In contrast to mechanisms mediating uptake of intracellular bacterial pathogens, bacterial egress and cell-to-cell transmission are poorly understood. Previously, we showed that the transmission of pathogenic mycobacteria between phagocytic cells also depends on nonlytic ejection through an F-actin based structure, called the ejectosome. How the host cell maintains integrity of its plasma membrane during the ejection process was unknown. Here, we reveal an unexpected function for the autophagic machinery in nonlytic spreading of bacteria. We show that ejecting mycobacteria are escorted by a distinct polar autophagocytic vacuole. If autophagy is impaired, cell-to-cell transmission is inhibited, the host plasma membrane becomes compromised and the host cells die. These findings highlight a previously unidentified, highly ordered interaction between bacteria and the autophagic pathway and might represent the ancient way to ensure nonlytic egress of bacteria.autophagy | Dictyostelium discoideum | Mycobacterium marinum | ejection

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of function from the maternal allele ofUBE3A, a gene encoding an E3 ubiquitin ligase.UBE3Ais only expressed from the maternally inherited allele in mature human neurons due to tissue-specific genomic imprinting. Imprinted expression ofUBE3Ais restricted to neurons by expression ofUBE3A antisense transcript(UBE3A-ATS) from the paternally inherited allele, which silences the paternal allele ofUBE3Aincis. However, the mechanism restrictingUBE3A-ATSexpression andUBE3Aimprinting to neurons is not understood. We used CRISPR/Cas9-mediated genome editing to functionally define a bipartite boundary element critical for neuron-specific expression ofUBE3A-ATSin humans. Removal of this element led to up-regulation ofUBE3A-ATSwithout repressing paternalUBE3A. However, increasing expression ofUBE3A-ATSin the absence of the boundary element resulted in full repression of paternalUBE3A, demonstrating thatUBE3Aimprinting requires both the loss of function from the boundary element as well as the up-regulation ofUBE3A-ATS. These results suggest that manipulation of the competition betweenUBE3A-ATSandUBE3Amay provide a potential therapeutic approach for AS.

Gene expression programs change during cellular transitions. It is well established that a network of transcription factors and chromatin modifiers regulate RNA levels during embryonic stem cell (ESC) differentiation, but the full impact of post-transcriptional processes remains elusive. While cytoplasmic RNA turnover mechanisms have been implicated in differentiation, the contribution of nuclear RNA decay has not been investigated. Here, we differentiate mouse ESCs, depleted for the ribonucleolytic RNA exosome, into embryoid bodies to determine to which degree RNA abundance in the two states can be attributed to changes in transcription versus RNA decay by the exosome. As a general observation, we find that exosome depletion mainly leads to the stabilization of RNAs from lowly transcribed loci, including several protein-coding genes. Depletion of the nuclear exosome cofactor RBM7 leads to similar effects. In particular, transcripts that are differentially expressed between states tend to be more exosome sensitive in the state where expression is low. We conclude that the RNA exosome contributes to down-regulation of transcripts with disparate expression, often in conjunction with transcriptional down-regulation.

T cell development depends on sequential interactions of thymocytes with cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells. PSMB11 is a catalytic proteasomal subunit present exclusively in cTECs. Because proteasomes regulate transcriptional activity, we asked whether PSMB11 might affect gene expression in cTECs. We report that PSMB11 regulates the expression of 850 cTEC genes that modulate lymphostromal interactions primarily via the WNT signaling pathway. cTECs from Psmb11−/− mice 1) acquire features of medullary thymic epithelial cells and 2) retain CD8 thymocytes in the thymic cortex, thereby impairing phase 2 of positive selection, 3) perturbing CD8 T cell development, and 4) causing dramatic oxidative stress leading to apoptosis of CD8 thymocytes. Deletion of Psmb11 also causes major oxidative stress in CD4 thymocytes. However, CD4 thymocytes do not undergo apoptosis because, unlike CD8 thymocytes, they upregulate expression of chaperones and inhibitors of apoptosis. We conclude that PSMB11 has pervasive effects on both CD4 and CD8 thymocytes via regulation of gene expression in cTECs.

24Tracking active transcription with the nuclear run-on (NRO) assays has been instrumental in 25 uncovering mechanisms of gene regulation. The coupling of NROs with high-throughput 26 sequencing has facilitated the discovery of previously unannotated or undetectable RNA classes 27 genome-wide. Precision run-on sequencing (PRO-seq) is a run-on variant that maps polymerase 28 active sites with nucleotide or near-nucleotide resolution. One main drawback to this and many 29 other nascent RNA detection methods is the somewhat intimidating multi-day workflow 30 associated with creating the libraries suitable for high-throughput sequencing. Here, we present 31an improved PRO-seq protocol where many of the enzymatic steps are carried out while the 32 biotinylated NRO RNA remains bound to streptavidin-coated magnetic beads. These 33 adaptations reduce time, sample loss and RNA degradation, and we demonstrate that the 34 resulting libraries are of the same quality as libraries generated using the original published 35protocol. The assay is also more sensitive which permits reproducible, high-quality libraries from 36 10 4 -10 5 cells instead of 10 6 -10 7 . Altogether, the improved protocol is more tractable allows for 37 nascent RNA profiling from small samples, such as rare samples or FACS sorted cell 38populations. 39 40