The chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus uncovers patterns of genome evolution in mosquitoes.

BACKGROUND: Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood. METHODS: In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus. RESULTS: We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus, and the vector of arboviruses Ae. aegypti. Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus, as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes. CONCLUSION: The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies.

The RNA-binding proteins CELF1 and ELAVL1 cooperatively control the alternative splicing of CD44.

CD44 mRNA contains nine consecutive cassette exons, v2 to v10. Upon alternative splicing, several isoforms are produced with different impacts on tumor biology. Here, we demonstrate the involvement of the RNA-binding proteins CELF1 and ELAVL1 in the control of CD44 splicing. We show by FRET-FLIM that these proteins directly interact in the nucleus. By combining RNAi-mediated depletion and exon array hybridization in HeLa cells, we observe that the exons v7 to v10 of CD44 are highly sensitive to CELF1 and ELAVL1 depletion. We confirm by RT-PCR that CELF1 and ELAVL1 together stimulate the inclusion of these exons in CD44 mRNA. Finally, we show in eight different tumor types that high expression of CELF1 and/or ELAVL1 is correlated with the inclusion of CD44 variable exons. These data point to functional interactions between CELF1 and ELAVL1 in the control of CD44 splicing in human cancers.

Chromatin loop anchors contain core structural components of the gene expression machinery in maize.

BACKGROUND: Three-dimensional chromatin loop structures connect regulatory elements to their target genes in regions known as anchors. In complex plant genomes, such as maize, it has been proposed that loops span heterochromatic regions marked by higher repeat content, but little is known on their spatial organization and genome-wide occurrence in relation to transcriptional activity. RESULTS: Here, ultra-deep Hi-C sequencing of maize B73 leaf tissue was combined with gene expression and open chromatin sequencing for chromatin loop discovery and correlation with hierarchical topologically-associating domains (TADs) and transcriptional activity. A majority of all anchors are shared between multiple loops from previous public maize high-resolution interactome datasets, suggesting a highly dynamic environment, with a conserved set of anchors involved in multiple interaction networks. Chromatin loop interiors are marked by higher repeat contents than the anchors flanking them. A small fraction of high-resolution interaction anchors, fully embedded in larger chromatin loops, co-locate with active genes and putative protein-binding sites. Combinatorial analyses indicate that all anchors studied here co-locate with at least 81.5% of expressed genes and 74% of open chromatin regions. Approximately 38% of all Hi-C chromatin loops are fully embedded within hierarchical TAD-like domains, while the remaining ones share anchors with domain boundaries or with distinct domains. Those various loop types exhibit specific patterns of overlap for open chromatin regions and expressed genes, but no apparent pattern of gene expression. In addition, up to 63% of all unique variants derived from a prior public maize eQTL dataset overlap with Hi-C loop anchors. Anchor annotation suggests that < 7% of all loops detected here are potentially devoid of any genes or regulatory elements. The overall organization of chromatin loop anchors in the maize genome suggest a loop modeling system hypothesized to resemble phase separation of repeat-rich regions. CONCLUSIONS: Sets of conserved chromatin loop anchors mapping to hierarchical domains contains core structural components of the gene expression machinery in maize. The data presented here will be a useful reference to further investigate their function in regard to the formation of transcriptional complexes and the regulation of transcriptional activity in the maize genome.

Mapping the genomic landscape of CRISPR-Cas9 cleavage.

RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis.

Regulation of alternative splicing is an important process for cell differentiation and development. Down-regulation of Ptbp1, a regulatory RNA-binding protein, leads to developmental skin defects in Xenopus laevis. To identify Ptbp1-dependent splicing events potentially related to the phenotype, we conducted RNAseq experiments following Ptbp1 depletion. We systematically compared exon-centric and junction-centric approaches to detect differential splicing events. We showed that the junction-centric approach performs far better than the exon-centric approach in Xenopus laevis. We carried out the same comparisons using simulated data in human, which led us to propose that the better performances of the junction-centric approach in Xenopus laevis essentially relies on an incomplete exonic annotation associated with a correct transcription unit annotation. We assessed the capacity of the exon-centric and junction-centric approaches to retrieve known and to discover new Ptbp1-dependent splicing events. Notably, the junction-centric approach identified Ptbp1-controlled exons in agfg1, itga6, actn4, and tpm4 mRNAs, which were independently confirmed. We conclude that the junction-centric approach allows for a more complete and informative description of splicing events, and we propose that this finding might hold true for other species with incomplete annotations.

The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA.

The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.

Eighteen New Candidate Effectors of the Phytonematode Heterodera glycines Produced Specifically in the Secretory Esophageal Gland Cells During Parasitism.

