The pioneer factor SOX9 competes for epigenetic factors to switch stem cell fates [CUT&RUN]

When stem cells activate a member of the SOX family of pioneer factors, they bind their cognate sequences within condensed chromatin, not only evicting the associated nucleosome, but also recruiting H3K4-monomethylases, which modify flanking nucleosomes to open chromatin and activate one lineage program; simultaneously, pioneer factors also silence alternative fates, but in this case act indirectly, through competing for H3K4-monomethylases pre-existing at the original fate genes. Overall design: Epidermal stem cells were FACS isolated as mentioned above and CUT&RUN was performed as previously described with minor modifications indicated in methods. CUT&RUN sequencing libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina. PCR-amplified libraries were purified using 1x ratio of SPRI beads and eluted in 15µL 0.1x TE buffer. All CUT&RUN libraries were sequenced on Illumina NextSeq using 40-bp paired-end reads.

当干细胞激活SOX家族（SOX family）的先驱因子（pioneer factor）成员后，该因子会结合浓缩染色质内的同源识别序列，不仅可驱逐与之结合的核小体（nucleosome），还能招募H3K4单甲基化酶（H3K4-monomethylases）；这类酶会修饰侧翼核小体以开放染色质结构，并激活特定谱系程序。与此同时，先驱因子还会沉默其他备选细胞命运，但该过程并非直接作用，而是通过竞争初始细胞命运基因位点上预先存在的H3K4单甲基化酶来间接实现。

总体实验设计：按照前述方法通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分离表皮干细胞（epidermal stem cells），并采用CUT&RUN技术进行实验，仅在方法部分注明的流程中进行了小幅优化。CUT&RUN测序文库的构建使用适配Illumina平台的NEBNext Ultra II DNA Library Prep Kit for Illumina以及NEBNext Multiplex Oligos for Illumina。经PCR扩增的文库采用1倍体积的SPRI磁珠（SPRI beads）进行纯化，并用15 μL 0.1× TE缓冲液洗脱。所有CUT&RUN文库均在Illumina NextSeq测序平台上进行测序，采用40 bp双端读长（paired-end reads）。