S5B and S6),60 while the reverse is not true (Fig. depletion suggests that the targeting of H3.3 to PML-NBs is implicated in pericentromeric heterochromatin business. Together, our results underline the importance of the replication-independent chromatin assembly pathway for histone replacement in non-dividing senescent cells and establish PML-NBs as important regulatory sites for the incorporation of new H3.3 into chromatin. Keywords: chromatin dynamics, H3.3, DAXX, senescence, PML-NBs Introduction Most mammalian cells only divide a limited number of times before they undergo terminal differentiation or enter the state of senescence. Cellular senescence may be brought on by numerous forms of stress stimuli. First described as the result of replicative exhaustion of cultured normal diploid cells, 1 senescence can also be induced by oxidative stress, activated oncogenes such as H-RasV12, or inadequate growth conditions.2-5 Oncogene-induced senescence (OIS) results from a DNA damage response (DDR) activated by aberrant DNA replication6-8 and may pose as an important anti-tumor barrier. Identification of senescent cells in benign or premalignant, but not malignant tissues or using various human and mouse model systems seems to support this hypothesis.9-13 Like terminal differentiation, senescence is characterized by irreversible cell cycle arrest and rigorous reorganization of cellular morphology, including the structure of the chromatin. Chromatin is comprised of nucleosomes that each consists of 147 base pairs of DNA wrapped around a core histone octamer. The histone octamer is composed of a central (H3-H4)2 tetramer flanked by 2 H2ACH2B histone dimers.14 Three principle mechanisms bring about chromatin alterations in eukaryotic cells: (1) post-translational modification of histone tails, (2) the action of chromatin remodeling enzymes, and (3) the replacement of canonical histone proteins by histone variants.14 Incorporation of histone variants into chromatin is orchestrated by a family of proteins called histone chaperones15 and may provide different biophysical properties to the chromatin fiber or (S,R,S)-AHPC hydrochloride different post-translational modification sites, thus influencing nucleosome stability and function.14,16 Histone H3.3 is a variant of histone H3 that differs by only 5 amino acids from the canonical replicative histone variant H3.1 and has emerged as a regulator of chromatin states.17 H3.3 is constitutively expressed throughout the cell cycle and in quiescence18 and is incorporated into chromatin in a DNA synthesis-independent manner.19,20 It is enriched within actively transcribed genes, 21-25 but also accumulates at pericentromeric and telomeric heterochromatin regions.26-28 While the histone chaperone HIRA, along with associated factors, ASF1a, Ubinuclein1, and Cabin1, is responsible for H3.3 deposition into active chromatin,19,20,27,29-32 the H3.3-specific chaperone DAXX in cooperation with the chromatin remodeler ATRX is essential for H3.3 deposition at heterochromatic loci.26,27,33 The ATRX/DAXX/H3.3 pathway has been implicated in the suppression of pancreatic neuroendocrine tumors (panNET) and pediatric glioblastomas,34-39 thus establishing its role in carcinogenesis. While establishment and maintenance of chromatin structure is central for genome function,40 how (S,R,S)-AHPC hydrochloride such a mechanism is achieved in senescent cells has remained unclear. Chromatin structure is extensively remodeled upon senescence entry, as exemplified by the formation of senescence-associated heterochromatin foci (SAHF), visible as microscopically discernible, punctate DNA foci in DAPI-stained senescent cells.41 These structures are thought to contribute to the senescence-associated cell cycle arrest in part by silencing proliferation-promoting genes through heterochromatinization.41 Moreover, oncogene-induced SAHF formation may protect premalignant cells to undergo apoptosis by limiting extensive DNA damage to sub-lethal levels.42 Little is known about the underlying mechanisms of the extensive chromatin reorganization observed in senescent cells. SAHF are enriched in markers of heterochromatin, including tri-methylated histone H3 at lysine 9 (H3K9me3), all HP1 isoforms, as well as HMGA proteins.41,43 In addition, SAHF are also enriched in the histone H2A variant macroH2A,44 a variant associated with gene silencing, as, for example, during X inactivation.45 Formation of MacroH2A- and HP1-containing SAHF is dependent on the 2 2 histone chaperones HIRA and ASF1a,44 suggesting that H3.3 may become enriched (S,R,S)-AHPC hydrochloride in SAHF during OIS.44,46 Interestingly, SAHF formation also depends on the prior localization of HIRA into promyelocytic leukemia (PML) nuclear bodies (PML-NBs),44,47 discrete foci, 0.2C1.0 m wide, that are present in most mammalian cell nuclei and stain positive for the tumor suppressor PML.48,49 PML-NBs have previously been implicated in the onset of OIS: they increase in Rabbit polyclonal to IL7R number and size upon overexpression of H-RasV12, and overexpression of PML triggers p53-dependent senescence.50,51 Thus, PML-NBs may represent important regulatory structures not only for the induction of OIS in general, but also for the establishment and maintenance of the specialized chromatin structure characteristic of the senescent state. In this study, we set out to investigate the.