MYC interaction with the tumor suppressive SWI/SNF complex member INI1 regulates transcription and cellular transformation.

MYC is a key driver of cellular transformation and is deregulated in most human cancers. Studies of MYC and its interactors have provided mechanistic insight into its role as a regulator of gene transcription. MYC has been previously linked to chromatin regulation through its interaction with INI1 (SMARCB1/hSNF5/BAF47), a core member of the SWI/SNF chromatin remodeling complex. INI1 is a potent tumor suppressor that is inactivated in several types of cancers, most prominently as the hallmark alteration in pediatric malignant rhabdoid tumors. However, the molecular and functional interaction of MYC and INI1 remains unclear. Here, we characterize the MYC-INI1 interaction in mammalian cells, mapping their minimal binding domains to functionally significant regions of MYC (leucine zipper) and INI1 (repeat motifs), and demonstrating that the interaction does not interfere with MYC-MAX interaction. Protein-protein interaction network analysis expands the MYC-INI1 interaction to the SWI/SNF complex and a larger network of chromatin regulatory complexes. Genome-wide analysis reveals that the DNA-binding regions and target genes of INI1 significantly overlap with those of MYC. In an INI1-deficient rhabdoid tumor system, we observe that with re-expression of INI1, MYC and INI1 bind to common target genes and have opposing effects on gene expression. Functionally, INI1 re-expression suppresses cell proliferation and MYC-potentiated transformation. Our findings thus establish the antagonistic roles of the INI1 and MYC transcriptional regulators in mediating cellular and oncogenic functions.

BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors.

The BioID proximity-based biotin labeling technique was recently developed for the characterization of protein-protein interaction networks [1]. To date, this method has been applied to a number of different polypeptides expressed in cultured cells. Here we report the adaptation of BioID to the identification of protein-protein interactions surrounding the c-MYC oncoprotein in human cells grown both under standard culture conditions and in mice as tumor xenografts. Notably, in vivo BioID yielded >100 high confidence MYC interacting proteins, including >30 known binding partners. Putative novel MYC interactors include components of the STAGA/KAT5 and SWI/SNF chromatin remodeling complexes, DNA repair and replication factors, general transcription and elongation factors, and transcriptional co-regulators such as the DNA helicase protein chromodomain 8 (CHD8). Providing additional confidence in these findings, ENCODE ChIP-seq datasets highlight significant coincident binding throughout the genome for the MYC interactors identified here, and we validate the previously unreported MYC-CHD8 interaction using both a yeast two hybrid analysis and the proximity-based ligation assay. In sum, we demonstrate that BioID can be utilized to identify bona fide interacting partners for a chromatin-associated protein in vivo. This technique will allow for a much improved understanding of protein-protein interactions in a previously inaccessible biological setting. BIOLOGICAL SIGNIFICANCE: The c-MYC (MYC) oncogene is a transcription factor that plays important roles in cancer initiation and progression. MYC expression is deregulated in more than 50% of human cancers, but the role of this protein in normal cell biology and tumor progression is still not well understood, in part because identifying MYC-interacting proteins has been technically challenging: MYC-containing chromatin-associated complexes are difficult to isolate using traditional affinity purification methods, and the MYC protein is exceptionally labile, with a half-life of only ~30 min. Developing a new strategy to gain insight into MYC-containing protein complexes would thus mark a key advance in cancer research. The recently described BioID proximity-based labeling technique represents a promising new complementary approach for the characterization of protein-protein interactions (PPIs) in cultured cells. Here we report that BioID can also be used to characterize protein-protein interactions for a chromatin-associated protein in tumor xenografts, and present a comprehensive, high confidence in vivo MYC interactome. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

Identification of c-MYC SUMOylation by mass spectrometry.

The c-MYC transcription factor is a master regulator of many cellular processes and deregulation of this oncogene has been linked to more than 50% of all cancers. This deregulation can take many forms, including altered post-translational regulation. Here, using immunoprecipitation combined with mass spectrometry, we identified a MYC SUMOylation site (K326). Abrogation of signaling through this residue by substitution with arginine (K326R) has no obvious effects on MYC half-life, intracellular localization, transcriptional targets, nor on the biological effects of MYC overexpression in two different cell systems assessed for soft agar colony formation, proliferation, and apoptosis. While we have definitively demonstrated that MYC SUMOylation can occur on K326, future work will be needed to elucidate the mechanisms and biological significance of MYC regulation by SUMOylation.

The role of ligand density and size in mediating quantum dot nuclear transport.

