KATNA1 and KATNB1 genes, respectively. Katanin-p60 has the enzymatic activity for microtubule severing, whereas katanin-p80 consists of multiple domains with different functions such as targeting katanin-p60 to the centrosome, augmenting microtubule severing by katanin-p60, and even suppressing microtubule severing. Despite the various important functions of katanin-p80, its transcriptional regulation has not been studied yet. Elk1 transcription factor has been shown to interact with microtubules and regulate the transcription of another microtubule severing protein, spastin. In spite of katanin’s importance, and structural and functional similarities to spastin, there is no study on the transcriptional regulation of katanin yet. In this study, we aimed to characterize KATNB1 promoter and analyze the effects of Elk1 on katanin-p80 expression. We identified a 518- bp TATA-less promoter including a critical CpG island and GC boxes as an optimal promoter, and sequential deletion of CpG island and the GC elements gradually decreased the KATNB1 promoter activity. In addition, we showed Elk1 binding on the KATNB1 promoter by EMSA. We found that Elk1 activated KATNB1 promoter, and increased both mRNA and protein levels of katanin-p80 in SH-SY5Y cells. On the other hand, KCl treatment increasing SUMOylation decreased KATNB1 promoter activity. Since microtubule severing is an important cellular mechanism of which malfunctions result in serious diseases such as spastic paraplegia, Alzheimer’s disease and cell cycle related disorders, identification of KATNB1 transcriptional regulation is crucial in understanding the coordination of microtubule severing activity by different proteins in the cells.Katanin is an ATPase family member protein that participates in microtubule severing. It has heterodimeric structure consisting of 60 kDa (katanin-p60) and 80 kDa (katanin-p80) subunits encoded by  KATNA1 (NM_007044) and KATNB1 (NM_005886) genes, respectively. Katanin-p60 contains a C-terminal AAA (ATPases Associated with various cellular Activities) domain which has an enzymatic activity that breaks microtubules while katanin-p80 has multiple domains  was identified as having the highest promoter activity and 160- bp region (between -1655 and -1496) was found to act as a minimal promoter for the KATNB1 gene promoter is located on chromosome 16 between CCDC135 and KATNB1 genes (ref. seq. NM_005886) according to NCBI Map Viewer database (http://www.ncbi.nlm.nih.gov/mapview/). The 1000- bp region of KANTB1 gene promoter (F1) (http://genome.ucsc.edu/cgi-bin/hgGateway). No TATA box or CAAT box was identified, but 4GC boxes and 1 CpG island were detected by bioinformatics analyses of the promoter  ((Putative ter (F1)  and 3 thgplot/)  led to a significant decrease in the promoter activity. Subsequent deletions of the promoter region from F2 to F3, F4, F5 and F6 regions caused continuous significant decreases in the promoter activity. Gradual removal of the CpG island with the deletion constructs (from F3 to F5: -1774 to -1655) caused a gradual decrease in the promoter activity. The subsequent deletion of the end of the CpG island (from F5 to F6: -1655 to -1518) caused a substantial decrease in the promoter activity. Therefore, the ∼ 160- bp region (located between -1655 and -1496) containing the terminal part of the CpG island (F5 construct) showed the minimal promoter activity. Deletion of the entire CpG island (F6 construct containing the region between -1518 and-1496) completely abolished the promoter activity .KATNB1 gene expression.These results indicate that the 2013- bp upstream of the first ATG acts as an optimal promoter and 1655- bp upstream of the first ATG acts as a minimal promoter for KATNB1 gene optimal promoter sequence (F2) (The newly identified nce (F2)  data hasKATNB1 promoter by using PROMO bioinformatics tool Homo sapiens. Bioinformatics analysis predicted only one Elk1 binding site positioned between -1652 and -1644 in the KATNB1 promoter  transcription factor binding sites (AC: T00250) on the promoter  and 3A.KATNB1 promoter was confirmed by EMSA  and mutated (Mut) oligonucleotides that contain the wild type consensus and mutated Elk1 DNA-binding sequences, respectively . The ElkIn the absence of cell extracts, no binding was observed  containing only the DNA binding domain of WT Elk1 phoresed . The comphoresed , lane 1 phoresed , lane 3.KATNB1 promoter was Elk1-db. Using anti-His tag antibody, the super-shifted complex was shown to contain the 6XHis tagged Elk1 recombinant protein (Elk1-db)  , lane 4.Since we observed an additional binding  or F1 construct and pCMV6-Elk1 vector (F1+Elk1) .SH-SY5Y cells were also co-transfected with either F2 construct  and pCMV-empty vector (F2) or F2 construct  and pCMV6-Elk1 vector (F2+Elk1) , II.Luciferase data showed that Elk1 increased promoter activity of both F1 and F2 constructs compared to control conditions , III, itAfter confirming Elk1 could actually activate katanin-p80 promoter, in order to further quantify whether Elk1’s binding to the katanin-p80 promoter would functionally reflect to any change at the mRNA level of katanin-p80, we performed qRT-PCR. In order to directly correlate if any possible change in katanin-p80 mRNA level could actually be based on Elk1 level, we first showed the overexpression of Elk1 in SH-SY5Y cells . For thiKATNB1 was performed. Relative quantification analysis of qRT-PCR data revealed an increase  in mRNA levels of katanin-p80 in Elk1 overexpressing SH-SY5Y cells indicating an up-regulation of the target gene expression  promoter activity compared to KCl untreated-Elk1 transfected cells (F2+Elk1) due to increased SUMO conjugation . CompareIn addition to luminometrical measurement analysis, Western blotting was performed  to evaluKATNB1 promoter and decreased katanin-p80 expression as confirmed with integrated pixel analysis  of F2 construct might contain possible inhibitor binding sites. On the other hand, cells that were induced with minimal promoter (F5) construct which included the terminal part of the CpG island and 2 GC boxes had lower promoter activity. Whereas, F6 construct which included a single GC box and no part of the CpG island showed almost no promoter activity, and it was about the same as basal level promoter activity. Therefore, the decrease in the number of GC boxes with the shortening constructs was associated with the decrease in the promoter activity  might have had the promoter activity as F2, as it would also include the entire CpG island and all 4 GC boxes. Besides GC boxes, the CpG island which is an important regulatory region was shown to be critical for the KATNB1 promoter, as well. Elk1 is one of the transcription factors that could bind to corresponding sequence within the CpG island.For the promoter characterization, the deletion constructs of activity . GC boxeKATNB1 promoter was confirmed by EMSA as seen that Elk1-DNA complex migrated more slowly than the free DNA and mutation of the binding sites prevented the complex formation. EMSA results also indicated that the additional binding observed  on April 17, 2013. This deposition indicates the presence of a splice variant form of Elk1 . The molecular weight of the complex is very similar to the complex including recombinant Elk1-db (96 amino acids) – WT oligonucleotide complex. The slight shift between the N terminal DNA binding domain including splice variant (95 amino acids) and the recombinant Elk1-db (96 amino acids) is also as a result of 6XHis tag attached to the recombinant Elk1-db. These data confirm that both full length Elk1 and Elk1-db splice variant could actually bind to the predicted binding region on the KATNB1 promoter, once again proving the specificity of our recombinant Elk1-db to the KATNB1 promoter.Elk1 transcription factor binding to observed  and Elk1observed . The cloobserved , lane 3 KATNB1 promoter (unpublished data). YY1 transcription factor is known to negatively regulate the activities of many genes  and the majority of YY1 binding sites in KATNB1 promoter are located in F1’s -2495 to -2013 region that is not included within the F2 construct. This region (482 bp) of F1 is highly conserved (∼%100) within the primates, and it is not conserved in other organisms, whereas F2 region is more conserved among different types of organisms. The F1 promoter region of human includes binding sites for transcription factors such as FOXP3, NF-1, RXR-alpha and RAR-beta that are mainly involved in differentiation, and these binding sites are absent in F2 region of the KATNB1 promoter.Binding of Elk1 to both F1 and F2 promoter constructs increased the promoter activity. Yet, the increase in F1 promoter region (∼1.5 fold) was not as high as the increase in F2 promoter region (∼2.5 fold). It is possible that the region from -2495 to -2013 (from F1 to F2) contains negative regulatory elements. Although both F1 and F2 promoter constructs contain the Elk1 binding site, some of the transcription factors binding sites that are present in F1 are not included in F2 promoter region. Based on our research, we know that YY1 transcription factor negatively regulates the Expression analysis revealed that Elk1 acted as an activator on katanin-p80 promoter, since we observed elevated mRNA levels in Elk1 overexpressing cells compared to control cells. Elk1 increased the activity of the optimal promoter about 2.5 fold in luciferase experiments 2+Elk1 whKATNB1 promoter, our group has previously shown that Elk1 has a repressive role on SPG4 gene promoter KATNB1 promoter, but not on SPG4 promoter. SRE consensus sequence CC(A/T)6GG resides at position from -1636 to -1627, at the juxtaposition of Elk1 binding site  construct would not include histones that would be the targets of HDAC. As a result, because there would not be any interaction with HDAC, the decrease is not reflected at that high level in the promoter activity measured by luminometer. Whereas in Western and immunocytochemistry experiments, overexpressed