We show that gene-rich chromosomes as well as areas of open and highly transcribed chromatins are rearranged to greater spatial proximity, thus enabling genes to share transcriptional machinery and regulatory elements. At a smaller scale, differentially interacting loci are enriched for cancer proliferation and estrogen-related genes. Moreover, these loci are correlated with higher ERα binding events and gene expression. Taken together these results reveal the role of a hormone - estrogen - on genome organization, and its effect on gene regulation in cancer.In the cell nucleus, each chromosome is confined to a chromosome territory. This spatial organization of chromosomes plays a crucial role in gene regulation and genome stability. An additional level of organization has been discovered at the chromosome scale: the spatial segregation into open and closed chromatins to form two genome-wide compartments. Although considerable progress has been made in our knowledge of chromatin organization, a fundamental issue remains the understanding of its dynamics, especially in cancer. To address this issue, we performed genome-wide mapping of chromatin interactions (Hi-C) over the time after estrogen stimulation of breast cancer cells. To biologically interpret these interactions, we integrated with estrogen receptor  This spatial organization of genome plays a crucial role in gene regulation and genome stability Hi-C is a recent high-throughput chromosome conformation capture technology for studying genome folding The analysis of Hi-C data is complex, and many statistical and computational methods have been recently developed to correct interaction heatmaps for several biases such as GC-content and distance between reads Although considerable progress has been made in our knowledge of global chromatin organization, a fundamental issue remains the understanding of its dynamics. There is a growing body of evidence that changes in transcriptional activity of genes is associated with repositioning of chromosomal regions relative to nuclear compartments and other genomic loci β-estradiol (E2) treatment. MCF-7 cell is an established cell system to investigate genome-wide estrogen mediated signaling pathways To answer these issues, we selected a breast cancer cell, MCF-7, and utilized Hi-C technology to capture chromatin organization before and after 17α binding, gene expression and multiple epigenetic marks. More specifically, gene-rich chromosomes as well as areas of open and highly transcribed chromatins are rearranged to greater spatial proximity. This phenomenon then allows genes to share transcriptional machinery and regulatory elements. At a smaller scale, loci that are differentially interacting show enrichment for cancer proliferation and E2-related genes. In addition, these loci are involved with higher ERα binding events and gene expression. Based on these results, our study demonstrates the role of a hormone - estrogen - on global genome organization and its link with gene regulantion in cancer.In this study, we show that time-series Hi-C data analysis is a promising methodology for better understanding global dynamics of chromatin and its link with gene regulation. Beside augmenting the number of long-range interactions, E2 induces a dynamic mechanism of global chromatin reorganization. To interpret this global chromatin reorganization, we compare Hi-C data with ERTFF1 using RT-qPCR data in three different batches of cells . Each biological replicate was then subjected to genome-wide chromosome conformation capture (Hi-C) as previously described For each time point, there are two biological replicate samples and two lanes per sample ( µg) was sheared to a size of 300–500 basepairs by a nebulizer supplied with the Illumina paired-end sample preparation kit. Fragmented DNA was end-paired using T4 DNA polymerase and Klenow polymerase with T4 polynucleotide kinase to phosphorylate the 5′ ends. A 3′ overhang was created using a 3′-5′ exonuclease-deficient Klenow fragment, and then subjected to immunoprecipitation by Dynabeads MyOne Streptavin C1 Beads (Invitrogen) in DNA LoBind tubes  with ligation of Illumina paired-end adaptor oligonucleotides. The ligation mixtures were electrophoresed on E-gel SizeSelect Sample preparation for paired-end sequencing was performed following the manufacturer's instructions. Briefly, ligated DNA (5http://mirnylab.bitbucket.org/hiclib/index.html). The five preprocessing steps are the following: (i) the raw sequences of both ends of Hi-C molecules are first mapped separately to the human genome reference hg19 using the Bowtie2 aligning software; (ii) after alignment, the Mirny's lab pipeline discards read pairs with two unmapped sides and removes read pairs corresponding to repeated instances of the same DNA molecule (which may result from PCR amplification). The pipeline then analyzes the position and direction of each mappable read from each read pair to separate molecular byproducts from informative double-sided (DS) reads; (iii) finally, all translocated and amplified regions From the Hi-C experiment, reads were preprocessed in five steps to calculate the interaction matrices. Steps (i), (ii) and (iv) were done using Mirny's lab library The number of reads obtained after Hi-C data preprocessing is summarized in Table S1a in http://www.ncbi.nlm.nih.gov/geo/) using accession number GSE51687.All Hi-C data are available at GEO (http://genome.ucsc.edu/): DNaseI hypersensitivity For H3K4Me2 binding data, we used ChIP-seq results from MCF-7 cells which were not stimulated with E2 Shannon entropy is calculated as:The interpretation of entropy is very simple. In statistics, it is used to evaluate the dispersion of a distribution. For Hi-C data analysis, the higher the entropy the higher is the spatial dispersion of interactions.Chromosomes which are close to each other are likely to interact more. To evaluate proximity, we consider the normalized frequency of interactions between two chromosomes To estimate the scaling coefficient, we first calculate the genome-wide average intrachromosomal interaction frequency http://www.people.fas.harvard.edu/~junliu/BACH/). The program uses a Bayesian probabilistic approach which assumes that local genomic region  of interest exhibits a consensus 3D chromosomal structure in a cell population. BACH relies on an efficient Markov chain Monte Carlo (MCMC) method to infer the underlying consensus 3D chromosomal structure.For 3D chromosome modeling, we use the recent program BACH vice versa), PC1 is constrained to be positively correlated with GC-content.We use principal component analysis to infer chromatin compartments To calculate correlations between chromatin compartmentalization  and genetic and epigenetic tracks, we proceed in the following way. We first bin at http://david.abcc.ncifcrf.gov/) determines functional enrichment clusters of gene ontology terms.To detect differentially interacting loci, we use the Wilcoxon test for paired samples to compare each interaction correlation matrix row between the time point t (α) binding events are mapped within a window of 250 kb around gene transcription start sites. Enrichment is assessed using an exact binomial test comparing counts of differentially versus non-differentially interacting genes which are bound by ERα. ERα ChIA-PET interaction events are mapped in the same manner and exact binomial test is used to compare counts of differentially versus non-differentially interacting genes which are ERα bound and are reported interacting with ChIA-PET. Microarray data are used to compare expression of differentially versus non-differentially interacting genes using Student's t-test. RNA polymerase II binding events are mapped within a window of 10 kb around gene transcription start sites. Enrichment is assessed using an exact binomial test comparing counts of differentially versus non-differentially interacting genes which are occupied by RNA polymerase II.Estrogen receptor alpha (ERhttp://www.cl.cam.ac.uk/~ys388/CytoHiC/) To visualize networks of interacting loci, we used the CytoHiC plug-in of Cytoscape ( µg) was reversely transcribed to cDNA with oligo-dT . RT-qPCR was performed by using SYBR Green dye chemistry (Applied Biosystems) on a StepOnePlus Real-Time PCR System apparatus (Applied Biosystems). Gene expression was measured by the β-actin as the internal control. Expression for the three genes CTSD, GREB1 and TFF1 are presented in Figure S2 in After 48 hours of hormone deprivation in 5% charcoal-dextran stripped serum media without phenol red, MCF-7 cells were stimulated with either 10 nM or 70 nM estrogen (E2) in different time periods  and then subjected to expression analysis by reverse transcription-quantitative PCR (RT-qPCR). Total RNA (2α (Santa Cruz). Pull-down DNA was subjected to quantitative PCR analysis using the SYBR Green-based detection method on a StepOnePlus Real-Time PCR System apparatus (Applied Biosystems). Quantitative values measured by a standard curve  of input DNA amplified with the same primer set. Results are presented as the mean of triplicates with standard derivation. ERα binding for the three estrogen-responsive genes CTSD, GREB1 and TFF1, and for one control gene GAPDH, are presented in Figure S3 in After 48 hours of hormone deprivation in 5% charcoal-dextran stripped serum media without phenol red, MCF-7 cells were stimulated with either 10 nM or 70 nM estrogen (E2) in different time periods  and then subjected to chromatin immunoprecipitation analysis. Immunoprecipitated DNA from treated MCF-7 cells was prepared according to the ChIP protocol published by Young and coworkers GAPDH loading control.Charcoal-stripped MCF-7 cells stimulated with E2 (70 nM) were collected at different time-points of treatment . Treated cells were then subjected to 3C-qPCR analyses as previously described THRAP1 (or MED13) and the associated interacting ERα binding site localized at 20q13 (20q13 DERE) were prepared from BACs . The BAC clones were purified using a large-construct DNA kit (Qiagen) and labeled by nick translation using the Nick Translation kit  following the manufacturer's recommendations. Briefly, 1 µg of the BAC clone was conjugated with either SpectrumGreen- or SpectrumRed-labeled dUTP, coprecipitated with 10X (v/v) human Cot-1 (Invitrogen), and dissolved in Hybridization Buffer (Sigma). The reaction was carried out for 8 h at 15°C and stopped by heating the sample to 70°C for 10 min. For interphase FISH, fixed cells were treated with 0.005% pepsin for digestion, following 0.5 h treatment of 1.9N HCl at room temperature for cell denaturation. Hybridization was performed overnight at 37°C with pre-hybridized labeled probes (150 ng per sample) and slides were washed in following solution: 2X SSC (37°C for 30 min), 2X SSC (room temperature for 30 min), and 1X SSC (room temperature for 30 min). Nuclei counterstained with DAPI (0.1 µg/mL) were placed on a polished concave slide with Vectashield Mounting Medium .E2-treated (70 nM) MCF-7 cells were fixed by Carnoy's fixative and then subjected to Interphase Fluorescence In Situ hybridization (FISH). The probe mapped to THRAP1 spots in 100 cells per treatment. There are around 40-50 copies of DERE and 10-12 copies of THRAP1. Colocalization between the two loci was defined based on pixel overlap.Images were captured by Olympus IX83 fluorescence microscope and analyzed with CellSens Dimension Imaging System. CellSens Dimension Imaging System (Olympus) was used to analyze colocalization of DERE and We report the analysis of Hi-C data from MCF-7 breast cancer cells: before  and iteratively corrected heatmaps for removing biases Interchromosomal interaction frequencies between pairs of chromosomes show that E2 has an effect on chromosome territories  and GREB1 (chromosome 2) et al. did not Hu We then studied the effect of E2 on the polymer behavior of chromosomes. For this purpose, we calculated the scaling