Genome-wide disclosure of the Nkx2.2 targets in oligodendrocytes and their progenitor cells suggests several novel target genes

The Nkx2.2 transcription factor contributes to regulate the timing of myelination in the CNS. Still, information about the regulon of Nkx2.2 in oligodendrocytes (Ols) and their progenitor cells (OPCs) is restricted to a few genes. Therefore we aimed to identify the entire regulon of Nkx2.2. We performed transcript profiling of postnatal Nkx2.2-/- mice, investigated the expression of selected transcripts in Ol lineage cells after siRNA induced Nkx2.2 knock-down, and identified the genome-wide active Nkx2.2 binding sites in murine OPCs and Ols by use of ChIP-sequencing technique. Thereby we have found 521 genes with active Nkx2.2 binding sites within 10,000 bp of their gene loci that furthermore are differently expressed in absence or lower expression of Nkx2.2. Functionally, the target genes are associated with mitosis, migration stop, protein transport, lipid metabolism, mitochondrial biogenesis, formation of Ca2+ waves, and WNT signalling. Furthermore, motif discovery techniques and tests for overlaps between sequencing reads suggest that MYRF, OLIG2, SOX10, and TCF3 interact or compete with Nkx2.2 in OPCs/Ols. However, the activator versus repressor activity of Nkx2.2 does not seem to depend on the potential interaction with either of the transcription factors. Test for overlaps with sites of specific histone 3 modifications show that the Nkx2.2 binding sites are more frequently associated H3K4me3 and H3K27ac in genes to which Nkx2.2 may act as an activator. This supports previous studies showing that Nkx2.2 acts in a HDAC1 dependent way. Together this provides new insight into how Nkx2.2 contributes to the timing of OPC differentiation and myelination in CNS. Genome-wide Nkx2.2 targets in mouse oligodendrocyte progenitor cells and in ex vivo differentiated oligodendrocytes examined by ChIP-sequencing technique.

Nkx2.2转录因子（Nkx2.2 transcription factor）可参与调控中枢神经系统（central nervous system, CNS）内髓鞘形成的时序进程。然而，目前关于少突胶质细胞（oligodendrocytes, Ols）及其祖细胞（oligodendrocyte progenitor cells, OPCs）中Nkx2.2调控子（regulon）的相关研究信息仅局限于少数基因。为此，本研究旨在完整鉴定Nkx2.2的调控网络。我们对出生后Nkx2.2基因敲除（Nkx2.2-/-）小鼠开展了转录谱分析，通过小干扰RNA（small interfering RNA, siRNA）介导的Nkx2.2敲低实验，检测了少突胶质细胞谱系细胞中特定转录本的表达水平，并利用染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）技术，鉴定了小鼠少突胶质细胞祖细胞与少突胶质细胞内全基因组范围的活性Nkx2.2结合位点。本研究共筛选得到521个基因，其基因座上下游10,000 bp范围内存在活性Nkx2.2结合位点，且在Nkx2.2缺失或表达下调的样本中呈现显著差异表达。功能富集分析显示，这些靶基因主要富集于有丝分裂、迁移停止、蛋白质转运、脂质代谢、线粒体生物发生、钙波形成以及Wnt信号通路（WNT signalling）等生物学过程。此外，基序发现技术与测序读段重叠性分析结果表明，MYRF、OLIG2、SOX10及TCF3可在少突胶质细胞祖细胞/少突胶质细胞中与Nkx2.2发生相互作用或竞争结合。值得注意的是，Nkx2.2的转录激活或抑制活性似乎并不依赖于与上述任一转录因子的潜在相互作用。对组蛋白3修饰位点的重叠性分析显示，在Nkx2.2发挥转录激活功能的靶基因中，Nkx2.2结合位点更频繁地与H3K4me3和H3K27ac修饰相关联，这与此前关于Nkx2.2以依赖于组蛋白去乙酰化酶1（histone deacetylase 1, HDAC1）的方式发挥调控功能的研究结论一致。综上，本研究为解析Nkx2.2如何参与调控中枢神经系统内少突胶质细胞祖细胞分化及髓鞘形成时序提供了全新的认知。本数据集涵盖通过染色质免疫共沉淀测序技术鉴定的小鼠少突胶质细胞祖细胞及体外（ex vivo）分化少突胶质细胞内全基因组范围的Nkx2.2靶基因信息。