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1.  Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts 
Molecular Cancer  2013;12:89.
Background
Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy.
Methods
To determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs null for Klf4 (Klf4−/−), we transfected Klf4−/−MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4+/+) and untransfected or mock-transfected Klf4−/−MEFs.
Results
We show that overexpression of Klf4 in Klf4−/−MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4−/−MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.
Conclusion
Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.
doi:10.1186/1476-4598-12-89
PMCID: PMC3750599  PMID: 23919723
KLF4; Genetic instability; DNA damage responses; Aneuploidy; Centrosome amplification; Mouse embryonic fibroblasts
2.  Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts 
Background
Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4.
Methods
Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels.
Results
One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data.
Conclusions
These data are not only consistent with previous functional studies of KLF4’s role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4’s functions.
PMCID: PMC3165197  PMID: 21892412
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
3.  Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts 
Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions.
PMCID: PMC3165197  PMID: 21892412
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
4.  Nodal signals mediate interactions between the extra-embryonic and embryonic tissues in zebrafish 
Developmental biology  2007;310(2):363-378.
In many vertebrates, extra-embryonic tissues are important signaling centers that induce and pattern the germ layers. In teleosts, the mechanism by which the extra-embryonic yolk syncytial layer (YSL) patterns the embryo is not understood. Although the Nodal-related protein Squint is expressed in the YSL, its role in this tissue is not known. We generated a series of stable transgenic lines with GFP under the control of squint genomic sequences. In all species, nodal-related genes induce their own expression through a positive feedback loop. We show that two tissue specific enhancers in the zebrafish squint gene mediate the response to Nodal signals. Expression in the blastomeres depends upon a conserved Nodal response element (NRE) in the squint first intron, while expression in the extra-embryonic enveloping layer (EVL) is mediated by an element upstream of the transcription start site. Targeted depletion experiments demonstrate that the zebrafish Nodal-related proteins Squint and Cyclops are required in the YSL for endoderm and head mesoderm formation. Thus, Nodal signals mediate interactions between embryonic and extra-embryonic tissues in zebrafish that maintain nodal-related gene expression in the margin. Our results demonstrate a high degree of functional conservation between the extra-embryonic tissues of mouse and zebrafish.
doi:10.1016/j.ydbio.2007.08.008
PMCID: PMC2044568  PMID: 17850782
Zebrafish; squint; cyclops; EVL; YSL; Nodal-related; mesoderm; endoderm; extra-embryonic; SB-505124
5.  Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish 
Background
The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-β superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.
Results
We show that two drugs, SB-431542 and SB-505124, completely block the response to Nodal signals when added to embryos after the mid-blastula transition. By blocking Nodal receptor activity at later stages, we demonstrate that Nodal signaling is required from the mid-to-late blastula period to specify sequentially, the somites, notochord, blood, Kupffer's vesicle, hatching gland, heart, and endoderm. Blocking Nodal signaling at late times prevents specification of cell types derived from the embryo margin, but not those from more animal regions. This suggests a linkage between cell fate and length of exposure to Nodal signals. Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure. Finally, cell fate specification is delayed in squint mutants and accelerated when Nodal levels are elevated.
Conclusion
We conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.
doi:10.1186/1471-213X-7-22
PMCID: PMC1851950  PMID: 17391517

Results 1-5 (5)