Article Figures & Data
Figures
- Figure 1
PHF8 and TIP60 colocalize and recruit each other in neuronal interchromatin space. A, Endogenous PHF8 immunostained with anti-PHF8 antibody (ab36068; Abcam) forms hundreds of discrete puncta that specifically localize to the interchromatin space in hippocampal neurons (representative z-slice of a hippocampal neuronal nucleus; left, widefield; right, SIM). Scale bar, 1 μm. B, Endogenous TIP60 forms puncta of roughly the same caliber as those of PHF8 above (left, hippocampal nucleus in widefield; right, SIM), which also localize to the interchromatin space. Scale bar, 1 μm. C, A representative field of hippocampal neurons stained with an antibody against the phosphorylated CTD of RNA polymerase II (YSPTSPS phospho S5, abbreviated to S5P), showing that S5P, a marker of the transcription initiation complex, localizes to the same nuclear compartment as PHF8 in the nucleus. Scale bar, 1 μm. D, A hippocampal neuronal nucleus outlined in blue, showing the localization of spectrally distinct PHF8−tdTomato and TIP60−CFP pseudo-colored in green and red, respectively, which overlapped completely in the nuclear interchromatin space (merge channel, yellow pixels indicate colocalization). DAPI was used to stain the DNA (blue). Scale bar, 0.2 μm. E, When TIP60 is overexpressed by itself in hippocampal neurons (middle, red), endogenous PHF8 (left, green) is seen to be recruited to the TIP60 puncta in hippocampal neurons (right, merge). DAPI was used to stain the DNA (blue). Scale bar, 0.2 μm. Asterisk (*) indicates endogenous protein staining. F, When PHF8 is overexpressed by itself (left, green) in hippocampal neurons, endogenous TIP60 (middle) is seen to be recruited to the PHF8 puncta (right, merge). DAPI was used to stain the DNA (blue). Scale bar, 0.2 μm. Asterisk (*) indicates endogenous protein staining. G. Endogenous TIP60 is located within 30 nm of PHF8 as shown by P-LISA, showing distinct areas where PHF8 interacts with endogenous TIP60 (red spots) on the border with DAPI-dense regions (blue). Scale bar, 0.5 μm. H, Two Hek293 nuclei are shown, one is positive for PHF8 shRNA (outlined in red) while the other is not (outlined in green). Positive PHF8−TIP60 interaction hotspots were stained as red punctae. Scale bar, 0.5 μm. I, Quantification of the number of hotspots found in cells transfected with PHF8, PHF8 shRNA, the mutant PHF8 F279S, or, in F279S-transfected cells expressing PHF8, shRNA were quantified using Blobfinder, and the means and standard errors are displayed in a bar grapht (triple asterisks indicating statistical significance using the unpaired t test; p ≤ 0.0001). J, Double immunofluorescence confirming the existence of PHF8 in the identified PLA hotspots where PHF8 and TIP60 interact. Scale bar, 0.5 μm.
- Figure 2
PHF8 and TIP60 physically associate to form a dual function chromatin-modifying complex. A, Coimmunoprecipitation of PHF8 and TIP60 in HEK293T nuclear extracts, where TIP60−YFP was pulled down with anti-GFP antibody and PHF8−FLAG was detected with anti-FLAG by Western blotting. B, Pulldown of PHF8−YFP showed that TIP60−FLAG was detected in the IP fraction but not in the YFP-only control lane. C, Endogenous coimmunoprecipitation of PHF8 and TIP60 in DIV12 cortical neuronal nuclear extracts, showing that PHF8 is able to be pulled down by both the anti-PHF8 antibody and anti-TIP60 antibody, but not the anti-GFP antibody. D, E, Truncated constructs of TIP60 protein (A to F) containing the indicated TIP60 domain (E) were fused to YFP and then cotransfected with full-length PHF8−FLAG and immunoprecipitated with an anti-GFP antibody. Western analysis was performed to detect PHF8−FLAG in the immunoprecipitates using the anti-FLAG antibody. A negative control of YFP only is denoted by (-), whereas full-length TIP60 served as a positive control (+). F, G, Total histones from HEK293 cells overexpressing PHF8, TIP60, or both were separated on TAU gels (F) or conventional SDS-PAGE (G). Overexpression of TIP60 alone increases H3K9 acetylation in HEK293 cells for both the H3.1 and H3.3 isoforms, whereas acetylation of the non-TIP60 substrate H2BK5 was not affected. Coexpression of PHF8 and TIP60 increases H3.3K9 acetylation to even higher levels. H, Chromatin immunoprecipitation using an antibody specific to H3K9me2, showing that overexpression of wild-type PHF8 but not the clinical mutant F279S (U, unbound or input levels of H3K9me2; B, bound or immunoprecipitated H3K9me2). I, J, ChIP assays of HEK293T cells transfected with PHF8, TIP60, or both analyzing histone tails positive for H3K4me3, the transcriptionally-activating histone mark that is known to be bound by PHF8, show that the increase in H3K9ac (I) and H3K14ac (J) is specific to histones carrying H3K4me3, and that this histone population was enriched in H3.3 (as shown by the more intense staining of this isoform on the TAU gel; asterisk). Western blot of the same lysates using an H3.3 antibody serves as loading control. The right panel shows bar graphs quantifying the increase in H3.3K9 and H3.3K14 acetylation, relative to the untransfected control (n = 3; p = 0.19 for PHF8 only, 0.04 for TIP60 only, 0.02 for PHF8+TIP60; asterisks indicates statistical significance: p < 0.05).
