Drosophila spermatogenesis cell differentiation pathway involves the activation of a large set of genes in primary spermatocytes. Most of these genes are activated by testis-specific TATA-binding protein associated factors (tTAFs). In the current model for the activation mechanism, Polycomb plays a key role silencing these genes in the germline precursors, and tTAF-dependent activation in primary spermatocytes involves the displacement of Polycomb from gene promoters. We investigated the genome-wide binding of Polycomb in wild type and tTAF mutant testes. According to the model we expected to see a clear enhancement in Polycomb binding at tTAF-dependent spermatogenesis genes in tTAF mutant testes. However, we find little evidence for such an enhancement in tTAF mutant testes compared to wild type. To avoid problems arising from cellular heterogeneity in whole testis analysis, we further tested the model by analysing Polycomb binding in purified germline precursors, representing cells before tTAF-dependent gene activation. Although we find Polycomb associated with its canonical targets, we find little or no evidence of Polycomb at spermatogenesis genes. The lack of Polycomb at tTAF-dependent spermatogenesis genes in precursor cells argues against a model where Polycomb displacement is the mechanism of spermatogenesis gene activation.The  Drosophila; the spermatogenesis genes. These studies indicated that removal of the Polycomb protein from spermatogenesis genes was a key event in their activation. This was interesting because Polycomb is a central component of a gene regulatory complex that works at the level of chromatin and is best known for its role in stable heritable gene silencing. We have further analysed this system by examining Polycomb binding at the genome-wide scale and specifically in the precursor cells where Polycomb is proposed to repress the spermatogenesis genes. Surprisingly, this cell-specific analysis found no clear evidence for Polycomb binding at the spermatogenesis genes before their activation. This argues against the current model where Polycomb removal is the key mechanism involved in the activation of these genes. Further work is needed to identify the actual mechanism of gene activation in this system; however, our results fit with previous work suggesting that lamin-association rather than Polycomb binding may be responsible for repression of spermatogenesis genes prior to their activation.How are developmental gene expression programs activated? An interesting activation mechanism has been suggested by studies of the switch-on of the genes required for sperm differentiation in  Drosophila spermatogenesis cell differentiation pathway involves the activation of over 1,000 genes in a testis-specific transcription program in primary spermatocytes. This represents a key developmental switch and provides an important model system for the analysis of regulatory mechanisms governing cell fate specification and pathway-specific gene expression programs (reviewed in IID complex containing testis-specific TATA-binding protein associated factors (tTAFs) The How tTAFs and tMAC interact to switch on the primary spermatocyte transcription program is unclear, however the current model of spermatogenesis gene activation As this model was based on the analysis of Pc binding at only a few selected spermatogenesis gene promoters, we decided to extend the investigation to a genomic scale and examine the genome-wide binding of Pc in wild type and tTAF mutant testes. Contrary to what one would expect from the current model, our results show little evidence for enhanced Pc binding at tTAF-dependent spermatogenesis gene promoters in tTAF mutant testes. Considering that cellular heterogeneity may complicate whole testis analysis, we further investigated Pc binding in purified germline precursors, where we find no clear evidence for the binding of Pc at tTAF-dependent spermatogenesis genes. This argues that Pc displacement is not the mechanism involved in tTAF-dependent gene activation, challenging the existing model and highlighting the benefit of performing genome-wide epigenetic analysis in a cell type-specific manner.can; FBgn0011569) mutant testes, we performed ChIP together with microarray analysis (ChIP-array) on chromatin from whole testes. For ChIP we followed the approach we used previously to investigate Pc binding in embryos and imaginal discs Pc-GFP fly line and anti-GFP antibody. For both wild type and homozygous can mutant testes, we find robust Pc binding at many known Pc target genes; e.g. the bithorax complex, the Antennapedia complex and the engrailed  locus (dj (FBgn0019828), fzo (FBgn0011596) and Mst87F (FBgn0002862) that were previously assayed by Chen et al. Mst87F in can mutant versus wild type testes Mst87F and ∼2-fold effects at dj and fzocan mutant testes; neither in the ChIP-array binding profiles (To generate a genome-wide view of Pc binding in wild type and tTAF (7) locus . The Pc profiles  nor in tprofiles .dj, fzo and Mst87F in a genomic context reveals a potential problem with the interpretation of ChIP enrichment at these loci. The small fzo transcription unit lies within an intron of the much larger cnc (FBgn0262975) gene and there is an extensive Pc binding domain that covers most of the cnc gene. It is thus difficult to know whether ChIP enrichment close to the TSS of fzo is functionally related to the regulation of fzo or cnc. A similar situation occurs at dj, which is associated with the Pc binding domain involved in the regulation of the Antennapedia complex.Visualising the Pc binding at sa; FBgn0002842) mutant testes Drosophila genome. Also included in the analysis is a set of canonical Pc target genes from the embryo data of Kwong et al. can mutant versus wild type testes , and for can mutant, the median log2 enrichment = 0.47 (1.39 fold enrichment). These small positive enrichment values are similar to the enrichments seen for a set of housekeeping genes, which are expressed independently of tTAFs (see 2 enrichment = 0.90 (1.87 fold enrichment) and the can mutant median log2 enrichment = 0.73 (1.66 fold enrichment), whereas for Pc binding at a set of top embryo Pc peaks (see 2 enrichment = 1.65 (3.14 fold enrichment) and the can mutant median log2 enrichment = 1.10 (2.15 fold enrichment). The level of Pc binding at the spermatogenesis genes is similar in wild type and can mutant testes, although there is a slightly enhanced level of Pc binding in the can mutant testes .For a more global view of Pc binding at tTAF-dependent spermatogenesis genes, we used a conservative set of 942 genes identified as genes with >4-fold down-regulation in tTAF  to technical difficulties in comparing the two different ChIP samples. Wild type and mutant testes differ in several ways and may not be directly comparable; can mutant testes are smaller in size and lack post-meiotic cells including mature sperm, have an expanded primary spermatocyte population, contain many dead and dying cells In summary, our data does not replicate the specific effects seen by Chen et al. at bam (FBgn0000158) mutant flies expressing the germline marker vas-GFP. In bam mutant testes, spermatogonia are prevented from differentiating into primary spermatocytes + cells in bam mutant testes represent the germline precursor population of stem cells and spermatogonia  in the bithorax complex, the Dfd PRE in the Antennapedia complex and the en PRE , but no evidence for Pc binding at the three selected spermatogenesis genes.Quantitative PCR analysis confirms these microarray results . There i2 enrichment = 0.02 (1.01 fold enrichment)) and no significant difference in the binding at spermatogenesis genes versus non-spermatogenesis genes. The canonical Pc target genes on the other hand show higher enrichment, assayed at either the TSS (median log2 enrichment = 0.28 (1.21 fold enrichment)) or at a set of top embryo Pc peaks (median log2 enrichment = 0.66 (1.58 fold enrichment)) although compared to whole testes the median enrichments are modest. We suggest that this is, at least in part, due to differences in Pc targets between germline precursors and the embryo. Accumulated binding profiles at the TSS support the lack of Pc binding at tTAF-dependent spermatogenesis genes, showing clear Pc binding at the canonical Pc targets, but Pc binding at the spermatogenesis genes at a background level similar to that for random gene sets ), and overall we see no correlation between can/wild type enrichment ratio and enrichment level in precursors (Pearson correlation = 0.07).In addition, we tested whether the tTAF-dependent spermatogenesis genes that showed most enhanced Pc binding in In summary, these results from our cell-defined analysis of Pc binding do not support the Chen et al. model, which predicts that Pc should be present at the tTAF-dependent spermatogenesis genes in the germline precursors.Pc-GFP testes, in very early primary spermatocytes we find Pc present on the chromosome masses, diffusely present in the nucleoplasm and excluded from the nucleolus  and bol , in primary spermatocyte nuclei. Although we find evidence of transcribed genes looping out a short distance from their chromosome territories, neither gene was transcribed at the nucleolar periphery  has been associated with the permanent heritable silencing of the Hox genes, it has more recently been shown to have a dynamic role in cell fate decisions and the regulation of lineage-specific gene expression programs  plus protease inhibitors (Sigma-Aldrich P8340) in medium for 5 min at room temperature. After washing in PBS, cells were extracted by vigorously pipetting for 1 min, using a narrow-ended P200 tip (Rainin RT-200F), in 100 µl PBS; and fixed by adding an equal volume of 2% formaldehyde (Sigma-Aldrich F8775) in medium, mixing thoroughly, and incubating for 15 min at 37°C in an Eppendorf Thermomixer at 700 rpm. Fixation was stopped by adding 400 µl ice-cold medium and placing the sample on ice. The sample was spun down in a swing-out rotor at 1,000 g for 5 min at 4°C, and the pellet snap frozen in liquid N2 and stored at −80°C. Approximately 1,000 testes were dissected for each ChIP-array replicate. Testes were dissected in batches of 70, and for each batch, the time lapsing from the start of dissection till fixation was approximately 1 hr.Testes were dissected in ice-cold Schneider's medium  and incubated with collagenase . Cells were sorted using a 100 µm nozzle on a MoFlo FACS machine (Beckman Coulter) equipped with a 488 nm argon laser (100 mW). Cells were sorted into a microfuge tube containing 700 µl PBS/0.01% Triton X-100. Events were triggered on forward scatter and GFPg for 15 min at 4°C, transferred to a thin-walled 0.5 ml microfuge tube (Axygen PCR-05-C), re-spun down and resuspended in 130 µl Lysis Buffer (17 mM Tris.HCl (pH 8), 3.4 mM EDTA.Na2, 0.34% SDS) containing protease inhibitors (Sigma-Aldrich P8340), and sonicated for 5 cycles at high setting using a Diagenode Bioruptor (1 cycle is 30 s ON and 30 s OFF). After sonication, the sample was centrifuged at 16,000 g for 15 min at 4°C, the chromatin-containing supernatant transferred to a fresh microfuge tube, and 70 µl RIPA buffer (36.7 mM Tris.HCl (pH 8), 2.5 mM EDTA.Na2, 0.01% SDS, 2.46% Triton X-100, 374 mM NaCl) containing protease inhibitors added to the chromatin sample. The ChIP reaction, washes and DNA purification were performed as in Dahl and Collas + sorted events). Beads were washed, chromatin eluted, RNA and proteins digested, and the DNA then purified by phenol/chloroform extraction and ethanol precipitation using linear acrylamide as carrier and