Recently, human PAIRED-LIKE homeobox transcription factor (TF) genes were discovered whose expression is limited to the period of embryo genome activation up to the 8-cell stage. One of these TFs is LEUTX, but its importance for human embryogenesis is still subject to debate. We confirmed that human LEUTX acts as a TAATCC-targeting transcriptional activator, like other K50-type PAIRED-LIKE TFs. Phylogenetic comparisons revealed that Leutx proteins are conserved across Placentalia and comprise two conserved domains, the homeodomain, and a Leutx-specific domain containing putative transcriptional activation motifs (9aaTAD). Examination of human genotype resources revealed 116 allelic variants in LEUTX. Twenty-four variants potentially affect function, but they occur only heterozygously at low frequency. One variant affects a DNA-specificity determining residue, mutationally reachable by a one-base transition. In vitro and in silico experiments showed that this LEUTX mutation (alanine to valine at position 54 in the homeodomain) results in a transactivational loss-of-function to a minimal TAATCC-containing promoter and a 36 bp motif enriched in genes involved in embryo genome activation. A compensatory change in residue 47 restores function. The results support the notion that human LEUTX functions as a transcriptional activator important for human embryogenesis.
The emergence of the COVID-19 outbreak at the end of 2019, caused by the novel coronavirus SARS-CoV-2, has, to date, led to over 13.6 million infections and nearly 600,000 deaths. Consequently, there is an urgent need to better understand the molecular factors triggering immune defense against the virus and to develop countermeasures to hinder its spread. Using in silico analyses, we showed that human major histocompatibility complex (MHC) class I cell-surface molecules vary in their capacity for binding different SARS-CoV-2-derived epitopes, i.e., short sequences of 8-11 amino acids, and pinpointed five specific SARS-CoV-2 epitopes that are likely to be presented to cytotoxic T-cells and hence activate immune responses. The identified epitopes, each one of nine amino acids, have high sequence similarity to the equivalent epitopes of SARS-CoV virus, which are known to elicit an effective T cell response in vitro. Moreover, we give a structural explanation for the binding of SARS-CoV-2-epitopes to MHC molecules. Our data can help us to better understand the differences in outcomes of COVID-19 patients and may aid the development of vaccines against SARS-CoV-2 and possible future outbreaks of novel coronaviruses.
We report the extent, specific sites and structural requirements of joint inflammation related citrullination in extracellular proteins. A total of 40 synovial fluid samples derived from chronically inflamed human joints were analysed by heparin-agarose fractionation and LC-MS/MS. Citrullination of 55 arginines in extracellular proteins was detected. Importantly, 20% of the sites have a characterized function related to the hallmarks of destructive joint inflammation. E.g. four arginine residues, shown here to be citrullinated, are also affected by mutations in inherited diseases causing haemolysis or blood clotting dysfunction. Citrullination of integrin ligands was selected for further studies since fibronectin R234 in isoDGR was among the most frequently citrullinated arginines in synovial fluid. Assays with synovial fibroblasts and integrin αVβ3 indicated decreased affinity to the enzymatically citrullinated integrin binding sites. To conclude, our data indicate that in inflamed joints extensive citrullination affects the functional arginine residues in extracellular proteins.
45During the human oocyte-to-embryo transition, the fertilized oocyte undergoes 46 final maturation and the embryo genome is gradually activated during the first 47 three cell divisions. How this transition is coordinated in humans is largely 48 unknown. We show that the double homeodomain transcription factor DUX4 49 contributes to this transition. DUX4 knockdown in human zygotes caused 50 insufficient transcriptome reprogramming as observed three days after 51 fertilization. Induced DUX4 expression in human embryonic stem cells activated 52 transcription of thousands of newly identified bi-directional transcripts, including 53 putative enhancers for embryonic genome activation genes such as LEUTX. DUX4 54 protein interacted with transcriptional modifiers that are known to couple 55 enhancers and promoters. Taken together, our results reveal that DUX4 is a 56 pioneer regulating oocyte-to-embryo transition in human through activation of 57 intergenic genome, especially enhancers, and hence setting the stage for early 58 human embryo development. 59 60 61 62 63 64 65 4 Mammalian pre-implantation development commences with oocyte-to-embryo 66 transition, which involves fundamental changes in the epigenetic landscapes, 67 modulation of cell cycle control, and translation or clearance of selected maternal 68 mRNAs, culminating to embryonic genome activation 1,2 . The pioneer regulators 69 orchestrating the oocyte-to-embryo transition and first embryo genome activation steps 70 in human remain poorly understood. The conserved double homeodomain transcription 71factor DUX4 represents a plausible candidate regulating the oocyte-to-embryo 72 transition in humans, given its capacity to activate germline genes and genomic repeat 73 elements 3-5 . Here we show that DUX4 is able to launch the first reprogramming steps 74 from oocyte to embryo in human by activating thousands of novel enhancers and 75 therein, modulating the transcriptome and chromatin. Human DUX4 knockdown 76 embryos are viable until the third day of development, but their transcriptome is 77 severely altered. Our proteomics approaches suggest that DUX4 binds to Mediator 78 complex and chromatin modifiers through its C-terminal domains, providing a likely 79 explanation to how DUX4 may extensively modulate the genome. This study implies a 80 wider role for DUX4 as a cellular gate keeper acting both as a general genomic modifier 81 of cell fate as well as a specific inducer of first wave embryo genome activation genes. 82 83Results 84 Quantification of DUX4 in human embryos 85The DUX4 induced gene network is highly conserved 6 and recent reports showed that 86 DUX4 is expressed in early human embryos 3,4 . However, details of this dynamic 87 process, including initiation of DUX4 expression, remained ambiguous. Therefore, we 88Given the short-term and precise manifestation of DUX4 mRNA and protein in human 107 zygotes and early cleavage stage embryos, we next asked how DUX4 regulates the first 108 steps of human embryo development. We microinjected either DUX4...
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