Project description:We uncovered an unexpected function of mixed-lineage leukemia (MLL) protein, which is a critical epigenetic factor and whose disruption leads to leukemogenesis, in the functional regulation of miRNAs. We reveal that the MLLC180 subunit alone can colocalize with miRNA-induced silencing complex (miRISC) components in cytoplasm processing bodies (P-bodies), where miRNA-mediated gene silencing takes place. In addition, we show that MLL was specifically required for miRNA-mediated translational repression, but not for miRNA or siRNA-mediated mRNA cleavage. Mechanistically, MLL was necessarily required for recruiting miRNA to the miRISC and P-body core, partly through its binding partner RAN. Furthermore, dysregulation of the miRNA repression function in MLL leukemic cells was essential for high-level expression of MYC, which ensured the survival of MLL leukemic cells. Thus, our investigation discovered that the MLL subunit alone can exert other functions in miRNA-mediated gene silencing apart from the epigenetic function of the whole MLL heterodimer. We also demonstrated as proof-of-principle that functional deregulation of miRNA may play an important role in cancer.
Project description:To demonstrate RIPSeeker program that is developed for RIP-seq analyses, we generated RIP-seq data corresponding to the protein CCNT1 in HEK293 cell line using standard RIP-seq protocols described in Zhao et al., (2010). We performed two in-house RIP-seq experiments both for CCNT1 in human HEK293 cells. Briefly, we generated tagged CCNT1 using a triple tag system that supports lentiviral stable expression and mammalian affinity purification (MAPLE) Mak et al (2010). The HEK293 cells stably expressing tagged CCNT1 was purified by M2 agarose beads, followed by RNA extraction by Trizol. The library synthesis was carried out according to the RIP-seq protocol described in Zhao et al., (2010) except that one of the two experiments was done with non-strand-specific sequencing.
Project description:WIN Site inhibitors bind the WIN Site of WDR5 resulting in decreased transcription of WDR5 target genes, many of which encode components of the protein synthesis machinery. In this study, we determined proteome alterations in an MLL-rearranged leukemia cell line treated for either 24 or 72 hours with a WIN Site inhibitor. The data from these studies, along with Ribo-Seq, RNA-Seq, and CRISPR screen experiments, guided us in assembling a collection of compounds that, when combined with WIN Site inhibitor, synergistically inhibit growth of MLL-rearranged leukemia cells.
Project description:To demonstrate RIPSeeker program that is developed for RIP-seq analyses, we generated RIP-seq data corresponding to the protein CCNT1 in HEK293 cell line using standard RIP-seq protocols described in Zhao et al., (2010).