Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells.

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.

A novel bioinformatic approach reveals cooperation between Cancer/Testis genes in basal-like breast tumors.

Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis, prompting the need for new tools to understand, detect, and treat these tumors. Certain germline-restricted genes show aberrant expression in tumors and are known as Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here we designed a new bioinformatic approach to examine Cancer/Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the two Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells and not by the microenvironment, and that they are not expressed by normal breast progenitors; in other words, their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Simultaneous expression of both genes in breast cells in vitro has a synergistic effect that increases stemness and activates a transcriptional profile also observed in double-positive tumors. Therefore, we reveal a functional cooperation between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worst outcomes.

[Using 2C-like cells to understand embryonic totipotency]. / Comprendre la totipotence embryonnaire à partir des cellules 2C-like.

Totipotency is the ability of a cell to generate a whole organism, a property that characterizes the first embryonic cells, such as the zygote and the blastomeres. This review provides a retrospective on the progress made in the last decade in the study of totipotency, especially with the discovery of mouse ES cells expressing markers of the 2-cell stage (2C-like cells). This model has greatly contributed to a better understanding of the molecular mechanisms involved in totipotency (pioneer factors, epigenetic regulation, splicing, nuclear maturation). 2C-like cells have also paved the way for the development of new cellular models of human totipotency.

Title: Comprendre la totipotence embryonnaire à partir des cellules 2C-like. Abstract: La totipotence est la capacité d'une cellule à générer un organisme entier, une propriété qui caractérise les premières cellules embryonnaires, comme le zygote et les blastomères. Dans cette revue, nous proposons une rétrospective des avancées réalisées au cours de la dernière décennie concernant l'étude de la totipotence avec, notamment, la découverte des cellules ES murines exprimant des marqueurs du stade 2-cellules (2CLC). Ce modèle a considérablement contribué à la meilleure compréhension des mécanismes moléculaires impliqués dans la totipotence (facteurs pionniers, régulation épigénétique, épissage, maturation nucléaire). Les cellules 2CLC ont aussi ouvert la voie au développement de nouveaux modèles cellulaires de totipotence humaine.

Locus-level L1 DNA methylation profiling reveals the epigenetic and transcriptional interplay between L1s and their integration sites.

Long interspersed element 1 (L1) retrotransposons are implicated in human disease and evolution. Their global activity is repressed by DNA methylation, but deciphering the regulation of individual copies has been challenging. Here, we combine short- and long-read sequencing to unveil L1 methylation heterogeneity across cell types, families, and individual loci and elucidate key principles involved. We find that the youngest primate L1 families are specifically hypomethylated in pluripotent stem cells and the placenta but not in most tumors. Locally, intronic L1 methylation is intimately associated with gene transcription. Conversely, the L1 methylation state can propagate to the proximal region up to 300 bp. This phenomenon is accompanied by the binding of specific transcription factors, which drive the expression of L1 and chimeric transcripts. Finally, L1 hypomethylation alone is typically insufficient to trigger L1 expression due to redundant silencing pathways. Our results illuminate the epigenetic and transcriptional interplay between retrotransposons and their host genome.

RSK3 switches cell fate: from stress-induced senescence to malignant progression.

BACKGROUND: TGFß induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGFß but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGFß-induced senescence escape. METHODS: In vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-ß-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections. RESULTS: A screen with active kinases in HMECs upon TGFß treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGFß-induced senescence. Interestingly, RSK3 expression decreased in response to TGFß in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGFß-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. Strikingly, senescent TGFß-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-κΒ in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland. CONCLUSIONS: We conclude that RSK3 switches cell fate from senescence to malignancy in response to TGFß signaling.

E4F1 and ZNF148 are transcriptional activators of the -57A > C and wild-type TERT promoter.

