Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces

During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.

Degradation of the Repetitive Genomic Landscape in a Close Relative of Caenorhabditis elegans

The abundance, diversity, and genomic distribution of repetitive elements is highly variable among species. These patterns are thought to be driven in part by reproductive mode and the interaction of selection and recombination, and recombination rates typically vary by chromosomal position. In the nematode Caenorhabditis elegans, repetitive elements are enriched at chromosome arms and depleted on centers, and this mirrors the chromosomal distributions of other genomic features such as recombination rate. How conserved is this genomic landscape of repeats, and what evolutionary forces maintain it? To address this, we compared the genomic organization of repetitive elements across five Caenorhabditis species with chromosome-level assemblies. As previously reported, repeat content is enriched on chromosome arms in most Caenorhabditis species, and no obvious patterns of repeat content associated with reproductive mode were observed. However, the fig-associated C. inopinata has experienced repetitive element expansion and reveals no association of global repeat density with chromosome position. Patterns of repeat superfamily specific distributions reveal this global pattern is driven largely by a few repeat superfamilies that in C. inopinata have expanded in number and have weak associations with chromosome position. Additionally, 15% of predicted protein-coding genes in C. inopinata align to transposon-related proteins. When these are excluded, C. inopinata has no enrichment of genes in chromosome centers, in contrast to its close relatives who all have such clusters. Forward evolutionary simulations reveal that chromosomal heterogeneity in recombination rate alone can generate structured repetitive genomic landscapes when insertions are weakly deleterious, whereas chromosomal heterogeneity in the fitness effects of transposon insertion can promote such landscapes across a variety of evolutionary scenarios. Thus, patterns of gene density along chromosomes likely contribute to global repetitive landscapes in this group, although other historical or genomic factors are needed to explain the idiosyncrasy of genomic organization of various transposable element taxa within C. inopinata. Taken together, these results highlight the power of comparative genomics and evolutionary simulations in testing hypotheses regarding the causes of genome organization.

Genetic Dissection of Grain Yield and Agronomic Traits in Maize under Optimum and Low-Nitrogen Stressed Environments

Understanding the genetic basis of maize grain yield and other traits under low-nitrogen (N) stressed environments could improve selection efficiency. In this study, five doubled haploid (DH) populations were evaluated under optimum and N-stressed conditions, during the main rainy season and off-season in Kenya and Rwanda, from 2014 to 2015. Identifying the genomic regions associated with grain yield (GY), anthesis date (AD), anthesis-silking interval (ASI), plant height (PH), ear height (EH), ear position (EPO), and leaf senescence (SEN) under optimum and N-stressed environments could facilitate the use of marker-assisted selection to develop N-use-efficient maize varieties. DH lines were genotyped with genotyping by sequencing. A total of 13, 43, 13, 25, 30, 21, and 10 QTL were identified for GY, AD ASI, PH, EH, EPO, and SEN, respectively. For GY, PH, EH, and SEN, the highest number of QTL was found under low-N environments. No common QTL between optimum and low-N stressed conditions were identified for GY and ASI. For secondary traits, there were some common QTL for optimum and low-N conditions. Most QTL conferring tolerance to N stress was on a different chromosome position under optimum conditions.

Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to disassemble bridging fibers, and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochores fibers.

Degradation of the repetitive genomic landscape in a close relative of C. elegans

The abundance, diversity, and genomic distribution of repetitive elements is highly variable among species. These patterns are thought to be driven in part by reproductive mode and the interaction of selection and recombination, and recombination rates typically vary by chromosomal position. In the nematode C. elegans, repetitive elements are enriched at chromosome arms and depleted on centers, and this mirrors the chromosomal distributions of other genomic features such as recombination rate. How conserved is this genomic landscape of repeats, and what evolutionary forces maintain it? To address this, we compared the genomic organization of repetitive elements across five Caenorhabditis species with chromosome-level assemblies. As previously reported, repeat content is enriched on chromosome arms in most Caenorhabditis species, and no obvious patterns of repeat content associated with reproductive mode were observed. However, the fig-associated Caenorhabditis inopinata has experienced rampant repetitive element expansion and reveals no association of global repeat content with chromosome position. Patterns of transposable element superfamily-specific distributions reveal this global pattern is driven largely by a few transposable element superfamilies that in C. inopinata have expanded in number and have weak associations with chromosome position. Additionally, 15% of predicted protein-coding genes in C. inopinata align to transposon-related proteins. When these are excluded, C. inopinata has no enrichment of genes in chromosome centers, in contrast to its close relatives who all have such clusters. Forward evolutionary simulations reveal that chromosomal heterogeneity in recombination rate is insufficient for generating structured genomic repetitive landscapes. Instead, heterogeneity in the fitness effects of transposable element insertion is needed to promote heterogeneity in repetitive landscapes. Thus, patterns of gene density along chromosomes are likely drivers of global repetitive landscapes in this group, although other historical or genomic factors are needed to explain the idiosyncrasy of genomic organization of various transposable element taxa within C. inopinata. Taken together, these results highlight the power of comparative genomics and evolutionary simulations in testing hypotheses regarding the causes of genome organization.

