Project description:MYC controls the transcription of large numbers of long non-coding RNAs. Since MYC is a ubiquitous oncoprotein, some of these long non-coding RNAs (lncRNAs) probably play a significant role in cancer. We applied CRISPRi to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 non-coding loci that play a positive role in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RTqPCR and in CRISPRi-competition assays with individual GFP-expressing sgRNA-constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, it is superior to the KRAB repressor domain which serves routinely as transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP allowing the construction of a doxycycline regulatable CRISPRi-system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.
Project description:MYC controls the transcription of large numbers of long non-coding RNAs. Since MYC is a ubiquitous oncoprotein, some of these long non-coding RNAs (lncRNAs) probably play a significant role in cancer. We applied CRISPRi to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 non-coding loci that play a positive role in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RTqPCR and in CRISPRi-competition assays with individual GFP-expressing sgRNA-constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, it is superior to the KRAB repressor domain which serves routinely as transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP allowing the construction of a doxycycline regulatable CRISPRi-system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.
Project description:The present study reveals LMYC and MXD1 as novel regulators of a transcriptional program that is modulated during the maturation of Batf3-dependent dendritic cells (also known as type I classical dendritic cells or cDC1s). We used microarray analysis with ERCC spike in controls to determine the transcriptional effects of MYCL and MXD1 deficiency at steady state and after activation with poly IC. Mycl-deficient mice are available from Jackson laboratories as B6.129S6(C)-Mycltm1.1Kmm/J. Mxd1-/- mice were provided by R. Eisenman.
Project description:White fat browning is a highly variable genetic trait in mice (Guerra et al., 1998). To gain an overview of strain variations in browning capacities, we performed transcriptome analysis of white fat browning in (1) 5 inbred mouse strains (C57BL/6J, 129S6sv/ev, A/J, AKR/J, and SWR/J) with distinct browning propensities in WAT, and (2) F1 hybrids derived from a high (129S6sv/ev) and low browning strain (C57BL/6J) cross. From our analyses, several transcription factors emerged as novel regulators of white fat browning, including the TFs Zfp521, Fhl1, and Mxd1. We further validated the role of these TFs and performed 3' RNA-seq experiments upon their knockdown, in order to characterize their mechanism of action.
Project description:Herpesviruses rely on host cell transcription and translation machineries for replication. Viral proteins thus function to redirect multiple cellular proteins for viral replication. In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here we show that an evolutionally-shaped viral protein sequence is a great starting material for unique drug development to modulate cellular transcription. Cellular c-Myc protein (MYC) is overexpressed in over 70% of all types of cancer cells and therefore a very attractive target to control cancer cell growth. We identified a small functional peptide derived from the Kaposi's sarcoma-associated herpesvirus transactivator (K-Rta), which strongly attenuates MYC expression, reduces cell proliferation, and selectively kills cancer cells in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide blocks promoter-enhancer interactions by preventing coactivator complex consisting of Nuclear receptor coactivator 2, p300, and SWI/SNF proteins from engaging the MYC promoter in leukemia cells. Target gene profiling with SLAM-seq suggests that the viral peptide attenuates MYC expression through a mechanism likely different from that of BET bromodomain inhibitors. Furthermore, fusing the 13 amino acids peptide with humanized anti-CD22 single chain armed the antibody drug with cell killing ability, and inhibited cell growth in soft agar. Our studies thus demonstrate the utility of the peptide sequence as a therapeutics module, which may be used to modulate MYC activity in a cell type-specific manner.
Project description:Herpesviruses rely on host cell transcription and translation machineries for replication. Viral proteins thus function to redirect multiple cellular proteins for viral replication. In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here we show that an evolutionally-shaped viral protein sequence is a great starting material for unique drug development to modulate cellular transcription. Cellular c-Myc protein (MYC) is overexpressed in over 70% of all types of cancer cells and therefore a very attractive target to control cancer cell growth. We identified a small functional peptide derived from the Kaposi's sarcoma-associated herpesvirus transactivator (K-Rta), which strongly attenuates MYC expression, reduces cell proliferation, and selectively kills cancer cells in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide blocks promoter-enhancer interactions by preventing coactivator complex consisting of Nuclear receptor coactivator 2, p300, and SWI/SNF proteins from engaging the MYC promoter in leukemia cells. Target gene profiling with SLAM-seq suggests that the viral peptide attenuates MYC expression through a mechanism likely different from that of BET bromodomain inhibitors. Furthermore, fusing the 13 amino acids peptide with humanized anti-CD22 single chain armed the antibody drug with cell killing ability, and inhibited cell growth in soft agar. Our studies thus demonstrate the utility of the peptide sequence as a therapeutics module, which may be used to modulate MYC activity in a cell type-specific manner.