Formation Of Senescence-associated Heterochromatin Foci Research Articles

Dynamic 3D genome reorganization during senescence: defining cell states through chromatin.

Cellular senescence, a cell state characterized by growth arrest and insensitivity to growth stimulatory hormones, is accompanied by a massive change in chromatin organization. Senescence can be induced by a range of physiological signals and pathological stresses and was originally thought to be an irreversible state, implicated in normal development, wound healing, tumor suppression and aging. Recently cellular senescence was shown to be reversible in some cases, with exit being triggered by the modulation of the cell's transcriptional program by the four Yamanaka factors, the suppression of p53 or H3K9me3, PDK1, and/or depletion of AP-1. Coincident with senescence reversal are changes in chromatin organization, most notably the loss of senescence-associated heterochromatin foci (SAHF) found in oncogene-induced senescence. In addition to fixed-cell imaging, chromatin conformation capture and multi-omics have been used to examine chromatin reorganization at different spatial resolutions during senescence. They identify determinants of SAHF formation and other key features that differentiate distinct types of senescence. Not surprisingly, multiple factors, including the time of induction, the type of stress experienced, and the type of cell involved, influence the global reorganization of chromatin in senescence. Here we discuss how changes in the three-dimensional organization of the genome contribute to the regulation of transcription at different stages of senescence. In particular, the distinct contributions of heterochromatin- and lamina-mediated interactions, changes in gene expression, and other cellular control mechanisms are discussed. We propose that high-resolution temporal and spatial analyses of the chromatin landscape during senescence will identify early markers of the different senescence states to help guide clinical diagnosis.

Protection against Microglia Senescence by the Dietary Supplement Dekosilhue® in BV2 Cells: A New Perspective for Obesity and Related Complications

Growing evidence indicates chronic low-grade systemic inflammation as a major pathophysiological mechanism of obesity. Systemic inflammation provokes an immune response in the brain through the activation of microglia that results in the development of neuroinflammation, cellular senescence, and occurrence of neurological dysfunction. In the efforts to identify an innovative intervention with potential efficacy on obesity and associated complications, our aim was to study the capability of the dietary supplement Dekosilhue® (DKS), successfully used for improving the control of body weight, to attenuate microglia senescent phenotype. Microglia senescence was induced by intermittent stimulation of BV2 cells with LPS 500 ng/mL every 72 h for 4 h/day, over a period of 10 days. DKS (100 µg/mL) treatment reduced ß-galactosidase activity and expression, the formation of senescence-associated heterochromatin foci to control levels, and increased cell viability of senescent BV2 (2 folds of control). DSK reduced the expression of Nuclear Factor-kB (NF-kB) (20% lower than control), a key molecule involved in the acquisition of the senescence-associated secretory phenotype (SASP). DKS promoted a neuroprotective effect by increasing the cell viability of SH-SY5Y neuronal cells exposed to the senescent BV2 conditioned medium to values of non-senescent cells. In conclusion, DKS attenuated the senescent microglia phenotype, showing senotherapeutic activity that might be further investigated as adjunctive intervention for obesity and obesity-related neurological disturbances.

Role of Histone Variant H2A.J in Fine-Tuning Chromatin Organization for the Establishment of Ionizing Radiation-Induced Senescence.

Radiation-induced senescence is characterized by profound changes in chromatin organization with the formation of Senescence-Associated-Heterochromatin-Foci (SAHF) and DNA-Segments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-Associated-Secretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in establishing radiation-induced senescence. Primary and genetically-modified lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy. Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry. Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory regions decisively modulates transcription factor recruitment and inflammatory gene expression, resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent H2A.J expression in response to radiation-induced DNA damage, therefore contributing to pro-inflammatory tissue reactions. The fine-tuned incorporation of H2A.J defines the epigenetic landscape for driving the senescence programme in response to radiation-induced DNA damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into DNA-damage triggered epigenetic mechanisms governing the biological processes of radiation-induced injury.

