Tudor Interacting Repair Regulator (TIRR) is an RNA-binding protein (RBP) that interacts directly with 53BP1, restricting its access to DNA double-strand breaks (DSBs) and its association with p53. We utilized iCLIP to identify RNAs that directly bind to TIRR within cells, identifying the long non-coding RNA NEAT1 as the primary RNA partner. The high affinity of TIRR for NEAT1 is due to prevalent G-rich motifs in the short isoform (NEAT1_1) region of NEAT1. This interaction destabilizes the TIRR/53BP1 complex, promoting 53BP1’s function. NEAT1_1 is enriched during the G1 phase of the cell cycle, thereby ensuring that TIRR-dependent inhibition of 53BP1’s function is cell cycle-dependent. TDP-43, an RBP that is implicated in neurodegenerative diseases, modulates the TIRR/53BP1 complex by promoting the production of the NEAT1 short isoform, NEAT1_1. Together, we infer that NEAT1_1, and factors regulating NEAT1_1, may impact 53BP1-dependent DNA repair processes, with implications for a spectrum of diseases. TIRR interacts directly with 53BP1, restricting its access to DNA double-strand breaks (DSBs) and its association with p53. Here, the authors show that the lncRNA NEAT1, regulated by TDP-43, destabilizes the TIRR/53BP1 complex in G1, promoting 53BP1’s function in DSB repair and p53 transactivation.

It is unclear whether metabolic health corresponds to reduced oncogenesis or vice versa. We study Tudor-interacting repair regulator (TIRR), an inhibitor of p53 binding protein 1 (53BP1)-mediated p53 activation, and the physiological consequences of enhancing tumor suppressor activity. Deleting TIRR selectively activates p53, significantly protecting against cancer but leading to a systemic metabolic imbalance in mice. TIRR-deficient mice are overweight and insulin resistant, even under normal chow diet. Similarly, reduced TIRR expression in human adipose tissue correlates with higher BMI and insulin resistance. Despite the metabolic challenges, TIRR loss improves p53 heterozygous (p53HET) mouse survival and correlates with enhanced progression-free survival in patients with various p53HET carcinomas. Finally, TIRR’s oncoprotective and metabolic effects are dependent on p53 and lost upon p53 deletion in TIRR-deficient mice, with glucose homeostasis and orexigenesis being primarily regulated by TIRR expression in the adipose tissue and the CNS, respectively, as evidenced by tissue-specific models. In summary, TIRR deletion provides a paradigm of metabolic deregulation accompanied by reduced oncogenesis.

Abstract Molecularly targeted therapies have reshaped oncology in recent decades, though not all patients are eligible for such treatments and resistance mechanisms limit clinical benefit. In response, combination therapies, which can induce potent synthetic lethalities, bypass drug resistance, and expand treatment options, are increasingly being developed. A major hurdle limiting novel combination therapy discovery is the sheer combinatorics associated with broadly testing two or more agents. Thus, there is a practical need for methodologies that prioritize test compounds and technologies for rapid and efficient screening. Towards these ends, we have developed a novel combination drug screening workflow that integrates machine learning-based drug synergy predictions with a high throughput droplet microfluidics-based screening platform. Our physical system, or FlowMatrix, consists of 9,216 nano wells arrayed in a 96 × 96 grid in which cells are incubated and drug compounds delivered via emulsified droplets along both its rows and columns to facilitate combination screening. Following multi-day incubation, live cells are imaged with fluorescence microscopy and drug combination synergies are calculated from cellular viabilities. In our current setup, which includes four dose drug treatments with replicates and controls, we capture complete data (control, single agent, and 4 × 4 drug-drug combination viability measurements) for up to 100 unique drug combinations within the 9,216 dual droplet loaded wells of one FlowMatrix. Our to-date screening efforts have focused on acute myeloid leukemia (AML) specifically, where we have profiled 12,700 AML cell line-drug combinations in our system. This encompasses >3,700 unique drug combinations profiled in 7 distinct AML cell lines with 183 total matrix runs. Among the strongest hits observed in our screens and confirmed with validation studies (~65% confirmation rate) are combinations currently being tested in clinical trials for other cancers and established therapies paired with non-cancer indicated drugs. In particular, we are interrogating combinations involving CDK12 inhibitors, which affect the transcription of DNA repair proteins, splicing, and cancer cell progression, including unexpected partner drugs that potentiate the effects of CDK12 blockade in AML cell lines. We have uncovered additional unexpected synergistic combinations with KIT inhibitors in RUNX1-RUNX1T1 altered AML lines, ATR inhibitors (including an established combination with Gemcitabine), and the BCL-2 inhibitor Venetoclax, including known partnerships with chemotherapies (e.g. Decitabine and Daunorubicin) and other targeted inhibitors. Deeper studies are underway to further validate and characterize these candidates towards expanding treatment options for AML. Ultimately, our platform may radically enhance drug combination screening potential. Citation Format: Anthony R. Soltis, Boryana Zhelyazkova, Pascal Drane, Efstathios Eleftheriadis, Andrew Ventresco, David A. Weitz, Arlinda Lee, Anthony J. Iafrate. A high-throughput platform identifies novel drug combinations towards acute myeloid leukemia therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3900.

