Coordinated control of senescence by lncRNA UCA1 and a novel CAPERα/TBX3 co-repressor
ABSTRACT: Coordination of a complex series of transcriptional, structural and signaling events culminates in cellular senescence, a crucial tumor suppressor mechanism. We have discovered a repressor complex composed of TBX3 and CAPERa which functions upstream of the RB and p53 effector pathways and is required to prevent senescence of primary cells and in mouse embryos. TBX3/ CAPERa directly binds and represses transcription and chromatin structure of genes in multiple senescence pathways and the LncRNA UCA1, which we have identified as a novel tumor suppressor. The TBX3/ CAPERa complex is physically disrupted in oncogene induced senescence, providing a new molecular mechanism for derepression of prosenescence pathways in this system. Our results provide new insight into the oncogenic properties of TBX3, and are the first demonstration of CAPERa and UCA1 function in vivo. mRNA Seq based gene differential expression analysis of two sample types (TBX3, Caper) relative to control and two sample types (pCDNA3.1, UCA1) relative to each other.
Project description:The CCR4-NOT complex, bearing poly(A) deadenylation activity, is a highly conserved regulator that is involved in biological control; however its action mechanisms and physiological targets remain unclear. Using genetic deletion of the CNOT3 subunit of this complex in early B cell progenitors, we show that CNOT3 plays a critical role in pro- to pre-B cell transition. CNOT3 participated in controlling germline transcription, compaction of the immunoglobulin heavy chain (Igh) locus, and Igh rearrangement, and in destabilizing tumor suppressor p53 mRNA. Moreover, by genetic ablation of p53 or introduction of pre-rearranged Igh transgene, the B cell developmental defect in the Cnot3 knockout background could be partly rescued, suggesting that CCR4-NOT complex exerts critical control in B cell differentiation processes by co-utilizing transcriptional and post-transcriptional mechanisms. Pro-B cells mRNA profiles of Mb1(cre/+) and Cnot3(fl/fl)Mb1(cre/+) mice were generated by deep sequencing using Illumina HiSeq 1500
Project description:This is the model described in: Feedback between p21 and reactive oxygen production is necessary for cell senescence.
Passos JF, Nelson G, Wang C, Richter T, Simillion C, Proctor CJ, Miwa S, Olijslagers S, Hallinan J, Wipat A, Saretzki G, Rudolph KL, Kirkwood TB, von Zglinicki T. ;Mol Sys Biol2010;6:347. Epub 2010 Feb 16. PMID:20160708 doi:10.1038/msb.2010.5;
Cellular senescence--the permanent arrest of cycling in normally proliferating cells such as fibroblasts--contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of 'deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFbeta. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.
Project description:Here we show that Tet1 is down-regulated in mouse nucleus accumbens (NAc), a key brain reward structure, by repeated cocaine administration which enhances behavioral responses to cocaine. Through genome-wide 5hmC profiling, we identified 5hmC changes selectively clustered in both enhancer and coding regions of genes with several annotated neural functions. By coupling with mRNA sequencing, we found cocaine-induced alterations in 5hmC correlate positively with alternative splicing. We also demonstrated that 5hmC alteration at certain genes lasts up to a month after cocaine exposure. RNA Nac samples were collected at various time points after 7 daily cocaoine ip administration for 5hmC and transcriptome analysis
Project description:Estrogen signaling in breast cancer cells relies on long-range chromatin interactions connecting distal regulatory elements bound by the estrogen receptor 1 (ESR1) to target gene promoters. This ensures stimulus and subtype-specific transcriptional responses. Expanding on the function of CTCF and the cohesin complex in breast cancer, we demonstrate that the chromatin-looping factor ZNF143 binds the promoter of most early-response estrogen target genes connected to distal regulatory elements in ESR1-positive breast cancer cells. Its chromatin occupancy is unaffected by estrogen stimulation, supporting a stable three-dimensional genomic architecture within the early response to estrogen. Its loss abrogates the estrogen-induced transcriptional response and growth of breast cancer cells. When taking into account CTCF, ZNF143 and cohesin complex subunits, we show that chromatin-looping factors are genetically altered in over 20% of ESR1-positive primary breast tumors. Furthermore, the overexpression of ZNF143, CTCF and RAD21, a cohesin complex subunit, in ESR1-positive breast tumors associates with a worse clinical outcome. Overall, our results suggest that ZNF143 is a new critical effector of the estrogen response and highlights the contribution of the chromatin looping machinery to ESR1-positive breast cancer development. mRNA profiles of MCF-7 cells (siCtl or siZNF143) under vehicle (EtOH) or E2 (10 uM 17-beta oestradiol) stimulation
Project description:RNA-seq was performed on LFS MDAH041 cells that were young (PD10-12), aging (PD17-19) and replicatively senescent (PD28-30), as well as spontaneously immortal cells and cells that were induced into senescence or quiescence, in order to profile the pathways common in all 4 types of sensecence and the pathways affected as a cell approaches senescence. RNA was sequenced in biological triplicates of each sample using the Illumina HiSeq2000
Project description:Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. Close to one third of these proteins, were neither previously annotated nor could be functionally predicted to bind RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of unexpected mRNA-binders with novel molecular functions participating in combinatorial post-transcriptional gene-expression networks. To obtain a more detailed picture of the RNA present in the pooled precipitates of four consecutive oligo(dT)-purifications, we constructed a cDNA library by random priming of 4-thiouridine (4SU)- and 6-thioguanosine (6SG)-labeled RNA derived from UV-irradiated (365 nm)and non-irradiated cells. Digital gene expression analysis of the cDNA library of non-irradiated cells, labeled with 4SU and 6SG, was performed. To monitor the incorporation of photoreactive nucleotides into mRNA, we isolated 4SU- and 6SG-labeled RNA from the oligo(dT) precipitate of non-crosslinked cells by biotinylation and streptavidin purification (Dolken et al., 2008).