Gold and silver plasmonic nanoprobes trace the positions of histone codes

We visualized the distribution of heterochromatin in a single nucleus using plasmonic nanoparticle-conjugated H3K9me3 and H3K27me3 antibodies. Due to distance-dependent plasmonic coupling effects between nanoprobes, their scattering spectra shift to longer wavelengths as the distance between heterochromatin histone markers reduced during oncogene-induced senescence (OIS). These observations were supported by simulating scattering profiles based on considerations of particle numbers, interparticle distances, and the spatial arrangements of plasmonic nanoprobes. Using this plasmon-based colourimetric imaging, we estimated changes in distances between H3K9me3 and H3K27me3 during the formation of senescence-associated heterochromatin foci in OIS cells. We anticipate that the devised analytical technique combined with high-spatial imaging and spectral simulation will eventually lead to a new means of diagnosing and monitoring disease progression and cellular senescence.

Radiation-Induced Osteocyte Senescence Alters Bone Marrow Mesenchymal Stem Cell Differentiation Potential via Paracrine Signaling.

Cellular senescence and its senescence-associated secretory phenotype (SASP) are widely regarded as promising therapeutic targets for aging-related diseases, such as osteoporosis. However, the expression pattern of cellular senescence and multiple SASP secretion remains unclear, thus leaving a large gap in the knowledge for a desirable intervention targeting cellular senescence. Therefore, there is a critical need to understand the molecular mechanism of SASP secretion in the bone microenvironment that can ameliorate aging-related degenerative pathologies including osteoporosis. In this study, osteocyte-like cells (MLO-Y4) were induced to cellular senescence by 2 Gy γ-rays; then, senescence phenotype changes and adverse effects of SASP on bone marrow mesenchymal stem cell (BMSC) differentiation potential were investigated. The results revealed that 2 Gy irradiation could hinder cell viability, shorten cell dendrites, and induce cellular senescence, as evidenced by the higher expression of senescence markers p16 and p21 and the elevated formation of senescence-associated heterochromatin foci (SAHF), which was accompanied by the enhanced secretion of SASP markers such as IL-1α, IL-6, MMP-3, IGFBP-6, resistin, and adiponectin. When 0.8 μM JAK1 inhibitors were added to block SASP secretion, the higher expression of SASP was blunted, but the inhibition in osteogenic and adipogenic differentiation potential of BMSCs co-cultured with irradiated MLO-Y4 cell conditioned medium (CM- 2 Gy) was alleviated. These results suggest that senescent osteocytes can perturb BMSCs’ differential potential via the paracrine signaling of SASP, which was also demonstrated by in vivo experiments. In conclusion, we identified the SASP factor partially responsible for the degenerative differentiation of BMSCs, which allowed us to hypothesize that senescent osteocytes and their SASPs may contribute to radiation-induced bone loss.

Fucoidan induces ROS-dependent epigenetic modulation in cervical cancer HeLa cell

Fucoidan is a sulfated polysaccharide obtained from marine algae and known for various pharmacological activities. In this study, we investigated the effect of Fucoidan on cell viability, redox balance, cytoskeletal component F-actin, HDAC inhibition, autophagy, and senescence phenomenon in human cervical cancer HeLa cell line in comparison to positive control suberoylanilide hydroxamic acid by flow cytometry, fluorescence microscopy, and western blotting. Our observations revealed that Fucoidan exposure induces cytotoxicity in HeLa cells via ROS and mitochondrial superoxide generation and loss of ATP. Colorimetrical studies suggested that Fucoidan impairs the function of HDAC expression. Fucoidan treatment also contributes to the change in the granularity of cells, senescence-associated heterochromatin foci formation that leads to senescence in HeLa cells. Moreover, we visualize that Fucoidan exhibits autophagosomes formation with monodansylcadaverine, and flow cytometry analysis by acridine orange further substantiates that Fucoidan triggers autophagy in HeLa cells. Additionally, the changes in the expression of proteins p21, p16, BECN1, and HDAC1 were seen as markers of senescence, autophagy, and HDAC inhibition by FACS and immunoblotting. Molecular docking study validates Fucoidan–HDAC1 association in corroboration with the experimental data. Collectively, these mechanistic studies demonstrated that Fucoidan could be a therapeutic molecule for targeting HDACs in cervical cancer.

