Molecular antagonism between X-chromosome and autosome signals determines nematode sex.

Sex is determined in Caenorhabditis elegans by the ratio of X chromosomes to the sets of autosomes, the X:A signal. A set of genes called X signal elements (XSEs) communicates X-chromosome dose by repressing the masculinizing sex determination switch gene xol-1 (XO lethal) in a dose-dependent manner. xol-1 is active in 1X:2A embryos (males) but repressed in 2X:2A embryos (hermaphrodites). Here we showed that the autosome dose is communicated by a set of autosomal signal elements (ASEs) that act in a cumulative, dose-dependent manner to counter XSEs by stimulating xol-1 transcription. We identified new ASEs and explored the biochemical basis by which ASEs antagonize XSEs to determine sex. Multiple antagonistic molecular interactions carried out on a single promoter explain how different X:A values elicit different sexual fates. XSEs (nuclear receptors and homeodomain proteins) and ASEs (T-box and zinc finger proteins) bind directly to several sites on xol-1 to counteract each other's activities and thereby regulate xol-1 transcription. Disrupting ASE- and XSE-binding sites in vivo recapitulated the misregulation of xol-1 transcription caused by disrupting cognate signal element genes. XSE- and ASE-binding sites are distinct and nonoverlapping, suggesting that direct competition for xol-1 binding is not how XSEs counter ASEs. Instead, XSEs likely antagonize ASEs by recruiting cofactors with reciprocal activities that induce opposite transcriptional states. Most ASE- and XSE-binding sites overlap xol-1's -1 nucleosome, which carries activating chromatin marks only when xol-1 is turned on. Coactivators and corepressors tethered by proteins similar to ASEs and XSEs are known to deposit and remove such marks. The concept of a sex signal comprising competing XSEs and ASEs arose as a theory for fruit flies a century ago. Ironically, while the recent work of others showed that the fly sex signal does not fit this simple paradigm, our work shows that the worm signal does.

Dose-dependent action of the RNA binding protein FOX-1 to relay X-chromosome number and determine C. elegans sex.

We demonstrate how RNA binding protein FOX-1 functions as a dose-dependent X-signal element to communicate X-chromosome number and thereby determine nematode sex. FOX-1, an RNA recognition motif protein, triggers hermaphrodite development in XX embryos by causing non-productive alternative pre-mRNA splicing of xol-1, the master sex-determination switch gene that triggers male development in XO embryos. RNA binding experiments together with genome editing demonstrate that FOX-1 binds to multiple GCAUG and GCACG motifs in a xol-1 intron, causing intron retention or partial exon deletion, thereby eliminating male-determining XOL-1 protein. Transforming all motifs to GCAUG or GCACG permits accurate alternative splicing, demonstrating efficacy of both motifs. Mutating subsets of both motifs partially alleviates non-productive splicing. Mutating all motifs blocks it, as does transforming them to low-affinity GCUUG motifs. Combining multiple high-affinity binding sites with the twofold change in FOX-1 concentration between XX and XO embryos achieves dose-sensitivity in splicing regulation to determine sex.

High throughput analysis of nuclear receptor-cofactor interactions.

Various assays have been employed to study the nuclear receptor/cofactor interactions. Coimmunoprecipitation protocols, both yeast and mammalian two-hybrid systems, and electrophoretic mobility shift/supershift assays are all commonly used. One of the most useful assays for studying direct protein-protein interactions is the glutathione-S-transferase "pulldown" assay. We have developed a high-throughput version of this assay that utilizes a filter microplate to allow parallel processing of many samples, significantly reducing the time and reagents required for the assay and increasing the sensitivity of the assay for weaker protein-protein interactions.

Strategies for Efficient Genome Editing Using CRISPR-Cas9.

The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and nonmodel organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes for both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-strand breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans We also devised strategies to insert long (10 kb) exogenous sequences and incorporate multiple nucleotide substitutions at a considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. We found a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9-targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.

Revisiting the X:A signal that specifies Caenorhabditis elegans sexual fate.

In Caenorhabditis elegans, sex is determined by the opposing actions of X-signal elements (XSEs) and autosomal signal elements (ASEs), which communicate the ratio of X chromosomes to sets of autosomes (X:A signal). This study delves more deeply into the mechanism by which XSEs transmit X chromosome dose. We determined the relative contributions of individual XSEs to the X:A signal and showed the order of XSE strength to be sex-1 > sex-2 > fox-1 > ceh-39 >/= region 1 XSE. sex-1 exerts a more potent influence on sex determination and dosage compensation than any other XSE by functioning in two separate capacities in the pathway: sex-1 acts upstream as an XSE to repress xol-1 and downstream as an activator of hermaphrodite development and dosage compensation. Furthermore, the process of dosage compensation affects expression of the very XSEs that control it; XSEs become fully dosage compensated once sex is determined. The X:A signal is then equivalent between XO and XX animals, causing sexual differentiation to be controlled by genes downstream of xol-1 in the sex-determination pathway. Prior to the onset of dosage compensation, the difference in XSE expression between XX and XO embryos appears to be greater than twofold, making X chromosome counting a robust process.

