A Connection By Fate Novel Pdf Download

[Eberardo Topher]

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The regeneration of new myocardium by stem or progenitor cells is an important therapeutic option. Cellular or nuclear fusion is considered as an alternative to cell reprogramming by transdifferentiation. However, the generation of hybrid cells may also be a consequence of a transient transmembrane exchange of proteins and organelles between cells. Therefore, we investigated the formation of intercellular connections, which may allow the transport of macromolecular structures between labeled adult human endothelial progenitor cells (EPC) and GFP-expressing neonatal rat cardiomyocytes (CM) in a coculture system. FACS analysis revealed that, 6 days after initiation of coculture, 2.1+/-0.4% of the cells stained positive for GFP and Dil-ac-LDL. 6 hours after initiation of the coculture, ultrafine intercellular structures between Dil-ac-LDL-labeled EPC and GFP-expressing CM were observed. The number of EPC, which established nanotubular connections with CM increased from 0.5+/-0.2% after 6 hours to 2.6+/-0.3% after 24 hours of coculture. The intercellular connections had a diameter from 50 to 800 nm, a length of 5 to 120 mum, and were only transiently established. To determine whether the nanotubular structures allowed the transport of organelles, we labeled CM with a mitochondrial live tracker (MitoTracker). Using time-lapse video microscopy, we observed the transport of stained complexes between CM and EPC resulting in up-take of MitoTracker-positive structures in EPC. Thus, the present study shows a novel type of cell-to-cell communication between progenitor cells and CM in vitro, which may contribute to the acquisition of a cardiomyogenic phenotype independent of permanent cellular or nuclear fusion.

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Meiotic recombination is initiated by the formation of double-strand breaks (DSBs), which are repaired as either crossovers (COs) or noncrossovers (NCOs). In most mammals, PRDM9-mediated H3K4me3 controls the nonrandom distribution of DSBs; however, both the timing and mechanism of DSB fate control remain largely undetermined. Here, we generated comprehensive epigenomic profiles of synchronized mouse spermatogenic cells during meiotic prophase I, revealing spatiotemporal and functional relationships between epigenetic factors and meiotic recombination. We find that PRDM9-mediated H3K4me3 at DSB hotspots, coinciding with H3K27ac and H3K36me3, is intimately connected with the fate of the DSB. Our data suggest that the fate decision is likely made at the time of DSB formation: earlier formed DSBs occupy more open chromatins and are much more competent to proceed to a CO fate. Our work highlights an intrinsic connection between PRDM9-mediated H3K4me3 and the fate decision of DSBs, and provides new insight into the control of CO homeostasis.

Meiosis is a specialized form of cell division that generates haploid gametes from diploid cells, and is essential for sexual reproduction and evolution1,2. Fundamental to meiosis is the process of meiotic recombination, which leads to the transmission of new combinations of linked alleles to the next generation1,2. Meiotic recombination is initiated by the introduction of programmed DNA double-strand breaks (DSBs) by the topoisomerase-like transesterases, SPO11 and TOPVIBL1,3,4,5,6. Following DSB formation, single-stranded DNA (ssDNA) ends are engaged in the process of repair, which results in the loading of RAD51 and DMC11,2,7. This facilitates the search for homologous chromosomes (homologs) in the majority of strand invasion and the formation of single-end invasion strand exchange intermediates (SEIs)2,8,9,10. SEIs can be resolved either by synthesis-dependent strand annealing (SDSA) to generate only noncrossover (NCO) recombinants, or by double Holliday junctions (dHJs) to generate crossover (CO)/NCO recombinants2,8,9,10,11,12,13,14,15,16,17,18,19. Only a small fraction of DSBs are subsequently repaired to produce COs, whereas the remaining ones lead to the formation of NCOs2,8. DSBs occur most often at preferred sites termed hotspots, whose locations become marked by H3K4me3; in most mammals, this is determined by the histone methyltransferase PRDM920,21,22,23,24. Despite recent advances, much remains to be known about the dynamic nature and function of critical epigenetic factors involved in the recombination process and in particular, in the DSB fate decision.