Bupleurum chinense Polysaccharide Improves LPS-Induced Senescence of RAW264.7 Cells by Regulating the NF-κB Signaling Pathway.

Macrophages are important inflammatory cells that play a vital role in inflamm-aging. Bupleurum chinense polysaccharide (BCP), an effective component of the Bupleurum chinense herb, exerts multiple beneficial pharmacological effects, such as improving immunity and antioxidant activity. However, the effects of BCP on macrophage-aging and inflamm-aging are yet to be established. In this study, we examined the effects of BCP on proliferation, inflammatory cytokines, β-galactosidase (SA-β-gal), senescence-associated heterochromatin foci (SAHF), reactive oxygen species (ROS), mitochondrial membrane potential, p53, p16, and p65/NF-κB signaling proteins in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. BCP significantly inhibited production of interleukin-1α (IL-1α), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), reduced the expression of SA-β-gal and formation of SAHF, as well as ROS level, and stabilized the mitochondrial membrane potential in RAW264.7 cells stimulated with LPS. Furthermore, BCP inhibited the expression of aging-related genes, p53 and p16, suppressed phosphorylation of p65 protein, and enhanced the expression of I-κBα protein through the NF-κB signaling pathway in LPS-stimulated RAW264.7 cells. Accordingly, we conclude that BCP effectively suppresses inflamm-aging by reducing inflammatory cytokine levels and oxidative stress production following activation of the NF-κB signaling pathway in RAW264.7 cells stimulated with LPS. Our collective findings support the utility of BCP as a novel pharmaceutical agent with potential anti-inflamm-aging effects.

Histone Variant H2A.J Marks Persistent DNA Damage and Triggers the Secretory Phenotype in Radiation-Induced Senescence.

Irreparable double-strand breaks (DSBs) in response to ionizing radiation (IR) trigger prolonged DNA damage response (DDR) and induce premature senescence. Profound chromatin reorganization with formation of senescence-associated heterochromatin foci (SAHF) is an essential epigenetic mechanism for controlling the senescence-associated secretory phenotype (SASP). To decipher molecular mechanisms provoking continuous DDR leading to premature senescence, radiation-induced DSBs (53BP1-foci) and dynamics of histone variant H2A.J incorporation were analyzed together with chromatin re-modeling in human fibroblasts after IR exposure. High-resolution imaging by transmission electron microscopy revealed that persisting 53BP1-foci developed into DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), consistently located at the periphery of SAHFs. Quantitative immunogold-analysis by electron microscopy revealed that H2A.J, steadily co-localizing with 53BP1, is increasingly incorporated into DNA-SCARS during senescence progression. Strikingly, shRNA-mediated H2A.J depletion in fibroblasts modified senescence-associated chromatin re-structuring and abolished SASP, thereby shutting down the production of inflammatory mediators. These findings provide mechanistic insights into biological phenomena of SASP and suggest that H2A.J inhibition could ablate SASP, without affecting the senescence-associated growth arrest.

4D Genome Rewiring during Oncogene-Induced and Replicative Senescence

SummaryTo understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.

Pak2 kinase promotes cellular senescence and organismal aging

Cellular senescence defines an irreversible cell growth arrest state linked to loss of tissue function and aging in mammals. This transition from proliferation to senescence is typically characterized by increased expression of the cell-cycle inhibitor p16INK4a and formation of senescence-associated heterochromatin foci (SAHF). SAHF formation depends on HIRA-mediated nucleosome assembly of histone H3.3, which is regulated by the serine/threonine protein kinase Pak2. However, it is unknown if Pak2 contributes to cellular senescence. Here, we show that depletion of Pak2 delayed oncogene-induced senescence in IMR90 human fibroblasts and oxidative stress-induced senescence of mouse embryonic fibroblasts (MEFs), whereas overexpression of Pak2 accelerated senescence of IMR90 cells. Importantly, depletion of Pak2 in BubR1 progeroid mice attenuated the onset of aging-associated phenotypes and extended life span. Pak2 is required for expression of genes involved in cellular senescence and regulated the deposition of newly synthesized H3.3 onto chromatin in senescent cells. Together, our results demonstrate that Pak2 is an important regulator of cellular senescence and organismal aging, in part through the regulation of gene expression and H3.3 nucleosome assembly.