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break. The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Isotype-restricted corepressor recruitment: a constitutively closed helix 12 conformation in retinoic acid receptors beta and gamma interferes with corepressor recruitment and prevents transcriptional repression.

Retinoic acid receptors (RARs) are ligand-regulated transcription factors that play multiple roles in vertebrate development and differentiation. RARs as a class are capable of both repressing and activating target gene expression. Transcriptional repression is mediated through the recruitment of corepressor proteins such as SMRT. Notably, vertebrates encode three major forms of RARs, alpha, beta, and gamma, and these distinct RAR isotypes differ in the ability to recruit a corepressor. RAR alpha strongly interacts with SMRT and can repress target gene transcription, whereas RAR beta and -gamma interact with SMRT only weakly and fail to repress. We report here the use of a genetic suppressor approach, based on a yeast two-hybrid interaction assay using Saccharomyces cerevisiae, for the isolation of RAR beta mutants that have gained the RAR alpha-like corepressor phenotype, i.e., a strong interaction with SMRT and the ability to repress gene expression in vertebrate cells. Analysis of these gain-of-function mutants indicates that the different corepressor interaction properties of RAR alpha, -beta and -gamma are determined by a gating mechanism through which amino acid differences in the helix 3 region of these receptors influence the position of the receptor C-terminal helix 12 domain. As a consequence, the RAR beta and RAR gamma receptors appear to adopt a constitutively closed helix 12 conformation in the absence of hormone that may approximate the conformation of RAR alpha when bound to hormone agonist. This closed helix 12 conformation in RAR beta and RAR gamma blocks corepressor binding, prevents repression, and permits significant levels of target gene activation even in the absence of hormone. We refer to this phenomenon as a "gate-latch" model of corepressor regulation.

Targeted genome editing in Caenorhabditis elegans using CRISPR/Cas9.

Utilization of programmable nucleases to generate DNA lesions at precise endogenous sequences has transformed the ability to edit genomes from microbes to plants and animals. This is especially true in organisms that previously lacked the means to engineer precise genomic changes, like Caenorhabditis elegans. C. elegans is a 1 mm long free-living, nonparasitic, nematode worm, which is easily cultivated in a laboratory. Its detailed genetic map and relatively compact genome (~100 megabases) helped make it the first metazoan to have its entire genome sequenced. With detailed sequence information came development of numerous molecular tools to dissect gene function. Initially absent from this toolbox, however, were methods to make precise edits at chosen endogenous loci. Adapting site-specific nucleases for use in C. elegans, revolutionized studies of C. elegans biology. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and then CRISPR-associated protein 9 (Cas9) were used to target specific endogenous DNA sequences to make double-strand DNA breaks (DSBs). Precise changes could be engineered by providing repair templates targeting the DSB in trans. The ease of programming Cas9 to bind and cleave DNA sequences with few limitations has led to its widespread use in C. elegans research and sped the development of strategies to facilitate mutant recovery. Numerous innovative CRISPR/Cas9 methodologies are now primed for use in C. elegans. WIREs Dev Biol 2017, 6:e287. doi: 10.1002/wdev.287 For further resources related to this article, please visit the WIREs website.

Pituitary resistance to thyroid hormone syndrome is associated with T3 receptor mutants that selectively impair beta2 isoform function.

Resistance to thyroid hormone (RTH) syndrome is an inherited inability to respond appropriately to T3 hormone. In generalized RTH, the T3 response of both the pituitary and periphery is disrupted. In pituitary (or central) RTH, the ability of the pituitary to sense (and down-regulate) elevated T3 is selectively impaired, whereas the periphery remains relatively T3 responsive, resulting in peripheral thyrotoxicity. Both forms of disease are linked to mutations in thyroid hormone receptor (TR)-beta. TRbeta is expressed by alternate mRNA splicing as two isoforms: TRbeta2, found primarily in the pituitary/hypothalamus, and TRbeta1, expressed broadly in many tissues. We report here that the wild-type TRbeta2 isoform displays an enhanced T3 response relative to the TRbeta1 isoform. Mutations associated with generalized RTH (P453S, G345S) impair both TRbeta2 and TRbeta1 function proportionally, whereas mutations associated with pituitary-specific RTH (R338L, R338W, R429Q) disproportionately disrupt TRbeta2 function. We propose that in the normal organism, and in generalized RTH, TRbeta2 in the pituitary can sense rising T3 levels in advance of TRbeta1 in the periphery, preventing thyrotoxicity. In contrast, the TRbeta mutations associated with pituitary RTH disproportionately disrupt the pituitary's ability to sense and suppress elevated T3 levels in advance of the periphery, producing symptoms of thyrotoxicity.

