Hugues Parrinello

Bio: Hugues Parrinello is an academic researcher. The author has contributed to research in topics: Biology & Medicine. The author has an hindex of 2, co-authored 2 publications receiving 1736 citations.

An ancient truncated duplication of the anti-Mullerian hormone receptor type 2 gene is a potential conserved master sex determinant in the Pangasiidae catfish family

Abstract: The evolution of sex determination (SD) mechanisms in teleost fishes is amazingly dynamic, as reflected by the variety of different master sex-determining genes identified, even sometimes among closely related species. Pangasiids are a group of economically important catfishes in many South-Asian countries, but little is known about their sex determination system. Here, we generated novel genomic resources for 12 Pangasiid species and provided a first characterization of their SD system. Based on an Oxford Nanopore long-read chromosome-scale high quality genome assembly of the striped catfish Pangasianodon hypophthalmus, we identified a duplication of the anti-Müllerian hormone receptor type II gene (amhr2), which was further characterized as being sex-linked in males and expressed only in testicular samples. These first results point to a male-specific duplication on the Y chromosome (amhr2by) of the autosomal amhr2a. Sequence annotation revealed that the P. hypophthalmus Amhr2by is truncated in its N-terminal domain, lacking the cysteine-rich extracellular part of the receptor that is crucial for ligand binding, suggesting a potential route for its neofunctionalization. Short-read genome sequencing and reference-guided assembly of 11 additional Pangasiid species, along with sex-linkage studies, revealed that this truncated amhr2by duplication is also conserved as a male-specific gene in many Pangasiids. Reconstructions of the amhr2 phylogeny suggested that amhr2by arose from an ancient duplication / insertion event at the root of the Siluroidei radiation that is dated around 100 million years ago. Altogether these results bring multiple lines of evidence supporting that amhr2by is an ancient and conserved master sex-determining gene in Pangasiid catfishes, a finding that highlights the recurrent usage of the transforming growth factor β pathway in teleost sex determination and brings another empirical case towards the understanding of the dynamics or stability of sex determination systems.

CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles

Abstract: The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications.We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion.Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles

Abstract: The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications.We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion.Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

Topological domains in mammalian genomes identified by analysis of chromatin interactions

Abstract: The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories, and numerous models have been proposed for how chromosomes fold within chromosome territories. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid advances in the study of three-dimensional genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide. Here we investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term 'topological domains', as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. Finally, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome.

Spatial partitioning of the regulatory landscape of the X-inactivation centre

Abstract: High-order chromatin folding in topologically associating domains has a critical role in proper long-range transcriptional control around the Xist locus, and presumably throughout the genome. The spatial organization of the genome is linked to biological function, and advances in genomic technologies are allowing the conformation of chromosomes to be assessed genome wide. Two groups present complementary papers on the subject. Bing Ren and colleagues use Hi-C, an adaption of the chromosome conformation capture (3C) technique, to investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types. Large, megabase-sized chromatin interaction domains, termed topological domains, are found to be a pervasive and conserved feature of genome organization. Edith Heard and colleagues use chromosome conformation capture carbon-copy (5C) technology and high-resolution microscopy to obtain a high-resolution map of the chromosomal interactions over a large region of the mouse X chromosome, including the X-inactivation centre. A series of discrete topologically associating domains is revealed, as is a previously unknown long intergenic RNA with a potential regulatory role. In eukaryotes transcriptional regulation often involves multiple long-range elements and is influenced by the genomic environment1. A prime example of this concerns the mouse X-inactivation centre (Xic), which orchestrates the initiation of X-chromosome inactivation (XCI) by controlling the expression of the non-protein-coding Xist transcript. The extent of Xic sequences required for the proper regulation of Xist remains unknown. Here we use chromosome conformation capture carbon-copy (5C)2 and super-resolution microscopy to analyse the spatial organization of a 4.5-megabases (Mb) region including Xist. We discover a series of discrete 200-kilobase to 1 Mb topologically associating domains (TADs), present both before and after cell differentiation and on the active and inactive X. TADs align with, but do not rely on, several domain-wide features of the epigenome, such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters. Disruption of a TAD boundary causes ectopic chromosomal contacts and long-range transcriptional misregulation. The Xist/Tsix sense/antisense unit illustrates how TADs enable the spatial segregation of oppositely regulated chromosomal neighbourhoods, with the respective promoters of Xist and Tsix lying in adjacent TADs, each containing their known positive regulators. We identify a novel distal regulatory region of Tsix within its TAD, which produces a long intervening RNA, Linx. In addition to uncovering a new principle of cis-regulatory architecture of mammalian chromosomes, our study sets the stage for the full genetic dissection of the X-inactivation centre.