Heterodera glycines, the soybean cyst nematode, is the number one pathogen of soybean (Glycine max). This nematode infects soybean roots and forms an elaborate feeding site in the vascular cylinder. H. glycines produces an arsenal of effector proteins in the secretory esophageal gland cells. More than 60 H. glycines candidate effectors were identified in previous gland-cell-mining projects. However, it is likely that additional candidate effectors remained unidentified. With the goal of identifying remaining H. glycines candidate effectors, we constructed and sequenced a large gland cell cDNA library resulting in 11,814 expressed sequence tags. After bioinformatic filtering for candidate effectors using a number of criteria, in situ hybridizations were performed in H. glycines whole-mount specimens to identify candidate effectors whose mRNA exclusively accumulated in the esophageal gland cells, which is a hallmark of many nematode effectors. This approach resulted in the identification of 18 new H. glycines esophageal gland-cell-specific candidate effectors. Of these candidate effectors, 11 sequences were pioneers without similarities to known proteins while 7 sequences had similarities to functionally annotated proteins in databases. These putative homologies provided the bases for the development of hypotheses about potential functions in the parasitism process.

Involvement of ITIH5, a candidate gene for congenital uterovaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome), in female genital tract development.

The ITI (inter-trypsine inhibitor) gene family includes five genes (ITIH1 to ITIH5) that encode proteins involved in the dynamics of the extracellular matrix (ECM). ITIH5 was found inactivated by partial deletion in a case of congenital uterovaginal aplasia, a human rare disease also called Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. The aim of the present study was to analyze the expression of ITIH5 in the uterus in adult life and during embryogenesis in order to establish the involvement of this gene in both normal and pathological conditions of uterus development. This was achieved in mice by reverse transcription-quantitative PCR, whole-mount hybridization, and Western blot analysis. Itih5 expression was much stronger in female genital tract primordia (Müllerian ducts) and derivatives than elsewhere in the body. This gene was strongly expressed during pregnancy and development of the female genital tract, indicating that the encoded protein probably had an important function in the uterus during these periods. Two different specific isoforms of the protein were detected in Müllerian derivatives during embryogenesis and in adults. Although ITIH genes are expected to be predominantly expressed in the liver, ITIH5 is mainly expressed in the uterus during development and adult life. This tends to indicate an additional and specific role of this gene in the female reproductive tract, and furthermore reinforces ITIH5 as a putative candidate gene for MRKH syndrome.

ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate.

The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible. We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells. Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.

The Splicing Factor PTBP1 Represses TP63 γ Isoform Production in Squamous Cell Carcinoma.

The TP63 gene encodes the p63 transcription factor. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, ß, γ, and δ. The regulatory functions of p63 are isoform specific. The α isoform inhibits the epithelial-to-mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Using The Cancer Genome Atlas data, we observed that a higher proportion of the TP63γ isoform is a detrimental factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC) and is accompanied by the downregulation of desmosomal genes. By a correlation-based approach, we investigated the regulation of the production of the TP63γ isoform. According to our analysis of GTEx data, the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ in several tissues. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines, keratinocyte or Xenopus embryos leads to an increase in TP63γ isoform abundance. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63γ-specific exon. Intronic regions around the TP63γ-specific exon were sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Together, these results identify TP63γ as an unfavorable prognostic marker in HNSCC, and identify PTBP1 as the first direct splicing regulator of TP63γ production and a potential route toward TP63 isoform control. Significance: Quantifying TP63γ isoforms in patients' tumors could allow for the early detection of patients with HNSCC with an early loss in desmosomal gene expression and poor prognostic. The identification of PTBP1 as a transacting factor controlling TP63γ production may allow to control TP63γ expression.

Involvement of ZFPIP/Zfp462 in chromatin integrity and survival of P19 pluripotent cells.

Toti- or pluripotent cells proliferation and/or differentiation have been shown to be strongly related to nuclear chromatin organization and structure over the last past years. We have recently identified ZFPIP/Zfp462 as a zinc finger nuclear factor necessary for correct cell division during early embryonic developmental steps of vertebrates. We thus questioned whether this factor was playing a general role during cell division or if it was somehow involved in embryonic cell fate or differentiation. To achieve this goal, we performed a knock-down experiment in the pluripotent P19 and differentiated 3T3 cell lines, both expressing endogenous ZFPIP/Zfp462. Using specific shRNA directed against ZFPIP/Zfp462 transcripts, we demonstrated that depletion of this protein induced cell death in P19 but had no effect in 3T3 cells. In addition, in the absence of the protein, the P19 cells exhibited a complete destructuration of pericentromeric domains associated with a redistribution of the HP1alpha proteins and an increase in DNA satellites transcribed RNAs level. These data suggested an instrumental role of ZFPIP/Zfp462 in maintaining the chromatin structure of pluripotent cells.

