An HP1 isoform-specific feedback mechanism regulates Suv39h1 activity under stress conditions.

The presence of H3K9me3 and heterochromatin protein 1 (HP1) are hallmarks of heterochromatin conserved in eukaryotes. The spreading and maintenance of H3K9me3 is effected by the functional interplay between the H3K9me3-specific histone methyltransferase Suv39h1 and HP1. This interplay is complex in mammals because the three HP1 isoforms, HP1α, β, and γ, are thought to play a redundant role in Suv39h1-dependent deposition of H3K9me3 in pericentric heterochromatin (PCH). Here, we demonstrate that despite this redundancy, HP1α and, to a lesser extent, HP1γ have a closer functional link to Suv39h1, compared to HP1β. HP1α and γ preferentially interact in vivo with Suv39h1, regulate its dynamics in heterochromatin, and increase Suv39h1 protein stability through an inhibition of MDM2-dependent Suv39h1-K87 polyubiquitination. The reverse is also observed, where Suv39h1 increases HP1α stability compared HP1β and γ. The interplay between Suv39h1 and HP1 isoforms appears to be relevant under genotoxic stress. Specifically, loss of HP1α and γ isoforms inhibits the upregulation of Suv39h1 and H3K9me3 that is observed under stress conditions. Reciprocally, Suv39h1 deficiency abrogates stress-dependent upregulation of HP1α and γ,  and enhances HP1β levels. Our work defines a specific role for HP1 isoforms in regulating Suv39h1 function under stress via a feedback mechanism that likely regulates heterochromatin formation

The tumor suppressor SirT2 regulates cell cycle progression and genome stability by modulating the mitotic deposition of H4K20 methylation

The establishment of the epigenetic mark H4K20me1 (monomethylation of H4K20) by PR-Set7 during G2/M directly impacts S-phase progression and genome stability. However, the mechanisms involved in the regulation of this event are not well understood. Here we show that SirT2 regulates H4K20me1 deposition through the deacetylation of H4K16Ac (acetylation of H4K16) and determines the levels of H4K20me2/3 throughout the cell cycle. SirT2 binds and deacetylates PR-Set7 at K90, modulating its chromatin localization. Consistently, SirT2 depletion significantly reduces PR-Set7 chromatin levels, alters the size and number of PR-Set7 foci, and decreases the overall mitotic deposition of H4K20me1. Upon stress, the interaction between SirT2 and PR-Set7 increases along with the H4K20me1 levels, suggesting a novel mitotic checkpoint mechanism. SirT2 loss in mice induces significant defects associated with defective H4K20me1-3 levels. Accordingly, SirT2-deficient animals exhibit genomic instability and chromosomal aberrations and are prone to tumorigenesis. Our studies suggest that the dynamic cross-talk between the environment and the genome during mitosis determines the fate of the subsequent cell cycle

Identificació de seqüències peptídiques específiques per a cèl·lules de càncer de pròstata metastàtic : direccionament de vectors adenovirals /

