Autophagy constitutes a major cell protective mechanism eliminating damaged components and maintaining energy homoeostasis via recycling nutrients under normal/stressed conditions. Although the core components of autophagy have been well studied, regulation of autophagy at the transcriptional level is poorly understood. Herein, we establish ZKSCAN3, a zinc-finger family DNA-binding protein, as a transcriptional repressor of autophagy. Silencing of ZKSCAN3 induced autophagy and increased lysosome biogenesis. Importantly, we show that ZKSCAN3 represses transcription of a large gene set (>60) integral to, or regulatory for, autophagy and lysosome biogenesis/function and a subset of these genes, including Map1lC3b and Wipi2 represent direct targets. Interestingly, ZKSCAN3 and TFEB are oppositely regulated by starvation and in turn oppositely regulate lysosomal biogenesis and autophagy, suggesting that they act in conjunction. Altogether, our study uncovers an autophagy master-switch regulating the expression of a transcriptional network of genes integral to autophagy and lysosome biogenesis/function.
Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep-sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome; this permits the systematic analysis of initiation, origin efficiency, fork progression and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.
Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110β catalytic subunit of the Class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes, and increases its interaction with the small GTPase Rab5. This p110β-Rab5 association maintains Rab5 in its GTP-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110β mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110β acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.
Splicing and translation are highly regulated steps of gene expression. Altered expression of proteins involved in these processes can be deleterious. Therefore, the cell has many safeguards against such misregulation. We report that the oncogenic splicing factor SRSF1, which is overexpressed in many cancers, stabilizes the tumor-suppressor protein p53 by abrogating its MDM2-dependent proteasomal degradation. We show that SRSF1 is a necessary component of an MDM2/ribosomal-protein complex—separate from the ribosome—that functions in a p53-dependent ribosomal-stress checkpoint pathway. Consistent with the stabilization of p53, increased SRSF1 expression in primary human fibroblasts decreases cellular proliferation and ultimately triggers oncogene-induced senescence (OIS). These findings underscore the deleterious outcome of SRSF1 overexpression and identify a cellular defense mechanism against its aberrant function. Furthermore, they implicate the RPL5-MDM2 complex in OIS, and demonstrate a link between spliceosomal and ribosomal components—functioning independently of their canonical roles—to monitor cellular physiology and cell-cycle progression.
ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a previously unknown mechanism for the recruitment of a chromatin remodeling enzyme and defines a novel function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes.
We recently reported that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5′-end capped mRNAs. Here we report that their mammalian homolog, Dom3Z, possesses pyrophosphohydrolase, decapping and 5′-3′ exoribonuclease activities, and will be referred to as DXO. Surprisingly, we find that DXO preferentially degrades defectively capped pre-mRNAs in cells. Further studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, in contrast to current understanding, and poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at up to 1.5Å resolution provide elegant insights into the catalytic mechanism and molecular basis for its three apparently distinct activities. Our data reveal a pre-mRNA 5′-end capping quality control mechanism in mammalian cells, with DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5′-end capping and subsequent pre-mRNA processing.
pre-mRNA quality control; pre-mRNA decapping; 5′-3′ exoribonuclease
Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using over 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed novel TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an 'anti-defense' protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.
In this issue of Molecular Cell, Jiao et al (2013) describe the mammalian enzyme DXO, which has pyrophosphohydrolase, decapping and 5′-3′ exoribonucease activity, and functions as an important checkpoint in co-transcriptional capping of RNA polymerase II (pol II) pre-mRNA transcripts.
The MRN (MRE11-RAD50-NBS1) complex has been implicated in many aspects of the DNA damage response. It has key roles in sensing and processing DNA double-strand breaks, as well as in activation of ATM (ataxia-telangiectasia mutated). We reveal a new function for MRN in ATR (ATM and RAD3-related) activation by using defined ATR-activating DNA structures in Xenopus egg extracts. Strikingly, we demonstrate that MRN is required to recruit TOPBP1 to an ATR-activating structure that contains a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) junction, and that this recruitment is necessary for phosphorylation of Chk1. We also show that the 911 (RAD9-RAD1-HUS1) complex is not required for TOPBP1 recruitment, but is essential for TOPBP1 function. Thus, whereas MRN is required for TOPBP1 recruitment at a ss/dsDNA junction, 911 is required for TOPBP1 “activation”. These findings provide new molecular insight into how ATR is activated.
How the cell recognizes cytosolic DNA including DNA based microbes to trigger host defense related gene activation remains to be fully resolved. Here, we demonstrate that STING (for Stimulator of Interferon Genes), an endoplasmic reticulum (ER) translocon associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self (apoptotic, necrotic) or pathogen related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and pro-inflammatory genes in addition to type I IFN. Our data indicates that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA.
Cullin 3, the core subunit of the CRL3 ubiquitin ligase family, is essential for development, but its substrates remain poorly defined. Here, Chen et al. (2009) report that CRL3BACURD targets the RhoA GTPase for degradation thereby maintaining actin cytoskeleton integrity.
