Lu Yi-Hsueh, Dallner Olof Stefan, Birsoy Kivanc, Fayzikhodjaeva Gulya, Friedman Jeffrey M
Laboratory of Molecular Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Laboratory of Molecular Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA ; Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Mol Metab. 2015 Feb 13;4(5):392-405. doi: 10.1016/j.molmet.2015.02.002. eCollection 2015 May.
Leptin gene expression is highly correlated with cellular lipid content in adipocytes but the transcriptional mechanisms controlling leptin expression in vivo are poorly understood. In this report, we set out to identify cis- and trans-regulatory elements controlling leptin expression.
Leptin-BAC luciferase transgenic mice combining with other computational and molecular techniques were used to identify transcription regulatory elements including a CCAAT-binding protein Nuclear Factor Y (NF-Y). The function of NF-Y in adipocyte was studied in vitro with 3T3-L1 cells and in vivo with adipocyte-specific knockout of NF-Y.
Using Leptin-BAC luciferase mice, we showed that DNA sequences between -22 kb and +8.8 kb can confer quantitative expression of a leptin reporter. Computational analysis of sequences and gel shift assays identified a 32 bp sequence (chr6: 28993820-2899385) consisting a CCAAT binding site for Nuclear Factor Y (NF-Y) and this was confirmed by a ChIP assay in vivo. A deletion of this 32 bp sequence in the -22 kb to +8.8 kb leptin-luciferase BAC reporter completely abrogates luciferase reporter activity in vivo. RNAi mediated knockdown of NF-Y interfered with adipogenesis in vitro and adipocyte-specific knockout of NF-Y in mice reduced expression of leptin and other fat specific genes in vivo. Further analyses of the fat specific NF-Y knockout revealed that these animals develop a moderately severe lipodystrophy that is remediable with leptin therapy.
These studies advance our understanding of leptin gene expression and show that NF-Y controls the expression of leptin and other adipocyte genes and identifies a new form of lipodystrophy.
瘦素基因表达与脂肪细胞中的细胞脂质含量高度相关，但对体内控制瘦素表达的转录机制了解甚少。在本报告中，我们着手鉴定控制瘦素表达的顺式和反式调节元件。
将瘦素 - BAC荧光素酶转基因小鼠与其他计算和分子技术相结合，以鉴定转录调节元件，包括CCAAT结合蛋白核因子Y（NF - Y）。在体外使用3T3 - L1细胞研究NF - Y在脂肪细胞中的功能，并在体内对NF - Y进行脂肪细胞特异性敲除来研究其功能。
使用瘦素 - BAC荧光素酶小鼠，我们发现 - 22 kb至 + 8.8 kb之间的DNA序列可赋予瘦素报告基因定量表达。对序列的计算分析和凝胶迁移试验鉴定出一个32 bp序列（chr6: 28993820 - 2899385），该序列包含核因子Y（NF - Y）的CCAAT结合位点，这在体内通过染色质免疫沉淀试验得到证实。在 - 22 kb至 + 8.8 kb瘦素 - 荧光素酶BAC报告基因中删除此32 bp序列会完全消除体内荧光素酶报告基因的活性。RNAi介导的NF - Y敲低在体外干扰脂肪生成，而在小鼠体内对NF - Y进行脂肪细胞特异性敲除会降低瘦素和其他脂肪特异性基因的表达。对脂肪特异性NF - Y敲除小鼠的进一步分析表明，这些动物会发展出中度严重的脂肪营养不良，可用瘦素治疗进行补救。
这些研究增进了我们对瘦素基因表达的理解，并表明NF - Y控制瘦素和其他脂肪细胞基因的表达，并鉴定出一种新形式的脂肪营养不良。
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