Alveolar rhabdomyosarcoma (ARMS) patients harboring PAX3-FOXO1 and PAX7-FOXO1 fusion proteins exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. To uncover mechanisms that contribute to tumor initiation, we developed a novel muscle progenitor model and used epigenomic approaches to unravel genome re-wiring events mediated by PAX3/7 fusion proteins. Importantly, these regulatory mechanisms are conserved across established ARMS cell lines, primary tumors, and orthotopic-patient derived xenografts.
View Article and Find Full Text PDFClustering of supernumerary centrosomes, which potentially leads to cell survival and chromosomal instability, is frequently observed in cancers. However, the molecular mechanisms that control centrosome clustering remain largely unknown. The centrosomal kinesin KIF24 was previously shown to restrain the assembly of primary cilia in mammalian cells.
View Article and Find Full Text PDFUsing Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterize at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. We identify key promoter-enhancer interaction motifs, namely, cliques and networks, and interactions that are dependent on Pax7 binding. Remarkably, Pax7 binds to a majority of super-enhancers, and together with a cadre of interacting transcription factors, assembles feed-forward regulatory loops.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
July 2019
Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors.
View Article and Find Full Text PDFLoss of primary cilia is frequently observed in tumor cells, including pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction and the inability to exit the cell cycle. However, the molecular mechanisms that explain how PDAC cells lose primary cilia are still ambiguous. In this study, we found that inhibition or silencing of histone deacetylase 2 (HDAC2) restores primary cilia formation in PDAC cells.
View Article and Find Full Text PDFIntraflagellar transport sub-complex A (IFT-A) is known to regulate retrograde IFT in the cilium. To rigorously assess its other possible roles, we knocked out an IFT-A subunit, IFT121/WDR35, in mammalian cells and screened the localization of more than 50 proteins. We found that Wdr35 regulates cilium assembly by selectively regulating transport of distinct cargoes.
View Article and Find Full Text PDFThe primary cilium is an antenna-like, immotile organelle present on most types of mammalian cells, which interprets extracellular signals that regulate growth and development. Although once considered a vestigial organelle, the primary cilium is now the focus of considerable interest. We now know that ciliary defects lead to a panoply of human diseases, termed ciliopathies, and the loss of this organelle may be an early signature event during oncogenic transformation.
View Article and Find Full Text PDFMany proteins are known to promote ciliogenesis, but mechanisms that promote primary cilia disassembly before mitosis are largely unknown. Here we identify a mechanism that favours cilium disassembly and maintains the disassembled state. We show that co-localization of the S/G2 phase kinase, Nek2 and Kif24 triggers Kif24 phosphorylation, inhibiting cilia formation.
View Article and Find Full Text PDFThe primary cilium is an antenna-like, microtubule-based organelle on the surface of most vertebrate cells for receiving extracellular information. Although primary cilia form in the quiescent phase, ciliary disassembly occurs when quiescent cells re-enter the proliferative phase. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for cell-proliferation-coupled primary cilia disassembly.
View Article and Find Full Text PDFAutophagy is the primary catabolic process triggered in response to starvation. Although autophagic regulation within the cytosolic compartment is well established, it is becoming clear that nuclear events also regulate the induction or repression of autophagy. Nevertheless, a thorough understanding of the mechanisms by which sequence-specific transcription factors modulate expression of genes required for autophagy is lacking.
View Article and Find Full Text PDFThe primary cilium acts as a cellular antenna, transducing diverse signaling pathways, and recent evidence suggests that primary cilia are important in development and cancer. However, a role for cilia in normal muscle development and rhabdomyosarcoma (RMS) has not been explored. Here we implicate primary cilia in proliferation, hedgehog (Hh) signaling, and differentiation of skeletal muscle cells.
View Article and Find Full Text PDFWe have identified Talpid3/KIAA0586 as a component of a CP110-containing protein complex important for centrosome and cilia function. Talpid3 assembles a ring-like structure at the extreme distal end of centrioles. Ablation of Talpid3 resulted in an aberrant distribution of centriolar satellites involved in protein trafficking to centrosomes as well as cilia assembly defects, reminiscent of loss of Cep290, another CP110-associated protein.
View Article and Find Full Text PDFCilia are evolutionarily conserved, membrane-bound, microtubular projections emanating from the cell surface. They are assembled on virtually all cell types in the human body, with very few exceptions, and several recent reviews have covered the topic in great detail. The cilium is assembled from mature (mother) centrioles or basal bodies, which serve to nucleate growth of axonemes that give rise to two structurally distinct variants, motile and nonmotile cilia.
