Genetic deletion of ZNF91 in human embryonic stem cells leads to ectopic activation of SVAs and collective upregulation of KRAB zinc finger gene clusters. Genetic deletion of ZNF91 in human embryonic stem cells leads to ectopic activation of SVAs and collective upregulation of KRAB zinc finger gene clusters

Transposable element (TE) invasions have shaped vertebrate genomes over the course of evolution. They have contributed an extra layer of species-specific gene regulation by providing novel transcription factor binding sites. In humans, SVA elements are one of three still active TE families, and approximately 2800 SVA insertions exist in the human genome, half of which are human-specific. TEs are often silenced by KRAB zinc finger (KZNF) proteins recruiting co-repressor proteins that establish a repressive chromatin state. A number of KZNFs have been reported to bind SVAs, but their individual contribution to repressing SVAs and their roles in suppressing SVA-mediated gene-regulatory effects remains elusive. We analyzed the genome-wide binding profile for ZNF91 in human cells and found that ZNF91 interacts with the VNTR region of SVAs. Through CRISPR-Cas9 mediated deletion of ZNF91 in human embryonic stem cells we established that loss of ZNF91 results in increased transcriptional activity of SVAs. In contrast, SVA activation was not observed upon genetic deletion of the ZNF611 gene encoding another strong SVA-interactor. Epigenetic profiling confirmed the loss of SVA repression in the absence of ZNF91 and revealed that mainly evolutionary young SVAs gain gene activation-associated epigenetic modifications. Genes close to activated SVAs showed a mild upregulation, indicating SVAs adopt properties of cis-regulatory elements in the absence of repression. Intriguingly, genome-wide de-repression of SVAs elicited the communal upregulation of KZNFs that reside in KZNF clusters. This phenomenon may provide new insights into the potential mechanisms utilized by the host genome to sense and counteract TE invasions. Overall design: Identification of ZNF91 binding sites in HEK293 cells using ZNF91-GFP overexpression followed by ChIP-seq. RNA seq and ChIP seq analysis of CRISPR/Cas9 ZNF91 wild type and knockout human embryonic stem cell lines

转座因子（Transposable Element, TE）入侵在演化进程中塑造了脊椎动物基因组。它们通过提供全新的转录因子结合位点，为物种特异性基因调控增添了一层额外的调控层级。在人类中，SVA因子是目前仍具有活性的三类转座因子家族之一，人类基因组中约存在2800个SVA插入序列，其中一半为人类特异性插入。
转座因子通常会被KRAB型锌指蛋白（KRAB Zinc Finger, KZNF）募集辅阻遏蛋白，进而建立阻遏性染色质状态，从而被沉默。已有多项研究报道多种KZNF可结合SVA因子，但它们在抑制SVA因子过程中的个体贡献，以及其在抵消SVA介导的基因调控效应中的具体作用仍不明确。
本研究分析了人类细胞中ZNF91的全基因组结合谱，发现ZNF91可与SVA的可变数目串联重复（Variable Number Tandem Repeat, VNTR）区域相互作用。通过在人类胚胎干细胞中利用CRISPR-Cas9介导敲除ZNF91，我们证实ZNF91的缺失会导致SVA的转录活性升高。与之相反，在编码另一种强效SVA结合蛋白的ZNF611基因被遗传敲除后，并未观察到SVA的激活。表观遗传谱分析证实，ZNF91缺失时SVA的阻遏状态被解除，且主要是演化上较年轻的SVA获得了与基因激活相关的表观遗传修饰。靠近被激活SVA的基因呈现轻度上调，表明在阻遏缺失的情况下，SVA可发挥顺式调控元件的特性。
有趣的是，SVA的全基因组去阻遏引发了位于KZNF簇内的KZNF的集体上调。这一现象或为宿主基因组感知并对抗转座因子入侵的潜在机制提供新的研究视角。
本研究总体实验设计如下：通过在HEK293细胞中过表达ZNF91-GFP并进行染色质免疫沉淀测序（Chromatin Immunoprecipitation Sequencing, ChIP-seq），鉴定ZNF91的全基因组结合位点；对CRISPR/Cas9介导的ZNF91野生型及敲除型人类胚胎干细胞系开展RNA测序（RNA Sequencing, RNA-seq）与ChIP-seq分析。