coefficient of intrachromosomal interaction frequency To better understand the effects of E2 on chromosome folding, we modeled the 3D polymer structure of chromatin with the recent BACH program It has been previously shown that at the megabase scale chromatin segregates into two spatial compartments A and B corresponding respectively to open and closed chromatin α binding, colocalize with compartment A. Absence of colocalization with microarray gene expression data is not surprising given the noisy nature of such data and the scale of analysis . It has recently been reported that ERα interacts with PAD2 and that PAD2-mediated citrullination leads to local chromatin decondensation and transcriptional activation of target genes α to bind to estrogen responsive elements (EREs) We next investigated the function of E2-induced differential interactions between genomic loci. For this purpose, we first assessed functional enrichments to draw a global picture. Differentially interacting loci were calculated for any time point t  and 4 h , and slight differences at 24 h   . As for α binding events to better understand the role of ERα-mediated E2 effect on functional chromatin interactions and conformation. The integration of ERα bound genes with Hi-C data reveals E2 effect on the network of interactions (EXOC2 and DUSP22 genes from chromosome 6 (light blue nodes framed in blue) connect a group of genes from chromosome 6 (yellow nodes) to the DQ601567 gene from chromosome 5, and to the DQ574804 and DQ575686 genes from chromosome 17. This change of interacting gene network configuration reveals the role of ERα enables higher interactions among genes of the same chromosome, while maintaining interchromosomal interactions through hub nodes connecting chromosomes.Interacting gene networks were built using CytoHiC with a resolution of 500 kb ractions . After EERα gene interacts with two tumor suppressor genes (EGR1 and TGFBR2). However after E2 stimulation, these genes don't interact anymore with ERα and their expressions are inhibited , and this can be a pathway to regulate cancer proliferation.We then sought to assess whether E2 induces changes in interacting networks of genes involved in a known E2-related biological process such as “regulation of cell death” (GO:0010941). E2 is a potent inhibitor of apoptosis and it regulates the expression of several apoptotic proteins nhibited . This fir BCL2L1 . These oTHRAP1 (located in 17q23) and DERE from 20q13  located in 17q23 and 20q13 loci have been recently shown to be involved in frequent amplification in MCF-7 cells. These DEREs remotely control the transcription of target genes present on different chromosomes through chromatin proximity om 20q13 . After EOur work shows that time-series Hi-C data analysis is a promising methodology for studying the global dynamic of chromatin and its impact on gene regulation. Our results reveal a dramatic structural effect of a hormone on genome folding, meaning that not only chromatin can be locally modified through loop formations but also the whole genome organization can be rearranged in a relatively short amount of time.To better understand the impact of E2 on genome organization, chromosome territories, polymer-like behavior and chromatin compartmentalization were studied. Analysis of interchromosomal interactions reveals that small gene-rich chromosomes (chromosomes 16-17 and 19–22) tend to interact more with each other after E2 stimulation. At the chromosome level, E2 affects the polymer-like behavior of chromatin from a more compact to a fractal model, thus facilitating gene regulation. Three dimension modeling shows a higher organization of chromatin compartmentalization where linear positions are in closer spatial proximity.α binding, gene expression and multiple epigenetic marks. For some chromosomes, such as chromosome 6, E2 induces a higher spatial compartmentalization of active and repressive marked, gene rich, highly organized and open chromatin regions. At a smaller scale, we observe that differential chromatin interactions are mostly localized in gene-rich regions after E2 stimulation. Moreover, most interacting genes are enriched for gene ontology terms known to be affected by estrogen and/or related to cancer progression. ERα binding is significantly associated with a large part of these interacting genes. Regarding expression, differential interactions appear to be correlated with regulation of gene transcription. Network analysis show that E2 induces interactions between ERα gene and apoptotic genes, which can be a pathway for E2 to regulate cancer cell proliferation through promoter-promoter interactions. In addition, analysis of interactions between DEREs located in 17q23 and 20q13 loci reveals their E2-induced spatial proximity and suggests coregulation of target genes over the genome. This E2-induced spatial proximity also provides an explanation for the observed high number of fusion events between the two loci.To biologically interpret this global change of genome organization, Hi-C data were integrated with ERα binding events represents an efficient approach to identify biologically relevant interactions. It is also worth noting that our Hi-C results make precise the large scale chromatin organization change previously observed using microscopy Hi-C interactions were validated using other techniques. For instance, Hi-C interactions between 17q23 and 20q13 at 1 and 4 h were confirmed by spatial proximity using FISH analysis. Similar interaction results between the two loci were reported using 3C-qPCR analysis α binding events and expression, but molecular functional studies will be required to reveal causality relations. Finally this work provides a global − These results are promising but there is still much to be done. Although the recent strings and binders switch model can explain the observed changes in polymer-like behavior of chromatin, it is not clear what it is the biological mechanism behind these changes. Regarding the functional relevance of interactions, our work only pointed out correlations between differential interactions, ERFile S1Figures S1-S9 and Table S1.(PDF)Click here for additional data file.


Seaweed genetic engineering is a transgenic expression system with unique features compared with those of heterotrophic prokaryotes and higher plants. This study discusses several newly sequenced seaweed nuclear genomes and the necessity that research on vector design should consider endogenous promoters, codon optimization, and gene copy number. Seaweed viruses and artificial transposons can be applied as transformation methods after acquiring a comprehensive understanding of the mechanism of viral infections in seaweeds and transposon patterns in seaweed genomes. After cultivating transgenic algal cells and tissues in a photobioreactor, a biosafety assessment of genetically modified (GM) seaweeds must be conducted before open-sea application. We propose a set of programs for the evaluation of gene flow from GM seaweeds to local/geographical environments. The effective implementation of such programs requires fundamentally systematic and interdisciplinary studies on algal physiology and genetics, marine hydrology, reproductive biology, and ecology. Chlorophyll a and b in green algae, and fucoxanthin in brown algae). They generally live attached to hard substrates (such as rocks) in coastal areas, although some brown algae in Laminariales and red algae in Corallinales can live at depths of several or occasionally nearly a hundred meters below the sea surface [Sargassum and Ulva) via changes of their intercellular gas sacs to maintain their favored depth in the water. There are approximately 10,000 species of seaweed throughout the world; however, seaweeds are generally regarded as a polyphyletic group that does not have a genetically common multicellular ancestor. They originated through multiple endosymbiotic events during the course of geological time. Generally speaking, green and red algae originated from a primary endosymbiosis when a eukaryotic host cell acquired an ancestral cyanobacterium as its plastid to form a primary symbiotic oxygenic eukaryote [Seaweeds  are plant-like organisms encompassing macroscopic, multicellular, benthic marine algae. The term comprises red, brown and green algae, which are classified according to the thallus color derived from their dominant pigments . The gametophyte and tetrasporophyte phases are usually morphologically similar [Saccorhiza polyschides, Laminaria), monophasic (Compsonema saxicola), and isomorphic types (Ectocarpus siliculosus), showing great variability between different groups [Laminaria, it consists of a macroscopic diploid generation, producing sporangium in which the cells then divide into haploid zoospores by meiosis prior to being released and growing to microscopic dissimilar male and female gametophytes [Ulva undertakes an alternation between macroscopic isomorphic diploid and haploid phases, however, in some species there can be twelve morphologically identical phases that differ in cytological and reproductive details [Unlike higher plants, seaweeds, most of which display alternation of generation, have far more complicated life histories due to the flexible relationship among morphological phases, cytological events, and genetic behaviors than generally realized . Many re similar , althoug similar . In some similar   and possesses rare large-scale gene duplications. Similar to the Ectocarpus genome, the Chondrus genome possesses halogen metabolism mechanisms for adaptation to the tidal coastal environment. The carbohydrate metabolism of Chondrus suggests the polyphyly of cellulose synthesis and the mannosylglycerate synthase in red algae potentially originates from a marine bacterium. In evolutionary history, red algae genomes have undergone loss and expansion including; the loss of genes, introns, and intergenic DNA by ecological forces, followed by an expansion of genome size resulting from the activities of transposable elements [Pyropia yezoensis, one of the most commercialized and well-cultivated seaweeds, has also been sequenced. In its 43 Mbp genome, 35% of the genes are functionally uncharacterized, and a second homolog of the phycobilisome-degradation gene, which had been assumed to be chloroplast derived, was found in the nuclear genome. This newly discovered gene may be involved either in phycobilisome photobleaching or in P. yezoensis nitrate metabolism [Saccharina japonica, whose genome size is 580–720 Mbp [Macrocystis pyrifera assessed gene expression under different abiotic factors such as, light, temperature and nutrients and revealed novel gene families in brown algae. The assembly of the 228 Mbp sequence revealed high genetic similarity between Macrocystis pyrifera and its brown seaweed relatives Ectocarpus and Laminaria [etc., which are essential in genetic engineering.There is plentiful and complete genomic information for microalgae, whereas the genomic knowledge of seaweed is very limited. The genome of scovered . For carscovered . The firelements . The gentabolism . With thtabolism ,35. Rece–720 Mbp , has bee–720 Mbp . In addi–720 Mbp ,32 and h–720 Mbp , the decaminaria . These bEctocarpus is regarded as the first promising candidate for consideration since its 214 Mbp genome has been assembled and analyzed [Ectocarpus genome is much smaller than its brown relatives Laminariales (580–720 Mbp) [Ectocarpus strains in several collection centers and a barcode system for those strains is under construction [Ectocarpus have also recently been published. In red seaweed, Pyropia yezoensis and other Porphyra spp. have been proposed as model organisms in red seaweed research of life history and ecophysiology [One bottleneck in seaweed genetic engineering is the diversity of genetic backgrounds and physiological activities (life history) among different seaweed strains. Both transient and stable genetic transformations have been accomplished in only a few strains of red, green, and brown seaweeds ,28. At panalyzed  and seveanalyzed ,40. The 720 Mbp)  and Fuca720 Mbp) ; thus ittruction . In the truction ,45,46,47ysiology ,48.Chlamydomonas [Genome engineering is focused on the development of methods to precisely manipulate nucleic acids in living cells. Targeted genome modification in plants has been regarded as an elusive goal . Howeverydomonas , howeverydomonas ,54. Hereydomonas ,51,55,56Xanthomonas, when they infect plants through the type III secretion pathway [Arabidopsis thaliana, gene knockouts in rice [Chlamydomonas have been established, fused with FokI nuclease, this technology could be utilized to create specific modifications in the Chlamydomonas genome [TALs  have rap pathway . These p pathway  and tran pathway . Due to  pathway , it has  pathway ,63, inse in rice , and the in rice . There a in rice . Severals genome ,67. Arabidopsis, the efficiency of HDR-mediated insertion is higher than NHEJ-mediated insertions [CRISPR has been hailed as a revolution in genomic engineering  Figure B. It is sertions . When CRsertions . This sysertions ,75, multsertions ,76, genosertions ,78, transertions , dynamicsertions , and evesertions . Presentsertions ,70.Chlamydomonas. Based on the assumption that closely phylogenetically related species may share similar genetic, biochemical and physiological and morphological features [Porphyridium purpureum [Thalassiosira pseudonana [Realistically, both the TAL and CRISPR techniques are not mature enough to be applied universally. Efforts must be made to resolve many fundamental problems in both methods; for example, the molecular structure and catalytic mechanism of CRISPR complex  and the features ; the esturpureum  in Rhodoeudonana  in HeterDrosophila [Arabidopsis promoters have TATA boxes [Eukaryotic promoters are generally described as having a core promoter near the site of transcription initiation and one or more enhancer elements that may be located more distantly . Promoteosophila , which aosophila . Neverthosophila  and onlyTA boxes . It seemTA boxes , and somTA boxes . When coP. yezoensis cells [fcp) and the endogenous 5′ upstream region of the actin 1 gene from P. yezoensis (PyAct1), have shown efficient activity when used to construct expression vectors. The former has been applied in L. japonica [Chlamydomonas reinhardtii [cis-regulatory code [The traditional viral promoters CaMV35S and SV40, commonly used in plant and animal genetic engineering, have been broadly applied in seaweed genetic engineering when there is sparse genetic information available ,93,94,95is cells . Some aljaponica , whereasjaponica ,101. Thejaponica , and it nhardtii . Increasnhardtii ,84,104. ory code , which wetc. At present, there are four prevailing strategies in rational promoter design; introducing random mutations into the promoter sequence by error-prone PCR, mutating non-consensus spacer nucleotide regions within a promoter via saturation mutagenesis, assembling different tandem upstream activation sequences to tune the core promoter and altering the structure of transcription factor-binding site sequences [de novo tunable promoter synthesis in seaweed genetic engineering, while more rational strategies will still rely on the increasing understanding of promoter sequence structure diversity from marine algae uncovered by NGS technologies. Natural promoters for use in seaweed genetic engineering can either be isolated from endogenous algal sequences or isolated from a heterologous viral promoter. Occasionally, heterologous viral or phage-derived promoters cannot regulate transcription levels, meanwhile, isolated endogenous promoters are commonly unable to maximize the transcriptional capacity of the host . These sequences . Schlabaequences . All of P. tenera and P. yezoensis has been successfully used in transgenic research; this fluorescent protein uses short emission wavelengths to diminish the background fluorescence interference from chlorophyll molecules in algal cells [de novo gene synthesis has become another powerful tool to express heterologous genes in host organisms. Designing an optimal gene requires a comprehensive inspection of codon usage, mRNA secondary structure, cryptic ribosome binding sites [Codon usage is another barrier to the transport of exogenous protein-coding genes into seaweeds. Exogenous proteins usually contain codons which are rarely used in the desired host, in addition to transcriptional regulation elements within their own coding sequence . Codon bal cells . Aside fng sites , and theng sites . These ong sites .Agrobacterium-mediated method yields high proportions of transformants that combine low copy number with the expression of the non-selected reporter gene, and bombardment particles with minimal cassettes yield high absolute transformation efficiencies [Agrobacterium-mediated method [Agrobacterium mechanism within the algal nuclear genome constrain the application of the Agrobacterium-mediated method in seaweed [Particle bombardment, electroporation, and glass beads are the three primary transformation methods applied in seaweed due to their mature technologies and their successful application in many phylums and classes of seaweeds  ,120,121 ciencies . Howeverd method  in seawe seaweed .Chlorella system and viruses in the brown seaweed system [Large dsDNA viruses known to infect eukaryotic algae are categorized into two groups: viruses in the unicellular microalgae d system . Nearly d system . Howeverd system . When cod system . The trad system . Thus, td system ,129, butd system . The secd system . The vird system . DetailsC. crispus, implies the existence of many transposon elements. The transposon component of this genome is quite complicated because of their huge number and significant divergence [Pyropia yezoensis genome [Transposons, a group of mobile DNA first discovered in maize, have been developed as powerful tools for genetic engineering in some microalgae ,133. Thevergence . Transpos genome ,134 as as genome .The initial debate about the risks from transgenic organisms emerged with the birth of the first genetically modified (GM) organism in the 1970s . The thretc.), life cycle, and maximum growth conditions will have a primary effect on the probability and degree of the gene flow from GM seaweeds; (2) Distribution medium of other aquatic organisms: The life cycle of seaweeds is so complex that any organism, including marine invertebrates, vertebrates, or even other algae and sea grasses, can assist GM seaweeds in the distribution of GM spores; (3) Hydrological structure of the local aquatic environment: The trial area should be a relatively stable environment; however, monthly or seasonal changes should be considered, and daily or weekly hydrological fluctuations must be noted to understand the time of the release of spores; (4) Genetic information exchanges with other related population or species: Seaweeds can “compare notes” on their respective genetic information by various sexual or asexual reproductive processes according to the degree of their genetic compatibility to other species or populations; (5) Life cycle and reproductive strategies of GM seaweeds: Close attention should be given to any genetic, physiological, or morphological changes in the life history of GM seaweeds, especially during their release of spores to the adjacent aquatic environment; (6) Survivability of algal propagules from GM seaweed: The fitness of any escaped vegetative fragments or sporangia of GM seaweeds to the environment outside the cultivation area must be determined; (7) Semi-quantification of gene flow: Several mathematical models specific to each GM species or population are required because the life cycles of seaweeds are extremely diverse; even within a genus, the life cycle can significantly differ under various conditions [We propose a set of rational assessment programs for evaluating gene flow from GM seaweeds to local or large-scale environment based on the assessment of gene flow probability from transgenic plants . Howevernditions . In the nditions  because nditions ,144; (8)i.e., endogenous promoters, reverse mutation as a selective marker, and the replacement of any non-algal viral DNA sequence with algal virus nucleotides [Another necessary step is to construct “complete marine algae-derived” vectors, leotides . Basing leotides , Gresselleotides .etc.). The final biosafety assessment of a GM seaweed demands multi-disciplinary research on algal genetics, physiology, reproductive biology, and ecology from the molecular to at least the local aquatic ecosystem level through mathematics, biology, chemistry, physics, and even sociology when considering environmental issues. These innovations will bring unlimited possibilities in seaweed genetic engineering, which is still in its infancy. Combining these innovations with the rapidly developing fields of systems biology and metabolic engineering will satisfy the demands related to energy, environment, and human health.Seaweed genetic engineering could bridge the gap between fundamental and applied studies in seaweed research. The decreasing cost of sequencing provides us with many opportunities to investigate the fine genetic structure of seaweeds and consequently identify innovative genetic transformation elements (promoters, transformation methods, selective markers,


Epstein-Barr Virus (EBV) transforms resting B-lymphocytes into proliferating lymphoblasts to establish latent infections that can give rise to malignancies. We show here that EBV-encoded transcriptional regulator EBNA2 drives the cooperative and combinatorial genome-wide binding of two master regulators of B-cell fate, namely EBF1 and RBP-jκ. Previous studies suggest that these B-cell factors are statically bound to target gene promoters. In contrast, we found that EBNA2 induces the formation of new binding for both RBP-jκ and EBF1, many of which are in close physical proximity in the cellular and viral genome. These newly induced binding sites co-occupied by EBNA2-EBF1-RBP-jκ correlate strongly with transcriptional activation of linked genes that are important for B-lymphoblast function. Conditional expression or repression of EBNA2 leads to a rapid alteration in RBP-jκ and EBF1 binding. Biochemical and shRNA depletion studies provide evidence for cooperative assembly at co-occupied sites. These findings reveal that EBNA2 facilitate combinatorial interactions to induce new patterns of transcription factor occupancy and gene programming necessary to drive B-lymphoblast growth and survival. Epstein-Barr Virus (EBV) reprograms host cell transcription through multiple mechanisms. Here, we show that EBV-encoded transcriptional co-activator EBNA2 drives the formation of new chromosome binding sites for host cell factors RBP-jκ and EBF1. The formation of these new sites is EBNA2-dependent. These newly formed sites have overlapping or neighboring consensus binding sites for these factors, but are only co-occupied in the presence of EBNA2. Newly formed, co-occupied binding sites are highly enriched at promoter and enhancer regulatory elements of genes activated by EBV and required for B-cell proliferation and survival. These findings indicate that EBNA2 drives cooperative and combinatorial transcription factor interactions on chromosomal DNA. We suggest that models depicting the static binding of master regulatory transcription factors to consensus binding sites be revised, and that co-activators, like EBNA2, induce dynamic and combinatorial selection of genome-wide binding sites to alter gene regulation. Tumor viruses encode many factors that mimic and alter host cell processes. Epstein-Barr Virus (EBV) is a human tumor virus associated with various lymphoid and epithelial cell malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, and lymphoproliferative disorders in the