CEA/DRF/IBFJ/iRCM/LRTS, 92265, Fontenay-aux-Roses Cedex, France.
Inserm U967, 92265, Fontenay-aux-Roses Cedex, France.
Epigenetics Chromatin. 2019 Jul 22;12(1):46. doi: 10.1186/s13072-019-0288-3.
Cell type-specific use of cis-acting regulatory elements is mediated by the combinatorial activity of transcription factors involved in lineage determination and maintenance of cell identity. In macrophages, specific transcriptional programs are dictated by the transcription factor PU.1 that primes distal regulatory elements for macrophage identities and makes chromatin competent for activity of stimuli-dependent transcription factors. Although the advances in genome-wide approaches have elucidated the functions of these macrophage-specific distal regulatory elements in transcriptional responses, chromatin structures associated with PU.1 priming and the underlying mechanisms of action of these cis-acting sequences are not characterized.
Here, we show that, in macrophages, FACT subunit SPT16 can bind to positioned nucleosomes directly flanking PU.1-bound sites at previously uncharacterized distal regulatory elements located near genes essential for macrophage development and functions. SPT16 can interact with the transcriptional co-regulator TRIM33 and binds to half of these sites in a TRIM33-dependent manner. Using the Atp1b3 locus as a model, we show that FACT binds to two positioned nucleosomes surrounding a TRIM33/PU.1-bound site in a region, located 35 kb upstream the Atp1b3 TSS, that interact with the Atp1b3 promoter. At this - 35 kb region, TRIM33 deficiency leads to FACT release, loss of the two positioned nucleosomes, RNA Pol II recruitment and bidirectional transcription. These modifications are associated with higher levels of FACT binding at the Atp1b3 promoter, an increase of RNA Pol II recruitment and an increased expression of Atp1b3 in Trim33 macrophages.
Thus, sequestering of SPT16/FACT by TRIM33 at PU.1-bound distal regions might represent a new regulatory mechanism for RNA Pol II recruitment and transcription output in macrophages.
细胞类型特异性顺式作用调控元件的使用是由参与谱系确定和细胞身份维持的转录因子的组合活性介导的。在巨噬细胞中，特定的转录程序由转录因子 PU.1 决定，PU.1 为巨噬细胞身份的远端调控元件做好准备，并使染色质有能力进行刺激依赖性转录因子的活性。尽管全基因组方法的进步已经阐明了这些巨噬细胞特异性远端调控元件在转录反应中的功能，但与 PU.1 引发相关的染色质结构和这些顺式作用序列的作用机制尚不清楚。
在这里，我们发现在巨噬细胞中，FACT 亚基 SPT16 可以直接结合到位于先前未表征的远端调控元件附近的、靠近对巨噬细胞发育和功能至关重要的基因的 PU.1 结合位点两侧的定位核小体。SPT16 可以与转录共调节剂 TRIM33 相互作用，并以 TRIM33 依赖的方式与这些位点的一半结合。使用 Atp1b3 基因座作为模型，我们表明 FACT 结合到围绕 Atp1b3 TSS 上游 35 kb 处与 Atp1b3 启动子相互作用的一个区域中的两个定位核小体，该区域包含一个 TRIM33/PU.1 结合位点。在这个 -35 kb 区域，TRIM33 缺陷导致 FACT 释放、两个定位核小体丢失、RNA Pol II 募集和双向转录。这些修饰与 Atp1b3 启动子处更高水平的 FACT 结合、RNA Pol II 募集的增加以及 Trim33 巨噬细胞中 Atp1b3 的表达增加有关。
因此，TRIM33 在 PU.1 结合的远端区域对 SPT16/FACT 的隔离可能代表了巨噬细胞中 RNA Pol II 募集和转录输出的一种新的调节机制。
