Telomeres expand sphere of influence: emerging molecular impact of telomeres in non-telomeric functions.

Although the impact of telomeres on physiology stands well established, a question remains: how do telomeres impact cellular functions at a molecular level? This is because current understanding limits the influence of telomeres to adjacent subtelomeric regions despite the wide-ranging impact of telomeres. Emerging work in two distinct aspects offers opportunities to bridge this gap. First, telomere-binding factors were found with non-telomeric functions. Second, locally induced DNA secondary structures called G-quadruplexes are notably abundant in telomeres, and gene regulatory regions genome wide. Many telomeric factors bind to G-quadruplexes for non-telomeric functions. Here we discuss a more general model of how telomeres impact the non-telomeric genome - through factors that associate at telomeres and genome wide - and influence cell-intrinsic functions, particularly aging, cancer, and pluripotency.

Correction: Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.

[This corrects the article DOI: 10.1371/journal.pgen.1007782.].

Non-duplex G-Quadruplex Structures Emerge as Mediators of Epigenetic Modifications.

The role of non-duplex DNA, the guanine-quadruplex structure in particular, is becoming widely appreciated. Increasing evidence in the last decade implicates quadruplexes in important processes such as transcription and replication. Interestingly, more recent work suggests roles for quadruplexes, in association with quadruplex-interacting proteins, in epigenetics through both DNA and histone modifications. Here, we review the effect of the quadruplex structure on post-replication epigenetic memory and quadruplex-induced promoter DNA/histone modifications. Furthermore, we highlight the epigenetic state of the telomerase promoter where quadruplexes could play a key regulatory role. Finally, we discuss the possibility that DNA structures such as quadruplexes, within a largely duplex DNA background, could act as molecular anchors for locally induced epigenetic modifications.

Non-duplex G-Quadruplex DNA Structure: A Developing Story from Predicted Sequences to DNA Structure-Dependent Epigenetics and Beyond.

The story of the non-duplex DNA form known as the G-quadruplex (G4) has traversed a winding path. From initial skepticism followed by debate to a surge in interest, the G4 story intertwines many threads. Starting with computational predictions of a gene regulatory role, which now include epigenetic functions, our group was involved in many of these advances along with many other laboratories. Following a brief background, set in the latter half of the last century when the concept of the G4 as a structure took ground, here we account the developments. This is through a lens that though focused on our groups' research presents work from many other groups that played significant roles. Together these provide a broad perspective to the G4 story. Initially we were intrigued on seeing potential G4 (pG4)-forming sequences, then known to be found primarily at the telomeres and immunoglobin switch regions, occurring throughout the genome and being particularly prevalent in promoters of bacteria. We further observed that pG4s were not only prevalent but also conserved through evolution in promoters of human, chimpanzee, mouse and rat genomes. This was between 2005 and 2007. Encouraged by these partly and partly in response to the view held by many that genome-wide presence of G4s were genomic "accidents", the focus shifted to seeking experimental evidence.In the next year, 2008, two independent findings showed promise. First, on treating human cancer cells with G4-binding ligands, we observed widespread change in gene expression. Second, our search for the missing G4-specific transcription factor, without which, importantly, G4s in promoters posed only half the story, yielded results. We determined how NM23-H2 (also known as NME2 or NDPK-B) interacts with G4s and how interaction of NM23-H2 with a G4 in the promoter of the oncogene c-myc was important for regulation of c-myc transcription. NM23-H2, and subsequently many other similar factors discovered by multiple groups, is possibly giving shape to what might be the "G4-transcriptome". Later, a close look at NM23-H2-G4 interaction in regulation of the human reverse transcriptase gene (hTERT) revealed the role of G4s in local epigenetic modifications. Meanwhile work from others showed how G4s impact histone modifications following replication. Together these show the intrinsic role of DNA sequence, through formation of DNA structure, in epigenetics.More recent work, however, was waiting to reveal aspects that tend to bring forth a completely new understanding of G4s. We observed that the telomere-repeat-binding-factor-2 (TRF2), known canonically to be telomere-associated, binds extensively outside telomeres throughout the genome. Moreover, a large fraction of the non-telomeric TRF2 sites comprise G4s. Second, the extent of non-telomeric TRF2 binding at promoters was dependent on telomere length. Thereby TRF2-induced epigenetic gene regulation was telomere-dependent. Together these implicate underlying connections that show signs of addressing an intriguing unanswered question that takes us back to the beginning: Why are G4s prevalent in two distinct regions, the telomeres and gene promoters?