resuspended in 10 µl PCR grade water. Approximately 10 µl of chromatin was retained as input and purified alongside the ChIP sample. Quantitative PCR primers used for Pc enrichment analysis are listed in Sorted cells were spun down in a swing-out rotor at 4,000 Pc-GFP or can mutant Pc-GFP flies and fixed using 1% formaldehyde in PBS for 15 min at 37°C. Fixation was stopped by washing in PBS/125 mM Glycine/0.01% Triton X-100. Chromatin preparation was performed as described above, except that before sonication testes were homogenized using a motor-driven plastic pestle (Sigma-Aldrich Z359947) in a thin-walled 0.5 ml tube containing 130 µl Lysis Buffer. The ChIP reaction, washes and DNA purification were performed as described above. The ChIP reaction used 1 µl of rabbit anti-GFP antibody A total of 100 testes were dissected from wild type ChIP and input DNA were amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (Sigma-Aldrich WGA4) following the manufacturer's instructions from the library preparation stage. Approximately 150 pg of DNA was used for amplification. Samples were amplified for 22 cycles (sorted cells) or 20 cycles (whole testes), and amplified DNA was purified using the QIAquick PCR Purification Kit (Qiagen). The amplified ChIP and input DNA  were labelled with Cy5 and Cy3 using the NimbleGen Dual-Color DNA Labeling Kit for sorted cells, or the BioPrime DNA Labeling Kit (Invitrogen) in the presence of Cy3- or Cy5-dCTP  for whole testes, and hybridised onto Nimblegen ChIP-chip 2.1M Whole-Genome Tiling Arrays according to the manufacturer's instructions.Pc-GFP whole testes where we performed three. A Cy3/Cy5 dye swap was performed for one biological replicate of each sample. For all ChIP-array experiments, input chromatin was used as the reference control to assay ChIP enrichment. Arrays were scanned and the scanned images processed using NimbleScan software to generate pair files, which were then converted into chromatin enrichment profiles (log2 ChIP/input ratio) using the TiMAT pipeline (http://bdtnp.lbl.gov/TiMAT/). Samples were median scaled at 500, quantile normalized (ChIP and input samples together), and window smoothed using a window size of 500 bp and a minimum number of oligos per window of 4. Enrichment profiles were visually examined using the Integrated Genome Browser (http://bioviz.org/igb/index.html). The ChIP-array data has been submitted to Gene Expression Omnibus under accession number GSE39935.We performed two biological replicates for each sample, except wild type Drosophila melanogaster genome. Gene lists for spermatogenesis genes were derived from Chen et al. sa mutant testes. The tMAC-dependent spermatogenesis genes  in aly (FBgn0004372) mutant testes. The embryo Pc target gene set (386 genes) is from Kwong et al. 2 ChIP enrichments >1.0 in Kwong et al. http://www.flymine.org/).All analyses are based on release 5.48 of the bol and fzo DNA fragments. The bol probe was generated from a 2,851 bp intron fragment PCR-amplified using the primers: 5′ AAACGATGGCAACAAAGGAG 3′ and 5′ TGGCACAGATACGAAGCAAG 3′. The fzo gene lacks introns and so the fzo probe was generated from a 1,264 bp exon fragment PCR-amplified using the primers: 5′ GGCCCTAAAACCCTCAACTC 3′ and 5′ TAAAACGGTGCCCAAGCTAC 3′. Sheep anti-digoxigenin primary antibody  and goat anti-sheep Alexa 568 secondary antibody  were used for detection.Immunolabelling was performed as in Redhouse et al. Figure S1can mutant whole testes, with embryo data en locus , and for the three tTAF-dependent spermatogenesis genes dj, fzo, and Mst87F .Linear scaled ChIP enrichment profiles. ChIP enrichment profiles for Pc in wild type and (TIF)Click here for additional data file.Figure S2+ germline precursors with A showing all events, B showing events after gating by R1 and C showing events after gating by both R1 and R2. Autofluorescence induced by the 488 nm laser is plotted against GFP fluorescence in order to discriminate between genuine GFP+ cells and autofluorescent events.  Sorted germline precursors; (D) phase contrast image, (E) fluorescence image. Sort purity >99%.FACS gating strategy used to sort the germline precursor population. (A–C) Gates R1, R2 and R3 were used to sort GFP(TIF)Click here for additional data file.Figure S3CycA). Note that Pc binding is observed at fzo in the GFP-negative somatic cells and not in the germline precursors. Error bars = 1 SD from 2 independent biological replicates .Quantitative PCR analysis of Pc enrichment in purified germline precursors and GFP-negative somatic cells. Pc enrichment is shown for the promoters of the three selected tTAF-dependent spermatogenesis genes calculated as fold enrichment relative to a negative control gene ((TIF)Click here for additional data file.Figure S4bol (A–C) and fzo (D–F) combined with histone immunolabelling in mature primary spermatocytes.  RNA in situ;  histone labelling;  merge. The asterisk marks the nucleolus. These single z sections show one or two puncta per nucleus and overall we see four puncta per nucleus corresponding to the four gene copies in these tetraploid G2 cells. Scale bars represent 8 µm.Localisation of transcribing spermatogenesis genes. Single z sections showing RNA fluorescent in situ hybridisation for (TIF)Click here for additional data file.Figure S5Spermatogenesis genes are preferentially associated with BLACK chromatin. Percentage of genes associated with each of the five chromatin domain types in Kc cells from Filion et al. (TIF)Click here for additional data file.Table S1en PRE were taken from Langlais et al. Quantitative PCR primers used for Pc enrichment analysis. The primer sequences used for the (DOCX)Click here for additional data file.


Transcription factors (TFs) and their binding sites (TFBSs) play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP). However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs.egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue.The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase), through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially in cancer biology. The established site-specific enzyme digestion enables a reliable and individual detection option for densely arranged binding motifs in vivo not provided by e.g. EMSA or in vivo footprinting. Given the important function of transcription factors in neoplastic mechanism, our method enables a broad diversity of application options for clinical studies. Transcription factors (TFs) are core elements of transcriptional regulation and play also an important role in the systems biology of cancer characterized by changes in the expression levels of certain genes . A complSo far, several methods have been developed for identification and analysis of TFBSs. A commonly used method to verify TFBSs is the electrophoretic mobility shift assay (EMSA) . Indeed,In contrast, the chromatin immunoprecipitation (ChIP) offers a distinct advantage over EMSA and in vivo footprinting, since the ChIP technique not only specifies which nucleotides are bound, but also identifies the interacting protein(s) in the context of in vivo samples . In thisThus, the aim of this study was to develop and optimize a ChIP technique for the specific and individual analysis of neighboring TFBSs at once in both cell culture and tissue material.In order to support the individual analysis of adjacent TFBSs, we have developed an improved ChIP assay by replacing the traditionally used random fragmentation step with a site-specific enzyme digestion. This site-specific ChIP allows the immunoprecipitation and enrichment of DNA fragments containing only one TFBS, thus permitting to distinguish between adjacent occupied and non- occupied binding sequences in vivo. The principle of the site-specific ChIP and its advantages over similar procedures like EMSA and in vivo footprinting is exemplified in Figure egfr) focusing on the well described transcription factor Sp1  can assure that up to now. So our approach reasonably approximates this completeness. Though, in combination with a preceded consensus analysis using an adequate algorithm (like MatInspector) our site-specific ChIP is an effective method for the selective identification of complex TFBSs structures.egfr, there were no complications.For all restriction sites we used restriction enzymes  which exhibit exactly the same digestion and buffering conditions . The concern that TFBSs might not be flanked by naturally occurring restriction enzyme sites and not separated by an appropriate distance from each other seems, in accordance to our experience, to be a rare event. At least in the case of the The suitability of the used anti-Sp1-Antibody for an application in the ChIP-procedure has been checked and assured by the supplier . The utilized buffers are common to all antibody based ChIP assays.By adjusting sample preparation and chromatin isolation procedures the technique is also applicable on tissue material, enabling a broad diversity of application options for clinical and molecular studies.in vivo. So, the main benefit of the site-specific ChIP lies in the investigation of specific regulatory regions in greater detail.The site-specific ChIP is not dedicated to high throughput screening approaches but instead supports the functional analysis of a complex regulation scheme of a single gene in a systems biology view - i.g. the interaction pattern between occupied TFBSs. The focus of this work is develop a method for a) the detection of new binding sites in combination with a preceded consensus analysis and b) the individual examination of single TFBSs allocation in a complex system of neighboring binding motifs of the same type. Hence, the site-specific ChIP technique does not provide an assessment of whether the TF-binding is associated with gene transcription, what is a common limitation of all ChIP-variations. For answering the question of functional relevance further methods have to be employed. E.g. site directed mutagenesis or deletion analysis for TFBSs and promoter activity investigation The site-specific ChIP, which uses an endonuclease-based TFBSs specific DNA fragmentation followed by a PCR-based enzyme digestion control, opens new possibilities for the functional investigation of complex neighboring TFBSs systems of genes, even within GC-rich regions. In combination with methods for TFBSs prediction -18, chip7 osteosarcoma cells which were fixated by a 1% formaldehyde solution. The Nuclei were isolated using cell lysis buffer, separated by centrifugation and resuspended in adequate standard restriction buffer . Chromatin was fragmented by subjecting the nuclei to restriction enzyme digestion according to Kang et al. [egfr intron 1. A complete DNA digestion was achieved by chromatin treatment with 200 U of each enzyme for 4 h at 37°C and further 100 U of each enzyme for additional 16 h at 37°C. The nuclei were then incubated with and 200 U aliquot of each enzyme and 200 U of RNase for 2 h at 37°C. Completion of restriction enzyme fragmentation was verified by electrophoresis separation on a 1.5% agarose gel. The optimization of the specific enzyme digestion based DNA fragmentation is shown on Additional file For the cell line experiments we used 10g et al. , includiThe tissues samples were processed as follows. 