Retinoic acid receptor-alpha is stabilized in a repressive state by its C-terminal, isotype-specific F domain.

Retinoic acid receptors (RARs) are hormone-regulated transcription factors that play multiple roles in vertebrate development and differentiation. Three isotypes of RARs, alpha, beta, and gamma, are encoded by distinct genetic loci and possess distinct transcriptional properties. Typically, RARalpha represses target gene transcription in the absence of hormone, whereas RARbeta and gamma fail to repress under these conditions. This inability of RARbeta and RARgamma to repress transcription is due to intramolecular interactions between helix 3 and helix 12 of the hormone binding domains of these isotypes that inhibit corepressor binding while favoring coactivator binding. We report here that the converse ability of RARalpha to repress requires the integrity of the receptor F domain, a domain that maps C-terminal to helix 12, varies in sequence among different nuclear receptors, and is of poorly understood function. The F domain appears to help stabilize helix 12 of RARalpha in a more open position that enhances corepressor binding and inhibits coactivator binding in the absence of hormone. Intriguingly, the RARalpha F domain is isotype autonomous in its function. We speculate that the RARalpha F domain may dock elsewhere on the receptor surface, and this intramolecular interaction may maintain RARalpha helix 12 in an open, repression-competent conformation.

Retinoic acid receptors beta and gamma do not repress, but instead activate target gene transcription in both the absence and presence of hormone ligand.

Retinoic acid receptors (RARs) are important mediators of retinoid signaling in morphogenesis, development, and cell differentiation. Three major isotypes of RARs, denoted alpha, beta, and gamma, have been identified, each encoded by a distinct genetic locus. Although RARalpha, RARbeta, and RARgamma share many structural and functional features, these three isotypes are known to play unique, as well as overlapping, roles in physiology and development. We report here that the three RAR isotypes display different transcriptional properties in the absence of hormone ligand; under these conditions, RARalpha is a strong repressor of target gene expression, whereas both RARbeta and RARgamma fail to repress and instead are able to mediate substantial levels of hormone-independent transcriptional activation. These differing transcriptional properties appear to reflect the differing abilities of the three RAR isotypes to interact with the SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor protein: RARalpha binds to SMRT strongly both in vitro and in vivo, whereas RARbeta and RARgamma interact only weakly with SMRT. The ability to repress or to activate transcription in the absence of hormone maps predominantly to isotype-specific differences in the sequence of helix 3 within the hormone binding domain of the RARs, and the transcriptional properties of one isotype can be exchanged with that of another by exchanging portions of helix 3. The different transcriptional properties of RARalpha, RARbeta, and RARgamma in the absence of hormone contribute to the distinctive biological functions of these proteins and provide a rationale for the strong conservation of the three distinct isotypes during the vertebrate evolutionary radiation.

Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design.

Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.

Power-line frequency electromagnetic fields do not induce changes in phosphorylation, localization, or expression of the 27-kilodalton heat shock protein in human keratinocytes.

The linkage of the exposure to the power-line frequency (50-60 Hz) electromagnetic fields (EMF) with human cancers remains controversial after more than 10 years of study. The in vitro studies on the adverse effects of EMF on human cells have not yielded a clear conclusion. In this study, we investigated whether power-line frequency EMF could act as an environmental insult to invoke stress responses in human keratinocytes using the 27-kDa heat shock protein (HSP27) as a stress marker. After exposure to 1 gauss (100 micro T) EMF from 20 min to 24 hr, the isoform pattern of HSP27 in keratinocytes remained unchanged, suggesting that EMF did not induce the phosphorylation of this stress protein. EMF exposure also failed to induce the translocation of HSP27 from the cytoplasm to the nucleus. Moreover, EMF exposure did not increase the abundance of HSP27 in keratinocytes. In addition, we found no evidence that EMF exposure enhanced the level of the 70-kDa heat shock protein (HSP70) in breast or leukemia cells as reported previously. Therefore, in this study we did not detect any of a number of stress responses in human keratinocytes exposed to power-line frequency EMF.

An improved high throughput protein-protein interaction assay for nuclear hormone receptors.

The Glutathione-S-Transferase (GST) "pulldown" assay has been used extensively to assay protein interactions in vitro. This methodology has been especially useful for investigating the interactions of nuclear hormone receptors with a wide variety of their interacting partners and coregulatory proteins. Unfortunately, the original GST-pulldown technique relies on multiple binding, washing and elution steps performed in individual microfuge tubes, and requires repeated centrifugation, aspiration, and suspension steps. This type of batch processing creates a significant liquid handling bottleneck, limiting the number of sample points that can be incorporated into one experiment and producing inherently less efficient washing and elution than would a flow-through methodology. In this manuscript, we describe the adaptation of this GST-pulldown assay to a 96-well filter plate format. The use of a multi-well filter plate makes it possible to assay more samples in significantly less time using less reagents and more efficient sample processing than does the traditional single tube assay.