Barbara J Meyer

Targeted Genome Editing Across Species Using ZFNs and TALENs

Engineered nucleases target specific DNA sequences for gene disruption in nonmodel organisms. Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species.

Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin

Synaptotagmin is an abundant synaptic vesicle-associated protein proposed to be involved in calcium-mediated neurotransmitter release. Our molecular and genetic results demonstrate that, although synaptotagmin is required for the proper function of the presynaptic nerve terminal in C. elegans, some neurotransmitter release persists in synaptogamin mutants. In C. elegans neurons, synaptotagmin is localized to regions known to be rich in synapses and appears to be associated with synaptic vesicles. Mutants defective in the synaptotagmin gene, called snt-1, exhibit severe behavioral abnormalities that are characteristic of deficiencies in synaptic function, including severe locomotion, feeding, and defecation defects. The mutants are defective in exocytosis, since they accumulate acetylcholine, and are resistant to cholinesterase inhibitors, but they nevertheless remain sensitive to cholinergic receptor agonists. In spite of these exocytic defects, snt-1 mutants are capable of coordinated motor movements, indicating that the mutants do not have a complete block of neurotransmitter release.

DPY-27: A chromosome condensation protein homolog that regulates C. elegans dosage compensation through association with the X chromosome

dpy-27 is an essential dosage compensation gene that acts to reduce expression of both hermaphrodite X chromosomes. The DPY-27 protein becomes specifically localized to the X chromosomes of wild-type XX embryos, but remains diffusely distributed throughout the nuclei of male (XO) embryos. In xol-1 mutant XO embryos that activate the XX mode of dosage compensation and die from inappropriately low X chromosome transcript levels, DPY-27 becomes localized to X. Therefore, sex specificity of the dosage compensation process is regulated at the step of DPY-27 X chromosome localization. DPY-27 exhibits striking similarity to proteins required for assembly and structural maintenance of Xenopus chromosomes in vitro and for segregation of yeast chromosomes in vivo. These findings suggest a link between global regulation of gene expression and higher order chromosome structure. We propose that DPY-27 implements dosage compensation by condensing the chromatin structure of X in a manner that causes general reduction of X chromosome expression.

Condensin and cohesin complexity: the expanding repertoire of functions

Condensin and cohesin complexes act in diverse nuclear processes in addition to their widely known roles in chromosome compaction and sister chromatid cohesion. Recent work has elucidated the contribution of condensin and cohesin to interphase genome organization, control of gene expression, metazoan development and meiosis. Despite these wide-ranging functions, several themes have come to light: both complexes establish higher-order chromosome structure by inhibiting or promoting interactions between distant genomic regions, both complexes influence the chromosomal association of other proteins, and both complexes achieve functional specialization by swapping homologous subunits. Emerging data are expanding the range of processes in which condensin and cohesin are known to participate and are enhancing our knowledge of how chromosome architecture is regulated to influence numerous cellular functions.

Condensin and cohesin: more than chromosome compactor and glue

Two related protein complexes, cohesin and condensin, are essential for separating identical copies of the genome into daughter cells during cell division. Cohesin glues replicated sister chromatids together until they split at anaphase, whereas condensin reorganizes chromosomes into their highly compact mitotic structure. Unexpectedly, mutations in the subunits of these complexes have been uncovered in genetic screens that target completely different processes. Exciting new evidence is emerging that cohesin and condensin influence crucial processes during interphase, and unforeseen aspects of mitosis. Each complex can perform several roles, and individual subunits can associate with different sets of proteins to achieve diverse functions, including the regulation of gene expression, DNA repair, cell-cycle checkpoints and centromere organization.