0.03 g frozen kidney carcinoma tissue was chopped into small pieces and thawed in freshly prepared PBS containing 1% formaldehyde for crosslinking. The tissue was homogenized to avoid cell clumps and the ChIP was performed as described above for the cell culture experiments.Taq DNA Polymerase, 1X PCR reaction buffer II, 400 μM of each dNTP (Applied Biosystems) and 1.3 mM MgCl2 were mixed for PCR amplification in a 25 μl reaction volume. A defined amount of 4 ng lymphocyte DNA was used for the internal control of each PCR reaction approach. The PCR was started with an initial denaturation of 95°C for 9 min, followed by a secondary denaturation at 98°C for 1 min. Next 40 cycles of denaturation at 98°C for 10 sec, annealing at 65°C for 2 min, and a single product extension step at 72°C for 7 min followed. The PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining.The detection of the immunoprecipitated Sp1 binding sites was done by PCR. For each sample : 2.5 μl ChIP-DNA template, 5 pmol of Sp1 binding site specific primer, 1 U of The control of completeness of the enzyme digestion control was also performed by PCR as described and carried out on DNA templates prepared from the Sp1 immunoprecipitated chromatin sample and the whole-cell sample (input control), as well as uncleaved HOS cells DNA from the same lysate (positive control).For the negative control, primers spanning an unrelated genomic region within the 3'-UTR of the GAPDH gene were used yielding a 97-bp product.Project name: Site-specific chromatin immunoprecipitation: A selective method to individually analyze neighboring transcription factor binding sites in vivoProject home page: noneOperating systems: noneProgramming language: nonehttp://www.genomatix.de)Other requirements: MatInspector V7.4.8 (License: noneAny restriction to use by non-academics: noneEgfr: Epidermal Growth Factor Receptor gene; ChIP: chromatin immunoprecipitation; TF: transcription factor; TFBSs: transcription factor binding sites.The authors declare that they have no competing interests.RS, KA, BB, HB, EK contributed to the final manuscript, provided clinical samples and shared knowledge on molecular methods; AN performed experiments; RS performed experiments and developed the given method. All authors read and approved the final manuscript.Data were analyzed anonymously. Nonetheless we have performed the study according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University of Muenster. We have acquired tissue samples only with the informed consent of the patients or patients' next of kin with the understanding by all parties that it may well be used for research. All patients provided written informed consent for the collection of samples and subsequent analysis.Figure S1. Optimization of the specific enzyme digestion based DNA fragmentation.Click here for file


Caenorhabditis elegans holocentromeres are organized as dispersed but discretely localized point centromeres, each forming a single cenH3-containing nucleosome. These centromeric sites co-localize with kinetochore components, and their occupancy is dependent on the cenH3 loading machinery. These sites coincide with non-specific binding sites for multiple transcription factors (‘HOT’ sites), which become occupied when cenH3 is lost. Our results show that the point centromere is the basic unit of holocentric organization in support of the classical polycentric model for holocentromeres, and provide a mechanistic basis for understanding how centromeric chromatin might be maintained.Centromeres vary greatly in size and sequence composition, ranging from ‘point’ centromeres with a single cenH3-containing nucleosome to ‘regional’ centromeres embedded in tandemly repeated sequences to holocentromeres that extend along the length of entire chromosomes. Point centromeres are defined by sequence, whereas regional and holocentromeres are epigenetically defined by the location of cenH3-containing nucleosomes. In this study, we show that DOI:http://dx.doi.org/10.7554/eLife.02025.001 During cell division, the chromosomes in the original cell must be replicated and these ‘sister chromosomes’ must then be divided equally between the two new daughter cells. At first, the sister chromosomes are held together near a region called the centromere, which is important because the microtubules that pull the sister chromosomes apart attach themselves to the centromere. In many cases, the centromere is a small region near the middle of the chromosomes, which produces a classic X shape. However, in some organisms centromeres span the entire length of the chromosomes. There are at least 13 plant and animal lineages with such holocentromeres.Inside the nucleus of cells, DNA is wrapped around molecules called histones. There are five major families of histones, and histones belonging to one of these families—the H3 histones—are replaced by cenH3 variant histones at both conventional centromeres and holocentromeres. There are many unanswered questions about holocentromeres. In particular, do holocentromeres truly extend along the full length of the chromosomes, or are they found at a large number of specific sites?C. elegans to investigate holocentromeres in greater detail. These experiments showed that the holocentromere in C. elegans is actually made of about 700 individual centromeric sites distributed along the length of the chromosomes. Each of these sites contains just one nucleosome that contains cenH3, and these sites are likely to be the sites that microtubules attach to during cell division. Surprisingly, the same sites can also act as so-called ‘HOT–sites’: these sites are bound by many proteins that are involved in regulating the process by which genes are expressed as proteins, which suggests a link between centromeres and these regulatory proteins.Now Steiner and Henikoff have studied the distribution of cenH3 in the genome of the worm The work of Steiner and Henikoff describes how centromeric nucleosomes are distributed across the genome, but why and how cenH3 ends up at these particular 700 sites remains an open question.DOI:http://dx.doi.org/10.7554/eLife.02025.002 C. elegans is amenable to address this question because the genome is repeat-poor, making it possible to precisely map centromeric regions.The centromere is a defining feature of eukaryotic chromosomes and is essential for the segregation of chromosomes during cell division, as it organizes the proteinaceous kinetochore for attachment to the spindle apparatus at mitosis. Centromeres are universally marked by the variant histone cenH3  that replaces canonical histone H3 in centromeric nucleosomes, and most commonly localize to a single position along the chromosome . HoweverC. elegans chromosomes are holocentric, whereby mitotic spindle fibers attach along the length of chromosomes and pull them to the poles as straight bars rather than from a single position that defines the more familiar monocentric chromosomes  localizes to a characteristic band along the length of the chromosome at mitosis . We found that cenH3 sites coincide with high CENP-C signal, indicating that they serve as attachment sites for the kinetochore, consistent with a polycentric organization of the chromosome. Individual sites resemble budding yeast point centromeres and coincide with transcription factor hotspots, which become occupied by transcription factors when cenH3 is lost, providing a clue as to how kinetochore sites might be selected and maintained.To identify potential kinetochore attachment sites in To precisely localize cenH3-containing nucleosomes and identify potential kinetochore attachment sites, we digested chromatin from mixed-stage embryos with micrococcal nuclease (MNase) and solubilized the majority of the chromatin by cavitation, a method adapted from C. elegans cenH3 using formaldehyde crosslinking (X-ChIP-chip)  . Replication-independent nucleosome turnover is mainly driven by transcription (As expected from a previous ChIP-microarray map of IP-chip) , we founIP-chip) . In C. ehromatin . Indeed, N-ChIP) . This is N-ChIP) . We ther N-ChIP) . Consistcription , consistcription .−16). The previously published data  embryos compared to wildtype (knl-2(RNAi) embryos (knl-2(RNAi) and R = 0.973 for wildtype vs wildtype; comparison of normalized fragment counts in 10-bp bins, N = 7633808).Incorporation of cenH3 into chromatin depends on the kinetochore protein KNL-2 . To testwildtype  for simiwildtype  and was  embryos , and theIt is conceivable that with a partial depletion of the factor required for cenH3-assembly into chromatin, relatively high cenH3 occupancy is maintained at the sites that are functional due to perdurance of the protein. Indeed, cenH3 is still locally enriched at cenH3 sites in the knockdown, albeit at much reduced levels, while the broad domains of weak enrichment are lost . These rknl-2(RNAi) embryos  sites (C. elegans transcription factors and are therefore expected to bind transcription factors with low affinity. We found that 117 out of 248 HOT sites coincided with cenH3 sites (−161). Although this degree of overlap is striking, the actual overlap of cenH3 sites and transcription factor hotspots is likely to be much larger, given the fact that that only 22 transcription factors have been used for the definition of HOT sites, but that there are 934 predicted transcription factors encoded in the C. elegans genome (GAGA-rich sequences are well-characterized targets for the GAGA factor (GAF/Trl) in nogaster . HoweverT) sites . These sT) sites . The seqs genome . We alsos genome  and wells genome . Moreoves genome . These dEmbryonic cells contain both cenH3 and transcription factors, thus complicating the analysis of the chromatin landscape at centromeric sites. To probe the chromatin landscape in cenH3-depleted cells, we analyzed our previously published data for affinity-purified adult muscle cells, where cenH3 protein is below detection levels , and cenC. elegans myoD homologue and is required for proper myogenesis  were found at many, if not all centromeric sites. Replacement of cenH3 nucleosomes by transcription factors at HOT sites upon exit from the cell cycle may then result in their reported enhancer activity .CenH3 protein has been shown to turn over completely during the mitotic cell cycle, to disappear during the pachytene stage of meiotic prophase, to reappear when nuclei progress into diplotene and to be absent from mature sperm . These oC. elegans holocentromeres are organized as dispersed point centromeres, consistent with the polycentromere model. Our discovery of the coincidence of centromeric sites with transcription factor hotspots points to a possible mechanism for centromeric site selection and maintenance.Our results resolve the long-standing question whether holocentromeres are polycentric or diffuse. We show that E. coli strain NA22. To deplete KNL-2 by RNAi, synchronized populations were grown on NA22 until fourth larval instar (L4), washed in M9 buffer and transferred to bacteria expressing dsRNA that targets knl-2 for 24 hr. Embryos were harvested from adults by sodium hypochlorite treatment.We used the standard wild-type strain N2 and OP64 grown at 20°C. Synchronized populations were cultured on peptone-rich plates seeded with Worms were decapitated with a razor blade in M9 buffer, freeze cracked on dry ice and fixed 2 min in methanol and 4 min in acetone at −20°C. Samples were incubated with anti-cenH3 (rabbit)  and anti2, 340 mM sucrose, 0.2 mM spermine, 0.5 mM spermidine, 0.5 mM phenylmethanesulfonate [PMSF]). Nuclei were isolated using a glass Dounce homogenizer with 15 strokes each of the loose- and tight-fitting inserts in buffer A supplemented with 0.1% Trition X-100 and 0.25% NP-40 substitute. The homogenate was diluted five times with buffer A, the debris were removed by spinning at 100×g for 2 min and nuclei were pelleted by spinning at 1000×g for 10 min. Nuclei were transferred to 1 ml 10 mM Tris pH7.5, 2 mM MgCl2, 0.5 mM PMSF and pre-warmed for 5 