Studying the effects of the physicochemical properties of nanomaterials on cellular uptake, toxicity, and exocytosis can provide the foundation for designing safer and more effective nanoparticles for clinical applications. However, an understanding of the effects of these properties on subcellular transport, accumulation, and distribution remains limited. The present study investigates the effects of surface density and particle size of semiconductor quantum dots on cellular uptake as well as nuclear transport kinetics, retention, and accumulation. The current work illustrates that cellular uptake and nuclear accumulation of nanoparticles depend on surface density of the nuclear localization signal (NLS) peptides with nuclear transport reaching a plateau at 20% surface NLS density in as little as 30 min. These intracellular nanoparticles have no effects on cell viability up to 72 h post treatment. These findings will set a foundation for engineering more sophisticated nanoparticle systems for imaging and manipulating genetic targets in the nucleus.

Identifying Myc interactors.

In this chapter, we discuss in detail two essential methods used to evaluate the interaction of Myc with another protein of interest: co-immunoprecipitation (Co-IP) and in vitro pull-down assays. Co-IP is a method that, by immunoaffinity, allows the identification of protein-protein interactions within cells. We provide methods to conduct Co-IPs from whole-cell extracts as well as cytoplasmic and nuclear-enriched fractions. By contrast, the pull-down assay evaluates whether a bait protein that is bound to a solid support can specifically interact with a prey protein that is in solution. We provide methods to conduct in vitro pull-downs and further detail how to use this assay to distinguish whether a protein-protein interaction is direct or indirect. We also discuss methods used to screen for Myc interactors and provide an in silico strategy to help prioritize hits for further validation using the described Co-IP and in vitro pull-down assays.

Optimization of experimental design parameters for high-throughput chromatin immunoprecipitation studies.

High-throughput, microarray-based chromatin immunoprecipitation (ChIP-chip) technology allows in vivo elucidation of transcriptional networks. However this complex is not yet readily accessible, in part because its many parameters have not been systematically evaluated and optimized. We address this gap by systematically assessing experimental-design parameters including antibody purity, dye-bias, array-batch, inter-day hybridization bias, amplification method and choice of hybridization control. The combined performance of these optimized parameters shows a 90% validation rate in ChIP-chip analysis of Myc genomic binding in HL60 cells using two different microarray platforms. Increased sensitivity and decreased noise in ChIP-chip assays will enable wider use of this methodology to accurately and affordably elucidate transcriptional networks.

Cancer therapeutics: targeting the dark side of Myc.

The potent Myc oncoprotein plays a pivotal role as a regulator of tumorigenesis in numerous human cancers of diverse origin. Experimental evidence shows that inhibiting Myc significantly halts tumour cell growth and proliferation. This review summarises recent progress in understanding the function of Myc as a transcription factor, with emphasis on key protein interactions and target gene regulation. In addition, major advances in drug development aimed at eliminating Myc are described, including antisense and triple helix forming oligonucleotides, porphyrins and siRNA. Future anti-Myc strategies are also discussed that inhibit Myc at the level of expression and/or function. Targeting the dark side of Myc with novel therapeutic agents promises to have a profound impact in combating cancer.

Identification of a novel c-Myc protein interactor, JPO2, with transforming activity in medulloblastoma cells.

c-myc oncogene activation is critical in the pathogenesis of a spectrum of human malignancies. The c-Myc NH2-terminal domain (MycNTD) is essential for cellular transformation, and mediates critical protein interactions that modulate c-Myc oncogenic properties. In medulloblastoma, the most common malignant pediatric brain tumor, deregulated c-myc expression is linked with poorer disease phenotypes and outcomes. The biological basis for these associations is, however, not well understood. To better understand mechanisms underlying Myc-mediated transformation of medulloblastoma, we sought to identify novel MycNTD protein interactors from a medulloblastoma cell line library using a unique two-hybrid system. We identified a novel MycNTD binding protein, JPO2, which shows nuclear colocalization with c-Myc, and interacts with c-Myc both in vitro and in mammalian cells. In Rat1a transformation assays, JPO2 potentiates c-Myc transforming activity, and can complement a transformation-defective Myc mutant. Immunohistochemical studies indicate tumor-specific JPO2 expression in human medulloblastoma, and an association of JPO2 expression with metastatic tumors. Significantly, JPO2 expression induces colony formation in UW228, a medulloblastoma cell line, whereas RNAi-mediated JPO2 knockdown impairs colony formation in UW228, and in Myc-transformed UW228 cells. These data provide evidence for biochemical and functional interaction between c-Myc and JPO2 in medulloblastoma transformation. JPO2 is closely related to JPO1, a Myc transcriptional target with transforming activity. As tumor-specific JPO1 expression in human and murine medulloblastoma has also been reported; these collective observations suggest important functional links between the novel JPO protein family and c-Myc in medulloblastoma transformation.

Trimeric G protein-dependent frizzled signaling in Drosophila.

Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. Here, we show roles for the Galpha(o) subunit (Go) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. Go is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (Go-GTP) can signal when the receptor is removed. Thus, Go is likely part of a trimeric G protein complex that directly transduces Fz signals from the membrane to downstream components.