Molecularly targeted therapies have reshaped cancer treatment in recent decades; however, not all patient cancers are eligible for or effectively treated by such approaches. In addition, resistance mechanisms (both pre-existing and acquired) limit clinical benefit. To these ends, combination therapies involving targeted agents are increasingly being developed for their potential to induce potent synthetic lethalities, bypass drug resistance, promote durable responses, limit adverse side effects via reduced dosing, and expand treatment options. In the context of acute myeloid leukemia (AML) specifically, several efficacious combination therapies have been approved for specific patient subsets, such as all-trans retinoic acid (ATRA) plus arsenic trioxide in the PML-RARA fusion acute promyelocytic subtype and Midostaurin plus Cytarabine and Daunorubicin in FLT3-mutant AML. Despite the availability of these and other therapies, there remains an unmet need for treatments against AML subtypes lacking actionable driver alterations or for which current therapies are insufficient. A major hurdle limiting novel combination therapy discovery is the sheer combinatorics associated with mixing two or more agents (e.g., screening all possible pairs of 100 drugs in a single cell line requires nearly 5,000 combination experiments). Thus, there is a practical need for methodologies that prioritize test compounds among broad candidate sets and high throughput technologies for rapid and efficient screening. We have developed a combination drug screening workflow that integrates machine learning-based predictions of potentially synergistic compound pairs with a droplet microfluidics-based screening platform. Our physical platform, termed the FlowMatrix, contains a series of nano wells arrayed in a 96 x 96 grid (for 9,216 total wells) in which cells are incubated and compounds delivered via emulsified droplets. To facilitate drug combination screening, the FlowMatrix is loaded column-wise with a 96-element library and subsequently loaded a second time along its rows with either the same or an independent drug library. In our current setup, one FlowMatrix can capture complete data for up to 100 unique drug pairs, which includes control, single-agent, and 4x4 dose combination measurements for each pair. Following multi-day incubation, viability for each well is assessed by the number of live cells imaged with fluorescence microscopy. Combinations with high scoring synergy based on Bliss independence criteria are then pushed to validation and further characterization pipelines. We are currently focused on identifying novel combination therapies for AML. Using small molecule libraries of diverse compound classes and machine learning to prioritize initial screening candidates, we have to-date profiled >8,500 AML cell line-drug combinations in our system. This encompasses >2,900 unique drug combinations profiled in 7 distinct AML cell lines. Among ~270 hits that emerged from these screens are established clinical and preclinical AML combination therapies, in addition to candidates that, to the best of our knowledge, are novel in the context of AML. Strong established hits in our screen include ATR inhibition plus Gemcitabine treatment as well as several combinations involving the BCL-2 inhibitor Venetoclax with chemotherapies (Decitabine and Daunorubicin), Quizartinib, Idasanutlin, and mTOR inhibitors. Thus, we have developed an efficient and cost-effective high throughput drug combinations profiling system that has uncovered candidate therapies that may expand treatment options for patients afflicted by AML.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1 - autoinhibited for chromatin binding - that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.