min at 37°C. CaCl2 to a final concentration of 2 mM, and 0.1 units of micrococcal nuclease  was added. After 1, 2, 5 or 10 min the reaction was stopped by the addition of ethylenediaminetetraacetic acid (EDTA) to a final concentration of 30 mM. A light MNase digestion corresponding to 2 min in this time-course experiment was used for all other experiments unless otherwise noted. Chromatin was solubilized by cavitation using needle extraction , a protocol modified from N2 embryos were treated in 0.1U/ml chitinase (Sigma) for 30–60 min and washed with buffer A  for 2 min. MNase was inactivated by addition of EGTA to 5 mM and nuclei were washed once in HM2. Chromatin was crosslinked in 1% formaldehyde for 10 min. Crosslinking was quenched by adding glycine to 125 mM for 10 min. Nuclei were washed with HM2 and lysed in 50 mM Tris pH7.5, 10 mM EDTA, 1% SDS, 0.5 mM PMSF by vortexing for 2 min. The lysate was diluted to 20 mM Tris pH7.5, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 0.5 mM PMSF and sonicated with a Sonic Dismembrator Model 500 (Fisher Scientific) for 40s at 30% amplitude. Debris removal, pre-clearing and CENP-C ChIP were done as for native ChIP, with the antibody from For CENP-C ChIP, nuclei were prepared and MNase treated as for native ChIP, except that MNase incubation was done in HM2 . Proteins were crosslinked with 1% formaldehyde for 15 min, the reaction was quenched with 125 mM glycine for 10 min, the volume increased to 50 ml, and chromatin pelleted by spinning at 2000×g for 10 min. The pellet was washed again in PBS, resuspended in 50 mM Tris pH7.5, 10 mM EDTA, 1% SDS containing 1x Complete Protease Inhibitor Cocktail (Roche), incubated 10 min at room temperature, diluted to 20 mM Tris pH7.5, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA containing 1x Complete Protease Inhibitor Cocktail (Roche) and sonicated with a Sonic Dismembrator Model 500 (Fisher Scientific) for 4 min at 30% amplitude. Debris was removed by spinning twice at maximum speed for 5 min. The extract was incubated with an anti-FLAG M2 antibody (Sigma) overnight at 4°C. Protein G beads pre-blocked with BSA and yeast tRNA were added for 4h. Beads were washed and DNA isolated as described for CENP-C ChIP above.Libraries were prepared using a modified Illumina paired-end library protocol as described in C. elegans genome release WS220 using Novoalign (http://www.novocraft.com) with default parameters, except that each multiple hit was mapped to one site chosen at random . The number of inserts aligned to each 10-bp interval of the genome was counted, and the interval counts were normalized by dividing by the total number of counts for all intervals, and then scaled by multiplying by the number of bases in the genome. We considered fragments >140 bp to represent nucleosomes, the in silico equivalent of excising a gel slice around the ∼150-bp size range from an MNase-digested chromatin ladder and extracting the DNA for single-end sequencing. As we use a modified paired-end sequencing protocol to include all fragments >25 bp , and peaks that exceeded 30 counts in at least one of the biological replicates were considered positive. For CENP-C peak calling, input counts were subtracted from CENP-C ChIP counts, and peaks that exceeded 20 counts were considered positive. For insoluble chromatin peak calling, peaks that exceeded 100 counts were considered positive.2-ratios only to compare to previously published array data. Otherwise, we consider input reads a separate “blank” experiment that we subtract from the ChIP counts.To normalize against input, we used log review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent . This study uses a different method (ChIP-Seq) to map cenH3 and comes to a very different conclusion: in addition to the broad domains, they identify ∼700 discrete locations for cenH3 and conclude that the centromeres of C. elegans are in fact similar to the point centromeres of yeast. The authors also describe that the centromeres correspond to the so-called “HOT” sites where transcription factors tend to accumulate in non-dividing cells. These observations are novel, resolve a long-standing question regarding the structure of holocentric chromosomes, and have intriguing implications for the formation and maintenance of centromeres.This report describes the distribution of centromeric chromatin in eLife. However, the consensus among the reviewers is that the following aspects of the paper must be strengthened before we proceed further. Here are the most important points that should be addressed upon revision.All three reviewers are generally interested in your work and find the study of significant importance to be suitable for publication in The authors should demonstrate more convincingly that cenH3 peaks and not broad cenH3 domains are the centromeres that build the kinetochore. To be more convincing they need to present the CENP-C X-chip in a more transparent and comprehensive manner:1) In - How many CENP-C sites are identified?- What sub-fraction of cenH3 peaks overlaps with what subfraction of the other sequences with centromere-like behavior: insoluble sites and CENP-C peaks . Most importantly, how does this compare to broad cenH3 domains?2) Given that the MNase digest is important to the argument that the identified cenH3 sites show the properties of point centromeres, the authors should show a control in 3) More information on the peak calling should be provided as this confused two reviewers. 1) In, only two regions of the CENP-C X-Chip are shown. To assess the quality of the CENP-C X-Chip it would be really important to see a larger region of DNA as it has been done in other figures of this paper.The quality of the CENP-C X-ChIP should be most evident from the heat map in - How many CENP-C sites are identified?- What sub-fraction of cenH3 peaks overlaps with what subfraction of the other sequences with centromere-like behavior: insoluble sites and CENP-C peaks . Most importantly, how does this compare to broad cenH3 domains?lower than within the rest of the genome. That is, our CENP-C ChIP data not only confirm that the cenH3 peaks are kinetochore sites, the data also argue against the hypothesis that broad domains harbor kinetochore sites. We have included these new data in We agree that a direct head-to-head comparison of cenH3 peaks and broad domains with respect to insoluble sites and CENP-C peaks is important to strengthen our conclusion that cenH3 sites correspond to kinetochore sites. There are 347 CENP-C peaks, of which 163 overlap with the cenH3 sites, whereas only 26 CENP-C peaks overlap with the domains. Based on the fact that the coverage of cenH3 peaks is only 0.3% that of broad domains, the enrichment of CENP-C at cenH3 peaks relative to cenH3 domains is (163/26)/0.003 ≈ 2000-fold. Similarly, there are 2060 peaks in the insoluble chromatin, 460 of which coincide with the cenH3 peaks, compared to 147 for the domains, which implies a relative enrichment of insoluble chromatin at cenH3 peaks to cenH3 broad domains of (147/460)/0.003 ≈ 800-fold. Importantly, the densities of both CENP-C and insoluble chromatin peaks within the domains are significantly 2) Given that the MNase digest is important to the argument that the identified cenH3 sites show the properties of point centromeres, the authors should show a control infor MNase digestion, such as an H3 nucleosome where the amplitude is less sensitive when compared to cenH3.We agree that we had not sufficiently described the characteristic MNase sensitivity of the point centromere sites. To strengthen this point, we have substantially revised To quantify the MNase-sensitivity of cenH3 nucleosomes, the nucleosomes flanking the cenH3 peaks and the +1 nucleosomes, we plotted the occupancy at these features relative to the occupancy of first time point. This clearly shows that the cenH3 peaks are more MNase-sensitive than both the flanking and +1 nucleosomes .3) More information on the peak calling should be provided as this confused two reviewers.We have extended the description of our very simple peak calling in the Methods section  and added a sentence in the Results section . We classified all sites that exceeded 30 normalized counts in at least one of two biological replicate (cenH3 ChIP minus input) as peaks. 30 counts is the equivalent of the genome-wide mean plus seven standard deviations.


The genes encoding nuclear receptors comprise multiple 5′untranslated exons, which give rise to several transcripts encoding the same protein, allowing tissue-specific regulation of expression. Both human and mouse peroxisome proliferator activated receptor (PPAR) α genes have multiple promoters, although their function is unknown. Here we have characterised the rat PPARα promoter region and have identified three alternative PPARα transcripts, which have different transcription start sites owing to the utilisation of distinct first exons. Moreover these alternative PPARα transcripts were differentially expressed between adipose tissue and liver. We show that while the major adipose (P1) and liver (P2) transcripts were both induced by dexamethasone, they were differentially regulated by the PPARα agonist, clofibric acid, and leptin. Leptin had no effect on the adipose-specific P1 transcript, but induced liver-specific P2 promoter activity via a STAT3/Sp1 mechanism. Moreover in Wistar rats, leptin treatment between postnatal day 3–13 led to an increase in P2 but not P1 transcription in adipose tissue which was sustained into adulthood. This suggests that the expression of the alternative PPARα transcripts are in part programmed by early life exposure to leptin leading to persistent change in adipose tissue fatty acid metabolism through specific activation of a quiescent PPARα promoter. Such complexity in the regulation of PPARα may allow the expression of PPARα to be finely regulated in response to environmental factors. PPARα is a ligand-activated transcription factor, which belongs to the nuclear hormone receptor superfamily PPARα is mainly expressed in tissues with high rates of fatty acid β oxidation such as liver, skeletal muscle, brown fat, heart, and kidneysThere is also evidence that PPARα gene transcription can be programmed by environmental factors in early life To date, little is known about of the molecular factors that mediate the tissue-specific regulation of PPARα, or its regulation by perinatal factors or leptin. The genes encoding nuclear receptors frequently comprise multiple 5′untranslated exons giving rise to transcripts, which are expressed differentially between tissues. The human All animal work was approved by the Animal Ethics Committee of the University of Auckland  and complied with the New Zealand Code of Ethical Conduct for the care and use of animals for scientific purposes .ad libitum throughout gestation. Litter size was adjusted to 8 pups at birth to ensure standardised nutrition until weaning. At postnatal day 3, female pups were randomized to receive either saline or recombinant rat leptin  (2.5 µg/g/day) for 10 days by subcutaneous injection (n = 16 per group). Dams were fed ad libitum until offspring were weaned on day 22. Saline or leptin-treated offspring were weaned onto standard rat chow. This produced 8 groups of female rats (n = 8 per group). On postnatal day 170, rats were fasted overnight, and killed by halothane anaesthesia followed by decapitation. Liver and retroperitoneal adipose tissue was removed immediately, frozen in liquid nitrogen and stored at −80°C.A detailed description of the study design has been published previously Total PPARα, P1 and P2 PPARα, AOX and