Nucleation and spreading maintain Polycomb domains every cell cycle.

Gene repression by the Polycomb pathway is essential for metazoan development. Polycomb domains, characterized by trimethylation of histone H3 lysine 27 (H3K27me3), carry the memory of repression and hence need to be maintained to counter the dilution of parental H3K27me3 with unmodified H3 during replication. Yet, how locus-specific H3K27me3 is maintained through replication is unclear. To understand H3K27me3 recovery post-replication, we first define nucleation sites within each Polycomb domain in mouse embryonic stem cells. To map dynamics of H3K27me3 domains across the cell cycle, we develop CUT&Flow (coupling cleavage under target and tagmentation with flow cytometry). We show that post-replication recovery of Polycomb domains occurs by nucleation and spreading, using the same nucleation sites used during de novo domain formation. By using Polycomb repressive complex 2 (PRC2) subunit-specific inhibitors, we find that PRC2 targets nucleation sites post-replication independent of pre-existing H3K27me3. Thus, competition between H3K27me3 deposition and nucleosome turnover drives both de novo domain formation and maintenance during every cell cycle.

Small RNA pathways in the nematode Ascaris in the absence of piRNAs.

Small RNA pathways play key and diverse regulatory roles in C. elegans, but our understanding of their conservation and contributions in other nematodes is limited. We analyzed small RNA pathways in the divergent parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that associate with secondary 5'-triphosphate 22-24G-RNAs. These small RNAs target repetitive sequences or mature mRNAs and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Even in the absence of a piRNA pathway, Ascaris CSR-1 may still function to "license" as well as fine-tune or repress gene expression. Ascaris ALG-4 and its associated 26G-RNAs target and likely repress specific mRNAs during testis meiosis. Ascaris WAGO small RNAs demonstrate target plasticity changing their targets between repeats and mRNAs during development. We provide a unique and comprehensive view of mRNA and small RNA expression throughout spermatogenesis. Overall, our study illustrates the conservation, divergence, dynamics, and flexibility of small RNA pathways in nematodes.

Comprehensive Chromosome End Remodeling during Programmed DNA Elimination.

Germline and somatic genomes are in general the same in a multicellular organism. However, programmed DNA elimination leads to a reduced somatic genome compared to germline cells. Previous work on the parasitic nematode Ascaris demonstrated that programmed DNA elimination encompasses high-fidelity chromosomal breaks and loss of specific genome sequences including a major tandem repeat of 120 bp and ~1,000 germline-expressed genes. However, the precise chromosomal locations of these repeats, breaks regions, and eliminated genes remained unknown. We used PacBio long-read sequencing and chromosome conformation capture (Hi-C) to obtain fully assembled chromosomes of Ascaris germline and somatic genomes, enabling a complete chromosomal view of DNA elimination. We found that all 24 germline chromosomes undergo comprehensive chromosome end remodeling with DNA breaks in their subtelomeric regions and loss of distal sequences including the telomeres at both chromosome ends. All new Ascaris somatic chromosome ends are recapped by de novo telomere healing. We provide an ultrastructural analysis of Ascaris DNA elimination and show that eliminated DNA is incorporated into double membrane-bound structures, similar to micronuclei, during telophase of a DNA elimination mitosis. These micronuclei undergo dynamic changes including loss of active histone marks and localize to the cytoplasm following daughter nuclei formation and cytokinesis where they form autophagosomes. Comparative analysis of nematode chromosomes suggests that chromosome fusions occurred, forming Ascaris sex chromosomes that become independent chromosomes following DNA elimination breaks in somatic cells. These studies provide the first chromosomal view and define novel features and functions of metazoan programmed DNA elimination.

Polyploidy and nuclear phenotype characteristics of cardiomyocytes from diabetic adult and normoglycemic aged mice.

The frequency of polyploid nuclei in the aging human heart is in sharp contrast with that in the human liver. An inverse pattern exists between the mouse heart and liver cells. Ploidy degrees in mouse hepatocytes under hyperglycemic conditions are elevated to higher levels than those in aged hepatocytes. In this study, image analysis cytometry was used to investigate the effect of diabetes and aging on Feulgen-DNA quantities, ploidy degrees, nuclear shapes and chromatin texture in mouse cardiomyocytes compared to previously reported data for mouse hepatocytes. Adult, non-obese diabetic (NOD) hyperglycemic and normoglycemic females and 56-week-old normoglycemic BALB/c females were used. A small percentage (∼7%) of the cardiomyocyte nuclei in severely hyperglycemic NOD adult mice possessed higher ploidy values than those in the 8-week-old normoglycemic mice. Surprisingly, the Feulgen-DNA values and the frequency of nuclei belonging to the 4C and 8C ploidy classes were even higher (∼6%) in normoglycemic NOD specimens than in age-matched hyperglycemic NOD specimens. Additionally, a pronounced elongated nuclear shape was observed especially in adult normoglycemic NOD mice. In conclusion, NOD mice, irrespective of their glycemic level, exhibit a moderate increase in ploidy degrees within cardiomyocyte nuclei during the adult lifetime. As expected, aging did not affect the Feulgen-DNA values and the ploidy degrees of cardiomyocytes in BALB/c mice. The differences in ploidy degrees and chromatin textures such as absorbance variability and entropy, between adult NOD and aged BALB/c mice are consistent with other reports, indicating dissimilarities in chromatin functions between diabetes and aging.

DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy.

Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of-CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for-CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than-CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.