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1.  Variant PRC1 Complex-Dependent H2A Ubiquitylation Drives PRC2 Recruitment and Polycomb Domain Formation 
Cell  2014;157(6):1445-1459.
Summary
Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.
Graphical Abstract
Highlights
•Variant PRC1 complex-dependent H2AK119ub1 leads to binding of PRC2 and H3K27me3•Canonical PRC1 complexes fail to efficiently deposit H2AK119ub1 and recruit PRC2•A variant KDM2B/PCGF1/PRC1 complex is required for polycomb domain formation at CGIs•Failure to target KDM2B/PCGF1/PRC1 causes polycomb phenotypes and lethality in mice
Formation of repressive polycomb domains depends on histone ubiquitination catalyzed by variant PRC1 complexes at unmethylated CpG islands followed by PRC2 recruitment and methylation, rather than exclusively through initial association of PRC2.
doi:10.1016/j.cell.2014.05.004
PMCID: PMC4048464  PMID: 24856970
2.  Hypoxic enhancement of exosome release by breast cancer cells 
BMC Cancer  2012;12:421.
Background
Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling.
Methods
Breast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant.
Results
Exposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions.
Conclusions
These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion.
doi:10.1186/1471-2407-12-421
PMCID: PMC3488584  PMID: 22998595
Hypoxia; Exosomes; Breast cancer cells; Nanoparticle tracking analysis; Nanosight; ExoquickTM

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