Landscape of Monoallelic DNA Accessibility and Gene Regulatory Networks during Reprogramming to Naive Pluripotency and X Chromosome Reactivation [10X]

X chromosome reactivation (XCR) represents a paradigm to study epigenetic regulation and the reversal of chromatin silencing, although how it is linked to the pluripotency gene regulatory network, pluripotency transcription factors (TFs) and chromatin remodelling processes remains largely unexplained. Several pluripotency TFs have been linked to XCR through binding to the long non-coding RNA gene Xist, but whether other regulatory elements are involved is unclear. Here, we show that reprogramming to iPSCs induces gradual acquisition of chromatin accessibility at specific sites on the inactive X chromosome (Xi) which is subsequently propagated to other regulatory elements including enhancers and promoters. Regions that are genomically closer to day 0 accessible regions become biallelically accessible earlier, than the more distant ones. For distinct categories of chromatin regions that become biallelically accessible at different times, different sets of transcription factor (TF) motifs are enriched, including the pluripotency TFs KLF4 and cMYC. In addition, regions that become bialleliacally accessible earlier also show increased TF pluripotency binding, supporting the link between pluripotency TFs in XCR. To further explore the relationship between pluripotency TFs and XCR, we construct the active Gene Regulatory Networks (GRNs) during iPSC reprogramming using scRNA-seq and computational analyses. We reveal gene targets of 311 TFs expressed across reprogramming to iPSCs. Using this single-cell regulatory atlas, we show that X-linked genes are preferentially targeted by several pluripotency TFs. Our results suggest a new model where XCR may be coordinated by direct targeting of regulatory elements on the X chromosome by pluripotency TFs, concomitant with step-wise acquisition of chromatin accessibility. Altogether, our results demonstrate how gradual acquisition of a new GRN during reprogramming of cellular identity is linked with dynamic induction of chromatin accessibility and overcomes stable chromatin silencing on the Xi. 10X single-cell RNA-seq

X染色体再激活（X chromosome reactivation, XCR）是研究表观遗传调控与染色质沉默逆转的经典研究范式，但其与多能性基因调控网络、多能性转录因子（transcription factors, TFs）以及染色质重塑过程之间的关联，目前仍尚未完全阐明。已有研究通过结合长链非编码RNA基因Xist，将数种多能性转录因子与XCR建立了关联，但尚不明确是否存在其他调控元件参与其中。本研究显示，诱导多能干细胞（induced pluripotent stem cells, iPSCs）重编程过程中，失活X染色体（inactive X chromosome, Xi）上的特定位点会逐渐获得染色质可及性，随后这一可及性状态会扩散至包括增强子与启动子在内的其他调控元件。相较于距离更远的区域，基因组上与第0天开放区域更为接近的区域会更早实现双等位基因开放。对于不同时间点实现双等位基因开放的各类染色质区域，其富集的转录因子（transcription factor, TF）基序组合各不相同，其中包括多能性转录因子KLF4与cMYC。此外，更早实现双等位基因开放的区域，其转录因子结合的多能性特征也更为显著，进一步佐证了多能性转录因子与XCR之间的关联。为进一步探究多能性转录因子与XCR之间的关联，本研究借助单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与计算分析手段，构建了iPSCs重编程过程中的活跃基因调控网络（Gene Regulatory Networks, GRNs）。本研究鉴定出了iPSCs重编程全过程中表达的311种转录因子的靶基因。借助这一单细胞调控图谱，本研究发现X连锁基因会被数种多能性转录因子优先靶向结合。本研究结果提出了一种全新的模型：XCR或可通过多能性转录因子直接靶向X染色体上的调控元件，并伴随染色质可及性的逐步获得来实现协调调控。综上，本研究阐明了细胞身份重编程过程中，新基因调控网络的逐步建立如何与染色质可及性的动态诱导相关联，并最终打破失活X染色体上稳定存在的染色质沉默状态。本研究采用10X单细胞RNA测序技术。