and SUMOylated Elk1 could interact with HDAC on the promoter present intrinsically in the cells, and the outcome is significantly reflected as a decrease in the endogenous katanin-p80 protein level.Once Elk1 is SUMOylated, it cannot be phosphorylated and it dramatically decreases the level of target gene expression. SUMOylation brings Histone deacetylases (HDAC) to the promoter region In a eukaryotic gene structure, in addition to the promoter region which is typically adjacent to the upstream of a gene, there are many regulatory elements that can be found in the downstream of promoters. For instance, 5′ untranslated region (UTR) and 5′-UTR introns (5′-UI) are the regions that can influence the level of gene expression. Approximately 35% of human genes  contain introns within the 5′-UTR KATNB1 promoter. We also showed that Elk1 has an activator role on the regulation of KATNB1 promoter. In addition, CpG island which includes the Elk1 binding site is important for the promoter activity and GC boxes play activatory role on KATNB1 gene expression.In conclusion, we identified the critical and optimal regions on KATNB1 promoter, it is likely that in addition to the transcriptional regulation, CpG island methylation, as in epigenetic level regulation, could also be important for katanin-p80 expression. The epigenetic level regulation of katanin promoter awaits for further investigation in order to better understand the microtubule severing activity and its effects within cells.This study would have a great impact to understand different gene expression patterns of katanin-p80, resulting different katanin-p60/katanin-p80 ratios both in development and different tissues, causing different degree of severing by katanin-p60. Therefore, it will also give insight into understanding of functional differences in different microtubule severing proteins, katanin and spastin. Based on the critical regions present on Dulbecco’s modified Eagle’s medium  and fetal bovine serum were obtained from Invitrogen Corp. . L-Glutamine and Penicillin/Streptomycin were provided from Biochrom . Elk1 and mutated Elk1 oligonucleotides were synthesized by Integrated DNA Technologies, Inc. . LightShift Chemiluminescent EMSA Kit, Biotin 3′ End DNA Labeling Kit and CL-XPosure Film were purchased from Thermo Scientific Pierce, Inc. . TransFast™ Transfection Reagent and Dual Luciferase Assay System were purchased from Promega Corp. .KATNB1 start codon into pGL3 vector (Promega) (Taq DNA polymerase enzyme (Fermentas) from genomic DNA and cloned into Hind III/KpnI sites of pGL3 vector. Other inserts named F2, F3, F4 and F5 were obtained from F1 by using appropriate forward primers and a common reverse primer (Katnb1 R). The smallest insert F6 was obtained by hybridization of its own forward and reverse primers in a thermocycler (All deletion constructs were obtained by cloning different portions of the human genomic DNA between 2495-1496 bp upstream (excluding 5′UTR region) of the Promega)  and 3 10mocycler .The pCMV6-Elk1 (encoding amino acids 1–428 of Elk1) was used in forced experiments, transfections for qRT-PCR and Western blotting. The pRL-TK  was used as an internal control in luciferase assays.KATNB1 1000-bp promoter was determined by using UCSC Genome Browser ‘Get Genomic Sequence Near Gene’ tool (http://genome.ucsc.edu/). The promoter was analyzed for the presence of TATA-box, CAAT-box and GC-box via EBI (The European Bioinformatics Institute) ClustalW sequence analysis tool . The promoter was also analyzed for the presence of CpG island via EBI, EMBOSS CpGPlot/Report tool (http://www.ebi.ac.uk/Tools/emboss/cpgplot/). To determine deletion constructs and predict the Elk1 binding site, possible protein-DNA interactions within the promoter were analyzed using PROMO bioinformatics tool from ALGGEN server by selecting both factor’s species and site species as Homo sapiens with the maximum matrix dissimilarity rate 0–3% DNA sequencing was performed by using 3100 Genetic Analyzer (Applied Biosystems) and BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) according to the manufacturer’s protocol.2. Cells were plated onto poly-L-lysine  coated glass coverslips at a density of 3×104 cells/well for immunocytochemistry experiments; 5×104 cells/well in 24-well culture dish for luciferase experiments; 1×106 cells/well in 6-well plate for qRT-PCR and Western blotting experiments.SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266™) were used in this study as they are the prime model system for the study of neuronal functions. SH-SY5Y human neuroblastoma cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and Penicillin/Streptomycin at a final concentration of 100 U/ml and 100 µg/ml, respectively. Cells were cultivated at 37°C in an atmosphere of 5% CORenilla for luciferase assay . At 48 h post-transfection, growth medium was removed from cultured cells and cell lysates were obtained by adding 60 µl 1X passive lysis buffer and scraping. Cell lysates were mixed sequentially with firefly and Renilla specific substrates in luminometer plates according to the manufacturer’s instructions. Luminometer was programmed to perform a 2 sec pre-measurement delay while shaking the plate, followed by a 10 sec measurement period for each reporter assay. The measured activity of the firefly luciferase was normalized to that of Renilla luciferase. All experiments were performed in triplicates and were repeated 6 times using different DNA preparations.6 cells, were prepared by using Mammalian Cell Extraction Kit  according to manufacturer’s instructions.Whole cell extracts of Elk1 overexpressing SH-SY5Y cells, which were transfected with 5 µg pCMV6-Elk1/10Production of recombinant Elk1 protein which includes ETS DNA binding domain (amino acids 1–96) was performed as previously described Sequences of wild type (WT) and mutated (Mut) oligonucleotides are shown in 6 cells were transfected with 2 µg of DNA (pCMV6-Elk1) by using Amaxa Nucleofector  and Nucleofector® Kit V reagent according to the manufacturer’s protocols. After 24 h of nucleofection, the cells were prepared for isolation of RNA. Total RNA was extracted from Elk1 transfected SH-SY5Y cells by using High Pure RNA Isolation Kit (Roche) in accordance with the manufacturer’s instructions. 1 µg of total RNA was reverse transcribed by using Random Hexamer primers and Reverse Transcriptase (Roche) according to the manufacturer’s instructions.1×10Specific katanin-p80 probe and primers were purchased from Universal Probe Library (Roche Applied Science). Beta-actin  gene was used as reference for relative expression analysis. Quantitative Real Time – PCR (qRT-PCR) reactions were performed with Light Cycler® 480 Probes Master qRT-PCR Kit using a Roche Light Cycler 480 according to the following program: initial denaturation at 95°C for 10 min for 1 cycle, denaturation at 95°C for 10 sec, amplification at 60°C for 30 sec, extension at 72°C for 1 sec for 45 cycles. ΔΔCt method was used to analyze qRT-PCR data. According to Schmittgen et al. To increase SUMOylation 4 cells/well on poly-L-lysine coated coverslips. Cells were then transfected with Elk1 expression vectors using TransFast™ Transfection Reagent according to the manufacturer’s instructions. Naive cells were used as negative control for examining endogenous katanin-p80 level. After 24 h of transfection, one group of Elk1 transfected cells was treated with KCl for 1 h to increase SUMO conjugation SH-SY5Y cells were plated at a density of 3×106 cells were transfected with 2 µg of DNA (pCMV6-Elk1) by nucleofection. After 24 h, total protein was extracted from cells. Equal amounts (2 µg protein/lane) of proteins obtained from whole cell extracts of untransfected and Elk1 transfected samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Membrane was blocked in 5% skim milk powder/TBST (TBS containing 0.1% Tween 20) for 2 h at room temperature, and then stained with the following primary antibodies at indicated dilutions overnight at 4°C; rabbit polyclonal anti-Elk1 antibody , rabbit polyclonal anti-katanin-p80 antibody  and rabbit monoclonal anti-β-actin  in 5% skim milk powder/TBST. Membrane was then incubated with HRP conjugated goat anti-rabbit IgG secondary antibody  for 2 h at room temperature. Bands were visualized using Super Signal West Femto Chemiluminescent Substrate (Thermo Scientific) and Kodak Medical X-ray Processor.1×10Total proteins obtained from whole cell extracts were used in immunoprecipitation experiments. Firstly, rabbit polyclonal anti-Elk1 antibody  was diluted in 1∶50 µg/ µl in PBS-T buffer and incubated with 50 µl Dynabeads Protein G (Invitrogen) for 10 min according to manufacturer’s protocol. Then, cross-linked beads were incubated with 1 mg total protein for 15 min at room temperature. Unbound proteins were washed 3 times with 200 µl PBS and bound proteins were eluted using 20 µl 50 mM Glycine buffer (pH 2.8). Eluted proteins were finally incubated for 3 min at 70°C in order to dissociate the complex. SUMOylation level of immunoprecipitated Elk1 was detected by Western blotting and rabbit monoclonal anti-SUMO-1 antibody  and rabbit polyclonal anti-SUMO-2/3 antibody  were used as primary antibody.Photoshop CS5 Software was used to analyze the relative intensity of the protein bands in Western blot images. The intensity value for each protein band was calculated by measuring the selected band area and pixel values. Obtained values were normalized against values of internal control. Immunocytochemistry analysis was also performed with the same software. Total fluorescence per cell and corrected integrated density for each cell were obtained by subtracting background from raw images which were taken under same microscopy settings.All the experiments were statistically analyzed by two-tailed unpaired student’s t test using GraphPad Instat 3 software. Error bars in the graphs were generated using ± SD values. P value under 0.05 was considered significant for all statistical analysis.


