Royalactin drives a ground-state-like pluripotency state in mESCs. All genes with mean normalized read counts larger than 10 were considered for principal component analysis PCA. GO term enrichment analysis found that genes involved in basic metabolism, transcription, and development were responsible for this separation Fig. We next wondered whether a homolog of Royalactin existed in mammals.
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However, the latter computational tool revealed that Royalactin is distantly related to an existing structure in the PDB database 25 , a secreted salivary gland protein SGP from the sand fly, L. Thus, NHLRC3 appears to be a mammalian pluripotency maintenance factor, whose existence demonstrates a remarkable conservation of macromolecular structure and function. The mammalian structural analog of Royalactin maintains naive and ground-state pluripotency in mouse embryonic stem cells. Superimposition of these models right demonstrates striking similarity between them.
In summary, our results demonstrate an unexpected capacity for Royalactin as a pluripotency factor that confers self-renewal and promotes emergence of the naive pluripotent gene regulatory network, and identify Regina as a factor that can promote ground-state pluripotency in mESCs. A better understanding of the interaction of Royalactin and Regina with genetic pathways and biochemical processes conserved in evolution, and how the different pluripotency networks function independently or synergistically to maintain stem cell self-renewal, will advance our efforts to better control stem cell fate, and provide a platform for dissection of the pluripotent state.
Our findings and the discovery of Regina thus support the intriguing idea that profound molecular conservation underlies even the most evolutionarily novel traits. Future work in dissecting the mechanistic action of Royalactin in mammalian cells, including further characterization of its mammalian structural analog Regina, will shed new light on mammalian pluripotency and provide additional means to enhance optimal maintenance and derivation of ESCs for therapeutic application and regenerative medicine. Chang were grown on 0.
Media and protein were changed daily. This sample was further polished by a second immobilized metal Ni affinity chromatography IMAC step using increasing concentrations of imidazole for elution. The filtered supernatant was concentrated fold and used directly in cell culture assays. N vector was a gift of Howard Y. Four weeks after injection, the mice were euthanized and the teratomas were harvested.
Analyses were performed by a board-certified veterinary pathologist. CGR 8. Media was changed daily. Cells were passaged using Accutase Stemcell Technologies and suspended in M2 media for injection. HRP-conjugated secondary antibodies were used at , Reads were aligned to the mouse reference genome build mm9 using Tophat. A maximum of a default 2 mismatches was allowed for read alignment. Gene counts were calculated using the HTSeq-count utility 29 and used as an input for differential gene expression analysis with DESeq version 1.
Genes with a p -value of 0. Validation of top differentially regulated genes was performed with quantitative reverse transcription polymerase chain reaction. Further network analysis on differentially significant genes was performed using NetworkAnalyst PCA analysis was performed using samples as indicated. Samples from different libraries were normalized using shifted log of normalized counts using DESeq.
Reads were aligned to the mouse reference genome build mm9 using Bowtie. The ATAC-seq regions were divided into separate analyses: correlation with closest TSS, correlation with traditional enhancer regions present in the mm9 genome, and correlation with super-enhancer regions discovered for the mm9 genome The heatmaps for TSS regions, traditional enhancers, super-enhancers, and differential peaks were produced using unsupervised clustering methods, which used the normalized signal values obtained by quantile normalization, to extract transitions between two states: upregulated and downregulated.
The peaks were filtered on the basis of a p -value threshold of 0. The significant GO terms were filtered to only include GO terms associated with pluripotency and GO terms associated with metabolism. For pluripotency related GO terms, biological processes including morphogenesis, development, proliferation, and stem cell processes were analyzed. For metabolic GO terms, biological processes that were related to metabolism and biosynthetic processes were chosen. Motif analysis for the differential peak lists was performed using HOMER with all differential peaks used as background.
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The structural models are available upon request. All relevant data are available from the authors. Li, M. Ground rules of the pluripotency gene regulatory network. Wu, D. A conserved mechanism for control of human and mouse embryonic stem cell pluripotency and differentiation by shp2 tyrosine phosphatase.
PloS ONE 4 , e Evans, M.
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Kamakura, M. Royalactin induces queen differentiation in honeybees. Inoue, S. Honda, Y. Lifespan-extending effects of royal jelly and its related substances on the nematode Caenorhabditis elegans. PloS ONE 6 , e Miyashita, A. Body-enlarging effect of royal jelly in a non-holometabolous insect species, Gryllus bimaculatus. Open 5 , — A hypopharyngeal gland protein of the worker honeybee Apis mellifera L. Protein Expr. Majtan, J.
Watanabe, K. Stimulation of cell growth in the U human myeloid cell line by honey royal jelly protein. Cytotechnology 26 , 23—27 Maleszka, R. Beyond Royalactin and a master inducer explanation of phenotypic plasticity in honey bees. Buenrostro, J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Methods 10 , — Marks, H.
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Berman, H. Contributor: Hassall, John, Contributor: James, Gilbert. Contributor: Moore-Park, Carton, Contributor: Smith, G. Contributor: Thompson, Margaret, active Contributor: Webb, Marie. Loading branch holdings The queen bee -- The anemones -- The mist -- The beech and the oak -- The dragon-fly and the water-lily -- The weeds -- The sparrow. Dulac, Edmund, Hassall, John, James, Gilbert. Moore-Park, Carton, Smith, G.
Thompson, Margaret, active