PLoS One. 2017 Apr 25;12(4):e0176190.
DOI: 10.1371/journal.pone.0176190.

Pax7 remodels the chromatin landscape in skeletal muscle stem cells.

KC Lilja, N Zhang, A. Magli, V. Gunduz, CJ Bowman, RW Arpke, R. Darabi, M Kyba, R. Perlingeiro and BD Dynlacht

Pluripotent stem cells (PSC) hold great promise for the treatment of human skeletal muscle diseases. However, it remains challenging to convert PSC to skeletal muscle cells, and the mechanisms by which the master regulatory transcription factor, Pax7, promotes muscle stem (satellite) cell identity are not yet understood. We have taken advantage of PSC-derived skeletal muscle precursor cells (iPax7), wherein the induced expression of Pax7 robustly initiates the muscle program and enables the in vitro generation of precursors that seed the satellite cell compartment upon transplantation. Remarkably, we found that chromatin accessibility in myogenic precursors pre-figures subsequent activation of myogenic differentiation genes. We also found that Pax7 binding is generally restricted to euchromatic regions and excluded from H3K27 tri-methylated regions in muscle cells, suggesting that recruitment of this factor is circumscribed by chromatin state. Further, we show that Pax7 binding induces dramatic, localized remodeling of chromatin characterized by the acquisition of histone marks associated with enhancer activity and induction of chromatin accessibility in both muscle precursors and lineage-committed myoblasts. Conversely, removal of Pax7 leads to rapid reversal of these features on a subset of enhancers. Interestingly, another cluster of Pax7 binding sites is associated with a durably accessible and remodeled chromatin state after removal of Pax7, and persistent enhancer accessibility is associated with subsequent, proximal binding by the muscle regulatory factors, MyoD1 and myogenin. Our studies provide new insights into the epigenetic landscape of skeletal muscle stem cells and precursors and the role of Pax7 in satellite cell specification.

Journal of Biological Chemistry, March 14, 2017
pii: jbc.M117.783662. doi: 10.1074/jbc.M117.783662. [Epub ahead of print]

Rnf11 sequestration of Smurf2 on membranes antagonizes Smad7 downregulation of TGFB signaling

Ryan J. Malonis, Wenxiang Fu, Mark J. Jelcic, Marae Thompson, Brian S. Canter, Mary Tsikitis, Francisco J. Esteva and Irma Sánchez

The activity of the E3 ligase, Smurf2, is antagonized by an intra-molecular, auto-inhibitory interaction between its C2 and Hect domains. Relief of Smurf2 auto-inhibition is induced by TGFβ and is mediated by the inhibitory SMAD, Smad7. In a proteomic screen for endo-membrane interactants of the RING-domain E3 ligase, Rnf11, we identified Smurf2, among a cohort of Hect E3 ligases previously implicated in TGFβ signaling. Reconstitution of the Smurf2/Rnf11 complex in vitro unexpectedly revealed robust Smurf2 E3 ligase activity, with biochemical properties previously restricted to the Smurf2/Smad7 complex. Using in vitro binding assays, we find that Rnf11 can directly compete with Smad7 for Smurf2 and that binding is mutually exclusive and dependent on a proline-rich domain. Moreover, we found that co-expression of Rnf11 and Smurf2 dramatically reduced Smurf2 ubiquitylation in the cell. This effect is strictly dependent on complex formation and sorting determinants that regulate the association of Rnf11 with membranes. Rnf11 is over-expressed in certain tumors, and, importantly, we find that depletion of this protein down-regulated gene expression of several TGFβ-responsive genes, dampened cell proliferation, and dramatically reduced cell migration in response to TGFβ. Our data suggest for the first time that choice of binding partners for Smurf2 can sustain or repress TGFβ signaling and Rnf11 may promote TGFβ-induced cell migration

