Super-enhancer landscape rewiring in cancer: The epigenetic control at distal sites.

Super-enhancers evolve as elements at the top of the hierarchical control of gene expression. They are important end-gatherers of signaling pathways that control stemness, differentiation or adaptive responses. Many epigenetic regulations focus on these regions, and not surprisingly, during the process of tumorigenesis, various alterations can account for their dysfunction. Super-enhancers are emerging as key drivers of the aberrant gene expression landscape that sustain the aggressiveness of cancer cells. In this review, we will describe and discuss about the structure of super-enhancers, their epigenetic regulation, and the major changes affecting their functionality in cancer.

TGS1 mediates 2,2,7-trimethyl guanosine capping of the human telomerase RNA to direct telomerase dependent telomere maintenance.

Pathways that direct the selection of the telomerase-dependent or recombination-based, alternative lengthening of telomere (ALT) maintenance pathway in cancer cells are poorly understood. Using human lung cancer cells and tumor organoids we show that formation of the 2,2,7-trimethylguanosine (TMG) cap structure at the human telomerase RNA 5' end by the Trimethylguanosine Synthase 1 (TGS1) is central for recruiting telomerase to telomeres and engaging Cajal bodies in telomere maintenance. TGS1 depletion or inhibition by the natural nucleoside sinefungin impairs telomerase recruitment to telomeres leading to Exonuclease 1 mediated generation of telomere 3' end protrusions that engage in RAD51-dependent, homology directed recombination and the activation of key features of the ALT pathway. This indicates a critical role for 2,2,7-TMG capping of the RNA component of human telomerase (hTR) in enforcing telomerase-dependent telomere maintenance to restrict the formation of telomeric substrates conductive to ALT. Our work introduces a targetable pathway of telomere maintenance that holds relevance for telomere-related diseases such as cancer and aging.

Chromatin Regulation at Parental Gene Promoters by Pseudogene Sense lncRNAs.

There is accumulating evidence that pseudogenes can produce functionally relevant lncRNAs in a tightly controlled manner. This class of transcripts has been demonstrated to play an important role in development and disease, by controlling parental gene expression. Classically, pseudogene derived lncRNAs compete with parental transcripts for miRNAs or factors that control parental mRNA metabolisms. Recently, pseudogene lncRNAs were demonstrated to take over the control of classic chromatin modifying enzymes and alter parental gene promoter activity or genome wide gene expression. Here, we discuss a new mechanism of parental gene expression controlled by the mOct4P4 lncRNA, a sense transcript derived from the murine Oct4 pseudogene 4. mOct4P4 lncRNA specifically interacts with the RNA binding protein FUS and the Histone Methyltransferase SUV39H1 to target heterochromatin formation at the parental Oct4 promoter in trans. In addition, we will address key issues for the functional dissection of epigenetic control of parental gene promoters by pseudogene lncRNAs.

FUS-dependent loading of SUV39H1 to OCT4 pseudogene-lncRNA programs a silencing complex with OCT4 promoter specificity.

The resurrection of pseudogenes during evolution produced lncRNAs with new biological function. Here we show that pseudogene-evolution created an Oct4 pseudogene lncRNA that is able to direct epigenetic silencing of the parental Oct4 gene via a 2-step, lncRNA dependent mechanism. The murine Oct4 pseudogene 4 (mOct4P4) lncRNA recruits the RNA binding protein FUS to allow the binding of the SUV39H1 HMTase to a defined mOct4P4 lncRNA sequence element. The mOct4P4-FUS-SUV39H1 silencing complex holds target site specificity for the parental Oct4 promoter and interference with individual components results in loss of Oct4 silencing. SUV39H1 and FUS do not bind parental Oct4 mRNA, confirming the acquisition of a new biological function by the mOct4P4 lncRNA. Importantly, all features of mOct4P4 function are recapitulated by the human hOCT4P3 pseudogene lncRNA, indicating evolutionary conservation. Our data highlight the biological relevance of rapidly evolving lncRNAs that infiltrate into central epigenetic regulatory circuits in vertebrate cells.

SFPQ and NONO suppress RNA:DNA-hybrid-related telomere instability.

