Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Wolynska 33, 60-637, Poznan, Poland.
Department of Animal Physiology and Biochemistry, Poznan University of Life Sciences, Wolynska 35, 60-637, Poznan, Poland.
Chromosome Res. 2019 Sep;27(3):271-284. doi: 10.1007/s10577-019-09604-2. Epub 2019 Jan 17.
Proper expression of the PPARG gene, which encodes a key transcription factor of adipogenesis, is indispensable in the formation of mature adipocytes. The positioning of a gene within the nuclear space has been implicated in gene regulation. We here report on the significance of the PPARG gene's nuclear positioning for its activity during in vitro adipogenesis in the pig. We used an established system of differentiation of mesenchymal stem cells derived from bone marrow and adipose tissue into adipocytes. The differentiation process was carried out for 7 days, and the cells were examined using the 3D DNA/immuno-FISH and RNA/DNA-FISH approaches. PPARG transcript level was measured using real-time PCR, and PPARγ activity was detected with colorimetric assay. Changes in the nuclear location of the PPARG gene were observed when we compared undifferentiated mesenchymal stem cells with mature adipocytes. The gene moved from the nuclear periphery to the nuclear center as its transcriptional activity increased. The RNA/DNA-FISH approach shows that differences in primary transcript production correlated with the allele's nuclear positioning. Transcriptionally active alleles preferentially occupy the central part of the nucleus, while inactive alleles are found on the nuclear periphery. We also show that transcription of PPARG begins with one allele, but that both alleles are active in later stages of differentiation. Our results provide evidence that functionally distinct alleles of the PPARG gene are positioned in different parts of the cell nucleus. This confirms the importance of nuclear architecture to the regulation of PPARG gene transcription, and thus to the fate of the adipose cell.
PPARG 基因的正确表达对于脂肪生成过程中成熟脂肪细胞的形成至关重要，该基因编码脂肪生成的关键转录因子。基因在核内的位置与基因调控有关。我们在此报告了 PPARG 基因在猪体外脂肪生成过程中的核定位对其活性的重要性。我们使用了已建立的骨髓和脂肪组织来源的间充质干细胞分化为脂肪细胞的系统。分化过程持续 7 天，并使用 3D DNA/免疫荧光原位杂交（FISH）和 RNA/DNA-FISH 方法检查细胞。使用实时 PCR 测量 PPARG 转录本水平，并用比色法检测 PPARγ 活性。当我们比较未分化的间充质干细胞和成熟脂肪细胞时，观察到 PPARG 基因的核定位发生了变化。随着转录活性的增加，该基因从核周向核中心移动。RNA/DNA-FISH 方法表明，初级转录产物的差异与等位基因的核定位相关。转录活性的等位基因优先占据核的中心部分，而无活性的等位基因位于核周缘。我们还表明，PPARG 的转录从一个等位基因开始，但在分化的后期两个等位基因都活跃。我们的结果提供了证据，表明 PPARG 基因的功能不同的等位基因定位于细胞核的不同部分。这证实了核结构对 PPARG 基因转录调控的重要性，从而对脂肪细胞的命运产生了重要影响。
