干细胞-胡萍

10.1101/gad.2021411Access the most recent version at doi: 2011 25: 569-580 originally published online February 28, 2011Genes Dev. Jie Yao, Richard D. Fetter, Ping Hu, et al. myogenesis Subnuclear segregation of genes and core promoter factors in Material Supplemental http://genesdev.cshlp.org/content/suppl/2011/02/28/gad.2021411.DC1.html References http://genesdev.cshlp.org/content/25/6/569.full.html#related-urls Article cited in: http://genesdev.cshlp.org/content/25/6/569.full.html#ref-list-1 This article cites 61 articles, 19 of which can be accessed free at: Open Access Freely available online through the Genes & Development Open Access option. service Email alerting click heretop right corner of the article or Receive free email alerts when new articles cite this article - sign up in the box at the http://genesdev.cshlp.org/subscriptions go to: Genes & DevelopmentTo subscribe to Copyright © 2011 by Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press on March 16, 2012 - Published by genesdev.cshlp.orgDownloaded from Today Typewritten Text 干细胞 Subnuclear segregation of genes and core promoter factors in myogenesis Jie Yao,1,2 Richard D. Fetter,1 Ping Hu,2 Eric Betzig,1 and Robert Tjian1,2,3 1Janelia Farm Research Campus, The Single Cell Biochemistry Consortium, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA; 2Department of Molecular and Cell Biology, Li Kashing Center For Biomedical and Health Sciences, CIRM Center of Excellence, University of California at Berkeley, Berkeley, California 94720, USA Recent findings implicate alternate core promoter recognition complexes in regulating cellular differentiation. Here we report a spatial segregation of the alternative core factor TAF3, but not canonical TFIID subunits, away from the nuclear periphery, where the key myogenic gene MyoD is preferentially localized in myoblasts. This segregation is correlated with the differential occupancy of TAF3 versus TFIID at theMyoD promoter. Loss of this segregation by modulating either the intranuclear location of the MyoD gene or TAF3 protein leads to altered TAF3 occupancy at the MyoD promoter. Intriguingly, in differentiated myotubes, the MyoD gene is repositioned to the nuclear interior, where TAF3 resides. The specific high-affinity recognition of H3K4Me3 by the TAF3 PHD (plant homeodomain) finger appears to be required for the sequestration of TAF3 to the nuclear interior. We suggest that intranuclear sequestration of core transcription components and their target genes provides an additional mechanism for promoter selectivity during differentiation. [Keywords: transcription; nucleus; MyoD; core promoter factors; superresolution microscopy] Supplemental material is available for this article. Received December 12, 2010; revised version accepted January 24, 2011. The regulation of gene transcription plays a seminal role in the development and differentiation of cell types in multicellular organisms. Significant progress has been made in the identification of transcription factors, and genome-wide mapping of their cognate binding sites has accelerated with the development of massively parallel DNA sequencing capabilities (Farnham 2009). Despite this rapid progress in dissecting the biochemistry of transcrip- tion, the question of how these gene regulatory factors find their target promoters in the cell nucleus remains poorly understood. Genomic DNA in eukaryotic cells is com- pacted by histone proteins to form chromatin—highly folded and condensed protein/DNA structures inside the nucleus (Cremer and Cremer 2001; Spector 2003). Live-cell imaging analysis suggests that many transcrip- tion factors rapidly diffuse across the nucleus and tran- siently bind to their target genes (Darzacq et al. 2009; Hager et al. 2009). Importantly, it has been recognized that genes are nonrandomly distributed in the nucleus and with respect to chromatin territories (Misteli 2007; Kumaran et al. 2008; Sinclair et al. 2010), and that gene activation and cellular differentiation may be accompa- nied by gene repositioning (Moen et al. 2004; Chuang et al. 2006; Meister et al. 2010). Although the position of a gene in the nucleus does not obligatorily determine its activity (Yao et al. 2007; Kumaran et al. 2008), transcrip- tion factors must be able to navigate the cell nucleus and access target genes in order to activate transcription. Thus, an important but challenging question that has largely escaped analysis is whether access and targeting of tran- scription factors to specific nuclear subcompartments can influence and regulate transcription output. From a techni- cal standpoint, although live-cell imaging provides some measurement of mobility and kinetics of populations of transcription factor molecules in the nucleus, individual transcription factor molecules are not readily visiblewithin the context of nuclear architecture using conventional light microscopy. Recent advances in fluorescence micros- copy with single-molecule resolution provide us an oppor- tunity to revisit this problem of transcription factor acces- sibility and selective utilization at target gene promoters. Another challenge to accurate subnuclear positioning of regulatory factors is the relative paucity of spatial landmarks within the nucleus. One readily recognizable positional element is the nuclear periphery, demarcated by structures at the inner surface of the nuclear envelope (Akhtar and Gasser 2007; Lusk et al. 2007). Interactions of chromatin domains with the nuclear lamina (NL) have been identified during embryonic stem cell differentiation 3Corresponding author. E-MAIL jmlim@berkeley.edu; FAX (510) 643-9547. Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.2021411. Freely available online through the Genes & Development Open Access option. GENES & DEVELOPMENT 25:569–580 � 2011 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/11; www.genesdev.org 569 Cold Spring Harbor Laboratory Press on March 16, 2012 - Published by genesdev.cshlp.orgDownloaded from (Peric-Hupkes et al. 2010). In yeast, components of the nuclear periphery have been implicated in both repres- sion and activation of gene transcription (Andrulis et al. 1998; Schmid et al. 2006). In mammalian cells, experi- ments that tether reporter genes to the nuclear periph- ery have resulted in differential expression of some, but not all, reporters, as well as adjacent endogenous genes (Finlan et al. 2008; Kumaran and Spector 2008; Reddy et al. 2008). We speculate that identifying transcription factors that exhibit differential access to the nuclear periphery may be informative in exploring the potential roles of nuclear organizations of genes and proteins as a mechanism of gene control. Our recent analysis of skeletal myogenesis suggests that alternate core promoter recognition factors may play a hitherto unappreciated role in regulating cell type- specific transcription (Deato and Tjian 2007; Deato et al. 2008; D’Alessio et al. 2009; Goodrich and Tjian 2010). During muscle formation, the MyoD gene is expressed in both myoblasts and later in differentiated myotubes and acts as a key regulatory factor driving myogenic differentiation (Tapscott et al. 1988). In con- trast, theMyogenin gene is turned on only after myocytes exit the cell cycle and begin to fuse, thus activating genes at later stages of differentiation (Edmondson and Olson 1989). Another recently uncovered transcriptional event associated with skeletal myogenesis was the unexpected loss of the prototypic core promoter recognition complex TFIID (Deato and Tjian 2007). It had beenwell established that, in rapidly growing cells, the multisubunit core transcription complex TFIID is essential for promoter rec- ognition and potentiating activated transcription from yeast to humans (Naar et al. 2001). TAF3 is a substoichio- metric TFIID subunit first identified in Drosophila (Gangloff et al. 2001), and later shown to interact with the histone mark H3K4Me3 (Vermeulen et al. 2007). Remarkably, during myoblast-to-myotube differentiation of mouse C2C12 cell culture and during muscle develop- ment in vivo, TFIID is largely eliminated, and, instead, TAF3 can be detected associated with the core promoter of the late-expressing Myogenin gene (Deato and Tjian 2007). Curiously, in myoblasts, TFIID and TAF3 are both present in the same nucleus, but how myogenic genes differentially use TFIID versus TAF3 in myoblasts posed an intriguing conundrum. Here we tracked two key myogenic genes—MyoD and Myogenin—and alternate core promoter recognition fac- tors by fluorescence in situ hybridization (FISH), immu- nofluorescence staining, and dual-color photoactivation localization microscopy (PALM). By employing ‘‘super- resolution’’ PALM-based cell imaging approaches, we more precisely localized individual transcription factor molecules within distinct nuclear regions. We found that, in myoblasts, canonical TFIID subunits are present at the nuclear periphery, where the MyoD gene preferentially resides, while TAF3 is largely segregated from the nuclear periphery; this differential subnuclear distribution of TFIID versus TAF3 is correlated with their selective occu- pancies at theMyoD promoter inmyoblasts. In contrast, in myotubes, where TFIID is lost, MyoD becomes reposi- tioned to the nuclear interior, where TAF3 resides, and this is accompanied by an increased occupancy of TAF3 at the MyoD promoter. Furthermore, by ectopically modulating the locations of theMyoD promoter and/or TAF3 protein, we show that their spatial segregation is functionally linked to the selective occupancy of TAF3 at the MyoD promoter. We also found that specific recognition and high-affinity binding by the TAF3 plant homeodomain (PHD) finger to the histone mark H3K4Me3 may be required for the sequestration of TAF3 to the nuclear interior. These studies suggest that differential