Project description:In Xenopus, establishment of the anterior-posterior axis involves two key signalling pathways, canonical Wnt and Nodal-related TGF-Î². There are also a number of transcription factors that feedback upon these pathways. The homeodomain protein Hex, an early marker of anterior positional information, acts as a transcriptional repressor suppressing induction and propagation of the Spemman organiser while specifying anterior identity. We show that Hex promotes anterior identity by amplifying the activity of canonical Wnt signalling. Hex exerts this activity by inhibiting the expression of Tle-4, a member of the Groucho family of transcriptional co-repressors that we identified as a Hex target in embryonic stem (ES) cells and Xenopus embryos. This Hex-mediated enhancement of Wnt signalling results in the up-regulation of the Nieuwkoop centre genes Siamois and Xnr-3 and the subsequent increased expression of the anterior endodermal marker Cerberus and other mesendodermal genes downstream of Wnt signalling. We also identified Nodal as a Hex target in ES cells. We demonstrate that in Xenopus, the Nodal-related genes Xnr-1 and 2, but not 5 and 6, are regulated directly by Hex. The identification of Nodal-related genes as Hex targets explains the ability of Hex to suppress induction and propagation of the organiser. Together these results support a model in which Hex acts early in development to reinforce a Wnt-mediated, Nieuwkoop-like signal to induce anterior endoderm, and later in this tissue to block further propagation of Nodal-related signals. The ability of Hex to regulate the same targets in both Xenopus and mouse implies this model is conserved. Experiment Overall Design: Identification of Hex target genes In Xenopus, the establishment of the anterior-posterior axis involves two key signalling pathways, the canonical Wnt and the Nodal-related TGF-Î² and a number of transcription factors that feedback upon these pathways. The homeodomain protein Hex, an early marker of anterior positional information, is a transcriptional repressor that suppresses the induction and propagation of the Spemman organiser while specifying anterior identity. Using microarray expression profiling we identified mouse Tle-4 as a Hex target in Embryonic Stem (ES) cells and showed that Tle-4 expression is directly regulated by Hex in Xenopus embryos. We also identified Nodal as a Hex target in ES cells. Taken together these results support a model in which Hex acts early in development to reinforce a Wnt-mediated, Nieuwkoop-like signal to induce anterior endoderm and later in this tissue to block further propagation of Nodal-related signals. The ability of Hex to regulate the same targets in both frog and mouse suggests that this model is conserved.

Project description:Lipid intermediates derived from sphingolipid metabolism are crucial regulators of mitochondrial function from yeast to humans. Among these intermediates, trans-2-hexadecenal (t-2hex) within the sphingolipid degradation pathway exhibits remarkable efficiency in inducing mitochondria-mediated cell death. In yeast cell cultures, the addition of t-2-hex triggers complete disintegration of the mitochondrial network, leading to subsequent cell death. This effect is particularly pronounced in yeast cells lacking the activity of the t-2-hex degrading enzyme, Hfd1. However, the molecular mechanisms of t-2-hex induction of mitochondrial dysfunction are completely unknown. In this project, we want to exploit the unprecedented power of yeast genetics to unveil novel genetic determinants involved in t-2-hex's pro-apoptotic function. To accomplish this, we employed the SATAY method, which combines saturated transposon mutagenesis with high-throughput sequencing to functionally explore the yeast genome. In our screening approach, hfd1 mutant cells harboring a plasmid-encoded inducible MiniDs transposon were induced by galactose, resulting in extensive integration of the transposon throughout the yeast genome. Cells with the plasmid excised and the transposon genomically integrated were pooled together, creating a high-density transposon library comprising approximately 2.3E+06 independent insertion mutants. Subsequently, the pooled mutant library was subjected to treatment with the mitochondria-mediated death inducer, t-2-hexadecenal. As a control, cells were also incubated with the solvent dimethyl sulfoxide (DMSO), in which hexadecenal is dissolved. Following the treatments, cells were collected for genomic DNA extraction and digestion, using restriction enzymes with frequent four-base pair recognition sites. The resulting library fragments were circularized using T4 DNA ligase, and the transposon-genome junctions were selectively amplified through PCR with outward-facing primers specific to the transposon. Finally, the pooled and purified amplicons were subjected to massive sequencing on an Illumina MiSeq platform. The obtained sequences were then aligned to the reference genome of Saccharomyces cerevisiae, allowing for the mapping of transposon insertions and the calculation of transposon counts per gene. This project enabled the identification of genes required for the resistance and toxicity to t-2hex.

Project description:We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1), but their biosynthesis remains elusive. Guided by the well-characterised truncation pathway in invertebrates and plants in which the N-acetyl-β-D-hexosaminidase (Hex) isoenzymes catalyse paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and β subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-/- and HEXB-/-) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, ELISA, proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly of Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilisation of a putative non-canonical truncation pathway in favour of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-deficient mutants and showed a pronounced switch for N-glycoproteins co-trafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and β mediate PMP formation via a putative non-canonical truncation pathway in neutrophils.

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