Activating mutations in NOTCH1, an essential regulator of T cell development, are frequently found in human T cell acute lymphoblastic leukemia (T-ALL). Despite important advances in our understanding of Notch signal transduction, the regulation of Notch functions in the nucleus remains unclear. Using immunoaffinity purification, we identified NOTCH1 nuclear partners in T-ALL cells and showed that, beyond the well-characterized core activation complex (ICN1-CSL-MAML1), NOTCH1 assembles a multifunctional complex containing the transcription coactivator AF4p12, the PBAF nucleosome remodeling complex, and the histone demethylases LSD1 and PHF8 acting through their demethylase activity to promote epigenetic modifications at Notch-target genes. Remarkably, LSD1 functions as a corepressor when associated with CSL-repressor complex and as a NOTCH1 coactivator upon Notch activation. Our work provides new insights into the molecular mechanisms that govern Notch transcriptional activity and represents glimpse into NOTCH1 interaction landscape, which will help in deciphering mechanisms of NOTCH1 functions and regulation.
HIV-1 transactivator Tat has greatly contributed to our understanding of transcription elongation by RNAPII. We purified HIV-1 Tat-associated factors from HeLa nuclear extract and show that Tat forms two distinct and stable complexes. Tatcom1 consists of the core active P-TEFb, MLL-fusion partners involved in leukemia (AF9, AFF4, AFF1, ENL, and ELL), and PAF1 complex. Importantly, Tatcom1 formation relies on P-TEFb while optimal CDK9 CTD-kinase activity is AF9 dependent. MLL-fusion partners and PAF1 are required for Tat transactivation. Tatcom2 is composed of CDK9, CycT1, and 7SK snRNP lacking HEXIM. Tat remodels 7SK snRNP by interacting directly with 7SK RNA, leading to the formation of a stress-resistant 7SK snRNP particle. Besides the identification of factors required for Tat transactivation and important for P-TEFb function, our data show a coordinated control of RNAPII elongation by different classes of transcription elongation factors associated in a single complex and acting at the same promoter.
Autophagy is a major catabolic pathway in eukaryotes associated with a broad spectrum of human diseases. In autophagy, autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. However, the molecular mechanism underlying autophagosome maturation is largely unknown. Here we report that TECPR1 binds to the Atg12-Atg5 conjugate and phosphatidylinositol 3-phosphate (PtdIns(3)P) to promote autophagosome-lysosome fusion. TECPR1 and Atg16 form mutually exclusive complexes with the Atg12-Atg5 conjugate; and TECPR1 binds PtdIns(3)P upon association with the Atg12-Atg5 conjugate. Strikingly, TECPR1 localizes to and recruits Atg5 to autolysosome membrane. Consequently, elimination of TECPR1 leads to accumulation of autophagosomes and blocks autophagic degradation of LC3-II and p62. Finally, autophagosome maturation marked by GFP-mRFP-LC3 is defective in TECPR1 deficient cells. Thus, we propose that the concerted interactions among TECPR1, Atg12-Atg5 and PtdIns(3)P provide the fusion specificity between autophagosomes and lysosomes and that the assembly of this complex initiates the autophagosome maturation process.
Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme catalyzing conversion of phosphoenolpyruvate (PEP) to pyruvate with transferring a phosphate from PEP to ADP. We report here that PKM2 localizes to the cell nucleus. The levels of nuclear PKM2 correlate with cell proliferation. PKM2 activates transcription of MEK5 by phosphorylating stat3 at Y705. In vitro phosphorylation assays show that PKM2 is a protein kinase using PEP as phosphate donor. ADP competes with the protein substrate binding, indicating that the substrate may bind to the ADP site of PKM2. Our experiments suggest that PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. Expression a PKM2 mutant that exists as a dimer promotes cell proliferation, indicating that protein kinase activity of PKM2 plays a role in promoting cell proliferation. Our study reveals an important link between metabolism alteration and gene expression during tumor transformation and progression.
Pyruvate kinase M2; Proliferation; stat3; protein kinase; glycolysis
A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked Inhibitor of Apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its anti-apoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it mono-ubiquitylates Groucho(Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing β-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program.