In vertebrates, the telomere repeat containing long, non-coding RNA TERRA is prone to form RNA:DNA hybrids at telomeres. This results in the formation of R-loop structures, replication stress and telomere instability, but also contributes to alternative lengthening of telomeres (ALT). Here, we identify the TERRA binding proteins NONO and SFPQ as novel regulators of RNA:DNA hybrid related telomere instability. NONO and SFPQ locate at telomeres and have a common role in suppressing RNA:DNA hybrids and replication defects at telomeres. NONO and SFPQ act as heterodimers to suppress fragility and homologous recombination at telomeres, respectively. Combining increased telomere fragility with unleashing telomere recombination upon NONO/SFPQ loss of function causes massive recombination events, involving 35% of telomeres in ALT cells. Our data identify the RNA binding proteins SFPQ and NONO as novel regulators at telomeres that collaborate to ensure telomere integrity by suppressing telomere fragility and homologous recombination triggered by RNA:DNA hybrids.

Epigenetic silencing of miR-296 and miR-512 ensures hTERT dependent apoptosis protection and telomere maintenance in basal-type breast cancer cells.

The catalytic subunit of the telomerase complex, hTERT, ensures unlimited proliferative potential of cancer cells by maintaining telomere function and protecting from apoptosis. Using a miRNA screening approach we identified miR-296-5p and miR-512-5p as miRNAs that target hTERT in breast cancer cells. Ectopic miR-296-5p and miR-512-5p reduce telomerase activity, drive telomere shortening and cause proliferation defects by enhancing senescence and apoptosis in breast cancer cells. In line with the relevance of hTERT expression for human cancer we found that miR-296-5p and miR-512-5p expression is reduced in human breast cancer. Accordingly, high expression of miR-296-5p and miR-512-5p target genes including hTERT is linked with significantly reduced distant metastasis free survival and relapse free survival of basal type breast cancer patients. This suggests relevance of the identified miRNAs in basal type breast cancer. Epigenetic silencing of miR-296 and miR-512 encoding genes is responsible for low levels of miR-296-5p and miR-512-5p expression in basal type breast cancer cells. Disrupting gene silencing results in a dramatic upregulation of miR-296-5p and miR-512-5p levels leading to reduced hTERT expression and increased sensitivity to the induction of apoptosis. Altogether, our data suggest that epigenetic regulatory circuits in basal type breast cancer may contribute to high hTERT levels by silencing miR-296-5p and miR-512-5p expression, thereby contributing to the aggressiveness of basal type breast cancer.

Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA.

Pseudogene-derived, long non-coding RNAs (lncRNAs) act as epigenetic regulators of gene expression. Here we present a panel of new mouse Oct4 pseudogenes and demonstrate that the X-linked Oct4 pseudogene Oct4P4 critically impacts mouse embryonic stem cells (mESCs) self-renewal. Sense Oct4P4 transcription produces a spliced, nuclear-restricted lncRNA that is efficiently upregulated during mESC differentiation. Oct4P4 lncRNA forms a complex with the SUV39H1 HMTase to direct the imposition of H3K9me3 and HP1α to the promoter of the ancestral Oct4 gene, located on chromosome 17, leading to gene silencing and reduced mESC self-renewal. Targeting Oct4P4 expression in primary mouse embryonic fibroblasts causes the re-acquisition of self-renewing features of mESC. We demonstrate that Oct4P4 lncRNA plays an important role in inducing and maintaining silencing of the ancestral Oct4 gene in differentiating mESCs. Our data introduces a sense pseudogene-lncRNA-based mechanism of epigenetic gene regulation that controls the cross-talk between pseudogenes and their ancestral genes.

Altered telomere homeostasis and resistance to skin carcinogenesis in Suv39h1 transgenic mice.

The Suv39h1 and Suv39h2 H3K9 histone methyltransferases (HMTs) have a conserved role in the formation of constitutive heterochromatin and gene silencing. Using a transgenic mouse model system we demonstrate that elevated expression of Suv39h1 increases global H3K9me3 levels in vivo. More specifically, Suv39h1 overexpression enhances the imposition of H3K9me3 levels at constitutive heterochromatin at telomeric and major satellite repeats in primary mouse embryonic fibroblasts. Chromatin compaction is paralleled by telomere shortening, indicating that telomere length is controlled by H3K9me3 density at telomeres. We further show that increased Suv39h1 levels result in an impaired clonogenic potential of transgenic epidermal stem cells and Ras/E1A transduced transgenic primary mouse embryonic fibroblasts. Importantly, Suv39h1 overexpression in mice confers resistance to a DMBA/TPA induced skin carcinogenesis protocol that is characterized by the accumulation of activating H-ras mutations. Our results provide genetic evidence that Suv39h1 controls telomere homeostasis and mediates resistance to oncogenic stress in vivo. This identifies Suv39h1 as an interesting target to improve oncogene induced senescence in premalignant lesions.

miR-155 drives telomere fragility in human breast cancer by targeting TRF1.