carnitine palmitoyltransferase (CPT)-1 mRNA concentrations were determined by real time RTPCR TGACTGAGGAGGGGCTGGAA 3′; outer gene specific primer 5′ AGCCTTCACATGCGTGGACT 3) and used with the nested forward 5′ RACE adaptor primers provided by the kit. Cycling conditions for both outer and nested PCR were initial denaturation of 94°C 3 minutes, followed by 35 cycles of 94°C 30 s, anneal at 60°C 30 sec, and extension at 72°C 1 minute 30 sec. Resulting PCR products were purified, cloned into pGem T-Easy (Promega) and sequenced. The sequence of the transcripts P1–3 was confirmed by sequencing multiple clones from liver and adipose tissue.RNA was amplified using a 5′ RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM RACE) kit (Ambion) according to manufacturer’s instructions. Briefly, 10 µg of total RNA from liver and adipose tissue was incubated with calf intestinal alkaline phosphatase to remove free 5′ phosphates, the 5′ CAP structure was then removed from the RNA with tobacco acid pyrophosphatase (TAP) and the TAP treated RNA ligated to the 5′ RACE adapter and reverse transcribed into cDNA using random hexamers. To amplify the 5′ region of PPARα mRNA, generated from the 5′RLM RACE, nested gene specific reverse primers for PPARα were designed . All primers contained either a SacI or XhoI restriction site at the 5′ end of the forward primer, and a HindIII restriction site at the 5′ end of the reverse primer to allow cloning of PCR fragments into the pGL3 Basic reporter vector (Promega). Cycling conditions for PCR were as follows; initial denaturation of 94°C 2 minutes, followed by 40 cycles of 95°C 45 s, anneal at a 60°C 45 sec, and extension at 72°C 5 minutes. Resulting PCR products were gel extracted, cloned into the pGL3 Basic reporter vector (Promega) and sequenced to confirm the presence of the insert. The Quik change method of mutagenesis (Stratagene) was used to create a P2 promoter construct with a mutated Sp1 site. Two complementary primers were designed using the QuikChange® Primer Design Program  to change the Sp1 consensus sequence GGGCGG to an EcoR1 restriction site GAATTC (Forward primer 5′ GCCTCAGGTGCCCAGGAATTCGAGGGCACGCGCGAGG 3′ and reverse complement 5′ CCTCGCGCGTGCCCTCGAATTCCTGGGCACCTGAGGC 3′). The primers (0.4 µM) were then annealed to 30 ng P2- pGL3 DNA and extended using Pfu polymerase. Cycling conditions for PCR were as follows; initial denaturation of 95°C 2 minutes, followed by 18 cycles of 95°C 30 s, anneal at a 60°C 1 min, and extension at 72°C 4 minutes, followed by a final extension at 72°C for 5 minutes. Resulting PCR products were digested with the restriction enzyme DpnI at 37°C for 1 hour and the product transformed into DH5α E.Coli. Clones were cut with EcoR1 and sequenced to confirm the presence of the mutation.The human hepatoma cell line HepG2 (ECACC- Sigma Aldrich) was cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 u/ml penicillin and 100 ug/ml streptomycin. HepG2 cells were transfected using calcium phosphate Statistical comparisons of mRNA expression and methylation levels between treatments relative to the untreated control were carried out using a Students unpaired t test. Statistical comparisons of luciferase activity levels between promoter constructs and treatments were determined by ANOVA followed by Bonferoni’s post hoc analysis. All values are plotted as the mean ±SEM.To investigate tissue specific 5′heterogeneity of rat PPARα mRNA, 5′RLM RACE PCR analysis was conducted on total RNA from adipose and liver tissue from adult Wistar rats. 5′RLM RACE revealed 2 different sized PCR products from the liver (723 bp and 463 bp) but only 1 from adipose tissue (465 bp) . The PCRhttp://www.ensembl.org/index.html), and gave rise to a distinct unique first exon of 60 bp termed exon 1A. The liver specific transcripts were termed P2 and P3. P2 comprises exon 1B, 2 and 3. Exon 1B (previously exon 1) is 67 bp longer than the original exon 1 at the 5′ end resulting in a 318 bp exon. The P3 transcript contains exon 2A which is a 145 bp novel exon located several Kb downstream from exon 2, and has been termed exon 2A. This exon is spliced directly onto Ensembl exon 3, and is the only transcript that does not contain exon 2 , and the remainder were exactly as identified by Ensembl (exons 2 and 3). The 5′UTR of the adipose specific transcript (P1) is encoded by exons 1A, 2 and 3. The transcription start site for this transcript is 257 bp upstream of the Ensembl published start site  were cloned into the reporter vector pGl3basic and transfected into HepG2 cells. This resulted in two partially overlapping promoter regions for P1 and P2 and a distinct downstream promoter for P3 . Both P1To determine whether the individual PPARα promoters are differentially regulated, the PPARα promoter-pGL3 luciferase reporter constructs  were transfected into HepG2 cells and treated for 24 hrs with clofibric acid and dexamethasone at a range of concentrations which have previously been reported to induce PPARα expression To determine whether leptin could induce transcription from the alternative PPARα promoters, HepG2 cells were transfected with the P1, P2 and P3 promoter constructs and treated with increasing concentrations of leptin for 24 hrs. We found that P2 promoter activity was significantly increased in the presence of 500 ng/ml and 1000 ng/ml leptin. In contrast, leptin had no effect on either P1 or P3 promoter activity . As leptLeptin modulation of transcription via STAT3 has been shown to occur either directly through the binding of STAT3 to its response element in the promoter of a gene or indirectly through a STAT3/Sp1 co-operative mechanism As there is evidence that hepatic PPARα gene transcription can be programmed by environmental factors in early life including leptin, we next investigated whether neonatal leptin treatment induced a persistent increase in PPARα expression in adipose tissue and whether the P1 and P2 transcripts were differentially affected. The expression of PPARα together with its target genes AOX and CPT-1 were examined in adipose tissue from PN170 rats treated with either saline or leptin (2.5 µg/g/day) by subcutaneous injection for 10 days from PN3-13. This dosage of leptin was used, as similar levels have been reported previously to induce leptin receptor signaling, alter neuropeptide expression To determine whether this persistent increase in transcription from the P2 promoter in response to neonatal leptin treatment was due to altered DNA methylation, sodium bisulfite pyrosequencing was performed using genomic DNA extracted from adipose tissue from neonatal saline and leptin treated adult female rats. The analysis of the region (−336 to −117 bp) immediately upstream of the PPARα P2 transcription start site (TSS) which contains the Sp1 response element in the P2 unique region showed that all CpGs within this region had a methylation level of below 10% regardless of treatment ., althouThe genomic organisation of the human and mouse PPARα genes indicate that PPARα, like other nuclear receptors, contains multiple 5′UTR variants and promoter regions Since the 5′ UTR can modulate RNA stabilityThe P1 and P2 transcripts did not contain ATG initiation codons in the sequence upstream of the previously reported ATG codon suggesting that they possess the same open reading frame as the previously reported transcript. The P3 transcript, however, contains four uATGs, 3 have adequate Kozak consensus sequences The analysis of the sequence upstream of the transcription start sites of the P1, P2 and P3 sites revealed that the P1/P2 promoters possess the characteristics of typical GC rich promoters common to nuclear hormone receptors such as the absence of TATA elements and the presence of CpG islands containing multiple Sp1 response elements. The P3 promoter, in contrast, did not contain any CpG islands or Sp1 response elements. Moreover, unlike the sequences upstream of the P1 and P2 TSS, which gave rise to high levels of promoter activity in HepG2 cells, the sequence upstream of the P3 start site was not active in HepG2 cells. This low activity may indicate that important regulatory elements outside the region cloned in this study are required for P3 expression or that the promoter is inactive in the absence of stimulatory factors that are not present in liver cell line HepG2.www.genomatix.de) of the promoter region of PPARα did not reveal any glucocorticoid response elements, but a putative NF-1 binding site was identified in the sequence shared by both P1 and P2. NF-1 is a transcription factor that has been shown to mediate GR responsiveness It has been reported previously that PPARα transcription is induced by CFA Leptin plays a critical role in maintaining energy balance In conclusion we show that the rat PPARα gene has multiple transcripts, which are expressed in a tissue specific manner and are differentially regulated by leptin. Moreover neonatal leptin exposure induces a persistent change in adipose tissue gene expression through specific activation of an otherwise quiescent PPARα promoter. It is possible that leptin may have similar effects in other species. For example, human PPARα also has multiple promoters and hence it is possible that long term modulation of PPARα activity, and hence lipid metabolism, by promoter switching as a result of leptin exposure in early life may also occur in humans. If the changes to gene expression in adipose tissue reported in the present study were to occur in humans, it may be expected that those who experience higher leptin exposure in early life would have greater capacity to meet the metabolic challenge of a high calorie diet after weaning, while a lower leptin exposure would result in a reduced capacity to regulate fatty acid deposition in adipose tissue. This may explain, at least in part, the observation that low umbilical cord blood leptin levels are associated with rapid postnatal weight gain