Abstract High-grade serous ovarian carcinoma (HGSOC) patients with germline mutations in BRCA1/2 exhibit high sensitivity and improved outcome to double strand DNA break (DSB)-inducing agents [i.e. platinum and Poly(ADP-ribose) polymerase inhibitors (PARPi)] due to underlying defects in DNA repair via homologous recombination (HR). Due to their effectiveness, three PARP inhibitors (olaparib, rucaparib, niraparib) have recently gained FDA approval for the treatment of HGSOCs. However, de novo and acquired resistance to these agents is common even in the BRCA mutation carriers, and pose a significant, and unsolved, clinical challenge. Therefore, we adopted a systematic approach to comprehensibly identify unexplored factors/pathways that could be responsible for PARPi/platinum resistance in BRCA-defective HGSOC patients. Here we identify DYNLL1 as a negative regulator of DNA end resection through a loss-of-function CRISPR screen in BRCA1-mutant ovarian carcinoma cells. DNA end resection is a vital process that initiates homologous recombination (HR)-mediated repair of double-stranded DNA breaks (DSBs), and consequently influences genome stability. In BRCA-defective HGSOC patients, DNA end resection is greatly compromised and contribute to the loss of HR and PARP inhibitor sensitivity. Loss of DYNLL1 allows DNA end resection and restores HR in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and PARP inhibitors. In primary ovarian carcinomas low BRCA1 expression correlates with increased chromosomal aberrations, and the junction sequences of somatic structural variants indicate the loss of HR. Concurrent decrease in DYNLL1 expression in BRCA1 low ovarian cancers ‘rescued' this phenotype with reduced genomic alterations and increased homology at putative lesions. DYNLL1 limits nucleolytic degradation of DNA ends by interacting with the DNA end resection machinery (MRN complex, BLM helicase and DNA2) in cells. The impact of DYNLL1 on end resection can be re-capitulated in vitro and this is dependent on direct interaction with MRE11. In the absence of exogenous stress, depletion of DYNLL1 slows DNA replication fork progression due to ectopic activity of MRE11. Therefore, we infer that DYNLL1 is an important anti-resection factor that significantly influences genomic stability and response to DNA damaging chemotherapy. Citation Format: Yizhou Joseph He, Khyati Meghani, Marie-Christine Caron, Chunyu Yang, Daryl A. Ronato, Jie Bian, Anchal Sharma, Jessica Miller, Niraj Joshi, Alexandre Detappe, John G. Doench, Gaelle Legube, David E. Root, Alan D. D'Andrea, Pascal Drané, Subhojyoti De, Panagiotis Konstantinopoulos, Jean-Yves Masson, and Dipanjan Chowdhury. DYNLL1 INHIBITS DNA END RESECTION IN BRCA1-DEFICIENT CELLS AND REGULATES PARP INHIBITOR SENSITIVITY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-027.

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.

BRCA1/2-mutated ovarian cancers (OCs) are defective in homologous recombination repair (HRR) of double-strand breaks (DSBs) and thereby sensitive to platinum and PARP inhibitors (PARPis). Multiple PARPis have recently received US Food and Drug Administration (FDA) approval for treatment of OCs, and resistance to PARPis is a major clinical problem. Utilizing primary and recurrent BRCA1/2-mutated carcinomas from OC patients, patient-derived lines, and an in vivo BRCA2-mutated mouse model, we identified a microRNA, miR-493-5p, that induced platinum/PARPi resistance exclusively in BRCA2-mutated carcinomas. However, in contrast to the most prevalent resistance mechanisms in BRCA mutant carcinomas, miR-493-5p did not restore HRR. Expression of miR-493-5p in BRCA2-mutated/depleted cells reduced levels of nucleases and other factors involved in maintaining genomic stability. This resulted in relatively stable replication forks, diminished single-strand annealing of DSBs, and increased R-loop formation. We conclude that impact of miR-493-5p on multiple pathways pertinent to genome stability cumulatively causes PARPi/platinum resistance in BRCA2 mutant carcinomas.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

