Cholesterol is required for transcriptional repression by BASP1

Lipids are present within the cell nucleus, where they engage with factors involved in gene regulation. Cholesterol associates with chromatin in vivo and stimulates nucleosome packing in vitro, but its effects on specific transcriptional responses are not clear. Here, we show that the lipidated Wilms tumor 1 (WT1) transcriptional corepressor, brain acid soluble protein 1 (BASP1), interacts with cholesterol in the cell nucleus through a conserved cholesterol interaction motif. We demonstrate that BASP1 directly recruits cholesterol to the promoter region of WT1 target genes. Mutation of BASP1 to ablate its interaction with cholesterol or the treatment of cells with drugs that block cholesterol biosynthesis inhibits the transcriptional repressor function of BASP1. We find that the BASP1–cholesterol interaction is required for BASP1-dependent chromatin remodeling and the direction of transcription programs that control cell differentiation. Our study uncovers a mechanism for gene-specific targeting of cholesterol where it is required to mediate transcriptional repression

Streamlined histone-based fluorescence lifetime imaging microscopy (FLIM) for studying chromatin organisation

Changes in chromatin structure are key determinants of genomic responses. Thus, methods that enable such measurements are instrumental for investigating genome regulation and function. Here, we report further developments and validation of a streamlined method of histone-based fluorescence lifetime imaging microscopy (FLIM) that robustly detects chromatin compaction states in fixed and live cells, in 2D and 3D. We present a quality-controlled and detailed method that is simpler and faster than previous methods, and uses FLIMfit open-source software. We demonstrate the versatility of this chromatin FLIM through its combination with immunofluorescence and implementation in immortalised and primary cells. We applied this method to investigate the regulation of chromatin organisation after genotoxic stress and provide new insights into the role of ATM in controlling chromatin structure independently of DNA damage. Collectively, we present an adaptable chromatin FLIM method for examining chromatin structure and establish its utility in mammalian cells

Ecological enhancement techniques to improve habitat heterogeneity on coastal defence structures

Sea level rise and higher storm frequency are increasing the need for the placement of hard coastal defences worldwide. The majority of these defences lack optimal habitats for intertidal species, resulting in low diversity and abundance. The construction of coastal defences within marine protected areas (MPA) is also increasing and this study investigates ways to limit the loss of species diversity and intertidal habitat caused by installing rock armour defence structures and other coastal developments. Arrays of holes and grooves were created on granite rock armour in the north of England at Runswick Bay, N. Yorkshire and limestone rock groynes in southern England at Boscombe, Poole Bay, Dorset. Runswick Bay is a Marine Conservation Zone (MCZ) designated for its intertidal habitat and Boscombe is located in close proximity to a Special Area of Conservation (SAC). After 12 months, the treatments had attracted new species to the defence structures and increased the overall diversity and abundance of organisms compared to control areas. Mobile fauna including crabs and fish were also recorded utilising the holes and grooves at Boscombe. Non-native species were recorded in grooves at one site however their abundance was not significantly different to that of control areas. At the southern site, species known to be spreading in response to climate change were found in treatments but not in control areas. The cost of the installation of these enhancement techniques was low in relation to that of the defence scheme and could be easily incorporated before, during or after construction. Through evaluation of the use of these ecological enhancement techniques on coastal structures, it is suggested that they have considerable potential to increase biodiversity on artificial structures, particularly when used within large-scale coastal engineering defence projects

CTLA4 depletes T cell endogenous and trogocytosed B7 ligands via cis-endocytosis.

CD28 and CTLA4 are T cell coreceptors that competitively engage B7 ligands CD80 and CD86 to control adaptive immune responses. While the role of CTLA4 in restraining CD28 costimulatory signaling is well-established, the mechanism has remained unclear. Here, we report that human T cells acquire antigen-presenting-cell (APC)-derived B7 ligands and major histocompatibility complex (MHC) via trogocytosis through CD28:B7 binding. Acquired MHC and B7 enabled T cells to autostimulate, and this process was limited cell-intrinsically by CTLA4, which depletes B7 ligands trogocytosed or endogenously expressed by T cells through cis-endocytosis. Extending this model to the previously proposed extrinsic function of CTLA4 in human regulatory T cells (Treg), we show that blockade of either CD28 or CTLA4 attenuates Treg-mediated depletion of APC B7, indicating that trogocytosis and CTLA4-mediated cis-endocytosis work together to deplete B7 from APCs. Our study establishes CTLA4 as a cell-intrinsic molecular sink that limits B7 availability on the surface of T cells, with implications for CTLA4-targeted therapy

The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis

Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a highrisk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the RasMEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers