TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs.

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.

DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells.

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.

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

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.

Dephosphorylation enables the recruitment of 53BP1 to double-strand DNA breaks.

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/R3ß 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 death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3.

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.

Endogenous p53 inhibitor TIRR dissociates systemic metabolic health from oncogenic activity.

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.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

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.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

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.

p53-dependent stimulation of redox-related genes in the lymphoid organs of gamma-irradiated--mice identification of Haeme-oxygenase 1 as a direct p53 target gene.

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.

Dysregulation of the peroxisome proliferator-activated receptor target genes by XPD mutations.

Mutations in the XPD subunit of TFIIH give rise to human genetic disorders initially defined as DNA repair syndromes. Nevertheless, xeroderma pigmentosum (XP) group D (XP-D) patients develop clinical features such as hypoplasia of the adipose tissue, implying a putative transcriptional defect. Knowing that peroxisome proliferator-activated receptors (PPARs) are implicated in lipid metabolism, we investigated the expression of PPAR target genes in the adipose tissues and the livers of XPD-deficient mice and found that (i) some genes are abnormally overexpressed in a ligand-independent manner which parallels an increase in the recruitment of RNA polymerase (pol) II but not PPARs on their promoter and (ii) upon treatment with PPAR ligands, other genes are much less induced compared to the wild type, which is due to a lower recruitment of both PPARs and RNA pol II. The defect in transactivation by PPARs is likely attributable to their weaker phosphorylation by the cdk7 kinase of TFIIH. Having identified the phosphorylated residues in PPAR isotypes, we demonstrate how their transactivation defect in XPD-deficient cells can be circumvented by overexpression of either a wild-type XPD or a constitutively phosphorylated PPAR S/E. This work emphasizes that underphosphorylation of PPARs affects their transactivation and consequently the expression of PPAR target genes, thus contributing in part to the XP-D phenotype.

Multifaceted Impact of MicroRNA 493-5p on Genome-Stabilizing Pathways Induces Platinum and PARP Inhibitor Resistance in BRCA2-Mutated Carcinomas.

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.

Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein.

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.

A comprehensive study of p53 transcriptional activity in thymus and spleen of gamma irradiated mouse: high sensitivity of genes involved in the two main apoptotic pathways.

PURPOSE: Gamma-irradiation leads to activation of p53 tumour suppressor gene and to p53-dependant stimulation of a large panel of cellular genes including proapoptotic genes involved in intrinsic and extrinsic pathways. Most in vivo published data referred to high (lethal) irradiation doses. The present study was performed to analyse the p53-dependent response to more relevant low irradiation doses. MATERIALS AND METHODS: Mice were whole body exposed to irradiation doses decreasing from 5 - 0.05 Gy. Gene expression was estimated by real time reverse transcriptase polymerase chain reaction measurements on RNA extracted from thymus and spleen. Apoptosis was evaluated by the percentage of either annexin V positive or sub-G1 cells. RESULTS: A 0.1 Gy irradiation dose already gives a significant stimulation of Puma (p53 up-regulated modulator of apoptosis), and 0.2 Gy of Bax (Bcl-2-associated X protein) and Killer/DR5 (Death Receptor 5). The expression of genes involved in the two apoptotic pathways was induced as soon as 1 h post-irradiation and reached a maximum at 3 h, the induction level depending on both the gene and the organ. A significant increase in the number of apoptotic cells is already detectable at 0.5 Gy with a maximum of induction at 6 h. CONCLUSIONS: Our results reveal the high in vivo sensitivity of p53-dependent transcriptional activation of genes involved in the two main apoptotic pathways, their stimulation preceding the induction of apoptosis.

Advanced multimodal nanoparticles delay tumor progression with clinical radiation therapy.

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.

Key clinical beam parameters for nanoparticle-mediated radiation dose amplification.

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.

Cockayne syndrome B protein regulates the transcriptional program after UV irradiation.

The phenotype of the human genetic disorder Cockayne syndrome (CS) is not only due to DNA repair defect but also (and perhaps essentially) to a severe transcription initiation defect. After UV irradiation, even undamaged genes are not transcribed in CSB cells. Indeed, neither RNA pol II nor the associated basal transcription factors are recruited to the promoters of the housekeeping genes, around of which histone H4 acetylation is also deficient. Transfection of CSB restores the recruitment process of RNA pol II. On the contrary, the p53-responsive genes do not require CSB and are transcribed in both wild-type and CSB cells upon DNA damage. Altogether, our data highlight the pivotal role of CSB in initiating the transcriptional program of certain genes after UV irradiation, and also may explain some of the complex traits of CS patients.

Selective regulation of vitamin D receptor-responsive genes by TFIIH.

Mutations in the XPD subunit of the transcription/repair factor TFIIH cause the Xeroderma pigmentosum disorder. We show that in some XP-D deficient cells, transactivation by the vitamin D receptor (VDR) is selectively inhibited for a subset of responsive genes, such as CYP24, and that the XPD/R683W mutation prevents VDR recruitment on its promoter. Contrary to other nuclear receptors, VDR, which lacks a functional A/B domain, is not phosphorylated and consequently not regulated by the cdk7 kinase of TFIIH. In fact, we demonstrate that the VDR transactivation defect resides in Ets1, another activator that cannot be phosphorylated by TFIIH in XP-D cells. Indeed, the phosphorylated Ets1 seems to promote the binding of VDR to its responsive element and trigger the subsequent recruitment of coactivators and RNA pol II. We propose a model in which TFIIH regulates the activity of nuclear receptors by phosphorylating either their A/B domain or an additional regulatory DNA binding partner.