53BP1 is a central player in the repair of DNA-double strand breaks (DSBs) as it orchestrates the choice between the DSB repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). 53BP1 promotes repair by NHEJ by countering the function of BRCA1 in the HR pathway. This antagonistic relationship of BRCA1 and 53BP1 has been revealed in the context of BRCA1 deficiency. BRCA1-mutant cells are deficient in HR-mediated DNA repair and exquisitely sensitive to treatment with Poly-ADP-Ribose Polymerase (PARP) inhibitors. Loss of 53BP1 almost completely abolishes the sensitivity of BRCA1-mutant cells to PARP inhibitors, which also correlates with the restoration of competent HR. This finding illustrates the fact that 53BP1 and BRCA1 compete to repair the same DSB. In a physiologic scenario, the BRCA1 and 53BP1 interplay ensures that the repair is accurate and restricted to the appropriate cellular contexts. The newly identified 53BP1-partner TIRR represents a pathway that modulates DNA repair by restricting the access of 53BP1 to DNA lesions. 53BP1 and TIRR form a stable nuclear soluble complex in undamaged cells. Following DNA damage, TIRR dissociates from 53BP1 through a mechanism involving the phosphorylation of the N-terminal of 53BP1 by the Ataxia Telangiectasia Mutated (ATM) kinase and the recruitment of Rap1-interacting factor 1 homolog (RIF-1). This dissociation allows nuclear soluble 53BP1 to localize to chromatin. At the molecular level, TIRR interacts with the Tudor domain of 53BP1. This domain is involved in 53BP1 recruitment to the damaged chromatin by recognition of histone H4 dimethylated in lysine K20 (H4K20me2). Structural evidence indicates that H4K20me2 and TIRR binding surfaces on 53BP1 Tudor domain overlap. However, the binding affinity of TIRR with Tudor is »25-fold stronger than the interaction of Tudor with H4K20me2 peptide. Intriguingly the dissociation of TIRR from 53BP1 appears to involve the N-terminal region and not the Tudor region. The in vitro and structural studies were restricted to the Tudor domains of 53BP1 therefore it is feasible that the N-terminal end of 53BP1 also directly interacts with TIRR (Fig. 1). The disruption of this interaction via the recruitment of RIF-1 to 53BP1 is sufficient for the release of TIRR. The importance of TIRR as a functional modulator of 53BP1 was clearly demonstrated in cells overexpressing TIRR. An excess of TIRR alters the stoichiometry and the dynamics of dissociation and consequently inhibits the recruitment of 53BP1 to DSBs. Physiological consequence of TIRR overexpression was observed in 2 scenarios, one it prevents class switching recombination in B lymphocytes a process that largely relies on the recruitment of 53BP1 to endogenous DSBs generated during the recombination process. Second, it impairs PARP inhibitor sensitivity in BRCA1-mutated cells, mimicking an absence of 53BP1. Conversely, loss-of-function studies also reveal the impact of TIRR on 53BP1 activity. TIRR depletion destabilizes 53BP1 but more importantly affects how 53BP1 interacts with its partners. Indeed, the absence of TIRR increases the DNA-damage dependent association of 53BP1 with its effector partners such as RIF-1 or PTIP. This functionally correlates with the fact that depletion of TIRR renders BRCA1-mutated cells even more sensitive to PARP inhibitor in a 53BP1-dependent manner. This “hyper-active” state of 53BP1 has been also observed previously in cells overexpressing the E3 ubiquitin-ligase RNF168, an enzyme involved in the recruitment of 53BP1 to the damage. Therefore, it is likely that TIRR protein level is tightly controlled in ‘normal’ cells to modulate 53BP1 function. Interestingly, a compilation of 50 studies in the Cancer Genome Atlas (TCGA) shows that the TIRR gene locus (alias Nudt16L1) is amplified in 29 out of the 34 different carcinomas. Therefore, one attractive possibility would be that amplification of the TIRR gene – that would result in TIRR protein level increase and consequently 53BP1 inactivation is one of the mechanisms by which BRCA1-mutant tumors acquire PARP inhibitor resistance. Future analysis of BRCA1-mutant tumors from ovarian or breast cancer patients resistant to PARP inhibitors may reveal the clinical relevance of TIRR in cancer therapy. The number of epigenetic reader proteins that are shown to be involved in disease development and progression is growing, and there is increasing knowledge about their involvement in the pathogenesis of various diseases. There is currently a

Radiation therapy is a major treatment regimen for more than 50% of cancer patients. The collateral damage induced on healthy tissues during radiation and the minimal therapeutic effect on the organ-of-interest (target) is a major clinical concern. Ultra-small, renal clearable, silica based gadolinium chelated nanoparticles (SiGdNP) provide simultaneous MR contrast and radiation dose enhancement. The high atomic number of gadolinium provides a large photoelectric cross-section for increased photon interaction, even for high-energy clinical radiation beams. Imaging and therapy functionality of SiGdNP were tested in cynomolgus monkeys and pancreatic tumor-bearing mice models, respectively. A significant improvement in tumor cell damage (double strand DNA breaks), growth suppression, and overall survival under clinical radiation therapy conditions were observed in a human pancreatic xenograft model. For the first time, safe systemic administration and systematic renal clearance was demonstrated in both tested species. These findings strongly support the translational potential of SiGdNP for MR-guided radiation therapy in cancer treatment.