Interestingly, our analysis of H3K4me1 revealed a similar trend but different dynamics. We found that very weak but detectable H3K4me1 signals started to appear even in type B spermatogonia; however, substantial H3K4me1 signals occurred in mid-preleptotene, and persisted until mid-pachytene, which lasted significantly longer than H3K4me3 signals. Although H3K4me1 peaks in mid-zygotene, similar to H3K4me3, we found that it covered a longer range, likely more nucleosomes, than H3K4me3 did (Fig. 2c; Supplementary information, Fig. S8f). Interestingly, most central DSB nucleosomes were converted from H3K4me1 to H3K4me3 in mid-zygotene and lasted till late-zygotene (Fig. 2c), consistent with the H3K4me3 pattern described above. Such patterns indicated that the chromatin containing DSB is subjected to a previously unappreciated dynamic regulation between H3K4me1 and H3K4me3, which might also be mediated by PRDM9. Supporting this, our in vitro enzymatic analyses found that PRDM9 mainly catalyzed generation of H3K4me1 and H3K4me3 in recombinant nucleosomes (Fig. 2d). Such intrinsic enzymatic features indicate that PRDM9-mediated H3K4me1 may also functionally contribute to DSB formation.

Altogether, these results reveal a specific spatiotemporal regulation of the hotspot-associated H3K4me3 dynamics during meiosis prophase I, which is intrinsically coupled with a previously unknown transition from H3K4me1 to H3K4me3 mediated by PRDM9, and indicate that they may play a role in the progression of recombination.

Little is known about the potential roles of PRDM9-mediated H3K4me3 during DSB repair; particularly how and when the cell determines which DSBs will be repaired as COs or NCOs remains unclear. Notably, we found that of the 10,137 hotspot-associated H3K4me3 marks generated specifically in mid-zygotene, 2369 were quickly erased during the mid-late zygotene stage (henceforth defined as fast-turnover H3K4me3), and a further 1036 were erased during the late-zygotene to early 1-pachytene stage (defined as slow-turnover H3K4me3); this period, from the mid-zygotene to early 1-pachytene stage, corresponds to DSB disappearance and NCO formation (Fig. 3a)9,10,34,35. Moreover, only 3 of the 7375 hotspot-associated H3K4me3 peaks persisting in early 1-pachytene were observed in mid-pachytene, during which the SEIs are thought to be converted to dHJs9,10,34,35. We therefore inferred that DSBs with H3K4me3 marks that disappear more quickly tend to be repaired as NCOs by the SDSA pathway during the zygotene stage, whereas more persistent H3K4me3 marks more likely denote sites of CO-designated recombination during the early-mid pachytene stage. These results indicated that the dynamics of the hotspot-associated H3K4me3 is in accordance with the reported kinetics of DSB formation and repair, such as the onset of SEI formation and the onset of dHJ formation34,35,36. It is tempting to speculate that hotspot-associated H3K4me3 generated at different times are distinct from one another, and that these varying types create corresponding environments that support either NCO differentiation or CO formation.

We also analyzed the DNA methylation levels in the DSB hotspots (defined by SPO11-oligo). We found that the DNA methylation levels around the DSB hotspots are similar to those in global genomic regions, suggesting that DNA methylation may not be involved in DSB formation (Supplementary information, Fig. S13g).

We further tested whether H3K27ac, H3K36me3, and chromatin state contributed to recombinational progression. We found that the width and density of H3K27ac and H3K36me3 peaks around the four groups of DSBs described above (Fig. 3c) were similar to those of the H3K4me3 peaks (Fig. 5a, b), indicating that the DSBs at sites of early-forming H3K4me3 also occupy extended and stronger H3K27ac and H3K36me3 signals. Moreover, DSB regions with early-forming H3K4me3 were also significantly enriched for NDRs (Fig. 5c), further confirming a connection between the active histone modification and open chromatin state at DSB sites. We then analyze the chromatin accessibility around PRDM9-binding sites (defined by the PRDM9-affinity-Seq) in these four clusters29. The results showed that there is a significant difference for chromatin accessibility between global genomic regions and early-forming H3K4me3 regions in zygotene stages. For the other three clusters, the differences are milder (Fig. 5d). In addition, the chromatin state around the 16 well-known CO hotspots (described above) was much more open than that around general DSB hotspots (defined by SPO11-oligo), or around the DSBs at the sites of fast-turnover, slow-turnover, or even persistent H3K4me3 marks (Supplementary information, Fig. S14). Taken together, these results indicate that H3K27ac and H3K36me3, coordinately with PRDM9-mediated H3K4me3, generate a robust and permissive chromatin environment at DSBs, in particular, the earlier formed DSBs, to support them to proceed to a CO event.

Our findings highlight potentially crucial roles of PRDM9-mediated H3K4me3 in directing DSB fate, provide new insight into CO homeostatic control, and call for future studies on how PRDM9 selects chromatin regions at an early stage to generate stronger and extended H3K4me3.

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