Drosophila cardiac tube organogenesis requires multiple phases of Hox activity.

The segmented Drosophila linear cardiac tube originates from two cell lineages that give rise to the anterior aorta (AA) and the posterior cardiac tube. The three Hox genes of the Bithorax Complex as well as Antennapedia (Antp) have been shown to be expressed in the posterior cardiac tube, while no Hox gene is expressed in the anterior aorta. We show that the cells of the whole tube adopt the anterior aorta identity in the complete absence of Hox function. Conversely, ectopic expression of Antp, Ultrabithorax (Ubx), or abdominal-A (abd-A) transformed the anterior aorta into posterior cardiac tube by all available criteria, indicating an equivalent early function in their ability to direct a posterior cardiac tube lineage. We further demonstrate that Hox genes act in a subsequent step during cardiac tube organogenesis, specifically on the differentiation of posterior cardiac tube myocytes. In addition, while some of these functions are fulfilled equally well by any one of the three Hox genes, some others are specific to a given Hox. Notably, the gene encoding the anion transporter Na+-Driven Anion Exchanger 1 behaves as a Hox differential transcriptional target and is activated by abd-A in the heart and repressed by Ubx in the posterior aorta. This analysis illustrates the mechanisms by which Hox genes can orchestrate organogenesis and, in particular, allows a clear uncoupling of the different phases of Hox activity in this process.

[Genetic control of cardiac tube formation in Drosophila]. / Contrôle génétique de la formation du tube cardiaque chez la Drosophile.

In Drosophila, the heart is composed of a simple linear tube constituted of 52 pairs of myoendothelial cells which differentiate during embryogenesis to build up a functional mature organ. The cardiac tube is a contractile organ with autonomous muscular activity which functions as a hemolymph pump in an open circulatory circuit. The cardiac tube is organized in metamers which contain six pairs of cardioblasts per segment. Within each metamer the cardioblasts express a combination of genetic markers underlying their functional diversity. For example, the two most posterior cardiac cells in segments A5 to A7 differentiate into ostiae which allow the inflow of hemolymph in the tube. An additional axial information along the anteroposterior axis orchestrates the subdivision of the cardiac tube into an "aorta" in the anterior region and a "heart" in the posterior region which behave as distinct functional entities. The major pacemaker activity is located in the most caudal part of the heart. This analysis has being made possible by the identification and the utilization of specific morphological and genetic markers and an in vivo observation of cardiac function in the embryo. Functional organogenesis of the cardiac tube is accurately controlled by genetic programs that have been in part identified. Hox genes are responsible for the axial subdivision of the tube into functional modules. They activate, in their specific domains of expression, target genes effectors of the terminal differentiation. On the other hand, part of the information required for segmental information is provided by Hedgehog, a morphogen secreted by dorsal ectoderm, whose activity triggers the ostiae formation in the heart domain.

Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the Bithorax Complex genes and hedgehog signaling.

The Drosophila larval cardiac tube is composed of 104 cardiomyocytes that exhibit genetic and functional diversity. The tube is divided into the aorta and the heart proper that encompass the anterior and posterior parts of the tube, respectively. Differentiation into aorta and heart cardiomyocytes takes place during embryogenesis. We have observed living embryos to correlate morphological changes occurring during the late phases of cardiogenesis with the acquisition of organ function, including functional inlets, or ostiae. Cardiac cells diversity originates in response to two types of spatial information such that cells differentiate according to their position, both within a segment and along the anteroposterior axis. Axial patterning is controlled by homeotic genes of the Bithorax Complex (BXC) which are regionally expressed within the cardiac tube in non-overlapping domains. Ultrabithorax (Ubx) is expressed in the aorta whereas abdominal A (abd-A) is expressed in the heart, with the exception of the four most posterior cardiac cells which express Abdominal B (Abd-B). Ubx and abd-A functions are required to confer an aorta or a heart identity on cardiomyocytes, respectively. The anterior limit of the expression domain of Ubx, abd-A and Abd-B is independent of the function of the other genes. In contrast, abd-A represses Ubx expression in the heart and ectopic overexpression of abd-A transforms aorta cells into heart cardiomyocytes. Taken together, these results support the idea that BXC homeotic genes in the cardiac tube conform to the posterior prevalence rule. The cardiac tube is also segmentally patterned and each metamere contains six pairs of cardioblasts that are genetically diverse. We show that the transcription of seven up (svp), which is expressed in the two most posterior pairs of cardioblasts in each segment, is dependent on hedgehog (hh) signaling from the dorsal ectoderm. In combination with the axial information furnished by abd-A, the segmental hh-dependent information leads to the differentiation of the six pairs of svp-expressing cells into functional ostiae.