Polycomb group (PcG) proteins are epigenetic gene silencers that are implicated in neoplastic development. Their oncogenic function might be associated with their well-established role in the ...maintenance of embryonic and adult stem cells. In this review, we discuss new insights into the possible mechanisms by which PcGs regulate cellular identity, and speculate how these functions might be relevant during tumorigenesis.
Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been ...of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ∼1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for crosstalk between neighboring genes and estimate that enhancers can influence gene expression on average over ∼20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation.
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•A method for parallel monitoring of thousands of reporters integrated in the genome•Genome-wide landscape of chromatin position effects in mouse embryonic stem cells•Attenuation of transcriptional activity in lamina-associated domains•• Enhancers and transcription units influence gene expression generally over ∼20 kb
A method is presented to map and monitor thousands of randomly integrated reporters into the mouse ESC genome in parallel. This generates genome-wide maps that link chromatin composition and structure to gene expression and other functions.
Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to ...regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N-bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.
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.
In mouse and human neural progenitor and glioblastoma “stem-like” cells, we identified key targets of the Polycomb-group protein BMI1 by combining ChIP-seq with in vivo RNAi screening. We discovered ...that Bmi1 is important in the cellular response to the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) and endoplasmic reticulum (ER) stress pathways, in part converging on the Atf3 transcriptional repressor. We show that Atf3 is a tumor-suppressor gene inactivated in human glioblastoma multiforme together with Cbx7 and a few other candidates. Acting downstream of the ER stress and BMP pathways, ATF3 binds to cell-type-specific accessible chromatin preloaded with AP1 and participates in the inhibition of critical oncogenic networks. Our data support the feasibility of combining ChIP-seq and RNAi screens in solid tumors and highlight multiple p16INK4a/p19ARF-independent functions for Bmi1 in development and cancer.
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•Crosstalk between Bmi1 and TGF-β/BMP pathways in self-renewal and differentiation•Bmi1 target gene Atf3 connects Polycomb gene repression with TGF-β and BMP networks•Atf3 is a tumor suppressor gene in brain tumors by inhibiting key oncogenic pathways•Cbx7 is a tumor suppressor in gliomagenesis
Bmi1 is required for efficient self-renewal of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs). In this study, we investigated whether leukemia-associated fusion proteins, which differ ...in their ability to activate
Hox expression, could initiate leukemia in the absence of
Bmi1. AML1-ETO and PLZF-RARα, which do not activate
Hox, triggered senescence in
Bmi1
−/−
cells. In contrast, MLL-AF9, which drives expression of
Hoxa7 and
Hoxa9, readily transformed
Bmi1
−/−
cells. MLL-AF9 could not initiate leukemia in
Bmi1
−/−Hoxa9
−/−
mice, which have further compromised HSC functions. But either gene could restore the ability of MLL-AF9 to establish LSCs in the double null background. As reported for Bmi1, Hoxa9 regulates expression of
p16
Ink4a/p19
ARF
locus and could overcome senescence induced by AML1-ETO. Together, these results reveal an important functional interplay between MLL/Hox and Bmi1 in regulating cellular senescence for LSC development, suggesting that a synergistic targeting of both molecules is required to eradicate a broader spectrum of LSCs.
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► MLL-AF9, but not AML1-ETO, is capable of establishing LSCs in the absence of Bmi1 ► Ablation of Hoxa9 abolishes Bmi1-independent establishment of MLL LSCs in
Bmi1
−/− cells ► Hoxa9 suppresses expression of p16
Ink4a and p19
Arf and senescence ► Synergistic targeting of Bmi1 and Hox pathways is required for certain LSCs
Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. Here, ...we adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. Our results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and we demonstrate that a topological rather than a sequence-based mechanism is responsible for this constraint. Our results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture.
The ability of retroviruses and transposons to insert their genetic material into host DNA makes them widely used tools in molecular biology, cancer research and gene therapy. However, these systems ...have biases that may strongly affect research outcomes. To address this issue, we generated very large datasets consisting of ~ 120,000 to ~ 180,000 unselected integrations in the mouse genome for the Sleeping Beauty (SB) and piggyBac (PB) transposons, and the Mouse Mammary Tumor Virus (MMTV). We analyzed ~ 80 (epi)genomic features to generate bias maps at both local and genome-wide scales. MMTV showed a remarkably uniform distribution of integrations across the genome. More distinct preferences were observed for the two transposons, with PB showing remarkable resemblance to bias profiles of the Murine Leukemia Virus. Furthermore, we present a model where target site selection is directed at multiple scales. At a large scale, target site selection is similar across systems, and defined by domain-oriented features, namely expression of proximal genes, proximity to CpG islands and to genic features, chromatin compaction and replication timing. Notable differences between the systems are mainly observed at smaller scales, and are directed by a diverse range of features. To study the effect of these biases on integration sites occupied under selective pressure, we turned to insertional mutagenesis (IM) screens. In IM screens, putative cancer genes are identified by finding frequently targeted genomic regions, or Common Integration Sites (CISs). Within three recently completed IM screens, we identified 7%-33% putative false positive CISs, which are likely not the result of the oncogenic selection process. Moreover, results indicate that PB, compared to SB, is more suited to tag oncogenes.
EZH2 is frequently overexpressed in glioblastoma (GBM), suggesting an oncogenic function that could be a target for therapeutic intervention. However, reduced EZH2 activity can also promote ...tumorigenesis, leading to concerns about the use of EZH2 inhibitors. Here, we provide further insight about the effects of prolonged Ezh2 inhibition in glioblastoma using preclinical mouse models and primary tumor-derived human GBM cell lines. Using doxycycline-inducible shRNAs that mimic the effects of a selective EZH2 inhibitor, we demonstrate that prolonged Ezh2 depletion causes a robust switch in cell fate, including significantly enhanced proliferation, DNA damage repair, and activation of part of the pluripotency network, resulting in altered tumor cell identity and tumor progression. Short-term Ezh2 depletion significantly improved survival without the tumor progression observed upon prolonged Ezh2 depletion, suggesting that precise dosing regiments are very important. These results could be of high clinical relevance with regard to how glioblastomas should be treated with epigenetic therapies.
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•Robust switch in cell fate upon prolonged Ezh2 depletion causes tumor progression•Ezh2 depletion results in modified differentiation status and tumor cell identity•Prolonged Ezh2 depletion in glioblastoma significantly enhances DNA damage repair•Effects of EZH2 inhibition in glioblastoma depend on well-balanced drug dosing
EZH2 is reported to have both oncogenic and tumorsuppressor functions, leading to concerns about the use of EZH2 inhibitors. de Vries et al. show that prolonged Ezh2 inhibition in glioblastoma results in an altered tumor cell identity and tumor progression, unlike short-term Ezh2 inhibition, suggesting that well-balanced drug dosing is important.
Polycomb group proteins (PcGs) are involved in gene repression through chromatin modifications and required for the maintenance of both embryonic and adult stem cells. Genome-wide studies demonstrate ...that genes targeted by PcG are predominantly developmental transcription factors. In embryonic stem cells, these genes carry not only a repressive PcG mark but also an activating mark, resulting in so-called ‘bivalent domains’. New data suggest that genes with bivalent domains are primed for differential expression upon differentiation. We propose that the resolution of a bivalent domain into either an active or repressed state constitutes a cell fate decision, and that by postponing these decisions PcG contributes to pluripotency.
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