DNA/RNA: Building Blocks of Life Under UV Irradiation

International audienceDuring the last 10 years, intense experimental and theoretical work has proven the existence of ultrafast nonradiative decay routes for UV-excited monomeric nucleic acid bases, accounting for their high photostability. This mechanism has been explained by the occurrence of easily accessible conical intersections connecting the first excited ππ* state with the ground state. However, recent studies of substituent and solvent effects indicate that the situation is more complicated than what was initially thought, notably by the presence of dark excited states. Moreover, the actual shape of the excited-state potential energy surface may induce nonexponential dynamics. Further efforts are needed in order to clarify how various environmental factors affect the structural and dynamical aspects of the nucleic acid base excited states

Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator–like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.Harvard University. Society of FellowsNational Human Genome Research Institute (U.S.) (Center for Excellence in Genomics Science P50 HG003170)United States. Dept. of Energy (Genomes to Life DE-FG02-02ER63445)United States. Defense Advanced Research Projects Agency (W911NF-08-1-0254, G.M.C.)Wyss Institute of Biologically Inspired EngineeringNational Institutes of Health (U.S.) (Transformative R01 (R01 NS073124-01))European School of Molecular Medicine (predoctoral fellowship

Writing and Rewriting the Epigenetic Code of Cancer Cells: From Engineered Proteins to Small Molecules

The epigenomic era has revealed a well-connected network of molecular processes that shape the chromatin landscape. These processes comprise abnormal methylomes, transcriptosomes, genome-wide histone post-transcriptional modifications patterns, histone variants, and noncoding RNAs. The mapping of these processes in large scale by chromatin immunoprecipitation sequencing and other methodologies in both cancer and normal cells reveals novel therapeutic opportunities for anticancer intervention. The goal of this minireview is to summarize pharmacological strategies to modify the epigenetic landscape of cancer cells. These approaches include the use of novel small molecule inhibitors of epigenetic processes specifically deregulated in cancer cells and the design of engineered proteins able to stably reprogram the epigenetic code in cancer cells in a way that is similar to normal cells

Tight-binding parameters for charge transfer along DNA

We systematically examine all the tight-binding parameters pertinent to charge transfer along DNA. The $\pi$ molecular structure of the four DNA bases (adenine, thymine, cytosine, and guanine) is investigated by using the linear combination of atomic orbitals method with a recently introduced parametrization. The HOMO and LUMO wavefunctions and energies of DNA bases are discussed and then used for calculating the corresponding wavefunctions of the two B-DNA base-pairs (adenine-thymine and guanine-cytosine). The obtained HOMO and LUMO energies of the bases are in good agreement with available experimental values. Our results are then used for estimating the complete set of charge transfer parameters between neighboring bases and also between successive base-pairs, considering all possible combinations between them, for both electrons and holes. The calculated microscopic quantities can be used in mesoscopic theoretical models of electron or hole transfer along the DNA double helix, as they provide the necessary parameters for a tight-binding phenomenological description based on the $\pi$ molecular overlap. We find that usually the hopping parameters for holes are higher in magnitude compared to the ones for electrons, which probably indicates that hole transport along DNA is more favorable than electron transport. Our findings are also compared with existing calculations from first principles.Comment: 15 pages, 3 figures, 7 table

Novel role of Engrailed 1 as a prosurvival transcription factor in basal-like breast cancer and engineering of interference peptides block its oncogenic function

