Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA.
J Biol Chem. 2010 Feb 26;285(9):6443-52. doi: 10.1074/jbc.M109.084285. Epub 2009 Dec 22.
The human negative elongation factor (NELF) is a four-subunit protein complex that inhibits the movement of RNA polymerase II (RNAPII) at an early elongation stage in vitro. NELF-mediated stalling of RNAPII also attenuates transcription of a number of inducible genes in human cells. To obtain a genome-wide understanding of human NELF-mediated transcriptional regulation in vivo, we carried out an exon array study in T47D breast cancer cells with transient small interfering RNA knockdown of individual NELF subunits. Upon depletion of NELF-A, -C, or -E, the vast majority of NELF-regulated genes were down-regulated. Many of the down-regulated genes encode proteins that play key roles in cell cycle progression. Consequently, NELF knockdown resulted in significant reduction in DNA synthesis and cell proliferation. Chromatin immunoprecipitation showed that NELF knockdown led to dissociation of RNAPII from the promoter-proximal region of the cell cycle-regulating genes. This was accompanied by decreased histone modifications associated with active transcription initiation (H3K9Ac) and elongation (H3K36Me3), as well as reduced recruitment of the general transcription factor TFIIB and increased overall histone occupancy at a subset of the down-regulated promoters. Lastly, our study indicates that NELF regulates alternative transcription initiation of BSG (Basigin) gene by differentially influencing RNAPII density at the two neighboring exons at the 5' end of the gene. Taken together, our data suggest a diverse transcriptional consequence of NELF-mediated RNAPII pausing in the human genome.
人类负延伸因子（NELF）是一个由四个亚基组成的蛋白质复合物，它在体外早期延伸阶段抑制 RNA 聚合酶 II（RNAPII）的移动。NELF 介导的 RNAPII 停滞也会减弱人类细胞中许多诱导基因的转录。为了在体内获得对人类 NELF 介导的转录调控的全基因组理解，我们在 T47D 乳腺癌细胞中进行了外显子数组研究，用单个 NELF 亚基的瞬时小干扰 RNA 敲低进行实验。在 NELF-A、-C 或 -E 耗尽后，绝大多数 NELF 调节的基因下调。许多下调的基因编码在细胞周期进展中起关键作用的蛋白质。因此，NELF 敲低导致 DNA 合成和细胞增殖显著减少。染色质免疫沉淀显示，NELF 敲低导致 RNAPII 从细胞周期调节基因的启动子近端区域解离。这伴随着与活性转录起始（H3K9Ac）和延伸（H3K36Me3）相关的组蛋白修饰减少，以及一般转录因子 TFIIB 的募集减少和一组下调启动子的总体组蛋白占有率增加。最后，我们的研究表明，NELF 通过在基因 5' 端的两个相邻外显子上影响 RNAPII 密度，调节 BSG（Basigin）基因的替代转录起始。总之，我们的数据表明，NELF 介导的 RNAPII 暂停在人类基因组中具有多样化的转录后果。
