consistent with direct transcriptional activation of TERT. Our results indicate that TERT is a target of c–MYC activity and identify a pathway linking cell proliferation and chromosome integrity in normal and neoplastic cells.

Non-transcriptional control of DNA replication by c-Myc

The c-Myc proto-oncogene encodes a transcription factor that is essential for cell growth and proliferation and is broadly implicated in tumorigenesis. However, the biological functions required by c-Myc to induce oncogenesis remain elusive. Here we show that c-Myc has a direct role in the control of DNA replication. c-Myc interacts with the pre-replicative complex and localizes to early sites of DNA synthesis. Depletion of c-Myc from mammalian (human and mouse) cells as well as from Xenopus cell-free extracts, which are devoid of RNA transcription, demonstrates a non-transcriptional role for c-Myc in the initiation of DNA replication. Overexpression of c-Myc causes increased replication origin activity with subsequent DNA damage and checkpoint activation. These findings identify a critical function of c-Myc in DNA replication and suggest a novel mechanism for its normal and oncogenic functions.

c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I

c-Myc coordinates cell growth and division through a transcriptional programme that involves both RNA polymerase (Pol) II- and Pol III-transcribed genes. Here, we demonstrate that human c-Myc also directly enhances Pol I transcription of ribosomal RNA (rRNA) genes. rRNA synthesis and accumulation occurs rapidly following activation of a conditional MYC-ER allele (coding for a Myc–oestrogen-receptor fusion protein), is resistant to inhibition of Pol II transcription and is markedly reduced by c-MYC RNA interference. Furthermore, by using combined immunofluorescence and rRNA-FISH, we have detected endogenous c-Myc in nucleoli at sites of active ribosomal DNA (rDNA) transcription. Our data also show that c-Myc binds to specific consensus elements located in human rDNA and associates with the Pol I-specific factor SL1. The presence of c-Myc at specific sites on rDNA coincides with the recruitment of SL1 to the rDNA promoter and with increased histone acetylation. We propose that stimulation of rRNA synthesis by c-Myc is a key pathway driving cell growth and tumorigenesis.

Direct activation of RNA polymerase III transcription by c-Myc

The proto-oncogene product c-Myc has a direct role in both metazoan cell growth and division. RNA polymerase III (pol III) is involved in the generation of transfer RNA and 5S ribosomal RNA, and these molecules must be produced in bulk to meet the need for protein synthesis in growing cells. We demonstrate here that c-Myc binds to TFIIIB, a pol III-specific general transcription factor, and directly activates pol III transcription. Chromatin immunoprecipitation reveals that endogenous c-Myc is present at tRNA and 5S rRNA genes in cultured mammalian cells. These results suggest that activation of pol III may have a role in the ability of c-Myc to stimulate cell growth.

Myc target genes

The myc family of proto-oncogenes belongs to the basic helix-loop-helix leucine-zipper (bHLHZ) class of transcription factors. Myc proteins function as transcriptional activators through heterodimerization with Max, but might also act as negative regulators of transcription. Identification of genes directly controlled by Myc-Max has proved difficult, but recent work is producing a growing list of candidates. Results to date suggest that Myc-Max influences cell growth and proliferation through direct activation of genes involved in DNA synthesis, RNA metabolism and cell-cycle progression.

Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

The c‐Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence‐specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti‐Max and anti‐Myc antibodies, we have identified a Myc‐regulated gene and genomic sites occupied by Myc‐Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity ‘canonical’ CACGTG sequence. However, the most common in vivo binding sites belonged to the group of ‘non‐canonical’ E box‐related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc‐estrogen receptor fusion protein (Myc‐ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc‐Max. In addition, as for c‐Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down‐regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc‐Max heterodimers interact in vivo with a specific set of E box‐related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism.

