Critical roles of RNA m5C-MBD6-H2AK119ub deubiquitylation axis in TET2-mediated HSC regulation - Abstract TET2 is commonly mutated in adult myeloid malignancies and normal individuals with clonal hematopoiesis. TET2 plays an important role in the regulation of hematopoietic stem/progenitor cells (HSCs/HPCs; HSPCs) as TET2 loss leads to hallmark HSC self-renewal enhancement with skewed differentiation towards monocytic lineage. Besides DNA 5-methylcytosine (5mC), TET2 also catalyzes RNA 5-methylcytosine (m5C) oxidation. The catalytic activity of TET2 is required for normal HSPC function, we therefore speculate that the enzymatic activity of TET2 on RNA also contributes to HSPC regulation. Indeed, TET2 can control chromatin state and transcription by binding to an RNA-binding protein PSPC1 and modulating RNA m5C oxidization in mouse embryonic stem cells (mESCs). Stimulated by a recent study discovering a novel mechanism of chromatin regulation through reversible N6-methyladenosine modification on chromatin-associated RNA (caRNA), we explored the potential chromatin regulation through the TET2-mediated caRNA m5C oxidation, which led to the following exciting findings: (i) TET2 oxidizes m5C on caRNAs to hm5C (5-hydroxymethylation ) with long terminal repeat (LTR) RNAs as the main substrates. (ii) MBD6 is a specific RNA m5C reader protein and NSUN2 is a main m5C writer protein for caRNAs. (iii) MBD6, via binding to m5C-modified LTR RNAs, recruits H2AK119ub deubiquitylase complex to mediate local chromatin activation in mESCs. Based on these findings, we hypothesize that TET2 exerts its regulatory function in HSPCs mainly through caRNA m5C oxidation, and deregulation of caRNA m5C- MBD6-H2AK119ub deubiquitylation axis upon TET2 loss/mutation leads to altered HSPC gene expression and function. To test our hypothesis, we will: (1) Define the importance of TET2-mediated caRNA m5C oxidation in the regulation of HSPC function. We will first identify in HSPCs the key caRNAs with altered m5C methylation upon TET2 loss and corelate with local gene expression. We will then examine the impact of caRNA m5C deregulation on TET2 deficiency-mediated abnormal HSPC function and gene expression by using Nsun2 knockout (normalize the caRNA m5C increases upon TET2-loss) as well as targeted demethylation (with dCas13Rx-TET2CD) and methylation blockage (with steric anti-sense oligo) of individual TET2 LTR RNA target. (2) Investigate the significance of RNA m5C reader, MBD6, in TET2-mediated regulation of HSPC by examining the impact of MBD6 loss on TET2 deficiency-mediated hallmark abnormal HSPC function and gene expression. 3) Validate the roles of caRNA m5C-MBD6-H2AK119ub deubiquitylation axis in human HSPCs. We will also investigate whether mutations of TET2 impair caRNA m5C-MBD6-H2AK119ub deubiquitylation axis in HSPCs from TET2-mutated myeloid malignancies. The sensitivity of TET2-mutated vs. TET2-WT HSPCs to MBD6 or NSUN2 loss will also be accessed. Success of the proposed studies will gain fundamental knowledge on HSPC biology by establishing caRNA m5C-MBD6-H2AK119ub deubiquitylation axis as a critical and novel pathway for TET2-mediated HSPC regulation. These studies can lead to novel strategies to target TET2-mutated HSPCs.