nuclear compartmentalization of target genes and regulatory fac- tors may provide an additional mechanism for promoter selectivity during differentiation of animal cells. Results Nuclear locations of key myogenic genes in myoblasts To begin this study, we determined the positions ofMyoD by immuno-DNA FISH in mouse C2C12 myoblasts. Visualization of the nuclear periphery by an antibody against nuclear Lamin B shows that the MyoD gene is preferentially localized to the nuclear periphery (Fig. 1A,B; Figure 1. FISH analysis of MyoD and Myoge- nin gene loci in C2C12 myoblasts. (A,B) DNA FISH of MyoD gene (red) in myoblasts. (A) The nuclear periphery is highlighted by anti-Lamin B (green). Other micrographs follow the same color scheme. (B) Frequency histogram versus the distance of MyoD genes from the NL. B, D, and F are frequency histograms versus distance from genes to the NL in the corresponding FISH experiments shown in A, C, and E, respectively. (C,D) RNA FISH of MyoD gene in myoblasts. Kolgomorov-Smirnov (K-S) test of distributions in B and D: P = 0.50. (E,F) DNA FISH of the Myogenin gene (red) in myoblasts. K-S test of distributions in B and F: P < 0.001. Fisher’s exact tests of MyoD and Myogenin genes that are located within 0.6-, 0.8-, or 1.0-mm distance to the NL: P < 0.0001 in all cases. Bars, 5 mm. Yao et al. 570 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press on March 16, 2012 - Published by genesdev.cshlp.orgDownloaded from Supplemental Fig. S1A), in agreement with a previous study (Lee et al. 2006). Importantly, nascent MyoD tran- scripts were visualized through immuno-RNA FISH, and these transcripts were also located at the nuclear periph- ery (Fig. 1C,D), confirming that the peripherally localized MyoD genes are transcriptionally active. In contrast, the Myogenin gene is inactive in myoblasts, and we found that this ‘‘later’’ gene is located largely to the nuclear interior in myoblasts (Fig. 1E,F; Supplemental Fig. S1B). Chromatin immunoprecipitation (ChIP) assays confirm that RNA polymerase II (Pol II) occupancy is enhanced at the MyoD promoter relative to the MyoG promoter in myoblasts (Supplemental Fig. S1C). These findings reveal that two key temporally regulated myogenic genes are differentially localized within the myoblast nucleus, posing intriguing potential mechanisms for their differ- ential regulation. Localizing general transcription factors in myoblasts The MyoD gene is actively transcribed in myoblasts by the codependent action of upstream activators and req- uisite core promoter recognition complexes (Hu et al. 2008). In the case ofMyoD transcription inmyoblasts, the prototypic core factor TFIID occupies the MyoD pro- moter. Because theMyoD gene is preferentially localized at the nuclear periphery in myoblasts (Fig. 1), we set out to visualize which components of the transcription apparatus are colocalized at the nuclear periphery. We investigated the localizations of Pol II and TFIID by immunofluorescence staining and high-resolution multi- color confocal microscopy. As expected, Pol II is diffusely localized throughout the nucleoplasm, including the zone at the nuclear periphery in myoblasts (Fig. 2A,B). Furthermore, TAF11, TAF4, and TBP subunits of TFIID Figure 2. Immunofluorescence staining of several components of core transcription machinery in C2C12 myoblasts. (A, panel i) RNA polymerase II stained with 4H8 anti- body (green) with anti-Lamin B (red). (Panel ii) The image in panel i superimposed by DNA staining with Hoechst 33342 (blue). (Panel iii) The radial intensity plot inte- grated over the entire contour of nuclear lamin from representative images (n = 8). Error bars are standard deviations. The same organization follows for the rest of sub- panels. (B) Ser5-phosphorylated RNA Pol II (n = 3). (C) TAF11 (n = 3). (D) TAF4 (n = 3). (E) TBP (n = 3). (F) TAF3 (n = 6). (G) Intensity plots of antibody staining signals in com- parison: (Panel i) TAF3, TAF4, and TAF11. One-way ANOVA test of GFP intensity values between 0 and 0.4 mm from the lamina: P < 0.001. (Panel ii) TAF3, Pol II (4H8), and Pol II (Ser5P): P < 0.04. (Panel iii) TAF4, Pol II (4H8), and Pol II (Ser5P): P < 0.04. Bars, 2 mm. Subnuclear locations of core promoter factors GENES & DEVELOPMENT 571 Cold Spring Harbor Laboratory Press on March 16, 2012 - Published by genesdev.cshlp.orgDownloaded from are diffusely localized inside the nucleoplasm of myo- blasts, and careful examination confirms that these immunostaining signals are still detectable at the bound- ary of the nucleus, labeled by an antibody against nuclear Lamin B (Fig. 2C–E). Because TAF4 is an essential component of the TFIID complex (Wright et al. 2006), it is likely that holo-TFIID is not only distributed through- out the nucleoplasm, but is also present at the nuclear periphery. Our DNA staining profiles usually show a peak close to the lamina signal (Supplemental Fig. S2), and this