Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein Nanog1–2, which plays an essential role in establishing ground state pluripotency during somatic cell reprogramming3–4. While the genomic occupancy of Nanog has been extensively investigated, comparatively little is known about Nanog-associated proteins5 and their contribution to the Nanog-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identified 27 high-confidence protein interaction partners of Nanog in mouse ES cells. These consist of 19 novel partners of Nanog that have not been reported before including the Ten eleven translocation (Tet) family methylcytosine hydroxylase Tet1. We confirmed physical association of Nanog with Tet1, and demonstrated that Tet1, in synergy with Nanog, enhances the efficiency of reprogramming. We also found physical association and reprogramming synergy of Tet2 with Nanog, and demonstrated that knockdown of Tet2 abolishes the reprogramming synergy of Nanog with a catalytically deficient mutant of Tet1 (Tet1Mut). These results indicate that the physical interaction between Nanog and Tet1/2 proteins facilitates reprogramming in a manner that is dependent on Tet1/2's catalytic activity. Tet1 and Nanog co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in ES cells, and Tet1 binding is reduced upon Nanog depletion. Co-expression of Nanog and Tet1 results in expression priming of and increased 5hmC levels at top ranked common targets Esrrb and Oct4 before reprogramming to naïve pluripotency. We propose that Tet1 is recruited by Nanog to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of Nanog and uncover a novel role for 5mC hydroxylases in the establishment of naïve pluripotency.

Key Points• RARa2 activates Wnt and hedgehog pathways in maintaining myeloma stem cell features and drug resistance.We previously demonstrated that RARa2 expression is increased in CD138 selected plasma cells of relapsed multiple myelomas (MMs), and increased expression was linked to poor prognosis in newly diagnosed MM patients. In the present study, we demonstrate that increased RARa2 confers myeloma stem cell features. Higher expression of RARa2 was identified in the multiple myeloma stem cell (MMSC) fraction. Overexpression of RARa2 in bulk MM cell lines resulted in: 1) increased drug resistance; 2) increased clonogenic potential; 3) activation of both Wnt and Hedgehog (Hh) pathways; 4) increased side population and aldehyde dehydrogenase levels; and 5) increased expression of embryonic stem cell genes. The opposite effects were seen with RARa2 knockdown. We demonstrate that RARa2 induces drug resistance by activating the drug efflux pump gene ABCC3 and anti-apoptotic Bcl-2 family members. Inhibition of Wnt signaling or ABCC3 function could overcome drug resistance in RARa2 overexpressing MM cells. We also showed that in the 5TGM1 mouse model, targeting of the Wnt and Hh pathways using CAY10404, cyclopamine, or itraconazole significantly reduced the myeloma tumor burden and increased survival. Targeting RARa2 or its downstream signaling pathways provides a potential strategy to eliminate

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Ibrutinib (Imbruvica®), a small-drug inhibitor of Bruton tyrosine kinase (BTK), is currently undergoing clinical testing in patients with multiple myeloma (MM), yet important questions on the role of BTK in myeloma biology and treatment are outstanding. Using flow-sorted side population (SP) cells from human myeloma cell lines (HMCLs) and MM primary samples as surrogate fort the elusive multiple myeloma stem cell (MMSC), we found that elevated expression of BTK in myeloma cells leads to AKT/WNT/β-catenin-dependent up-regulation of key stemness genes (OCT4, SOX2, NANOG, MYC) and enhanced self-renewal. Enforced transgenic expression of BTK in myeloma cells increased features of cancer stemness, including clonogenicity and resistance to widely used myeloma drugs, whereas inducible knockdown of BTK abolished them. Furthermore, over-expression of BTK in myeloma cells promoted tumor growth in laboratory mice and rendered SP-derived tumors that contained high levels of BTK more sensitive to the selective, second-generation BTK inhibitor, CGI1746, than SP-derived tumors that harbored low levels of BTK. Taken together, these findings implicate BTK as a positive regulator of myeloma stemness and provide additional support for the clinical testing of BTK-targeted therapies in patients with myeloma.

Specific recognition of CN(-) in sodium cyanide solution was achieved using two imidazole-based receptors (A and B). Visually detectable color changes were associated with the formation of hydrogen bonded adducts, A.CN(-) and B.CN(-). Ratiometric fluorescence response was achieved for receptor A on binding to CN(-), and this reagent was used for imaging bacterial cells pre-exposed to 1.42 microM CN(-) solution.

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis , an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models. Electronic supplementary material The online version of this article (10.1186/s13059-019-1776-2) contains supplementary material, which is available to authorized users.

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Background: Nanog is a core factor that is required for the maintenance of embryonic stem (ES) cell pluripotency and self-renewal. Results: Alternative splicing results in Nanog proteins with different capacities for maintaining the undifferentiated ES cell state. Conclusion:The Nanog N-terminal domain is regulated by post-transcriptional modification. Significance: Nanog protein variants either fully support the maintenance of pluripotency or facilitate differentiation.