Nuclear pore density controls heterochromatin reorganization during senescence.

During oncogene-induced senescence (OIS), heterochromatin is lost from the nuclear periphery and forms internal senescence-associated heterochromatin foci (SAHFs). We show that an increased nuclear pore density during OIS is responsible for SAHF formation. In particular, the nucleoporin TPR is necessary for both formation and maintenance of SAHFs. Loss of SAHFs does not affect cell cycle arrest but abrogates the senescence-associated secretory phenotype-a program of inflammatory cytokine gene activation. Our results uncover a previously unknown role of nuclear pores in heterochromatin reorganization in mammalian nuclei and demonstrate the importance of heterochromatin organization for a specific gene activation program.

The influence and the mechanism of enhancer of zeste homolog 2 and mammalian target of rapamycin in senescence of human gastric cancer cell line AGS

Objective To explore the influence and the mechanism of enhancer of zeste homolog 2 (EZH2) and mammalian target of rapamycin (mTOR) in senescence of human gaistric cancer cell line AGS. Methods EZH2 was downregulated by infected with lentivirus silencing EZH2. Senescence-associated β-galactosidase (SA-β-gal) and formation of senescence-associated heterochromatinfoci (SAHF) were used to identify cell senescence. flow cytometry and proliferation cell nuclear antigen (Ki-67) analysis were employed to investigate the effects of EZH2 depletion on the cell cycle and proliferation. Changing in p53-p21 axis activation was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. Results The percentage of SA-β-gal and SAHF positive cell increased after silencing EZH2, the percentage of SA-β-gal increased from (4.83±0.71)% to (35.22±2.67)% and the percentage of SAHF positive cell increased from (5.20±1.48)% to (25.68±2.53)%, flow cytometry showed that silencing EZH2 increased the percentage of G0/G1-phase cells, the data of control group was (41.20±3.70)%, and which increased to (57.60±2.97)% after silencing EZH2. and the expressions of p53 and p21 were significantly increased, the relative expression level of mRNA increased 2.18±0.29 times and 3.06±0.28 times comparing to control group. However, the influence were decreased by rapamycin, which is mTOR inhibitor, the percentage of SA-β-gal and SAHF positive cell decreased from (40.06±3.89)% to (26.44±3.82)% and from (32.38±3.89)% to (20.48±3.44)% respectively. and the percentage of G0/G1-phase, S-phase and G2/M-phase cells changed significantly. The G0/G1-phase cell percentage was (27.15±3.16)% and (56.03±5.29)%, the S-phase cell percentage was (50.63±2.97)% and (32.25±3.35)%, and G2/M-phase cell percentage was (44.28±3.16)% and (28.37±3.17)%. Conclusion Silencing EZH2 could promote senescence of human gaistric cancer cell line AGS by regulating p53 and p21 expression, and the process need the senescence-transformation of mTOR related signaling pathways. Key words: Enhancer of zeste homolog 2; Mammalian target of rapamycin; Senescence; Gaistric cancer

Preclinical characterization of abemaciclib in hormone receptor positive breast cancer.

Abemaciclib is an ATP-competitive, reversible kinase inhibitor selective for CDK4 and CDK6 that has shown antitumor activity as a single agent in hormone receptor positive (HR+) metastatic breast cancer in clinical trials. Here, we examined the mechanistic effects of abemaciclib treatment using in vitro and in vivo breast cancer models. Treatment of estrogen receptor positive (ER+) breast cancer cells with abemaciclib alone led to a decrease in phosphorylation of Rb, arrest at G1, and a decrease in cell proliferation. Moreover, abemaciclib exposure led to durable inhibition of pRb, TopoIIα expression and DNA synthesis, which were maintained after drug removal. Treatment of ER+ breast cancer cells also led to a senescence response as indicated by accumulation of β-galactosidase, formation of senescence-associated heterochromatin foci, and a decrease in FOXM1 positive cells. Continuous exposure to abemaciclib altered breast cancer cell metabolism and induced apoptosis. In a xenograft model of ER+ breast cancer, abemaciclib monotherapy caused regression of tumor growth. Overall these data indicate that abemaciclib is a CDK4 and CDK6 inhibitor that, as a single agent, blocks breast cancer cell progression, and upon longer treatment can lead to sustained antitumor effects through the induction of senescence, apoptosis, and alteration of cellular metabolism.

