Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C.

Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs.

Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

Cladogenesis and reticulation in Cuscuta sect. Denticulatae (Convolvulaceae).

As traditionally circumscribed, Cuscuta sect. Denticulatae is a group of three parasitic plant species native to the deserts of Western USA (Cuscuta denticulata, Cuscuta nevadensis) and the central region of Baja California, Mexico (Cuscuta veatchii). Molecular phylogenetic studies confirmed the monophyly of this group and suggested that the disjunct C. veatchii is a hybrid between the other two species. However, the limited sampling left the possibility of alternative biological and methodological explanations. We expanded our sampling to multiple individuals of all the species collected from across their entire geographical ranges. Sequence data from the nuclear and plastid regions were used to reconstruct the phylogeny and find out if the topological conflict was maintained. We obtained karyotype information from multiple individuals, investigated the morphological variation of the group thorough morphometric analyses, and compiled data on ecology, host range, and geographical distribution. Our results confirmed that C. veatchii is an allotetraploid. Furthermore, we found previously unknown autotetraploid population of C. denticulata, and we describe a new hybrid species, Cuscuta psorothamnensis. We suggest that this newly discovered natural hybrid is resulting from an independent (and probably more recent) hybridization event between the same diploid parental species as those of C. veatchii. All the polyploids showed host shift associated with hybridization and/or polyploidy and are found growing on hosts that are rarely or never frequented by their diploid progenitors. The great potential of this group as a model to study host shift in parasitic plants associated with recurrent allopolyploidy is discussed.

Defects in tRNA Anticodon Loop 2'-O-Methylation Are Implicated in Nonsyndromic X-Linked Intellectual Disability due to Mutations in FTSJ1.

tRNA modifications are crucial for efficient and accurate protein synthesis, and modification defects are frequently associated with disease. Yeast trm7Δ mutants grow poorly due to lack of 2'-O-methylated C32 (Cm32 ) and Gm34 on tRNA(Phe) , catalyzed by Trm7-Trm732 and Trm7-Trm734, respectively, which in turn results in loss of wybutosine at G37 . Mutations in human FTSJ1, the likely TRM7 homolog, cause nonsyndromic X-linked intellectual disability (NSXLID), but the role of FTSJ1 in tRNA modification is unknown. Here, we report that tRNA(Phe) from two genetically independent cell lines of NSXLID patients with loss-of-function FTSJ1 mutations nearly completely lacks Cm32 and Gm34 , and has reduced peroxywybutosine (o2yW37 ). Additionally, tRNA(Phe) from an NSXLID patient with a novel FTSJ1-p.A26P missense allele specifically lacks Gm34 , but has normal levels of Cm32 and o2yW37 . tRNA(Phe) from the corresponding Saccharomyces cerevisiae trm7-A26P mutant also specifically lacks Gm34 , and the reduced Gm34 is not due to weaker Trm734 binding. These results directly link defective 2'-O-methylation of the tRNA anticodon loop to FTSJ1 mutations, suggest that the modification defects cause NSXLID, and may implicate Gm34 of tRNA(Phe) as the critical modification. These results also underscore the widespread conservation of the circuitry for Trm7-dependent anticodon loop modification of eukaryotic tRNA(Phe) .

E151 (sym15), a pleiotropic mutant of pea (Pisum sativum L.), displays low nodule number, enhanced mycorrhizae, delayed lateral root emergence, and high root cytokinin levels.

In legumes, the formation of rhizobial and mycorrhizal root symbioses is a highly regulated process which requires close communication between plant and microorganism. Plant mutants that have difficulties establishing symbioses are valuable tools for unravelling the mechanisms by which these symbioses are formed and regulated. Here E151, a mutant of Pisum sativum cv. Sparkle, was examined to characterize its root growth and symbiotic defects. The symbioses in terms of colonization intensity, functionality of micro-symbionts, and organ dominance were compared between the mutant and wild type. The endogenous cytokinin (CK) and abscisic acid (ABA) levels and the effect of the exogenous application of these two hormones were determined. E151 was found to be a low and delayed nodulator, exhibiting defects in both the epidermal and cortical programmes though a few mature and functional nodules develop. Mycorrhizal colonization of E151 was intensified, although the fungal functionality was impaired. Furthermore, E151 displayed an altered lateral root (LR) phenotype compared with that of the wild type whereby LR emergence is initially delayed but eventually overcome. No differences in ABA levels were found between the mutant and the wild type, but non-inoculated E151 exhibited significantly high CK levels. It is hypothesized that CK plays an essential role in differentially mediating the entry of the two micro-symbionts into the cortex; whereas it would inhibit the entry of the rhizobia in that tissue, it would promote that of the fungus. E151 is a developmental mutant which may prove to be a useful tool in further understanding the role of hormones in the regulation of beneficial root symbioses.

Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging (SNP-CLING).

Imaging and chromatin capture techniques have provided important insights into our understanding of nuclear organization. A limitation of these techniques is the inability to resolve allele-specific spatiotemporal properties of genomic loci in living cells. Here, we describe an allele-specific CRISPR live-cell DNA imaging technique (SNP-CLING) to provide the first comprehensive insights into allelic positioning across space and time in mouse embryonic stem cells and fibroblasts. With 3D imaging, we studied alleles on different chromosomes in relation to one another and relative to nuclear substructures such as the nucleolus. We find that alleles maintain similar positions relative to each other and the nucleolus; however, loci occupy unique positions. To monitor spatiotemporal dynamics by SNP-CLING, we performed 4D imaging and determined that alleles are either stably positioned or fluctuating during cell state transitions, such as apoptosis. SNP-CLING is a universally applicable technique that enables the dissection of allele-specific spatiotemporal genome organization in live cells.

A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus.

The binding of the transcriptional regulator CTCF to the genome has been implicated in the formation of topologically associated domains (TADs). However, the general mechanisms of folding the genome into TADs are not fully understood. Here we test the effects of deleting a CTCF-rich locus on TAD boundary formation. Using genome-wide chromosome conformation capture (Hi-C), we focus on one TAD boundary on chromosome X harboring ~ 15 CTCF binding sites and located at the long non-coding RNA (lncRNA) locus Firre. Specifically, this TAD boundary is invariant across evolution, tissues, and temporal dynamics of X-chromosome inactivation. We demonstrate that neither the deletion of this locus nor the ectopic insertion of Firre cDNA or its ectopic expression are sufficient to alter TADs in a sex-specific or allele-specific manner. In contrast, Firre's deletion disrupts the chromatin super-loop formation of the inactive X-chromosome. Collectively, our findings suggest that apart from CTCF binding, additional mechanisms may play roles in establishing TAD boundary formation.

Function and evolution of local repeats in the Firre locus.

More than half the human and mouse genomes are comprised of repetitive sequences, such as transposable elements (TEs), which have been implicated in many biological processes. In contrast, much less is known about other repeats, such as local repeats that occur in multiple instances within a given locus in the genome but not elsewhere. Here, we systematically characterize local repeats in the genomic locus of the Firre long noncoding RNA (lncRNA). We find a conserved function for the RRD repeat as a ribonucleic nuclear retention signal that is sufficient to retain an otherwise cytoplasmic mRNA in the nucleus. We also identified a repeat, termed R0, that can function as a DNA enhancer element within the intronic sequences of Firre. Collectively, our data suggest that local repeats can have diverse functionalities and molecular modalities in the Firre locus and perhaps more globally in other lncRNAs.