Cell Reports, November 1, 2016 : 17(6): 1505-1517

Role for IFT-A complex in selective transport in the primary cilium

Wenxiang Fu, Lei Wang, Sehyun Kim, Ji Li and Brian D. Dynlacht

Intraflagellar 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. Beyond its role in retrograde transport, we show that Wdr35 functions in fusion of Rab8 vesicles at the nascent cilium, protein exit from the cilium, and centriolar satellite organization. Furthermore, we show that Wdr35 is essential for entry of many membrane proteins into the cilium through robust interactions with cargoes and other IFT-A subunits, but the actin network functions to dampen this transport. Wdr35 is mutated in several ciliopathies, and we find that certain disease mutations impair interactions with cargo and other IFT-A subunits. Together, our data link defects in IFT-A mediated cargo transport with disease.

eLife 2016;5:e12950
Published May 5, 2016

Tethering of an E3 ligase by PCM1 regulates the abundance of centrosomal KIAA0586/Talpid3 and promotes ciliogenesis

Lei Wang Kwanwoo Lee Ryan Malonis Irma Sánchez Brian D Dynlacht

To elucidate the role of centriolar satellites in ciliogenesis, we deleted the gene encoding the PCM1 protein, an integral component of satellites. PCM1 null human cells show marked defects in ciliogenesis, precipitated by the loss of specific proteins from satellites and their relocation to centrioles. We find that an amino-terminal domain of PCM1 can restore ciliogenesis and satellite localization of certain proteins, but not others, pinpointing unique roles for PCM1 and a group of satellite proteins in cilium assembly. Remarkably, we find that PCM1 is essential for tethering the E3 ligase, Mindbomb1 (Mib1), to satellites. In the absence of PCM1, Mib1 destabilizes Talpid3 through poly-ubiquitylation and suppresses cilium assembly. Loss of PCM1 blocks ciliogenesis by abrogating recruitment of ciliary vesicles associated with the Talpid3-binding protein, Rab8, which can be reversed by inactivating Mib1. Thus, PCM1 promotes ciliogenesis by tethering a key E3 ligase to satellites and restricting it from centrioles.

PLOS Genetics : January 14, 2016 DOI:10.1371/journal.pgen.1005794

PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation

Yang Yan, Li W, Hoque M. ,Liming H, Shen Steven, Tian Bian, and Brian D. Dynlacht

Gene transcription can be regulated through multiple mechanisms, such as histone modifications that create structural changes of the chromatin leading to gene activation or suppression, or regulation of the 3. cleavage site of the mRNA, known as alternative cleavage and polyadenylation (APA), resulting in the generation of transcript isoforms with various lengths. Here we present genome-wide subunit-specific roles of the PAF complex (Paf1C) related to both mechanisms of transcriptional regulation. Using mouse muscle cells, we show contrasting results with yeast, namely, that depletion of Paf1C subunits does not affect certain histone modifications previously associated with this complex and that the complex exhibits subunit-specific functions. We also discovered a novel role of Paf1C in APA, wherein genome-wide transcript shortening occurs after depletion of three of the subunits. However, APA varies after depletion of certain subunits, reinforcing our conclusions regarding subunit specificity. Furthermore, by comparing depletions of two subunits, we show that the accumulation of RNA polymerase II (PolII) near the transcription start site (TSS) is specifically associated with the activation of TSS-proximal pA sites observed in one depletion but not the other.

Nature Communications: 2015 Aug 20;6:8087. doi:10.1038/ncomms9087

Nek2 activation of Kif24 ensures cilium disassembly during the cell cycle

Kim, Sehyun, Lee Kwanwoo, Choi JH, Ringstad Niels and Dynlacht Brian David.

Many 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. We show that Kif24, a microtubule depolymerizing kinesin, is phosphorylated by Nek2, which stimulates its activity and prevents the outgrowth of cilia in proliferating cells, independent of Aurora A and HDAC6. Our data also suggest that cilium assembly and disassembly are in dynamic equilibrium, but Nek2 and Kif24 can shift the balance toward disassembly. Further, Nek2 and Kif24 are overexpressed in breast cancer cells, and ablation of these proteins restores ciliation in these cells, thereby reducing proliferation. Thus, Kif24 is a physiological substrate of Nek2, which regulates cilia disassembly through a concerted mechanism involving Kif24-mediated microtubule depolymerization.

