Klf4 Directly Interacts with Oct4 and Sox2 to Promote Reprogramming

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
Stem Cells (Impact Factor: 6.52). 12/2009; 27(12):2969-78. DOI: 10.1002/stem.231
Source: PubMed


Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by ectopic expression of specific sets of transcription factors. Oct4, Sox2, and Klf4, factors that share many target genes in embryonic stem (ES) cells, are critical components in various reprogramming protocols. Nevertheless, it remains unclear whether these factors function together or separately in reprogramming. Here we show that Klf4 interacts directly with Oct4 and Sox2 when expressed at levels sufficient to induce iPS cells. Endogenous Klf4 also interacts with Oct4 and Sox2 in iPS cells and in mouse ES cells. The Klf4 C terminus, which contains three tandem zinc fingers, is critical for this interaction and is required for activation of the target gene Nanog. In addition, Klf4 and Oct4 co-occupy the Nanog promoter. A dominant negative mutant of Klf4 can compete with wild-type Klf4 to form defective Oct4/Sox2/Klf4 complexes and strongly inhibit reprogramming. In the absence of Klf4 overexpression, interaction of endogenous Klf4 with Oct4/Sox2 is also required for reprogramming. This study supports the idea that direct interactions between Klf4, Oct4, and Sox2 are critical for somatic cell reprogramming.

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    • "There is ample evidence for possible involvement of several other proteins in organization of long-range interactions, including the transcription factor SP1 that contains C2H2-type zinc finger DNA-binding domain and glutamine-rich dimerization domain (Courey et al., 1989; Mastrangelo et al., 1991; Su et al., 1991), the transcription factor Klf4 (Wei et al., 2013) that interact with many transcription regulators, including Oct4 and Sox2 (Wei et al., 2009), general activator p300/CBP, and repressors such as HDAC and CtBP (Swamynathan, 2010), MAR-binding protein SATB1 (Cai et al., 2006; Gong et al., 2011), TFIIIC (Kirkland et al., 2013), and condensins (D’Ambrosio et al., 2008). In any case however, it would be premature to arrive at any definitive conclusions about the role of these proteins in the chromosome architecture. "
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    • "RNA interference experiments confirm that Klf4 is redundant with two other family members, Klf2 and Klf5, in regulating expression of pluripotency related genes. In ES cells, Klf4 has been shown to be important to activate Lefty1 together with Oct4 and Sox2 (Chan et al., 2009; Nakatake et al., 2006; Wei et al., 2009). Genome-wide chromatin immunoprecipitation with microarray analysis (ChIP-Chip) demonstrates that the DNA binding profile of Klf4 overlaps with that of Oct4 and Sox2 on promoters of genes specifically underlying establishment of iPSCs (Nakatake et al., 2006), suggesting transcriptional synergy among these factors. "
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    ABSTRACT: Embryonic stem (ES) cells are pluripotent cells with the capacity for unlimited self-renewal or differentiation. Inhibition of MAPK pathways enhances mouse ES cell pluripotency characteristics. Compared to wildtype ES cells, jnk2(-/-) ES cells displayed a much higher growth rate. To determine whether JNKs are required for stem cell self-renewal or differentiation, we performed a phosphorylation kinase array assay to compare mouse ES cells under LIF+ or LIF- culture conditions. The data showed that activation of JNKs was induced by LIF withdrawal. We also found that JNK1 or 2 phosphorylated Klf4 at threonines 224 and 225. Activation of JNK signaling and phosphorylation of Klf4 inhibited Klf4 transcription and transactivation activity. Importantly, jnk1(-/-) and jnk2(-/-) murine embryonic fibroblasts (MEFs) exhibited a significantly greater potency in the ability to increase the number of iPS colonies compared with jnk wildtype MEFs. Overall, our results demonstrated that JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.
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    • "This result probably occurred because endogenous Lefty1 expression is maintained by epigenetic regulation and other transcription factors including Oct3/4 and Sox2 [34]. Klf4 interacts with various factors, including Oct3/4, Sox2, and Glis1 [22] [39], and these interactions promote somatic cell reprogramming [22] [39]. Here, we identified Zfp296 as a novel Klf4- interacting protein. "
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    ABSTRACT: Pluripotency and self-renewing ability of embryonic stem (ES) cells are regulated by several transcription factors, including Oct3/4, Sox2, Kruppel-like factor 4 (Klf4), and c-Myc. These transcription factors reprogram somatic cells into induced pluripotent stem (iPS) cells. Zinc finger protein (Zfp) 296 has been reported to enhance iPS cell formation. Here we found that Zfp296 interacts with Klf4. A maltose-binding protein pull-down assay demonstrated that Klf4 binds to the Zfp296 158-483 amino acid region, and that Zfp296 binds to the Klf4 DNA- binding domain (DBD). A quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of Zfp296 and Klf4 decreased during differentiation of E14 and ZHBTc4 ES cells. We also found that green fluorescent protein-labeled Zfp296 and Klf4 were localized to the nucleus. Because Zfp296 bound to the Klf4 DBD, we next examined the influence of Zfp296 on Klf4 DNA-binding activity. A biotin DNA pull-down assay showed that Klf4 binds to the Lefty1 promoter region, and that binding activity was sustained even in the presence of Zfp296. In contrast, a reporter assay showed that the Lefty1 promoter was activated by Klf4, and that the enhanced activity was repressed by Zfp296. These findings suggest that Zfp296 is a functional regulator of Klf4 in ES cells.
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