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 (0.4% formaldehyde in 1× PBS) 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 (50 mM Tris pH8.0, 10 mM EDTA, 1% SDS) 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.
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 (http://liulab.dfci.harvard.edu/MACS/) , and the peaks annotated using GPAT (, 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/).
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 from the mock silenced GC1-spg data sets in the GEO data base (GSE19355). Excel files with the normalised data were compared to those comprising a list of CREM (or CREB) and H3K4me3-occupied promoters, thus assigning an expression value to each of the corresponding genes.
The following antibodies were used: the anti-CREMtau rabbit polyclonal antibody has been previously described [14, 48], CREB (06-863, Upstate), H3K4me3 (MC315 04-745, Millipore), Pol II (H-224 Sc-9001 Santa Cruz). Real-time PCR was performed on a Roche Lightcycler using Roche SYBR Green mix. The following primer sequences were used. Ift172 : 5'-TTTTGCCTCTCGTAGCACCT-3', 5'-ATGCAAACCTAACGCAAACC-3'
Gosr2 : 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'