E4F1 deficiency results in oxidative stress–mediated cell death of leukemic cells

Deletion of E4F1 inflicts mitochondrial damage and oxidative stress on murine and human myeloid leukemia cells but not healthy macrophages

The Transcription Factor E4F1 Coordinates CHK1-Dependent Checkpoint and Mitochondrial Functions

Recent data support the notion that a group of key transcriptional regulators involved in tumorigenesis, including MYC, p53, E2F1, and BMI1, share an intriguing capacity to simultaneously regulate metabolism and cell cycle. Here, we show that another factor, the multifunctional protein E4F1, directly controls genes involved in mitochondria functions and cell-cycle checkpoints, including Chek1, a major component of the DNA damage response. Coordination of these cellular functions by E4F1 appears essential for the survival of p53-deficient transformed cells. Acute inactivation of E4F1 in these cells results in CHK1-dependent checkpoint deficiency and multiple mitochondrial dysfunctions that lead to increased ROS production, energy stress, and inhibition of de novo pyrimidine synthesis. This deadly cocktail leads to the accumulation of uncompensated oxidative damage to proteins and extensive DNA damage, ending in cell death. This supports the rationale of therapeutic strategies simultaneously targeting mitochondria and CHK1 for selective killing of p53-deficient cancer cells

E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis.

The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis

Etude de la voie de modification post-traductionnelle SUMO (implication dans la régulation transcriptionnelle)

SUMO (Small Ubiquitin-related Modifier) est une protéine apparentée à l'Ubiquitine. A ce jour, une centaine de protéines ont été décrites comme étant sumoylées. Les fonctions biologiques liées à la sumoylation des protéines ne sont pas clairement comprises. Récemment, 3 familles distinctes de SUMO E3 ligase ont été découvertes ; les PIAS (Protein Inhibitor of Activated STAT), la nucléoporine RanBP2 et le co-répresseur Polycomb2 (Pc2). Afin de définir plus précisément le rôle de la voie SUMO dans la régulation transcriptionnelle, nous avons recherché de nouveaux substrats de SUMO et étudié l'activité des nouveaux facteurs SUMO E3 ligase. Par l'étude des protéines HDAC4 (Histone DeAcetylase 4) et MR (Mineralocorticoïd Receptor), nous avons pu montrer le rôle majeur de la voie SUMO dans les mécanismes de répressions transcriptionnelles. Ces deux protéines sont modifiées in vivo et in vitro par SUMO et leur modification est associée à la répression de la transcription. La modulation de leurs activités transactivatrices par la voie SUMO fait intervenir des mécanismes moléculaires différents, dépendants de leur état de sumoylation, de la nature du promoteur et des activités SUMO E3 ligase et transcriptionnelles des PIAS. Ces travaux ont également mis en évidence l'activité SUMO E3 ligase de RanBP2 sur HDAC4 et des PIAS sur MR. L'activité SUMO E3 ligase de RanBP2 sur HDAC4 indique également que la sumoylation et l'import nucléaire sont peut-être couplés. A l'échelle cellulaire, la sumoylation de PML est nécessaire à la formation de corps nucléaires normaux. En outre la sur-expression de PML dans des fibroblastes primaires induit leur sénescence prématurée, un processus capable de protéger les cellules contre la transformation tumorale induite par certains oncogènes. En cherchant à savoir si ces processus étaient liés, nous avons pu montrer que ni la sumoylation de PML, ni l'intégrité des corps nucléaires ne sont nécessaires à l'induction de la sénescence prématurée.Sumoyiation is a novel post-translational modification pathway that resembles Ubiquitylation. SUMO (Small Ubiquitin-like Modifier) is a small 101 amino-acid protein structurally related to Ubiquitin that is covalently linked to a lysine residue side chain of a target protein. To date, about one hundred proteins, including a large number of transcription factors, are known to be modified by SUMO, yet the functions of this modification remain obscure. Very recently, three different families of SUMO E3 ligases have been described; the Protein Inhibitor of Activated STAT (PIASs) family, the nucleoporin RanBP2 and the co-repressor Polycomb2 (Pc2). To better understand the role of sumoylation in transcriptional regulation, we studied the effect of sumoylation on the transcriptional activities of two substrates, the histone deacetylase HDAC4 and mineralocorticoîd Receptor (MR). We show that in both cases, sumoylation is associated with transcriptional repression, although SUMO-mediated repression by HDAC4 and MR are driven by different molecular mechanisms that likely depend on protein's sumoylation level, the promoter context and E3 ligase (PIAS) activities. Furthermore, we show that the RanBP2 provides E3 ligase activity for the modification of HDAC4 and PIAS proteins for MR. The RanBP2 SUMO E3 ligase activity led us to propose a model whereby sumoylation and nuclear import are coupled events. At the cellular level, PML's sumoylation was shown to be determinant for nuclear bodies assembly. It was also previously demonstrated that PML over-expression in human primary fibroblasts induces premature senescence. This process is suspected to be a protection against oncogenes-induced transformation. We were interested in studying the roles of PML's sumoylation and nuclear bodies integrity during on PML-induced premature senescence. Our results indicates that neither the sumoylation of PML nor the integrity of the nuclear bodies is necessary for this process.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Mechanisms of DNA Methyltransferase Recruitment in Mammals

