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The transcriptional mechanisms driving lineage specification during development are still largely unknown, as the interplay of multiple transcription factors makes it difficult to dissect these molecular events. Using a cell-based differentiation platform to probe transcription function, we investigated the role of the key paraxial mesoderm and skeletal myogenic commitment factors-mesogenin 1 (Msgn1), T-box 6 (Tbx6), forkhead box C1 (Foxc1), paired box 3 (Pax3), Paraxis, mesenchyme homeobox 1 (Meox1), sine oculis-related homeobox 1 (Six1), and myogenic factor 5 (Myf5)-in paraxial mesoderm and skeletal myogenesis. From this study, we define a genetic hierarchy, with Pax3 emerging as the gatekeeper between the presomitic mesoderm and the myogenic lineage. By assaying chromatin accessibility, genomic binding and transcription profiling in mesodermal cells from mouse and human Pax3-induced embryonic stem cells and Pax3-null embryonic day (E)9.5 mouse embryos, we identified conserved Pax3 functions in the activation of the skeletal myogenic lineage through modulation of Hedgehog, Notch, and bone morphogenetic protein (BMP) signaling pathways. In addition, we demonstrate that Pax3 molecular function involves chromatin remodeling of its bound elements through an increase in chromatin accessibility and cooperation with sine oculis-related homeobox 4 (Six4) and TEA domain family member 2 (Tead2) factors. To our knowledge, these data provide the first integrated analysis of Pax3 function, demonstrating its ability to remodel chromatin in mesodermal cells from developing embryos and proving a mechanistic footing for the transcriptional hierarchy driving myogenesis.

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.

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.

The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and RNA polymerase II (PolII) elongation. Here, we provide the first genome-wide profiles for the distribution of the entire complex in mammalian cells using chromatin immunoprecipitation and high throughput sequencing. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3' ends, a profile that sharply contrasted with the yeast complex. Remarkably, we identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription using RNAi coupled with deep sequencing of the 3' ends of transcripts. Moreover, we found that depletion of Paf1C subunits resulted in the accumulation of PolII over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits led to global loss of histone H2B ubiquitylation, although there was little impact of Paf1C depletion on other histone modifications, including tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and indicate that Paf1C subunits could play roles in controlling transcript length through suppression of PolII accumulation at transcription start site (TSS)-proximal pA sites and regulating pA site choice in 3'UTRs.

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies.

Abstract Background Selective proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during eukaryotic development, generating a cleaved histone H3 (H3cl) product within a small, but significant, portion of the genome. Although increasing evidence supports a regulatory role for H3NT proteolysis in gene activation, the nuclear H3NT proteases and the biological significance of H3NT proteolysis remain largely unknown. Results In this study, established cell models of skeletal myogenesis were leveraged to investigate H3NT proteolysis. These cells displayed a rapid and progressive accumulation of a single H3cl product within chromatin during myoblast differentiation. Using conventional approaches, we discovered that the canonical extracellular matrix (ECM) protease, matrix metalloproteinase 2 (MMP-2), is the principal H3NT protease of myoblast differentiation that cleaves H3 between K18-Q19. Gelatin zymography demonstrated progressive increases in nuclear MMP-2 activity, concomitant with H3cl accumulation, during myoblast differentiation. RNAi-mediated depletion of MMP-2 impaired H3NT proteolysis and resulted in defective myogenic gene activation and myoblast differentiation. Supplementation of MMP-2 ECM activity in MMP-2-depleted cells was insufficient to rescue defective H3NT proteolysis and myogenic gene activation. Conclusions This study revealed that MMP-2 is a novel H3NT protease and the principal H3NT protease of myoblast differentiation. The results indicate that myogenic signaling induces MMP-2-dependent H3NT proteolysis at early stages of myoblast differentiation. Importantly, the results support the necessity of nuclear MMP-2 H3NT protease activity, independent of MMP-2 activity in the ECM, for myogenic gene activation and proficient myoblast differentiation.

We present a fast, versatile and adaptive-multiscale algorithm for analyzing a wide-variety of DNA microarray data. Its primary application is in normalization of array data as well as subsequent identification of ‘enriched targets’, e.g. differentially expressed genes in expression profiling arrays and enriched sites in ChIP-on-chip experimental data.We show how to accommodate the unique characteristics of ChIP-on-chip data, where the set of ‘enriched targets’ is large, asymmetric and whose proportion to the whole data varies locally.Contact:lerman@umn.eduSupplementary information: Supplementary figures, related preprint, free software as well as our raw DNA microarray data with PCR validations are available at http://www.math.umn.edu/~lerman/supp/bioinfo06 as well as Bioinformatics online. [ABSTRACT FROM AUTHOR]

Abstract The study of global transcriptional regulatory networks in eukaryotes has advanced rapidly in the last five years with the advent of genomic microarrays that allow investigators to comprehensively scrutinize the structure and composition of transcriptomes and chromatin. Many facets of this field have benefited from an examination of the location of proteins associated with chromatin. We describe here how we have used genomic arrays to perform location analysis (or ChIP-on-chip analysis) of regulatory factors that control the complex process of cell cycle progression and differentiation, in order to identify key direct transcriptional targets. Combining this information with data obtained through expression profiling and computational analysis of transcription factor binding sites has also allowed us to devise transcriptional regulatory networks governing these processes. [ABSTRACT FROM AUTHOR]

Chromatin looping allows for enhancer–promoter interactions that regulate transcription. Here the authors show that activation of embryonic myogenesis is associated with establishment of long-range chromatin interactions centred on Pax3-bound loci and find that Ldb1 functions as the mediator of looping interactions at a subset of Pax3 binding sites.

Removal of daughter centriole-specific/enriched proteins (DCPs) and assembly of distal appendages (DA) are important for centrosome asymmetry. Here, the authors report that a centriolar distal end protein network regulates DCP removal, DA assembly, and ciliary vesicle docking.