Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150Glued bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)–LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7–RILP complex to remove p150Glued and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

The small GTPase Rab7 controls late endocytic transport by the minus end–directed motor protein complex dynein–dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP–Rab7–ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150Glued, which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150Glued recruitment by Rab7–RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as βIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150Glued dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7–RILP is transferred by ORP1L to βIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with βIII spectrin, which requires the activities of Rab7–RILP and ORP1L.

Estrogen receptor-α (ER) is the driving transcription factor in most breast cancers, and its associated proteins can influence drug response, but direct methods for identifying interacting proteins have been limited. We purified endogenous ER using an approach termed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) and discovered the interactome under agonist- and antagonist-liganded conditions in breast cancer cells, revealing transcriptional networks in breast cancer. The most estrogen-enriched ER interactor is GREB1, a potential clinical biomarker with no known function. GREB1 is shown to be a chromatin-bound ER coactivator and is essential for ER-mediated transcription, because it stabilizes interactions between ER and additional cofactors. We show a GREB1-ER interaction in three xenograft tumors, and using a directed protein-protein approach, we find GREB1-ER interactions in half of ER(+) primary breast cancers. This finding is supported by histological expression of GREB1, which shows that GREB1 is expressed in half of ER(+) cancers, and predicts good clinical outcome. These findings reveal an unexpected role for GREB1 as an estrogen-specific ER cofactor that is expressed in drug-sensitive contexts.

DNA topoisomerase II inhibitors are a major class of cancer chemotherapeutics, which are thought to eliminate cancer cells by inducing DNA double-strand breaks. Here we identify a novel activity for the anthracycline class of DNA topoisomerase II inhibitors: histone eviction from open chromosomal areas. We show that anthracyclines promote histone eviction irrespective of their ability to induce DNA double-strand breaks. The histone variant H2AX, which is a key component of the DNA damage response, is also evicted by anthracyclines, and H2AX eviction is associated with attenuated DNA repair. Histone eviction deregulates the transcriptome in cancer cells and organs such as the heart, and can drive apoptosis of topoisomerase-negative acute myeloid leukaemia blasts in patients. We define a novel mechanism of action of anthracycline anticancer drugs doxorubicin and daunorubicin on chromatin biology, with important consequences for DNA damage responses, epigenetics, transcription, side effects and cancer therapy.