immunosuppressed , 2. EBV EBV encodes several nuclear proteins that modulate host and viral gene transcription . EBNA2 iPatterns of transcription factor binding to chromosomal regulatory elements is a primary determinant of cell fate and identity , 15, 16.Transcription factors with sequence-specific DNA binding capability are known to recognize a distinct primary DNA sequence element. However, not all consensus sequences are occupied by these transcription factors. Epigenetic factors, including DNA methylation, nucleosome positioning, heterochromatin, and competitive transcription factor binding may influence the occupancy at different chromosomal locations in cell-type dependent manners. It is also known that some chromosome occupancy may depend on cooperative binding with additional factors, including sequence specific factors and non-DNA binding co-regulatory factors. Here, we present evidence that EBNA2 is one such co-regulatory factor that alters the genome wide distribution of sequence-specific B-cell master regulatory factors EBF1 and RBP-jκ.To investigate the landscape of transcription factor binding on identical genomes in two closely related cell types, we first compared the binding patterns of EBF1 and RBP-jκ on the EBV genomes in B-cells with distinct latency types. We compared an EBV positive lymphoblastoid cell line (LCL) with a type III latency expression pattern (EBNA2 positive) to an EBV positive Burkitt lymphoma (BL) cell line (Mutu I) with a type I latency expression pattern (EBNA2 negative) Figs  and S1. To determine if the cell-type specific binding of EBF1 and RBP-jκ extended to the cellular genome, we compared ChIP-Seq data for cellular genome binding patterns in LCL and Mutu I cells . We founEBV-encodes several transcriptional co-regulators that binds RBP-jκ and are expressed in type III, but not type I latency. We therefore aligned our ChIP-Seq data sets for RBP-jκ and EBF1 with several published ChIP-Seq data sets for EBNA2, EBNA3C, and EBNALP from both LCL and Mutu III cells , 18–20 . The de The convergent binding of EBNA2, EBF1, and RBP-jκ was further analyzed for colocalizations with other well-established histone modifications and transcription factors . We gene−12 by Fisher exact test, To investigate the functional significance of the co-occupancy of EBNA2 with EBF1 and RBP-jκ, we analyzed the mRNA expression of LCL and Mutu I cells using Illumina microarrays Figs  and S4. Correlations studies suggest that EBNA2 may facilitate binding and co-occupancy of RBP-jκ and EBF1 in LCLs. To experimentally determine if EBNA2 can alter EBF1 and RBP-jκ chromosome occupancy, we utilized an EBNA2-inducible cell line, EREB2.5, that contains an EBNA2 protein fused to the estrogen receptor (ER) . The ER-in vitro DNA affinity assays followed by Western blotting  transformed with Mutu I virus. EBV positive BL cell lines Mutu I, Kem I, and Kem III were obtained from Dr. Jeffrey Sample, Penn State University Hershey Medical School, PA. LCLs, Mutu I, Kem I and Kem III cells, were maintained in RPMI containing 12% FBS and antibiotics (penicillin and streptomycin). EREB 2.5, a lymphoblastoid cell line expressing the estrogen-inducible EBNA2-estrogen receptor (ER) fusion protein , was mai7 cells per assay with 10 μg of either rabbit anti- EBF1 (EMD Millipore AB10523), RBP-jκ Abcam AB25949), histone H3K4me3 (EMD Millipore 07–473), H3K4me1 (Active Motif 39297), H3K27me3 (Active Motif 39155) antibodies or control rabbit IgG (Santa Cruz Biotechnology sc-2027). ChIP was performed by as described previously with some modifications , e.g for 10kb from TSS, a window of [3.16kb to 31.6kb] was generated. TSS distance X for the plot varied from log10X = 2 to log10X = 5.2 using step of 0.1.RNA samples extracted from three independently cultured Mutu I or LCL cells then further treated with DNase I by using DNase treatment and removal kit (Ambion). RNA quality was determined using the Bioanalyzer (Agilent). Only samples with RIN numbers >7.5 were used for further studies. Equal amounts (400ng) of total RNA was amplified as recommended by Illumina and hybridized to the HumanHT-12 v4 human whole genome bead arrays. Illumina BeadStudio v.3.0 software was used to export expression levels and detection p-values for each probe of each sample. Arrays were quantile-normalized and filtered to remove probes with a detection p-value>0.05 in all samples. Expression level comparisons between the two cell lines were done using two sample t-test and correction for multiple testing to estimate False Discovery Rate (FDR) was performed . FDR<5% S1 Fig(A) Magnified view of ChIP-Seq peaks mapped to EBV genome for EBF1, RBP-jκ, and EBNA2 at OriP and Cp regions (left) or LMP2A/ LMP1 promoter regulatory regions (right). (B) Sequence analysis of EBF1 and RBP-jκ peak centers and consensus binding sites at Cp, LMP1, and LMP2A.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(A) Cellular ChIP-Seq tracks for genes with LCL-specific co-occupied sites for EBF1, RBP-jκ, and EBNA2. Tracks shown for IL7, HES1, FCER2, and ICA1. (B) Cellular ChIP-Seq tracks for genes with Mutu I-specific co-occupied sites for EBF1 and RBP-jκ. Tracks shown for ZNF595, MIR4325, and RNF144B. Primer positions are highlighted in magenta.(TIF)Click here for additional data file.S4 Fig(A) Number of genes with RNA-expression differences between LCL (L) and Mutu I (M) were calculated for genes with cell-type specific EBF1 peaks. Genes with EBF1-specific binding sites in LCL were 94.5% likely to have greater transcription levels in LCL. (B) Heat map of the top 20 genes with LCL (red) or Mutu I (blue)-specific gene expression and cell-type specific EBF1 binding sites near the TSS. (C) RT-qPCR analysis for genes with cell-type specific binding for EBF1 in LCL (red), or for Mutu I (blue). (D) Functions enriched and predicted to be activated by genes with cell-specific EBF1 binding and transcription in LCL.(TIF)Click here for additional data file.S5 FigA), RBP-jκ (B), or EBNA2 (C) are shown for EREB2.5 cells treated with estradiol (blue), or 24 (pink), or 48 hrs (red) after withdrawal of estradiol. ChIP was assayed for sites associated with genes indicated below each bar graph. Asterisk indicates p < 0.05.Cells were treated as in main text (TIF)Click here for additional data file.S6 FigEREB2.5 cells treated continuously with estradiol (E2) or at 24 (middle) or 48 hrs (lower panel) after estradiol removal. FACS intensity after propidium iodide incorporation shown in the X-axis and cell number is shown in the Y-axis.(TIF)Click here for additional data file.S7 FigEREB2.5 cells were depleted of EBNA2 by withdrawal of estradiol for 72 hrs (grey), followed by re-addition of estradiol for 72 hrs (black). Cells were then subject to ChIP assays for EBF1 (top panels) or RBP-jκ (lower panels). Cellular binding sites for EBNA2 co-occupied sites is shown in left panel, non-EBNA2 co-occupied sites (middle panel), and viral genome sites (right panel). Asterisk indicates p < 0.05.(TIF)Click here for additional data file.S8 FigMutuI (M) or LCL (L) cell extracts were subject to IP with control IgG, anti-EBF1, or anti-RBP-jκ, and then assayed by Western blot for EBNA2, EBF1, or RBP-jκ as indicated. Input represents 2% of the total starting lysate for IP. Arrow indicates the RBP-jκ band above the background cross-reacting IgG heavy chain band.(TIF)Click here for additional data file.S9 FigSequence organization of the consensus RBP-jκ and EBF1binding sites in LMP1, IL7, and HES1 promoters used for DNA-affinity assays shown in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Parkinson’s disease (PD) is one of the major neurodegenerative disorders. Mitochondrial malfunction is implicated in PD pathogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced putative kinase 1 (PINK1), a serine/threonine kinase, plays an important role in the quality control of mitochondria and more than 70 PINK1 mutations have been identified to cause early-onset PD. However, the regulation of PINK1 gene expression remains elusive. In the present study, we identified the transcription start site (TSS) of the human PINK1 gene using switching mechanism at 5’end of RNA transcription (SMART RACE) assay. The TSS is located at 91 bp upstream of the translation start site ATG. The region with 104 bp was identified as the minimal promoter region by deletion analysis followed by dual luciferase assay. Four functional cis-acting nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-binding sites within the PINK1 promoter were identified. NFκB overexpression led to the up-regulation of PINK1 expression in both HEK293 cells and SH-SY5Y cells. Consistently, lipopolysaccharide (LPS), a strong activator of NFκB, significantly increased PINK1 expression in SH-SY5Y cells. Taken together, our results clearly suggested that PINK1 expression is tightly regulated at its transcription level and NFκB is a positive regulator for PINK1 expression. Parkinson’s disease (PD) is the second most common neurodegenerative disease . PD patiPhosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced putative kinase 1 (PINK1), initially was identified as a downstream molecule of PTEN in cancer cells . PINK1 iNuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) is a family of diametric transcription factors, regulating numerous genes involving in cell survival, inflammation and immunity. The NFκB family consists of five members, p50, p52, p65 (RelA), RelB and C-Rel. All of them share an N-terminal Rel homology domain (RHD) responsible for DNA binding and homo- or hetero-dimerization. Only three members including p65, RelB and C-Rel contain transcription activation domain (TAD) necessary for the activation of target genes. NFκB exists in forms of homo- or hetero-dimer and the most abundant form is the p65/p50 heterodimer. NFκB dimers bind with inhibitor of κB (IκB) proteins in cytoplasm, preventing its nuclear translocation and DNA binding. Once IκB protein degradation is induced, NFκB dimers are released from IκB-NFκB complex and subsequently translocated from cytoplasm to nucleus , thus trAlthough the function of PINK1 is well studied in terms of mitochondrial quality control, the regulation of PINK1 gene expression is barely explored. In the present study, we aimed to understand the transcriptional regulation of the PINK1 gene. For the first time, we identified that the transcription start site (TSS) of PINK1 is located 91 bp upstream of the translation start site (ATG). The region of 104 bp (−78 to +26) contains the minimal promoter with functional transcriptional activity for the PINK1 gene. Furthermore, we showed that the PINK1 gene promoter contains 4 functional cis-acting NFκB-binding sites, which promote PINK1 expression. Taken together, our results demonstrated that PINK1 expression is tightly regulated at its transcriptional level and that NFκB is a positive regulator for PINK1 expression.The human PINK1 gene spans a region of 18,056 bp on chromosome 1p36. The human PINK1 gene transcript (NCBI Reference Sequence: NM_032409) is 2680-bp long and composed of 8 exons . However, additional deletion of 31 bp from pPINK1-G to pPINK1-H (−47 to +26) significantly reduced the luciferase activity to 11.46 ± 2.50 RLU (p < 0.05). These data suggest that the region from −78 to +26, containing the transcription start site, possesses the minimal promoter activity.To determine the functional promoter region of PINK1 gene, we sub-cloned the 1,825 bp of 5’ flanking region of the PINK1 gene into the promoterless plasmid vector pGL3-basic. The pGL3-basic vector lacks eukaryotic promoter and enhancer sequences upstream of a reporter luciferase gene. The luciferase activity in cells transfected with this plasmid depends on the presence and proper orientation of a functional promoter upstream of the luciferase gene. The pPINK1-A plasmid was constructed to contain the 1,825 bp fragment from −1799 to +26 bp of PINK1 gene upstream