The Histone Chaperone FACT Coordinates H2A.X-Dependent Signaling and Repair of DNA Damage.

Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression.

Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

Inhibition of retrograde transport protects mice from lethal ricin challenge.

Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.

Absolute Quantification of Drug Vector Delivery to the Cytosol.

Macromolecular drugs inefficiently cross membranes to reach their cytosolic targets. They require drug delivery vectors to facilitate their translocation across the plasma membrane or escape from endosomes. Optimization of these vectors has however been hindered by the difficulty to accurately measure cytosolic arrival. We have developed an exceptionally sensitive and robust assay for the relative or absolute quantification of this step. The assay is based on benzylguanine and biotin modifications on a drug delivery vector of interest, which allow, respectively, for selective covalent capture in the cytosol with a SNAP-tag fusion protein and for quantification at picomolar sensitivity. The assay was validated by determining the absolute numbers of cytosolic molecules for two drug delivery vectors: the B-subunit of Shiga toxin and the cell-penetrating peptide TAT. We expect this assay to favor delivery vector optimization and the understanding of the enigmatic translocation process.

Targeted Shiga toxin-drug conjugates prepared via Cu-free click chemistry.

The main drawback of the anticancer chemotherapy consists in the lack of drug selectivity causing severe side effects. The targeted drug delivery appears to be a very promising strategy for controlling the biodistribution of the cytotoxic agent only on malignant tissues by linking it to tumor-targeting moiety. Here we exploit the natural characteristics of Shiga toxin B sub-unit (STxB) as targeting carrier on Gb3-positive cancer cells. Two cytotoxic conjugates STxB-doxorubicin (STxB-Doxo) and STxB-monomethyl auristatin F (STxB-MMAF) were synthesised using copper-free 'click' chemistry. Both conjugates were obtained in very high yield and demonstrated strong tumor inhibition activity in a nanomolar range on Gb3-positive cells.

Analysis of articulation between clathrin and retromer in retrograde sorting on early endosomes.

Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans-Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes-TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069-7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.

Imaging local deposition of newly synthesized histones in UVC-damaged chromatin.

DNA damage not only jeopardizes genome integrity but also challenges the well-organized association of DNA with histone proteins into chromatin, which is key for regulating gene expression and cell functions. The extent to which the original chromatin structure is altered after repair of DNA lesions is thus a critical issue. Dissecting histone dynamics at sites of DNA damage has provided mechanistic insights into chromatin plasticity in response to genotoxic stress. Here, we present an experimental protocol for visualizing the deposition of newly synthesized histone H3 variants at sites of UVC damage in human cells that couples SNAP-tag based labeling of new histones with local UVC irradiation of cells through micropore filters.

A new delivery system for auristatin in STxB-drug conjugate therapy.

A key challenge in anticancer therapy is to gain control over the biodistribution of cytotoxic drugs. The most promising strategy consists in conjugating drugs to tumor-targeting carriers, thereby combining high cytotoxic activity and specific delivery. To target Gb3-positive cancer cells, we exploit the non-toxic B-subunit of Shiga toxin (STxB). Here, we have conjugated STxB to highly potent auristatin derivatives (MMA). A former linker was optimized to ensure proper drug-release upon reaching reducing environments in target cells, followed by a self-immolation step. Two conjugates were successfully obtained, and in vitro assays demonstrated the potential of this targeting system for the selective elimination of Gb3-positive tumors.