Accumulating evidence has shown that dysfunctional mitochondria can be selectively removed by mitophagy. Dysregulation of mitophagy is implicated in the development of neurodegenerative disease and metabolic disorders. How individual mitochondria are recognized for removal and how this process is regulated remain poorly understood. Here we report that FUNDC1, an integral mitochondrial outer-membrane protein, is a receptor for hypoxia-induced mitophagy. FUNDC1 interacted with LC3 through its typical LC3-binding motif Y(18)xxL(21), and mutation of the LC3-interaction region impaired its interaction with LC3 and the subsequent induction of mitophagy. Knockdown of endogenous FUNDC1 significantly prevented hypoxia-induced mitophagy, which could be reversed by the expression of wild-type FUNDC1, but not LC3-interaction-deficient FUNDC1 mutants. Mechanistic studies further revealed that hypoxia induced dephosphorylation of FUNDC1 and enhanced its interaction with LC3 for selective mitophagy. Our findings thus offer insights into mitochondrial quality control in mammalian cells.

The functional importance of gene enhancers in regulated gene expression is well established(1–3). In addition to widespread transcription of long non-coding RNAs (ncRNA) in mammalian cells(4–6), bidirectional ncRNAs referred to as eRNAs are transcribed on enhancers(7–9). However, it has remained unclear whether these eRNAs are functional, or merely a reflection of enhancer activation. Here, we report that 17β-estradiol (E2)-bound estrogen receptor α (ERα) on enhancers causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, appear to exert important roles for the observed ligand-dependent induction of target coding genes, causing an increased strength of specific enhancer:promoter looping initiated by ERα binding. Cohesin, present on many ERα-regulated enhancers even prior to ligand treatment, apparently contributes to E2-dependent gene activation, at least in part, by stabilizing E2/ERα/eRNA-induced enhancer:promoter looping. Our data indicate that eRNAs are likely to exert important functions in many regulated programs of gene transcription.

SUMMARY Distal enhancers characterized by H3K4me1 mark play critical roles in developmental and transcriptional programs. However, potential roles of specific distal regulatory elements in regulating RNA Polymerase II (Pol II) promoter-proximal pause release remain poorly investigated. Here we report that a unique cohort of jumonji C domain-containing protein 6 (JMJD6) and bromodomain-containing protein 4 (Brd4) co-bound distal enhancers, termed anti-pause enhancers (A-PEs), regulate promoter-proximal pause release of a large subset of transcription units via long-range interactions. Brd4-dependent JMJD6 recruitment on A-PEs mediates erasure of H4R3me2(s), which is directly read by 7SK snRNA, and decapping/demethylation of 7SK snRNA, ensuring the dismissal of the 7SKsnRNA/HEXIM inhibitory complex. The interactions of both JMJD6 and Brd4 with the P-TEFb complex permit its activation and pause release of regulated coding genes. The functions of JMJD6/ Brd4-associated dual histone and RNA demethylase activity on anti-pause enhancers have intriguing implications for these proteins in development, homeostasis and disease.

Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of smallcell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.cancer diagnostics | personalized therapy A s a genomic disease, cancer involves a series of changes in the genome, starting from primary tumors, via circulating tumor cells (CTCs), to metastases that cause the majority of mortalities (1-3). These genomic alterations include copy number variations (CNVs), single-nucleotide variations (SNVs), and insertions/deletions (INDELs). Regardless of the concentrated efforts in the past decades, the key driving genomic alterations responsible for metastases are still elusive (1).For noninvasive prognosis and diagnosis of cancer, it is desirable to monitor genomic alterations through the circulatory system. Genetic analyses of cell-free DNA fragments in peripheral blood have been reported (4-6) and recently extended to the whole-genome scale (7-9). However, it may be advantageous to analyze CTCs, as they represent intact functional cancer cells circulating in peripheral blood (10). Although previous studies have shown that CTC counting was able to predict progression and overall survival of cancer patients (11,12), genomic analyses of CTCs could provide more pertinent information for personalized therapy (13). However, it is difficult to probe the genomic changes in DNA obtainable from the small number of captured CTCs. To meet this challenge, a single-cell whole-genome amplification (WGA) method, multiple annealing and loopingbased amplification cycles (MALBAC) (14), has been developed to improve the amplification uniformity across the entire genome over previous methods (15,16), allowing precise determination of CNVs and detection of SNVs with a low false-positive rate in a single cell. Here, we present genomic analyses of CTCs from...

SummaryNatural variations in gene expression provide a mechanism for multiple phenotypes to arise in an isogenic bacterial population. In particular, a sub-group termed persisters show high tolerance to antibiotics. Previously, their formation has been attributed to cell dormancy. Here we demonstrate that bacterial persisters, under β-lactam antibiotic treatment, show less cytoplasmic drug accumulation as a result of enhanced efflux activity. Consistently, a number of multi-drug efflux genes, particularly the central component TolC, show higher expression in persisters. Time-lapse imaging and mutagenesis studies further establish a positive correlation between tolC expression and bacterial persistence. The key role of efflux systems, among multiple biological pathways involved in persister formation, indicates that persisters implement a positive defense against antibiotics prior to a passive defense via dormancy. Finally, efflux inhibitors and antibiotics together effectively attenuate persister formation, suggesting a combination strategy to target drug tolerance.

Graphical Abstract Highlights d The degree of drug-tolerant cells being dormant can be measured by ''dormancy depth'' d Cellular dark foci, proved to be protein aggresomes, indicate dormancy depth d Depletion of intracellular ATP is the major force driving aggresomes formation d DnaK is vital in the disaggregation of aggresomes when a dormant cell resuscitates In this work, Pu et al. introduced a concept of ''dormancy depth'' that provides a unifying framework for understanding both persisters and viable but non-culturable cells. Subsequent mechanistic investigations revealed how ATP-dependent dynamic protein aggregation regulates cellular dormancy and resuscitation, the fine control of which facilitates bacterial drug tolerance. SUMMARYCell dormancy is a widespread mechanism used by bacteria to evade environmental threats, including antibiotics. Here we monitored bacterial antibiotic tolerance and regrowth at the single-cell level and found that each individual survival cell shows different ''dormancy depth,'' which in return regulates the lag time for cell resuscitation after removal of antibiotic. We further established that protein aggresome-a collection of endogenous protein aggregates-is an important indicator of bacterial dormancy depth, whose formation is promoted by decreased cellular ATP level. For cells to leave the dormant state and resuscitate, clearance of protein aggresome and recovery of proteostasis are required. We revealed that the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the lag time for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.

Autophagy is an evolutionarily conserved catabolic process involved in several physiological and pathological processes1,2. Although primarily cytoprotective, autophagy can also contribute to cell death; it is thus important to understand what distinguishes the life or death decision in autophagic cells3. Here, we report that induction of autophagy is coupled to reduction of histone H4 lysine 16 acetylation (H4K16ac) through downregulation of the histone acetyltransferase hMOF/KAT8/MYST1, and demonstrate that this histone modification regulates the outcome of autophagy. At a genome-wide level we find that H4K16 deacetylation is associated predominantly with the downregulation of autophagy-related genes. Antagonizing H4K16ac downregulation upon autophagy induction results in the promotion of cell death. Our findings establish that alteration in a specific histone posttranslational modification during autophagy, affects the transcriptional regulation of autophagy-related genes and initiates a regulatory feedback loop, which serves as a key determinant of survival versus death responses upon autophagy induction.

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of