Open Access Highly Accessed Research article

Genome-wide search identifies Ccnd2 as a direct transcriptional target of Elf5 in mouse mammary gland

Rosalba Escamilla-Hernandez, Rumela Chakrabarti, Rose-Anne Romano, Kirsten Smalley, Qianqian Zhu, William Lai, Marc S Halfon, Michael J Buck and Satrajit Sinha*

Author Affiliations

Department of Biochemistry, State University of New York at Buffalo, Center for Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14203, USA

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BMC Molecular Biology 2010, 11:68  doi:10.1186/1471-2199-11-68

Published: 10 September 2010

Additional files

Additional file 1:

Table 1 List of putative Elf5 target genes. Shown are the genes that are located within 100 Kb of the isolated Elf5-ChIPed DNAs and the size of the immunoprecipitated DNA fragment that was sequenced.

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Additional file 2:

Figure S1 Relative location and the genomic context of the Elf5 ChIPed segment. Select groups of putative Elf5 target genes chosen for further evaluation are highlighted with their genomic organization, chromosome number and the position of the ChIPed DNA fragment.

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Additional file 3:

Figure S2 Sequence conservation of the upstream Elf5-ChIPed region. A 10 kb region 5' of the CCND2 gene was compared between different species by using the GenomeVISTA program http://genome.lbl.gov/vista/index.shtml webcite. The segment bound by Elf5 was highly conserved among several species as indicated by the box.

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Additional file 4:

Figure S3 Sequence of the proximal CcnD2 promoter and the upstream region. The core GGAA Elf5-binding sequence is boxed.

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Additional file 5:

Figure S4 Purified GST-Elf5 WT and the two DNA-binding deficient mutants. GST-Elf5 wildtype and GST-Elf5 mutants were purified using GST-agarose and eluted samples were run on a SDS-PAGE gel to assess purity and amount of the proteins. The WT1 and WT2 samples represent two independently purified fractions. The mutants, MT1 and MT2 were K to A substitution at amino acid 216 and R to A substitution at amino acid 219 respectively, of the mouse Elf5 protein.

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Additional file 6:

Figure S5 Western blot demonstrating the expression of HA-Elf5 and HA-Elf3 in transient transfection experiments. The expression of the HA-epitope tagged Ets proteins was detected by anti-HA antibodies.

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Additional file 7:

Figure S6 Increase in Ccnd2 expression in the absence of Elf5. Elf5f/f primary mammary epithelial cells (MECs) were transduced with Ad-Cre in suspension and plated on BM matrix (A). Western blot analysis of protein lysates from Elf5f/f MECs transduced with Ad-Cre resulted in increased Ccnd2 expression. Elf5 is absent in Ad-Cre-Elf5f/f cells (B).

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Additional file 8:

Table 2. Primer sequences used for occupancy analysis of putative Elf5 target genes, real time RT-PCR and gelshift experiments.

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