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CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3' end processing.

Youngblood BA, Grozdanov PN, MacDonald CC - Nucleic Acids Res. (2014)

Bottom Line: Similarly, the role of 3' end processing in regulation of ESC pluripotency and cell cycle is poorly understood.However, τCstF-64 only partially compensates for lost CstF-64 function, despite being recruited to the histone mRNA 3' end-processing complex.Reduction of τCstF-64 in CstF-64-deficient ESCs results in even greater levels of histone mRNA polyadenylation, suggesting that both CstF-64 and τCstF-64 function to inhibit polyadenylation of histone mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430-6540, USA.

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Loss of CstF-64 results in diminished pluripotent state, decreased cell proliferation and disrupted cell cycle. Alkaline phosphatase staining of (A) WT ESCs, (B) Cstf2E6 ESCs and (C) WT ESCs cultured without LIF for 96 h, 100X magnification. (D) Relative mRNA expression of the pluripotency regulators, Pou5f1 (Oct4), Nanog, Klf4 and Lefty2 and (E) differentiation markers, T (Brachyury), Olig2 and Nkx6-3, representing mesoderm, ectoderm and endoderm germ layers, respectively. * denotes P < 0.05 and ** denotes P < 0.01. (F) Growth rate analysis of WT and Cstf2E6 ESCs. Cells were plated in triplicate and data points represent the average cell count. Standard deviation is also shown. The growth rates on days 3, 4 and 5 between the WT and Cstf2E6 ESCs were statistically significant at P < 0.05 using a one-tailed t-test. (G) Cell cycle analysis of WT and Cstf2E6 ESCs stained with PI and analyzed using flow cytometry. For the cell cycle analysis, a two-tailed t-test was performed to obtain significance on triplicate samples. The P values for both G1 and G2/M phases is P < 0.05.
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Figure 2: Loss of CstF-64 results in diminished pluripotent state, decreased cell proliferation and disrupted cell cycle. Alkaline phosphatase staining of (A) WT ESCs, (B) Cstf2E6 ESCs and (C) WT ESCs cultured without LIF for 96 h, 100X magnification. (D) Relative mRNA expression of the pluripotency regulators, Pou5f1 (Oct4), Nanog, Klf4 and Lefty2 and (E) differentiation markers, T (Brachyury), Olig2 and Nkx6-3, representing mesoderm, ectoderm and endoderm germ layers, respectively. * denotes P < 0.05 and ** denotes P < 0.01. (F) Growth rate analysis of WT and Cstf2E6 ESCs. Cells were plated in triplicate and data points represent the average cell count. Standard deviation is also shown. The growth rates on days 3, 4 and 5 between the WT and Cstf2E6 ESCs were statistically significant at P < 0.05 using a one-tailed t-test. (G) Cell cycle analysis of WT and Cstf2E6 ESCs stained with PI and analyzed using flow cytometry. For the cell cycle analysis, a two-tailed t-test was performed to obtain significance on triplicate samples. The P values for both G1 and G2/M phases is P < 0.05.

Mentions: Compared to wild type mouse ESCs (Figure 2A), the Cstf2E6 cells displayed a different phenotype that consisted of a flattened morphology and decreased propensity to grow in tightly packed clusters (Figure 2B). To assess whether these morphological differences were accompanied by changes in pluripotency markers, we stained wild type and Cstf2E6 ESCs with alkaline phosphatase, an enzyme that is not expressed in differentiated ESCs (37). Wild type ESCs displayed intense staining for alkaline phosphatase, while the Cstf2E6 cells displayed less staining, suggesting diminished pluripotency (Figure 2A, B). This reduction is similar to wild type ESCs grown in the absence of LIF for 96 h displayed reduced staining (Figure 2C).


CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3' end processing.

Youngblood BA, Grozdanov PN, MacDonald CC - Nucleic Acids Res. (2014)

Loss of CstF-64 results in diminished pluripotent state, decreased cell proliferation and disrupted cell cycle. Alkaline phosphatase staining of (A) WT ESCs, (B) Cstf2E6 ESCs and (C) WT ESCs cultured without LIF for 96 h, 100X magnification. (D) Relative mRNA expression of the pluripotency regulators, Pou5f1 (Oct4), Nanog, Klf4 and Lefty2 and (E) differentiation markers, T (Brachyury), Olig2 and Nkx6-3, representing mesoderm, ectoderm and endoderm germ layers, respectively. * denotes P < 0.05 and ** denotes P < 0.01. (F) Growth rate analysis of WT and Cstf2E6 ESCs. Cells were plated in triplicate and data points represent the average cell count. Standard deviation is also shown. The growth rates on days 3, 4 and 5 between the WT and Cstf2E6 ESCs were statistically significant at P < 0.05 using a one-tailed t-test. (G) Cell cycle analysis of WT and Cstf2E6 ESCs stained with PI and analyzed using flow cytometry. For the cell cycle analysis, a two-tailed t-test was performed to obtain significance on triplicate samples. The P values for both G1 and G2/M phases is P < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117776&req=5

Figure 2: Loss of CstF-64 results in diminished pluripotent state, decreased cell proliferation and disrupted cell cycle. Alkaline phosphatase staining of (A) WT ESCs, (B) Cstf2E6 ESCs and (C) WT ESCs cultured without LIF for 96 h, 100X magnification. (D) Relative mRNA expression of the pluripotency regulators, Pou5f1 (Oct4), Nanog, Klf4 and Lefty2 and (E) differentiation markers, T (Brachyury), Olig2 and Nkx6-3, representing mesoderm, ectoderm and endoderm germ layers, respectively. * denotes P < 0.05 and ** denotes P < 0.01. (F) Growth rate analysis of WT and Cstf2E6 ESCs. Cells were plated in triplicate and data points represent the average cell count. Standard deviation is also shown. The growth rates on days 3, 4 and 5 between the WT and Cstf2E6 ESCs were statistically significant at P < 0.05 using a one-tailed t-test. (G) Cell cycle analysis of WT and Cstf2E6 ESCs stained with PI and analyzed using flow cytometry. For the cell cycle analysis, a two-tailed t-test was performed to obtain significance on triplicate samples. The P values for both G1 and G2/M phases is P < 0.05.
Mentions: Compared to wild type mouse ESCs (Figure 2A), the Cstf2E6 cells displayed a different phenotype that consisted of a flattened morphology and decreased propensity to grow in tightly packed clusters (Figure 2B). To assess whether these morphological differences were accompanied by changes in pluripotency markers, we stained wild type and Cstf2E6 ESCs with alkaline phosphatase, an enzyme that is not expressed in differentiated ESCs (37). Wild type ESCs displayed intense staining for alkaline phosphatase, while the Cstf2E6 cells displayed less staining, suggesting diminished pluripotency (Figure 2A, B). This reduction is similar to wild type ESCs grown in the absence of LIF for 96 h displayed reduced staining (Figure 2C).

Bottom Line: Similarly, the role of 3' end processing in regulation of ESC pluripotency and cell cycle is poorly understood.However, τCstF-64 only partially compensates for lost CstF-64 function, despite being recruited to the histone mRNA 3' end-processing complex.Reduction of τCstF-64 in CstF-64-deficient ESCs results in even greater levels of histone mRNA polyadenylation, suggesting that both CstF-64 and τCstF-64 function to inhibit polyadenylation of histone mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430-6540, USA.

Show MeSH
Related in: MedlinePlus