Developmental Control of Polycomb Subunit Composition Mediates a Switch to Non-Canonical Functions during Hematopoiesis

The epigenetic machinery plays crucial roles in hematopoiesis, and its deregulation drives the pathogenesis of blood disorders. Polycomb Repressive Complex 2 (PRC2) is a major class of epigenetic repressor that catalyzes the di/tri-methylation of histone H3 lysine 27 (or H3K27me2/3). The canonical PRC2 complex consists of EED, SUZ12, and the histone methyltransferase EZH2. The functions of PRC2 in hematopoiesis remain elusive due in large to the existence of two highly related enzymatic subunits EZH1 and EZH2. While amplification or overexpression of PRC2 proteins is common in many cancers, inactivating mutation in PRC2 is frequently found in hematopoietic malignancies, indicating that PRC2 can be oncogenic or tumor suppressive in different cellular contexts. In light of recent efforts to therapeutically target EZH2 enzyme activities or canonical EZH2-PRC2 functions in various hematopoietic malignancies, it will be critical to fully assess the context-specific activity of this epigenetic complex in normal and malignant developmental processes.

The molecular mechanisms by which PRC2 regulates normal and neoplastic hematopoiesis is unclear, as are the non-redundant effects of canonical versus non-canonical PRC2 functions, which are mediated by EZH1 or EZH2 independent of H3K27me2/3. In this study, we demonstrate that the PRC2 enzymatic subunits EZH1 and EZH2 undergo an expression switch during hematopoiesis. EZH2 is highly expressed in primary human CD34+ hematopoietic stem/progenitor (HSPC) cells and progressively downregulated during erythroid and lymphoid specification, whereas EZH1 is significantly upregulated during differentiation. We next examined the in vivo stoichiometry of the PRC2 complexes by quantitative proteomics and revealed the existence of an EZH1-SUZ12 sub-complex lacking EED subunit in human erythroid cells. Through genome scale chromatin occupancy (by ChIP-seq) and transcriptional profiling (by RNA-seq) analyses, we provide evidence that EZH1 together with SUZ12 form a non-canonical PRC2 complex, occupy active chromatin domains marked by H3K4me3 and H3K27me1, and positively regulate gene expression. Furthermore, loss of EZH2 expression leads to global repositioning of EZH1 chromatin occupancy to EZH2 targets, and EZH1 complements EZH2 loss within canonical PRC2 target genes.

To elucidate the regulatory networks underlying the developmental control of EZH1/2 switch, we profiled the histone modifications and chromatin accessibility surrounding the EZH1 gene in both CD34+ HSPCs and committed erythroid cells. We identified and characterized an erythroid-selective enhancer element that is indispensable for the transcriptional activation of EZH1. Loss of function analysis using CRISPR/cas9-mediated enhancer deletion results in markedly decrease in EZH1 expression in human erythroid cells. Moreover, a switch from GATA2 to GATA1 expression controls the developmental EZH1/2 switch by differential association with distinct EZH1 enhancers during erythroid differentiation. Thus, the lineage- and developmental stage-specific regulation of PRC2 subunit composition leads to a switch from canonical silencing to non-canonical PRC2 functions. Our study also establishes a molecular link between the switch of master lineage regulators and developmental control of PRC2 composition, providing a means to coordinate linage-specific transcription and accompanying changes in the epigenetic landscape during blood stem cell specification.