Systematic genetic approaches have provided deep insight into the molecular and cellular mechanisms that operate in simple unicellular organisms. For multicellular organisms, however, the pleiotropy of gene function has largely restricted such approaches to the study of early embryogenesis. With the availability of genome-wide transgenic RNA interference (RNAi) libraries in Drosophila, it is now possible to perform a systematic genetic dissection of any cell or tissue type at any stage of the lifespan. Here we apply these methods to define the genetic basis for formation and function of the Drosophila muscle. We identify a role in muscle for 2,785 genes, many of which we assign to specific functions in the organization of muscles, myofibrils or sarcomeres. Many of these genes are phylogenetically conserved, including genes implicated in mammalian sarcomere organization and human muscle diseases.

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C) 1 , 2 has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells 3 – 7 yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells1–3. Assembly of mitotic chromosomes involves DNA looping by condensin4–8 and chromatin compaction by global histone deacetylation9–13. Although condensin confers mechanical resistance to spindle pulling forces14–16, it is not known how histone deacetylation affects material properties and, as a consequence, segregation mechanics of mitotic chromosomes. Here we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with the physical characteristics necessary for their precise movement during cell division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. By contrast, hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin phase separation to genome segregation in dividing cells.

BackgroundSystematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.Methodology/Principal FindingsWe show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.Conclusions/SignificanceThe Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

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Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells. Assembly of mitotic chromosomes involves DNA looping by condensin and chromatin compaction by global histone deacetylation. While condensin confers mechanical resistance towards spindle pulling forces, it is not known how histone deacetylation affects material properties and segregation mechanics of mitotic chromosomes. Here, we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with specific characteristics necessary for their precise movement during cellular division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. Hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules, and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin-intrinsic phase separation to genome segregation in dividing cells.

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C) 1-3 has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells 4-11 yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

Genetic information is stored in linear DNA molecules, which are highly folded inside cells. DNA replication along the folded template path yields two sister chromatids that initially occupy the same nuclear region in an intertwined arrangement. Dividing cells must disentangle and condense the sister chromatids into separate bodies such that a microtubule‐based spindle can move them to opposite poles. While the spindle‐mediated transport of sister chromatids has been studied in detail, the chromosome‐intrinsic mechanics presegregating sister chromatids have remained elusive. Here, we show that human sister chromatids resolve extensively already during interphase, in a process dependent on the loop‐extruding activity of cohesin, but not that of condensins. Increasing cohesin's looping capability increases sister DNA resolution in interphase nuclei to an extent normally seen only during mitosis, despite the presence of abundant arm cohesion. That cohesin can resolve sister chromatids so extensively in the absence of mitosis‐specific activities indicates that DNA loop extrusion is a generic mechanism for segregating replicated genomes, shared across different Structural Maintenance of Chromosomes (SMC) protein complexes in all kingdoms of life.