ZFPIP/Zfp462 is maternally required for proper early Xenopus laevis development.

ZFPIP (Zinc Finger Pbx1 Interacting Protein) has been recently identified in our laboratory in a yeast two hybrid screen using an embryonic mouse cDNA library and PBX1 as a bait. This gene encodes a large protein (250 kDa) that contains a bipartite NLS, numerous C2H2 zinc fingers and is highly conserved amongst vertebrates. In order to address the role of ZFPIP during embryonic development, we analysed the expression pattern of the gene and performed morpholinos injections into Xenopus laevis embryos. We first showed that the ZFPIP protein was maternally present in oocytes. Then, ZFPIP was detected from morula to neurula stages in the nucleus of the cells, with a gradient from animal to vegetal pole. By injection of ZFPIP morpholinos, we showed that morphant embryos were unable to undergo proper gastrulation and subsequently exhibited a persistent opened blastopore. Analysis of molecular and cellular events that were altered in morphant embryos highlighted an impairment of cell division processes as illustrated by atypical mitosis with aberrant metaphase, anaphase or telophase, incomplete chromosome segregation or conjointed nuclei. The overall data presented here demonstrated that ZFPIP was a major developing gene that acts in the very first steps of embryonic development of Xenopus laevis.

The Beginning of the End: A Chromosomal Assembly of the New World Malaria Mosquito Ends with a Novel Telomere.

Chromosome level assemblies are accumulating in various taxonomic groups including mosquitoes. However, even in the few reference-quality mosquito assemblies, a significant portion of the heterochromatic regions including telomeres remain unresolved. Here we produce a de novo assembly of the New World malaria mosquito, Anopheles albimanus by integrating Oxford Nanopore sequencing, Illumina, Hi-C and optical mapping. This 172.6 Mbps female assembly, which we call AalbS3, is obtained by scaffolding polished large contigs (contig N50 = 13.7 Mbps) into three chromosomes. All chromosome arms end with telomeric repeats, which is the first in mosquito assemblies and represents a significant step toward the completion of a genome assembly. These telomeres consist of tandem repeats of a novel 30-32 bp Telomeric Repeat Unit (TRU) and are confirmed by analyzing the termini of long reads and through both chromosomal in situ hybridization and a Bal31 sensitivity assay. The AalbS3 assembly included previously uncharacterized centromeric and rDNA clusters and more than doubled the content of transposable elements and other repetitive sequences. This telomere-to-telomere assembly, although still containing gaps, represents a significant step toward resolving biologically important but previously hidden genomic components. The comparison of different scaffolding methods will also inform future efforts to obtain reference-quality genomes for other mosquito species.

Interaction of ZFPIP with PBX1 is crucial for proper expression of neural genetic markers during Xenopus development.

ZFPIP/Zfp462 has been recently identified as a new vertebrate zinc finger encoding gene whose product interacts with Pbx1. Previous work indicates that ZFPIP is maternally expressed in Xenopus laevis oocytes and plays a key role during the cleavage phase of embryogenesis. This early expression is followed by a zygotic expression which overlaps with the neural Pbx1 expression pattern, suggesting an interaction between these two partners during Xenopus neurogenesis. In order to test the physiological interaction between ZFPIP and Pbx1, we carried out a dominant negative assay in which the Pbx1 interacting domain of ZFPIP (ZFPIPp) was overexpressed in Xenopus laevis embryos. We observed that ZFPIPp ectopic expression led to abnormal en2 and N-cam expression patterns, whereas krox-20 expression was not affected. Furthermore, we showed that while ZFPIPp alone was localized in the nucleus of Cos-7 cells, additional expression of Pbx1 induced a location of ZFPIPp at the perinuclear region of the cells. These overall data suggest that ZFPIP and Pbx1 could be partners and cooperate in the regulation of essential neural genes during Xenopus development.

PBX proteins: much more than Hox cofactors.

Pre-B cell leukaemia transcription factors (PBXs) were originally identified as Hox cofactors, acting within transcriptional regulation complexes to regulate genetic programs during development. Increasing amount of evidence revealed that PBX function is not restricted to a partnership with Hox or homeodomain proteins. Indeed, PBXs are expressed throughout murine embryonic development and are involved in several developmental pathways including Hox-independent mechanisms. This review summarizes what is known about PBX partnerships and proposes to position PBXs as central developmental factors whose role consists of integrating transduction signals, in order to regulate gene expression programs during development.

