DICER1 Is Essential for Self-Renewal of Human Embryonic Stem Cells.

MicroRNAs (miRNAs) are the effectors of a conserved gene-silencing system with broad roles in post-transcriptional regulation. Due to functional overlaps, assigning specific functions to individual miRNAs has been challenging. DICER1 cleaves pre-miRNA hairpins into mature miRNAs, and previously Dicer1 knockout mouse embryonic stem cells have been generated to study miRNA function in early mouse development. Here we report an essential requirement of DICER1 for the self-renewal of human embryonic stem cells (hESCs). Utilizing a conditional knockout approach, we found that DICER1 deletion led to increased death receptor-mediated apoptosis and failure of hESC self-renewal. We further devised a targeted miRNA screening strategy and uncovered essential pro-survival roles of members of the mir-302-367 and mir-371-373 clusters that bear the seed sequence AAGUGC. This platform is uniquely suitable for dissecting the roles of individual miRNAs in hESC self-renewal and differentiation, which may help us better understand the early development of human embryos.

TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells.

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP-seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

Author Correction: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells.

In the version of this article initially published, in the Methods, the Gene Expression Omnibus accession code for H3K36me3 ChIP-seq data was incorrectly given as GSM1003585 instead of GSM733725. The error has been corrected in the HTML, PDF and print versions of the article.

Publisher Correction: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells.

The version of the Supplementary Text and Figures file initially posted was missing Supplementary Tables 1-6 and the Supplementary Note and used incorrect versions of the supplementary figures.

CSF-1R inhibition alters macrophage polarization and blocks glioma progression.

Glioblastoma multiforme (GBM) comprises several molecular subtypes, including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumor-associated macrophages and microglia (TAMs). Macrophages depend on colony stimulating factor-1 (CSF-1) for differentiation and survival. We used an inhibitor of the CSF-1 receptor (CSF-1R) to target TAMs in a mouse proneural GBM model, which significantly increased survival and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, TAMs were not depleted in treated mice. Instead, glioma-secreted factors, including granulocyte-macrophage CSF (GM-CSF) and interferon-γ (IFN-γ), facilitated TAM survival in the context of CSF-1R inhibition. Expression of alternatively activated M2 markers decreased in surviving TAMs, which is consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in patients with proneural GBM. Our results identify TAMs as a promising therapeutic target for proneural gliomas and establish the translational potential of CSF-1R inhibition for GBM.

Inhibitory effect of a short Z-DNA forming sequence on transcription elongation by T7 RNA polymerase.

DNA sequences capable of forming unusual secondary structures can be a source of genomic instability. In some cases that instability might be affected by transcription, as recently shown for the Z-DNA forming sequence (CG)(14), which causes genomic instability both in mammalian cells and in bacteria, and this effect increases with its transcription. We have investigated the effect of this (CG)(14) sequence on transcription with T7 RNA polymerase in vitro. We detected partial transcription blockage within the sequence; the blockage increased with negative supercoiling of the template DNA. This effect was not observed in a control self-complementary sequence of identical length and base composition as the (CG)(14) sequence, when the purine-pyrimidine alternation required for Z-DNA formation was disrupted. These findings suggest that the inhibitory effect on T7 transcription results from Z-DNA formation in the (CG)(14) sequence rather than from an effect of the sequence composition or from hairpin formation in either the DNA or the RNA product.