Basal-like breast tumors are aggressive cancers associated with high proliferation and metastasis. Chemotherapy is currently the only treatment option; however, resistance often occurs resulting in recurrence and patient death. Some extremely aggressive cancers are also associated with hypoxia, inflammation and high leukocyte infiltration. Herein, we discovered that the neural-specific transcription factor, Engrailed 1 (EN1), is exclusively overexpressed in these tumors. Short hairpin RNA (shRNA)-mediated knockdown of EN1 triggered potent and selective cell death. In contrast, ectopic overexpression of EN1 in normal cells activated survival pathways and conferred resistance to chemotherapeutic agents. Exogenous expression of EN1 cDNA reprogrammed the breast epithelial cells toward a long-lived, neural-like phenotype displaying dopaminergic markers. Gene expression microarrays demonstrated that the EN1 cDNA altered transcription of a high number of inflammatory molecules, notably chemokines and chemokine receptors, which could mediate prosurvival pathways. To block EN1 function, we engineered synthetic interference peptides (iPeps) comprising the EN1-specific sequences that mediate essential protein-protein interactions necessary for EN1 function and an N-terminal cell-penetrating peptide/nuclear localization sequence. These EN1-iPeps rapidly mediated a strong apoptotic response in tumor cells overexpressing EN1, with no toxicity to normal or non EN1-expressing cells. Delivery of EN1-iPeps into basal-like cancer cells significantly decreased the fifty percent inhibitory concentrations (IC50) of chemotherapeutic drugs routinely used to treat breast cancer. Lastly, matrix-assisted laser desorption/ionization-time of flight mass spectrometry and immunoprecipitation assays demonstrated that EN1-iPeps captured targets involved in transcriptional and post-transcriptional regulation. Importantly, the EN1-iPeps bound the glutamyl-prolyl tRNA synthetase (EPRS) target, which has been associated with the transcript-specific translational control of inflammatory proteins and activation of amino-acid stress pathways. This work unveils EN1 as an activator of intrinsic inflammatory pathways associated with prosurvival in basal-like breast cancer. We further build upon these results and describe the engineering of iPeps targeting EN1 (EN1-iPeps) as a novel and selective therapeutic strategy to combat these lethal forms of breast cancer

Reprogramming epigenetic silencing: artificial transcription factors synergize with chromatin remodeling drugs to reactivate the tumor suppressor mammary serine protease inhibitor

Mammary serine protease inhibitor (maspin) is an important tumor suppressor gene whose expression is associated not only with tumor growth inhibition but also with decreased angiogenesis and metastasis. Maspin expression is down-regulated in metastatic tumors by epigenetic mechanisms, including aberrant promoter hypermethylation. We have constructed artificial transcription factors (ATFs) as novel therapeutic effectors able to bind 18-bp sites in the maspin promoter and reactivate maspin expression in cell lines that harbor an epigenetically silenced promoter. In this article, we have investigated the influence of epigenetic modifications on ATF-mediated regulation of maspin by challenging MDA-MB-231 breast cancer cells, comprising a methylated maspin promoter, with different doses of ATFs and chromatin remodeling drugs: the methyltransferase inhibitor 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor suberoylanilide hydroxamic acid. We found that the ATFs synergized with both inhibitors in reactivating endogenous maspin expression. The strongest synergy was observed with the triple treatment ATF-126 + 5-aza-2′-deoxycytidine + suberoylanilide hydroxamic acid, in which the tumor suppressor was reactivated by 600-fold. Furthermore, this combination inhibited tumor cell proliferation by 95%. Our data suggest that ATFs enhance the efficiency of chromatin remodeling drugs in reactivating silenced tumor suppressors. Our results document the power of a novel therapeutic approach that combines both epigenetic and genetic (sequence-specific ATFs) strategies to reactivate specifically silenced regions of the genome and reprogram cellular phenotypes

Different fatty acid metabolism effects of (−)-epigallocatechin-3-gallate and C75 in adenocarcinoma lung cancer

Background Fatty acid synthase (FASN) is overexpressed and hyperactivated in several human carcinomas, including lung cancer. We characterize and compare the anti-cancer effects of the FASN inhibitors C75 and (−)-epigallocatechin-3-gallate (EGCG) in a lung cancer model. Methods We evaluated in vitro the effects of C75 and EGCG on fatty acid metabolism (FASN and CPT enzymes), cellular proliferation, apoptosis and cell signaling (EGFR, ERK1/2, AKT and mTOR) in human A549 lung carcinoma cells. In vivo, we evaluated their anti-tumour activity and their effect on body weight in a mice model of human adenocarcinoma xenograft. Results C75 and EGCG had comparable effects in blocking FASN activity (96,9% and 89,3% of inhibition, respectively). In contrast, EGCG had either no significant effect in CPT activity, the rate-limiting enzyme of fatty acid β-oxidation, while C75 stimulated CPT up to 130%. Treating lung cancer cells with EGCG or C75 induced apoptosis and affected EGFR-signaling. While EGCG abolished p-EGFR, p-AKT, p-ERK1/2 and p-mTOR, C75 was less active in decreasing the levels of EGFR and p-AKT. In vivo, EGCG and C75 blocked the growth of lung cancer xenografts but C75 treatment, not EGCG, caused a marked animal weight loss. Conclusions In lung cancer, inhibition of FASN using EGCG can be achieved without parallel stimulation of fatty acid oxidation and this effect is related mainly to EGFR signaling pathway. EGCG reduce the growth of adenocarcinoma human lung cancer xenografts without inducing body weight loss. Taken together, EGCG may be a candidate for future pre-clinical development

Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability

BACKGROUND: Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. RESULTS: Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. CONCLUSIONS: These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-022-02728-5

Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer

With the recent comprehensive mapping of cancer genomes, there is now a need for functional approaches to edit the aberrant epigenetic state of key cancer drivers to reprogram the epi-pathology of the disease. In this study we utilized a programmable DNA-binding methyltransferase to induce targeted incorporation of DNA methylation (DNAme) in the SOX2 oncogene in breast cancer through a six zinc finger (ZF) protein linked to DNA methyltransferase 3A (ZF-DNMT3A). We demonstrated long-lasting oncogenic repression, which was maintained even after suppression of ZF-DNMT3A expression in tumor cells. The de novo DNAme was faithfully propagated and maintained through cell generations even after the suppression of the expression of the chimeric methyltransferase in the tumor cells. Xenograft studies in NUDE mice demonstrated stable SOX2 repression and long-term breast tumor growth inhibition, which lasted for >100 days post implantation of the tumor cells in mice. This was accompanied with a faithful maintenance of DNAme in the breast cancer implants. In contrast, downregulation of SOX2 by ZF domains engineered with the Krueppel-associated box repressor domain resulted in a transient and reversible suppression of oncogenic gene expression. Our results indicated that targeted de novo DNAme of the SOX2 oncogenic promoter was sufficient to induce long-lasting epigenetic silencing, which was not only maintained during cell division but also significantly delayed the tumorigenic phenotype of cancer cells in vivo, even in the absence of treatment. Here, we outline a genome-based targeting approach to long-lasting tumor growth inhibition with potential applicability to many other oncogenic drivers that are currently refractory to drug design

Expression of the pluripotency transcription factor OCT4 in the normal and aberrant mammary gland

Breast cancers with lactating features, some of which are associated with pregnancy and lactation, are often poorly differentiated, lack estrogen receptor, progesterone receptor, and HER2 expression and have high mortality. Very little is known about the molecular mechanisms that drive uncontrolled cell proliferation in these tumors and confer lactating features. We have recently reported expression of OCT4 and associated embryonic stem cell self-renewal genes in the normal lactating breast and breastmilk stem cells (hBSCs). This prompted us to examine OCT4 expression in breast cancers with lactating features and compare it with that observed during normal lactation, using rare specimens of human lactating breast. In accordance with previous literature, the normal resting breast (from non-pregnant, non-lactating women) showed minimal OCT4 nuclear expression (0.9%). However, this increased in the normal lactating breast (11.4%), with further increase in lactating adenomas, lactating carcinomas, and pregnancy-associated breast cancer (30.7-48.3%). OCT4 was expressed in the epithelium and at lower levels in the stroma, and was co-localized with NANOG. Comparison of normal non-tumorigenic hBSCs with OCT4-overexpressing tumorigenic breast cell lines (OTBCs) demonstrated upregulation of OCT4, SOX2, and NANOG in both systems, but OTBCs expressed OCT4 at significantly higher levels than SOX2 and NANOG. Similar to hBSCs, OTBCs displayed multi-lineage differentiation potential, including the ability to differentiate into functional lactocytes synthesizing milk proteins both in vitro and in vivo. Based on these findings, we propose a hypothesis of normal and malignant transformation in the breast, which centers on OCT4 and its associated gene network. Although minimal expression of these embryonic genes can be seen in the breast in its resting state throughout life, a controlled program of upregulation of this gene network may be a potential regulator of the normal remodeling of the breast toward a milk-secretory organ during pregnancy and lactation. Deregulation of this gene network either within or outside pregnancy and lactation may lead to aberrant breast cell proliferation and malignant transformation, suggesting a role of these genes in both normal lactation and breast oncogenesis