Open Access Highly Accessed Research article

Direct targets of Klf5 transcription factor contribute to the maintenance of mouse embryonic stem cell undifferentiated state

Silvia Parisi12*, Luca Cozzuto1, Carolina Tarantino12, Fabiana Passaro3, Simona Ciriello1, Luigi Aloia12, Dario Antonini12, Vincenzo De Simone3, Lucio Pastore13 and Tommaso Russo13*

Author Affiliations

1 CEINGE Biotecnologie Avanzate, Via Gaetano Salvatore 482, 80145 Naples, Italy

2 European School of Molecular Medicine (SEMM), Via Gaetano Salvatore 482, 80145 Naples, Italy

3 Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II", Via Sergio Pansini 5, 80131 Naples, Italy

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BMC Biology 2010, 8:128  doi:10.1186/1741-7007-8-128

Published: 27 September 2010

Additional files

Additional file 1:

Additional Table 1. Gene expression profile by microarray analysis upon Klf5 KD in ESCs. Fold change is calculated by comparing data from siKlf5 cells over the control (siNS). Cut-off >1.25- and <0.75-fold changes were used. Probes with FDR <0.1 were selected.

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Additional file 2:

Additional Figure 1. GO analysis of Klf5 targets and Klf expression profile during ESC differentiation. (a) Gene ontology (GO) annotation of the selected probe sets according to DAVID "Biological Process Classification" tool (Laboratory of Immunopathogenesis and Bioinformatics, Clinical Services Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD, USA). (b) Expression levels of Klf2, Klf4 and Klf5 were measured by qPCR in undifferentiated (t0) and 4-day (4d) and 7-day (7d) differentiated ESCs. The data are represented as fold changes relative to undifferentiated cells.

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Additional file 3:

Additional Table 2. Microarray data were compared with published results. Column siKlf5/siNS is referred to our microarray data (see also Additional Table 1). Differences with published data are highlighted in red. dw: downregulated gene, up: upregulated gene; na: data not available.

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Additional file 4:

Additional Figure 2. ChIP-seq validation by ChIP-qPCR. (a) Expression level of FLAG-Klf5 stable clone pools used to prepare chromatin for ChIP-seq experiment. Western blot was stained with an anti-FLAG and anti-Klf5. (b) List of peaks validated by ChIP-qPCR. Peaks with different numbers of tags were chosen. Peak location are indicated (chr, chromosome). (c) ChIP-seq validation was performed by ChIP-qPCR using anti-FLAG antibody and IgG, as control, with extracts derived from FLAG-Klf5 and Mock transfected ESCs. The data are expressed as the amount of precipitated DNA calculated relative to the total input chromatin. Samples from 1 to 15 correspond to regions close to the following genes: Agap1, Lamc2, Fcgr3, 170009P17Rik, Tgfβ2, Smx16, Nlgn1, Epha2 (upstream region), Epha2 (downstream region), Igfbp7, Serpine1, Cyp2s1, 4930467E23Rik, AC152164, Inpp4b, respectively. Three different control regions were chosen (samples 16, 17 and 18): chr1:10573933-10573984, chr1:71481391-71481461, chr3:12034661-12034625, respectively, where no significant peaks were found. Bars represent SD of triplicates.

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Additional file 5:

Additional Table 3. Results of ChIP-seq analysis. The putative binding sites of Klf5 are reported with relative number of tags for each peak and FDR. Match with gene microarray data is shown. The distance from the 5' and 3' boundaries of the Klf5-regulated genes is indicated.

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Additional file 6:

Additional Figure 3. Klf5 binding motifs identified with CisFinder via 200-bp sequences centered at binding peaks (E-score > 22).

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Additional file 7:

Additional Figure 4. qPCR validation of microarray data. Sixty Klf5 target genes were analyzed by qPCR to confirm the microarray data. Probe set of both downregulated (a) and upregulated (b) genes upon Klf5 KD is shown. Black bars represent not validated probes. The data are expressed as fold change relative to siNS transfected cells. Validated probes showed a P < 0.01.

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Additional file 8:

Additional Figure 5. KD of a subset of Klf5-target genes. (a) ESCs were stably transfected with shRNA plasmids for selected Klf5-target genes or with control shRNA (shGFP) and KD was verified by qPCR. The results are represented as fold changes relative to shGFP-transfected cells. SD of triplicates is reported. (b) Percentage of undifferentiated (blue) and differentiated (red) colonies observed by AP staining upon KD of eight Klf5-target genes with a second independent shRNA. *P < 0.01. (c) Expression levels of Oct3/4 and Nanog upon KD of eight Klf5-target genes with a second independent shRNA. The data are represented as fold changes relative to shGFP-transfected cells.

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Additional file 9:

Additional Figure 6. Expression of early differentiation markers of endoderm (Sox17), mesoderm (Brachyury) and ectoderm (Fgf5) upon KD of eight Klf5-target genes.

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Additional file 10:

Additional Figure 7. Klf5 KD in primary keratinocytes and Klf2 and Klf4 KD in ESCs. (a) Klf5 or NS siRNA were transfected in primary keratinocytes and Klf5 expression level was measured 12 hr after transfection by Western blot with anti-Klf5 antibody. (b) Expression levels of Klf2, Klf4 and Klf5 were measured by qPCR in ESCs 12 hours after siRNA transfection. The results are represented as fold changes. Bars represent SD of triplicates. P < 0.01.

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Additional file 11:

Additional Table 4. Sequences of shRNAs.

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Additional file 12:

Additional Table 5. Primers used for qPCR.

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Additional file 13:

Additional Table 6. Primers used for ChIP-qPCR.

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