Takifugu rubripes), and have cloned and characterized a functional promoter of this gene for the first time in a non-mammalian vertebrate. In silico analysis of the Fugu GLUT4 promoter identified potential binding sites for transcription factors such as SP1, C/EBP, MEF2, KLF, SREBP-1c and GC-boxes, as well as a CpG island, but failed to identify a TATA box. In vitro analysis revealed three transcription start sites, with the main residing 307 bp upstream of the ATG codon. Deletion analysis determined that the core promoter was located between nucleotides -132/+94. By transfecting a variety of 5´deletion constructs into L6 muscle cells we have determined that Fugu GLUT4 promoter transcription is regulated by insulin, PG-J2, a PPARγ agonist, and electrical pulse stimulation. Furthermore, our results suggest the implication of motifs such as PPARγ/RXR and HIF-1α in the regulation of Fugu GLUT4 promoter activity by PPARγ and contractile activity, respectively. These data suggest that the characteristics and regulation of the GLUT4 promoter have been remarkably conserved during the evolution from fish to mammals, further evidencing the important role of GLUT4 in metabolic regulation in vertebrates. The glucose transporter 4 (GLUT4) plays a key role in glucose uptake in insulin target tissues. This transporter has been extensively studied in many species in terms of its function, expression and cellular traffic and complex mechanisms are involved in its regulation at many different levels. However, studies investigating the transcription of the GLUT4 gene and its regulation are scarce. In this study, we have identified the GLUT4 gene in a teleost fish, the Fugu ( In mammals, the glucose transporter 4 (GLUT4) is the main facilitative glucose carrier responsible for the insulin-regulated glucose uptake in skeletal muscle and adipose tissue . GLUT4 eTakifugu rubripes), a teleost fish. Fugu was chosen as a model organism for this study because it was the second vertebrate genome to be sequenced, after the human genome, and its compact genome has proven to be a useful reference for the identification of genes and regulatory elements in other vertebrate genomes.The regulation of the expression of the GLUT4 gene is governed by complex mechanisms as it is subjected to both tissue-specific and hormonal metabolic regulation ,6,7 due in silico analysis. Moreover, we show that the regulatory region of the Fugu GLUT4 gene presents most of the binding motifs described as important for the transcriptional regulation of GLUT4 in mammals. In addition, we provide evidence that the transcription of the Fugu GLUT4 gene in skeletal muscle cells is inhibited by insulin and stimulated in response to PG-J2 (a PPARγ agonist) and to electrical stimulation. Transient transfection of various 5´deletion constructs showed that the response to PG-J2 depends on the number of PPARγ binding motifs and suggests the implication of HIF-1α in the regulation of the transcriptional response of the Fugu GLUT4 gene to contractile activity. In this study, we report on the identification of the GLUT4 gene in Fugu and on the characterization of a functional promoter region of the Fugu GLUT4 gene. We have identified three transcription start sites (TSSs) and demonstrated the homology of this regulatory region with that of the GLUT4 gene in other teleost species by 12,14-prostaglandin J2 (PG-J2) were purchased from Sigma . α-MEM, fetal bovine serum (FBS) and other tissue culture reagents were purchased from Invitrogen .Human recombinant insulin and 15-deoxy-Δslc2a4) gene was identified by searching the Fugu genomic sequence database in Ensembl . In order to clone the Fugu GLUT4 promoter region, we designed gene specific primers incorporating the KpnI and XhoI restriction sites  in Ensembl and their conservation profiles were analyzed with zPicture software (zpicture.dcode.org [www.genomatix.com [The genomic sequence corresponding to the Fugu GLUT4 (on sites  to amplicode.org ). Analysatix.com ) and Aliatix.com ). CpG isatix.com ). The gePhotinus pyralis) luciferase pGL3 expression vector system (Promega) was used as a reporter system to evaluate the transcriptional activity of the 5´-flanking region of the Fugu GLUT4 gene. The 1.3 kb Fugu GLUT4 promoter fragment was excised from the pGEM-T Easy vector by digestion with KpnI and XhoI  and ligated into the pGL3-basic vector digested with the same restriction enzymes to allow transcription of the firefly luciferase gene under the control of the Fugu GLUT4 promoter (pGL3-FuguGLUT4P). Following the same strategy, a series of unidirectional 5′ deletion constructs were generated by PCR using specific primers with the sequence of the KpnI and XhoI restriction sites incorporated (Renilla reniformis) luciferase under the control of the HSV-thymidine kinase promoter, was also used as an internal control for transfection efficiency (Promega). A reporter pGL3-Control vector (firefly luciferase gene under the control of the SV40 promoter) was used as a positive control of luciferase luminescence (Promega). A rat GLUT4 promoter construct (pGL3-ratGLUT4) containing a 2.2 kb DNA fragment upstream of the TSS of the rat GLUT4 gene, kindly donated by Dr. Rafael Salto  [The firefly  , was use2 at 37 °C. At confluence, C2C12 myoblasts were induced to differentiate into myotubes by culturing the cells for at least 5 days in α-MEM with the percentage of FBS reduced to 2%.The rodent-derived skeletal muscle cells lines L6  and C2C5 L6 myoblasts per well were plated in 24-well dishes and 2.5 x 105 C2C12 myoblasts were plated per well in 12-well dishes. Cells were cultured for 24 hours until 80-90% confluence and were transfected with the plasmids described above (1μg/well) using the Lipofectamine2000TM reagent (Invitrogen), following the manufacturer’s indications. Briefly, cells were plated at 90 % confluence and cultured overnight with α-MEM containing 10 % FBS without antibiotics at 37 °C. Lipofectamine2000 reagent was mixed with α-MEM without FBS/antibiotics (4.5 μl/150 μl medium), incubated for 5 min at room temperature, combined with construct DNA (1.5 μg/150 μl for pGL3 and 0.5 μg/150 μl for pRL-TK) and further incubated for 20-30 min at room temperature. Cells were washed once with PBS and a total of 100 μl of DNA-Lipofectamine2000 reagent mixture was added to each well (in triplicate) and incubated for 2-4 h under 5 % CO2 at 37 °C. After this period, 250 μl of α-MEM-20 % FBS without antibiotics were added to each well. Cells were incubated for 24 h and observed for evidence of cytotoxicity under the microscope. After transfection, L6 myoblasts were cultured in the absence or presence of human recombinant insulin  or PG-J2 (10 μM) for 18 h. Time-course experiments were performed with L6 myoblasts transfected with the pGL3-FuguGLUT4P construct in the absence or presence of insulin (100 nM) for 0.5, 1, 2, 4, 8 and 18 h. Transiently transfected C2C12 myotubes plated in 12 well dishes were subjected to electrical pulse stimulation using an Electrical Stimulator of Cultured Cell System (ESCC), as described by Marotta and co-workers [2, at 40 V with a pulse duration of 30 ms and a frequency of 3 Hz. Control C2C12 myotubes were incubated in the absence of electrical pulse stimulation. After stimulation, L6 myoblasts and C2C12 myotubes were lysed and luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s instructions. Luciferase activity measurements were performed with an Infinite® M200 reader . The level of firefly luciferase activity was normalized to that of sea pansy luciferase activity for each transfection.Approximately 1 x 10®Taq DNA Polymerase (Invitrogen). PCR conditions were as follows: 94°C for 3 min followed by 35 cycles of 94°C for 45 s, 60°C for 30 s and 72°C for 1 min, and a final step of 72°C for 10 min. Fragments were ligated into pCR®4-TOPO (Invitrogen) and transformed into One Shot® TOP10 competent cells (Invitrogen).The TSS of Fugu GLUT4 gene was determined using the GeneRacer Kit (Invitrogen) following the manufacturer’s indications. This technique is based on RNA ligase-mediated rapid amplification of 5´and 3´cDNA ends (RLM-RACE) and oligo-capping rapid amplification cDNA ends (RACE) methods. Nested PCR was performed using gene specific primers (GSPs)  and totap < 0.05.Statistical analyses were performed using SPSS11 . Statistical differences were analyzed by Kruskal–Wallis and Mann–Whitney non-parametric tests and were considered to be significant at A single GLUT4 gene insilico in the Fugu genome sequence database. The Fugu GLUT4 gene maps to Scaffold_63 and its structure consists of 11 exons and 10 introns, spanning approximately 4.8 kb . The translation initiation codon ATG and the termination codon TGA were located in exons 1 and 11, respectively. We determined the synteny between the human and the Fugu GLUT4 genes by identifying genes flanking the GLUT4 loci in the human and Fugu genomes  with homologs on human chromosome 17. The similarities between the Fugu and human GLUT4 loci with respect to the nature of the genes flanking the GLUT4 gene evidence a high degree of conservation in the organization of this important locus during evolution from fish to mammals. 4.8 kb 1A, B. Agenomes 1C. AnalWe determined experimentally the position of the TSS of the Fugu GLUT4 gene by 5´RACE. This approach yielded three PCR product sizes of 452, 307 and 261 bp . AnalysiIn silico analysis of the cloned 1.3 kb 5´-flanking region of the Fugu GLUT4 gene revealed the presence of multiple putative binding sites for TFs such as MEF2, SREBP, KLF, SP1/GC-box, NF-Y, E-box, PPAR-γ, PPAR-RXR and HIF-1 . Using sequence-specific primers , a DNA fnd HIF-1  and 2. Hnd HIF-1 .Tetraodon and Fugu showed two major regions, between -786/-334 and -234/+182 nucleotides, that were highly conserved, with a similarity higher than 70%,  into L6 myoblasts. This cell line was chosen because of its extensive use in the study of GLUT4 biology in skeletal muscle and because of the lack of a suitable fish muscle cell line. In parallel, a similar luciferase construct containing 2.2 kb of the rat GLUT4 promoter was transiently transfected as a control for the activity of the cloned Fugu GLUT4 promoter. Our results indicated that the Fugu GLUT4 promoter was functional and that its basal activity was significantly higher than that of the rat GLUT4 promoter .in silico predictions that identified PPAR-γ, HIF1, SP1/GC-box and NF1 TF binding sites as well as a CpG island within this region where the TSS+1 is located. Therefore, these results strongly suggest that the core sequence of the basal promoter is localized in the region -132 to +94.In order to further characterize the promoter regions of the Fugu GLUT4 promoter that are responsible for its basal activity, we generated a set of six luciferase reporter gene constructs each containing serial deletions of the FuguGLUT4P-1132 construct  and we tIn order to study whether insulin exerts a regulatory role on the expression of the Fugu GLUT4 gene at the level of transcription, we transiently transfected L6 myoblasts with the Fugu GLUT4 promoter or with the rat GLUT4 promoter, used as control, and stimulated transfected cells with human recombinant insulin (100 nM) for 18 h. Insulin treatment significantly decreased the activity of the Fugu and rat GLUT4 promoters by approximately 50% , confirmNext, we set out to identify the regions potentially involved in the regulation of the activity