of the luciferase reporter gene. Plasmid DNA was transfected into HEK293 cells, and luciferase activity was measured by a luminometer to reflect promoter activity. Compared with cells transfected with an empty vector pGL3-basic, pPINK1-A-transfected cells showed a robust luciferase activity (113.47 ± 10.63 RLU) . These results suggest that NFκB activates PINK1 gene transcription and the PINK1 promoter fragment contains functional NFκB sites.Computer-based transcription factor binding site analysis revealed four putative NFκB elements in the 1.8 kb promoter region of the human PINK1 gene  served as a positive binding probe and a mutant NFκB binding probe (NFκB-Mu-oligo) which loses the NFκB binding ability served as a negative binding probe . NFκB-WtTo further confirm the binding between PINK1-NFκB-1 in the human PINK1 promoter region and NFκB, we performed EMSA with IRDye-700 labelled PINK1-NFκB-1. The probe showed a shifted band Figure E lane 2 p < 0.01)  , respectively   Figure B in the y Figure C. Furthe) Figure D, respecMitochondrial dysfunction plays a critical role in the development of PD and other neurodegenerative diseases such as Alzheimer’s disease -29] and and29] aTo investigate the transcription and translation of the human PINK1 gene, we first mapped the adenine 91 bp upstream of the first ATG in exon 1 as the transcription start site. Next we cloned the 1.8 kb fragment of 5’ UTR of the human PINK1 gene into a promoterless vector upstream of a reporter luciferase gene. The luciferase assay showed that this 1.8 kb fragment has strong promoter activity, indicating that the 1.8 kb fragment functions as the human PINK1 gene promoter. Subsequently, by deletion analysis we found that the 104 bp fragment from −78 to 26 serves as the minimal promoter region. The human PINK1 gene promoter does not have high GC content (<50%) like the human BACE1 and TMP21 promoter ,41]. Co. Co41]. in vivo, promoting the investigation of PD pathogenesis and drug development. Moreover, as SH-SY5Y cells are human dopaminergic cells and the dopaminergic neurons are mainly affected in PD patients, the results from SH-SY5Y cells can better represent the possible PINK1 regulation pathway in the brain of PD patients.In this study, we clearly showed that NFκB significant upregulates PINK1 expression in both human and rodent cell lines, indicating that same regulation pathway exists in both human and rodents. It provides the strong evidence to support the use of rodent models to study the regulation of PINK1 expression N-terminal proteolysis of FL-PINK1 produces two fragments, ∆1-PINK1 and ∆2-PINK1. After processing the proteolysis productions translocate to cytosol and undergo proteasome degradation ,7],42],[42]7],7],. It has Our results demonstrate that PINK1 expression is tightly regulated at its transcription level and NFκB signaling is a positive regulator for PINK1 gene expression.To amplify the 5’-untranslated region of the PINK1 gene from DNA extracted from the human neuroblastoma cells SH-SY5Y, a forward primer (5’-cgcgagctcgttgcccaggctggtcttg) corresponding to −1799 bp of the transcriptional start site on exon 1 and a reverse primer (5’-cccaagcttcacaacaaacttggggcggtgcc) corresponding to +26 bp of the transcriptional start site were used. The PINK1 DNA fragment was cloned into a pGL3-basic vector upstream of a luciferase reporter gene (Promega) to construct pPINK1-A. Primers were designed to include restriction enzyme sites so that the PCR-amplified fragments can be inserted into the multiple cloning sites of the pGL3-basic vector. A series of deletion mutations  of PINK1 promoter were constructed utilizing primers listed as following: 26 F-cccaagcttcacaacaaacttggggcggtgcc, and 1799R- cccaagcttgttgcccaggctggtcttg; −795 F -ggcatggggatccaccatcttg, and -339 F-ccgctcgaggacctcgaatgctgccc; −103 F-ccgctcgagaaagtcactgctagaggc, and -50R-cccaagcttcgactcggcgcgtggggg; −78 F-ccgctcgagccagcatagcgcccccac, and -47 F -ccgctcgaggaactgccgcgggggccg, and -19 F-ccgctcgagccagcgcctgcgcctgcg, respectively.Total RNA was extracted from SH-SY5Y cells using TRI reagent (Sigma) following the manufacturer’s protocol. SMART-RACE was performed using the SMARTer™ RACE cDNA Amplification Kit (Clontech) following the user’s manual. Simply, the first stand cDNA was synthesized from total RNA extracted from SH-SYHY cells with oligo (dT) primer in the presence of SMARTer IIA oligonucleotide (5’-aagcagtggtatcaacgcagagtacxxxxx (X is undisclosed base in the proprietary SMARTer oligo sequence). The SMARTer IIA oligonucleotide is able to anneal to the 5’-end of the first stand cDNA and serve as template to extend the 5’-end cDNA tail. A PINK1 reverse primer (5’-ccgaagcttgccctgcaagcgtctcgtgt) was specifically designed to recognize the +431 to +450 bp of human PINK1 gene downstream of the translation start site (ATG). The PCR products containing PINK1promoter region were amplified using SMARTer IIA oligonucleotide and PINK1 reverse primer and the first stand cDNA as template. The resulting PCR products were cloned into pcDNA4/myc-His A vector for sequencing and the first nucleotide linking with the adapter sequence was identified as the transcription start site of the human PINK1 gene.2. 500 ng plasmid DNA per well of 24-well-plate for luciferase assay or 2 μg plasmid DNA per 35-mm-diameter plate for RNA extraction and Western blot analysis were used for cell transfection, respectively. For luciferase assay, pCMV-Rluc  was co-transfected as a control to normalize the transfection efficiency. The cells were transfected using PEI reagent  or Lipofectamine-™ 2000 reagent (Invitrogen). Cells were harvested 24 hrs after transfection and lysed with 100 μl 1× passive lysis buffer (Promega) per well. Firefly luciferase activities and Renilla luciferase activities of the same sample were sequentially assayed on a luminometer (Turner Designs Model 20/20) following the protocol of the Dual-luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalized to the Renilla luciferase activity and the resulted value reflected the promoter activity.Human embryonic kidney HEK293, mouse neuroblastoma N2A and human neuroblastoma SH-SY5Y cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine, 50 units/ml penicillin G sodium, and 50 μg/ml streptomycin sulfate (Invitrogen). All cells were maintained in a humidified 37°C incubator containing 5% COHEK293 cells were transiently transfected with NFκB p65 and nuclear extracts were collected. Cells were rinsed and harvested with 1 × phosphate-buffered saline. After centrifugation, cell pellets were resuspended with 5 × volume of buffer A . Cells were pipetted up and down gently and maintained on ice for 15 minutes. The cell suspension was transferred to a Kontes all glass Dounce tissue grinder and ruptured by 10 strokes. 10% NP40 was added into the cell suspension for a final concentration of 0.5%. The samples were placed on ice for 15 minutes and stroked 5 more times. Crude nuclei were collected by centrifugation at 2000 g for 10 minutes and washed three times with buffer A containing 0.5% NP40 and resuspended in buffer C  at 4°C for 15 min. The samples were centrifuged at 12000 g for 4 min at 4°C, and the supernatant containing nuclear proteins was collected. EMSA was performed as previously described . NF. NF48]. The authors declare that they have no competing interests.XD and WS conceived and designed the experiments; XD, JT, QX, YW performed the experiments; XD, YW, TL and WS analyzed the data; FC, WS contributed reagents/materials/analysis tools; XD, YW and WS wrote the paper. All authors read and approved the final manuscript.


Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair (TCR) has been described to stop transcription and quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. Moreover, numerous efforts investigated the fate of blocked RNA polymerase II (Pol II) during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed and degraded or bypass the lesions to allow TCR. We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We found that the used UVB dose did not induce Pol II degradation however surprisingly at about 93% of the promoters of all expressed genes Pol II occupancy was seriously reduced 2–4 hours following UVB irradiation. The presence of Pol II at these cleared promoters was restored 5–6 hours after irradiation, indicating that the negative regulation is very dynamic. We also identified a small set of genes , where the UVB-induced Pol II clearing did not operate. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH, but not TBP, follows the behavior of Pol II, suggesting that at these genes upon UVB treatment TFIIH is sequestered for DNA repair by the TCR machinery. In agreement, in cells where the TCR factor, the Cockayne Syndrome B protein, was depleted UVB did not induce Pol II and TFIIH clearance at promoters. Thus, our study reveals a UVB induced negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed gene promoters, and a small subset of genes, where Pol II escapes this negative regulation. Our genome is continuously exposed to genotoxic attacks that generate aberrant DNA structures. These can block the transcribing DNA-dependent RNA polymerase II (Pol II) enzyme and can lead to deleterious cellular processes. Cells have developed several mechanisms to stop Pol II, repair the roadblocks and to restore normal polymerase traffic. Numerous efforts investigated the fate of blocked Pol II during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed or bypass the lesions to allow repair. We carried out a genome-wide analysis of Pol II behavior upon a DNA damaging stress, UVB, which is relevant from the public health standpoint. Thus, we could follow UVB-induced Pol II behavior changes on every human gene over time. We uncovered a novel UV induced negative regulatory mechanism, which inhibits the recruitment of Pol II to the promoters of about 93% of all transcribed genes, and a small subset of gene  that escapes this negative regulation, probably because their gene products are required during/after UVB irradiation. Thus, we uncover how a cell induces a global negative regulation at the level of transcription initiation in response to a genotoxic stress. Analyses of short transcribed RNA molecules showed that these arrested polymerases are predominantly in a transcriptionally engaged state end of the annotated genes (EAGs) representing Pol IIs that have finished transcribing the pre-mRNA and are slowly transcribing and approaching the termination site often 4–6 kb away from the 3′end of the gene Proper cell homeostasis and function requires expression of the DNA encoded information. Maintenance of genome integrity and accurate replication is crucial for correctly regulated gene expression. Transcription of thousands of coding and non-coding RNAs by the RNA polymerase II (Pol II) is a regulated multistep process that can be divided into five stages: pre-initiation, initiation, promoter clearance, elongation and termination. Based on numerous genome-wide studies analyzing Pol II transcription in several metazoan organisms using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) it is now clear that on different regions of an expressed gene, distinct types of Pol II occupancy signals can be detected. The “canonical” Pol II occupancy ChIP-seq profile on an average expressed gene displays Pol II molecules engaged in the major phases of transcription Damage or alterations of the DNA structure can threaten the progression of transcription. Indeed, Pol II driven transcription has been reported to be disturbed by “roadblocks” on the DNA template, which arises from both environmental and endogenous sources, such as special DNA sequences, non-canonical DNA structures, topological constrains and DNA lesions UV light is one of the most genotoxic environmental sources of transcription-blocking DNA damages. Different wavelengths of the UV light can generate a wide range of lesions in the