observation is consistent with earlier studies visualizing dense DNA structures at the nuclear periphery by elec- tron microscopy (Davies 1967), and serves as a control to ascertain appropriate alignment of multiple image chan- nels in our analysis. It was reported that heterochromatin regions allow the placement of macromolecules with molecular weights of ;500 kDa (Bancaud et al. 2009), which is consistent with our observations that TAF4 and Pol II are present at the nuclear periphery. In contrast to Pol II and TFIID, myoblasts stained with TAF3 antibodies revealed a clearly distinguishable region immediately adjacent to the NL with measurably lower immunofluorescence levels (Fig. 2F). The various anti- bodies against TAF3 used in these nuclear staining studies were first affinity-purified and extensively char- acterized (Supplemental Fig. S3A,B). Integrated radial intensity profiles indicate that the distance of half-max- imum of TAF3 signals to the center of the lamin signal is ;400 nm (Fig. 2F, panel iii); this distance is larger than those measured for TFIID subunits or Pol II (;100–200 nm) (Fig. 2A–E), and is well within our optical resolution limits. We further found statistically significant differ- ences among the mean intensity values at the nuclear periphery between TAF3 versus TAF4/TAF11 subunits (Fig. 2G, panel i), as well as between TAF3 versus Pol II (Fig. 2G, panel ii). These cell imaging results are consis- tent with our previous biochemical observation that TAF3 is a substoichiometric subunit associated with the TFIID complex (Liu et al. 2008). Interestingly, the TAF4 signal appears to be distinct from that of Pol II and distributes ‘‘closer’’ to the nuclear periphery (Fig. 2G, panel iii). We also found that H3K9Me3 staining is detectably enriched at the nuclear periphery in myo- blasts, while H3K4Me3 staining is slightly shifted to the nuclear interior (Supplemental Fig. S4A). Taken together, our imaging analysis demonstrates significantly differential distributions for core transcription compo- nents at the nuclear periphery in C2C12 myoblasts, and we observed that a shell or region directly adjacent to the nuclear periphery is substantially depleted of TAF3 relative to the nuclear interior. Extending our studies to living cells, we directly visualized a distinct layer of reduced GFP-TAF3 fluores- cence intensity at the nuclear periphery (Supplemental Fig. S5A, panels iv,v). In contrast, GFP-tagged Rpb9, TAF11, and human TAF1 each showed fluorescence rela- tively uniformly distributed throughout the nucleoplasm, including the region adjacent to or at the nuclear periphery (Supplemental Fig. S5A, panels i–iii). These live-cell imag- ing studies are consistent with our immunofluorescence results, and support the notion that the lower levels of TAF3 observed at the nuclear periphery likely reflect its steady-state intranuclear distributions in living myo- blasts. Immunostaining of TRF3 proteins suggests that they might also distribute to the nuclear interior (Sup- plemental Fig. S3F). In primary myoblasts, the MyoD gene is also largely localized adjacent to the nuclear periphery and the Myogenin gene is largely localized adjacent to the nuclear interior, while TAF3 is localized largely to the nuclear interior and shows no signifi- cant difference from its distribution in C2C12 cells (Supplemental Fig. S6). There is also a high enrichment of H3K9Me3 and a slight decrease of Pol II levels at the nuclear periphery (Supplemental Fig. S6). Hence, the spatial distributions of MyoD gene and core tran- scription components in the in vivo-derived primary cells are largely consistent with our findings in C2C12 myoblasts. The observation that Pol II and canonical TFIID sub- units are present at the nuclear periphery, where TAF3 is underrepresented, supports the notion that the alter- native core factor TAF3 may be spatially segregated from the peripherally localized, actively transcribedMyoD gene. This intriguing finding leads us to speculate whether the subnuclear segregation of TAF3 from the MyoD gene may influence or perhaps preclude its association with the MyoD promoter. PALM imaging of transcription factor localization in myoblasts The nuclear periphery is a challenging subdomain of the nucleus to target for optical microscopy because its radial dimensions and lateral microdomains approach the dif- fraction limit of conventional optical microscopy. With three-dimensional (3D) structured illumination micros- copy, nuclear periphery components could be better resolved in both radial and lateral dimensions compared with confocal or deconvolution microscopy (Schermelleh et al. 2008). However, precisely comparing locations of distinct transcription factors at the nuclear periphery would best be served by methods that can visualize and resolve individual molecules within the context of the nucleus. We adapted dual-color PALM (Betzig et al. 2006; Bates et al