We performed a yeast two-hybrid screen using a truncated version of Bck2 and discovered six novel Bck2-binding partners including Mcm1, an essential protein that binds to and activates M/G1 promoters through Early Cell cycle Box (ECB) elements as well as to G2/M promoters. At M/G1 promoters Mcm1 is inhibited by association with two repressors, Yox1 or Yhp1, and gene activation ensues once repression is relieved by an unknown activating signal. Here, we show that Bck2 interacts physically with Mcm1 to activate genes during G1 phase. We used chromatin immunoprecipitation (ChIP) experiments to show that Bck2 localizes to the promoters of M/G1-specific genes, in a manner dependent on functional ECB elements, as well as to the promoters of G1/S and G2/M genes. The Bck2-Mcm1 interaction requires valine 69 on Mcm1, a residue known to be required for interaction with Yox1. Overexpression of BCK2 decreases Yox1 localization to the early G1-specific CLN3 promoter and rescues the lethality caused by overexpression of YOX1. Our data suggest that Yox1 and Bck2 may compete for access to the Mcm1-ECB scaffold to ensure appropriate activation of the initial suite of genes required for cell cycle commitment.The Bck2 protein is a potent genetic regulator of cell-cycle-dependent gene expression in budding yeast. To date, most experiments have focused on assessing a potential role for Bck2 in activation of the G 1 transition, many of the up-regulated genes encode key regulators of DNA replication (CDC6) and cyclins that initiate the events of cell cycle commitment . The promoters of genes activated at M/G1 contain a cis-regulatory sequence called the early cell cycle box (ECB), which is bound by the MADS-box transcription factor Mcm1, as well as the repressor Yox1 or Yhp1. The ECB cluster of genes defines a crucial cell cycle window during which a cell may change its fate; yet how the regulators that appear to act at ECBs are linked to cell cycle position is unclear, and coregulators, which experience tells us must exist, were unknown. Here, we describe our discovery that Bck2, a potent cell-cycle-regulator whose function has remained obscure, functions as a cofactor for Mcm1, to induce ECB–dependent gene expression. We also show that Bck2 has a role in promoting expression of late G1 and M/G2 genes. Our genetic and biochemical experiments reveal a new pathway for regulating gene expression associated with early cell cycle commitment, a process that is highly conserved.Cell-cycle-dependent gene expression is a universal feature of cell cycles, with clear transcriptional programs in yeast, bacteria, and metazoans. At the M/G Control over Cdc28 activity is exerted at several levels, including transcriptional induction of cyclin gene expression; the G1/S phase transition features activation of a massive transcriptional program of ∼120 genes, including the G1 cyclin genes CLN1 and CLN21/S cluster: SBF  binds the so-called SCB promoter element  found upstream of the cyclin genes and cell wall biosynthetic genes, while MBF  preferentially acts through a distinct element called the MCB (MluI cell cycle box) found mostly upstream of DNA replication and repair genes.The temporal control of transcription is likely a universal feature of cell cycles, with clear transcriptional programs in yeast, bacteria and metazoans 1/S transition is well established, deletion of Swi6, the common subunit of both complexes, does not cause cell cycle arrest, suggesting alternative pathways for activating G1 transcription. One of these pathways is defined by Bck2, a poorly understood cell cycle regulator whose deletion causes dramatic cell cycle phenotypes in certain genetic contexts. For example, deletion of both CLN3, which encodes a cyclin that activates Cdc28 at M/G1, and BCK2 causes synthetic lethality BCK2 is one of only two genes whose overexpression is known to bypass the lethality caused by mutation of all three G1-specific Cdc28 cyclins  pkc1 itself) and G1 cyclin deficiencies 1/S genes 1/S genes by Bck2 depends partly, but not wholly, on SBF and MBF and Bck2 appears capable of activating G1/S transcription in the absence of Cdc28, indicating SBF- and MBF-independence CLN2, termed UAS2, which completely lacks SCB or MCB elements, is activated by BCK2 overexpression CDC28-independent manner Although the role of SBF and MBF at the G1 and G2/M. During M/G1, Mcm1 functions as a critical constituent of a complex that forms on ECB (Early Cell cycle Box) elements in promoters of genes expressed at the M/G1 phase transition such as CLN3 and SWI42/M phase transition to control the CLB2 cluster of genes, including the G2 cyclin gene CLB2 and CDC20, which encodes an activator of the anaphase promoting complex CLB2 gene cluster is activated by the Clb-Cdc28 and Cdc5 kinases CLB2 cluster genes, but not CLB2 itself, contain hybrid elements composed of an Mcm1-binding site flanked by Yox1- and Fkh2-binding sites CLB2 cluster genes that contain such elements, Yox1 and Fkh2 compete for binding to DNA-bound Mcm1 despite the spatial separation of their DNA recognition elements 1/S gene CLN2, where it contributes to the presence of a nucleosome depleted region that is needed for reliable “on/off” expression once per cell cycle The MADS box transcription factor Mcm1 has an important regulatory function at two points in the cell cycle – M/G1 genes CLN3 and SWI4, through an interaction with Mcm1 on ECB elements in the promoters of these genes. Moreover, increased Bck2 dosage leads to decreased Yox1 binding at M/G1 promoters and overproduction of BCK2 rescues the lethality caused by overexpression of YOX1, indicating that Bck2 and Yox1 may compete for access to Mcm1 on promoters. Consistent with this hypothesis, mutation of a key residue on Mcm1 known to prevent interaction with Yox1 also prevents interaction with Bck2. In addition, we show that Bck2 interacts with the promoters of the G1/S gene CLN2 and the G2/M gene CLB2. Our experiments reveal a previously unappreciated function for Bck2 as a cofactor for Mcm1 and suggest a more general role for Bck2 in regulating cell-cycle gene expression.In this study, we show that Bck2 activates expression of the M/G1 progression. Bck2 is difficult to work with biochemically  genes DAN/TIR group of genes that encode cell wall mannoproteins during anaerobic growth TOR pathway for nutrient sensing SWI4 or MBP1 in our Y2H screen, but a direct test revealed a weak interaction between Bck2 and Swi4 (We used the ORFeome Y2H screening method and Swi4 . The ide1 phase: (1) mRNA from the M/G1 gene SWI4 accumulates much more slowly in bck2Δ than WT cells in synchronized cultures, whereas SWI4 is upregulated in cells that overexpress BCK2BCK2 stimulates the expression of the Mcm1-dependent reporter gene, P-lacZBCK2 causes increased transcription of CLN3 and SWI4 by microarray analysis BCK2 and M/G1 genes has been established. To evaluate the significance of the Mcm1-Bck2 physical interaction in vivo, we first assessed the effect of BCK2 deletion on expression of lacZ reporter genes whose expression was dependent on either multiple Mcm1-binding sites (4 x P-sites) 1 phase – CLN3, CDC6, CDC47, SWI4BCK2 had no effect on expression of a control ACT1-lacZ reporter gene. However, we saw a pronounced reduction in expression of the CLN3-lacZ, CDC6-lacZ, CDC47-lacZ, SWI4-lacZ, and P-lacZ reporter genes in the bck2Δ strain . However, CLN2 transcription was highly periodic in both WT and bck2Δ cells, indicating that these cultures were synchronously released from the mitotic block. Consistent with previous reports, CLN2 transcript was reduced in the bck2Δ strain ALG9) were unaffected. Strikingly, the accumulation of CLN3 and SWI4 mRNAs was significantly reduced in bck2Δ cells, and peak expression was also delayed, at least for the CLN3 transcript. In wild-type cells, CLB2 mRNA peaked after G1 transcripts as expected. However, in bck2Δ cells CLB2 transcripts were delayed and only began to accumulate near the end of the time-course, after the peak of CLB2 expression seen in wild-type cells. Thus, BCK2 is required for the appropriate expression of the M/G1 genes CLN3 and SWI4, the G1/S gene CLN2, and the G2/M gene CLB2.Given the clear roles for Mcm1 in cell-cycle-dependent gene expression, we chose to focus our follow-up analysis on the Mcm1-Bck2 interaction. In addition to the Y2H interaction between Bck2 and Mcm1, several observations from earlier studies implicate Bck2 in the activation of Mcm1 target genes in M/GΔ strain . These rΔ strain . Since M1 genes that contain Mcm1-binding sites suggested that Bck2 may function through ECB elements. To test this hypothesis, we assayed the effect of BCK2 deletion on expression of lacZ reporter plasmids containing either a WT CLN3 promoter that has intact ECB elements or a mutated CLN3 promoter that lacks functional ECB elements BCK2 in combination with mutation of ECB elements caused a level of reporter gene expression similar to that seen with either perturbation alone swi4(ecb)) CLN3 and SWI4 transcript levels in a WT strain, but not the cln3(ecb)swi4(ecb) mutant strain  and G2/M-regulated (CLB2) genes by a mechanism that does not depend on its effect on early G1 genes.As previously seen, overproduction of Bck2 also increased romoters . This reromoters , again s1-phase genes. We first used chromatin immunoprecipitation (ChIP) with a strain carrying a TAP-tagged allele of Bck2 to assess association of Bck2 with various promoters. We detected a reproducible enrichment of promoter DNA in the Bck2 ChIP, but the signal was very low relative to Swi4 or Mcm1 ChIPs (data not shown). To improve our assay, we repeated the ChIP experiment using a strain in which a FLAG-tagged derivative of Bck2 was conditionally overproduced  (CLN3 promoter DNA compared to SWI4 promoter DNA in Bck2 IPs likely reflects the presence of more ECB elements in the CLN3 promoter (6 versus 1). Association of Bck2 with the CLN3 and SWI4 promoters was entirely dependent on the presence of ECB elements, while association with the CLN2 promoter was unaffected, consistent with our gene expression analysis (CLN3 and SWI4 in a manner that depends on ECB elements.Since Bck2 functions through ECB elements  and physproduced . Under iffinose)  or vectoanalysis . Our finCLN2 promoter in our ChIP assays. The CLN2 promoter contains three SCB elements, which are required for cell-cycle-specific gene expression, and two Mcm1 binding sites, which contribute to the presence of a nucleosome depleted region but have no effect on average expression levels or cell-cycle-specific expression CLN2 promoter in strains that contained mutations in either set of elements scb*) or the Mcm1-binding sites (mcm1*) led to a modest reduction in Bck2 localization to the CLN2 promoter . Regardless, the mechanism of action of Bck2 at G2/M promoters must differ from that at M/G1 promoters where Bck2 appears to act on Mcm1 alone. Unlike the G2/M promoters, M/G1 promoters 1We present several lines of evidence that suggest a role for Bck2 in activation of Gpression . Second,promoter , in a maer  activation of genes involved in mating together with Ste12; (2) regulation of cell-type-specific genes together with α1 or α2; and (3) control of genes involved in arginine metabolism together with Arg80  Yeast strains used in this study  were derBCK2 gene were amplified from genomic DNA using PCR primers designed to be compatible with the Gateway system of recombinational cloning +20 µl 0.1% SDS+40 µl chloroform for 45 seconds. After 5 min, 0.2 ml of o-nitrophenylgalactoside  solution was added and the reaction was incubated at 30°C until a slight yellowing was observed. The reactions were stopped by the addition of 0.5 ml of 1 M Na2CO3, and the samples were centrifuged for 3 minutes at 13,000× g. The A420 of the supernatant was determined. β-Galactosidase units were calculated using the following formula: units = (A420)*(1000)/*(volume of extract in assay = 1 ml)*. For each time point, the assays were performed on three separate cultures, and the average is reported. Qualitative β-galactosidase overlay assays were performed as described Quantitative β-galactosidase assays were performed as follows. Exponentially growing cells at an optical density at 600 nm of 0.2 to 0.25 were harvested. Extracts were prepared by vortexing the cells in 1 ml Z-buffer (0.1 M NaPOcln3Δbck2Δ pGAL-CLN3 -URA3 strain (BY3015) bearing ADH1-GAL4 DBD-BCK2-LEU2 (1–20) plasmids were grown in plasmid selective medium, prepared at equivalent optical density and spotted in serial 10-fold dilutions onto plasmid selective medium containing either galactose lacking 5′FOA or glucose+5′FOA, and incubated for 48 h at 30°C.Transformants of a 2O. PCR reactions were performed in 25 µl volumes containing 5 µl of the yeast cell preparation described. PCR reactions were performed using 5 minute extension times in order to ensure that large ORF inserts were isolated. PCR reactions were electrophoresed on agarose gels, purified using the PureLink kit (Invitrogen) and sent for sequencing analysis.The Y2H ORFeome method ADH1-GAL4 DBD  or ADH1-GAL4 DBD-BCK2 Fragment 11 (Bck2) in a two-hybrid bait strain (Y8930) were mated to yeast transformants of a two-hybrid prey strain (Y8800) bearing specific ADH1-GAL4-AD-ORF-TRP plasmids. Diploids were selected by streaking cells onto double plasmid selection medium (SD – Leu – Trp). Diploids of equivalent optical density were spotted in serial 10-fold dilutions on double plasmid selection medium (SD – Leu – Trp), or medium where growth was proportional to transcription of the ADE2 gene (SD – Leu – Trp - Ade), and incubated for 48 h at 30°C. Six diploid strains carrying different combinations of AD and DBD ORF-fusions Yeast transformants carrying cdc20-3 temperature-sensitive strain . RNA was transcribed into cDNA using the Superscript II Reverse Transcriptase kit (Invitrogen) and RNA was then removed by addition of NaOH. Reactions were run on the ABI 7500 system (Applied Biosystems) using standard Q-PCR conditions. Data were analyzed using ABI7500 system software. VIC and FAM labelled fluorogenic primers (ABI), used to detect V69E yeast two-hybrid prey plasmid, the Mcm1WT prey plasmid was subject to in vitro mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene).To construct the Mcm1CLN2, CLN3, CLB2 and SWI4 promoter DNA by TaqMan Q-PCR (Applied Biosystems) using primers with homology to target gene promoter DNA  or galactose-containing medium (inducing conditions) to mid-log phase. Cultures were harvested and anti-FLAG ChIPs were analyzed for oter DNA . Enrichmoter DNA .Figure S1BCK2 truncations fused to the GAL4 DNA binding domain. (A) Transcriptional activation by BCK2 truncations in lacZ reporter assays. Yeast-two-hybrid bait strains, in which GAL4 UAS elements drive expression of ADE2, HIS3, and lacZ reporter genes (Y8930), bearing truncated versions of the BCK2 gene, ADH1-GAL4 DB-BCK2 (F1-20) (LEU2) plasmids were spotted onto plasmid selection medium, incubated for 48 h at 30°C, overlaid with a top agar solution containing X-Gal, and incubated at 30°C until blue color was seen (inset). To quantify the differences in transcriptional activity observed by the overlay assay, three independent isolates from a single transformation reaction were grown to mid-log phase in plasmid selective medium and subjected to quantitative β-galactosidase liquid assays to measure lacZ expression. Values are expressed in Miller Units. (B) Transcriptional activation by BCK2 truncations in ADE2 reporter assays. A yeast-two-hybrid bait strain where GAL4 UAS elements drive expression of ADE2, HIS3, and lacZ reporter genes (Y8930) bearing ADH1-GAL4 DB-BCK2 (1–20) (LEU2) plasmids were spotted in serial 10-fold dilutions on plasmid-selection medium or medium where growth is proportional to transcription of the ADE2 gene. Plates were incubated for 48 h at 30°C. (C) Complementation of a cln3Δbck2ΔpGAL-CLN3 strain growth defect by BCK2 truncation derivatives. Transformants of a cln3Δbck2Δ GAL-CLN3 (URA3) strain (BY3015) bearing ADH1-GAL4 DBD-BCK2 (1–20) plasmids (LEU2) were grown in plasmid selective medium, grown to equivalent optical density and spotted in serial 10-fold dilutions onto plasmid selective medium containing either galactose without 5-FOA or glucose+5-FOA. Plates were incubated for 48 h at 30°C.Assays with (PDF)Click here for additional data file.Figure S2CLN2 promoter. WT (BY4741) and swi4Δ, swi6Δ and mbp1Δ strains from the deletion set containing pGAL-BCK2-FLAG were grown in inducing conditions and Bck2-FLAG (left panel) and Swi4 (right panel) recruitment to the CLN2 promoter were assayed by ChIP.Localization of Bck2-Flag and Swi4 to the (PDF)Click here for additional data file.Figure S3CLB2 promoter. WT (BY4741) and a fkh2Δ strain from the deletion set containing vector or pGAL-BCK2-FLAG were grown in inducing conditions and Bck2-FLAG recruitment to the CLB2 promoter were assayed by ChIP.Localization of Bck2-FLAG to the (PDF)Click here for additional data file.Table S1BCK2 Truncation Primers were used in constructing plasmids (underlined regions indicate BCK2 sequence). Y2H AD Sequencing Primers were used to confirm that the sequence was correct. Q-PCR analysis of RNA and chromatin ChIP DNA was done using TaqMan with the primers and fluorogenic probes shown. The mcm1-V69E mutant was constructed with the mutagenic primers shown.Oligonucleotides used in this study. Y2H AD PCR Primers and (XLS)Click here for additional data file.


Luciferase reporter assays showed a retinoblastoma-independent role for E2F2 in the negative regulation of these target genes. Importantly, we show that the DNA binding activity of the transcription factor CREB contributes to E2F2-mediated repression of Mcm5 and Chk1 promoters. siRNA-mediated CREB knockdown, expression of a dominant negative KCREB mutant or disruption of CREB binding by mutating a CRE motif on Mcm5 promoter, relieved E2F2-mediated transcriptional repression. Taken together, our data uncover a new regulatory mechanism for E2F-mediated transcriptional control, whereby E2F2 and CREB cooperate in the transcriptional repression of a subset of E2F2 target genes.E2F2 is essential for the maintenance of T lymphocyte quiescence. To identify the full set of E2F2 target genes, and to gain further understanding of the role of E2F2 in transcriptional regulation, we have performed ChIP-chip analyses across the genome of lymph node–derived T lymphocytes. Here we show that during quiescence, E2F2 binds the promoters of a large number of genes involved in DNA metabolism and cell cycle regulation, concomitant with their transcriptional silencing. A comparison of ChIP-chip data with expression profiling data on resting  Indeed, accumulating evidence suggests that most E2Fs can function both as activators as well as repressors of target genes, including those coding for microRNAs  family of tumor suppressor proteins , althougin vivo requires the identification of the full range of genes regulated by each individual E2F. Our group and others have reported that −E2f2/− and compound −E2f1/−−/E2f2/− mice exhibit defects in the proliferative properties of hematopoietic cells  mice (C57Bl6:129Sv background) were maintained on a normal light/dark cycle in cages with microisolator lids, and were genotyped by standard polymerase chain reaction (PCR) technology, as previously described  were stimulated for the indicated times with immobilized antibodies against CD3 . Human embryonic kidney (HEK) 293 T cells and human U2OS osteosarcoma cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS.T cell preparation and culture were carried out in complete medium . Lymph nodes were extracted from 4-week-old WT and For cell synchronization at mitosis, U2OS cultures were incubated with thymidine (2 mM) for 18 h. Subsequently, cells were washed and cultured for an additional 20 h in fresh medium. Nocodazole (100 ng/ml) was added to the cultures for the last 16 h. Cells at M phase were collected by shaking off the plates and seeded in complete medium for subsequent analyses. To assess cell cycle distribution, cells were fixed with chilled 70% ethanol, treated with RNAse, stained with 50 µg/ml propidium iodide and analyzed by flow cytometry . Cell cycle distribution analysis was performed with ModFit LT software, and data were represented with WinMDI2.8 software.Mcm5 genomic region was cloned into the pCR2.1-TOPO vector using the ‘TOPO TA cloning’ kit (Invitrogen). After digestion with MluI and BglII, the generated insert was cloned into the pGL3-basic luciferase reporter vector (Promega).Plasmid constructs pChk1-Luc (encompassing the −613 to +1664 genomic region of human Chk1), pE2F-Luc , pCRE-Luc , pRc-CMV-HA-E2F2 and pCREB-VP16 have been described previously . PlasmidMcm5-Luc, and E2F motifs in pMcm5-Luc and in pChk1-Luc reporter plasmids was carried out using either the QuikChange Lightning Site-Directed Mutagenesis Kit or the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Stratagene), following manufacturer’s directions (See Supplementary Table S1 for nucleotide sequences of the primer sets used for mutagenesis).Site-directed mutagenesis of the CRE motif in pDNA fragments encoding HA-tagged E2F2 deletion mutants lacking the transactivation domain (HA-E2F2-ΔTRD) or the DNA binding domain (HA-E2F2-ΔDBD) were amplified by PCR using the pRc-CMV-HA-E2F2 vector as template. To amplify the fragment encoding HA-E2F2-ΔTRD (corresponding to amino acids 1–358), the forward primer (5′-CTGCAAGGGCCCCGGGCCT-3′) in combination with the reverse primer (5′-CTGCTGGGGGGTTGGCGCTGGT-3′) were used. To amplify the fragment encoding HA-E2F2(ΔDBD) (corresponding to amino acids 196–437), the forward primer (5′-TTTGAAGACCCCACCA-3′) and the reverse primer (5′-ATTAATCAACAGGTCCCCAAGG-3′) were used. PCR products were digested with BamHI and HindIII and ligated into the pEYFP-C1 vector (Clontech) to generate the corresponding pEYFP-HA-E2F2 deletion mutant. To create pEYFP-HA-E2F2-ΔDD (lacking the dimerization domain encompassing amino acids 197–358), pUC57-HA-E2F2-ΔDD was obtained (Genscript), and the insert was subcloned into the pEYFP-C1 vector using the BamHI/HindIII restriction sites. The orientation and integrity of all constructions were confirmed by DNA sequencing.Transient transfections were performed by the calcium phosphate method in HEK293T cells, or with Fugene (Promega) in U2OS cells.For knockdown of endogenous E2F2 and CREB, siRNA oligonucleotides were transfected at a final concentration of 10 nM using Lipofectamine RNAiMAX (Life Technologies) following the manufacturer’s recommendation.Renilla luciferase reporter vector (pRL-TK) and empty vector to 2 µg of DNA per transfection. Using the Dual-Luciferase Reporter Assay System (Promega), the reporter firefly luciferase activity was measured 48 h after transfection and was normalized to the transfection efficiency estimated by the activity of Renilla luciferase in each sample. Results were calculated as fold induction over the luciferase expression in cells that were transfected only with the appropriate reporter vector.For luciferase activity assays, cells at 40% confluency in six-well plates were transfected with 200 ng of the firefly luciferase reporter vector, 20 ng of the Supplementary Table S2.RNA extraction was performed with TRIzol Reagent (Life Technologies). RNA was purified with the RNeasy Mini Kit (Qiagen) and reverse transcribed into cDNA with the High-Capacity cDNA RT Kit (AB). Quantitative real-time PCR was performed as described previously . Sequenc4H, pH 7.2; 1 mM EDTA; 1 mM EGTA; 150 mM NaCl; 1% NP-40 and a cocktail of protease and phosphatase inhibitors (Roche). Protein concentrations in supernatants were determined using a commercially available kit (DC Protein Assay from Bio-Rad). Twenty to fifty micrograms of protein were loaded per lane, fractionated in 10–12% sodium dodecyl sulphate-polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad). Antibodies against the following proteins were used: CREB , p-CREB , E2F2  and β-Actin . Immunocomplexes were visualized with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Amersham), followed by chemiluminiscence detection  with a ChemiDoc camera (Bio-Rad).For western blot analyses, cells were lysed in buffer containing 10 mM NaPOSupplementary Table S3. Antibodies used were E2F2 , CREB  and SV40Tag .Chromatin immunoprecipitations and the quantification of immunoprecipitate-enriched DNA sequences by real-time PCR were performed as described previously . SequencIn ChIP-chip experiments, DNA obtained by standard ChIP experiments together with 20 ng of total input DNA was amplified by ligation mediated-polymerase chain reaction (LM-PCR), following manufacturer’s recommendation (Roche NimbleGen). Labeling, hybridization and detection of protein binding sites was performed by NimbleGen Systems Inc. Briefly, ChIP samples and total input DNA were labeled with Cy5 and Cy3, respectively, and co-hybridized to the Mouse ChIP-chip 385 K RefSeq Promoter Array, 