Supplementary Material for: An Alternative Transcription Start Site Yields Estrogen-Unresponsive <b><i>Kiss1</i></b> mRNA Transcripts in the Hypothalamus of Prepubertal Female Rats

The importance of the <i>Kiss1</i> gene in the control of reproductive development is well documented. However, much less is known about the transcriptional regulation of <i>Kiss1</i> expression in the hypothalamus. Critical for these studies is an accurate identification of the site(s) where <i>Kiss1</i> transcription is initiated. Employing 5′-RACE PCR, we detected a transcription start site (TSS1) used by the hypothalamus of rats, mice, nonhuman primates and humans to initiate <i>Kiss1</i> transcription. In rodents, an exon 1 encoding 5′-untranslated sequences is followed by an alternatively spliced second exon, which encodes 5′-untranslated regions of two different lengths and contains the translation initiation codon (ATG). In nonhuman primates and humans, exon 2 is not alternatively spliced. Surprisingly, in rat mediobasal hypothalamus (MBH), but not preoptic area (POA), an additional TSS (TSS2) located upstream from TSS1 generates an exon 1 longer (377 bp) than the TSS1-derived exon 1 (98 bp). The content of TSS1-derived transcripts increased at puberty in the POA and MBH of female rats. It also increased in the MBH after ovariectomy, and this change was prevented by estrogen. In contrast, no such changes in TSS2-derived transcript abundance were detected. Promoter assays showed that the proximal TSS1 promoter is much more active than the putative TSS2 promoter, and that only the TSS1 promoter is regulated by estrogen<i>. </i>These differences appear to be related to the presence of a TATA box and binding sites for transcription factors activating transcription and interacting with estrogen receptor-α in the TSS1, but not TSS2, promoter.

<i>Kiss1</i>基因在生殖发育调控中的重要性已得到充分证实。然而，目前对于下丘脑内<i>Kiss1</i>基因表达的转录调控机制，我们的认知仍较为匮乏。此类研究的核心前提是精准鉴定<i>Kiss1</i>转录的起始位点。本研究采用5′-RACE PCR（5'末端快速扩增聚合酶链式反应），在大鼠、小鼠、非人灵长类及人类的下丘脑组织中，检测到了一种用于启动<i>Kiss1</i>转录的转录起始位点（transcription start site, TSS1）。在啮齿类动物中，编码5′非翻译序列的外显子1之后，衔接一个经可变剪接的第二外显子：该外显子可编码两种不同长度的5′非翻译区，且包含翻译起始密码子（translation initiation codon, ATG）。而在非人灵长类与人类中，外显子2不存在可变剪接现象。令人意外的是，仅在大鼠下丘脑内侧基底部（mediobasal hypothalamus, MBH）而非视前区（preoptic area, POA）中，存在一个位于TSS1上游的额外转录起始位点（TSS2）：该位点介导产生的外显子1长度为377 bp，较TSS1衍生的外显子1（98 bp）更长。雌性大鼠视前区与下丘脑内侧基底部中，TSS1衍生的转录本丰度在青春期显著升高。该转录本在下丘脑内侧基底部的水平同样会在卵巢切除术后上升，且这一变化可被雌激素阻断。与之相反，未检测到TSS2衍生的转录本丰度存在此类变化。启动子活性实验结果显示，近端TSS1启动子的活性远高于推测的TSS2启动子，且仅TSS1启动子可受雌激素调控。上述差异似乎与TSS1启动子中存在TATA盒（TATA box）、激活转录的转录因子结合位点以及可与雌激素受体α（estrogen receptor-α）结合的序列相关，而TSS2启动子则不具备此类调控元件。

搜集汇总

背景与挑战

背景概述

该数据集是Kiss1基因在雌性青春期前大鼠下丘脑转录调控研究的补充材料，主要关注两个转录起始点（TSS1和TSS2）的差异。研究发现TSS1衍生的转录本受青春期和雌激素调控，而TSS2衍生的转录本则无此响应，揭示了Kiss1基因转录的复杂性和组织特异性机制。数据集包括原始实验数据，支持神经内分泌学领域的研究，并遵循CC BY 4.0许可协议。