The transcription factor Krüppel-like factor 4 (KLF4) is an important regulator of cell fate decision, including cell cycle regulation, apoptosis, and stem cell renewal, and plays an ambivalent role in tumorigenesis as a tissue specific tumor suppressor or oncogene. Here we report that the Von Hippel-Lindau gene product, pVHL, physically interacts with KLF4 and regulates its rapid turnover observed in both differentiated and stem cells. We provide mechanistic insights into KLF4 degradation and show that pVHL depletion in colorectal cancer cells leads to cell cycle arrest concomitant with increased transcription of the KLF4-dependent p21 gene. Finally, immunohistochemical staining revealed elevated pVHL and reduced KLF4 levels in colon cancer tissues. We therefore propose that unexpectedly pVHL, via the degradation of KLF4, is a facilitating factor in colorectal tumorigenesis.
Cells transiently adapt to hypoxia by globally decreasing protein translation. However, specific proteins needed to respond to hypoxia evade this translational repression. The mechanisms of this phenomenon remain unclear. We screened for and identified small molecules that selectively decrease HIF-2a translation in an mTOR independent manner, by enhancing the binding of Iron Regulatory Protein 1 (IRP1) to a recently reported Iron-Responsive Element (IRE) within the 5’-untranslated region (UTR) of the HIF-2a message. Knocking down the expression of IRP1 by shRNA abolished the effect of the compounds. Hypoxia de-represses HIF-2a translation by disrupting the IRP1- HIF-2a IRE interaction. Thus, this chemical genetic analysis describes a molecular mechanism by which translation of the HIF-2a message is maintained during conditions of cellular hypoxia through inhibition of IRP-1 dependent repression. It also provides the chemical tools for studying this phenomenon.
The ability of cells to respond to changes in nutrient availability is critical for an adequate control of metabolic homeostasis. Mammalian target of rapamycin complex 1 (mTORC1) is a central complex kinase in these processes. The signaling adaptor p62 binds raptor, and integral component of the mTORC1 pathway. p62 interacts with TNF receptor associated factor 6 (TRAF6) and is required for mTORC1 translocation to the lysosome and its subsequent activation. Here we show that TRAF6 is recruited to and activates mTORC1 through p62 in amino acid-stimulated cells. We also show that TRAF6 is necessary for the translocation of mTORC1 to the lysosomes and that the TRAF6-catalyzed K63 ubiquitination of mTOR regulates mTORC1 activation by amino acids. TRAF6, through its interaction with p62 and activation of mTORC1, modulates autophagy and is an important mediator in cancer cell proliferation. Interfering with the p62-TRAF6 interaction serves to modulate autophagy and nutrient sensing.
The Cul4-Cdt2 (CRL4Cdt2) E3 ubiquitin ligase is a master regulator of cell cycle progression and genome stability. Despite its central role in the degradation of many cell-cycle regulators, e.g. Cdt1, p21 and Pr-Set7/Set8, little is known about the regulation of its activity. We report that Cdt2 is autoubiquitylated by the CRL4A E3 ubiquitin ligase. Cdt2 is additionally polyubiquitylated and degraded by Cul1-FBXO11 (CRL1FBXO11). CRL1FBXO11-mediated degradation of Cdt2 stabilizes p21 and Set8, and this is important during the response to TGF-beta, with the Set8 induction being important for turning off the activation of Smad2. The migration of epithelial cells is also stimulated by CRL1FBXO11-mediated downregulation of Cdt2 and the consequent stabilization of Set8. This is a novel example of cross-regulation between specific cullin 4 and cullin 1 E3 ubiquitin ligases and highlights the role of ubiquitylation in regulating cellular responses to TGF-beta and the migration of epithelial cells.
Cdt2; Set8; CRL4; FBXO11; Cullin; Ubiquitylation
Cells regulate adhesion in response to internally-generated and externally-applied forces. Integrins connect the extracellular matrix to the cytoskeleton and provide cells with mechanical anchorages and signaling platforms. Here we show that cyclic forces applied to a fibronectin–integrin α5β1 bond switch the bond from a short-lived state with 1-s lifetime to a long-lived state with 100-s lifetime. We term this phenomenon “cyclic mechanical reinforcement” as the bond strength remembers the history of force application, accumulates over repeated cycles, but does not require force to be sustained. Cyclic mechanical reinforcement strengthens the fibronectin–integrin α5β1 bond through the RGD binding site of the ligand with the synergy binding site greatly facilitating the process. A flexible integrin hybrid domain is also important for cyclic mechanical reinforcement. Our results reveal a mechanical regulation of receptor–ligand interactions and identify a molecular mechanism for cell adhesion strengthening by cyclic forces.
Crosstalk between H2B ubiquitylation (H2Bub) and H3 K4 methylation plays important roles in coordinating functions of diverse cofactors during transcription activation. The underlying mechanism for this trans-tail signaling pathway is poorly defined in higher eukaryotes. Here, we show that 1) ASH2L in the MLL complex is essential for H2Bub-dependent H3 K4 methylation. Deleting or mutating K99 of the N-terminal winged helix (WH) motif in ASH2L abrogates H2Bub-dependent regulation; 2) crosstalk can occur in trans and does not require ubiquitin to be on nucleosomes or histones to exert regulatory effects; 3) Trans-regulation by ubiquitin promotes MLL activity for all three methylation states; and 4) MLL3, an MLL homolog, does not respond to H2Bub, highlighting regulatory specificity for MLL family histone methyltransferases. Altogether, our results potentially expand the classic histone crosstalk to nonhistone proteins, which broadens the scope of chromatin regulation by ubiquitylation signaling.