The Candida albicans biofilm gene circuit modulated at the chromatin level by a recent molecular histone innovation.

Histone H3 and its variants regulate gene expression but the latter are absent in most ascomycetous fungi. Here, we report the identification of a variant histone H3, which we have designated H3VCTG because of its exclusive presence in the CTG clade of ascomycetes, including Candida albicans, a human pathogen. C. albicans grows both as single yeast cells and hyphal filaments in the planktonic mode of growth. It also forms a three-dimensional biofilm structure in the host as well as on human catheter materials under suitable conditions. H3VCTG null (hht1/hht1) cells of C. albicans are viable but produce more robust biofilms than wild-type cells in both in vitro and in vivo conditions. Indeed, a comparative transcriptome analysis of planktonic and biofilm cells reveals that the biofilm circuitry is significantly altered in H3VCTG null cells. H3VCTG binds more efficiently to the promoters of many biofilm-related genes in the planktonic cells than during biofilm growth, whereas the binding of the core canonical histone H3 on the corresponding promoters largely remains unchanged. Furthermore, biofilm defects associated with master regulators, namely, biofilm and cell wall regulator 1 (Bcr1), transposon enhancement control 1 (Tec1), and non-dityrosine 80 (Ndt80), are significantly rescued in cells lacking H3VCTG. The occupancy of the transcription factor Bcr1 at its cognate promoter binding sites was found to be enhanced in the absence of H3VCTG in the planktonic form of growth resulting in enhanced transcription of biofilm-specific genes. Further, we demonstrate that co-occurrence of valine and serine at the 31st and 32nd positions in H3VCTG, respectively, is essential for its function. Taken together, we show that even in a unicellular organism, differential gene expression patterns are modulated by the relative occupancy of the specific histone H3 type at the chromatin level.

Extra-telomeric impact of telomeres: Emerging molecular connections in pluripotency or stemness.

Telomeres comprise specialized nucleic acid-protein complexes that help protect chromosome ends from DNA damage. Moreover, telomeres associate with subtelomeric regions through looping. This results in altered expression of subtelomeric genes. Recent observations further reveal telomere length-dependent gene regulation and epigenetic modifications at sites spread across the genome and distant from telomeres. This regulation is mediated through the telomere-binding protein telomeric repeat-binding factor 2 (TRF2). These observations suggest a role of telomeres in extra-telomeric functions. Most notably, telomeres have a broad impact on pluripotency and differentiation. For example, cardiomyocytes differentiate with higher efficacy from induced pluripotent stem cells having long telomeres, and differentiated cells obtained from human embryonic stem cells with relatively long telomeres have a longer lifespan. Here, we first highlight reports on these two seemingly distinct research areas: the extra-telomeric role of telomere-binding factors and the role of telomeres in pluripotency/stemness. On the basis of the observations reported in these studies, we draw attention to potential molecular connections between extra-telomeric biology and pluripotency. Finally, in the context of the nonlocal influence of telomeres on pluripotency and stemness, we discuss major opportunities for progress in molecular understanding of aging-related disorders and neurodegenerative diseases.

Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.

Telomere-binding proteins constituting the shelterin complex have been studied primarily for telomeric functions. However, mounting evidence shows non-telomeric binding and gene regulation by shelterin factors. This raises a key question-do telomeres impact binding of shelterin proteins at distal non-telomeric sites? Here we show that binding of the telomere-repeat-binding-factor-2 (TRF2) at promoters ~60 Mb from telomeres depends on telomere length in human cells. Promoter TRF2 occupancy was depleted in cells with elongated telomeres resulting in altered TRF2-mediated transcription of distal genes. In addition, histone modifications-activation (H3K4me1 and H3K4me3) as well as silencing marks (H3K27me3)-at distal promoters were telomere length-dependent. These demonstrate that transcription, and the epigenetic state, of telomere-distal promoters can be influenced by telomere length. Molecular links between telomeres and the extra-telomeric genome, emerging from findings here, might have important implications in telomere-related physiology, particularly ageing and cancer.