Telomeres consist of DNA tandem repeats that recruit the multiprotein complex shelterin to build a chromatin structure that protects chromosome ends. Although cancer formation is linked to alterations in telomere homeostasis, there is little understanding of how shelterin function is limited in cancer cells. Using a small-scale screening approach, we identified miR-155 as a key regulator in breast cancer cell expression of the shelterin component TERF1 (TRF1). miR-155 targeted a conserved sequence motif in the 3'UTR of TRF1, resulting in its translational repression. miR-155 was upregulated commonly in breast cancer specimens, as associated with reduced TRF1 protein expression, metastasis-free survival, and relapse-free survival in estrogen receptor-positive cases. Modulating miR-155 expression in cells altered TRF1 levels and TRF1 abundance at telomeres. Compromising TRF1 expression by elevating miR-155 increased telomere fragility and altered the structure of metaphase chromosomes. In contrast, reducing miR-155 levels improved telomere function and genomic stability. These results implied that miR-155 upregulation antagonizes telomere integrity in breast cancer cells, increasing genomic instability linked to poor clinical outcome in estrogen receptor-positive disease. Our work argued that miRNA-dependent regulation of shelterin function has a clinically significant impact on telomere function, suggesting the existence of "telo-miRNAs" that have an impact on cancer and aging.

An Oct4-pRb axis, controlled by MiR-335, integrates stem cell self-renewal and cell cycle control.

The pluripotency of mouse embryonic stem cells (mESCs) is controlled by a network of transcription factors, mi-RNAs, and signaling pathways. Here, we present a new regulatory circuit that connects miR-335, Oct4, and the Retinoblastoma pathway to control mESC self-renewal and differentiation. Oct4 drives the expression of Nipp1 and Ccnf that inhibit the activity of the protein phosphatase 1 (PP1) complex to establish hyperphosphorylation of the retinoblastoma protein 1 (pRb) as a hallmark feature of self-renewing mESCs. The Oct4-Nipp1/Ccnf-PP1-pRb axis promoting mESC self-renewal is under control of miR-335 that regulates Oct4 and Rb expression. During mESC differentiation, miR-335 upregulation co-operates with the transcriptional repression of Oct4 to facilitate the collapse of the Oct4-Nipp1/Ccnf-PP1-pRb axis, pRb dephosphorylation, the exit from self-renewal, and the establishment of a pRb-regulated cell cycle program. Our results introduce Oct4-dependent control of the Rb pathway as novel regulatory circuit controlling mESC self-renewal and differentiation.

miR-335 directly targets Rb1 (pRb/p105) in a proximal connection to p53-dependent stress response.

Loss-of-function mutations of retinoblastoma family (Rb) proteins drive tumorigenesis by overcoming barriers to cellular proliferation. Consequently, factors modulating Rb function are of great clinical import. Here, we show that miR-335 is differentially expressed in human cancer cells and that it tightly regulates the expression of Rb1 (pRb/p105) by specifically targeting a conserved sequence motif in its 3' untranslated region. We found that by altering Rb1 (pRb/p105) levels, miR-335 activates the p53 tumor suppressor pathway to limit cell proliferation and neoplastic cell transformation. DNA damage elicited an increase in miR-335 expression in a p53-dependent manner. miR-335 and p53 cooperated in a positive feedback loop to drive cell cycle arrest. Together, these results indicate that miR-335 helps control proliferation by balancing the activities of the Rb and p53 tumor suppressor pathways. Further, they establish that miR-335 activation plays an important role in the induction of p53-dependent cell cycle arrest after DNA damage.

TRF1 controls telomere length and mitotic fidelity in epithelial homeostasis.