CRISPR-Cas9 Editing in Maize: Systematic Evaluation of Off-target Activity and Its Relevance in Crop Improvement.

CRISPR-Cas9 enabled genome engineering has great potential for improving agriculture productivity, but the possibility of unintended off-target edits has evoked some concerns. Here we employ a three-step strategy to investigate Cas9 nuclease specificity in a complex plant genome. Our approach pairs computational prediction with genome-wide biochemical off-target detection followed by validation in maize plants. Our results reveal high frequency (up to 90%) on-target editing with no evidence of off-target cleavage activity when guide RNAs were bioinformatically predicted to be specific. Predictable off-target edits were observed but only with a promiscuous guide RNA intentionally designed to validate our approach. Off-target editing can be minimized by designing guide RNAs that are different from other genomic locations by at least three mismatches in combination with at least one mismatch occurring in the PAM proximal region. With well-designed guides, genetic variation from Cas9 off-target cleavage in plants is negligible, and much less than inherent variation.

Identification of a new type of PBX1 partner that contains zinc finger motifs and inhibits the binding of HOXA9-PBX1 to DNA.

PBX1 belongs to the TALE-class of homeodomain protein and has a wide functional diversity during development. Indeed, PBX1 is required for haematopoiesis as well as for multiple developmental processes such as skeletal patterning and organogenesis. It has furthermore been shown that PBX1 functions as a HOX cofactor during development. More recent data suggest that PBX1 may act even more broadly by modulating the activity of non-homeodomain transcription factors. To better understand molecular mechanisms triggered by PBX1 during female genital tract development, we searched for additional PBX1 partners that might be involved in this process. Using a two hybrid screen, we identified a new PBX1 interacting protein containing several zinc finger motifs that we called ZFPIP for Zinc Finger PBX1 Interacting Protein. We demonstrated that ZFPIP is expressed in embryonic female genital tract but also in other PBX1 expression domains such as the developing head and the limb buds. We further showed that ZFPIP is able to bind physically and in vivo to PBX1 and moreover, that it prevents the binding of HOXA9/PBX complexes to their consensus DNA site. We suggest that ZFPIP is a new type of PBX1 partner that could participate in PBX1 function during several developmental pathways.

A chromosome-scale assembly of the sorghum genome using nanopore sequencing and optical mapping.

Long-read sequencing technologies have greatly facilitated assemblies of large eukaryotic genomes. In this paper, Oxford Nanopore sequences generated on a MinION sequencer are combined with Bionano Genomics Direct Label and Stain (DLS) optical maps to generate a chromosome-scale de novo assembly of the repeat-rich Sorghum bicolor Tx430 genome. The final assembly consists of 29 scaffolds, encompassing in most cases entire chromosome arms. It has a scaffold N50 of 33.28 Mbps and covers 90% of the expected genome length. A sequence accuracy of 99.85% is obtained after aligning the assembly against Illumina Tx430 data and 99.6% of the 34,211 public gene models align to the assembly. Comparisons of Tx430 and BTx623 DLS maps against the public BTx623 v3.0.1 genome assembly suggest substantial discrepancies whose origin remains to be determined. In summary, this study demonstrates that informative assemblies of complex plant genomes can be generated by combining nanopore sequencing with DLS optical maps.

Bruno-3 regulates sarcomere component expression and contributes to muscle phenotypes of myotonic dystrophy type 1.

Steinert disease, or myotonic dystrophy type 1 (DM1), is a multisystemic disorder caused by toxic noncoding CUG repeat transcripts, leading to altered levels of two RNA binding factors, MBNL1 and CELF1. The contribution of CELF1 to DM1 phenotypes is controversial. Here, we show that the Drosophila CELF1 family member, Bru-3, contributes to pathogenic muscle defects observed in a Drosophila model of DM1. Bru-3 displays predominantly cytoplasmic expression in muscles and its muscle-specific overexpression causes a range of phenotypes also observed in the fly DM1 model, including affected motility, fiber splitting, reduced myofiber length and altered myoblast fusion. Interestingly, comparative genome-wide transcriptomic analyses revealed that Bru-3 negatively regulates levels of mRNAs encoding a set of sarcomere components, including Actn transcripts. Conversely, it acts as a positive regulator of Actn translation. As CELF1 displays predominantly cytoplasmic expression in differentiating C2C12 myotubes and binds to Actn mRNA, we hypothesize that it might exert analogous functions in vertebrate muscles. Altogether, we propose that cytoplasmic Bru-3 contributes to DM1 pathogenesis in a Drosophila model by regulating sarcomeric transcripts and protein levels.