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Upon WNT/beta-catenin pathway activation, stabilized beta-catenin travels to the nucleus where it associates with the TCF/LEF transcription factors, constitutively bound to genomic Wnt-responsive elements (WREs), to activate target gene transcription. Discovering the binding profile of beta-catenin is therefore required to unambiguously assign direct targets of WNT signaling. Cleavage under targets and release using nuclease (CUT&RUN) has emerged as prime technique for mapping the binding profile of DNA-interacting proteins. Here, we present a modified version of CUT&RUN, named LoV-U (low volume and urea), that enables the robust and reproducible generation of beta-catenin binding profiles, uncovering direct WNT/beta-catenin target genes in human cells, as well as in cells isolated from developing mouse tissues. CUT&RUN-LoV-U outperforms original CUT&RUN when targeting co-factors that do not bind the DNA, can profile all classes of chromatin regulators and is well suited for simultaneous processing of several samples. We believe that the application of our protocol will allow the detection of the complex system of tissue-specific WNT/beta-catenin target genes, together with other non-DNA-binding transcriptional regulators that act downstream of ontogenetically fundamental signaling cascades., Funding Agencies|Cancerfonden [CAN 2018/542, 21 1572 Pj]; Vetenskapsradet [2021-03075]; Linkopings Universitet; Knut och Alice Wallenberg Stiftelse

Alphaviruses are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the Alphavirus chikungunya virus (CHIKV) has spread explosively in the last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.

The purpose of this study is to use a qualitative approach to get an understanding of what is expected of social workers targeted towards women managers in social services. Nine social workers were interviewed. The empirical data was then analyzed on the basis of gender theory. Respondents did not see their supervisor as a manager but more as a social worker with special tasks. Our empirical data indicates that there is a strong belief that men and women possess different properties. The social workers thought that men were likely to make a career and women were considered to work in the area of social care. It was considered to have a different leadership in social services than in other organizations.