Sperm chromatin proteomics identifies evolutionarily conserved fertility factors

Male infertility is a long-standing enigma of significant medical concern. The integrity of sperm chromatin is a clinical indicator of male fertility and in vitro fertilization potential: chromosome aneuploidy and DNA decondensation or damage are correlated with reproductive failure. Identifying conserved proteins important for sperm chromatin structure and packaging can reveal universal causes of infertility. Here we combine proteomics, cytology and functional analysis in Caenorhabditis elegans to identify spermatogenic chromatin-associated proteins that are important for fertility. Our strategy employed multiple steps: purification of chromatin from comparable meiotic cell types, namely those undergoing spermatogenesis or oogenesis; proteomic analysis by multidimensional protein identification technology (MudPIT) of factors that co-purify with chromatin; prioritization of sperm proteins based on abundance; and subtraction of common proteins to eliminate general chromatin and meiotic factors. Our approach reduced 1,099 proteins co-purified with spermatogenic chromatin, currently the most extensive catalogue, to 132 proteins for functional analysis. Reduction of gene function through RNA interference coupled with protein localization studies revealed conserved spermatogenesis-specific proteins vital for DNA compaction, chromosome segregation, and fertility. Unexpected roles in spermatogenesis were also detected for factors involved in other processes. Our strategy to find fertility factors conserved from C. elegans to mammals achieved its goal: of mouse gene knockouts corresponding to nematode proteins, 37% (7/19) cause male sterility. Our list therefore provides significant opportunity to identify causes of male infertility and targets for male contraceptives.

Sex-Specific Assembly of a Dosage Compensation Complex on the Nematode X Chromosome

In nematodes, flies, and mammals, dosage compensation equalizes X-chromosome gene expression between the sexes through chromosome-wide regulatory mechanisms that function in one sex to adjust the levels of X-linked transcripts. Here, a dosage compensation complex was identified in the nematode Caenorhabditis elegans that reduces transcript levels from the two X chromosomes in hermaphrodites. This complex contains at least four proteins, including products of the dosage compensation genes dpy-26 and dpy-27. Specific localization of the complex to the hermaphrodite X chromosomes is conferred by XX-specific regulatory genes that coordinately control both sex determination and dosage compensation.

MIX-1 : AN ESSENTIAL COMPONENT OF THE C. ELEGANS MITOTIC MACHINERY EXECUTES X CHROMOSOME DOSAGE COMPENSATION

We show that a functional component of the C. elegans mitotic machinery regulates X chromosome gene expression. This protein, MIX-1, is a member of the dosage compensation complex that associates specifically with hermaphrodite X chromosomes to reduce their gene expression during interphase. MIX-1 also associates with all mitotic chromosomes to ensure their proper segregation. Both dosage compensation and mitosis are severely disrupted by mix-1 mutations. MIX-1 belongs to the SMC protein family required for mitotic chromosome condensation and segregation in yeast and frogs. Thus, an essential, conserved component of mitotic chromosomes has been recruited to the dosage compensation process. Rather than dosage compensation and mitosis being achieved by two separate sets of related genes, these two processes share an identical component, indicating a common mechanism for establishing higher order chromosome structure and proper X chromosome gene expression.

Condensins Regulate Meiotic DNA Break Distribution, thus Crossover Frequency, by Controlling Chromosome Structure

Meiotic crossover (CO) recombination facilitates evolution and accurate chromosome segregation. CO distribution is tightly regulated: homolog pairs receive at least one CO, CO spacing is nonrandom, and COs occur preferentially in short genomic intervals called hotspots. We show that CO number and distribution are controlled on a chromosome-wide basis at the level of DNA double-strand break (DSB) formation by a condensin complex composed of subunits from two known condensins: the C. elegans dosage compensation complex and mitotic condensin II. Disruption of any subunit of the CO-controlling condensin dominantly changes DSB distribution, and thereby COs, and extends meiotic chromosome axes. These phenotypes are cosuppressed by disruption of a chromosome axis element. Our data implicate higher-order chromosome structure in the regulation of CO recombination, provide a model for the rapid evolution of CO hotspots, and show that reshuffling of interchangeable molecular parts can create independent machines with similar architectures but distinct biological functions.

Condensin-driven remodelling of X chromosome topology during dosage compensation

The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (∼1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.