DNA methylation is an essential epigenetic mark in mammals. The proper distribution of this mark depends on accurate deposition and maintenance mechanisms, and underpins its functional role. This, in turn, depends on the precise recruitment and activation of de novo and maintenance DNA methyltransferases (DNMTs). In this review, we discuss mechanisms of recruitment of DNMTs by transcription factors and chromatin modifiers—and by RNA—and place these mechanisms in the context of biologically meaningful epigenetic events. We present hypotheses and speculations for future research, and underline the fundamental and practical benefits of better understanding the mechanisms that govern the recruitment of DNMTs

Description of an optimized ChIP-seq analysis pipeline dedicated to genome wide identification of E4F1 binding sites in primary and transformed MEFs

International audienceThis Data in Brief report describes the experimental and bioinformatic procedures that we used to analyze and interpret E4F1 ChIP-seq experiments published in Rodier et al. (2015) [10]. Raw and processed data are available at the GEO DataSet repository under the subseries # GSE57228. E4F1 is a ubiquitously expressed zinc-finger protein of the GLI-Kruppel family that was first identified in the late eighties as a cellular transcription factor targeted by the adenoviral oncoprotein E1A13S (Ad type V) and required for the transcription of adenoviral genes (Raychaudhuri et al., 1987) [8]. It is a multifunctional factor that also acts as an atypical E3 ubiquitin ligase for p53 (Le Cam et al., 2006) [2]. Using KO mouse models we then demonstrated that E4F1 is essential for early embryonic development (Le Cam et al., 2004), for proliferation of mouse embryonic cell (Rodier et al., 2015), for the maintenance of epidermal stem cells (Lacroix et al., 2010) [6], and strikingly, for the survival of cancer cells (Hatchi et al., 2007) [4]; (Rodier et al., 2015) [10]. The latter survival phenotype was p53-independent and suggested that E4F1 was controlling a transcriptional program driving essential functions in cancer cells. To identify this program, we performed E4F1 ChIP-seq analyses in primary Mouse Embryonic Fibroblasts (MEF) and in p53(-/-), H-Ras(V12)-transformed MEFs. The program directly controlled by E4F1 was obtained by intersecting the lists of E4F1 genomic targets with the lists of genes differentially expressed in E4F1 KO and E4F1 WT cells (Rodier et al., 2015). We describe hereby how we improved our ChIP-seq analyses workflow by applying prefilters on raw data and by using a combination of two publicly available programs, Cisgenome and QESEQ