Point mutations within the TERT promoter are the most recurrent somatic noncoding mutations identified across different cancer types, including glioblastoma, melanoma, hepatocellular carcinoma, and bladder cancer. They are most abundant at -146C > T and -124C > T, and rarer at -57A > C, with the latter originally described as a familial case, but subsequently shown also to occur somatically. All three mutations create de novo E26-specific (ETS) binding sites and result in activation of the TERT gene, allowing cancer cells to achieve replicative immortality. Here, we used a systematic proteomics screen to identify transcription factors preferentially binding to the -146C > T, -124C > T, and -57A > C mutations. Although we confirmed binding of multiple ETS factors to the mutant -146C > T and -124C > T sequences, we identified E4F1 as a -57A > C-specific binder and ZNF148 as a TERT wild-type (WT) promoter binder that showed reduced interaction with the -124C > T allele. Both proteins are activating transcription factors that bind specifically to the -57A > C and WT (at position 124) TERT promoter sequence in corresponding cell lines, and up-regulate TERT transcription and telomerase activity. Our work describes new regulators of TERT gene expression with possible roles in cancer.

A genetic screen identifies BEND3 as a regulator of bivalent gene expression and global DNA methylation.

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements and higher-order chromatin structures. Consequently, these chromatin marks are indispensable for mammalian development and alterations often lead to disease, such as cancer. Bivalent promoters are especially important during differentiation and development. Here we used a genetic screen to identify new regulators of a bivalent repressed gene. We identify BEND3 as a regulator of hundreds of bivalent promoters, some of which it represses, and some of which it activates. We show that BEND3 is recruited to a CpG-containg consensus site that is present in multiple copies in many bivalent promoters. Besides having direct effect on the promoters it binds, the loss of BEND3 leads to genome-wide gains of DNA methylation, which are especially marked at regions normally protected by the TET enzymes. DNA hydroxymethylation is reduced in Bend3 mutant cells, possibly as consequence of altered gene expression leading to diminished alpha-ketoglutarate production, thus lowering TET activity. Our results clarify the direct and indirect roles of an important chromatin regulator, BEND3, and, more broadly, they shed light on the regulation of bivalent promoters.

A genome-wide screen reveals new regulators of the 2-cell-like cell state.

In mammals, only the zygote and blastomeres of the early embryo are totipotent. This totipotency is mirrored in vitro by mouse '2-cell-like cells' (2CLCs), which appear at low frequency in cultures of embryonic stem cells (ESCs). Because totipotency is not completely understood, we carried out a genome-wide CRISPR knockout screen in mouse ESCs, searching for mutants that reactivate the expression of Dazl, a gene expressed in 2CLCs. Here we report the identification of four mutants that reactivate Dazl and a broader 2-cell-like signature: the E3 ubiquitin ligase adaptor SPOP, the Zinc-Finger transcription factor ZBTB14, MCM3AP, a component of the RNA processing complex TREX-2, and the lysine demethylase KDM5C. All four factors function upstream of DPPA2 and DUX, but not via p53. In addition, SPOP binds DPPA2, and KDM5C interacts with ncPRC1.6 and inhibits 2CLC gene expression in a catalytic-independent manner. These results extend our knowledge of totipotency, a key phase of organismal life.

Unchanged PCNA and DNMT1 dynamics during replication in DNA ligase I-deficient cells but abnormal chromatin levels of non-replicative histone H1.

DNA ligase I (LigI), the predominant enzyme that joins Okazaki fragments, interacts with PCNA and Pol δ. LigI also interacts with UHRF1, linking Okazaki fragment joining with DNA maintenance methylation. Okazaki fragments can also be joined by a relatively poorly characterized DNA ligase IIIα (LigIIIα)-dependent backup pathway. Here we examined the effect of LigI-deficiency on proteins at the replication fork. Notably, LigI-deficiency did not alter the kinetics of association of the PCNA clamp, the leading strand polymerase Pol ε, DNA maintenance methylation proteins and core histones with newly synthesized DNA. While the absence of major changes in replication and methylation proteins is consistent with the similar proliferation rate and DNA methylation levels of the LIG1 null cells compared with the parental cells, the increased levels of LigIIIα/XRCC1 and Pol δ at the replication fork and in bulk chromatin indicate that there are subtle replication defects in the absence of LigI. Interestingly, the non-replicative histone H1 variant, H1.0, is enriched in the chromatin of LigI-deficient mouse CH12F3 and human 46BR.1G1 cells. This alteration was not corrected by expression of wild type LigI, suggesting that it is a relatively stable epigenetic change that may contribute to the immunodeficiencies linked with inherited LigI-deficiency syndrome.

Structural basis for activation of DNMT1.

DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human DNMT1 bound to its two natural activators: hemimethylated DNA and ubiquitinated histone H3. We find that a hitherto unstudied linker, between the RFTS and CXXC domains, plays a key role for activation. It contains a conserved α-helix which engages a crucial "Toggle" pocket, displacing a previously described inhibitory linker, and allowing the DNA Recognition Helix to spring into the active conformation. This is accompanied by large-scale reorganization of the inhibitory RFTS and CXXC domains, allowing the enzyme to gain full activity. Our results therefore provide a mechanistic basis for the activation of DNMT1, with consequences for basic research and drug design.

Context-dependent CpG methylation directs cell-specific binding of transcription factor ZBTB38.

DNA methylation on CpGs regulates transcription in mammals, both by decreasing the binding of methylation-repelled factors and by increasing the binding of methylation-attracted factors. Among the latter, zinc finger proteins have the potential to bind methylated CpGs in a sequence-specific context. The protein ZBTB38 is unique in that it has two independent sets of zinc fingers, which recognize two different methylated consensus sequences in vitro. Here, we identify the binding sites of ZBTB38 in a human cell line, and show that they contain the two methylated consensus sequences identified in vitro. In addition, we show that the distribution of ZBTB38 sites is highly unusual: while 10% of the ZBTB38 sites are also bound by CTCF, the other 90% of sites reside in closed chromatin and are not bound by any of the other factors mapped in our model cell line. Finally, a third of ZBTB38 sites are found upstream of long and active CpG islands. Our work therefore validates ZBTB38 as a methyl-DNA binder in vivo and identifies its unique distribution in the genome.

Large-Scale Chromatin Rearrangements in Cancer.

Epigenetic abnormalities are extremely widespread in cancer. Some of them are mere consequences of transformation, but some actively contribute to cancer initiation and progression; they provide powerful new biological markers, as well as new targets for therapies. In this review, we examine the recent literature and focus on one particular aspect of epigenome deregulation: large-scale chromatin changes, causing global changes of DNA methylation or histone modifications. After a brief overview of the one-dimension (1D) and three-dimension (3D) epigenome in healthy cells and of its homeostasis mechanisms, we use selected examples to describe how many different events (mutations, changes in metabolism, and infections) can cause profound changes to the epigenome and fuel cancer. We then present the consequences for therapies and briefly discuss the role of single-cell approaches for the future progress of the field.

Structure-based screening combined with computational and biochemical analyses identified the inhibitor targeting the binding of DNA Ligase 1 to UHRF1.

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.

Staying true to yourself: mechanisms of DNA methylation maintenance in mammals.

DNA methylation is essential to development and cellular physiology in mammals. Faulty DNA methylation is frequently observed in human diseases like cancer and neurological disorders. Molecularly, this epigenetic mark is linked to other chromatin modifications and it regulates key genomic processes, including transcription and splicing. Each round of DNA replication generates two hemi-methylated copies of the genome. These must be converted back to symmetrically methylated DNA before the next S-phase, or the mark will fade away; therefore the maintenance of DNA methylation is essential. Mechanistically, the maintenance of this epigenetic modification takes place during and after DNA replication, and occurs within the very dynamic context of chromatin re-assembly. Here, we review recent discoveries and unresolved questions regarding the mechanisms, dynamics and fidelity of DNA methylation maintenance in mammals. We also discuss how it could be regulated in normal development and misregulated in disease.

Lysine Methylation Regulators Moonlighting outside the Epigenome.

Landmark discoveries made nearly two decades ago identified known transcriptional regulators as histone lysine methyltransferases. Since then, the field of lysine methylation signaling has been dominated by studies of how this small chemical posttranslational modification regulates gene expression and other chromatin-based processes. However, recent advances in mass-spectrometry-based proteomics have revealed that histones are just a subset of the thousands of eukaryotic proteins marked by lysine methylation. As the writers, erasers, and readers of histone lysine methylation are emerging as a promising therapeutic target class for cancer and other diseases, a key challenge for the field is to define the full spectrum of activities for these proteins. Here we summarize recent discoveries implicating non-histone lysine methylation as a major regulator of diverse cellular processes. We further discuss recent technological innovations that are enabling the expanded study of lysine methylation signaling. Collectively, these findings are shaping our understanding of the fundamental mechanisms of non-histone protein regulation through this dynamic and multi-functional posttranslational modification.