The nuclear matrix associated hnRNP U/SAF-A protein has been implicated in diverse pathways from transcriptional regulation to telomere length control to X inactivation, but the precise mechanism underlying each of these processes has remained elusive. Here, we report hnRNP U as a regulator of SMN2 splicing from a custom RNAi screen. Genome-wide analysis by CLIP-seq reveals that hnRNP U binds virtually to all classes of regulatory non-coding RNAs, including all snRNAs required for splicing of both major and minor classes of introns, leading to the discovery that hnRNP U regulates U2 snRNP maturation and Cajal body morphology in the nucleus. Global analysis of hnRNP U-dependent splicing by RNA-seq coupled with bioinformatic analysis of associated splicing signals suggests a general rule for splice site selection through modulating the core splicing machinery. These findings exemplify hnRNP U/SAF-A as a potent regulator of nuclear ribonucleoprotein particles in diverse gene expression pathways.
The connection between cancer and inflammation is widely recognized; yet the underlying molecular mechanisms are poorly understood. We report here that TIPE2 provides a molecular bridge from inflammation to cancer by targeting the Ras signaling pathway. TIPE2 binds the Ras-interacting domain of the RalGDS family of proteins, which are essential effectors of activated Ras. This binding prevented Ras from forming an active complex, thereby inhibiting the activation of the downstream signaling molecules Ral and AKT. Consequently, TIPE2 deficiency led to heightened activation of Ral and AKT, resistance to cell death, increased migration, and dysregulation of exocyst complex formation. Conversely, TIPE2 overexpression induced cell death and significantly inhibited Ras-induced tumorigenesis in mice. Importantly, TIPE2 expression was either completely lost or significantly down-regulated in human hepatic cancer. Thus, TIPE2 is an inhibitor of both inflammation and cancer, and potential drug target for inflammatory and neoplastic diseases.
Ras; apoptosis; oncogenesis; inflammation; cytoskeleton
Proper assembly of kinetochores (KTs) during mitosis is required for bipolar attachment of spindle microtubules (MTs) and the accumulation of spindle assembly checkpoint (SAC) components. Here we show that testis expressed protein 14 (Tex14), which has been implicated in midbody function, is recruited to KTs by Plk1 in a Cdk1-dependent manner during early mitosis. Exclusion of Tex14 from kinetochores results in an inability to efficiently localize outer KT components, impaired KT-MT attachment, chromosome congression defects and whole chromosome instability. In addition, we demonstrate that phosphorylation of Tex14 by Plk1 during metaphase promotes APCCdc20-mediated Tex14 degradation. Inhibition of this phosphorylation event causes retention of Tex14 at KTs and results in delayed metaphase to anaphase transition and chromosome segregation defects. Our findings identify Tex14 as an important mediator of KT structure and function and the fidelity of chromosome separation.
Tex14; kinetochore; Plk1; spindle assembly checkpoint
During embryonic cell cycles, B-cyclin-CDKs function as the core component of an autonomous oscillator. Current models for the cell-cycle oscillator in non-embryonic cells are slightly more complex, incorporating multiple G1, S-phase, and mitotic cyclin-CDK complexes. However, periodic events persist in yeast cells lacking all S-phase and mitotic B-cyclin genes, challenging the assertion that cyclin-CDK complexes are essential for oscillations. These and other results led to the proposal that a network of sequentially activated transcription factors functions as an underlying cell-cycle oscillator. Here we examine the individual contributions of a transcription-factor network and cyclin-CDKs to the maintenance of cell-cycle oscillations. Our findings suggest that while cyclin-CDKs are not required for oscillations, they do contribute to oscillation robustness. A model emerges in which cyclin expression (thereby, CDK activity) is entrained to an autonomous transcriptional oscillator. CDKs then modulate oscillator function and serve as effectors of the oscillator.
The ARF tumor suppressor is a central component of the cellular defense against oncogene activation in mammals. p14ARF activates p53 by binding and inhibiting HDM2, resulting, inter alia, in increased transcription and expression of the cyclin-dependent kinase inhibitor p21 and consequent cell-cycle arrest. We analyzed the effect of p14ARF induction on nucleolar protein dynamics using SILAC mass spectrometry and have identified the human Formin-2 (FMN2) protein as a component of the p14ARF tumor suppressor pathway. We show that FMN2 is increased upon p14ARF induction at both the mRNA and the protein level via a NF-κB-dependent mechanism that is independent of p53. FMN2 enhances expression of the cell-cycle inhibitor p21 by preventing its degradation. FMN2 is also induced by activation of other oncogenes, hypoxia, and DNA damage. These results identify FMN2 as a crucial component in the regulation of p21 and consequent oncogene/stress-induced cell-cycle arrest in human cells.