Descripció del recurs: el 18-08-2008Consultable des del TDXTítol obtingut de la portada digitalitzadaEl càncer de pròstata és el càncer majoritàriament diagnosticat en homes adults i la segona causa de mort, després del càncer de pulmó. Els tractaments convencionals com la prostatectomia radical, radioteràpia i quimioteràpia tan sols funcionen en la malaltia localitzada, essent el desenvolupament de metàstasis la principal causa de mort dels pacients. Per tant, és necessària la cerca de noves teràpies que millorin l'eficàcia dels tractaments del càncer de pròstata, sobretot en presència de focus metastàtics en altres òrgans. En aquest sentit, la teràpia gènica - que consisteix en la transferència de material genètic per a substituir, restablir o potenciar les funcions biològiques de cèl·lules i teixits- s'ha proposat com una alternativa al tractament del càncer de pròstata. La majoria dels protocols de teràpia gènica en càncer de pròstata utilitzen la injecció intratumoral, que limita la teràpia als tumors localitzats, sent necessària l'administració sistèmica del vector terapèutic per tal d'arribar a la malaltia disseminada. Per a poder administrar sistèmicament un vector cal evitar la transferència del transgen als teixits i cèl·lules no diana i, per tant, aquest vector haurà d'estar específicament dirigit a aquests. Els Adenovirus són els vectors més utilitzats tant en teràpia gènica del càncer com en teràpia gènica del càncer de pròstata ja que presenten una elevada transferència del transgen sense introduir el seu genoma al de la cèl·lula hoste, poden transportar fragments grans de DNA i es poden infectar una gran varietat de cèl·lules i teixits. Aquest extens tropisme és alhora que una avantatge una limitació ja que es pot produir la infecció de cèl·lules i teixits no diana degut a l'expressió ubiqua del seu receptor primari: el Coxsackie's and Adenovirus Receptor (CAR). Així doncs, el direccionament específic d'aquests vectors és de gran interès. En aquest treball, mitjançant el cribratge in vitro de la llibreria de Phage Display PhD-C7C sobre cèl·lules de càncer de pròstata metastàtic (PC3MM2) hem seleccionat 3 seqüències peptídiques (CTPQNTTMC, CYPSRSPLC i CPLHQRPMC) que han demostrat una major eficiència d'unió a les variants metastàtiques de càncer de pròstata que a les altres línies cel·lulars analitzades. Per tal de determinar si aquestes seqüències podrien ser funcionals pel direccionament específic de vectors terapèutics, les hem introduït a l'HI loop de la fibra de l'Adenovirus 5. Hem obtingut 3 Adenovirus recombinants: l'AdTLCTPQ (amb la seqüència CTPQNTTMC a l'HI loop), l'AdTLCYPS (amb la seqüència CYPSRSPLC a l'HI loop) i l'AdTLCPLH (amb la seqüència CPLHQRPMC a l'HI loop) i hem comprovat la seva infectivitat tant en sistemes in vitro com in vivo. Tot i que no hem obtingut diferències estadísticament significatives en la infecció de tumors i metàstasis en sistemes in vivo, els resultats obtinguts in vitro demostren que l'AdTLCTPQ i l'AdTLCPLH poden infectar les cèl·lules emprant un receptor diferent a CAR i que, per tant, utilitzant un vector terapèutic adequat, aquestes seqüències podrien ser funcionals pel direccionament de vectors terapèutics.Prostate cancer is most commonly diagnosed in adult men and is the second cause of death after lung cancer. Radical prostatectomy, radiotherapy and chemotherapy only are effective in localized disease, being metastasis the principal cause of patient's death. Therefore, it's necessary the development of novel therapies to improve prostate cancer treatment. Gene therapy -which consists in the transfer of genetic material to restore, promote or replace biological functions of cells and tissues- has been proposed as an alternative to prostate cancer treatment. The major part of prostate cancer gene therapy studies use intratumoral injection, limiting the therapy to localized disease and being necessary the systemic administration to reach disseminated disease. In order to administrate systemically a vector it is necessary to avoid transgen transfer to non-target cells, and so it will have to be specifically directed to target cells. Adenoviruses are the vectors more used in cancer gene therapy and in prostate cancer gene therapy because present an elevated transgen transfer, can accommodate large DNA fragments and can infect a large variety of cells and tissues. This extended tropism is an advantage but a limitation too because non-specific transgen transfer due to ubiquitous expression of the primary receptor: Coxsackie's and Adenovirus Receptor (CAR). So on, there's a big interest in specific targeting of these vectors. In this work, through the screening in vitro of the Phage Display library PhD-C7C on metastatic prostate cancer cells PC3MM2, we have selected 3 peptide sequences (CTPQNTTMC, CYPSRSPLC and CPLHQRPMC) that have demonstrate a higher binding efficiency to metastatic prostate cancer cells than to the rest of cell lines analyzed. To determine if these peptide sequences could be functional in specific targeting of therapeutic vectors we have introduced them into the HI loop of Ad5 fiber. We have generate 3 recombinant adenoviruses: AdTLCTPQ (CTPQNTTMC sequence into the HI loop), AdTLCYPS (CYPSRSPLC sequence into the HI loop) and AdTLCPLH (CPLHQRPMC sequence into the HI loop) and analyzed its infectivity in vitro and in vivo. Although we have not observed and increase in tumor and metastasis infectivity in vivo, in vitro results demonstrate that AdTLCTPQ and AdTLCPLH can transduce cells using a CAR independent pathway and, so on, using an adequate therapeutic vector these sequences could functions as target specific peptides

Shikimic acid protects skin cells from UV-induced senescence through activation of the NAD+-dependent deacetylase SIRT1