of the Fugu GLUT4 promoter by insulin. For this purpose, we transiently transfected rat L6 myoblasts with the various deletion constructs and incubated the cells for 18 h in the absence or presence of insulin (100 nM). L6 myoblasts transiently transfected with any of the six deletion constructs showed a significant reduction in their transcriptional activity in response to insulin . The twoGiven the presence of putative binding sites for PPARs (PPARγ and PPAR-RXR) in the Fugu GLUT4 promoter sequence, we next asked whether PPARγ could be involved in the regulation of the Fugu GLUT4 gene at the transcriptional level by testing the effects of PG-J2, a natural PPARγ ligand. For this purpose, we stimulated L6 myoblasts that were transiently transfected with the FuguGLUT4P-1132 construct in the absence or presence of PG-J2 (10 μM) for 18 h. Our results show that treatment with PG-J2 significantly increased the transcriptional activity of the Fugu GLUT4 promoter . To idenIn vitro differentiated mouse C2C12 myotubes, contrary to L6 myotubes, develop a contractile apparatus of sarcomere units and are able to contract in response to electrical pulse [We next asked whether contractile activity could regulate the transcriptional activity of the Fugu GLUT4 gene in mouse C2C12 myotubes. al pulse ,29 and cal pulse . TherefoThe GLUT4 gene is widely considered essential for the control of glucose homeostasis in mammals. Although the GLUT4 gene is present in the sequenced genomes of most non-mammalian vertebrate species, no information is available on its characterization and its transcriptional regulation outside of mammals. Given our previous studies on the regulation of the expression of the GLUT4 gene at the mRNA and protein levels in teleost fish ,15,31-33From a functional point of view, we have demonstrated that the Fugu GLUT4 promoter is active when expressed in the rat muscle cell line L6. Furthermore, by generating a series of 5´-deletion constructs of the Fugu GLUT4 upstream regulatory region, we have determined that the +1 TSS as well as the presumptive core promoter are indeed located between nucleotides -132 and +93, matching completely the position of the predicted CpG island. The -901 and -826 constructs were the only deletions showing a significant increase in basal activity over that of the -1132 promoter construct. This finding suggests the existence of an enhancer region between nucleotides -901 and -669, as also supported by the presence of a tandem of binding sites for Klf15 between nucleotides -665 and -677. In mammals, Klf15 strongly induces GLUT4 transcription by synergizing with MEF2 . Insulin is an effective inducer of GLUT4 mRNA and protein expression as well as of GLUT4 translocation in fish ,15,31-33in vitro effects of PPARγ activation. PPARs are ligand–activated transcription factors from the nuclear receptor family. Three PPAR isoforms  have been described, and they differ in their tissue distribution and ligand specificity [In order to study other mechanisms potentially involved in the regulation of the Fugu GLUT4 promoter activity, we first investigated the cificity . In partcificity  and is ecificity . Furthercificity , while tcificity . It is scificity . PG-J2 hcificity ,52. Our cificity . Althougcificity -56, inclcificity . Furthercificity ,58,59 orcificity  to PPARγin vitro. Our results clearly show that electrical stimulation of skeletal muscle cells results in an increase in Fugu GLUT4 gene transcription. Interestingly, all the deletion constructs except the one lacking the HIF-1 motif (+94) showed a significant increase in the transcriptional activity in response to electrical pulse stimulation. This result is in agreement with previous data indicating that HIF-1 binding factor participates in contraction-induced GLUT4 transcriptional activity [Muscle contraction and chronic contractile activity of skeletal muscle cells have also been reported to stimulate GLUT4 gene transcription -64. In tactivity ,63. Furtactivity  and provactivity . Our groactivity  and hypoactivity  could beIn summary, in the present study we have cloned and characterized the first functional GLUT4 gene promoter in a non-mammalian species. The Fugu GLUT4 gene promoter presents most of the binding sites described as important for the transcriptional regulation of the mammalian GLUT4 gene. Moreover, in agreement with data available in mammals, the Fugu GLUT4 gene promoter is regulated by insulin, PPARγ and contractile activity, suggesting evolutionarily conserved mechanisms leading to the regulation of the expression of the main insulin-regulated glucose transporter, GLUT4. Given that insulin also stimulates the translocation of the GLUT4 protein to the plasma membrane in fish skeletal muscle cells ,68, as iFigure S1Genomic structure and chromosomal localization of the Fugu GLUT4 (slc2a4) gene. (A) Organization of the Fugu GLUT4 gene. Exons are numbered and indicated by boxes and introns are indicated by lines. (B) Position of the genomic region containing the human GLUT4 gene (indicated by a red box) in human chromosome 17 (HsCh.17) (top) and localization of the Fugu GLUT4 gene in Scaffold_63, as indicated by a red box (bottom). (C) Synteny of the regions containing the GLUT4 gene in HsCh.17 and Fugu Scaffold_63. Surrounding slc2a4, the genes ybx2 (Y box binding protein 2), eif5a (eukaryotic translation initiation factor 5A), gps2 (G protein pathway suppressor 2), neurl4 , acap1 , ctdnep1 (CTD nuclear envelope phosphatase 1) and gabarap (GABA(A) receptor-associated protein) appear in both species but arranged in slightly different order.(TIF)Click here for additional data file.Figure S2Prediction of a CpG island within the 5´ cloned region of the Fugu GLUT4gene. Black arrows indicate the positions delimiting the CpG island relative to the +1 TSS. Blue area indicates the position of the CpG island.(TIF)Click here for additional data file.


H3f3b.The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b knockout (KO) mice exhibit a semilethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in mouse embryonic fibroblasts (MEFs) with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile.Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development. The nucleosome is a dynamic structure whose constituent histones undergo a vast number of posttranslational modifications (PTMs), which influence nucleosomal function. In addition, the histones can be entirely replaced by deposition of variant histones carrying their own unique complements of PTMs. For example, canonical histone H3 proteins, H3.1 and H3.2, can be replaced by the histone variant, H3.3. Deposition of H3.3 is thought to produce a nucleosomal state that is less structured and associated with a more transcriptionally active state . One notAnother H3 variant, CENP-A is essential for centromere function where a role for histone H3.3 is also implicated . H3.3 maThe unique functions of the histone H3.3 protein are conferred by the few amino acids by which it varies from canonical histone H3 proteins, and by the distinct expression of H3.3 throughout the cell cycle. H3.3 protein differs by only four and five unique residues from histones H3.2 and H3.1, respectively. Residues S31, A87, I89, and G90 are completely unique to H3.3. Histone H3.3 phosphorylated at Serine31 (H3.3S31P) is enriched at domains bordering centromeres during metaphase and may function in mitosis , while aH3f3a and H3f3b (H3.3A and H3.3B in Drosophila), encode the H3.3 protein, while more than a dozen histone H3.1 and H3.2 genes encode the canonical histone H3.1 and H3.2 proteins. The H3f3a and H3f3b genes also are uniquely located outside canonical histone gene clusters, and their gene structure is distinct from that of canonical histone genes in that they have introns and they also produce mRNAs with polyadenylated tails. When compared to each other, H3f3a and H3f3b also have distinct gene expression patterns, untranslated regions and promoters  and [NCBI-GEO:GSE35321], respectively.in-situ hybridization; FL: Floxed; H3K4me3: Histone H3 lysine 4 tri-methylation; H3K9ac: Histone H3 lysine 9 acetylation; H3.3S31P: Histone H3.3 phosphorylated at serine 31; ICC-IC: Immunocytochemistry-Immunofluorescence; KO: Knockout; MCM: Minichromosome maintenance complex; MEF: Mouse embryonic fibroblasts; MFI: Mean fluorescence intensity; MSC: Mesenchymal stem cell; PML: Promyelocytic leukemia; PTM: Posttranslational modification; RD: Replication dependent; RI: Replication independent; RT: Room temperature; TSS: Transcription start site; WT: Wildtype.ChIP-Seq: Chromatin immunoprecipitation-sequencing; ES: Embryonic stem cells; FISH: Fluorescent The authors declare that they have no competing interests.KB conducted all mouse breeding and genotyping to produce the H3f3b knockout strain. KB also did the majority of experiments conducted that are reported in this manuscript. BY conducted most of the immunostaining studies of MEFs, did the kinetochore staining and quantitative analysis, and aided in data interpretation. BB conducted data analysis from the ChIP-Seq studies and conducted other experiments including cell cycle and cell shape analysis, JR aided in expression microarray analysis, HO conducted the ChIP-Seq sequencing and aided in data analysis, RC prepared ChIPs and aided in the ChIP-Seq studies, and PK conceived of the project, supervised the studies, did experimental design and data interpretation, and together with KB did the majority of the writing of the manuscript. All authors read, helped write and edit, and have approved the manuscript.A) Genomic location, (B) genotyping genomic PCR, and (C) southern blotting assays/probes for H3f3b.(Click here for fileA-B) H3f3a mRNA levels in (A) other knockout (KO) mouse embryonic fibroblasts (MEF) lines produced and (B) KO embryonic tissue. (C) H3.3 protein levels of total H3.3 protein levels in other MEF lines produced. (D) Western quantification of H3.3 protein levels in KO 1, 2, 49 and 52. (E) Western quantification of H3.3S31P protein levels in KO1, 2, 49 and 52 relative to H3.3 protein levels.(Click here for fileA) (top) Example of normal wildtype (WT) karyotype. (Bottom) Example of knockout 1 (KO1) karyotype exhibiting breaks. (B) Abnormal shape factor and evidence of elevated levels of endoreduplication in the KO, DAPI staining and quantitation. Arrows indicate nuclei with possible endoreduplication. (C) Metaphase spread of mouse embryonic fibroblasts (MEF) line KO2 show endoreduplication. (D) DAPI/CREST staining for mouse acrocentric chromosomes and centromeric regions in WT 63 and KO 56. Scale bar = 10 um. (E) Immunostaining for total H3 in WT 46 and KO 49 (MEFs). Scale bar = 20 um.(Click here for filein-situ hybridization (FISH) (green) on wildtype (WT) and H3f3b knockout (KO) cells either arrested in (i) metaphase or (ii) unsynchronized in interphase.Telomere fluorescent Click here for fileA) Total RNA levels in wildtype (WT) and knockout (KO) mouse embryonic fibroblasts (MEFs). (B) qPCR validation of genes downregulated on microarray. (C) Histone genes with upregulated expression in constitutive KO – array data. (D) Immunoblots of histone H2b and H4. (E) Western blot and associated quantification of CENP-A protein in KO 49 and 52 versus mean WT.