Emerging Molecular Connections between NM23 Proteins, Telomeres and Telomere-Associated Factors: Implications in Cancer Metastasis and Ageing.

The metastasis suppressor function of NM23 proteins is widely understood. Multiple enzymatic activities of NM23 proteins have also been identified. However, relatively less known interesting aspects are being revealed from recent developments that corroborate the telomeric interactions of NM23 proteins. Telomeres are known to regulate essential physiological events such as metastasis, ageing, and cellular differentiation via inter-connected signalling pathways. Here, we review the literature on the association of NM23 proteins with telomeres or telomere-related factors, and discuss the potential implications of emerging telomeric functions of NM23 proteins. Further understanding of these aspects might be instrumental in better understanding the metastasis suppressor functions of NM23 proteins.

Telomere repeat-binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters.

The role of the telomere repeat-binding factor 2 (TRF2) in telomere maintenance is well-established. However, recent findings suggest that TRF2 also functions outside telomeres, but relatively little is known about this function. Herein, using genome-wide ChIP-Seq assays of TRF2-bound chromatin from HT1080 fibrosarcoma cells, we identified thousands of TRF2-binding sites within the extra-telomeric genome. In light of this observation, we asked how TRF2 occupancy is organized within the genome. Interestingly, we found that extra-telomeric TRF2 sites throughout the genome are enriched in potential G-quadruplex-forming DNA sequences. Furthermore, we validated TRF2 occupancy at several promoter G-quadruplex motifs, which did adopt quadruplex forms in solution. TRF2 binding altered expression and the epigenetic state of several target promoters, indicated by histone modifications resulting in transcriptional repression of eight of nine genes investigated here. Furthermore, TRF2 occupancy and target gene expression were also sensitive to the well-known intracellular G-quadruplex-binding ligand 360A. Together, these results reveal an extensive genome-wide association of TRF2 outside telomeres and that it regulates gene expression in a G-quadruplex-dependent fashion.

Insights about genome function from spatial organization of the genome.

Over the last 15 years, development of chromosome conformation capture (3C) and its subsequent high-throughput variants in conjunction with the fast development of sequencing technology has allowed investigators to generate large volumes of data giving insights into the spatial three-dimensional (3D) architecture of the genome. This huge data has been analyzed and validated using various statistical, mathematical, genomics, and biophysical tools in order to examine the chromosomal interaction patterns, understand the organization of the chromosome, and find out functional implications of the interactions. This review summarizes the data generated by several large-scale high-throughput chromosome conformation capture studies and the functional implications obtained from the data analyses. We also discuss emerging results on factors (both CCCTC binding factor (CTCF) related and CTCF independent) that could contribute to looping interactions.

Promise of G-Quadruplex Structure Binding Ligands as Epigenetic Modifiers with Anti-Cancer Effects.

Evidences from more than three decades of work support the function of non-duplex DNA structures called G-quadruplex (G4) in important processes like transcription and replication. In addition, G4 structures have been studied in connection with DNA base modifications and chromatin/nucleosome arrangements. Recent work, interestingly, shows promise of G4 structures, through interaction with G4 structure-interacting proteins, in epigenetics-in both DNA and histone modification. Epigenetic changes are found to be intricately associated with initiation as well as progression of cancer. Multiple oncogenes have been reported to harbor the G4 structure at regulatory regions. In this context, G4 structure-binding ligands attain significance as molecules with potential to modify the epigenetic state of chromatin. Here, using examples from recent studies we discuss the emerging role of G4 structures in epigenetic modifications and, therefore, the promise of G4 structure-binding ligands in epigenetic therapy.

Extratelomeric Binding of the Telomere Binding Protein TRF2 at the PCGF3 Promoter Is G-Quadruplex Motif-Dependent.

Telomere repeat binding factor 2 (TRF2) is critical for the protection of chromosome ends. Mounting evidence suggests that TRF2 associates with extratelomeric sites and TRF2 functions may not be limited to telomeres. Here, we show that the PCGF3 promoter harbors a sequence capable of forming the DNA secondary structure G-quadruplex motif, which is required for binding of TRF2 at the PCGF3 promoter. We demonstrate that promoter binding by TRF2 mediates PCGF3 promoter activity, and both the N-terminal and C-terminal domains of TRF2 are necessary for promoter activity. Altogether, this shows for the first time that a telomere binding factor may regulate a component of the polycomb group of proteins.

Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex-dependent fashion.

Transcriptional activation of the human telomerase reverse transcriptase (hTERT) gene, which remains repressed in adult somatic cells, is critical during tumorigenesis. Several transcription factors and the epigenetic state of the hTERT promoter are known to be important for tight control of hTERT in normal tissues, but the molecular mechanisms leading to hTERT reactivation in cancer are not well-understood. Surprisingly, here we found occupancy of the metastasis suppressor non-metastatic 2 (NME2) within the hTERT core promoter in HT1080 fibrosarcoma cells and HCT116 colon cancer cells and NME2-mediated transcriptional repression of hTERT in these cells. We also report that loss of NME2 results in up-regulated hTERT expression. Mechanistically, additional results indicated that the RE1-silencing transcription factor (REST)-lysine-specific histone demethylase 1 (LSD1) co-repressor complex associates with the hTERT promoter in an NME2-dependent way and that this assembly is required for maintaining repressive chromatin at the hTERT promoter. Interestingly, a G-quadruplex motif at the hTERT promoter was essential for occupancy of NME2 and the REST repressor complex on the hTERT promoter. In light of this mechanistic insight, we studied the effects of G-quadruplex-binding ligands on hTERT expression and observed that several of these ligands repressed hTERT expression. Together, our results support a mechanism of hTERT epigenetic control involving a G-quadruplex promoter motif, which potentially can be targeted by tailored small molecules.

NM23/NDPK proteins in transcription regulatory functions and chromatin modulation: emerging trends.

NM23/NDPK proteins have been studied for their metastasis suppressor role but the molecular pathways involved in this process are not very vivid. Nucleotide binding and kinase activities of NM23 proteins implicated in anti-metastatic effects have been widely studied. In addition to these, transcriptional regulation adds another arm to the versatility of NM23 proteins that together with the other functions may contribute to better understanding of underlying mechanisms. In this review we discuss emerging reports describing the role of NM23 proteins in gene regulation and chromatin modulation in association with other factors or on their own.

QuadBase2: web server for multiplexed guanine quadruplex mining and visualization.

DNA guanine quadruplexes or G4s are non-canonical DNA secondary structures which affect genomic processes like replication, transcription and recombination. G4s are computationally identified by specific nucleotide motifs which are also called putative G4 (PG4) motifs. Despite the general relevance of these structures, there is currently no tool available that can allow batch queries and genome-wide analysis of these motifs in a user-friendly interface. QuadBase2 (quadbase.igib.res.in) presents a completely reinvented web server version of previously published QuadBase database. QuadBase2 enables users to mine PG4 motifs in up to 178 eukaryotes through the EuQuad module. This module interfaces with Ensembl Compara database, to allow users mine PG4 motifs in the orthologues of genes of interest across eukaryotes. PG4 motifs can be mined across genes and their promoter sequences in 1719 prokaryotes through ProQuad module. This module includes a feature that allows genome-wide mining of PG4 motifs and their visualization as circular histograms. TetraplexFinder, the module for mining PG4 motifs in user-provided sequences is now capable of handling up to 20 MB of data. QuadBase2 is a comprehensive PG4 motif mining tool that further expands the configurations and algorithms for mining PG4 motifs in a user-friendly way.

BreCAN-DB: a repository cum browser of personalized DNA breakpoint profiles of cancer genomes.

BreCAN-DB (http://brecandb.igib.res.in) is a repository cum browser of whole genome somatic DNA breakpoint profiles of cancer genomes, mapped at single nucleotide resolution using deep sequencing data. These breakpoints are associated with deletions, insertions, inversions, tandem duplications, translocations and a combination of these structural genomic alterations. The current release of BreCAN-DB features breakpoint profiles from 99 cancer-normal pairs, comprising five cancer types. We identified DNA breakpoints across genomes using high-coverage next-generation sequencing data obtained from TCGA and dbGaP. Further, in these cancer genomes, we methodically identified breakpoint hotspots which were significantly enriched with somatic structural alterations. To visualize the breakpoint profiles, a next-generation genome browser was integrated with BreCAN-DB. Moreover, we also included previously reported breakpoint profiles from 138 cancer-normal pairs, spanning 10 cancer types into the browser. Additionally, BreCAN-DB allows one to identify breakpoint hotspots in user uploaded data set. We have also included a functionality to query overlap of any breakpoint profile with regions of user's interest. Users can download breakpoint profiles from the database or may submit their data to be integrated in BreCAN-DB. We believe that BreCAN-DB will be useful resource for genomics scientific community and is a step towards personalized cancer genomics.