DNA template. Based on its wavelength, UV light can be divided into UVA (315–400 nm), UVB (280–315 nm) and UVC (<280 nm). UVC has higher energy level than UVA and UVB due to its shorter wavelength; however, UVC radiation is not relevant from a public health standpoint as it is absorbed by the ozone layer. UV-related lesions in living organisms are induced mostly by UVA and UVB.The ionizing energy of UVB and UVC radiation is directly absorbed by the DNA and can cause cyclobutane pyrimidine dimers (CPDs), pyrimidine 6-4 pyrimidone photoproducts (6-4PPs), which are the most persistent and predominant lesions In vitro the half-life of an arrested Pol II at a CPD can be ∼20 hours, and it covers 10 nt upstream and 25 nt downstream of the CPD It seems that cells have evolved several solutions to deal with the persistently stopped Pol II elongation complexes: i) Pol II can bypass the lesions, but this process is slow and extremely inefficient Our knowledge about Pol II transcription inhibition after UV irradiation is based on studies carried out on a few model genes and in experiments often using lethal UVC doses. Moreover, many studies have investigated the fate of Pol II elongation complexes during TCR, but very little information has been obtained on how TCR influences the whole Pol II transcription cycle during TCR. Therefore, the mechanism by which Pol II transcription is affected genome-wide upon sublethal doses of UV irradiation is not yet well understood. To investigate the fate of Pol II during TCR processes, we have investigated Pol II occupancy at a genome wide level following UVB treatment over time in human MCF7 cells. Our results show that on about 93% of the promoters of expressed genes Pol II occupancy is seriously reduced 2–4 hours following UVB irradiation, and that the presence of Pol II is restored to “normal”, or even sometimes higher, levels 5–6 hours after irradiation. We also identified a smaller set of genes, where the presence of Pol II at the promoter regions does not decrease, but rather increases at the promoters and also throughout the entire transcription units of these genes after UVB irradiation. Thus, our study reveals a global negative regulatory mechanism that targets RNA polymerase II transcription initiation on the large majority of transcribed genes following sublethal UV irradiation and a small subset of key regulatory genes, where Pol II escapes the negative regulation.2 of UVB irradiation on the DNA damage response proficient MCF7 human breast cancer cell line, containing wild type p53 , along the GBs of the genes (from TSS +100 bp to EAG) and downstream from EAG (from EAG to EAG +4 kb) regions of the 4500 expressed genes from the control data set and the six UVB irradiated samples . In addiFrom the heat maps it is visible that the generated clusters represent genes with distinct, unique Pol II transcription responses following UVB treatment, as we can observe well-defined differences in the changes of Pol II distribution at the different regions of the annotated genes. We found that the 4500 expressed genes can be sorted into two main groups, hereafter called A and B. Group A contains about 93% of the examined genes  and showInterestingly, in many sub-clusters Pol II occupancy increased at 1 hour and then again at 5 and 6 hours following UVB treatment at distinct regions of the transcription units . CompareIn contrast to group A, the relatively small set of genes in group B  and genes, out of which many regulate DNA damage response, signal transduction by p53 and apoptosis, where Pol II presence is increased in the TSS, GB and slightly at EAG+4000 regions . Note however that most of the NER factors are abundant in the nucleus DNA ligase IV (ATP-dependent), cyclin D2, p21 , GADD45A and GADD45B, and TP53AIP1, SESN2, FAS, BBC3, which are regulators of repair, cell growth or survival pathways further validating the biological significance of the present Pol II occupancy study.In the above detailed global analysis of Pol II behavior on expressed genes upon UVB treatment we have observed that many genes in group B, belonging to the GO categories ‘response to UV’ and ‘DNA damage response’, have increased Pol II occupancy throughout their whole transcription unit. Thus, we analyzed Pol II distribution at annotated repair and UV-responsive genes existing in the KEGG database To evaluate whether the above observed massive Pol II promoter clearance at 2–4 hour time points in Group A  is due tAdditionally we tested the level of the phosphorylation of the CTD of Rpb1 using antibodies that recognize different and specifically phosphorylated forms of the CTD heptapeptide repeats . In thisTo test whether the degradation of additional PIC subunits would be responsible for the important Pol II clearance from the promoters upon UVB irradiation, we tested the global protein levels of TBP, TFIIB and the kinase subunit of TFIIH, CDK7, which is known to phosphorylate the CTD of Pol II 2 UVB irradiation. Pol II signals were quantified on the promoters of two randomly selected genes from group A (rplp1 and ubc) and B (p21 and wdr24). Negative/mock control ChIP was carried out with Sepharose G beads alone (NoAb), and for an additional control, oligonucleotides were designed to target an intergenic region, where no Pol II binding is expected . To this end we used anti-Pol II ChIP coupled qPCR detection on non-treated samples and on samples incubated for 3 h and 6 h after 55 J/mexpected .p21) or increased (wdr24) Pol II enrichment at the promoters at 3 hours after UVB treatment compared to the control. In the case of wdr24 gene Pol II enrichment increases up to 6 hours post UVB treatment. These ChIP-qPCR validations are in good agreement with our ChIP-seq, bioinformatics and IF results.The ChIP-qPCR experiment confirmed the different Pol II behavior patterns at the promoters of the selected genes. At the promoters of genes from group A we observed a decreased Pol II occupancy in the sample that was harvested 3 hours following UVB irradiation when compared to the control . As expeNext, to better understand the mechanisms that may regulate the opposite Pol II behavior on gene promoters belonging to either group A or B, we investigated whether certain PIC subunit enrichments would also be affected upon UVB irradiation. To this end we carried out ChIP-qPCR detection using antibodies against subunits of two GTFs, the TATA-box binding protein (TBP) a subunit of TFIID, and p62, a subunit of the TFIIH . SurprisCSB is known to trigger the recruitment of NER factors, including TFIIH, to UV-induced DNA lesions to carry out the repair process see . Thus, tIn the past decades a lot of efforts have been devoted to understand how Pol II transcription is recovered after genotoxic stress, such as UV irradiation. However, our knowledge about Pol II transcription inhibition during TCR and the subsequent recovery of RNA synthesis is based only on a handful of model genes. Note that most of the previous studies that investigated the link between repair and transcription used much higher, often lethal, UVC doses 2 dose of UVB irradiation used had a wide effect on the genome of MCF7 cells, as it induced CPD lesions genome-wide, which persisted up to, or even longer than 24 hours  Pol II assembled in a PIC and pausing at or close to the TSSs runs in the gene body, ii) new PIC formation does not occur, or iii) existing PICs containing paused Pol II are actively removed from TSSs. Nevertheless, our observations strongly suggest the existence of a rapid negative regulatory mechanism, which seems to act on almost all expressed gene promoters during TCR to either remove existing PICs, containing paused Pol IIs, and/or prevent Pol II to enter in initiation, or re-initiation, complexes at promoters, when transcription-blocking lesions may still be present on the ORFs. The fact that promoter-proximal pausing of Pol II at group A genes 2–4 h after UVB irradiation decreases everywhere , indepenIn agreement, an active global transcriptional repression process, rather than a physical blocking of transcription, has already been suggested It is crucial for the cell that DNA lesions are repaired in the different transcription units with equal efficiencies, in order to avoid that short genes with low damage probability are repaired and transcribed quicker than long genes with a high likelihood of carrying many UV-induced CPD lesions. We do not know whether this negative “preventive” transcription regulatory mechanism can operate at several levels separately (i.e. remove existing PICs containing paused Pol II and/or block Pol II incorporation in new PICs) or whether it would inhibit both mechanisms by blocking the function of the same factor for example. Interestingly, it has been observed that the loss of de novo RNA synthesis occurs only at the UV-irradiated area of the nucleus The transcription factor TFIIH was shown to play an essential role in both transcription initiation and DNA repair In line with the sequestration model by the TC-NER machinery, earlier work showed that TFIIH engagement in transcription initiation takes only a few seconds, whereas when it engages in NER it takes minutes for every NER event, which may explain why the pool of TFIIH is quickly depleted and shifted towards repair The detected synchronized Pol II reappearance on the TSSs of most of the transcribed genes 5–6 hours after UVB irradiation suggests that transcription-coupled repair is largely completed by this time in MCF7 cells; TFIIH complexes are released from the repair sites and again available for new PIC formation on the transcribed genes. Moreover, our results investigating the presence of TBP, a DNA-binding component of TFIID, showed that the presence of TBP at the TSSs of genes is not changing dramatically over time after UVB irradiation. This observation suggests that on the transcribed genes during UVB irradiation, and following the potential TFIIH sequestration by the repair machinery, a minimal TFIID/TBP-containing re-initiation complex may stay bookmarking the TSSs where transcription has to be resumed once TCR is completed.While the Pol II clearance and recovery seems to be synchronized at the promoters of group A genes, we found very diverse patterns of Pol II behavior on the GB and on the EAG+4000 regions of the expressed genes during recovery after UVB irradiation . Based oAnother common characteristic exists on the GBs of group A transcription units, namely that in most of the cases there is an increase of Pol II occupancy at 1 hour after irradiation on the GBs of these genes, which show Pol II clearance from their promoters  and 3. TMoreover, Pol II changes on the GBs and EAG+4000 regions did not always parallel the homogenous decrease in Pol II binding at the promoters of group A genes following UVB irradiation. This suggests that the UVB-induced global negative regulatory mechanisms affecting Pol II transcription initiation, elongation and termination may be distinct and are not always interconnected.We also detected a set of genes, which show no decreased Pol II presence at their promoter regions, but rather a general increase of Pol II density throughout their three analyzed gene regions after UVB irradiation . Genes bp21/cdkn1a model gene that p53 uses unorthodox mechanisms to activate p21 when Pol II CTD phosphorylating kinases are inhibited, the recruitment of specific elongation factors are defective and mRNA synthesis from certain individual genes is inhibited It is known that p53 can regulate a subset of genes under compromised physiological conditions, when for example DNA is damaged. It is has been suggested using the Studies investigating the effect of genotoxic stresses on transcription reported two possible pathways by which Pol II protein levels may be affected: either the hyperphosphorylation of the CTD or the degradation of the largest subunit of Pol II Surprisingly, none of the antibodies