Nanobody-mediated control of gene expression and epigenetic memory.

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

High-Throughput Discovery and Characterization of Human Transcriptional Effectors.

Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.

To Break or Not To Break: Sex Chromosome Hemizygosity During Meiosis in Caenorhabditis.

Meiotic recombination establishes connections between homologous chromosomes to promote segregation. Hemizygous regions of sex chromosomes have no homologous chromosome to recombine with, yet must be transmitted through meiosis. An extreme case of hemizygosity exists in the genus Caenorhabditis, where males have a single X chromosome that completely lacks a homologous partner. To determine whether similar strategies have evolved to accommodate hemizygosity of the X during male meiosis in Caenorhabditis with distinct modes of sexual reproduction, we examined induction and processing of meiotic double strand breaks (DSBs) in androdioecious (hermaphrodite/male) Caenorhabditis elegans and C. briggsae, and gonochoristic (female/male) C. remanei and C. brenneri Analysis of the recombinase RAD-51 suggests more meiotic DSBs are induced in gonochoristic vs. androdioecious species. However, in late prophase in all species, chromosome pairs are restructured into bivalents around a single axis, suggesting that the holocentric nature of Caenorhabditis chromosomes dictates a single crossover per bivalent regardless of the number of DSBs induced. Interestingly, RAD-51 foci were readily observed on the X chromosome of androdioecious male germ cells, while very few were detected in gonochoristic male germ cells. As in C. elegans, the X chromosome in C. briggsae male germ cells undergoes transient pseudosynapsis and flexibility in DSB repair pathway choice. In contrast, in C. remanei and C. brenneri male germ cells, the X chromosome does not undergo pseudosynapsis and appears refractory to SPO-11-induced breaks. Together our results suggest that distinct strategies have evolved to accommodate sex chromosome hemizygosity during meiosis in closely related Caenorhabditis species.

Plasticity in the Meiotic Epigenetic Landscape of Sex Chromosomes in Caenorhabditis Species.

During meiosis in the heterogametic sex in some species, sex chromosomes undergo meiotic sex chromosome inactivation (MSCI), which results in acquisition of repressive chromatin and transcriptional silencing. In Caenorhabditis elegans, MSCI is mediated by MET-2 methyltransferase deposition of histone H3 lysine 9 dimethylation. Here we examined the meiotic chromatin landscape in germ lines of four Caenorhabditis species; C. remanei and C. brenneri represent ancestral gonochorism, while C. briggsae and C. elegans are two lineages that independently evolved hermaphroditism. While MSCI is conserved across all four species, repressive chromatin modifications are distinct and do not correlate with reproductive mode. In contrast to C. elegans and C. remanei germ cells where X chromosomes are enriched for histone H3 lysine 9 dimethylation, X chromosomes in C. briggsae and C. brenneri germ cells are enriched for histone H3 lysine 9 trimethylation. Inactivation of C. briggsae MET-2 resulted in germ-line X chromosome transcription and checkpoint activation. Further, both histone H3 lysine 9 di- and trimethylation were reduced in Cbr-met-2 mutant germ lines, suggesting that in contrast to C. elegans, H3 lysine 9 di- and trimethylation are interdependent. C. briggsae H3 lysine 9 trimethylation was redistributed in the presence of asynapsed chromosomes in a sex-specific manner in the related process of meiotic silencing of unsynapsed chromatin. However, these repressive marks did not influence X chromosome replication timing. Examination of additional Caenorhabditis species revealed diverse H3 lysine 9 methylation patterns on the X, suggesting that the sex chromosome epigenome evolves rapidly.

Pseudosynapsis and decreased stringency of meiotic repair pathway choice on the hemizygous sex chromosome of Caenorhabditis elegans males.

During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes.