Building a schizophrenia genetic network: Transcription Factor 4 regulates genes involved in neuronal development and schizophrenia risk. Building a schizophrenia genetic network: Transcription Factor 4 regulates genes involved in neuronal development and schizophrenia risk

The transcription factor 4 (TCF4) locus is a robust association finding with schizophrenia (SZ), but little is known about the genes regulated by the encoded transcription factor. Therefore, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) of TCF4 in neural-derived (SH-SY5Y) cells to identify genome-wide TCF4 binding sites, followed by data integration with SZ association findings. We identified 11,322 TCF4 binding sites overlapping in two ChIP-seq experiments. These sites are significantly enriched for the TCF4 Ebox binding motif (>85% having ≥1 Ebox) and implicate a gene set enriched for genes down-regulated in TCF4 siRNA knockdown experiments, indicating the validity of our findings. The TCF4 gene set was also enriched among 1) Gene Ontology categories such as axon/neuronal development, 2) genes preferentially expressed in brain, in particular pyramidal neurons of the somatosensory cortex, and 3) genes down-regulated in post-mortem brain tissue from SZ patients (OR=2.8, permutation p<4x10-5). Considering genomic alignments, TCF4 binding sites significantly overlapped those for neural DNA binding proteins such as FOXP2 and the SZ-associated EP300. TCF4 binding sites were modestly enriched among SZ risk loci from the Psychiatric Genomic Consortium (OR=1.56, p=0.03). In total, 130 TCF4 binding sites occurred in 39 of the 108 regions published in 2014. Thirteen genes within the 108 loci had both a TCF4 binding site ±10kb and were differentially expressed in siRNA knockdown experiments of TCF4, suggesting direct TCF4 regulation. These findings confirm TCF4 as an important regulator of neural genes and point towards functional interactions with potential relevance for SZ. Overall design: ChIP-seq for TCF4 (long isoform) using two different antibodies in SH-SY5Y cells, using IgG ChIP as control

转录因子4（TCF4）基因座是与精神分裂症（SZ）存在强关联的遗传位点，但目前对该基因编码的转录因子所调控的靶基因仍所知甚少。为此，我们对神经来源的SH-SY5Y细胞中的TCF4开展染色质免疫沉淀测序（ChIP-seq），以鉴定全基因组范围内的TCF4结合位点，随后将测序数据与精神分裂症关联研究结果进行整合分析。
我们在两次独立的ChIP-seq实验中鉴定出11322个重合的TCF4结合位点。这些位点显著富集TCF4的Ebox结合基序（超过85%的位点包含至少一个Ebox基序），且其所关联的基因集在TCF4小干扰RNA（siRNA）敲低实验中呈现显著下调，证实了本研究结果的可靠性。
该TCF4靶基因集还显著富集于以下类别：1）基因本体论（Gene Ontology，GO）功能分类，如轴突/神经元发育相关条目；2）脑内优先表达的基因，尤其是体感皮层的锥体神经元；3）精神分裂症患者死后脑组织中呈现下调表达的基因（优势比OR=2.8，置换检验p<4×10^-5）。
通过基因组比对分析发现，TCF4结合位点与FOXP2等神经DNA结合蛋白以及精神分裂症关联的EP300的结合位点存在显著重叠。TCF4结合位点在精神病基因组学联盟（Psychiatric Genomic Consortium，PGC）鉴定的精神分裂症风险基因座中呈现轻度富集（OR=1.56，p=0.03）。
总计有130个TCF4结合位点分布于2014年已发表的108个精神分裂症关联区域中的39个区域内。在这108个基因座中，有13个基因既存在±10kb范围内的TCF4结合位点，又在TCF4的siRNA敲低实验中呈现差异表达，提示TCF4对这些基因存在直接调控作用。
本研究结果证实TCF4是神经基因的重要调控因子，并揭示了其与精神分裂症潜在相关的功能互作机制。
总体实验设计：在SH-SY5Y细胞中，采用两种不同抗体靶向TCF4长亚型进行ChIP-seq测序，并以IgG免疫沉淀作为对照。