Project description:DallePazze2014 - Cellular senescene-induced
This model is described in the article:
Dynamic modelling of
pathways to cellular senescence reveals strategies for targeted
Dalle Pezze P, Nelson G, Otten EG,
Korolchuk VI, Kirkwood TB, von Zglinicki T, Shanley DP.
PLoS Comput. Biol. 2014 Aug; 10(8):
Cellular senescence, a state of irreversible cell cycle
arrest, is thought to help protect an organism from cancer, yet
also contributes to ageing. The changes which occur in
senescence are controlled by networks of multiple signalling
and feedback pathways at the cellular level, and the interplay
between these is difficult to predict and understand. To
unravel the intrinsic challenges of understanding such a highly
networked system, we have taken a systems biology approach to
cellular senescence. We report a detailed analysis of
senescence signalling via DNA damage, insulin-TOR, FoxO3a
transcription factors, oxidative stress response, mitochondrial
regulation and mitophagy. We show in silico and in vitro that
inhibition of reactive oxygen species can prevent loss of
mitochondrial membrane potential, whilst inhibition of mTOR
shows a partial rescue of mitochondrial mass changes during
establishment of senescence. Dual inhibition of ROS and mTOR in
vitro confirmed computational model predictions that it was
possible to further reduce senescence-induced mitochondrial
dysfunction and DNA double-strand breaks. However, these
interventions were unable to abrogate the senescence-induced
mitochondrial dysfunction completely, and we identified
decreased mitochondrial fission as the potential driving force
for increased mitochondrial mass via prevention of mitophagy.
Dynamic sensitivity analysis of the model showed the network
stabilised at a new late state of cellular senescence. This was
characterised by poor network sensitivity, high signalling
noise, low cellular energy, high inflammation and permanent
cell cycle arrest suggesting an unsatisfactory outcome for
treatments aiming to delay or reverse cellular senescence at
late time points. Combinatorial targeted interventions are
therefore possible for intervening in the cellular pathway to
senescence, but in the cases identified here, are only capable
of delaying senescence onset.
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Project description:The AF4/FMR2 proteins AFF1 and AFF4 act as a scaffold to assemble the Super Elongation Complex (SEC) that strongly activates transcriptional elongation of HIV-1 and cellular genes. Although they can dimerize, it is unclear whether the dimers exist and function within a SEC in vivo. Furthermore, it is unknown whether AFF1 and AFF4 function similarly in mediating SEC-dependent activation of diverse genes. Providing answers to these questions, our current study shows that AFF1 and AFF4 reside in separate SECs that display largely distinct gene target specificities. While the AFF1-SEC is more potent in supporting HIV-1 transactivation by the viral Tat protein, the AFF4-SEC is more important for HSP70 induction upon heat shock. The functional difference between AFF1 and AFF4 in Tat-transactivation has been traced to a single amino acid variation between the two proteins, which causes them to enhance the affinity of Tat for P-TEFb, a key SEC component, with different efficiency. Finally, genome-wide analysis confirms that the genes regulated by AFF1- and AFF4-SEC are largely non-overlapping and perform distinct functions. Thus, the SEC represents a family of related complexes that exist to increase the regulatory diversity and gene control options during transactivation of diverse cellular and viral genes. RNA-seq in HeLa cells of wild-type and after RNAi of AFF1 or AFF4.
Project description:Cellular senescence is a program of irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress. The underlying signalling pathways, potentially of major clinicopathological relevance, are unknown. A major stumbling block to studying senescence has been the absence of suitable model systems because of the asynchrony of this process in heterogeneous cell populations. To simplify this process many investigators study oncogene-induced senescence due to expression of activated oncogenes where senescence occurs prematurely without telomere attrition and can be induced acutely in a variety of cell types. We have taken a different approach by making use of the finding that reconstitution of telomerase activity by introduction of the catalytic subunit of human telomerase alone is incapable of immortalising all human somatic cells, but inactivation of the p16-pRB and p53-p21 pathways are required in addition. The ability of SV40 large T antigen to inactivate the p16-pRB and p53-p21 pathways has enabled us to use a thermolabile mutant of LT antigen, in conjunction with hTERT, to develop conditionally immortalised human (HMF3A) fibroblasts that are immortal but undergo an irreversible growth arrest when the thermolabile LT antigen is inactivated leading to activation of pRB and p53. When these cells cease dividing, senescence-associated- b-galactosidase activity is induced and the growth-arrested cells have morphological features and express genes in common with senescent cells. Since these cells growth arrest in a synchronous manner they are an excellent starting point for dissecting the pathways that underlie cellular senescence and act downstream of p16-pRB and p53-p21 pathways. We have combined genome-wide expression profiling with genetic complementation to undertake identification of genes that are differentially expressed when these conditionally immortalised human fibroblasts undergo senescence upon activation of the p16-pRB and p53-p21 tumour suppressor pathways. Genes differentially expressed upon senescence will be identified by comparing arrays from growing versus senescent cells. Changes in gene expression due to the temperature shift will be eliminated by comparing with array data from the non-conditional HMF3S cells grown at 34°C ±0.5°C and 38°C ±0.5°C. To determine if the changes in gene expression upon senescence are specific and reversible, the set of differential genes will then be overlaid with array data from cells in which senescence has been bypassed by inactivation of the p16-pRB and p53-p21 tumour suppressor pathways