(Click here for fileP <0.05).Gene expression analysis on constitutive and conditional mouse embryonic fibroblasts (MEFs) for genes that were up- or downregulated by at least 2 fold.  for genes that were up- or downregulated by at least 1.5 fold.Click here for fileOntological expression analysis on constitutive mouse embryonic fibroblasts (MEFs) for genes upregulated.Click here for fileOntological expression analysis on constitutive mouse embryonic fibroblasts (MEFs) for genes downregulated.Click here for fileBasic statistics from the alignment and peak calling portion of the chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis. Sequencing reads were aligned using Bowtie, followed by peak calling using both PeakRanger and Sole Search.Click here for fileA) Chromatin immunoprecipitation-sequencing (ChIP-Seq) global peak overlap between two biological replicates for each condition. (B) Average profile of ChIP-Seq peaks in the region from -1000 to +1000 bp around the transcription start site (TSS) of genes.(Click here for fileChromatin immunoprecipitation-sequencing (ChIP-Seq) differential peak analysis. Differential H3K4me3 and H3K9ac peaks were identified using R package DiffBind with False Discovery Rate < 0.1. Peak annotations within 30 kb of each peak were generated using Galaxy Cistrome tool peak2gene.Click here for fileOntology of genes within 30 kb of H3K4me3 peaks that are significantly different in knockout (KO) mouse embryonic fibroblasts (MEFs) 1 and 2 versus wildtype (WT).Click here for fileHistogram of H3K4me3 peaks that are significantly reduced (top) or increased (bottom) in the knockout (KO) compared to wildtype (WT), based on two biological replicates for each genotype. Histograms are divided into one plot for each chromosome. Chromosomal location is shown on the x-axis, scaled from 0 (centromere) to 1 (telomere); the y-axis shows fold change of significantly changed peaks (FDR <0.1). Graphs were produced using the R package ggplot2.Click here for fileSupplemental Methods.Click here for fileAntibody list.Click here for file


Alad), but not 5-aminolevulinate synthase gene (Alas2) or porphobilinogen deaminase (Pbgd). Similar to its role at the β-globin promoter, KLF1 induces factor recruitment and chromatin changes at the Alad1b promoter in a temporally-specific manner. In contrast to these changes, we observed a distinct mechanism of histone eviction at the Alad1b promoter. Furthermore, KLF1-dependent events were not modulated by GATA1 factor promoter co-occupancy alone. These results not only enhance our understanding of erythroid-specific modulation of heme biosynthetic regulation by KLF1, but provide a model that will facilitate the elucidation of novel KLF1-dependent events at erythroid gene loci that are independent of GATA1 activity.Krüppel-like factor 1(KLF1) is a hematopoietic-specific zinc finger transcription factor essential for erythroid gene expression. In concert with the transacting factor GATA1, KLF1 modulates the coordinate expression of the genes encoding the multi-enzyme heme biosynthetic pathway during erythroid differentiation. To explore the mechanisms underpinning KLF1 action at the gene loci regulating the first 3 steps in this process, we have exploited the K1-ERp erythroid cell line, in which KLF1 translocates rapidly to the nucleus in response to treatment with 4-OH-Tamoxifen (4-OHT). KLF1 acts as a differentiation-independent transcriptional co-regulator of delta-aminolevulinic acid dehydratase ( Genetic analysis has demonstrated that a small group of hematopoietic-specific transacting factors are essential for effective erythroid-specific gene transcription β-globin gene KLF1 is essential for definitive murine erythropoiesis, KLF1-null mice dying of anemia at E15.5 of gestation The heme biosynthesis pathway is critical for the development of the appropriate oxygen-carrying capacity of the erythrocyte. Coordinate expression of gene loci expressing 8 enzymes is required for effective heme synthesis. Fetal liver erythroblasts derived from KLF1-null mice demonstrate greatly diminished, but not absent, mRNA levels of the first three enzymes of the pathway β-globin gene cluster have been confounded by the variable influence of differentiation status on erythroid-specific gene transcription. In contrast, a clear understanding of the essential role of the ‘master’ regulator GATA1 in erythroid specification, differentiation and tissue-specific gene expression has been facilitated by the use of inducible cell lines derived from GATA1 null erythroblasts Alad gene in vitro and in vivo. Unlike Alas2 or Pbgd, KLF1 induced a rapid and substantial increase in Alad mRNA transcripts, enhancing the transcriptional rate being independent of cell differentiation. Our studies allow the separation of the role of GATA1 from KLF1 in vivo, characterizing key molecular events triggered specifically by KLF1 binding.Studies to address the precise role of KLF1 in modulating transcription at gene loci outside the Alad gene transcription in erythroid cells is regulated by a tissue-specific promoter (Alad1b) harboring consensus-binding sequences for GATA1 as well as a conserved CACC element Alas-2 and Pbgd gene promoters Alas-2 and Pbgd promoters was weaker, suggesting that KLF1 may play a more prominent role in the regulation of Alad1b-mediated transcription.Examination of global gene expression in KLF1-null murine fetal liver erythroblasts revealed that mRNA of the first three enzymes of the heme biosynthesis pathway was underrepresented, consistent with KLF1-regulated expression of these genes Alad, Alas-2 and Pbgd transcription in K1-ERp cells. This erythroid model, derived from KLF1-null fetal erythroblasts, expresses a transgenic KLF1 cDNA, fused in frame to sequences encoding a hemagglutinin (HA) epitope, and regulated by an estrogen receptor-dependent regulatory sequence Alad1b mRNA levels over a 6 h time period as measured by semi-quantitative real-time reverse transcription PCR (Q-RT-PCR)  . This inell line , consistAlad mRNA levels is a result of changes in transcription efficiency, or of mRNA stability, we monitored the induction of primary RNA transcripts from the Alad locus. We observed a rapid and significant increase in Alad primary transcript levels upon 4-OHT treatment of K1-ERp cells  and highly variant motifs as seen at the Ahsp erythroid gene locus Alad probe. Binding activity is lost with co-incubation of excess unlabeled probe. In contrast, a mutated CACC element probe failed to influence complex binding.To demonstrate binding of KLF1 to the CACC element of the Alad1b promoter in vivo, we performed KLF1-specific ChIP assays in K1-ERp cells. Enrichment of the Alad1b promoter was detected in 4-OHT treated KLF1 transgenic cells only  was cloned into pGL3-Basic vector and co-transfected into K562 cells (which express low endogenous levels of KLF1), with a renilla luciferase expression construct and either a KLF1 expression vector (pCMV-KLF1) or a vector control (pCMV). The HS2-β-Luc construct was used as a positive control for KLF1-dependent transactivation β-Luc and the Alad1b promoter were activated only in the presence of KLF1 . This particular observation further validates the specificity of the system as chromatin remodeling at the β-globin promoter is extremely sensitive to KLF1 Alad1b promoter 6hrs after exposure to 4-OHT compared to vehicle control cells.KLF1 binding at various erythroid-specific regulatory elements induces a change in chromatin architecture, as measured by DNaseI hypersensitivity β-globin locus is associated with recruitment of a SWI/SNF-encoding multiprotein complex β-globin promoter as well as the Alad1b promoter in the presence of KLF1 only  at the ed cells . HoweverAlad1b promoter. KLF1 is known to interact with three histone acetyltransferases (HATs) in vitro, namely CBP, p300 and P/CAF Alad1b promoter. As shown in Alad1b promoter prior to KLF1 induction by 4-OHT. In contrast, promoter occupancy by p300 was substantial in control cells, and underwent further enhancement after 4-OHT treatment . Taking advantage of our inducible cell-system, we show here that the Alad1b promoter is the first heme-regulatory sequence activated by KLF1 binding. Interestingly, over the 6 hours of 4-OHT exposure, levels of Alas2 mRNA, a marker of ongoing erythroid differentiation, remain unchanged. In contrast, Alad1b transcription increases significantly. Thus, the Alad1b transcriptional response to KLF1 binding is both direct and terminal differentiation-independent.In KLF1-null fetal erythroblasts, heme biosynthesis enzyme levels are reduced globally, but not abolished Alad mRNA levels are influenced by complex regulatory signals in the first 6 hours after induction of KLF1. Alad primary transcript levels increased initially but were diminished 2 hrs after 4-OHT exposure. This decrease in transcriptional rate does not correlate with changes in KLF1 promoter occupancy or the levels of Alad mRNA. These observations suggest that activating and repressive mechanisms influence Alad1b transcription. We also observed a significant increase in PBGD protein levels that does not correlate with Pgbd mRNA transcripts, suggesting that KLF1 may play a role in the translational regulation of Pbgd. These observations emphasize the complexity of the regulatory mechanisms influencing heme biosynthesis.Our studies suggest that Alad mRNA levels were induced 2.5 fold when compared to uninduced cells, suggesting that the Alad deficiency observed in KLF1-null erythrocytes contributes to the anemic phenotype. Support for this conclusion is provided by the hypochromic anemia phenotype in fish deficient in ALAD Alad gene transcription, a significant difference from its essential requirement for effective β-globin gene transcription. It is possible that KLF1 plays a similar role in chromatin remodeling, an early step in differentiation required at all erythroid loci β-globin gene, we observed only a modest increase in DNaseI sensitivity with KLF1 binding at the already hypersensitive Alad1b proximal regulatory sequence. In addition, and in contrast to the β-globin promoter, KLF1 binding to Alad1b induces a significant decrease in histone H3 promoter occupancy, consistent with the idea that KLF1 binding induces a local chromatin structural reconfiguration. This undescribed mechanism of KLF1 mediated chromatin remodeling, histone eviction, suggests more complex and context-specific mechanism(s) of KLF1 action than previously appreciated.We show that a role of KLF is to augment rather than induce Alad1b promoter DNaseI hypersensitivity predating KLF1-mediated recruitment of Brg1. Instead, these early events are associated with GATA1 and SCL/TAL-1 complex binding. Although the latter factors represent attractive candidates for regulating DNaseI hypersensitivity, it remains a possibility that their binding is not related causally to promoter accessibility. It will be important to explore the role of Brg1 thoroughly in histone eviction at the Alad1b promoter. Equally, it is possible that the role of Brg1 may vary completely, being required perhaps to enhance transcriptional initiation by recruitment of an upstream enhancer The SWI/SNF subunit Brg1 is a strong candidate for modulating KLF1-mediated histone eviction. Strong support for such a model comes from the essential role of KLF1:Brg1 interaction in nucleosomal phasing and induction of DNaseI hypersensitivity at the β-globin promoter Alad1b promoter may also be involved in histone eviction. We corrected the level of histone modification for total histone H3, our results suggesting that histone