Deconstructing PML-induced premature senescence

In this study, we investigated the subcellular and molecular mechanisms underlying promyelocytic leu kemia (PML)-induced premature senescence. We demonstrate that intact PML nuclear bodies are not required for the induction of senescence. We have determined further that of seven known PML isoforms, only PML IV is capable of causing premature senescence, providing the first evidence for functional differences among these isoforms. Of interest is the fact that in contrast to PML(+/+) fibroblasts, PML(–/–) cells are resistant to PML IV-induced senescence. This suggests that although PML IV is necessary for this process to occur, it is not sufficient and requires other components for activity. Finally, we provide evidence that PML IV-induced senescence involves stabilization and activation of p53 through phosphorylation at Ser46 and acetylation at Lys382, and that it occurs independently of telomerase and differs from that elicited by oncogenic Ras. Taken together, our data assign a specific pro-senescent activity to an individual PML isoform that involves p53 activation and is independent from PML nuclear bodies

On the Electro-optical Properties of Metallo-supramolecular Polyelectrolytes (MEPEs) Containing Copper(I) and Their Electrochromic Applications

In this thesis, a new field in electrochromic materials combining inorganic metal ions with organic ligands, which is called metallo-supramolecular materials, is introduced and discussed. The metallo-supramolecular polyelectrolytes (MEPEs) containing copper(I) metal ion, MEPE-Cu(I), is first synthesized and applied in electrochromism. To investigate different roles in electrochromic device, polyaniline-carbon nanotube (PANI-CNT) and poly(3,3-diethyl-3,4-dihydro-2H-thieno-[3,4-b][1,4]dioxepine) (PProDOT-Et2) are chosen with MEPE-Cu(I) for device fabrications. We choose 4’-4’’’’-(1,4-phenylene) bis (2,2’:6’,2’’-terpyridine) as the ligand and copper(I) acetate as metal ion center to synthesize the MEPE-Cu(I). Due to metal-to-ligand charge transfer (MLCT) and redox reaction of Cu(I)/Cu(II), the color could be change by applying potential to influence the interaction between Cu(I) and terpyridine ligands and becomes as a new cathodic coloration material. All the MEPE-Cu(I) thin films are prepared by drop-coating with a concentration of 1 mg/mL. By varying the volume per drop, four different thicknesses of MEPE-Cu(I) films (MEPE-Cu(I) 100 μL to 400 μL) are prepared and compared. And the limitation of thickness for MEPE-Cu(I) have been found. The cyclic-voltammetry (CV) pretreatment between 0.0 V and 1.5 V (vs. Ag/Ag+) for 100 cycles is applied for preparation of MEPE-Cu(I) thin film, which enhance the optical performance and reduce resistance of thin film in electrochemical reaction to reach stability of the film. It is also proved by SEM image for surface morphology and EQCM for mass change. MEPE-Cu(I) has transmittance change of above 20%, less 1s of response time and the coloration efficiency of around 260 cm2/C. With ultra-fast response time and higher coloration efficiency, MEPE-Cu(I) could be chosen to apply for electrochromic applications. Two electrochromic devices based on MEPE-Cu(I) with TBAP/ACN as electrolyte have been fabricated. In MEPE-Cu(I)/PANI-CNT ECD system, PANI-CNT as an anodic coloration material is selected as an ionic storage layer with higher charge capacity, which provide better ability of electron transfer for MEPE-Cu(I) and enhance the electrochromic properties. The transmittance change at 580 nm is similar to MEPE-Cu(I) thin film. However, the response time could be shortened to less than 0.5 s. In the long-term stability experiment, the device could be operated for 1500 cycles and maintains above 99% of original performance. Besides, in MEPE-Cu(I)/PProDOT-Et2 ECD, PProDOT-Et2 is selected as the main electrochromic layer to fabricated with MEPE-Cu(I) because of relatively smaller transmittance change. MEPE-Cu(I) could also act as an ionic storage layer and assist the electron transfer of PProDOT-Et2, which provides higher transmittance above 35%, fast response time of less than 1 s. In spite of larger operating potential window of 3.5 V, the device could be operated for 1000 cycles with only 6.3% of decay. Both these two devices prove that MEPE-Cu(I) could be a stable electrochromic material and worth applying for other applications