View Article and Find Full Text PDFCentrosome duplication is critical for cell division, and genome instability can result if duplication is not restricted to a single round per cell cycle. Centrosome duplication is controlled in part by CP110, a centriolar protein that positively regulates centriole duplication while restricting centriole elongation and ciliogenesis. Maintenance of normal CP110 levels is essential, as excessive CP110 drives centrosome over-duplication and suppresses ciliogenesis, whereas its depletion inhibits centriole amplification and leads to highly elongated centrioles and aberrant assembly of cilia in growing cells.
View Article and Find Full Text PDFWe have identified a protein, Kif24, that shares homology with the kinesin-13 subfamily of motor proteins and specifically interacts with CP110 and Cep97, centrosomal proteins that play a role in regulating centriolar length and ciliogenesis. Kif24 preferentially localizes to mother centrioles. Loss of Kif24 from cycling cells resulted in aberrant cilia assembly but did not promote growth of abnormally long centrioles, unlike CP110 and Cep97 depletion.
View Article and Find Full Text PDFWe have examined changes in the chromatin landscape during muscle differentiation by mapping the genome-wide location of ten key histone marks and transcription factors in mouse myoblasts and terminally differentiated myotubes, providing an exceptionally rich dataset that has enabled discovery of key epigenetic changes underlying myogenesis. Using this compendium, we focused on a well-known repressive mark, histone H3 lysine 27 trimethylation, and identified novel regulatory elements flanking the myogenin gene that function as a key differentiation-dependent switch during myogenesis. Next, we examined the role of Polycomb-mediated H3K27 methylation in gene repression by systematically ablating components of both PRC1 and PRC2 complexes.
View Article and Find Full Text PDFCurrent models posit that E2F transcription factors can be divided into members that either activate or repress transcription, in part through collaboration with the retinoblastoma (pRb) tumor suppressor family. The E2f3 locus encodes E2f3a and E2f3b proteins, and available data suggest that they regulate cell cycle-dependent gene expression through opposing transcriptional activating and repressing activities in growing and quiescent cells, respectively. However, the role, if any, of E2F proteins, and in particular E2f3, in myogenic differentiation is not well understood.
View Article and Find Full Text PDFThe multisubunit Sin3 corepressor complex regulates gene transcription through deacetylation of nucleosomes. However, the full range of Sin3 activities and targets is not well understood. Here, we have investigated genome-wide binding of mouse Sin3 and RBP2 as well as histone modifications and nucleosome positioning as a function of myogenic differentiation.
View Article and Find Full Text PDFPrimary cilia are nonmotile organelles implicated in signaling and sensory functions. Understanding how primary cilia assemble could shed light on the many human diseases caused by mutations in ciliary proteins. The centrosomal protein CP110 is known to suppress ciliogenesis through an unknown mechanism.
View Article and Find Full Text PDFMdm2, as the most important negative regulator of p53, plays an important homeostatic role in regulating cell division and the cellular response to DNA damage, oncogenic insult and other forms of cellular stress. We discovered that the DNA damaging agent adriamycin (doxorubicin) induces a novel aberrantly spliced Mdm2 mRNA which incorporates 108 bp of intronic sequence not normally found in the Mdm2 mature mRNA. Accordingly, we term this Mdm2 splice variant Mdm2(+108).
View Article and Find Full Text PDFSgo1 plays a key role in protecting sister chromatid cohesion during mitosis. In this issue of Developmental Cell, Wang et al. describe a shorter splice variant of Sgo1 (sSgo1) that functions specifically in centriole cohesion.
View Article and Find Full Text PDFCyclin A is a key regulator of DNA replication and mitosis, and cyclin A/Cdk2 activity is critical for progression of cells from G(1)/S to M phase of the cell cycle. There is abundant evidence that cyclin A is predominantly localized to, and functions in, the nucleus, and a number of nuclear cyclin A/Cdk2 substrates have been identified. Evidence supporting the presence of cyclin A and its associated activity in the cytoplasm have also been reported, but the biological significance of this cyclin/Cdk pool during cell cycle progression remains controversial.
View Article and Find Full Text PDFIn the current issue of Cancer Cell, Hallstrom et al. show that a subset of targets of the growth regulatory transcription factor E2F1 are repressed by a serum-induced PI3K activation, explaining how apoptosis can be suppressed while simultaneously engaging a proliferation program.
View Article and Find Full Text PDFThe retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes.
View Article and Find Full Text PDFMammalian centrioles play a dynamic role in centrosome function, but they also have the capacity to nucleate the assembly of cilia. Although controls must exist to specify these different fates, the key regulators remain largely undefined. We have purified complexes associated with CP110, a protein that plays an essential role in centrosome duplication and cytokinesis, and have identified a previously uncharacterized protein, Cep97, that recruits CP110 to centrosomes.
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