As nanoparticle solutions move towards human clinical trials in radiation therapy, the influence of key clinical beam parameters on therapeutic efficacy must be considered. In this study, we have investigated the clinical radiation therapy delivery variables that may significantly affect nanoparticle-mediated radiation dose amplification. We found a benefit for situations which increased the proportion of low energy photons in the incident beam. Most notably, "unflattened" photon beams from a clinical linear accelerator results in improved outcomes relative to conventional "flat" beams. This is measured by significant DNA damage, tumor growth suppression, and overall improvement in survival in a pancreatic tumor model. These results, obtained in a clinical setting, clearly demonstrate the influence and importance of radiation therapy parameters that will impact clinical radiation dose amplification with nanoparticles.

Excluding 53BP1 from chromatin is required to attenuate the DNA damage response during mitosis, yet the functional relevance and regulation of this exclusion are unclear. Here we show that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, located in its well-conserved ubiquitination-dependent recruitment (UDR) motif. Phosphorylating these sites blocks the interaction of the UDR motif with mononuclesomes containing ubiquitinated histone H2A and impedes binding of 53BP1 to mitotic chromatin. Ectopic recruitment of 53BP1-T1609A/S1618A to mitotic DNA lesions was associated with significant mitotic defects that could be reversed by inhibiting nonhomologous end-joining. We also reveal that protein phosphatase complex PP4C/R3β dephosphorylates T1609 and S1618 to allow the recruitment of 53BP1 to chromatin in G1 phase. Our results identify key sites of 53BP1 phosphorylation during mitosis, identify the counteracting phosphatase complex that restores the potential for DDR during interphase, and establish the physiological importance of this regulation.

The histone variant H3.3 marks active chromatin by replacing the conventional histone H3.1. In this study, we investigate the detailed mechanism of H3.3 replication-independent deposition. We found that the death domain-associated protein DAXX and the chromatin remodeling factor ATRX (alpha-thalassemia/mental retardation syndrome protein) are specifically associated with the H3.3 deposition machinery. Bacterially expressed DAXX has a marked binding preference for H3.3 and assists the deposition of (H3.3-H4)(2) tetramers on naked DNA, thus showing that DAXX is a H3.3 histone chaperone. In DAXX-depleted cells, a fraction of H3.3 was found associated with the replication-dependent machinery of deposition, suggesting that cells adapt to the depletion. The reintroduced DAXX in these cells colocalizes with H3.3 into the promyelocytic leukemia protein (PML) bodies. Moreover, DAXX associates with pericentric DNA repeats, and modulates the transcription from these repeats through assembly of H3.3 nucleosomes. These findings establish a new link between the PML bodies and the regulation of pericentric DNA repeat chromatin structure. Taken together, our data demonstrate that DAXX functions as a bona fide histone chaperone involved in the replication-independent deposition of H3.3.

Recent data showed that p53 stimulates the expression of genes encoding not only pro-but also antioxidant enzymes. It was suggested that antioxidant genes could be induced under physiologic levels of stress while the prooxidant ones respond to higher level of stress. Results presented in this article illustrate an additional degree of complexity. We show that the expression of Haeme-oxygenase 1 (HO-1), a stress-inducible gene that codes for an enzyme having antioxidant properties, is stimulated in a p53-dependent manner in the thymus and spleen of irradiated mice. We prove that HO-1 is a direct p53 target gene by showing that the p53RE identified within human and mouse genes is specifically bound by p53. The threshold of irradiation dose required to induce a significant response of HO-1 in the lymphoid organs of the irradiated mice is higher than that for Waf1/p21 that encodes an universal inhibitor of cell cycle. Moreover, induction of HO-1 occurs later than that of Waf1/p21. Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.