eviction may be associated with replacement by a histone variant β-globin promoter upon MEL cell erythroid differentiation Changes in covalent histone modifications  at the Alad1b promoter does not require KLF1. Similarly, GATA1 is present at the Alad1b promoter along with p300 and low levels of RNA Pol-II in the absence of KLF1. This is consistent with the idea that p300 but not CBP activity is required for differentiation-specific hematopoietic gene expression Alad1b promoter is enhanced in the presence of KLF1. In a second step, recruitment of p45NF-E2, Brg1 and CBP occurs in a KLF1-dependent manner. This two-phase model of factor recruitment suggests that each factor combination plays a unique role in achieving maximal gene transcription.The K1-ERp cell system is particularly attractive for study as it allows us to distinguish between GATA1- versus KLF1-dependent during erythropoietic differentiation. For example, Ldb1 recruitment to the in vitro and in vivo with GATA1 and KLF1, yet they are not mobilized in a similar manner to the Alad1b promoter Alad locus to a subnuclear compartment rich in shared transcription factors Both HATs interact physically Alad transcription requires binding of KLF1 to its cognate promoter, occurring in the absence of ongoing erythroid differentiation. In contrast, Alas2 and Pbgd transcription is KLF1- independent. We report a novel mode of chromatin remodeling triggered by KLF1 binding, with histone depletion. In addition, we have demonstrated a two-step mechanism of transcriptional activation of the Alad locus, with key transactivators requiring KLF1-binding. These observations provide further insight into the complex mechanisms of erythroid gene expression, and a platform for studies of the temporal events necessary for tissue-specific gene transcription.In conclusion, we show here that maximal 6 cells/ml, in D15puro (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% Fetal Calf Serum (FCS), 1% penicillin/Streptomycin and 1 µg/ml of puromycin (Invivogen)) at 37°C, 5% CO2. 2 units/ml of erythropoietin was added directly to the cells 3 days prior to cell harvest. The nuclear translocalization of KLF1 in K1-ERP cell was induced by the addition of 4-OH-Tamoxifen (4-OHT) to a final concentration of 100 nM. Murine Erythroleukemia cells (MEL) 2.K1-ERp cells RNA was extracted using the Trizol method and cDNA was prepared using the Superscript II First Strand System (Invitrogen) following the manufacturer’s protocols. RNA (10 µg) was treated with Turbo DNase (2 µL) (Ambion) in a 50 µL reaction to remove genomic DNA contamination. DNase-treated RNA (2 µg) was used for first strand DNA synthesis. The resulting cDNA was diluted 1∶50 for mRNA analysis and 1∶2 for primary transcripts analysis.Alad1b primary transcripts amplify the exon2-intron2 junction: Forward 5′ GCCTCCAACCTCATCTATCC-3′ and Reverse 5′- TCCCATTGCCTGTTCCAGTC-3′. Primers used to measure mRNA were as followed: Alas2 Forward 5′-CTTTG ATCTCAGGACTGCTG-3′ and Reverse 5′-AACAGCTGCGGTGCAAAGTA-3′, Alad1b Forward 5′-CACCTATGCTTAAGGAGCCA-3′ and reverse 5′-CAGAAGCACACAGGAAAGCA-3′, Hprt Forward 5′- GCAGTACAGCCCCAAAA TGG-3′ and Reverse 5′-AACAAAGTCTGGCCTGTATCCAA-3′, Pbgd Forward 5′- TACTTCTGGCTTCCAAGTGC-3′ and Reverse 5′-CAAGGTGAGGCATATCTTCC-3′.2 µl of the cDNA product was subjected to semi-quantitative analysis using real-time PCR (Applied Biosystems Prism 7900) with the appropriate primer pairs. Products were quantified using SYBR green fluorescence in 20 µl reactions. Primer pairs were designed using the Clone Manager software to obtain PCR products of 50–150 bp. Primers used to detect Whole cell extracts from ethanol treated and 4-OHT treated K1-ERp cells were obtained by lysis in 1% NP-40 lysis buffer. 50 µg of lysate was diluted with 6X SDS-loading buffer , denatured by boiling for 3 minutes and resolved on a 10% or 12% SDS-PAGE gel. Proteins were then transferred to a nitrocellulose membrane by wet transfer procedure. Primary antibody incubation with relevant antibodies was carried out overnight at 4°C. Secondary antibody incubation was carried out at a dilution of 1: 3,000 of anti-rabbit or 1: 10,000 anti-goat horseradish peroxidase (HRP) conjugated antibodies in 5% milk in 1X PBS, 0.1% Tween-20 for 1 hr. Peroxidase activity was detected by enhanced chemiluminescence (ECL) using detection reagents from Pierce followed by exposure to X-ray films.2, 20 fmol of Biotin-End labeled probe and dH2O to a final volume of 20 µl. A separate control reaction was prepared omitting the nuclear extract. After 20 min incubation at room temperature, 5 µl of 5X loading buffer was added and samples were loaded onto a 5% polyacrylamide gel. Samples were separated by electrophoresis in 0.5X TBE buffer. To assess binding specificity, a second control was prepared adding a 200X molar excess of unlabeled CACC proximal probe for competition. A third control was prepared adding a 200X molar excess of the non-specific mutated CACC proximal probe –CCCATTTAGTTTTGTGGTGG-. Mutated nucleotides are underlined.Nuclear extracts from MEL cells were prepared using a modified Dignam’s protocol 7 cells per immunoprecipitation condition were resuspended in cold cell lysis buffer  supplemented with protease inhibitors and incubated on ice for 10 min. Nuclei were then resuspended in 300 µl of nuclear lysis buffer  supplemented with protease inhibitors and incubated for 10 min at 4°C. Chromatin was sheared by sonication using the Bioruptor sonicator (Diagenode) to achieve an average DNA fragment size of 500–2000 bp. Soluble chromatin was collected and diluted 1 to 4 with cold immunoprecipitation buffer  supplemented with protease inhibitors. Samples were pre-cleared by adding 50 µl of salmon sperm DNA/protein A/G agarose beads (Millipore) and 5 µl/ml of rabbit serum. Antibody (2–5 µg), or appropriate normal IgG for control, were added to the soluble chromatin overnight at 4°C. Immune complexes were adsorbed to 70 µl of salmon sperm DNA/protein A/G agarose beads (Millipore) for 2 hrs at 4°C. Beads were washed twice in cold wash buffer 1  followed by 1 wash with cold wash buffer 2  and 2 washes with Tris-EDTA buffer. Protein-bound DNA was eluted from the beads by 2 sequential incubations of 15 min each with elution buffer . Crosslinks from immunoprecipitation samples and inputs were reversed by the addition of 1 µl of 20 mg/ml RNaseA (Ambion) and overnight incubation at 65°C. DNA purification was carried out using a Qiaquick PCR purification kit (QIAGEN) following manufacturer’s instructions. Analysis of the purified DNA was achieved using real-time PCR (Applied Biosystems Prism 7900) with the appropriate primer pairs. Products were quantified using SYBR green fluorescence in relation to a standard curve generated from serial dilutions of an input sample. PCR efficiency and specificity was determined by analysis of the standard curve and the dissociation curve respectively. Primer pairs were designed using CloneManager software to obtain PCR products of 50–150 bp. Primer sequences are presented in ChIP assays were performed as previously described, with slight modifications 6 K562 cells were co-transfected with pTk-Rn (0.04 µg) and pGL3-basic or pGL3-basic containing ALAD1b promoter sequence (0.5 µg) with either pCMV or pCMV-KLF1 (1 µg). Firefly luciferase activity was assayed 24 hr after transfection and corrected to Renilla luciferase activity. An HS2-β-Luc construct was used as a positive control for a KLF1 dependent promoter sequence Promoter-reporter assays were conducted using the Dual-Luciferase Reporter assay system from Promega following manufacturer’s instructions. 0.5×107) were washed once with cold PBS and resuspended in 14 ml RSB (10 mM Tris HCl (pH 7.5), 3 mM MgCl2, and 10 mM NaCl). Cells were lysed in the presence of 0.25% (v/v) NP-40, and nuclei were collected and resuspended in 1.5 mL RSB. The nuclei mixture (200 µL) was added to 30 µL of an enzymatic mix containing 0–18 units of DNaseI (ROCHE). DNaseI digestion was carried out at 37°C for 10 minutes and terminated by the addition of an equal volume of stop buffer  SDS, 20 mM Tris-HCl (pH 7.5), and 10 mM EDTA). The samples were subjected to proteinase K (500 µg/mL) treatment overnight at 55°C. DNA was recovered by extraction with phenol/chloroform followed be ethanol precipitation. DNA analysis was performed using real-time PCR with SYBR green fluorescence detection. Quantification of the undigested template was achieved in relation to a standard curve generated from serial dilutions of undigested genomic DNA. Results were normalized to those obtained for a previously determined DNaseI insensitive gene, NF-M Nf-m forward: 5′- GCTGGGTGATGCTTACGACC-3′ and reverse: 5′- GCGGCATTTGAACCCTCTT-3′DNaseI sensitivity assays were performed essentially as described with slight modifications Antibodies used for western blotting are as follows: anti-HSC70 (K-19) at a dilution of 1∶2000, anti-ALAS2 (D-4), anti-ALAD (A-7) and anti-PBGD (A-16) at a dilution of 1∶500 (SantaCruz Biotech). Antibodies used for chromatin immunoprecipitation studies are as follows: anti-HA (Y11), anti-GATA1 (N16), anti-Clim2 (N18), anti-NFE2 (C19), anti-CBP (A22), anti-p300 (N15), normal rabbit IgG, normal mouse IgG, normal goat IgG (SantaCruz Biotech.); anti-Brg1, anti-AcH3, anti-H3K14Ac, anti-H3K27Me (Millipore); anti-Pol II (4H8), anti-Ser5 Pol II, anti-H3, anti-H3K4Me3 (Abcam).Figure S1Alad mRNA levels in J2e and K1-ERp cells. Relative mRNA levels of Alad in the K1-ERp parental J2e cell line and K1-ERp cells treated with ethanol or 4-OHT, as determined by Q-RT-PCR. Represented mRNA levels were corrected to Hprt mRNA levels. RNA was isolated at 6 h after treatment. *p value≤0.05 by Student’s t-test. Data shown represents the average of at least 3 independent experiments (mean ± Std dev).(PDF)Click here for additional data file.Figure S2p18 Ink4c RNA levels in K562 cells. Relative mRNA levels of p18 Ink4c in K562 cells treated with Hemin to induce terminal differentiation (positive control) and transfected with vector plasmid or KLF1 expression plasmid, as determined by Q-RT-PCR. Represented mRNA levels were corrected to Hprt mRNA levels and relative expression determined by the ΔΔCt method. RNA was isolated at 48 h after treatment or transfection. *p value≤0.05 by Student’s t-test. Data shown represents the average of at least 3 independent experiments (mean ± Std dev).(PDF)Click here for additional data file.Figure S3Gata-1, Ldb1 and p45Nf-e2 mRNA levels in K1-ERp cells. Relative mRNA levels of Gata-1, Ldb1 and p45Nf-e2 in K1-ERp cells treated with ethanol or 4-OHT, as determined by semi-quantitative real-time PCR. Represented mRNA levels were corrected to Hprt mRNA levels. RNA was isolated 6 h after treatment. *p value≤0.05 by Student’s t-test. Data shown represents the average of at least 3 independent experiments (mean ± Std dev).(PDF)Click here for additional data file.Figure S4Alad1b promoter primers. *p value≤0.05. **p value ≤0.01 by Student’s t-test. Data shown represents the average of at least 3 independent experiments (mean ± SEM).KLF1 enhances the deposition of histone modification associated with active genes. ChIP performed on chromatin derived from K1-ERp cells treated with either vehicle control or 4-OHT. Antibodies were specific against (A) histone H3K4Me3, (B) acetylated histone H3. Target DNA enrichment relative to input was determined by Q-PCR using (PDF)Click here for additional data file.