raised against different phosphorylated forms of the CTD hepta-peptide repeat  revealed increased levels of phosphorylation of the Rpb1 CTD upon UVB stress that would explain the shift from the Pol IIA to the Pol IIO form . These rTaken together all our observations it seems that the massive Pol II clearance on group A genes upon UVB irradiation is not due to Pol II degradation, but due to the incapability of the Pol II complex to initiate transcription. It seems that during TCR the general transcription factor TFIIH is sequestered away from the TSSs by the repair machinery and this in turn would either dismantle genome-wide existing PICs and/or block the formation of new PICs on the majority of the transcribed genes. In contrast, on a small subset of key regulatory genes this negative mechanism does not work either because transcription initiation on these genes is not dependent on the repair-competent form of TFIIH or these genes are repaired much faster than the inhibited genes. Further experiments, will be needed to distinguish amongst all these different exciting possibilities. In conclusion, our study for the first time gives a genome-wide view about the mechanistic details of UVB induced Pol II transcription changes during TCR.The MCF7 human cell line  was grown in Dulbecco's Modified Eagle Medium  supplemented with 10% foetal calf serum (FCS). The medium contained insulin (0.6 µg/ml) and gentamicin (40 µg/ml). For assessing global and ongoing transcription cells were incubated with 1 mM 5-fluorouridine (Sigma-Aldrich) for 20 minutes. Incorporation of the modified nucleotide was monitored by indirect immunofluorescence using an anti 5-FU antibody from Sigma-Aldrich.2 UVB dose at 312 nm wavelength (Vilber T-15M lamp) or with with 55, 100 and 200 Joules/m2 , GAPD and non-targeting control  were transfected to MCF7 cells using Dharmafect Transfection Reagent 1  according to the manufacturers protocol. Cells were harvested for western blot analysis or ChIP 72 hours after the transfection.MCF7 cells were plate into a 6 well plate (300 cells/well) one day prior to UV treatment. In the following days, the survived cells will form colonies. 7 days after UV irradiation, the cells were washed with PBS then stained with Crystal violet solution . The staining was removed from the cells with 1% sodium dodecyl sulphate (SDS) containing MQ water. The crystal violet amount in both UV treated and in control samples were measured with spectrophotometer at 595 nm. Results were calculated from biological triplicatesCells were plated in 6 cm plates and UV-treated at 80–90% confluence. After incubation for 1, 2, 4, 6, 8, 16, 24 hours DNA was extracted from samples as well as from non-treated control with SIGMA GenElute Mammalian Genomic DNA Miniprep Kit. DNA content was quantified with Nanodrop. 250 ng of DNA was diluted in 2× SSC  buffer from each sample and was transferred to Amersham Hybond-N+ membrane with Slot-blot vacuum chamber. The membrane was treated with Denaturizing buffer  then Neutralizing buffer . The membrane was then treated according to Western blot protocol: blocked with PBS +2% milk, and then incubated with mouse IgG monoclonal anti-CPD (TDM2)  primary antibody overnight in 4°C.2 UVB. The irradiated and control cells 1, 2, 3, 4, 5, 6, hours later after incubation were washed twice with ice-cold PBS containing complete protease inhibitor cocktail (1×), phenylmethylsulfonyl fluoride (PMFS)(0.5 mM), β-glycerophosphate (10 mM), sodium orthovanadate (1 mM), sodium fluoride (20 mM) inhibitors. Cells were scraped in PBS containing protease inhibitor cocktail and frozen-thawed three times. Samples were sonicated for 10 cycles  (Bioruptor) then boiled for 5 min in loading solution. Protein samples were separated by a 6–10% SDS-PAGE, transferred and western blot assays were carried out by using standard methods. The following antibodies were used: RNA polymerase II N-terminal H-224× (Santa Cruz), Pol II Ser2, AB: Covance, (MMS-129R), Pol II Ser5 AB: Abcam, (ab5131) Pol II Ser 7 AB: MCF7 cells were plated in 6 cm plates prior treatment. At 80–90% confluence, cells were treated with 55 J/mUbc/NM_021009, Rplp1/NM_001003, p21/cdkn1a/NM_000389 and WDR24/NM_032259 genes. Intergenic region was selected as a negative control region. The ChIP experiments were repeated at least twice, and all the qPCR reactions were done in triplicates.MCF7 cells were plated in 15 cm dishes. At 80–90% confluence cells were UVB treated as described above. After 1, 2, 3, 4, 5, or 6 hours of incubation cells were quickly washed with PBS, and cross-linked with 1% formaldehyde for 20 minutes at room temperature. ChIP experiments were carried out as described earlier http://www.ncbi.nlm.nih.gov/gds) database. The 32 base pair tags generated from Hi-seq 2000 were mapped to the human genome (UCSC hg19) using the eland program allowing two mismatches. Only sequences that mapped uniquely to the genome with maximum of two mismatches were used for further analysis. To eliminate non-specific binding sites, we used our control ChIP-seq dataset, which was generated using an antibody that does not recognize any human proteins (GSE34001).The sample preparation for ChIP-seq was the same as described https://genome.ucsc.edu/), human genome Build 37 (hg19 assembly). Gene definitions were given by the refseq genes track Genome annotations were downloaded from the UCSC Genome Browser (http://www.bioconductor.org/packages/2.9/bioc/vignettes/DESeq/inst/doc/DESeq.pdf). We carried out analyses on all genes found in the refseq database and on 4500 expressed genes, based on the published RNA-seq dataset for MCF-7 cell line Intergenic regions (around 8000 regions) were selected that are far away from genes about 20 kb and tag numbers were counted on 2 kb interval in the middle of them for all the 7 samples. These values were used as an input for DESeq Bioconductor package, which normalizes the samples based on their median values.  was used for GO analyses and gene ID conversion. Further GO enrichment analyses were also performed in Manteia using its statistical module (http://manteia.igbmc.fr/). During the analyses only GO categories with lower than 0.01 p-values  were considered as positive results.The Database for Annotation, Visualization and Integrated Discovery (D.A.V.I.D.)  together with the survival of non-treated/control cells. (B) Slot Blot assay was carried out with a CPD specific antibody to test the genotoxicity of 55 J/m2 of UVB irradiation. The appearance and the persistence of the CPDs were tested at the indicated time points up to 24 hours after UVB treatment. (C) Western blot assays were carried out to test whether 55 J/m2 UVB induces detectable DNA-repair response in MCF7 cells. We tested the induction of two well-characterized regulators of the UV-response at the indicated time points: phospho-Chk1 and phospho-p53, respectively. Tubulin-α was used as loading control.Effects of sublethal dose of UVB on MCF7 cells. Experiments were carried out to find and characterize the sublethal dose of UVB irradiation for our study. (A) Crystal violet assay was carried out to compare the effect of different doses of UVB on MCF7 cells. The results are represented as a bar chart. The % of surviving cells are shown corresponding to each dose of UVB irradiation (55 J/m(PDF)Click here for additional data file.Figure S2Global Pol II tag density changes dynamically in the gene body regions of all transcribed genes following UVB irradiation. Mean Pol II read values (Y axis) were calculated for each ChIP-seq sample (as indicated on the x axis) from the GB region of the 4500 expressed genes. Each P value was calculated applying pairwise T-tests between control sample and each of the UVB treated samples (as indicated).(PDF)Click here for additional data file.Figure S3(First and second part): Different Pol II behavior patterns in time after UVB irradiation on selected genes from each subgroup of (PDF)Click here for additional data file.Figure S4CSB depletion using siRNA transfection. Scrambled siRNA (lane 1) and ERCC6 (CSB) siRNA (lane 2) transfection in MCF7 cells was carried out as described in (PDF)Click here for additional data file.Table S1Results of gene ontology analyses done with the Manteia software to identify the potential differential gene-function categories for the detected gene groups with distinct Pol II behavior patterns from (PDF)Click here for additional data file.Table S2List of genes represented in (XLSX)Click here for additional data file.


MEN1 gene that codes for the protein menin. Menin interacts with proteins that regulate transcription, DNA repair and processing, and maintenance of cytoskeletal structure. We describe the identification of FBP1 as an interacting partner of menin in a large-scale pull-down assay that also immunoprecipitated RBBP5, ASH2, and LEDGF, which are members of complex proteins associated with SET1 (COMPASS), a protein complex that methylates histone H3. This interaction was confirmed by coimmunoprecipitation and Flag-pull-down assays. Furthermore, menin localized to the FUSE site on the  MYC promoter, a site that is transactivated by FBP1. This investigation therefore places menin in a pathway that regulates MYC gene expression and has important implications for the biological function of menin.Multiple endocrine neoplasia type 1 (MEN1) is a syndrome characterized by tumors in multiple endocrine tissues such as the parathyroid glands, the pituitary gland, and the enteropancreatic neuroendocrine tissues. MEN1 is usually caused by mutations in the  MEN1 gene. Its inactivation has been implicated in tumors of many endocrine tissues. Germline inactivating mutations of MEN1 gene are the main cause of multiple endocrine neoplasia type 1 (MEN1), a syndrome characterized by tumors in the pituitary gland, the parathyroid glands, and the enteropancreatic neuroendocrine tissues. Around 70% of familial MEN1 index cases have an identifiable germline mutation in the MEN1 gene , 2120, 2 MYC promoter. Since our studies indicated that FBP1 interacts with menin, we wanted to determine if menin, like FBP1, is able to bind to the MYC promoter. To determine if menin is recruited to the FUSE region of the MYC promoter, we performed chromatin immunoprecipitation assays in HEK 293 and U2OS cells. Menin-bound DNA was immunoprecipitated and subjected to PCR using primers designed for the FUSE region [FBP1 binds to the FUSE DNA element on theE region . Anti-meE region . MYC promoter. We used a MYC promoter luciferase construct to examine if menin is able to regulate expression from the MYC promoter. For this assay, HEK293 cells were transfected with plasmids carrying FBP1 or menin or both. These cells were also transfected with MYC promoter luciferase constructs. A luciferase assay was conducted to examine regulation of the MYC promoter by FBP1 and menin (P < 0.05). Stimulation by menin was not increased further by addition of FBP1. FBP1 data was unexpected because previous studies with FBP1 demonstrated that it induced expression from a reporter with a MYC promoter chloramphenicol acetyltransferase chimeric construct by five-fold [ MYC by FBP1. For example, FBP1 expression did not correlate with MYC expression in hepatocellular carcinoma tissues [ MYC expression. For example, binding of FBP1 to FUSE brings together additional cis-elements and their respective transcription factors to maintain optimal expression of MYC [FBP1 is a single stranded DNA binding protein that induces expression of MYC by binding to the FUSE site on thend menin . We founive-fold . Howeverive-fold . These r tissues , 24. Addn of MYC , 26.In some contexts such as HL-60 leukemic cells, FBP1 is a transactivator of the MYC oncogene . The intp27Kip1 and p18Inc4c [ Men1 or Mll knockout mouse embryonic fibroblasts have reduced expression of p27Kip1 and p18Inc4c and higher cell proliferation compared to their wild type controls. Furthermore, the recruitment