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538 Polycomb Repressive Complex 2 Regulates Differentiation-Stage-Specific Autophagy Gene Expression and Safeguards Normal Erythropoiesis

Program: Oral and Poster Abstracts
Type: Oral
Session: 101. Red Cells and Erythropoiesis, Excluding Iron: Regulation of Erythropoiesis
Hematology Disease Topics & Pathways:
Research, Fundamental Science
Sunday, December 8, 2024: 12:45 PM

Jiazhuo Li, PhD1, Panpan Zhu1*, Zhiling Shi1*, Longping Wen2*, Stuart H Orkin, MD3,4,5 and Huafeng Xie1*

1Center for Medical Research on Innovation and Translation, Department of Hematology, Institute of Clinical Medicine, the Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou, China
2Medical Research Institute, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University,, Guangzhou, China
3Dana-Farber Cancer Institute, Inc., Boston, MA
4Department of Hematology/Oncology, Harvard Medical School/Boston Children's Hospital, Boston, MA
5Howard Hughes Medical Institute, Boston

Erythropoiesis is a complex yet tightly controlled process encompassing distinct stages, during which autophagy plays important roles. Autophagy is essential in the removal of organelles such as mitochondria and ribosomes, facilitating the transition from nucleated precursors to anucleate erythrocytes. While the importance of autophagy in erythropoiesis has been recognized, the dynamic changes in autophagy gene expression and activity during erythropoiesis remain to be characterized, and the regulatory mechanisms controlling their gene expression remain poorly understood.

We generated a conditional autophagy activity reporter mouse, ROSA-mCherry-EGFP-LC3/Vav-Cre, and observed basal levels of autophagy flux at the R2 stage (CD71+Ter119-), which increased at R3 (CD71+Ter119+) and culminated at R4 (CD71LowTer119+). To determine the expression of autophagy genes, erythroid precursor cells at R2, R3, and R4 stages were sorted for RNA sequencing. We identified a group of autophagy genes, including Ulk1, Pik3r1, and Map1lc3b, whose expression increases during cell maturation. Polycomb repressive complex 2 (PRC2) plays important roles in controlling differentiation-stage-specific gene expression. Deletion of PRC2 genes leads to defective erythropoiesis and anemia, as found in our previous studies. We crossed ROSA-mCherry-EGFP-LC3/Vav-Cre and Ezh2f/f mice and found that in the Ezh2 KO mouse (Ezh2f/f/Vav-Cre), autophagic activity is higher in erythroid precursors than in the WT cells at the same stages. RNA sequencing analysis revealed increased autophagy gene expression in KO cells compared to their WT counterparts, suggesting that PRC2 suppresses autophagy gene expression. For validation, we measured the 3mH3K27 marks by cut&tag assay in erythroid precursors and confirmed that erythroid differentiation-stage-specific autophagy genes were marked by 3mH3K27, with low levels at the R2 stage and increased levels in R3 cells.

Ezh2 KO leads to severe defects in erythropoiesis in neonatal and juvenile mice. Precursor cells accumulated before the R3 stage, and mice developed severe anemia. Proliferation of KO R2 and R3 cells dropped dramatically, from 60-70% of proliferating cells in WT mice to 20-30% in KO mice, as measured by the EdU incorporation assay. Cell death in early precursors also increased upon the loss of Ezh2. As autophagy plays important regulatory roles in cell proliferation and survival, we investigated whether increased autophagy flux in early precursors contributes to their proliferation and survival defects. Activating autophagy in G1ER cells, a GATA1-null erythroid precursor cell line expressing GATA1ER, by rapamycin or trehalose treatment induced cell apoptosis and decreased proliferation. Interestingly,β-Est treatment, thus maturation of G1ER cells, rendered cells resistant to rapamycin or trehalose -induced cell death and proliferation inhibition, suggesting that prematurely increased autophagy activity in early erythroid precursors leads to cell death and reduced proliferation.

Enucleation, followed by organelle extrusion, marks the terminal differentiation of erythroid precursors. In WT mice, enucleation occurs at the R4 stage, and organelle extrusion at R5, as determined by Hoechst 33342 and MitoTracker/LysoTracker staining, respectively. Premature elevation of autophagy genes in Ezh2 KO mice led to premature clearance of organelles, which was almost complete in Ezh2 KO R4 cells, while most WT R4 cells still retained mitochondria and lysosomes. Additionally, we found that more than 90% of R4 cells had already lost their nuclei compared to about 30% in WT R4 cells. This is consistent with the premature elevation of enucleation-related genes in Ezh2 KO mice, such as Riok3, E2f2 and others.Erythropoiesis defects in Ezh2 KO mice can be partly rescued by the administration of chloroquine or the ULK1 inhibitor MRT68921.

In summary, we found that the expression of autophagy genes and autophagy activity during erythropoiesis is regulated by PRC2. Deregulated autophagy gene expression in Ezh2 KO mice leads to reduced cell proliferation and increased apoptosis in R2 and R3 cells, as well as premature enucleation and organelle clearance in late precursors. Collectively, these factors lead to insufficient expansion of erythroid precursor cells and poorly prepared mature cells for oxygen carrying, resulting in severe anemia in Ezh2 KO mice.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH