Roles of Klf5 Acetylation in the Self-Renewal and the Differentiation of Mouse Embryonic Stem Cells

<div><p>Transcription factor Krüppel-like factor 5 (Klf5) plays important roles in the formation of the inner cell mass (ICM) and the trophectoderm during embryogenesis, as well as the self-renewal and the differentiation of mouse embryonic stem cells (ESCs). Acetylation of KLF5 has been shown to reverse the transcriptional activity of KLF5 in human epidermal cells and prostate cancer cells. Whether Klf5 acetylation contributes to the lineage specification in the blastocyst and pluripotency maintenance in ESCs remains unexplored. Here, we showed the ubiquitous expression of acetylated Klf5 in the ICM and the trophectoderm, ruling out the possibility that differential acetylation status of Klf5 leads to the lineage specification in the blastocyst. We found that K358Q mutation, mimicking acetylation, enhances the transcriptional activity of Klf5 for pluripotency genes in ESCs, and that K358Q Klf5 is more potent in pluripotency maintenance and in somatic cell reprogramming, compared to K358R Klf5. In ESCs, Klf5 acetylation, stimulated by TGF-β signaling, is involved in enhancing Sox2 expression. Moreover, upon ESC differentiation, acetylation of Klf5 facilitates the suppression of many differentiation genes, except for that K358Q Klf5 activates <i>Cdx2</i>, promoting trophectodermal differentiation. In summary, our results revealed the regulatory functions of Klf5 acetylation in ESC self-renewal and differentiation.</p></div

Regional classification of mainland China.

<p>Regional classification of mainland China.</p

Decomposition of the first hierarchy from the supply side (%).

<p>Decomposition of the first hierarchy from the supply side (%).</p

Intermediate demand structure from the demand side of production (%).

<p>Intermediate demand structure from the demand side of production (%).</p

Functions of acetylated Klf5 in mouse ESC differentiation.

<p>(A) Expression of total, Ac-, and unAc-Klf5 during ESC differentiation. The nonspecific band in the anti-Klf5 blot is marked by an asterisk. V6.5 ESCs were cultured without LIF and feeder cells. Cells were harvested on day 0, 2, 4, and 6, and subjected to Western blot assay. (B) Klf5 knockdown affects the expression of pluripotency genes and differentiation genes in day 4 EBs. Day 4 EBs from shGFP control ESCs and two independent stable Klf5 knockdown ESC clones were harvested for quantitative RT-PCR analysis. Averages and standard deviations from three independent experiments were plotted. (C) Rescue effect of WT, KR, and KQ Klf5 in <i>Klf5</i> knockdown ESCs. <i>shKlf5</i> targeting sequences in WT, KR, and KQ Klf5 were mutated to render them resistant to <i>shKlf5</i> knockdown. Stable ESC lines overexpressing shKlf5-immune WT, KR, and KQ Klf5 were established in the Klf5 knockdown ESCs. Day 4 EBs from these ESCs were harvested for quantitative RT-PCR analysis. Averages and standard deviations from three independent experiments were plotted. P values were calculated by reusable two-factor analysis of variance.</p

Utilization efficiency of R&D internal expenditure.

<p>Utilization efficiency of R&D internal expenditure.</p

Decomposition of the second and third hierarchies from the demand side (%).

<p>Decomposition of the second and third hierarchies from the demand side (%).</p

Klf5 acetylation, stimulated by TGF-β signaling, facilitates pluripotency maintenance in mouse ESCs.

<p>(A) TGF-β signaling promotes Klf5 acetylation. V6.5 ESCs were treated with 10 μg/ml TGF-β or 5 μM SB525334 (SB) for 48 hours, and then harvested for subsequent Western blot experiments. The nonspecific band in the anti-Klf5 blot is marked by an asterisk. The expression levels of Ac-Klf5 were quantified and normalized to Klf5. Averages and standard deviations of Ac-Klf5 expression from three experiments are shown. (B) Overexpression of KQ Klf5 mutant antagonizes the suppression effect of TGF-β signaling on Sox2 protein expression. Empty pCAGIPuro, Flag-tagged WT and KQ Klf5 overexpression plasmids were transfected into V6.5 ESCs. Cells were cultured in mouse ESC medium with or without 5 μM SB525334 for 2 days, and then harvested for subsequent Western blot experiments. The nonspecific band in the anti-Klf5 blot is marked by an asterisk. (C) The effect of WT and KQ Klf5 overexpression on the repression of pluripotency gene transcription by TGF-β signaling. Cells were treated as described in (B), and harvested for RNA purification and quantitative RT-PCR analysis. Averages and standard deviations from three independent experiments were plotted.</p

Decomposition of R&D internal expenditure from the supply and demand sides.

<p>Abbreviations are used for each region, and “NT” means national level.</p

Growth of R&D internal expenditure and output value of new products.

<p>Growth of R&D internal expenditure and output value of new products.</p