UV radiation is one of the main contributors to skin photoaging by promoting the accumulation of cellular senescence, which in turn induces a proinflammatory and tissue-degrading state that favors skin aging. The members of the sirtuin family of NAD + -dependent enzymes play an anti-senescence role and their activation suggests a promising approach for preventing UV-induced senescence in the treatment of skin aging. A two-step screening designed to identify compounds able to protect cells from UV-induced senescence through sirtuin activation identified shikimic acid (SA), a metabolic intermediate in many organisms, as a bona-fide candidate. The protective effects of SA against senescence were dependent on specific activation of SIRT1 as the effect was abrogated by the SIRT1 inhibitor EX-527. Upon UV irradiation SA induced S-phase accumulation and a decrease in p16 INK4A expression but did not protect against DNA damage or increased polyploidies. In contrast, SA reverted misfolded protein accumulation upon senescence, an effect that was abrogated by EX-527. Consistently, SA induced an increase in the levels of the chaperone BiP, resulting in a downregulation of unfolded protein response (UPR) signaling and UPR-dependent autophagy, avoiding their abnormal hyperactivation during senescence. SA did not directly activate SIRT1 in vitro, suggesting that SIRT1 is a downstream effector of SA signaling specifically in the response to cellular senescence. Our study not only uncovers a shikimic acid/SIRT1 signaling pathway that prevents cellular senescence, but also reinforces the role of sirtuins as key regulators of cell proteostasis

A complex interplay between H2A.Z and HP1 isoforms regulates pericentric heterochromatin

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2017-88975R, PID2020-117284RB-I00) (AV), and cofounded by FEDER funds/European Regional Development Fund (ERDF)-A Way to Build Europe; the Catalan Government Agency AGAUR (2017-SGR-148, 2021-SGR-01378 to AV) (FI-AGAUR fellowship 2022 FI_B 00924 to AG-S). We also thank the CERCA Programme/Generalitat de Catalunya for institutional support. Work from the PS lab is funded by Nazarbayev University Faculty Development Grant 021220FD2451. The work was also supported by the European Commission's Horizon 2020 research and innovation programme (BNV; Marie Słodowska-Curie grant # 895979).Pericentric heterochromatin (PCH) plays an essential role in the maintenance of genome integrity and alterations in PCH have been linked to cancer and aging. HP1 α, β, and γ, are hallmarks of constitutive heterochromatin that are thought to promote PCH structure through binding to heterochromatin-specific histone modifications and interaction with a wide range of factors. Among the less understood components of PCH is the histone H2A variant H2A.Z, whose role in the organization and maintenance of PCH is poorly defined. Here we show that there is a complex interplay between H2A.Z and HP1 isoforms in PCH. While the loss of HP1α results in the accumulation of H2A.Z.1 in PCH, which is associated with a significant decrease in its mobile fraction, H2A.Z.1 binds preferentially to HP1β in these regions. Of note, H2A.Z.1 downregulation results in increased heterochromatinization and instability of PCH, reflected by accumulation of the major epigenetic hallmarks of heterochromatin in these regions and increased frequency of chromosome aberrations related to centromeric/pericentromeric defects. Our studies support a role for H2A.Z in genome stability and unveil a key role of H2A.Z in the regulation of heterochromatin-specific epigenetic modifications through a complex interplay with the HP1 isoforms

SIRT6-dependent cysteine monoubiquitination in the PRE-SET domain of Suv39h1 regulates the NF-κB pathway

Sirtuins are NAD+-dependent deacetylases that facilitate cellular stress response. They include SirT6, which protects genome stability and regulates metabolic homeostasis through gene silencing, and whose loss induces an accelerated aging phenotype directly linked to hyperactivation of the NF-κB pathway. Here we show that SirT6 binds to the H3K9me3-specific histone methyltransferase Suv39h1 and induces monoubiquitination of conserved cysteines in the PRE-SET domain of Suv39h1. Following activation of NF-κB signaling Suv39h1 is released from the IκBα locus, subsequently repressing the NF-κB pathway. We propose that SirT6 attenuates the NF-κB pathway through IκBα upregulation via cysteine monoubiquitination and chromatin eviction of Suv39h1. We suggest a mechanism based on SirT6-mediated enhancement of a negative feedback loop that restricts the NF-κB pathway