Astral microtubule forces alter nuclear organization and inhibit DNA repair in budding yeast

Dividing cells must balance the maintenance of genome integrity with the generation of cytoskeletal forces that control chromosome position. In this study, we investigate how forces on astral microtubules impact the genome during cell division by using live-cell imaging of the cytoskeleton, chromatin, and DNA damage repair in budding yeast. Our results demonstrate that dynein-dependent forces on astral microtubules are propagated through the spindle during nuclear migration and when in excess can increase the frequency of double-stranded breaks (DSBs). Under these conditions, we find that homology-directed repair of DSBs is delayed, indicating antagonism between nuclear migration and the mechanism of homology-directed repair. These effects are partially rescued by mutants that weaken pericentric cohesion or mutants that decrease constriction on the nucleus as it moves through the bud neck. We propose that minimizing nuclear movement aids in finding a donor strand for homologous recombination.

Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

ABSTRACTAchieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC containing the mouseMsh3-Dhfrlocus (DHFR BAC). Here we extend this “BAC TG-EMBED” approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01-5 fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes expressed on a single BAC. Second, we show small variability in both the expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making choice of BACs more flexible. Third, we describe an intriguing phenomenon in which BAC transgenes are maintained as episomes in a large fraction of stably selected clones. Finally, we demonstrate the utility of BAC TG-EMBED by simultaneously labeling three nuclear compartments in 94% of stable clones using a multi-reporter DHFR BAC, constructed with a combination of synthetic biology and BAC recombineering tools. Our extended BAC TG-EMBED method provides a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies.

Automated nuclear cartography reveals conserved sperm chromosome territory localization across 2 million years of mouse evolution

Measurements of nuclear organization in asymmetric nuclei in 2D images have traditionally been manual. This is exemplified by attempts to measure chromosome position in sperm samples, typically by dividing the nucleus into zones, and manually scoring which zone a FISH signal lies in. This is time consuming, limiting the number of nuclei that can be analyzed, and prone to subjectivity. We have developed a new approach for automated mapping of FISH signals in asymmetric nuclei, integrated into an existing image analysis tool for nuclear morphology. Automatic landmark detection defines equivalent structural regions in each nucleus, then dynamic warping of the FISH images to a common shape allows us to generate a composite of the signal within the entire cell population. Using this approach, we mapped the positions of the sex chromosomes and two autosomes in three mouse lineages (Mus musculus domesticus, Mus musculus musculus and Mus spretus). We found that in all three, chromosomes 11 and 19 tend to interact with each other, but are shielded from interactions with the sex chromosomes. This organization is conserved across 2 million years of mouse evolution.

Modulation of RNA-dependent interactions in stress granules prevents persistent TDP-43 accumulation in ALS/FTD

ABSTRACTHuman genetic variants are usually represented by four values with variable length: chromosome, position, reference and alternate alleles. Thereis no guarantee that these components are represented in a consistent way across different data sources, and processing variant-based data can be inefficient because four different comparison operations are needed for each variant, three of which are string comparisons. Working with strings, in contrast to numbers, poses extra challenges on computer memory allocation and data-representation. Existing variant identifiers do not typicallyrepresent every possible variant we may be interested in, nor they are directly reversible. To overcome these limitations, VariantKey, a novel reversible numerical encoding schema for human genetic variants, is presented here alongside a multi-language open-source software implementation (http://github.com/genomicspls/variantkey). VariantKey represents variants as single 64 bit numeric entities, while preserving the ability to be searched and sorted by chromosome and position. The individual components of short variants can be directly read back from the VariantKey, while long variants are supported with a fast lookup table.Highlights~100 compounds identified by high-content screen inhibit SGs in HEK293, NPCs and iPS-MNs.ALS-associated RBPs are recruited to SGs in an RNA-dependent mannerMolecules with planar moieties prevent recruitment of ALS-associated RBPs to SGsCompounds inhibit TDP-43 accumulation in SGs and in TARDBP mutant iPS-MNs.