The role of MLL1-MENIN complex in somatic cell reprogramming

Expression of OCT4, SOX2, KLF4 and MYC (OSKM) can reprogram somatic cells to pluripotency. This process involves a complete reset of the somatic cell identity. However, chromatin-based mechanisms that safeguard cellular identities act as barriers to reprogramming, resulting in low efficiency of cell fate conversions. To identify such barriers, a loss of function genetic screen has previously been performed and MLL1 (KMT2A) was identified as a significant roadblock for reprogramming. In the scope of this work, we employed chemical and genetic approaches to characterize the effects of MLL1 in complex with MENIN on reprogramming of human somatic cells. Firstly, we validated the barrier function of this complex via utilizing an inhibitor, VTP50469, that selectively targets MLL1-MENIN complex, which led to increased reprogramming efficiency. Next, we generated fibroblast cell lines that express Cas9 and gRNAs targeting MLL1 and MEN1 genes. Reprogramming experiments with these cell lines showed that deletion of both MEN1 and MLL1 increase the efficiency of reprogramming significantly. To further understand the barrier function of this complex, we tested the rescue capabilities different MEN1 point mutants by overexpressing their cDNAs in MEN1 KO cells. These experiments showed that while MLL1 interaction is crucial for MENIN barrier function, MENIN might be interacting with additional proteins such as JUND to prevent reprogramming. Furthermore, we observed that DOT1L inhibition does not further enhance the reprogramming efficiency when combined with VTP50469 treatment or MEN1 depletion. This suggests that DOT1L-related mechanisms might be involved in the prevention of somatic cell reprogramming. Moreover, we performed RNA-sequencing to identify the transcriptional changes that place in MENIN inhibition and depletion. RNA-sequencing results showed that, compared to the controls, several of pluripotency genes which include genes that are usually expressed in late reprogramming are upregulated both in the context of MENIN inhibition and depletion. In line with that, we observed that a large set of fibroblast-specific genes are downregulated in the absence of MENIN activity. Additionally, we also showed that this complex imposes a barrier function during the resetting of primed pluripotent stem cells to naive pluripotency. The supplementation of the naive culture conditions with VTP50469 enhanced the induction naive pluripotent stem cells. This suggests that MLL1-MENIN inhibition is a feasible target for modulating human naive pluripotency. Taken together, the results represented herein shows that MLL1-MENIN complex imposes barriers in the acquisition of cell identities of higher developmental potential.