Functional genomics identifies therapeutic targets for MYC-driven cancer

MYC oncogene family members are broadly implicated in human cancers, yet are considered “undruggable” as they encode transcription factors. MYC also carries out essential functions in proliferative tissues, suggesting that its inhibition could cause severe side effects. We elected to identify synthetic lethal interactions with c-MYC overexpression (MYC-SL) in a collection of ∼3,300 druggable genes, using high-throughput siRNA screening. Of 49 genes selected for follow-up, 48 were confirmed by independent retesting and approximately one-third selectively induced accumulation of DNA damage, consistent with enrichment in DNA-repair genes by functional annotation. In addition, genes involved in histone acetylation and transcriptional elongation, such as TRRAP and BRD4, were identified, indicating that the screen revealed known MYC-associated pathways. For in vivo validation we selected CSNK1e, a kinase whose expression correlated with MYCN amplification in neuroblastoma (an established MYC-driven cancer). Using RNAi and available small-molecule inhibitors, we confirmed that inhibition of CSNK1e halted growth of MYCN-amplified neuroblastoma xenografts. CSNK1e had previously been implicated in the regulation of developmental pathways and circadian rhythms, whereas our data provide a previously unknown link with oncogenic MYC. Furthermore, expression of CSNK1e correlated with c-MYC and its transcriptional signature in other human cancers, indicating potential broad therapeutic implications of targeting CSNK1e function. In summary, through a functional genomics approach, pathways essential in the context of oncogenic MYC but not to normal cells were identified, thus revealing a rich therapeutic space linked to a previously “undruggable” oncogene.

Direct Regulation of RNA Polymerase III Transcription by RB, p53 and c-Myc

The synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III is cell cycle regulated in higher organisms. Overexpression of pol III products is a general feature of transformed cells. These observations may be explained by the fact that a pol III-specific transcription factor, TFIIIB, is strongly regulated by the tumour suppressors RB and p53, as well as the proto-oncogene product c-Myc. RB and p53 repress TFIIIB, but this restraint can be lost in tumours through a variety of mechanisms. In contrast, c-Myc binds and activates TFIIIB, causing potent induction of pol III transcription. Using chromatin immunoprecipitation and RNA interference, we show that c-Myc interacts with tRNA and 5S rRNA genes in transformed cervical cells, stimulating their expression. Availability of pol III products may be an important determinant of a cells capacity to grow. The ability to regulate pol III output may therefore be integral to the growth control functions of RB, p53 and c-Myc.

Modulation of T-lymphocyte development, growth and cell size by the Myc antagonist and transcriptional repressor Mad1

Activated lymphocytes must increase in size and duplicate their contents (cell growth) before they can divide. The molecular events that control cell growth in proliferating lymphocytes and other metazoan cells are still unclear. Here, we utilized transgenesis to provide evidence suggesting that the basic helix–loop– helix–zipper (bHLHZ) transcriptional repressor Mad1, considered to be an antagonist of Myc function, inhibits lymphocyte expansion, maturation and growth following pre‐T‐cell receptor (pre‐TCR) and TCR stimulation. Furthermore, we utilized cDNA microarray technology to determine that, of the genes repressed by Mad1, the majority (77%) are involved in cell growth, which correlates with a decrease in size of Mad1 transgenic thymocytes. Over 80% of the genes repressed by Mad1 have previously been found to be induced by Myc. These results suggest that a balance between Myc and Mad levels may normally modulate lymphocyte proliferation and development in part by controlling expression of growth‐regulating genes.

c-Myc accelerates S-phase and requires WRN to avoid replication stress.

c-Myc interacts with components of the pre-replication complex and directly regulates DNA replication [1]. However the consequences of this novel c-Myc function on cell cycle dynamics and replication-associated damage are unknown. Here, we show that c-Myc overexpression in primary human fibroblasts markedly accelerates S-phase while c-Myc deficient fibroblasts exhibit a prolonged S-phase. We also show that the Werner DNA helicase protein (WRN) plays a critical role in supporting c-Myc-driven S-phase, as depletion of WRN in c-Myc overexpressing cells increases DNA damage specifically at sites of DNA synthesis. This excess DNA damage activates a “replication stress” pathway involving ATR, CHK1, CHK2, and p53, leading to rapid senescence of WRN deficient c-Myc overexpressing cells. Indeed, depletion of p53 rescues this senescence response. We propose that WRN functions to repair abnormal replication structures caused by the acceleration of DNA replication by c-Myc. This work provides an additional mechanistic explanation for c-Myc-induced DNA damage and senescence, and reveals a vulnerability of c-Myc overexpressing cells that could potentially be exploited in cancer therapy.