Several studies have described functional peptides encoded in RNA that are considered to be noncoding. Telomerase RNA together with telomerase reverse transcriptase and regulatory proteins make up the telomerase complex, the major component of the telomere length-maintaining machinery. In contrast to protein subunits, telomerase RNA is expressed constitutively in most somatic cells where telomerase reverse transcriptase is absent. We show here that the transcript of human telomerase RNA codes a 121 amino acid protein (hTERP). The existence of hTERP was shown by immunoblotting, immunofluorescence microscopy and mass spectroscopy. Gain-of-function and loss-of-function experiments showed that hTERP protects cells from drug-induced apoptosis and participates in the processing of autophagosome. We suggest that hTERP regulates crosstalk between autophagy and apoptosis and is involved in cellular adaptation under stress conditions.

Homologous recombination provides an effective way to repair DNA double-strand breaks (DSBs) and is required for genetic recombination. During the process of homologous recombination, a heteroduplex DNA structure, or a 'Holliday junction' (HJ), is formed. The movement, or branch migration, of this junction is necessary for recombination to proceed correctly. In prokaryotes, the RecQ protein or the RuvA/RuvB protein complex can promote ATP-dependent branch migration of Holliday junctions. Much less is known about the processing of Holliday junctions in eukaryotes. Here, we identify RecQL1 as a predominant ATP-dependent, HJ branch migrator present in human nuclear extracts. A reduction in the level of RecQL1 induced by RNA interference in HeLa cells leads to an increase in sister chromatid exchange. We propose that RecQL1 is involved in the processing of Holliday junctions in human cells.

T cells are central to the immune response against various pathogens and cancer cells. Complex networks of transcriptional and post-transcriptional regulators, including microRNAs (miRNAs), coordinate the T cell activation process. Available miRNA datasets, however, do not sufficiently dissolve the dynamic changes of miRNA controlled networks upon T cell activation. Here, we established a quantitative and time-resolved expression pattern for the entire miRNome over a period of 24 h upon human T-cell activation. Based on our time-resolved datasets, we identified central miRNAs and specified common miRNA expression profiles. We found the most prominent quantitative expression changes for miR-155-5p with a range from initially 40 molecules/cell to 1600 molecules/cell upon T-cell activation. We established a comprehensive dynamic regulatory network of both the up- and downstream regulation of miR-155. Upstream, we highlight IRF4 and its complexes with SPI1 and BATF as central for the transcriptional regulation of miR-155. Downstream of miR-155-5p, we verified 17 of its target genes by the time-resolved data recorded after T cell activation. Our data provide comprehensive insights into the range of stimulus induced miRNA abundance changes and lay the ground to identify efficient points of intervention for modifying the T cell response.

DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase IIα with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase IIα stimulates the decatenation activity of Topoisomerase IIα. Consistent with these observations, RECQL5 co-localizes with Topoisomerase IIα during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process.

Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic treatments. Moreover, FCCS employing minute amounts of cells closely corroborated previously reported RNA dependent interactions and provided estimates of the relative overlap between factors in the mRNPs, thus depicting their heterogeneity. The described lysate-based FCS and FCCS analysis may not only complement current biochemical approaches but also provide novel opportunities for the quantitative analysis of the molecular composition and dynamics of single mRNPs.

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3' UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner.

ALT tumor cells often contain abundant DNA damage foci at telomeres and rely on the alternative lengthening of telomeres (ALT) mechanism to maintain their telomeres. How the telomere chromatin is regulated and maintained in these cells remains largely unknown. In this study, we present evidence that heterochromatin protein 1 binding protein 3 (HP1BP3) can localize to telomeres and is particularly enriched on telomeres in ALT cells. HP1BP3 inhibition led to preferential growth inhibition of ALT cells, which was accompanied by telomere chromatin decompaction, increased presence of C-circles, more pronounced ALT-associated phenotypes and elongated telomeres. Furthermore, HP1BP3 appeared to participate in regulating telomere histone H3K9me3 epigenetic marks. Taken together, our data suggest that HP1BP3 functions on telomeres to maintain telomere chromatin and represents a novel target for inhibiting ALT cancer cells.

The oncogenic transformation of normal cells into malignant, rapidly proliferating cells requires major alterations in cell physiology. For example, the transformed cells remodel their metabolic processes to supply the additional demand for cellular building blocks. We have recently demonstrated essential metabolic processes in tumor progression through the development of a methodological analysis of gene expression. Here, we present the Metabolic gEne RApid Visualizer (MERAV, http://merav.wi.mit.edu), a web-based tool that can query a database comprising ∼4300 microarrays, representing human gene expression in normal tissues, cancer cell lines and primary tumors. MERAV has been designed as a powerful tool for whole genome analysis which offers multiple advantages: one can search many genes in parallel; compare gene expression among different tissue types as well as between normal and cancer cells; download raw data; and generate heatmaps; and finally, use its internal statistical tool. Most importantly, MERAV has been designed as a unique tool for analyzing metabolic processes as it includes matrixes specifically focused on metabolic genes and is linked to the Kyoto Encyclopedia of Genes and Genomes pathway search.

