EZH2 is required for breast and pancreatic cancer stem cell maintenance and can be used as a functional cancer stem cell reporter.

Although cancer is largely seen as a disease stemming from genetic mutations, evidence has implicated epigenetic regulation of gene expression as a driving force for tumorigenesis. Epigenetic regulation by histone modification, specifically through polycomb group (PcG) proteins such as EZH2 and BMI-1, is a major driver in stem cell biology and is found to be correlated with poor prognosis in many tumor types. This suggests a role for PcG proteins in cancer stem cells (CSCs). We hypothesized that epigenetic modification by EZH2, specifically, helps maintain the CSC phenotype and that in turn this epigenetic modifier can be used as a reporter for CSC activity in an in vitro high-throughput screening assay. CSCs isolated from pancreatic and breast cancer lines had elevated EZH2 levels over non-CSCs. Moreover, EZH2 knockdown by RNA interference significantly reduced the frequency of CSCs in all models tested, confirming the role of EZH2 in maintenance of the CSC population. Interestingly, genes affected by EZH2 loss, and therefore CSC loss, were inversely correlated with genes identified by CSC enrichment, further supporting the function of EZH2 CSC regulation. We translated these results into a novel assay whereby elevated EZH2 staining was used as a reporter for CSCs. Data confirmed that this assay could effectively measure changes, both inhibition and enrichment, in the CSC population, providing a novel approach to look at CSC activity. This assay provides a unique, rapid way to facilitate CSC screening across several tumor types to aid in further CSC-related research.

Distinct Ldb1/NLI complexes orchestrate γ-globin repression and reactivation through ETO2 in human adult erythroid cells.

The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the ß-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the ß-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the (A)γ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing ß-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and ß-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.