The chromatin template imposes an epigenetic barrier during the process of somatic cell reprogramming. Here, using fibroblasts derived from macroH2A double knockout mice we show that these histone variants act cooperatively as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, we further demonstrate that macroH2A2 is the predominant barrier to reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together with H3K27me3, co-occupy pluripotency genes in wild type fibroblasts. In particular, we find macroH2A isoforms to be highly enriched at target genes of the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming. Finally, while macroH2A double knockout induced pluripotent cells are able to differentiate properly in vitro and in vivo, such differentiated cells retain the ability to return to a stem-like state. Therefore, we propose that macroH2A isoforms provide a redundant silencing layer or terminal differentiation ‘lock’ at critical pluripotency genes that presents as an epigenetic barrier when differentiated cells are challenged to reprogram.

Recent genome sequencing efforts in a variety of cancers have revealed mutations and/or structural alterations in and, which together encode a complex that deposits histone variant H3.3 into repetitive heterochromatin. These regions include retrotransposons, pericentric heterochromatin, and telomeres, the latter of which show deregulation in /-mutant tumors. Interestingly, and mutations are often found in pediatric tumors, suggesting a particular developmental context in which these mutations drive disease. Here we review the functions of ATRX and DAXX in chromatin regulation as well as their potential contributions to tumorigenesis. We place emphasis on the chromatin remodeler ATRX, which is mutated in the developmental disorder for which it is named, α-thalassemia, mental retardation, X-linked syndrome, and at high frequency in a number of adult and pediatric tumors.

Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings, however, requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study, we show that once specified to neuro-epithelial lineage, hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However, our analyses of 2 hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes, namely, migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes, a new potential source for therapeutic use for brain tumors and other neurological diseases.

SUMMARY Chromatin-mediated processes influence the development and progression of breast cancer. Using murine mammary carcinoma-derived tumorspheres as a functional readout for an aggressive breast cancer phenotype, we performed a loss-of-function screen targeting sixty epigenetic regulators. We identified the Polycomb protein Cbx8 as a key regulator of mammary carcinoma both in vitro and in vivo. Accordingly, Cbx8 is overexpressed in human breast cancer and correlates with poor survival. Our genomic analyses revealed that Cbx8 positively regulates Notch signaling by maintaining H3K4me3 levels on Notch-network gene promoters. Ectopic expression of Notch receptors partially rescues tumorsphere formation in Cbx8-depleted cells. We find that Cbx8 associates with non-PRC1 complexes containing the H3K4 methyltransferase complex component WDR5, which together regulate Notch gene expression. Thus, our study implicates a key non-canonical role for Cbx8 in promoting breast tumorigenesis.

Evidence has pointed to brain tumor stem cells (BTSC) as culprits behind human high-grade glioma (hHGG) resistance to standard therapy. Pre-clinical rodent models are the mainstay for testing of new therapeutic strategies. The typical model involves the intracranial injection of human glioma cells into immunocompromised hosts, hindering the evaluation of tumor-host responses and resulting in non-infiltrative tumors. The CT-2A model is an immunocompetent mouse model with potential to overcome these disadvantages. In this study, we confirmed the highly infiltrative nature of intracranial CT-2A tumors and optimized reproducible injection parameters. We then generated neurospheres and established, for the first time, the stemness of this model. CT-2A expression of the BTSC marker, CD133, increased from 2% in monolayer cells to 31% in fully-formed neurospheres. Investigation of three stem cell markers (Oct4, Nanog and Nestin) revealed a distinct stemness signature with monolayer cells expressing Oct4 and Nestin (no Nanog), and neurospheres expressing all three. Additionally, CT-2A cells were more proliferative and invasive than U87 cells, while CT-2A neurospheres were significantly more proliferative and invasive than either monolayer cells in vitro. Taken together, our results show that this model is a valuable tool for pre-clinical testing of novel therapeutics against hHGG and also affords the opportunity for investigation of BTSC in an immunocompetent setting.