Multi-feature clustering of CTCF binding creates robustness for loop extrusion blocking and Topologically Associating Domain boundaries.

Topologically Associating Domains (TADs) separate vertebrate genomes into insulated regulatory neighborhoods that focus genome-associated processes. TADs are formed by Cohesin-mediated loop extrusion, with many TAD boundaries consisting of clustered binding sites of the CTCF insulator protein. Here we determine how this clustering of CTCF binding contributes to the blocking of loop extrusion and the insulation between TADs. We identify enrichment of three features of CTCF binding at strong TAD boundaries, consisting of strongly bound and closely spaced CTCF binding peaks, with a further enrichment of DNA-binding motifs within these peaks. Using multi-contact Nano-C analysis in cells with normal and perturbed CTCF binding, we establish that individual CTCF binding sites contribute to the blocking of loop extrusion, but in an incomplete manner. When clustered, individual CTCF binding sites thus create a stepwise insulation between neighboring TADs. Based on these results, we propose a model whereby multiple instances of temporal loop extrusion blocking create strong insulation between TADs.

The Drosophila Fab-7 boundary modulates Abd-B gene activity by guiding an inversion of collinear chromatin organization and alternate promoter use.

Hox genes encode transcription factors that specify segmental identities along the anteroposterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, a feature known as collinearity. In Drosophila, the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive changes in histone modifications, chromatin architecture, and use of boundary elements and cis-regulatory regions. To dissect functional hierarchies, we compare chromatin organization in cell lines and larvae, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary for insulation between 3D domains carrying different histone modifications. Interestingly, we detect a non-canonical inversion of collinear chromatin dynamics at Abd-B, with the domain of active histone modifications progressively decreasing in size. This dynamic chromatin organization differentially activates the alternative promoters of the Abd-B gene, thereby expanding the possibilities for fine-tuning of transcriptional output.

Eye morphogenesis in the blind Mexican cavefish.

The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Disturbances in these events can lead to developmental defects and blindness. Here, we have deciphered the sequence of defective events leading to coloboma in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified defects in migratory cell behaviours during evagination that participate in the reduced optic vesicle size in cavefish, without proliferation defect. Further, impaired optic cup invagination shifts the relative position of the lens and contributes to coloboma in cavefish. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis.

Fgfr3 Is a Positive Regulator of Osteoblast Expansion and Differentiation During Zebrafish Skull Vault Development.

Gain or loss-of-function mutations in fibroblast growth factor receptor 3 (FGFR3) result in cranial vault defects highlighting the protein's role in membranous ossification. Zebrafish express high levels of fgfr3 during skull development; in order to study FGFR3's role in cranial vault development, we generated the first fgfr3 loss-of-function zebrafish (fgfr3lof/lof ). The mutant fish exhibited major changes in the craniofacial skeleton, with a lack of sutures, abnormal frontal and parietal bones, and the presence of ectopic bones. Integrated analyses (in vivo imaging and single-cell RNA sequencing of the osteoblast lineage) of zebrafish fgfr3lof/lof revealed a delay in osteoblast expansion and differentiation, together with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis. We conclude that changes in the extracellular matrix within growing bone might impair cell-cell communication, mineralization, and new osteoblast recruitment. © 2020 American Society for Bone and Mineral Research.

TRIM8 is required for virus-induced IFN response in human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) play a crucial role in antiviral innate immunity through their unique capacity to produce large amounts of type I interferons (IFNs) upon viral detection. Tripartite motif (TRIM) proteins have recently come forth as important modulators of innate signaling, but their involvement in pDCs has not been investigated. Here, we performed a rationally streamlined small interfering RNA (siRNA)-based screen of TRIM proteins in human primary pDCs to identify those that are critical for the IFN response. Among candidate hits, TRIM8 emerged as an essential regulator of IFN regulatory factor 7 (IRF7) function. Mechanistically, TRIM8 protects phosphorylated IRF7 (pIRF7) from proteasomal degradation in an E3 ubiquitin ligase-independent manner by preventing its recognition by the peptidyl-prolyl isomerase Pin1. Our findings uncover a previously unknown regulatory mechanism of type I IFN production in pDCs by which TRIM8 and Pin1 oppositely regulate the stability of pIRF7.

Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile.

In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at postembryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used three-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the postembryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. Stem Cells 2017;35:1505-1518.

Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases.

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

Identification of the CCR5-Delta32 HIV resistance allele and new mutations of the CCR5 gene in different Tunisian populations.

Polymorphisms in some chemokine receptor genes are associated with susceptibility to and progression of human immunodeficiency virus-1 (HIV-1) infection. Most mutations detected in the CC-chemokine receptor 5 (CCR5) gene are specific to different populations. In this study, we focused on polymorphisms of the CCR5 coding region in three healthy populations from Tunisia, corresponding to a cosmopolitan population from Tunis, and two isolated Berber populations. In addition to the CCR5-Delta32 deletion, eleven single nucleotide polymorphisms were detected. Some of these point mutations were associated with the same genotype and even the same haplotype. The (L55Q-C101X), I124, V131F, T143N, A159V, I237, T239A and G301R alleles have not been described previously, whereas the CCR5-Delta32, L55Q, A335V and Y339F variants have already been reported in the literature. The distribution and frequency of these variants were different among the three groups studied, a result in agreement with the mosaic genetic structure of the Tunisian population. To determine whether these alleles affect HIV-1 transmission, we compared allele frequencies between healthy and HIV-1 infected individuals from Tunis. The frequency of the CCR5-Delta32 variant was significantly different between the two groups, leading us to conclude that this mutation might confer protection against HIV infection in Tunisian populations.