SUMMARY Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a cofactor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer.

The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here we report that upregulation of the hyaluronan synthase HAS2 occurs in highly metastatic breast stem-like cancer cells (CSCs) defined by CD44+/CD24−/ESA+ phenotype, where it plays a critical role in the generation of a pro-metastatic microenvironment in breast cancer. HAS2 was critical for interaction of CSCs with tumor associated macrophages (TAMs), leading to enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan synthases which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings demonstrate a critical role for HAS2 in the development of a pro-metastatic microenvironment and suggest that HAS2 inhibitors can act as anti-metastatic agents that disrupt a paracrine growth factor loop within this microenvironment.

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Active DNA demethylation in mammals involves ten-eleven translocation (TET) family protein-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC) (an oxidized derivative of 5mC) at the single-cell level remain unexplored. Here, we present "CLEVER-seq" (chemical-labeling-enabled C-to-T conversion sequencing), which is a single-cell, single-base resolution 5fC-sequencing technology, based on biocompatible, selective chemical labeling of 5fC and subsequent C-to-T conversion during amplification and sequencing. CLEVER-seq shows intrinsic 5fC heterogeneity in mouse early embryos, Epi stem cells (EpiSCs), and embryonic stem cells (ESCs). CLEVER-seq of mouse early embryos also reveals the highly patterned genomic distribution and parental-specific dynamics of 5fC during mouse early pre-implantation development. Integrated analysis demonstrates that promoter 5fC production precedes the expression upregulation of a clear set of developmentally and metabolically critical genes. Collectively, our work reveals the dynamics of active DNA demethylation during mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells.

Active DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present a bisulfite-free method for whole-genome analysis of 5fC, based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps reveal limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones.

High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples remains challenging. Here, we present hmC-CATCH, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. hmC-CATCH is based on selective 5hmC oxidation, chemical labeling and subsequent C-to-T transition during PCR. Requiring only nanoscale input genomic DNA samples, hmC-CATCH enabled us to detect genome-wide hydroxymethylome of human embryonic stem cells in a cost-effective manner. Further application of hmC-CATCH to cell-free DNA (cfDNA) of healthy donors and cancer patients revealed base-resolution hydroxymethylome in the human cfDNA for the first time. We anticipate that our chemical biology approach will find broad applications in hydroxymethylome analysis of limited biological and clinical samples.

Abstract:A long-standing question in molecular biology relates to why the testes express the largest number of genes relative to all other organs. Here, we report a detailed gene expression map of human spermatogenesis using single-cell RNA-Seq. Surprisingly, we found that 20 spermatogenesis-expressed genes contain significantly fewer germline mutations than unexpressed genes, with the lowest mutation rates on the transcribed DNA strands. These results suggest a model of 'transcriptional scanning' to reduce germline mutations by correcting DNA damage. This model also explains the rapid evolution in sensory-and immune-defense related genes, as well as in male reproduction genes. Collectively, our results indicate that widespread 25 expression in the testes achieves a dual mechanism for maintaining the DNA integrity of most genes, while selectively promoting variation of other genes.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/282129 doi: bioRxiv preprint first posted online Mar. 14, 2018; 2 Main Text:Human tissues and organs are distinguished by the genes that they express and those that they do not 1,2 . Tissues have transcriptomes of different complexities in terms of uniquely-expressed genes, as well as those genes expressed at differential levels [3][4][5][6] . One overarching goal in the life sciences is to characterize the specific transcriptomic signatures of all human tissues, and 5 ultimately each different cell type at the single-cell level 7 .In males, the testis is unique in comparison with somatic tissues in that it contains germ cells which pass the genetic information on to the next generation 8 . Interestingly, it has been known for many years that the testis stands out as having the most complex transcriptome with the highest number of expressed genes [9][10][11][12] . Widespread transcription in the testes has been 10 reported to account for an amazing expression of over 80% of all our protein-coding genes 10,11,13 , as well as across many other mammals 3,10 .Several hypotheses have been proposed to explain this observation. Widespread expression may represent a functional requirement for the gene-products in question 12 .However, other more complex organs such as the brain do not exhibit a corresponding number of 15 expressed genes despite the fact that they consist of a substantially greater number of distinct cell types 3,10,14-16 . Moreover, recent animal studies have shown that many testis-enriched and evolutionarily-conserved genes are not required for male fertility in mice 17 . A second hypothesis implicates leaky transcription during the massive chromatin remodeling that occurs throughout spermatogenesis 12,18,19 . However, this model predicts more expression during later stages of 20 spermatogenesis -when the genome is undergoing the most chromatin changes -contradicting ...

Translational efficiency correlates with longevity, yet its role in lifespan determination remains unclear. Using ribosome profiling, translation efficiency is globally reduced during replicative aging in budding yeast by at least two mechanisms: Firstly, Ssd1 is induced during aging, sequestering mRNAs to P-bodies. Furthermore, Ssd1 overexpression in young cells reduced translation and extended lifespan, while loss of Ssd1 reduced the translational deficit of old cells and shortened lifespan. Secondly, phosphorylation of eIF2α, mediated by the stress kinase Gcn2, was elevated in old cells, contributing to the global reduction in translation without detectable induction of the downstream Gcn4 transcriptional activator. tRNA overexpression activated Gcn2 in young cells and extended lifespan in a manner dependent on Gcn4. Moreover, overexpression of Gcn4 sufficed to extend lifespan in an autophagy-dependent manner in the absence of changes in global translation, indicating that Gcn4-mediated autophagy induction is the ultimate downstream target of activated Gcn2, to extend lifespan.