The pattern of DNA methylation at cytosine bases in the genome is tightly linked to gene expression, and DNA methylation abnormalities are often observed in diseases. The ten eleven translocation (TET) enzymes oxidize 5-methylcytosines (5mCs) and promote locus-specific reversal of DNA methylation. TET genes, and especially TET2, are frequently mutated in various cancers, but how the TET proteins contribute to prevent the onset and maintenance of these malignancies is largely unknown. Here, we highlight recent advances in understanding the physiological function of the TET proteins and their role in regulating DNA methylation and transcription. In addition, we discuss some of the key outstanding questions in the field.

Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3′ terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicer-deficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.

The process of erythropoiesis must be efficient and robust to supply the organism with red bloods cells both under condition of homeostasis and stress. The microRNA (miRNA) pathway was recently shown to regulate erythroid development. Here, we show that expression of the locus encoding miR-144 and miR-451 is strictly dependent on Argonaute 2 and is required for erythroid homeostasis. Mice deficient for the miR-144/451 cluster display a cell autonomous impairment of late erythroblast maturation, resulting in erythroid hyperplasia, splenomegaly, and a mild anemia. Analysis of gene expression profiles from wild-type and miR-144/451–deficient erythroblasts revealed that the miR-144/451 cluster acts as a “tuner” of gene expression, influencing the expression of many genes. MiR-451 imparts a greater impact on target gene expression than miR-144. Accordingly, mice deficient in miR-451 alone exhibited a phenotype indistinguishable from miR-144/451–deficient mice. Thus, the miR-144/451 cluster tunes gene expression to impart a robustness to erythropoiesis that is critical under conditions of stress.

DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis.

Transcription factors (TFs) regulate many cellular processes and can therefore serve as readouts of the signaling and regulatory state. Yet for many TFs, the mode of action-repressing or activating transcription of target genes-is unclear. Here, we present diffTF (https://git.embl.de/grp-zaugg/diffTF) to calculate differential TF activity (basic mode) and classify TFs into putative transcriptional activators or repressors (classification mode). In basic mode, it combines genome-wide chromatin accessibility/activity with putative TF binding sites that, in classification mode, are integrated with RNA-seq. We apply diffTF to compare (1) mutated and unmutated chronic lymphocytic leukemia patients and (2) two hematopoietic progenitor cell types. In both datasets, diffTF recovers most known biology and finds many previously unreported TFs. It classifies almost 40% of TFs based on their mode of action, which we validate experimentally. Overall, we demonstrate that diffTF recovers known biology, identifies less well-characterized TFs, and classifies TFs into transcriptional activators or repressors.

Male fertility requires the continuous production of high quality motile spermatozoa in abundance. Alterations in all three metrics cause oligoasthenoteratozoospermia, the leading cause of human sub/infertility. Post-mitotic spermatogenesis inclusive of several meiotic stages and spermiogenesis (terminal spermatozoa differentiation) are transcriptionally inert, indicating the potential importance for the post-transcriptional microRNA (miRNA) gene-silencing pathway therein. We found the expression of miRNA generating enzyme Dicer within spermatogenesis peaks in meiosis with critical functions in spermatogenesis. In an expression screen we identified two miRNA loci of the miR-34 family (miR-34b/c and miR-449) that are specifically and highly expressed in post-mitotic male germ cells. A reduction in several miRNAs inclusive of miR-34b/c in spermatozoa has been causally associated with reduced fertility in humans. We found that deletion of both miR34b/c and miR-449 loci resulted in oligoasthenoteratozoospermia in mice. MiR-34bc/449-deficiency impairs both meiosis and the final stages of spermatozoa maturation. Analysis of miR-34bc^−/−;449^−/− pachytene spermatocytes revealed a small cohort of genes deregulated that were highly enriched for miR-34 family target genes. Our results identify the miR-34 family as the first functionally important miRNAs for spermatogenesis whose deregulation is causal to oligoasthenoteratozoospermia and infertility. The sustained production of functional motile sperm is critical for male fertility. In recent years, a dramatic increase of cases of male infertility were reported, with the most common cause represented by the production of morphologically abnormal spermatozoa with low motility. Several genetic and environmental factors have been proven to impact on sperm development. In particular, preliminary studies on samples from fertile and sterile individuals suggested that the deregulation of a class of small noncoding RNAs, called microRNAs, might be detrimental for sperm formation. To this end, we investigated the expression of Dicer, a core microRNA pathway component, in male germ cells and observed a peak of expression during meiosis. We performed a microRNA-expression screening and identified 5 members of the miR-34 family (miR-34bc and miR-449abc) as highly expressed from late meiosis to the sperm stage. Deletion of miR-34bc and miR-449 leads to sterility due to the production of abnormal spermatozoa with reduced motility. Thus our work proves for the first time the importance of a microRNA family in sperm formation and male fertility.