Techniques to Induce and Quantify Cellular Senescence.

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated β-galactosidase activity (SA-β-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including γ-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-β-gal, γ-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Loss of BRG1 induces CRC cell senescence by regulating p53/p21 pathway

Brahma-related gene-1 (BRG1) is the specific ATPase of switch/sucrose nonfermentable chromatin-remodeling complex that is aberrantly expressed or mutated in various cancers. However, the exact role of BRG1 in oncogenesis remains unknown. In this study, we demonstrate that the knockdown (KD) of BRG1 promotes cellular senescence by influencing the SIRT1/p53/p21 signal axis in colorectal cancer (CRC). In particular, we reveal that the expression level of BRG1 is inversely correlated with p21, one of the classic senescence regulators, and is decreased in senescent CRC cells. KD of BRG1 promoting senescence is indicated by the increase of senescence-associated β-galactosidase (SA-β-gal) activity, inhibition of cell proliferation, induction of cell cycle arrest, and formation of senescence-associated heterochromatin foci. BRG1 binds to SIRT1 and interferes with SIRT1-mediated deacetylation of p53 at K382. Rescue experiments by co-silencing p53 or treatment with EX527, a SIRT1-specific inhibitor, abrogated the cellular senescence induced by KD of BRG1. BRG1 KD cells resulted in smaller tumor formation than that in control cells in vivo. Collectively, our study shows that BRG1 has an important role in cellular senescence and tumor growth. The BRG1/SIRT1/p53 signal axis is a novel mechanism of cell senescence in CRC and is a new potential target for cancer therapy.

ZEB1-induced tumourigenesis requires senescence inhibition via activation of DKK1/mutant p53/Mdm2/CtBP and repression of macroH2A1

ObjectiveUnderstand the role of ZEB1 in the tumour initiation and progression beyond inducing an epithelial-to-mesenchymal transition.DesignExpression of the transcription factor ZEB1 associates with a worse prognosis in most cancers, including...

Pro-Proliferative Function of Mitochondrial Sirtuin Deacetylase SIRT3 in Human Melanoma

Melanoma, the most aggressive form of skin cancer, is often fatal if not treated early. Therefore, novel target-based strategies are required to combat this neoplasm. The objective of this study was to determine the role and functional significance of the mitochondrial sirtuin 3 (SIRT3) in melanoma. We found that compared with normal primary and immortalized human melanocytes, SIRT3 is significantly overexpressed in multiple human melanoma cells at mRNA and protein levels. Further, employing human tissue microarray, we found that SIRT3 is significantly upregulated in clinical melanoma tissues, compared with melanocytic nevi tissues. Furthermore, a short hairpin RNA-mediated knockdown of SIRT3 in human melanoma cells resulted in (i) a decrease in cellular proliferation, colony formation, and cellular migration; (ii) induction of senescence as shown by an increase in senescence-associated beta-galactosidase activity and formation of senescence-associated heterochromatin foci as well as an increase in mRNA and protein levels of p16(INK4a) and p21(Waf1); (iii) G1-phase arrest of the cell cycle; and (iv) decreases in mRNA and protein levels of cyclins (D1, E1) and cyclin-dependent kinases (2, 4, and 6). Conversely, forced exogenous overexpression of SIRT3 promoted an increase in proliferative potential of Hs294T melanoma cells and normal immortalized Mel-ST melanocytes. Finally, we found that SIRT3 knockdown significantly inhibited tumorigenesis in a xenograft model invivo. To our knowledge, this is the first study supporting the pro-proliferative function of SIRT3 in melanoma.

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein.

Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which can stabilize the state of senescence. The retinoblastoma (RB) protein has an important role in SAHF; cells that lack active RB pathway fail to form SAHF. It has been known that the posttranslational modifications of RB, for example, phosphorylation, regulate its function. To date, whether methylation of RB impacts on the SAHF formation is unknown. Here we report that JMJD3, a histone demethylase catalyzing the tri-methylation of H3K27 (H3K27me3), can demethylate the non-histone protein RB at the lysine810 residue (K810), which is a target of the methyltransferase Set7/9. We detected a significant upregulation of JMJD3 during cellular senescence and SAHF formation in WI38 cells induced by H-RasV(12), and we found that ectopic expression of JMJD3 promoted cellular senescence and SAHF formation in WI38 cells. Furthermore, during the process of SAHF assembly, JMJD3 was transported to the cytoplasm and interacted with RB through its demethylase domain JmjC. Significantly, our data demonstrated that the JMJD3-mediated demethylation of RB at K810 impeded the interaction of RB with the protein kinase CDK4 and resulted in reduced level of phosphorylation of RB at Serine807/811 (S807/811), implicating an important role of the interplay between the demethylation and phosphorylation of RB in SAHF assembly. This study highlights the role of JMJD3 as a novel inducer of SAHF formation through demethylating RB and provides new insights into the mechanisms of cellular senescence and SAHF assembly.

Abstract 2247: Histone H3 cleavage via Cathepsin L drives a cellular senescence program

Abstract Oncogene-induced senescence (OIS) is characterized by irreversible loss of proliferative capacity. It has been proposed that OIS functions as a tumor suppressor mechanism, and several studies have demonstrated induction of senescence markers in pre-malignant lesions. In addition to profound morphological changes, significant alterations in chromatin structure occur during senescence including formation of senescence-associated heterochromatin foci (SAHF). We have used well-characterized senescence models in human primary lung fibroblasts and melanocytes to study how chromatin alterations contribute to the senescence phenotype. We discovered that the N'-terminal tail of histone H3 is proteolytically cleaved by the lysosomal protease Cathepsin L1 (CTSL1). We show that CTSL1 is induced during senescence both at the mRNA and protein levels, and that it is present in the chromatin fraction of senescent cells. Chemical and shRNA-mediated inhibition of CTSL1 activity effectively block H3 tail cleavage and leads to a SAHF formation defect. We identified two distinct cleaved forms of H3 in senescent cells, which are associated with chromatin and incorporated into nucleosomes. Using antibodies directed against specific post-translational modifications (PTMs) of the histone H3 tail, we have mapped the approximate locations of the cleavage sites. Furthermore, overexpression of flag-tagged cleaved versions of H3, but not the full-length histone, trigger robust senescence in IMR90 cells accompanied by a modest induction of SAHFs. Interestingly, we also detect cleaved H3 in chromatin extracted from benign nevi tissue samples, providing evidence of H3 tail cleavage in senescence models in vivo. Collectively, our results suggest that H3 tail cleavage may contribute significantly to the senescence phenotype, potentially through transcriptional regulation of key senescence target genes. This area of investigation is currently ongoing. Citation Format: Luis F. Duarte, Andrew R. Young, Hsan-Au Wu, Taniya Panda, Zichen Wang, Alexandre G. Maia, Dan Hasson, Avnish Kapoor, Masashi Narita, Emily Bernstein. Histone H3 cleavage via Cathepsin L drives a cellular senescence program. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2247. doi:10.1158/1538-7445.AM2014-2247

Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells.

Senescence is a stable proliferation arrest characterized by profound changes in cellular morphology and metabolism as well as by extensive chromatin reorganization in the nucleus. One particular hallmark of chromatin changes during senescence is the formation of punctate DNA foci in DAPI-stained senescent cells that have been called senescence-associated heterochromatin foci (SAHF). While many advances have been made concerning our understanding of the effectors of senescence, how chromatin is reorganized and maintained in senescent cells has remained largely elusive. Because chromatin structure is inherently dynamic, senescent cells face the challenge of developing chromatin maintenance mechanisms in the absence of DNA replication in order to maintain the senescent phenotype. Here, we summarize and review recent findings shedding light on SAHF composition and formation via spatial repositioning of chromatin, with a specific focus on the role of lamin B1 for this process. In addition, we discuss the physiological implication of SAHF formation, the role of histone variants, and histone chaperones during senescence and also elaborate on the more general changes observed in the epigenome of the senescent cells.