► Proteomic analysis reveals that ARF induces FMN2 ► ARF, DNA damage, and hypoxia induce FMN2 transcription in a p53-independent manner ► FMN2 promoter is negatively regulated by NF-κB and E2F1 ► FMN2 regulates p21 protein levels by forming a complex and preventing its degradation
The appropriate execution of DNA double-strand break (DSB) repair is critical for genome stability and tumor avoidance. 53BP1 and BRCA1 directly influence DSB repair pathway choice by regulating 5′ end resection, but how this is achieved remains uncertain. Here we report that Rif1−/− mice are severely compromised for 53BP1-dependent class switch recombination (CSR) and fusion of dysfunctional telomeres. The inappropriate accumulation of RIF1 at DSBs in S phase is antagonized by BRCA1, and deletion of Rif1 suppresses toxic nonhomologous end joining (NHEJ) induced by PARP inhibition in Brca1-deficient cells. Mechanistically, RIF1 is recruited to DSBs via the N-terminal phospho-SQ/TQ domain of 53BP1, and DSBs generated by ionizing radiation or during CSR are hyperresected in the absence of RIF1. Thus, RIF1 and 53BP1 cooperate to block DSB resection to promote NHEJ in G1, which is antagonized by BRCA1 in S phase to ensure a switch of DSB repair mode to homologous recombination.
► RIF1 is essential for 53BP1-dependent CSR and fusion of dysfunctional telomeres ► BRCA1 antagonizes RIF1 in S phase to prevent error-prone repair by toxic NHEJ ► N-terminal phospho-SQ/TQ domain of 53BP1 interacts with and recruits RIF1 to DSBs ► RIF1 and 53BP1 promote NHEJ in G1 by blocking 5′ end resection of DSBs
The proapoptotic Bcl-2 protein Bax is predominantly found in the cytosol of nonapoptotic cells and is commonly thought to translocate to mitochondria following an apoptotic stimulus. The current model for Bax activation is that BH3 proteins bind to cytosolic Bax, initiating mitochondrial targeting and outer-membrane permeabilization. Here, we challenge this and show that Bax is constitutively targeted to mitochondria but in nonapoptotic cells is constantly translocated back to the cytosol. Using live-cell spinning-disk confocal imaging with a combination of FLIP, FRAP, and photoactivatable GFP-Bax, we demonstrate that disrupting adhesion-dependent survival signals slows the rate of Bax’s dissociation from mitochondria, leading to its accumulation on the outer mitochondrial membrane. The overall accumulation of mitochondrial Bax following loss of survival signaling sensitizes cells to proapoptotic BH3 proteins. Our findings show that Bax is normally in a dynamic equilibrium between cytosol and mitochondria, enabling fluctuations in survival signals to finely adjust apoptotic sensitivity.
► Bax exists in equilibrium between the cytosol and mitochondria ► The retrotranslocation of Bax is independent of direct-activator BH3 proteins ► Changes in survival signaling modulate the dissociation rate of mitochondrial Bax ► Accumulation of Bax on mitochondria sensitizes cells to apoptosis
Mechanisms through which long intergenic noncoding RNAs (ncRNAs) exert regulatory effects on eukaryotic biological processes remain largely elusive. Most studies of these phenomena rely on methods that measure average behaviors in cell populations, lacking resolution to observe the effects of ncRNA transcription on gene expression in a single cell. Here, we combine quantitative single-molecule RNA FISH experiments with yeast genetics and computational modeling to gain mechanistic insights into the regulation of the Saccharomyces cerevisiae protein-coding gene FLO11 by two intergenic ncRNAs, ICR1 and PWR1. Direct detection of FLO11 mRNA and these ncRNAs in thousands of individual cells revealed alternative expression states and provides evidence that ICR1 and PWR1 contribute to FLO11’s variegated transcription, resulting in Flo11-dependent phenotypic heterogeneity in clonal cell populations by modulating recruitment of key transcription factors to the FLO11 promoter.
Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, providing a molecular basis for coupling mRNA processing to transcription.
Transcription is coupled with pre-mRNA splicing in metazoans. In the current issue of Molecular Cell, Huang et al. shows that Med23 interacts with the RNA binding protein hnRNP L to regulate alternative splicing, thus expanding mediator’s function beyond transcriptional control.
In a recent issue of Molecular Cell, Witcher and Emerson (2009) demonstrate that CTCF loss of function, via defective steps in poly (ADP-ribosyl)ation, may predispose key genes in cancer cells to addition of abnormal promoter DNA methylation and silencing.
Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2’-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a halfmer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modelling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and that methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs.
