EZH2 controls mammary differentiation independently of its methyltransferase activity through controlling genomic STAT5 access

Mammary development is characterized by the proliferation and progressive differentiation of alveolar epithelium during pregnancy, culminating in lactation. These processes are largely controlled by hormones through transcription factors. We now explore the contributions of histone methyltransferases, which establish H3K27me3 marks, in the temporally-regulated differentiation of mammary epithelium. Loss of EZH2, but not EZH1, resulted in precocious mammary differentiation, which was facilitated by STAT5 binding to specific target genes and their activation. Mammary stem cells were not compromised in the absence of EZH2. Genome-wide H3K27me3 patterns remained intact in the absence of EZH2. Mammary-specific loci were devoid of H3K27me3 marks in mammary progenitor and mature cells, suggesting no regulatory role for this repressive mark. Lastly, the combined absence of EZH1 and EZH2 inhibited the formation of alveoli. Taken together, EZH2 controls temporally-restricted differentiation of mammary epithelium through H3K27me3-independent mechanisms. mRNA-seq and ChIP-seq in MMTV-Cre (Control), E1-/- (E1KO), E1+/-;E2f/f;control (E1+/-E2KO) and Ezh2f/f;control (E2KO) mammary gland tissues or MECs (purified mammary epithelial cells). H3K27me3 and STAT5 ChIP-seqs in mammary tissues at p13; H3K4me3 ChIP-seq in MECs (mammary epithelial cells) at p13; RNA-seqs at mature virgin (with/without prolactin injection), p13 and p18 mammary tissues.乳腺发育以妊娠期间腺泡上皮的增殖与渐进性分化为特征，最终以泌乳作为发育终点。上述过程主要由激素通过转录因子进行调控。本研究聚焦于建立三甲基化组蛋白H3赖氨酸27（H3K27me3）修饰的组蛋白甲基转移酶，在乳腺上皮细胞时序调控分化中的作用。研究发现，敲除EZH2而非EZH1会导致乳腺上皮提前分化，该过程可通过STAT5结合特定靶基因并激活其转录得以促进。敲除EZH2并不会对乳腺干细胞造成损伤。在EZH2缺失的情况下，全基因组范围内的H3K27me3修饰模式仍保持完整。乳腺祖细胞与成熟细胞中的乳腺特异性基因座缺失H3K27me3修饰，提示该抑制性修饰并无调控作用。最后，同时敲除EZH1与EZH2会抑制腺泡的形成。综上，EZH2通过不依赖于H3K27me3的机制调控乳腺上皮细胞的时序限制性分化。 本研究采用MMTV-Cre（对照组）、E1-/-（E1敲除组，E1KO）、E1+/-;E2f/f;对照（E1+/-E2KO组）以及Ezh2f/f;对照（E2KO组）的乳腺组织或纯化乳腺上皮细胞（mammary epithelial cells，简称MECs），开展mRNA测序（messenger RNA sequencing，mRNA-seq）与染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing，ChIP-seq）。此外，本研究对妊娠第13天（p13）的乳腺组织进行了H3K27me3与STAT5染色质免疫共沉淀测序；对妊娠第13天的纯化乳腺上皮细胞进行了H3K4me3染色质免疫共沉淀测序；并对成熟未孕小鼠（经催乳素注射/未注射）、妊娠第13天及第18天的乳腺组织进行了RNA测序（RNA sequencing，RNA-seq）。