Variants in an Hdac9 intronic enhancer plasmid impact Twist1 expression in vitro.
Variants in an Hdac9 intronic enhancer plasmid impact Twist1 expression in vitro.
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DOI：
10.1007/s00335-015-9618-3
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发表时间：
2016-04
期刊：
影响因子：
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通讯作者：
Toland AE
中科院分区：
文献类型：
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作者：
Siekmann TE;Gerber MM;Toland AE
皮肤肿瘤易感性5 （Skts5）先前通过对皮肤肿瘤易感小家鼠（NIH/Ola-S）和皮肤肿瘤抗性近交小家鼠（SPRET/Out-R）小鼠的连锁分析定位到小鼠12号染色体上。基于保守的非同义序列变异和表达分析，Hdac9被确定为Skts5的潜在候选基因。其他人的研究在人类HDAC9中发现了一个与TWIST1表达相关的增强子。我们从人类HDAC9增强子的同源区域中鉴定出NIH/Ola-S和SPRET/Out-R小鼠之间的45个序列变异。定位于内含子18的变异对荧光素酶的体外表达有差异影响。NIH/Ola-S克隆的荧光素酶表达量比单独载体或来自SPRET/Out-R-R的等效克隆增加了约1.7倍。此外，转染了部分NIH/Ola-S内含子的细胞诱导Twist1表达增加2.2倍，但来自SPRET/Out-R小鼠的相同区域并未导致Twist1表达上调。在硅转录因子分析中，发现了多个转录因子预测会差异结合NIH/Ola-S和SPRET/Out-R多态性位点。两种转录因子Gata3和Oct1的染色质免疫沉淀研究证实了NIH/Ola-S和SPRET/Out-R质粒之间的差异结合，证实了计算机预测。总之，这些研究提供了证据，证明小鼠与人类HDAC9增强子的同源区域也可以作为小鼠Twist1的转录增强子。由于NIH/Ola-S和SPRET/Out-R之间的异位序列变异差异影响荧光素酶的表达，与体外Twist1表达相关，并影响Gata3和Oct1的结合，这些变异可能解释了NIH/Ola-S和SPRET/Out-R之间皮肤肿瘤易感Skts5的部分差异。
Skin tumorsusceptibility 5 (Skts5) was previously mapped to mouse chromosome 12 through linkage analysis of skin tumor susceptible Mus musculus (NIH/Ola-S) and skin tumor resistant outbred Mus spretus (SPRET/Out-R) mice. Hdac9 was identified as a potential candidate for Skts5 based on conserved non-synonymous sequence variants and expression analyses. Studies by others identified an enhancer in human HDAC9 that correlated with TWIST1 expression. We identified 45 sequence variants between NIH/Ola-S and SPRET/Out-R mice from the orthologous region of the human HDAC9 enhancer. Variants mapping to intron 18 differentially affected luciferase expression in vitro. NIH/Ola-S clones showed an approximate 1.7-fold increased luciferase expression relative to vector alone or the equivalent clones from SPRET/Out-R-R. Furthermore, cells transfected with a portion of the NIH/Ola-S intron induced 2.2-fold increases in Twist1 expression, but the same region from SPRET/Out-R mice resulted in no up-regulation of Twist1. In silico transcription factor analyses identified multiple transcription factors predicted to differentially bind NIH/Ola-S and SPRET/Out-R polymorphic sites. Chromatin immunoprecipitation studies of two transcription factors, Gata3 and Oct1, demonstrated differential binding between NIH/Ola-S and SPRET/Out-R plasmids that corroborated the in silico predictions. Together these studies provide evidence that the murine orthologous region to a human HDAC9 enhancer also acts as a transcriptional enhancer for mouse Twist1. As ectopic sequence variants between NIH/Ola-S and SPRET/Out-R differentially impacted luciferase expression, correlated with Twist1 expression in vitro and affected Gata3 and Oct1 binding, these variants may explain part of the observed differences in skin tumor susceptibility at Skts5 between NIH/Ola-S and SPRET/Out-R.