Genetic screens reveal mechanisms for the transcriptional regulation of tissue-specific genes in normal cells and tumors.

The proper tissue-specific regulation of gene expression is essential for development and homeostasis in metazoans. However, the illegitimate expression of normally tissue-restricted genes-like testis- or placenta-specific genes-is frequently observed in tumors; this promotes transformation, but also allows immunotherapy. Two important questions are: how is the expression of these genes controlled in healthy cells? And how is this altered in cancer? To address these questions, we used an unbiased approach to test the ability of 350 distinct genetic or epigenetic perturbations to induce the illegitimate expression of over 40 tissue-restricted genes in primary human cells. We find that almost all of these genes are remarkably resistant to reactivation by a single alteration in signaling pathways or chromatin regulation. However, a few genes differ and are more readily activated; one is the placenta-expressed gene ADAM12, which promotes invasion. Using cellular systems, an animal model, and bioinformatics, we find that a non-canonical but druggable TGF-ß/KAT2A/TAK1 axis controls ADAM12 induction in normal and cancer cells. More broadly, our data show that illegitimate gene expression in cancer is an heterogeneous phenomenon, with a few genes activatable by simple events, and most genes likely requiring a combination of events to become reactivated.

Structure of the UHRF1 Tandem Tudor Domain Bound to a Methylated Non-histone Protein, LIG1, Reveals Rules for Binding and Regulation.

The protein UHRF1 is crucial for DNA methylation maintenance. The tandem Tudor domain (TTD) of UHRF1 binds histone H3K9me2/3 with micromolar affinity, as well as unmethylated linker regions within UHRF1 itself, causing auto-inhibition. Recently, we showed that a methylated histone-like region of DNA ligase 1 (LIG1K126me2/me3) binds the UHRF1 TTD with nanomolar affinity, permitting UHRF1 recruitment to chromatin. Here we report the crystal structure of the UHRF1 TTD bound to a LIG1K126me3 peptide. The data explain the basis for the high TTD-binding affinity of LIG1K126me3 and reveal that the interaction may be regulated by phosphorylation. Binding of LIG1K126me3 switches the overall structure of UHRF1 from a closed to a flexible conformation, suggesting that auto-inhibition is relieved. Our results provide structural insight into how UHRF1 performs its key function in epigenetic maintenance.

Mechanisms of DNA Methyltransferase Recruitment in Mammals.

DNA methylation is an essential epigenetic mark in mammals. The proper distribution of this mark depends on accurate deposition and maintenance mechanisms, and underpins its functional role. This, in turn, depends on the precise recruitment and activation of de novo and maintenance DNA methyltransferases (DNMTs). In this review, we discuss mechanisms of recruitment of DNMTs by transcription factors and chromatin modifiers-and by RNA-and place these mechanisms in the context of biologically meaningful epigenetic events. We present hypotheses and speculations for future research, and underline the fundamental and practical benefits of better understanding the mechanisms that govern the recruitment of DNMTs.

Depletion of ZBTB38 potentiates the effects of DNA demethylating agents in cancer cells via CDKN1C mRNA up-regulation.

DNA methyltransferase inhibitor (DNMTi) treatments have been used for patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and have shown promising beneficial effects in some other types of cancers. Here, we demonstrate that the transcriptional repressor ZBTB38 is a critical regulator of the cellular response to DNMTi. Treatments with 5-azacytidine, or its derivatives decitabine and zebularine, lead to down-regulation of ZBTB38 protein expression in cancer cells, in parallel with cellular damage. The depletion of ZBTB38 by RNA interference enhances the toxicity of DNMTi in cell lines from leukemia and from various solid tumor types. Further we observed that inactivation of ZBTB38 causes the up-regulation of CDKN1C mRNA, a previously described indirect target of DNMTi. We show that CDKN1C is a key actor of DNMTi toxicity in cells lacking ZBTB38. Finally, in patients with MDS a high level of CDKN1C mRNA expression before treatment correlates with a better clinical response to a drug regimen combining 5-azacytidine and histone deacetylase inhibitors. Collectively, our results suggest that the ZBTB38 protein is a target of DNMTi and that its depletion potentiates the toxicity of DNMT inhibitors in cancer cells, providing new opportunities to enhance the response to DNMT inhibitor therapies in patients with MDS and other cancers.