CREB and CREM are closely related factors that regulate transcription in response to various stress, metabolic and developmental signals. The CREMτ activator isoform is selectively expressed in haploid spermatids and plays an essential role in murine spermiogenesis.We have used chromatin immunoprecipitation coupled to sequencing (ChIP-seq) to map CREM and CREB target loci in round spermatids from adult mouse testis and spermatogonia derived GC1-spg cells respectively. We identify more than 9000 genomic loci most of which are cell-specifically occupied. Despite the fact that round spermatids correspond to a highly specialised differentiated state, our results show that they have a remarkably accessible chromatin environment as CREM occupies more than 6700 target loci corresponding not only to the promoters of genes selectively expressed in spermiogenesis, but also of genes involved in functions specific to other cell types. The expression of only a small subset of these target genes are affected in the round spermatids of CREM knockout animals. We also identify a set of intergenic binding loci some of which are associated with H3K4 trimethylation and elongating RNA polymerase II suggesting the existence of novel CREB and CREM regulated transcripts.in vivo and represents the most comprehensive experimental analysis of CREB/CREM regulatory potential to date.We demonstrate that CREM and CREB occupy a large number of promoters in highly cell specific manner. This is the first study of CREM target promoters directly in a physiologically relevant tissue  Cyclic AMP response element (CRE) binding protein (CREB) and cyclic AMP response element modulator (CREM) are highly related bZIP proteins that regulate transcription in response to various stress, metabolic and developmental signals ,2. CREB Fhl5 gene [Multiple isoforms of CREM have been described that act either as repressors or activators . Folliclhl5 gene  specifichl5 gene -16. WhilHere we have used chromatin immunoprecipitation coupled to massive parallel sequencing (ChIP-seq) to identify CREM target genes in round spermatids in testis and CREB target genes in spermatogonia derived GC1-spg cells. We identify more than 9000 genomic loci most of which are cell-specifically bound. Of these, more than 6700 target loci are occupied by CREM in testis, yet the expression of only a small subset of these genes are affected upon CREM knock out. We also identify intergenic CREB and CREM binding loci some of which are associated with H3K4 trimethylation suggesting the existence of novel CREB and CREM regulated transcripts.As previously reported, in testis, the CREMτ isoform is strongly and selectively expressed in round spermatids ,15, whilBmp2k) gene as a negative control and the testis-specific angiotensin I converting enzyme (Ace) promoter [Bmp2k control. Strong occupancy of the intraflagellar transport 172 homolog (Ift172) and small nuclear ribonucleoprotein polypeptide A (Snrpa) loci was observed, but no significant occupancy of the surfeit gene 6 (Surf6) locus was seen, when compared to the background of the anti-CREM antibody as defined by the signal seen using the Bmp2k control. All loci defined by more than 40 clustered reads were significantly enriched in qPCR, not all loci defined by under 40 reads showed enrichment. This number was therefore used as a cut off to obtain a high confidence list.We used ChIP-qPCR to validate CREM binding to sites that showed high, medium and low occupancy in the ChIP-seq experiments. We compared CREM ChIP to control ChIP with anti-GFP antibody at a series of identified binding sites and with an intragenic region from the bone morphogenic 2 inducible kinase . Trimethylation of this residue is tightly associated with transcription activity, a high density of this mark overlapping the 5'end of the gene indicates either its transcription or the presence of a poised preinitiation complex . CompariAs mentioned above CREB is not expressed in round spermatids, but is expressed in spermatogonia and in the somatic cells in testis. Due to its expression in several of the cell types that make up the testis, we cannot use total testis chromatin, even from early post-natal animals, for the ChIP-seq as we would be unable to distinguish in which cells CREB was occupying a given promoter. We therefore used GC1-spg cells that are immortalised spermatogonia  and exprThese results were confirmed by ChIP-qPCR where we observed significant, but variable occupation of all of the tested promoters. In this case even loci with small numbers of clustered reads showed significant enrichment with respect to the Bmp2k control Fig. . The majSimilar to what was observed with CREM, most CREB binding sites were observed in the promoters of annotated genes, but a signficant number of intra- and intergenic sites were also observed. Bioinformatic analysis showed that only a small number comprise full cannonical CREs and we identify a subset of sites with half-CRE or no identifiable CRE Fig. . Also siTo assess the association of CREB loci occupancy with transcriptional activity, we further performed H3K4me3 and pol II ChIP-seq in GC1-spg cells. Comparison with CREB occupancy shows that around 60% of CREB-occupied loci are associated with high levels of pol II and H3K4me3 in these cells  is occupied by CREM and active in testis, but not by CREB in GC1-spg cells, while the opposite is observed for the natriuretic peptide precursor type B (Nppb) locus  promoter [Sst is not expressed in testis or in GC1-spg cells as shown by the absence of H3K4me3 and pol II (data not shown), and the promoter CRE is not occupied in either cell type. However, CREB selectively occupies an alternative and conserved intragenic site in GC1-spg cells . These results show the existence of different classes of binding sites, those that are rather generally accessible, and a larger set that are occupied in a cell type-specific manner.In total, 9037 distinct CREB and CREM binding loci can be identified combining the ChIP-seq in round spermatids and GC1-spg cells. Despite the wide occupancy of potential binding sites by CREM in haploid cells, not all possible sites (i.e. full CREs) are occupied as the CRE element in the http://www.dkfz.de/tbi_old/crem/cremgenes.txt). Of these, only 58 are direct CREM targets with one or several CREM binding sites in their promoters.  2 genes are expressed in both testis and GC1-spg cells, yet their promoters are specifically occupied only in round spermatids by CREM . Mmu-mir-9-3 is a miRNA involved in silencing the neuronal transcription factor REST [Mmu-mir-132 and mmu-mir-219-1 are two CREB regulated brain-enriched miRNAs that regulate circadian rythmn and in the case of mir-132, neuronal morphogenesis ,28. At ttor REST . In conthttp://david.abcc.ncifcrf.gov/) to make a more comprehensive identification of potential CREB/CREM target genes in physiological functions , p300 (Ep300) and TORC1 (Crtc1) are expressed in GC1-spg cells and testis, but their promoters are occupied only in haploid cells by CREM  loci  CRE correlates with its expression. In our experiments, the Sst locus is expressed neither in testis nor in GC1-spg cells and the promoter CRE is not occupied by CREB or CREM. In contrast, CREB occupies a novel and conserved intronic site in GC1-spg cells that does not contain any CRE-related element. However, a more global analysis of our CREB data set shows that a majority of CREB occupied promoters are also characterised by high pol II occupancy and H3K4 trimethylation. Thus the idea that CREB preferentially occupies promoters of expressed genes is generally valid. Similarly, CREM-occupied genes in haploid cells are over-represented amongst the highly expressed gene set On the other hand, there are both promoter and intergenic sites that are not closely associated with H3K4 trimethylation. Thus, expression is neither an absolute pre-requisite nor outcome of CREB/CREM occupancy. Moreover as discussed above, there are promoters that are active in both testis and GC1-spg cells, but bound only by CREM in testis. There must therefore be additional factors that influence occupancy, such as DNA methylation or competition with other factors whose binding sites overlap that of CREB [Cha-Molstad et al., also suggested that CREB preferentially occupies loci that are expressed and marked with H3K4 methylation. As an example, they show that occupancy of the well studied somatostatin  a histone H1 variant specifically expressed in haploid cells [H1fnt promoter is occupied by CREM, selectively activated in round spermatids and is therefore a direct CREM target. The full data set of Beissbarth et al actually comprises more than 367 regulated transcripts many of which are only identifed as ESTs. If the proportion of direct CREM targets from the Affymetrix data can be extended to the full set, this would suggest that CREM directly regulates between 150-200 genes in male germ cells. This number still represents only a small fraction of the CREM bound promoters. CREM clearly occupies many more promoters than it actually regulates. Neither the presence of a full CRE nor a TATA element are pre-requisites to predict whether a gene will be regulated by CREM. The criteria that determine whether a promoter will be regulated or not by CREM remain to be determined.Strikingly, only a very restricted set of CREM-occupied genes are affected by CREM inactivation. Comparison of the 127 genes whose expression is affected with those that are bound by CREM identifies 58 genes as direct targets. For this analysis, we used only the Affymetrix data of Beissbarth et al  that mayid cells . The H1fBcl2 and Bcl6b (BAZF) may contribute to germ cell apoptosis. Bcl2 is not a direct CREM occupied promoter , but Bcl6b is a direct target. Apoptosis may potentially result from direct deregulation of this gene or of one or several of the other direct CREM target genes involved in signal transduction and metabolic processes.Inactivation of CREM leads to an early apoptosis of haploid cells around step 4 of their development ,15. AmonTogether our results have identified a large repertoire of CREM bound loci in haploid cells of which a small subset involved in signalling and metabolic processes are deregulated upon CREM inactivation and may explain the haploid cell apoptosis. In contrast to previous studies on smaller data sets, we have shown that promoter occupancy by CREB and CREM is highly cell specific in haploid and GC1 cells. This is the most comprehensive evaluation of CREB/CREM regulatory potential to date and we have identified a large set of promoters that may be regulated by these factors in different