Nature Cell Biology: online 17 November 2014; DOI: 10.1038/ncb3062

Foxk proteins repress the initiation of starvation-induced atrophy and autophagy programs

Bowman, CJ, Ayer DE , and Dynlacht ,BD

Autophagy 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. Here, we identify Foxk proteins (Foxk1 and Foxk2) as transcriptional repressors of autophagy in muscle cells and fibroblasts. Interestingly, Foxk1/2 serve to counter-balance another forkhead transcription factor, Foxo3, which induces an overlapping set of autophagic and atrophic targets in muscle. Foxk1/2 specifically recruits Sin3A–HDAC complexes to restrict acetylation of histone H4 and expression of critical autophagy genes. Remarkably, mTOR promotes the transcriptional activity of Foxk1 by facilitating nuclear entry to specifically limit basal levels of autophagy in nutrient-rich conditions. Our study highlights an ancient, conserved mechanism whereby nutritional status is interpreted by mTOR to restrict autophagy by repressing essential autophagy genes through Foxk–Sin3-mediated transcriptional control.

PNAS 2014 ; published ahead of print June 9, 2014, doi:10.1073/pnas.1323265111

Primary cilia control Hedgehog signaling during muscle differentiationand are deregulated in rhabdomyosarcoma

Fu W, Asp P, Canter B and Dynlacht BD

The primary cilium is an organelle emanating from the cell surface, and recent evidence suggests that it regulates various cellular signaling pathways and development and that aberrations in its assembly and function could result in tumorigenesis. Skeletal muscle differentiation proceeds through a temporally defined series of events to form multinucleated myofibers, and in rhabdomyosarcomas (RMS), myoblasts fail to differentiate. However, whether primary cilia are functionally linked to normal muscle differentiation or RMS is not known. In this study we show that in skeletal myoblasts, primary cilia are important for proliferation, hedgehog signaling, and differentiation. Furthermore, the deregulation of cilia and hedgehog in RMS could suggest the utility of therapeutically targeting ciliary components in these tumors.

Journal of Cell Biology, January 13, 2014, doi10.1083/jcb.201304153

The CP110-interacting proteins Talpid3 and Cep290 play overlapping roles in cilia assembly. 

Kobayashi T, Kim S, Lin YC, Inoue T, Dynlacht BD.

We 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. Talpid3 depletion also led to mislocalization of Rab8a, a small GTPase thought to be essential for ciliary vesicle formation. Expression of activated Rab8a suppressed cilia assembly defects provoked by Talpid3 depletion, suggesting that Talpid3 affects cilia formation through Rab8a recruitment and/or activation. Remarkably, ultrastructural analyses showed that Talpid3 is required for centriolar satellite dispersal, which precedes the formation of mature ciliary vesicles, a process requiring Cep290. These studies suggest that Talpid3 and Cep290 play overlapping and distinct roles in ciliary vesicle formation through regulation of centriolar satellite accretion and Rab.

Molecular Cell, March 20, 2014, Vol 53 No. 6, pg 979-992

A Role for H3K4 Monomethylation in Gene Repression and Partitioning of Chromatin Readers

Cheng J, Blum R, Bowman, C, Hu D, Shilatifard A, Shen S, Dynlacht BD

Monomethylation of lysine 4 on histone H3 (H3K4me1) is a well-established feature of enhancers and promoters, although its function is unknown. Here, we uncover roles for H3K4me1 in diverse cell types. Remarkably, we find that MLL3/4 provokes monomethylation of promoter regions and the conditional repression of muscle and inflammatory response genes in myoblasts. During myogenesis, muscle genes are activated, lose MLL3 occupancy, and become H3K4-trimethylated through an alternative COMPASS complex. Monomethylation-mediated repression was not restricted to skeletal muscle. Together with H3K27me3 and H4K20me1, H3K4me1 was associated with transcriptional silencing in embryonic fibroblasts, macrophages, and human embryonic stem cells (ESCs). On promoters of active genes, we find that H3K4me1 spatially demarcates the recruitment of factors that interact with H3K4me3, including ING1, which, in turn, recruits Sin3A. Our findings point to a unique role for H3K4 monomethylation in establishing boundaries that restrict the recruitment of chromatin-modifying enzymes to defined regions within promoters.