05545021001, a microarray containing the promoters of 19 000 mouse genes (each promoter encompassing a DNA sequence from −2000 to +500 bp). Data were extracted and peaks were detected with the NimbleScan Program, according to standard operating procedures by NimbleGen Systems Inc. This program also calculates False Discovery Rate (FDR) values for each peak.http://ceas.cbi.pku.edu.cn (http://david.abcc.ncifcrf.gov/ (P < 0.05) enrichment over the background category lists  were considered.Identification of overrepresented motifs in peaks detected by ChIP-chip was performed using the cis-regulatory element annotation system (CEAS) server at u.edu.cn . The locu.edu.cn . Cutoffsu.edu.cn ’ backgrocrf.gov/ . The part-test. A P < 0.05 was considered statistically significant.Data are presented as mean ± SD. The significance of the difference between two groups was assessed using the Student two-tailed n = 4 mice per experiment; three independent experiments). After ChIP with an anti-E2F2 antibody, we prepared amplicons by LM-PCR, and probed promoter microarrays representing 19 000 mouse genes. As a negative control, we carried out parallel ChIP-chip experiments with an irrelevant anti-SV40TAg antibody (anti-T). Using the NimbleScan program, we selected those E2F2-binding sites identified in at least two of the three immunoprecipitations performed with anti-E2F2 antibody. A total of 839 genes (Supplementary Table S4) were found to be selectively enriched with anti-E2F2 antibodies (FDR < 0.05).We have previously reported that E2F2 is essential for the maintenance of lymphocyte quiescence . To furt+/+E2f2 and −E2f2/− mice. From the ChIP-chip–enriched gene set, we selected six genes known to harbor E2F sites in their promoter . The β-actin gene, whose promoter lacks active E2F sites .To validate the results of the ChIP-chip analysis, we performed conventional chromatin immunoprecipitation with E2F2 antibodies, followed by quantitative PCR (ChIP-Q-PCR) in lymphocytes obtained from promoter A. This sind E2F2 , and fiv2F sites , was useRbbp4, Parp1, Ard1, Rpa2), DNA damage and repair , DNA replication  and mitotic cell cycle  were the most overrepresented biological processes among the 839 genes enriched in the ChIP-chip assay, with enrichment ratios >2 (Supplementary Table S5). Other categories such as RNA processing  and RNA splicing  were also overrepresented, albeit with enrichment ratios <2 (Supplementary Table S5).Gene Ontology analysis revealed that DNA metabolism (e.g. P < 10−10), with enrichment values of 5, 6 and 8, respectively.We hypothesized that E2F binding motifs would be overrepresented in the promoters of the E2F2-bound gene set identified by ChIP-chip. To test this hypothesis, we used CEAS , a progrTo identify potential E2F motifs in the E2F2-bound promoters, we made use of the MotifLocator tool of the program TOUCAN . Using a−E2f2/− lymphocytes (Polr2a and Prkdc) is not deregulated on E2F2 gene inactivation, suggesting that their transcription is redundantly regulated by other E2F family members. However, we identified a set of 51 E2F2-bound genes whose expression is deregulated (38 upregulated and 13 downregulated) in quiescent −E2f2/− lymphocytes , but not by a promoter carrying three mutated E2F binding sites (3X-mtE2F-Luc) (Mcm5 and Chk1 promoter activity up to ∼40% (P < 0.05) compared with cells transfected with empty plasmid  A. By con plasmid B. To exaromoters C. In lucromoters D, and, apromoter E. These Chk1-Luc or Mcm5-Luc reporter constructs, and luciferase reporter assays were carried out. As shown in P < 0.05), further demonstrating the repressive effect of E2F2 on these target promoters.Next, E2F2 expression was knocked down using RNA interference in U2OS cells, which express detectable levels of endogenous E2F2. A substantial reduction of E2F2 expression by two independent siRNA molecules was demonstrated by western blot analysis F. As a cE2F, Mcm5 and Chk1 promoters. Three expression plasmids coding for E2F2 deletion mutants lacking the DNA binding domain (E2F2-ΔDBD), the marked box and dimerization domain (E2F2-ΔDD) or the transactivation and RB binding domain (E2F2-ΔTRD) were generated . These results suggest that all three domains of E2F2 are necessary for efficient transcriptional activation as well as for repression of E2F2 target promoters.To elucidate the structural domains of E2F2 involved in transcriptional repression, we examined the effect of several E2F2 deletion mutants on the activity of 3X-enerated A. None o. Included in this set of genes are Chk1, previously shown to be bound by CREB  exhibited both motifs separated by <500 bp , but not to the promoter of β-actin, used as negative promoter control , in both +/+E2f2 and −E2f2/− lymphocytes . Furthermore, western blot analyses demonstrated that neither the expression level nor the phosphorylation kinetics of CREB, which is known to regulate its activity , changedcontrols E, suggesMcm5 and Chk1, constitutively active CREB did significantly reduce basal Chk1 or Mcm5 promoter activity  in HEK293T cells . Additionally, overexpression of a constitutively active mutant form of CREB (CREB-VP16) in which the full-length CREB protein is fused to the transactivation domain of the viral transcriptional coactivator VP16, further increased the activity of the synthetic promoter harboring four CRE motifs A, as preactivity A. Furthe3T cells B and C. nockdown C, suggesMcm5 and Chk1 mRNA levels . As expected, expression of these target genes was also higher in cells depleted of E2F2 by RNA interference . ChIP experiments revealed that E2F2 recruitment to Mcm5 promoter was not significantly altered on disruption of CREB binding sites compared with recruitment to WT Mcm5 promoter . In luciferase assays, transfection of KCREB into HEK293T cells increased significantly the basal transcriptional activity driven by Mcm5 promoter, and abrogated E2F2-mediated transcriptional repression  in combination with expression profiling and classical biochemical approaches, we have uncovered a novel partner in the regulation of E2F2-mediated transcriptional repression. We show that E2F2 and CREB are recruited to a subset of E2F target genes to co-regulate their expression at the transcriptional level, providing new insights into E2F regulatory complexity.in vitro , supporting the notion of functional redundancy in the family. The E2F2-targeted set is enriched in genes related to DNA replication and repair, DNA damage response, mitosis and RNA processing, suggesting that E2F2 is primarily involved in cell growth control in T lymphocytes. Interestingly, genes belonging to these functional categories are also recruited by other E2F members, and may thus constitute a set of ‘classical’ E2F targets. Many other E2F2-bound genes are involved in a wide variety of functions, such as sensory perception, cognition or response to stress, among others, although no enriched functional categories were observed in our analysis. Further ChIP validation assays performed in a diversity of cell types should provide a more comprehensive list of genes that can recruit E2F2 to their promoters, and may help define additional E2F2-regulated cell functions.Our data indicate that E2F2 is predominantly recruited to the proximal promoter  of both positively and negatively regulated target genes, similarly to E2F1, E2F4 and E2F7 ,37,38. Fin vitro , but motMcm5 promoter that exhibit high similarity to the canonical site (threshold of 0.9) led to a significant, albeit not complete, reversal of E2F2-mediated repression of luciferase activity. Of note, a search for E2F sites at a lower threshold level (0.85) revealed the existence of two additional putative E2F sites in Mcm5 promoter. These less-similar E2F sites might account for the residual binding and repressive activity that we find with the Mcm5 promoter mutated in the two canonical E2F sites (The capacity of E2Fs, including E2F2, to activate transcription has been shown in numerous studies using synthetic promoters carrying E2F sites to drive the expression of the luciferase reporter gene ,32. Howe2F sites D and E. cdc6 and Polα, respectively (E2F3b and E2F4 are also known to repress transcription by RB-independent mechanisms in some contexts. E2F3b represses Arf expression in MEFs, and attenuates gene transcription in myoblasts and myotubes in the absence of RB ,41. Simiectively ,17. Howe−E2f2/− cells did not influence CREB binding to these promoters, and conversely, E2F2 recruitment was not dependent on CREB binding activity. Nevertheless, these results do not preclude the possibility of a weak or transient physical interaction between E2F2 and CREB, or the existence of other factors that may connect both proteins on binding to their specific DNA motifs, and thereby regulate transcription.Our work has revealed that as many as 10% of the genes that bind E2F2 also harbor putative cis-regulatory elements composed of E2F and CREB binding motifs. Furthermore, we show that both E2F2 and CREB are bound to these motifs in quiescent T lymphocytes, coinciding with transcriptional silencing of target genes involved in DNA replication and cell cycle regulation. During G1/S transition, E2F2 binding to target promoters remains unchanged or is slightly increased, whereas CREB binding is diminished. We still do not know how these variations of E2F2 and CREB binding activity are related to gene expression. Although we initially hypothesized that E2F2 could interact physically with CREB, co-immunoprecipitation experiments did not reveal such interaction. Furthermore, lack of binding of E2F2 to target promoters in Creb has not been able to confirm these results. Creb knockout mice display reduction in thymic cellularity only after concomitant inactivation of Atf1, and lymphocyte proliferation is unaltered in these mice (Mcm5 or Chk1. These results imply the existence of a set of genes required for cellular proliferation that are not under CREB control.The role of CREB on lymphocyte proliferation remains controversial. Early work in transgenic mice that express a dominant-negative form of CREB showed that thymocytes and T cells display a proliferative defect characterized by decreased IL-2 levels, G1 cell cycle arrest and apoptosis, suggesting that CREB is required for cell cycle progression of T lymphocytes . Howeverese mice . In agreIL-2, c-jun or loricrin expression through the arrangement of repressive heterodimers with other CREB family members such as CREM (Mcm5 and Chk1 promoters by E2F2 and CREB described here. It is likely that future work will uncover further examples of genes that are regulated coordinately by E2F members and CREB.Earlier work had already reported enrichment in CRE motifs among E2F target genes, but the functional relevance of this finding was unknown ,44,45. F as CREM . Our ChI as CREM . This fiTaken together, our data reveal a novel layer of complexity in the regulation of E2F-responsive genes. We have shown that E2F2 functions not only as a transcriptional activator but also as a repressor. We have identified a novel molecular mechanism by which two oncogenic transcription factors, E2F2 and CREB, cooperate to negatively regulate a subset of genes involved in the processes of DNA metabolism and cell cycle control. Identifying the components of this novel repressor complex will be the subject of future investigations.Supplementary Data are available at NAR Online.Spanish Ministry of Economy and Competitiveness ; Basque Government ; University of the Basque Country [UFI11/20 to A.M.Z.]. Funding for open access: University of the Basque Country [UFI11/20].Conflict of interest statement. None declared.