SNP rs1049430 in the 3'-UTR of SH3GL2 regulates its expression: Clinical and prognostic implications in head and neck squamous cell carcinoma.

Single nucleotide polymorphisms (SNPs) in the 3'-UTR region are emerging cis-regulatory factors associated with the occurrences of several human diseases. SH3GL2, which is located at chromosome 9p21-22, is associated with hyperplastic/mildly dysplastic lesions of the head and neck and has a long 3'-UTR with multiple SNPs. The aim of the present study was to determine the susceptible allele(s) in the 3'-UTR SNPs of SH3GL2 in head and neck squamous cell carcinoma (HNSCC). First, we screened the genotypes of all SNPs located in the 3'-UTR of SH3GL2 in 110 controls and 147 cases in Indian populations by sequencing. A SNP (rs1049430:>G/T) that showed only heterozygosity was further confirmed by genotyping with an Illumina GoldenGate platform in 530 controls and 764 cases. Genotype-specific survival analysis of the HNSCC patients was performed. In addition, genotype-specific mRNA stability, isoform expression and protein expression were analyzed. SNP rs1049430 was not associated with disease occurrence, but it was associated with poor patient outcome. The G allele was associated with decreased SH3GL2 mRNA stability, differential splicing and low protein expression. Thus, our data demonstrate that the presence of the susceptible G allele in SNP rs1049430 is associated with the inactivation of SH3GL2 and could be used as a prognostic marker of HNSCC.

Fluorescent Dansyl-Guanosine Conjugates that Bind c-MYC Promoter G-Quadruplex and Downregulate c-MYC Expression.

The four-stranded G-quadruplex present in the c-MYC P1 promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Small-molecule compounds capable of inhibiting the c-MYC promoter activity by stabilising the c-MYC G-quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl-guanosine conjugates through one-pot modular click reactions. The dansyl-guanosine conjugates can selectively detect c-MYC G-quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c-MYC G-quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c-MYC G-quadruplex. A dual luciferase assay and qRT-PCR revealed that a dansyl-bisguanosine ligand represses the c-MYC expression, possibly by stabilising the c-MYC G-quadruplex.

Engineered reversal of drug resistance in cancer cells--metastases suppressor factors as change agents.

Building molecular correlates of drug resistance in cancer and exploiting them for therapeutic intervention remains a pressing clinical need. To identify factors that impact drug resistance herein we built a model that couples inherent cell-based response toward drugs with transcriptomes of resistant/sensitive cells. To test this model, we focused on a group of genes called metastasis suppressor genes (MSGs) that influence aggressiveness and metastatic potential of cancers. Interestingly, modeling of 84 000 drug response transcriptome combinations predicted multiple MSGs to be associated with resistance of different cell types and drugs. As a case study, on inducing MSG levels in a drug resistant breast cancer line resistance to anticancer drugs caerulomycin, camptothecin and topotecan decreased by more than 50-60%, in both culture conditions and also in tumors generated in mice, in contrast to control un-induced cells. To our knowledge, this is the first demonstration of engineered reversal of drug resistance in cancer cells based on a model that exploits inherent cellular response profiles.

Promoter-proximal transcription factor binding is transcriptionally active when coupled with nucleosome repositioning in immediate vicinity.

Previous studies have analyzed patterns of transcription, transcription factor (TF) binding or mapped nucleosome occupancy across the genome. These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF. Therefore, several aspects remain unclear: does TF binding influence nucleosome (re)organizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reorganization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression? With these in mind, following characterization of two states (before and after induction of a single TF of choice) we determined: (i) genomic binding sites of the TF, (ii) promoter nucleosome occupancy and (iii) transcriptome profiles. Results demonstrated that promoter-proximal TF binding influenced expression of the target gene when it was coupled to nucleosome repositioning at or close to its binding site in most cases. In contrast, only in few cases change in target gene expression was found when TF binding occurred without local nucleosome reorganization.