Open Access Research article

Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells

Igor Martianov1, Mohamed-Amin Choukrallah1, Arnaud Krebs1, Tao Ye1, Stephanie Legras1, Erikjan Rijkers2, Wilfred Van Ijcken2, Bernard Jost1, Paolo Sassone-Corsi3 and Irwin Davidson1*

Author Affiliations

1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS, 1 Rue Laurent Fries, 67404 Illkirch Cédex., France

2 Erasmus Medical Center, Dr Molewaterplein 50, 3015GE Rotterdam, the Netherlands

3 Department of Pharmacology, 2115 Gillespie Neuroscience, University of California, Irvine, California 92697-4625, USA

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BMC Genomics 2010, 11:530  doi:10.1186/1471-2164-11-530

Published: 29 September 2010

Additional files

Additional file 1:

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.

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Additional file 2:

Figure 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.

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Additional file 3:

Table 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.

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Additional file 4:

Table 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.

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Additional file 5:

Figure 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.

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Additional file 6:

Table 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.

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Additional file 7:

Table 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 webcite) 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.

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Additional file 8:

Figure 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.

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Additional file 9:

Table 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/ webcite). 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.

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Additional file 10:

Table 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.

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Additional file 11:

Figure 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.

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