Abstract. Members of the cytokine receptor family are composed of several noncovalently linked chains with sequence and structure homologies in their extracellular domain. Receptor subfamily members share at least one component: thus the receptors for interleukin (IL) 2 and ILl5 have common ~ and 3" chains, while those for IL2, 4, 7, and 9 have a common 3' chain. The intracellular pathway followed by IL2 receptors after ligand binding and endocytosis was analyzed by immunofluorescence and confocal microscopy in a human T lymphocytic cell line. Surprisingly, the a, ~, and 3' chains had different intracellular localizations after being endocytosed together. The ct chain was always in transferl-in-positive compartments (early/recycling endosomes), both at early and late internalization times, but was never detected in rab7-positive compartments (late endosomes). On the other hand, at late internalization times, the/3 and 3, chains were excluded from transferrin-positive organelles and did not colocalize with ~. Furthermore, /3 could be found in rab7-positive vesicles. These differences suggest that the ot chain recycles to the plasma membrane, while the ~ and 3' chains are sorted towards the degradation pathway. The half-lives of these three chains on the cell surface also reflect their different intracellular fates after endocytosis. The and 3' chains are very short-lived polypeptides since their half-life on the surface is only =1 h, whereas c~ is a much more stable surface protein. This shows for the first time that components of a multimeric receptor can be sorted separately along the endocytic pathway.

Understanding the pathways that are targeted by cancer drugs is instrumental for their rational use in a clinical setting. Inhibitors of histone deacetylases (HDACI) selectively inhibit proliferation of malignant cells and are used for the treatment of cancer, but their cancer selectivity is understood poorly. We conducted a functional genetic screen to address the mechanism(s) of action of HDACI. We report here that ectopic expression of two genes that act on retinoic acid (RA) signaling can cause resistance to growth arrest and apoptosis induced by HDACI of different chemical classes: the retinoic acid receptor ␣ (RAR␣) and preferentially expressed antigen of melanoma (PRAME), a repressor of RA signaling. Treatment of cells with HDACI induced RA signaling, which was inhibited by RAR␣ or PRAME expression. Conversely, RAR-deficient cells and PRAME-knockdown cells show enhanced sensitivity to HDACI in vitro and in mouse xenograft models. Finally, a combination of RA and HDACI acted synergistically to activate RA signaling and inhibit tumor growth. These experiments identify the RA pathway as a rate-limiting target of HDACI and suggest strategies to enhance the therapeutic efficacy of HDACI.biomarker ͉ chromatin modification ͉ drug resistance ͉ epigenetics ͉ nuclear hormone receptor E pigenetic DNA and histone modifications are appreciated as major determinants in the control of gene activity, and they are extensively deregulated in cancer. Histone acetylation is regulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs), which catalyze the addition and removal of acetyl groups to histones, respectively, and to a growing list of nonhistone substrates (1). The activities of HATs and HDACs are altered in several human cancers, and modulation of these classes of enzymes provides a potentially attractive therapeutic modality (2, 3). Several classes of HDAC inhibitors (HDACI) have been identified that block enzyme activity, resulting in global histone hyperacetylation. A wide array of literature on HDACI exists, describing their various effects, including G 1 and G 2 /M cell cycle arrests, apoptosis, and differentiation, and several HDACI have entered clinical trials (2-4). Gene expression profiling studies revealed that HDACI treatment induces alterations in transcription of Ͻ5% to Ϸ20% of expressed genes (5, 6) and have not elucidated a consistent picture of the pathway(s) or target(s) that are modulated by HDACI and, consequently, have not provided a comprehensive explanation for their anticancer effects.To identify cellular targets of HDACI action in transformed cells, we used the approach of large-scale functional genetic screening. In this screen we asked which genes or pathways could confer cellular resistance to HDACI. The present work provides evidence that the retinoic acid receptor (RAR) pathway is targeted by HDACI and that the cytotoxic effects of HDACI in solid tumor cells are, at least in part, through derepression of retinoic acid (RA) signaling. ResultsGe...

Cell lineages, which shape the body architecture and specify cell functions, derive from the integration of a plethora of cell intrinsic and extrinsic signals. These signals trigger a multiplicity of decisions at several levels to modulate the activity of dynamic gene regulatory networks (GRNs), which ensure both general and cell-specific functions within a given lineage, thereby establishing cell fates. Significant knowledge about these events and the involved key drivers comes from homogeneous cell differentiation models. Even a single chemical trigger, such as the morphogen all-trans retinoic acid (RA), can induce the complex network of gene-regulatory decisions that matures a stem/precursor cell to a particular step within a given lineage. Here we have dissected the GRNs involved in the RA-induced neuronal or endodermal cell fate specification by integrating dynamic RXRA binding, chromatin accessibility, epigenetic promoter epigenetic status, and the transcriptional activity inferred from RNA polymerase II mapping and transcription profiling. Our data reveal how RA induces a network of transcription factors (TFs), which direct the temporal organization of cognate GRNs, thereby driving neuronal/endodermal cell fate specification. Modeling signal transduction propagation using the reconstructed GRNs indicated critical TFs for neuronal cell fate specification, which were confirmed by CRISPR/Cas9-mediated genome editing. Overall, this study demonstrates that a systems view of cell fate specification combined with computational signal transduction models provides the necessary insight in cellular plasticity for cell fate engineering. The present integrated approach can be used to monitor the in vitro capacity of (engineered) cells/tissues to establish cell lineages for regenerative medicine.

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