TRF1 is a component of the shelterin complex at mammalian telomeres; however, a role for TRF1 in telomere biology in the context of the organism is unclear. In this study, we generated mice with transgenic TRF1 expression targeted to epithelial tissues (K5TRF1 mice). K5TRF1 mice have shorter telomeres in the epidermis than wild-type controls do, and these are rescued in the absence of the XPF nuclease, indicating that TRF1 acts as a negative regulator of telomere length by controlling XPF activity at telomeres, similar to what was previously described for TRF2-overexpressing mice (K5TRF2 mice). K5TRF1 cells also show increased end-to-end chromosomal fusions, multitelomeric signals, and increased telomere recombination, indicating an impact of TRF1 on telomere integrity, again similar to the case in K5TRF2 cells. Intriguingly, K5TRF1 cells, but not K5TRF2 cells, show increased mitotic spindle aberrations. TRF1 colocalizes with the spindle assembly checkpoint proteins BubR1 and Mad2 at mouse telomeres, indicating a link between telomeres and the mitotic spindle. Together, these results demonstrate that TRF1, like TRF2, negatively regulates telomere length in vivo by controlling the action of the XPF nuclease at telomeres; in addition, TRF1 has a unique role in the mitotic spindle checkpoint.

Role of TRF2 in the assembly of telomeric chromatin.

Telomeric chromatin is composed of TTAGGG repeats bound by the shelterin complex. Mammalian telomeres contain arrays of nucleosomes enriched in histone heterochromatic marks, and are associated with long non-coding telomeric RNAs. In addition, the adjacent subtelomeric DNA repeats are heavily methylated. The repressive chromatin environment at telomeres is thought to suppress the expression of closely located genes, a phenomenon known as "telomere position effect" (TPE), which can be influenced by telomere length and the telomeric chromatin structure. Here, we address the role of TRF2, a major telomere component, in the epigenetic regulation of telomeres. To this end, we took advantage of transgenic mice that overexpress TRF2 under the keratin 5 (K5) promoter (K5TRF2 mice). K5TRF2 mice have very short telomeres and an increased susceptibility to develop skin cancer. Interestingly, K5TRF2 cells show an aberrant nucleosomal organization at telomeres. In particular, K5TRF2 cells display a loss of heterochromatin marks at telomeric and subtelomeric repeats, which is concomitant with decreased abundance of core histones H3 and H4 and increased nucleosomal spacing at telomeres. Telomere shortening in K5TRF2 mice is also accompanied by decreased abundance of telomeric transcripts. Together, these findings indicate a previously unnoticed role for TRF2 in the assembly of telomeric chromatin.

A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases.

Dicer initiates RNA interference by generating small RNAs involved in various silencing pathways. Dicer participates in centromeric silencing, but its role in the epigenetic regulation of other chromatin domains has not been explored. Here we show that Dicer1 deficiency in Mus musculus leads to decreased DNA methylation, concomitant with increased telomere recombination and telomere elongation. These DNA-methylation defects correlate with decreased expression of Dnmt1, Dnmt3a and Dnmt3b DNA methyltransferases (Dnmts), and methylation levels can be recovered by their overexpression. We identify the retinoblastoma-like 2 protein (Rbl2) as responsible for decreased Dnmt expression in Dicer1-null cells, suggesting the existence of Dicer-dependent small RNAs that target Rbl2. We identify the miR-290 cluster as being downregulated in Dicer1-deficient cells and show that it silences Rbl2, thereby controlling Dnmt expression. These results identify a pathway by which miR-290 directly regulates Rbl2-dependent Dnmt expression, indirectly affecting telomere-length homeostasis.

Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination.

Mammalian telomeres have heterochromatic features, including trimethylated histone H3 at lysine 9 (H3K9me3) and trimethylated histone H4 at lysine 20 (H4K20me3). In addition, subtelomeric DNA is hypermethylated. The enzymatic activities responsible for these modifications at telomeres are beginning to be characterized. In particular, H4K20me3 at telomeres could be catalyzed by the novel Suv4-20h1 and Suv4-20h2 histone methyltransferases (HMTases). In this study, we demonstrate that the Suv4-20h enzymes are responsible for this histone modification at telomeres. Cells deficient for Suv4-20h2 or for both Suv4-20h1 and Suv4-20h2 show decreased levels of H4K20me3 at telomeres and subtelomeres in the absence of changes in H3K9me3. These epigenetic alterations are accompanied by telomere elongation, indicating a role for Suv4-20h HMTases in telomere length control. Finally, cells lacking either the Suv4-20h or Suv39h HMTases show increased frequencies of telomere recombination in the absence of changes in subtelomeric DNA methylation. These results demonstrate the importance of chromatin architecture in the maintenance of telomere length homeostasis and reveal a novel role for histone lysine methylation in controlling telomere recombination.

Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres.