Superfamily ATPases in Type IV pili (T4P), Type 2 secretion (T2S), and archaella (formerly archaeal flagella) employ similar sequences for distinct biological processes. Here we structurally and functionally characterize prototypical superfamily ATPase FlaI from Sulfolobus acidocaldarius showing FlaI activities in archaeal swimming organelle assembly and movement. FlaI solution X-ray scattering and crystal structures with and without nucleotide reveal a hexameric crown assembly with key cross-subunit interactions: rigid building blocks form between N-terminal domains (points) and neighboring subunit C-terminal domains (crown ring). Upon nucleotide binding, these six cross-subunit blocks move with respect to each other distinctly from secretion and pilus ATPases. Crown interactions and conformations regulate assembly, motility and force direction by a basic-clamp switching mechanism driving conformational changes between stable, backbone-interconnected moving blocks. Collective structural and mutational results identify in vivo functional components for assembly and motility, phosphate triggered rearrangements by ATP-hydrolysis, and molecular predictors for distinct ATPase superfamily functions.
Past studies have documented a cross-talk between H2B ubiquitylation (H2Bub) and H3K4 methylation, but little, if any, direct evidence exists explaining the mechanism underlying H2Bub-dependent H3K4 methylation on chromatin templates. Here, we took advantage of an in vitro histone methyltransferase assay employing a reconstituted yeast Set1 complex (ySet1C) and a recombinant chromatin template containing fully ubiquitylated H2B to gain valuable insights. Combined with genetic analyses, we demonstrate that the n-SET domain within Set1, but not Swd2, is essential for H2Bub-dependent H3K4 methylation. Spp1, a homolog of human CFP1, is conditionally involved in this cross-talk. Our findings extend to the human Set1 complex, underscoring the conserved nature of this disease-relevant, cross-talk pathway. As not all members of the H3K4 methyltransferase family contain n-SET domains, our studies call attention to the n-SET domain as being a predictor of H2B ubiquitylation ‘sensing’ in bringing about downstream H3K4 methylation.
TXNIP is an α-arrestin family protein that is induced in response to glucose elevation. It has been shown to provide a negative feedback loop to regulate glucose uptake into cells, though the biochemical mechanism of action has been obscure. Here, we report that TXNIP suppresses glucose uptake directly by binding to the glucose transporter, Glut1, inducing Glut1 internalization through clathrin coated pits, as well as indirectly by reducing the level of Glut1 mRNA. In addition, we show that energy stress results in phosphorylation of TXNIP by AMP-dependent protein kinase (AMPK), leading to its rapid degradation. This suppression of TXNIP results in an acute increase in Glut1 function and an increase in Glut1 mRNA (hence total protein levels) for long-term adaptation. The glucose influx through GLUT1 restores ATP/ADP ratios in the short run and ultimately induces TXNIP protein production to suppress glucose uptake once energy homeostasis is reestablished.
Faithful duplication of the genome in S phase followed by its accurate segregation in mitosis is essential to maintain genomic integrity. Recent studies have suggested that proteins involved in DNA transactions are also required for whole chromosome stability. Here we demonstrate that the MRN (Mre11, Rad50, and Nbs1) complex and CtIP are required for accurate chromosome segregation. Depletion of Mre11 or CtIP, antibody-mediated inhibition of Mre11, or small molecule inhibition of MRN using mirin results in metaphase chromosome alignment defects in Xenopus egg extracts. Similarly, loss of MRN function adversely affects spindle assembly around DNA-coated beads in egg extracts. Inhibition of MRN function in mammalian cells triggers a metaphase delay and disrupts the RCC1-dependent RanGTP gradient. Addition of the Mre11 inhibitor mirin to egg extracts and mammalian cells reduces RCC1 association with mitotic chromosomes. Thus, the MRN-CtIP pathway contributes to Ran-dependent mitotic spindle assembly by modulating RCC1 chromosome association.
A new study (Zemach et al., 2013) suggests that the chromatin remodeling ATPase, DDM1 is specifically required for cytosine methylation at linker histone H1-associated heterochromatin, facilitating access by three cytosine methyltransferases, including a previously uncharacterized CHH methylase, CMT2.
Two new studies in this issue of Molecular Cell (Kim et al., 2013 and Wu et al., 2013) provide new insights and reignite debate over how histone H2B ubiquitination promotes methylation of histone H3 lysine 4.
In a recent issue of Molecular Cell, Zheng et al. (2008) demonstrate that human DNA2, originally identified in yeast as a nuclear DNA replication and repair factor, functions exclusively in mammalian mitochondria in the recently discovered long-patch base excision repair pathway.