Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin.

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct territories. Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin

Polycomb-Dependent Chromatin Looping Contributes to Gene Silencing during Drosophila Development.

International audienceInterphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development. VIDEO ABSTRACT

Genes involved in CoQ synthesis in <i>S. cerevisiae, S. pombe</i>, humans, and <i>A. thaliana</i>.

<p>Genes involved in CoQ synthesis in <i>S. cerevisiae, S. pombe</i>, humans, and <i>A. thaliana</i>.</p

Functional Conservation of Coenzyme Q Biosynthetic Genes among Yeasts, Plants, and Humans

<div><p>Coenzyme Q (CoQ) is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of <i>Escherichia coli</i> and <i>Saccharomyces cerevisiae</i>; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast <i>Schizosaccharomyces pombe</i>. Here, we expand these findings by identifying ten genes (<i>dps1, dlp1, ppt1</i>, and <i>coq3–9</i>) that are required for CoQ synthesis. CoQ10-deficient <i>S. pombe coq</i> deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (<i>Arabidopsis thaliana</i>) to functionally complement the <i>S. pombe coq</i> deletion strains was determined. With the exception of <i>COQ9</i>, expression of all other human and plant <i>COQ</i> genes recovered CoQ10 production by the fission yeast <i>coq</i> deletion strains, although the addition of a mitochondrial targeting sequence was required for human <i>COQ3</i> and <i>COQ7</i>, as well as <i>A. thaliana COQ6</i>. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants.</p></div

Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin

International audienceMembraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct "territories." Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin

Complementation of the <i>S. pombe coq</i> deletion strains by <i>A. thaliana COQ</i> genes.

<p>(A–E) HPLC analyses of lipid extracts from the KH3 (<i>Δcoq3</i>) strain expressing <i>S. pombe coq3</i> or At<i>COQ3</i> (A); the KH4 (<i>Δcoq4</i>) strain expressing <i>S. pombe coq4</i> or At<i>COQ4</i> (B); the KH5 (<i>Δcoq5</i>) strain expressing <i>S. pombe coq5</i> or At<i>COQ5</i> (C); the KH6 (<i>Δcoq6</i>) strain expressing <i>S. pombe coq6</i>, At<i>COQ6</i>, or At<i>COQ6</i> fused with a mitochondrial targeting sequence (Mt-Signal) (D); and the KH8 (<i>Δcoq8</i>) strain expressing <i>S. pombe coq8</i> or At<i>COQ8</i> (E). DMQ10, demethoxyubiquinone.</p

The proposed CoQ biosynthesis pathway in <i>S. cerevisiae</i>, <i>S. pombe</i>, humans, and <i>A. thaliana</i>.

<p>At least ten genes, three of which have unassigned roles, are responsible for CoQ biosynthesis in <i>S. pombe</i>. All of these <i>S. pombe</i> enzymes have human counterparts, but <i>A. thaliana</i> lacks Coq7 and a component of the prenyl diphosphate synthase in this plant species differs from that in <i>S. pombe</i> and humans. The functions of Coq4 and Coq9 are currently unknown. Coq8 is a protein kinase that regulates the stability of Coq proteins in <i>S. cerevisiae</i>. The involvement of pABA in the CoQ pathway in <i>S. pombe</i>, human and <i>A. thaliana</i> has not yet been established. For simplicity, <i>ARH1</i> and <i>YAH1</i>, which are involved in CoQ synthesis through the regulation of Coq6 in <i>S. cerevisiae</i>, are not shown.</p

Strains used in this study.

<p>Strains used in this study.</p

Functional complementation of the <i>S. pombe Δdps1</i> and <i>Δdlp1</i> strains by Hs<i>DPS1</i> and Hs<i>DLP1</i>.

<p>HPLC analyses of lipid extracts from the RM19 (<i>Δdlp1</i>) strain expressing <i>S. pombe dlp1</i> or Hs<i>DLP1</i> and the LA1 (<i>Δdps1-Δdlp1</i>) strain expressing <i>S. pombe dps1</i> and <i>dlp1</i> or Hs<i>DPS1</i> and Hs<i>DLP1</i>.</p