The selective prolyl hydroxylase inhibitor IOX5 stabilizes HIF-1α and compromises development and progression of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.

C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line.

In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation.

SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation.

In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation1. In the male germline, RNA-directed DNA methylation silences young, active transposable elements2-4. The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements3,5. piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation.

TEX15 is an essential executor of MIWI2-directed transposon DNA methylation and silencing.

The PIWI protein MIWI2 and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of young active transposable elements (TEs) in the male germline. piRNAs are proposed to recruit MIWI2 to the transcriptionally active TE loci by base pairing to nascent transcripts, however the downstream mechanisms and effector proteins utilized by MIWI2 in directing de novo TE methylation remain incompletely understood. Here, we show that MIWI2 associates with TEX15 in foetal gonocytes. TEX15 is predominantly a nuclear protein that is not required for piRNA biogenesis but is essential for piRNA-directed TE de novo methylation and silencing. In summary, TEX15 is an essential executor of mammalian piRNA-directed DNA methylation.

A programmed wave of uridylation-primed mRNA degradation is essential for meiotic progression and mammalian spermatogenesis.

Several developmental stages of spermatogenesis are transcriptionally quiescent which presents major challenges associated with the regulation of gene expression. Here we identify that the zygotene to pachytene transition is not only associated with the resumption of transcription but also a wave of programmed mRNA degradation that is essential for meiotic progression. We explored whether terminal uridydyl transferase 4- (TUT4-) or TUT7-mediated 3' mRNA uridylation contributes to this wave of mRNA degradation during pachynema. Indeed, both TUT4 and TUT7 are expressed throughout most of spermatogenesis, however, loss of either TUT4 or TUT7 does not have any major impact upon spermatogenesis. Combined TUT4 and TUT7 (TUT4/7) deficiency results in embryonic growth defects, while conditional gene targeting revealed an essential role for TUT4/7 in pachytene progression. Loss of TUT4/7 results in the reduction of miRNA, piRNA and mRNA 3' uridylation. Although this reduction does not greatly alter miRNA or piRNA expression, TUT4/7-mediated uridylation is required for the clearance of many zygotene-expressed transcripts in pachytene cells. We find that TUT4/7-regulated transcripts in pachytene spermatocytes are characterized by having long 3' UTRs with length-adjusted enrichment for AU-rich elements. We also observed these features in TUT4/7-regulated maternal transcripts whose dosage was recently shown to be essential for sculpting a functional maternal transcriptome and meiosis. Therefore, mRNA 3' uridylation is a critical determinant of both male and female germline transcriptomes. In conclusion, we have identified a novel requirement for 3' uridylation-programmed zygotene mRNA clearance in pachytene spermatocytes that is essential for male meiotic progression.

Derivation of Human Trophoblast Stem Cells.

Trophoblast cells play an essential role in the interactions between the fetus and mother. Mouse trophoblast stem (TS) cells have been derived and used as the best in vitro model for molecular and functional analysis of mouse trophoblast lineages, but attempts to derive human TS cells have so far been unsuccessful. Here we show that activation of Wingless/Integrated (Wnt) and EGF and inhibition of TGF-ß, histone deacetylase (HDAC), and Rho-associated protein kinase (ROCK) enable long-term culture of human villous cytotrophoblast (CT) cells. The resulting cell lines have the capacity to give rise to the three major trophoblast lineages, which show transcriptomes similar to those of the corresponding primary trophoblast cells. Importantly, equivalent cell lines can be derived from human blastocysts. Our data strongly suggest that the CT- and blastocyst-derived cell lines are human TS cells, which will provide a powerful tool to study human trophoblast development and function.

Roles of MIWI, MILI and PLD6 in small RNA regulation in mouse growing oocytes.

The mouse PIWI-interacting RNA (piRNA) pathway produces a class of 26-30-nucleotide (nt) small RNAs and is essential for spermatogenesis and retrotransposon repression. In oocytes, however, its regulation and function are poorly understood. In the present study, we investigated the consequences of loss of piRNA-pathway components in growing oocytes. When MILI (or PIWIL2), a PIWI family member, was depleted by gene knockout, almost all piRNAs disappeared. This severe loss of piRNA was accompanied by an increase in transcripts derived from specific retrotransposons, especially IAPs. MIWI (or PIWIL1) depletion had a smaller effect. In oocytes lacking PLD6 (or ZUCCHINI or MITOPLD), a mitochondrial nuclease/phospholipase involved in piRNA biogenesis in male germ cells, the piRNA level was decreased to 50% compared to wild-type, a phenotype much milder than that in males. Since PLD6 is essential for the creation of the 5΄ ends of primary piRNAs in males, the presence of mature piRNA in PLD6-depleted oocytes suggests the presence of compensating enzymes. Furthermore, we identified novel 21-23-nt small RNAs, termed spiRNAs, possessing a 10-nt complementarity with piRNAs, which were produced dependent on MILI and independent of DICER. Our study revealed the differences in the biogenesis and function of the piRNA pathway between sexes.