ABSTRACT
BMI1 is a core protein of the polycomb repressive complex 1 (PRC1) that is overexpressed in several cancer types, making it a promising target for cancer therapies. However, the underlying mechanisms and interactions associated with BMI1-induced tumorigenesis are often context-dependent and complex. Here, we performed a drug resistance screen on mutagenized human haploid HAP1 cells treated with BMI1 inhibitor PTC-318 to find new genetic and mechanistic features associated with BMI1-dependent cancer cell proliferation. Our screen identified NUMA1-mutations as the most significant inducer of PTC-318 cell death resistance. Independent validations on NUMA1-proficient HAP1 and non-small cell lung cancer cell lines exposed to BMI1 inhibition by PTC-318 or BMI1 knockdown resulted in cell death following mitotic arrest. Interestingly, cells with CRISPR-Cas9 derived NUMA1 knockout also showed a mitotic arrest phenotype following BMI1 inhibition but, contrary to cells with wildtype NUMA1, these cells were resistant to BMI1-dependent cell death. The current study brings new insights to BMI1 inhibition-induced mitotic lethality in cancer cells and presents a previously unknown role of NUMA1 in this process.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinogenesis/genetics , Cell Cycle Proteins/genetics , Drug Resistance, Neoplasm/genetics , Neoplasms/genetics , Polycomb Repressive Complex 1/metabolism , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Apoptosis/genetics , CRISPR-Cas Systems/genetics , Carcinogenesis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Gene Knockout Techniques , Humans , M Phase Cell Cycle Checkpoints/drug effects , Mutation , Neoplasms/drug therapy , Neoplasms/pathology , Polycomb Repressive Complex 1/antagonists & inhibitors , Polycomb Repressive Complex 1/genetics , RNA, Small Interfering/metabolismABSTRACT
Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR-Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. Here we leverage our TRIP technology to integrate ~ 1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5' overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency.
Subject(s)
CRISPR-Cas Systems/genetics , DNA/genetics , Gene Editing/methods , RNA, Guide, Kinetoplastida/genetics , Animals , Cell Line , DNA Mutational Analysis , Genes, Reporter/genetics , Genetic Loci/genetics , Genetic Vectors/genetics , Mice , Mouse Embryonic Stem Cells , Mutation Rate , Plasmids/geneticsABSTRACT
Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large-scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses (ERVs) silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of ERVs, thus facilitating the transition through reprogramming. Stem Cells 2017;35:147-157.
Subject(s)
Cellular Reprogramming , Epigenesis, Genetic , Pluripotent Stem Cells/metabolism , Tripartite Motif-Containing Protein 28/metabolism , Animals , Cell Proliferation , Cellular Reprogramming/genetics , Chromatin/metabolism , Endogenous Retroviruses/metabolism , Gene Knockdown Techniques , Histone-Lysine N-Methyltransferase , Histones/metabolism , Lysine/metabolism , Methylation , Mice, Transgenic , Models, Biological , Pluripotent Stem Cells/cytology , RNA, Small Interfering/metabolism , Up-Regulation/geneticsABSTRACT
Aging causes profound effects on the hematopoietic stem cell (HSC) pool, including an altered output of mature progeny and enhanced self-propagation of repopulating-defective HSCs. An important outstanding question is whether HSCs can be protected from aging. The signal adaptor protein LNK negatively regulates hematopoiesis at several cellular stages. It has remained unclear how the enhanced sensitivity to cytokine signaling caused by LNK deficiency affects hematopoiesis upon aging. Our findings demonstrate that aged LNK-/- HSCs displayed a robust overall reconstitution potential and gave rise to a hematopoietic system with a balanced lineage distribution. Although aged LNK-/- HSCs displayed a distinct molecular profile in which reduced proliferation was central, little or no difference in the proliferation of aged LNK-/- HSCs was observed after transplantation when compared to aged WT HSCs. This coincided with equal telomere maintenance in WT and LNK-/- HSCs. Collectively, our studies suggest that enhanced cytokine signaling can counteract functional age-related HSC decline.