of Mll to the promoters of p27Kip1 and p18Inc4c is dependent on menin [p27Kip1 and p18Inc4c promoters was associated with increased expression of the target genes. These prior data demonstrated that menin recruits MLL to the promoters of p27 and p18 to induce their expression and thereby inhibit cell proliferation. In this model, MLL contributes to the tumor suppressor function of menin. Like menin, FBP1 is also a tissue specific tumor suppressor having recently shown to be mutated with LOH in oligodendrogliomas [In contrast to this oncogenic function, menin and MLL can inhibit cell growth. This is supported by the discovery that menin and MLL localize on the promoters of cyclin-dependent kinase inhibitors, p18Inc4c . These gon menin . LocalizRecently, the structure of menin was solved with MLL1, JUND, or MLL1-LEDGF heterodimer , 29. TheOur data have important implications in the study of menin's functions because menin has been shown to regulate expression of genes encoding p27 and p18 and has been found to be localized to the promoters of target genes. However, since crystal structures of menin have not identified a DNA binding region, it could bind to a promoter in association with a DNA binding protein such as FBP1 . In our  MYC. Since menin has not been shown to have a DNA binding region, the association with the FUSE region might be through interaction with DNA binding proteins such as FBP1. However, further experiments are needed to confirm this hypothesis. The interaction of menin with FBP1 could therefore contribute to the tumor suppressor function of menin or to the oncogenic function of menin depending on the targets that it is recruited to and the cell types that it is expressed in.In conclusion, we have identified FBP1 as an interacting partner for menin. Like FBP1, menin binds the FUSE region of


Time resolved transcriptional studies for two VDR target genes revealed selective attenuation and repression of VDR transcriptional responses in PC-3 cells. For example, responses in PC-3 cells revealed suppressed responsiveness of IGFBP3 and G0S2. Furthermore, Chromatin Immunoprecipitation (ChIP) assays revealed that suppressed transcriptional responses in PC-3 cells of IGFBP3 and G0S2 were associated with selective VDR-induced NCOR1 enrichment at VDR-binding regions on target-gene promoter regions. We propose that VDR inappropriately recruits co-repressors in prostate cancer cells. Subsequent direct and indirect mechanisms may induce local DNA methylation and stable transcriptional silencing. Thus a transient epigenetic process mediated by co-repressor binding, namely, the control of H3K9 acetylation, is distorted to favor a more stable epigenetic event, namely DNA methylation.The current study aimed to examine the gene specific mechanisms by which the actions of the vitamin D receptor (VDR) are distorted in prostate cancer. Transcriptional responses toward the VDR ligand, 1α,25(OH) CDKN1A (encodes p21(waf1/cip1)), IGFBP3 [G0S2 [Epigenetic mechanisms are central to the evolution of malignant phenotypes. The androgen receptor (AR)  exerts a, IGFBP3 –11 and GP3 [G0S2 ,13. In pThe impact of ADT on the malignant cell presents a formidable environment that cancer cells must adapt to. This process is multifaceted and includes loss of mitogenic signals downstream of the AR, triggering apoptosis, hypoxia  and inflammation that has an associated mileu of cytokine and other signals. Central aspects of the escape mechanisms to this restraint include increasing intrinsic AR ligand production and AR signaling capacity. However the transcriptional actions of the AR in ADT-RCaP are not merely a re-iteration of the normal AR transcriptome, but rather represent a fundamentally different transcriptome. Epigenetic events are central to the evolution of the altered AR signaling capacity.TMPRSS2 promoter is sustained in an AR responsive state. More recently genome-wide ChIP-chip and ChIP-Seq approaches have revealed considerable variability in the targeted transcriptional networks [The AR transcriptional program evolves toward increased targeting of proliferative gene promoters and decreased targeting of pro-differentiation genes ,15. For networks –19. For networks .These events are not unique to CaP. In a range of solid tumors and myeloid leukemia, nuclear receptors that normally exert mitotic restraint, such as the VDR, RARs and PPARs, become skewed, with selective suppression of gene targets associated with antiproliferative actions –26. ThusLoss and gain of function of transcriptional co-activators and co-repressors associates with transcriptional rigidity. Co-activators and co-repressors each display both loss and gain of function, and can result in similar phenotypes. Thus the loss of a co-activator can lead to suppressed ability of a transcription factor to trans-activate a given target. Similarly the gain of function of co-repressors can limit transactivation ability and enhance trans-repression. The opposite patterns will in turn enhance the trans-activation function. Compared to their co-activator cousins, the co-repressors are somewhat under-explored. Ambiguity remains over how and to what extent these actions are distorted in cancer (reviewed in Ref. ). The shNcor1−/− and Ncor2/Smrt−/− mice [The proto-typical co-repressors NCOR1 and NCOR2/SMRT were cloned in 1995 using nuclear receptor as bait ,35, and −/− mice .Evidence has also emerged that NCOR1 and NCOR2/SMRT complexes are dynamically recruited to activated transcription factors leading to active transrepression , for exaWell-established oncogenic roles for NCOR1 and NCOR2/SMRT have been elucidated in acute promyelocytic leukemia that results from a fusion between RARα, and either the promyelocytic leukemia (PML) or promyelocytic leukemia zinc finger (PLZF) genes . Both che.g. 1α,25(OH)2D3) plus HDAC inhibitors resulting in selective re-expression of VDR target genes, notably VDUP1 and GADD45A [Expression profiling in solid tumors has revealed altered NCOR1 and NCOR2/SMRT expression and localization, for example in breast, bladder, and prostate cancers ,26,47,48 GADD45A . Togethe GADD45A ,38,51,522D3 in non-malignant and malignant prostate epithelial cells.In contrast, a parallel literature has revealed loss of NCOR1 and NCOR2/SMRT is associated with the ADT-RCaP phenotype and enhances AR transcriptional programs ,54. Simi2D3. Non-malignant prostate epithelial RWPE-1 cell is highly responsive toward 1α,25(OH)2D3 [2D3 [Non-malignant and malignant prostate cell lines display a range of anti-proliferative responses toward 1α,25(OH)5(OH)2D3  whereas 5(OH)2D3 , is reca2D3 [2D3 ,32,56.2D3 actions, VDR-mediated gene regulatory actions were examined in RWPE-1 and PC-3 cells. Time-resolved regulation studies were undertaken with IGFBP3 and G0S2. The time-resolved kinetics in RWPE-1 and PC-3 cells for the genes are shown in IGFBP3 and G0S2 mRNA regulation were highly pronounced in RWPE-1 cells. In contrast, the mRNA was significantly reduced in PC-3 at multiple time points. Together these data indicate that gene regulation by 1α,25(OH)2D3 was most dynamic in cells that were most responsive to the anti-proliferative effects (RWPE-1 cells). By comparison, in PC-3 cells, the mRNA regulation profiles were selectively attenuated.As a functional indicator of 1α,25(OH)G0S2 and a well-characterized VDRE on IGFBP3 [2D3-regulated NCOR1 recruitment was evident in 1α,25(OH)2D3-recalcitrant PC-3 cells compared to 1α,25(OH)2D3-sensitive RWPE-1 at these regions examined. This occurred rapidly on both genes in PC-3 cells within the first hour of exposure to 1α,25(OH)2D3, compared to RWPE-1 cells, NCOR1 appeared to cycle off and be subsequently recruited back at later time points; at 24 h on the IGFBP3 response element and at 2 and 12 h on the TSS of G0S2 (data not shown). These findings suggest the underlying mechanisms of recruitment of NCOR1 in response to VDR activation differ significantly between the two cell types.Q-ChIP was undertaken to examine recruitment of NCOR1 to the TSS of n IGFBP3  in PC-3 2D3-recalcitrant phenotype is of potential clinical significance. To address this aim, we examined whether the co-repressor protein NCOR1 was differentially recruited to target genes that are known to regulate these anti-mitotic transcriptional programs.The VDR governs and influences anti-mitotic and prodifferentiation transcriptional programs and these actions are distorted in CaP cells . Therefo2D3 regulated expression was attenuated and even repressed compared to vehicle controls in PC-3 cells that are recalcitrant to the anti-mitotic actions of 1α,25(OH)2D3, compared to RWPE-1 cells. Building on these studies, we examined the binding of NCOR1 following VDR activation and revealed that 1α,25(OH)2D3 induced greater NCOR1 association on the IGFBP3 and G0S2 promoters in PC-3 cells, compared to RWPE-1 cells. Thus NCOR1 was sustained and enriched at VDR binding sites to different extents and at different time points. Probably reflecting looping events, the TSS showed sustained NCOR1 enrichment throughout the time course [As a starting point to these questions the current study examined differential mRNA regulation of two VDR target genes in different prostate cell models. These approaches revealed that 1α,25(OH)e course .2D3 [3 and cancer risk and advanced disease [in vitro, in vivo and epidemiological data support links between replete VDR signaling, growth restraint and broad anticancer activities, clinical exploitation of this receptor has been limited. A significant impediment to translation remains the inability to predict accurately which patients will respond to either chemoprevention or chemotherapy strategies centered on vitamin D compounds. The mechanisms that drive this resistant phenotype are often illusive and probably involve multiple aspects of disruption. Key mechanisms include gene amplification of the 1α,25(OH)2D3 metabolizing enzyme CYP24A1 [It is now over 30 years since the initial reports demonstrated the anticancer actions of 1α,25(OH)2D3 –61. Foll2D3 –64, as w2D3 ,66. As t disease –75. Howe CYP24A1  and repr CYP24A1 . The pro CYP24A1 .The process of inappropriate co-repressor recruitment may lead to stable gene silencing and in turn shed light on why the VDR and other nuclear receptors are expressed in non-malignant and retained in malignant prostate epithelial cells seemingly independent of the antiproliferative response .The differential recruitment of co-repressors also addresses another ambiguity in their cancer biology, namely the impact of altered expression of co-repressors. Increased NCOR1 and NCOR2/SMRT expression occurs also in breast and bladder cancer and suppressed the responsiveness of nuclear receptors that exert mitotic restraint, such as VDR and PPARα,γ ,47–49,78There is compelling evidence that histone and DNA methylation processes disrupt transcriptional actions, both alone and together. For example, one consequence of NCOR1 and NCOR2/SMRT association at target genes is the loss of H3K9ac and accumulation of H3K9me2, allowing the potential for hypermethylation at adjacent CpG regions. Further links exist between NCOR1 and DNA methylation through its interaction with KAISO . Similar2D3  stored as 1 mM stocks in ethanol.1α,25(OH)®) machine using TaqMan assays. Measurements were performed in technical and biological triplicate. The statistical significance was calculated using Student’s t-test.RNA was isolated using TRizol (Invitrogen). Target gene expression was quantitated on an ABI 7900 . Complexes were recovered using magnetic beads, washed, cross-linking was reversed and further cleared DNA was recovered by standard precipitation approaches. 25 ng DNA was used per Q-PCR reaction using SYBRgreen with pre-optimized primers as described previously [t-test was used to calculate the significant enrichment.X-ChIP was used to measure the association of NCOR1 binding as described previously . Brieflyeviously . Student