The CRISPR/Cas has been recently shown to be a powerful genome-editing tool in a variety of organisms. However, these studies are mainly focused on protein-coding genes. The present study aims to determine whether this technology can be applied to non-coding genes. One of the challenges for knockout of non-coding genes is that a small deletion or insertion generated by the standard CRISPR/Cas system may not necessarily lead to functional loss of a given non-coding gene because of lacking an open reading frame, especially in polyploidy human cell lines. To overcome this challenge, we adopt a selection system that allows for marker genes to integrate into the genome through homologous recombination (HR). Moreover, we construct a dual guide RNA vector that can make two cuts simultaneously at designated sites such that a large fragment can be deleted. With these approaches, we are able to successfully generate knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 in various human cell lines. Finally, we show that the HR-mediated targeting efficiency can be further improved by suppression of the non-homologous end joining pathway. Together, these results demonstrate the feasibility of knockout for non-coding genes by the CRISPR/Cas system in human cell lines.

The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.

Myocyte enhancer factor 2 (MEF2) is a family of transcription factors that regulates many processes, including muscle differentiation. Due to its many target genes, MEF2D requires tight regulation of transcription activity over time and by location. Epigenetic modifiers have been suggested to regulate MEF2-dependent transcription via modifications to histones and MEF2. However, the modulation of MEF2 activity by lysine methylation, an important posttranslational modification that alters the activities of transcription factors, has not been studied. We report the reversible lysine methylation of MEF2D by G9a and LSD1 as a regulatory mechanism of MEF2D activity and skeletal muscle differentiation. G9a methylates lysine-267 of MEF2D and represses its transcriptional activity, but LSD1 counteracts it. This residue is highly conserved between MEF2 members in mammals. During myogenic differentiation of C2C12 mouse skeletal muscle cells, the methylation of MEF2D by G9a decreased, on which MEF2D-dependent myogenic genes were upregulated. We have also identified lysine-267 as a methylation/demethylation site and demonstrate that the lysine methylation state of MEF2D regulates its transcriptional activity and skeletal muscle cell differentiation.

Recycling and de-novo deposition of histones during DNA replication is a critical challenge faced by eukaryotic cells and is coordinated by histone chaperones. Spermatogenesis is highly regulated sophisticated process necessitating not only histone modification but loading of testis specific histone variants. Here, we show that Germ Cell Nuclear Acidic protein (GCNA), a germ cell specific protein in adult mice, can bind histones and purified GCNA exhibits histone chaperone activity. GCNA associates with the DNA replication machinery and supports progression through S-phase in murine undifferentiated spermatogonia (USGs). Whilst GCNA is dispensable for embryonic germ cell development, it is required for the maintenance of the USG pool and for long-term production of sperm. Our work describes the role of a germ cell specific histone chaperone in USGs maintenance in mice. These findings provide a mechanistic basis for the male infertility observed in patients carrying GCNA mutations.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a well-characterized transcription factor that protects cells against oxidative and electrophilic stresses. Emerging evidence has suggested that NRF2 protects cells against DNA damage by mechanisms other than antioxidation, yet the mechanism remains poorly understood. Here, we demonstrate that knockout of NRF2 in cells results in hypersensitivity to ionizing radiation (IR) in the presence or absence of reactive oxygen species (ROS). Under ROS scavenging conditions, induction of DNA double-strand breaks (DSBs) increases the NRF2 protein level and recruits NRF2 to DNA damage sites where it interacts with ATR, resulting in activation of the ATR-CHK1-CDC2 signaling pathway. In turn, this leads to G2 cell cycle arrest and the promotion of homologous recombination repair of DSBs, thereby preserving genome stability. The inhibition of NRF2 by brusatol increased the radiosensitivity of tumor cells in xenografts by perturbing ATR and CHK1 activation. Collectively, our results reveal a novel function of NRF2 as an ATR activator in the regulation of the cellular response to DSBs. This shift in perspective should help furnish a more complete understanding of the function of NRF2 and the DNA damage response.

Transcription factors and chromatin remodeling proteins control the transcriptional variability for ESC lineage commitment. During ESC differentiation, chromatin modifiers are recruited to the regulatory regions by transcription factors, thereby activating the lineage-specific genes or silencing the transcription of active ESC genes. However, the underlying mechanisms that link transcription factors to exit from pluripotency are yet to be identified. In this study, we show that the Ctbp2-interacting zinc finger proteins, Zfp217 and Zfp516, function as linkers for the chromatin regulators during ESC differentiation. CRISPR-Cas9-mediated knock-outs of both Zfp217 and Zfp516 in ESCs prevent the exit from pluripotency. Both zinc finger proteins regulate the Ctbp2-mediated recruitment of the NuRD complex and polycomb repressive complex 2 (PRC2) to active ESC genes, subsequently switching the H3K27ac to H3K27me3 during ESC differentiation for active gene silencing. We therefore suggest that some zinc finger proteins orchestrate to control the concise epigenetic states on active ESC genes during differentiation, resulting in natural lineage commitment.

Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ∼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.

The tumor suppressor p53 contributes to the cellular fate after genotoxic insults, mainly through the regulation of target genes, thereby allowing e.g. repair mechanisms resulting in cell survival or inducing apoptosis. Unresolved so far is the issue, which exact mechanisms lead to one or the other cellular outcome. Here, we describe the interferon regulatory factor-2-binding protein-2 (IRF2BP2) as a new direct target gene of p53, influencing the p53-mediated cellular decision. We show that upregulation of IRF2BP2 after treatment with actinomycin D (Act.D) is dependent on functional p53 in different cell lines. This occurs in parallel with the down-regulation of the interacting partner of IRF2BP2, the interferon regulatory factor-2 (IRF2), which is known to positively influence cell growth. Analyzing the molecular functions of IRF2BP2, it appears to be able to impede on the p53-mediated transactivation of the p21- and the Bax-gene. We show here that overexpressed IRF2BP2 has an impact on the cellular stress response after Act.D treatment and that it diminishes the induction of apoptosis after doxorubicin treatment. Furthermore, the knockdown of IRF2BP2 leads to an upregulation of p21 and faster induction of apoptosis after doxorubicin as well as Act.D treatment.

The protein arginine methyltransferase 6 (PRMT6) is a coregulator of gene expression and executes its repressing as well as activating function by asymmetric dimethylation of histone H3 at R2 (H3 R2me2a). Given that elevated expression levels of PRMT6 have been reported in various cancer types, we explore here its role in cell proliferation and senescence. We find that knockdown of PRMT6 results in proliferation defects of transformed as well as non-transformed cells, causes G1-phase arrest and induces senescence. This phenotype is accompanied by transcriptional upregulation of important cell cycle regulators, most prominently the cyclin-dependent kinase (CDK) inhibitor gene p21 (p21(CIP1/WAF1), CDKN1A) and p16 (p16(INK4A), CDKN2A). Chromatin immuno-precipitation analysis reveals that the p21 gene is a direct target of PRMT6 and the corresponding histone mark H3 R2me2a. Using a cell model of oncogene-induced senescence (OIS), in which p21 is an essential activator of the senescent phenotype, we show that PRMT6 expression declines upon induction of senescence and conversely p21 gene expression increases. Moreover, overexpression of PRMT6 leads to reduced levels of OIS. These findings indicate that the transcriptional repressor activity of PRMT6 facilitates cell proliferation and blocks senescence by regulation of tumor suppressor genes and that this might contribute to the oncogenic capacity of PRMT6.

Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5΄ selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3΄ selective approaches which just provide internal sequences close to the 3΄ end. The only currently existing 5΄ selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5΄ selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.

We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using 29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat concatemers due to strand slippage. Instead, we used a combination of in vitro ligation and 29 DNA polymerase to amplify DNA. Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA). In the presence of two non-complementary primers, hybridizing to either strand of DNA, ligations can be amplified to generate microgram quantities of repeat containing DNA. Additionally, expanded repeats generated by rolling circle amplification can be produced in vectors for expression of expanded CUG (CUG(exp)) RNA capable of sequestering MBNL1 protein in cell culture. Amplification of dimerized expanded repeats (ADER) opens new possibilities for studies of repeat instability and pathogenesis in myotonic dystrophy, a neurological disorder caused by an expanded CTG repeat.

Posttranslational modifications of the Forkhead family transcription factor, FOXO1, have been known to have important regulatory implications in its diverse activities. Several types of modifications of FOXO1, including acetylation, phosphorylation, and ubiquitination, have been reported. However, lysine methylation of FOXO1 has not yet been identified. Here, we reported that FOXO1 is methylated by G9a at K273 residue in vitro and in vivo. Methylation of FOXO1 by G9a increased interaction between FOXO1 and a specific E3 ligase, SKP2, and decreased FOXO1 protein stability. In addition, G9a expression was increased by insulin and resulted in insulin-mediated FOXO1 degradation by K273 methylation. Tissue array analysis indicated that G9a was overexpressed and FOXO1 levels decreased in human colon cancer. Cell proliferation assays revealed that G9a-mediated FOXO1 methylation increased colon cancer cell proliferation. Fluorescence-activated cell sorting (FACS) analysis indicated that apoptosis rates were higher in the presence of FOXO1 than in FOXO1 knock-out cells. Furthermore, we found that G9a protein levels were elevated and FOXO1 protein levels were decreased in human colon cancer patients tissue samples. Here, we report that G9a specific inhibitor, BIX-01294, can regulate cell proliferation and apoptosis by inhibiting G9a-mediated FOXO1 methylation.