Mammalian telomeres have epigenetic marks of constitutive heterochromatin. Here, we study the impact of telomere length on the maintenance of heterochromatin domains at telomeres. Telomerase-deficient Terc(-/-) mice with short telomeres show decreased trimethylation of histone 3 at Lys9 (H3K9) and histone 4 at Lys20 (H4K20) in telomeric and subtelomeric chromatin as well as decreased CBX3 binding accompanied by increased H3 and H4 acetylation at these regions. Subtelomeric DNA methylation is also decreased in conjunction with telomere shortening in Terc(-/-) mice. In contrast, telomere repeat factors 1 and 2 show normal binding to telomeres independent of telomere length. These results indicate that loss of telomeric repeats leads to a change in the architecture of telomeric and subtelomeric chromatin consisting of loss of heterochromatic features leading to a more 'open' chromatin state. These observations highlight the importance of telomere repeats in the establishment of constitutive heterochromatin at mammalian telomeres and subtelomeres and point to histone modifications as important in counting telomere repeats.

Telomerase regulation and stem cell behaviour.

Telomerase expression is restricted to a few cell types of the adult organism, most notably germ cells and stem/progenitor cells. Telomerase activity in germ cells is sufficient to prevent telomere shortening with age. Stem cells, however, do not have sufficient telomerase to prevent telomere shortening associated with continuous tissue renewal with increasing age. Indeed, telomerase levels in the adult organism are thought to be rate-limiting for longevity. This is supported by rare human syndromes caused by mutations in telomerase components, which are characterized by premature loss of tissue renewal and premature death. More recently, the role of telomerase and telomere length in stem cells is starting to be elucidated.

The calpain system is involved in the constitutive regulation of beta-catenin signaling functions.

Beta-catenin is a multifunctional protein serving both as a structural element in cell adhesion and as a signaling component in the Wnt pathway, regulating embryogenesis and tumorigenesis. The signaling fraction of beta-catenin is tightly controlled by the adenomatous polyposis coli-axin-glycogen synthase kinase 3beta complex, which targets it for proteasomal degradation. It has been recently shown that Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Here we have highlighted the critical relevance of constitutive calpain pathway in the control of beta-catenin levels and functions, showing that small interference RNA knock down of endogenous calpain per se (i.e. in the absence of external stimuli) induces an increase in the free transcriptional competent pool of endogenous beta-catenin. We further characterized the role of the known calpain inhibitors, Gas2 and Calpastatin, demonstrating that they can also control levels, function, and localization of beta-catenin through endogenous calpain regulation. Finally we present Gas2 dominant negative (Gas2DN) as a new tool for regulating calpain activity, providing evidence that it counteracts the described effects of both Gas2 and Calpastatin on beta-catenin and that it works via calpain independently of the classical glycogen synthase kinase 3beta and proteasome pathway. Moreover, we provide in vitro biochemical evidence showing that Gas2DN can increase the activity of calpain and that in vivo it can induce degradation of stabilized/mutated beta-catenin. In fact, in a context where the classical proteasome pathway is impaired, as in colon cancer cells, Gas2DN biological effects accounted for a significant reduction in proliferation and anchorage-independent growth of colon cancer.

hGTSE-1 expression stimulates cytoplasmic localization of p53.

hGTSE-1 (human G(2) and S phase-expressed-1) is a cell cycle-regulated protein mainly localized in the cytoplasm and apparently associated with the microtubules. hGTSE-1 is able to down-regulate levels and activity of the p53 tumor suppressor protein: it binds the C-terminal region of p53 and represses its ability to induce apoptosis after DNA damage. Here we report that, after DNA damage, hGTSE-1 becomes stabilized in a p53-independent way and accumulated in the nucleus. Further characterization of hGTSE-1 localization revealed increased nuclear staining in unstressed cells after treatment with the nuclear export inhibitor leptomycin B, or when a nuclear export signal (NES) located in its C-terminal region was mutated. Finally, we provide evidence that hGTSE-1 ectopic expression, in addition to p53 protein levels down-regulation, is able to enhance cytoplasmic localization of p53. Interestingly, NES-mutated hGTSE-1 accumulates in the nucleus, binds p53 but looses its ability to enhance cytoplasmic redistribution of p53 and to regulate p53 protein levels. Similarly, when wild type hGTSE-1 functions on p53 were analyzed in cells lacking Mdm2, it failed in regulating both p53 localization and protein levels, thus indicating that hGTSE-1 requires an intact NES and functional Mdm2 for the regulation of p53. Our results provide new insights into the mechanism of hGTSE-1 function, whereby its characterized nucleo-cytoplasmic shuttling ability is required to regulate p53.