Class switch recombination (CSR) has the potential to generate genomic instability in B cells as activation-induced cytidine deaminase (AID), which mediates this process, is known to target many sites outside Igh. Nonetheless we do not fully understand what factors influence AID targeting genome-wide. Given that errors in CSR can lead to dangerous, oncogenic chromosomal translocations it is important to identify the elements that determine which genes are at risk of being ‘hit’ and could be involved in aberrant rearrangements. Here we have investigated the influence of nuclear organization in determining ‘off-target’ activity and the choice of fusion partners. Our studies indicate that the vast majority of known AID-mediated Igh translocation partners are found in chromosomal domains that contact this locus during class switching. Further, these interaction domains can be used to identify other genes that are hit by AID.
Igh; AID; translocation; genome stability; class switch recombination; nuclear organization; 4C-seq
Small RNAs target invaders for silencing in the CRISPR-Cas pathways that protect bacteria and archaea from viruses and plasmids. The CRISPR RNAs (crRNAs) contain sequence elements acquired from invaders that guide CRISPR-associated (Cas) proteins back to the complementary invading DNA or RNA. Here, we have analyzed essential features of the crRNAs associated with the Cas RAMP module (Cmr) effector complex, which cleaves targeted RNAs. We show that Cmr crRNAs contain an 8-nucleotide 5’ sequence tag (also found on crRNAs associated with other CRISPR-Cas pathways) that is critical for crRNA function and can be used to engineer crRNAs that direct cleavage of novel targets. We also present data that indicates that the Cmr complex cleaves an endogenous complementary RNA in Pyrococcus furiosus, providing direct in vivo evidence of RNA targeting by the CRISPR-Cas system. Our findings indicate that the CRISPR RNA-Cmr protein pathway may be exploited to cleave RNAs of interest.
Decapping represents a critical control point in regulating expression of protein coding genes. Here we demonstrate that decapping also modulates expression of long non-coding RNAs (lncRNAs). Specifically, levels of >100 lncRNAs in yeast are controlled by decapping and are degraded by a pathway that occurs independent of decapping regulators. We find many lncRNAs degraded by DCP2 are expressed proximal to inducible genes. Of these, we show several genes required for galactose utilization are associated with lncRNAs that have expression patterns inversely correlated with their mRNA counterpart. Moreover, decapping of these lncRNAs is critical for rapid and robust induction of GAL gene expression. Failure to destabilize a lncRNA known to exert repressive histone modifications results in perpetuation of a repressive chromatin state that contributes to reduced plasticity of gene activation. We propose that decapping and lncRNA degradation serve a vital role in transcriptional regulation specifically at inducible genes.
Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study, we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ, and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiency generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via Polycomb Repressive Complex 2 (PRC2) independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.
DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3′-phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and co-localize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway.
epigenetics; DNA methylation; active DNA demethylation; ROS1; gene silencing
The heterogeneous nature of mammalian PRC1 complexes has hindered our understanding of their biological functions. Here, we present a comprehensive proteomic and genomic analysis that uncovered six major groups of PRC1 complexes each containing a distinct PCGF subunit, a RING1A/B ubiquitin ligase, and a unique set of associated polypeptides. These PRC1 complexes differ in their genomic localization and only a small subset co-localize with H3K27me3. Further biochemical dissection revealed that the six PCGF-RING1A/B combinations form multiple complexes through association with RYBP or its homolog YAF2, which prevents the incorporation of other canonical PRC1 subunits such as CBX, PHC and SCM. Although both RYBP/YAF2- and CBX/PHC/SCM-containing complexes compact chromatin, only RYBP stimulates the activity of RING1B toward H2AK119ub1, suggesting a central role in PRC1 function. Knockdown of RYBP in ES cells compromised their ability to form embryoid bodies, likely because of defects in cell proliferation and maintenance of H2AK119ub1 level.
Ubiquitylation entails the concerted action of E1, E2 and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C-terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.
The cellular response to DNA damage employs multiple dynamic protein modifications to exert rapid and adaptable effects. Substantial work has detailed the roles of canonical checkpoint-mediated phosphorylation in this program. Recent studies have also implicated sumoylation in the DNA damage response; however, a systematic view of the contribution of sumoylation to replication and repair and its interplay with checkpoints is lacking. Here, using a biochemical screen in yeast, we establish that DNA damage-induced sumoylation occurs on a large scale. We identify MRX (Mre11-Rad50-Xrs2) as a positive regulator of this induction for a subset of repair targets. In addition, we find that defective sumoylation results in failure to complete replication of a damaged genome and impaired DNA end processing, highlighting the importance of the SUMO-mediated response in genome integrity. We also show that DNA damage-induced sumoylation does not require Mec1 checkpoint signaling, and the presence of both enables optimal DNA damage resistance.