Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in “A compartments” at active enhancers, which lose H3K27ac but not H3K4me1. Affected positions include hundreds of sites identified as superenhancers in many tissues. Dominant-negative mutation induced pro-oncogenic expression changes, including increased expression of Myc and its target genes. Together, our data suggest that disruption of enhancer accessibility represents a key source of altered function in SMARCA4-mutated disorders in a wide variety of tissues.

Adult stem cells reside in specialized niches where they receive environmental cues to maintain tissue homeostasis. In mammals, the stem cell niche within hair follicles is home to epithelial hair follicle stem cells and melanocyte stem cells, which sustain cyclical bouts of hair regeneration and pigmentation1–4. To generate pigmented hairs, synchrony is achieved such that upon initiation of a new hair cycle, stem cells of each type activate lineage commitment2,5. Dissecting the inter-stem-cell crosstalk governing this intricate coordination has been difficult, because mutations affecting one lineage often affect the other. Here we identify transcription factor NFIB as an unanticipated coordinator of stem cell behaviour. Hair follicle stem-cell-specific conditional targeting of Nfib in mice uncouples stem cell synchrony. Remarkably, this happens not by perturbing hair cycle and follicle architecture, but rather by promoting melanocyte stem cell proliferation and differentiation. The early production of melanin is restricted to melanocyte stem cells at the niche base. Melanocyte stem cells more distant from the dermal papilla are unscathed, thereby preventing hair greying typical of melanocyte stem cell differentiation mutants. Furthermore, we pinpoint KIT-ligand as a dermal papilla signal promoting melanocyte stem cell differentiation. Additionally, through chromatin-immunoprecipitation with high-throughput-sequencing and transcriptional profiling, we identify endothelin 2 (Edn2) as an NFIB target aberrantly activated in NFIB-deficient hair follicle stem cells. Ectopically induced Edn2 recapitulates NFIB-deficient phenotypes in wild-type mice. Conversely, endothelin receptor antagonists and/or KIT blocking antibodies prevent precocious melanocyte stem cell differentiation in the NFIB-deficient niche. Our findings reveal how melanocyte and hair follicle stem cell behaviours maintain reliance upon cooperative factors within the niche, and how this can be uncoupled in injury, stress and disease states.

The syndecan Sds2 and the innate immunity inhibitor Sarm1 function together and in distinct pathways to promote proper neuronal morphogenesis.

Resolution and formation of facultative heterochromatin is essential to development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood due to the inability to study heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription, indicating that changes in DNA topology through (de-)catenation rather than release of torsional stress through swiveling is necessary for heterochromatin resolution. In turn, TOP2 and BAF cooperate to recruit pluripotency factors, explaining some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2, also plays a role in the reformation of facultative heterochromatin, suggesting that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which may be critical to their structures.

The simultaneous acquisition of PET and MRI data shows promise to provide powerful capabilities to study disease processes in human subjects, guide the development of novel treatments, and monitor therapy response and disease progression. A brain-size PET detector ring insert for an MRI system is being developed that, if successful, can be inserted into any existing MRI system to enable simultaneous PET and MRI images of the brain to be acquired without mutual interference. The PET insert uses electro-optical coupling to relay all the signals from the PET detectors out of the MRI system using analog modulated lasers coupled to fiber optics. Because the fibers use light instead of electrical signals, the PET detector can be electrically decoupled from the MRI making it partially transmissive to the RF field of the MRI. The SiPM devices and low power lasers were powered using non-magnetic MRI compatible batteries. Also, the number of laser-fiber channels in the system was reduced using techniques adapted from the field of compressed sensing. Using the fact that incoming PET data is sparse in time and space, electronic circuits implementing constant weight codes uniquely encode the detector signals in order to reduce the number of electro-optical readout channels by 8-fold. Two out of a total of sixteen electro-optical detector modules have been built and tested with the entire RF-shielded detector gantry for the PET ring insert. The two detectors have been tested outside and inside of a 3T MRI system to study mutual interference effects and simultaneous performance with MRI. Preliminary results show that the PET insert is feasible for high resolution simultaneous PET/MRI imaging for applications in the brain.

Autophagy is a catabolic membrane-trafficking mechanism conserved in all eukaryotic cells. In addition to the nonselective transport of bulk cytosol, autophagy is responsible for efficient delivery of the vacuolar enzyme Ape1 precursor (prApe1) in the budding yeast Saccharomyces cerevisiae, suggesting the presence of a prApe1 sorting machinery. Sequential interactions between Atg19-Atg11 and Atg19-Atg8 pairs are thought responsible for targeting prApe1 to the vesicle formation site, the preautophagosomal structure (PAS), and loading it into transport vesicles, respectively. However, the different patterns of prApe1 transport defect seen in the atg11⌬ and atg19⌬ strains seem to be incompatible with this model. Here we report that prApe1 could not be targeted to the PAS and failed to be delivered into the vacuole in atg8⌬ atg11⌬ double knockout cells regardless of the nutrient conditions. We postulate that Atg19 mediates a dual interaction prApe1-sorting mechanism through independent, instead of sequential, interactions with Atg11 and Atg8. In addition, to efficiently deliver prApe1 to the vacuole, a proper interaction between Atg11 and Atg9 is indispensable. We speculate that Atg11 may elicit a cargo-loading signal and induce Atg9 shuttling to a specific PAS site, where Atg9 relays the signal and recruits other Atg proteins to induce vesicle formation.

Mutations in ARID1A rank amongst the most common molecular aberrations in human cancer.However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout in primary TP53 -/human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation. Genetic Wnt/-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation.ARID1A mutation induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin.Convergently, high-throughput compound screening indicated selective vulnerability of ARID1Adeficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wntinhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells.Research.

Purpose A brain sized radio-frequency (RF)-penetrable PET insert has been designed for simultaneous operation with MRI systems. This system takes advantage of electro-optical coupling and battery power to electrically float the PET insert relative to the MRI ground, permitting RF signals to be transmitted through small gaps between the modules that form the PET ring. This design facilitates the use of the built-in body coil for RF transmission, and thus could be inserted into any existing MR site wishing to achieve simultaneous PET/MR imaging. The PET detectors employ non-magnetic silicon photomultipliers in conjunction with a compressed sensing signal multiplexing scheme, and optical fibers to transmit analog PET detector signals out of the MRI room for decoding, processing, and image reconstruction. Methods The PET insert was first constructed and tested in a laboratory benchtop setting, where tomographic images of a custom resolution phantom were successfully acquired. The PET insert was then placed within a 3T body MRI system, and tomographic resolution/contrast phantom images were acquired both with only the B0 field present, and under continuous pulsing from different MR imaging sequences. Results The resulting PET images have comparable contrast-to-noise ratios (CNR) under all MR pulsing conditions: the maximum percent CNR relative difference for each rod type among all four PET images acquired in the MRI system has a mean of 14.0±7.7%. MR images were successfully acquired through the RF-penetrable PET shielding using only the built-in MR body coil, suggesting that simultaneous imaging is possible without significant mutual interference. Conclusions These results show promise for this technology as an alternative to costly integrated PET/MR scanners; a PET insert that is compatible with any existing clinical MRI system could greatly increase the availability, accessibility, and dissemination of PET/MR.