tissues and physiological processess. Correlation between the CREM/CREB, H3K4me3 and pol II data sets has identified many novel potential CREB regulated transcripts, and revealed the presence of CREB at novel start sites and distal regulatory elements.Chromatin was prepared from the testes of 12 week old adult 129/SV mice. Animal breeding and manipulation were performed according to French national ethical standards using approved protocols. The animals were sacrificed and the testes removed and opened directly in 20 ml fixing solution  in a Petri dish. Seminiferous tubules were fixed for 10 minutes with agitation. The cross-linking reaction was stopped by adding 2 ml 2 M glycine solution. Seminiferous tubules were then centrifuged at 4000 rpm and the pellet was lysed with 2 ml cell lysis buffer  for 15 minutes. GC1-spg cells were obtained from the ATCC collection and cultured in DMEM with 10% foetal calf serum. Cells were grown to 90% confluency and crosslinked with formaldehyde 0.4% for 10 min at room temperature and the reaction was stopped by adding glycine to final concentration 0.2 M for 10 minutes at room temperature. Fixed cells were rinsed twice with PBS and resuspended in lysis buffer. Lysate from cells or testis was sonicated 30 min (10 sec on/30 sec off) in Diagenode water bath-sonicator and centrifuged at 14000 rpm for 10 min. The cleared supernatant was used immediately in ChIP experiments or stored at -80°C. ChIP was performed by standard procedures.http://liulab.dfci.harvard.edu/MACS/) [http://bips.u-strasbg.fr/GPAT/Gpat_home.html). Peak detection with MACS was performed using the GFP-ChIP as negative control. Clustering was performed by first generating density (.wig format files) counting the number of tags in a 25 pb sliding window for each ChIP-seq data set. The coordinates of the CREB binding sites were used as reference, and the tag density for the Pol II and H3K4me3 data sets were extracted from the. wig files in the 10 kb around the CREB site. A matrix of binding sites and densities was generated and subjected to K-means clustering using the Cluster 3.0 software (http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/software.htm#ctv). The clustered matrix was visualized using Java TreeView. (http://jtreeview.sourceforge.net/).ChIP-seq was performed using an Illumina GAII sequencer and the raw data analysed by the Illumina Eland pipeline. Peak detection was performed using the MACS software (u/MACS/) , and theu/MACS/) , http://Comparison of the ChIP-seq data with the array expression data was carried out by first performing an RMA normalisation of the mouse spermatid expression. Cel files from  and fromIft172: 5'-TTTTGCCTCTCGTAGCACCT-3', 5'-ATGCAAACCTAACGCAAACC-3'The following antibodies were used: the anti-CREMtau rabbit polyclonal antibody has been previously described ,48, CREBGosr2: 5'-TAAAACTGGGAGGGATGCAG-3', 5'-CTAGGCCCATCTCATTTCCA-3'SnrpA: 5'-CACAACGCTCTTGAAGGTCA-3', 5'-GGAACGGACCAATCAAAGAA-3'Cdc6: 5'-AGACCTGGGGCTGTCCTATT-3', 5'-CCAAAGCCGCTCTACTTCAG-3' Camk2d: 5'-AAGCACGAGCACATAAGCAA-3', 5'-GCAGATTTAGAGGCCTACGG-3'Cd63: 5'-ACCTGGTTTTGCCATCTCTG-3', 5'-AAGCCTTAGAGCTCCCTTGC-3'Ace: 5'-GCTGGCACATTGCTCTATGA-3', 5'-AGCAGAGCAGCAGAAAGAGG-3'Ankrd52: 5'-TGACGTCAGGGAGAGAGCTT-3', 5'-CCCCATCACAAGGAGAGAAA-3'SURF6: 5'-GCCTTTCCCTTCATTTCCTC-3', 5'-GACCTGAGTATGGCGTGGAT-3'Bmp2k intragenic: 5'-TCCCTGCACGTACTTTAGGC-3', 5'-TTGGAGCACTCAAAGGTGTG-3'Rtbdn: 5'-TCCACGGTGCTGAAATATGA-3', 5'-CCAGGAACCGAGTTACAGGA-3'Stat3: 5'-CTCCCCCACGCAATCTAGTA-3', 5'-AAGCATTTGGGTTTGTGGAG-3'Pcna: 5'-GGGTTGGTAGTTGTCGCTGT-3', 5'-AGCACCTTCTTCAGGATGGA-3'Cfos: 5'-TTAGGACATCTGCGTCAGCA-3', 5'-CCCGTCTTGGCATACATCTT-3'Fn1: 5'-CCTCCCTTTCCTTCGAGTCT-3', 5'-GCCAGGTCTGGGACTAGAGA-3'B2m: 5'-CTGACCGGCCTGTATGCTAT-3', 5'-TCCACCCTGTAGCCTCAAAG-3'Crebzf: 5'-CCGGGTAGTACCGATGAGTG-3', 5'-CTTCGCTCCAGCCAGAGTAT-3'Klf7: 5'-CCAGCGTGTACAGTGCAGAT-3', 5'-TGTCAGTGAGTTTGCGTTCC-3'http://meme.nbcr.net/meme4_4_0/cgi-bin/meme.cgi) [Detection of CRE and half CRE elements in the CREB and CREM bound loci was performed by first extracting a region of 500 nucleotides centered on the peak summit. These sequences were then analysed using the motif search programme of Genomatix software. Additional analysis of the bound regions was performed using the Meme programme (eme.cgi) .IM designed and performed all of the ChIP assays, the immunostaining and immunoblot experiments, MAC, AK and TY developped and applied the algorithm for read density matrix clustering and AK also developped the ChIP-seq peak annotation programme, SL and ER performed the bioinformatics analyses of the ChIP-seq data, peak detection, motif detection, comparison with transcriptome data, WVI and BJ performed ChIP-seq library preparation and Illumina sequencing, PSC and ID concieved the project designed the experiments and ID wrote the manuscript. All authors have read and approved the final manuscript.Figure S1: Expression of the CREB and CREMτ proteins in adult mouse testis.A. Immunofluoresence on sections from adult mouse testis with the anti-CREB and CREMτ antibodies. The signal for the antibodies in red, the Hoechst stained nuclei and the merged views are show as indicated. Representative examples of the different cell types are indicated by arrows. L; Leydig cells, S; Sertoli cells; Sp; spermatogonia; P; pachytene spermatocytes, I; intertubular cells, R; round spermatids, E; elongated spermatids. These results reveal a distinct expression of CREB and CREMτ in the testis, where CREB is present in the spermatogonia, the Sertoli, Leydig and intertubular cells, while CREMτ is present in haploid round spermatids. 20 fold magnification. B. Western blots with the CREMτ and CREB antibodies on 20 ug of extracts from the indicated cells or tissues. ES is undifferentiated mouse E14 embryonic stem cells, COS is COS-1, and 3T3 is NIH3T3. The CREB antibody detects a single polypeptide corresponding to CREB in each of the extracts, while the CREMτ antibody detects the CREMτ proteins only in the testis extract.Click here for fileFigure S2: Example of ChIP-seq data over an 11,191,448 bp region of mouse chromosome 3qA3-3qB. The results for CREB and CREM in GC1 and haploid cells respectively are shown along with the ChIP-seq for H3K4me3 in GC1 cells (K4-GC1) and testis (K4-T). Representative CREB, CREM binding sites are indicated. Active promoters marked by H3K4me3 are also indicated. The * in the CREB and CREM chanels show examples of loci that are specifically occupied GC1 cells or haploid cells respectively. The * in the K4-GC1 and K4-T show examples promoters that are specifically or preferentially active in GC1 cells or testis, respectively.Click here for fileTable S1: CREM bound loci in mouse male haploid germ cells. Excel table of annotated loci bound by CREM. Page 1 shows annotation of bound loci to RefSeq genes. N is number, Chr chromosome, start and end shows the coordinates of the binding sites and summit, the coordinates of the center of the site. The number of clustered reads and the pvalue and the local fold enrichment are indicated. For annotation, the RefSeq transcript ID, the gene symbol, and TSS coordinates are indicated along with the distance between the summit of the peak and the TSS. No match signifies that there was no RefSeq annotation within 5 kb from the peak summit position. The presence of a perfect CRE, a CRE with 0 or 1 mismatch or a half CRE are then indicated. The H3K4me3 status of the loci is also indicated. Page 2 shows annotation of bound loci to Ensembl transcripts. Layout is as in page 1 except that the RefSeq transcripts are replaced by Ensembl annotation.Click here for fileTable S2: MiRNAs that are targets of CREM in mouse male haploid germ cells. Excel table of CREM bound miRNAs in haploid cells. Layout is essentially as in Table 1.Click here for fileFigure S3: Comparison of CREM and H3K4me3 ChIP-seq with adult mouse round spermatid gene expression.A. The probe set expression values were divided into classes of ≤ 10, 11-50, 51-500 and ≥501 and the % of the total probe sets in each category are represented by the black bars as a % of the total. The % of the total number of CREM and H3K4me3 occupied promoters in each expression category is shown with the shaded bars. B. A similar representation is shown for the probe set expression values and CREB and H3K4me3 occupied promoters in GC1-spg cells.Click here for fileTable S3: CREB bound loci in GC1 cells. Excel table of binding sites. Page 1 shows annotation of bound loci to RefSeq genes. Page 2 shows annotation of bound loci to Ensembl transcripts. Layout is as in Table S1.Click here for fileTable S4: Comparison of genes whose expression is affected by CREM inactivation in testis and CREM binding. Page 1 of the Excel table reproduces the Affymetrix data of Beissbarth et al., (1) (http://www.dkfz.de/tbi_old/crem/cremgenes.txt) concerning genes whose expression is deregulated in the testis of CREM knock-out mice. Page 2 shows the data for CREM occupancy of the subset of loci that map to the promoters of the genes in page 1. Note that some promoters contain several CREM occupied sites; Page 3 shows the list of the genes whose expression is deregulated from page 1 and whose promoters are occupied by CREM.Click here for fileFigure S4: UCSC web browser graphic view of CREB and CREM binding to the indicated loci.A-B. Selective binding of CREM to the Clock and Per 2 promoters. C. CREB, CREM occupancy and H3K4me3 over the Crem locus.Click here for fileTable S5: Functional annotation genes whose promoters are occupied by CREM (page 1) and CREB (page 2). Functional annotation was performed using the KEGG pathway of David (http://david.abcc.ncifcrf.gov/). In addition to the list of genes in each identified functional category, are shown the number of genes (gene count) the % of the total number of genes, and pvalue.Click here for fileTable S6: Excel table of CREM and CREB bound sites with no annotation to RefSeq genes or Ensembl transcripts. Pages 1 and 2 show the data concerning CREM and CREB binding sites for which no RefSeq annotation was found within 5 Kb of the peak summit and which were designated 'no match' in Tables S1 and S3. Layout is as in Table S1.Click here for fileFigure S5: A-B. UCSC web browser graphic view of CREB, H3K4me3 and pol II occupancy of intergenic sites upstream of annotated genes. In panel A, no elongating pol II is seen between the upstream site and the annotated promoter, while in panel B, a low level of elongating pol II is observed, suggesting the existence of an alternative promoter. C. Multiple CREB binding sites and high pol II occupancy of the locus encoding the Malat1 and MENε/β non-coding RNAs.Click here for file