- Figure 3
PHF8 removes the repressive histone mark H3K9me2 and associates with the activating histone mark H3K9ac. A, A representative hippocampal neuronal nucleus outlined in blue, transfected with PHF8−CFP (pseudo-colored green), showing a marked decrease in the repressive chromatin mark H3K9me2 (pseudo-colored blue) in the nuclear domains occupied by PHF8, which is not seen when the neuron is untransfected (B) or when the mutant PHF8−F279S was transfected (C). D, The same hippocampal nucleus depicted in A, showing the association of PHF8 puncta with the histone acetylation mark H3K9ac (arrows point to regions of close apposition between PHF8 and H3K9ac, six of which are shown at higher magnification by the insets on the right; green+red = yellow). E, Quantitation of the intensity of H3K9me2 staining in each nucleus (each symbol marks the H3K9me2 density of a single neuron), showing that PHF8-expressing neurons have significantly lower H3K9me2 density, whereas mutant PHF8 F279S-transfected neurons show the opposite effect (*** p < 0.0001; ns, not significant).
- Figure 4
Neuronal activity reorganizes PHF8 and TIP60 in the nucleus and effectuate histone methylation and acetylation changes. A, A representative image of a pair of hippocampal neuronal nuclei during the first 5 min of 4AP+Bic+Fors treatment and then at 45 min, showing the activity-dependent increase of PHF8 and TIP60 protein in the nucleus. B, Neural network activity visualized by Ca2+ imaging (gCamp6 intensity over time), with each different-colored line representing individual neurons, before (left) and after (right) treatment with 4-AP+Bic. C, Dot plot of nuclear levels of PHF8 and TIP60 (unpaired t test; *** p ≤ 0.0001; one-way ANOVA: F = 33.23, R 2 = 0.3693). Each symbol represents the intensity of PHF8 or TIP60 staining from a single neuronal nucleus, lines correspond to the mean and SEM of all neurons imaged at the indicated time points. D, mRNA levels of PHF8 show a biphasic peak with time of chemLTP, whereas TIP60 shows an initial upregulation but a return to baseline within 45 min of sustained activity. E, Time-courses of chromatin modification of neurons imaged using high-content screening (n = 500 − 1000/site, 6 sites/well, 96-well; ImageXpress Micro, Molecular Devices) show an activity-dependent decrease in the overall per-nucleus intensity of H3K9me2 staining in neural networks treated with chemLTP, which coincides with a robust increase in H3K9acS10P. F, Graphs of nuclear PHF8 as a function of nuclear TIP60 levels at 0, 5, and 45 min of neural network activation in ARC-positive versus ARC-negative neurons, showing two identifiable distinct populations of neurons. The bar graph indicates levels of TIP60 (red) and PHF8 (green) as a function of time of synaptic activation (in minutes; y-axis). PHF8 and TIP60 are both highly induced within as early as 5 min of synaptic activation (p = 0.00001).
- Figure 5
PHF8 and TIP60 modulate neuronal activity-induced histone acetylation at H3K9acS10P and activation of the Arc gene. A, Representative microscopic field of hippocampal neurons after 1 h of network activation by chemLTP, showing a positive correlation between the expression of Arc (red) and Tip60 (blue) with the phosphoacetylation mark H3K9acS10P (green). The bottom panels show three different neurons that induced varying amounts of ARC protein. The neuron expressing the highest amount of ARC (3) also has high amounts of H3K9acS10P. B, Quantification of 20 immunofluorescence-analyzed fields exemplified in A, showing a statistically significant increase in H3K9acS10P as well as endogenous TIP60 in ARC-expressing neurons (n = 347 neurons; *** p = 0.00001). C, Fusion constructs of PHF8 and its mutant F279S were individually expressed in hippocampal neurons and the next day the neuronal network was activated using ChemLTP (4AP+Bic+Fors). After 1 h of upregulated synaptic activity, the expression of PHF8, but not its mutant F279S, significantly increases histone acetylation at H3K9acS10P (n = 397 neurons; p = 0.00001). D, A representative microscopic field of neuronal nuclei after 1 h of ChemLTP, with neuronal nuclei stained by DAPI outlined in magenta, showing the induction of ARC protein expression in a small subset of neurons, one of which had been transfected with PHF8−CFP (blue), and is high in H3K9acS10P (green). E, A representative z-plane of a 3D SIM image of a neuronal nucleus after 1 h of chemLTP treatment, showing endogenous nuclear PHF8 puncta (green) and endogenous TIP60 (red) associating with the histone acetylation mark H3K9acS10P (blue). Arrows mark three selected regions, which are shown at higher magnification in the bottom panels, showing strong association between the PHF8−TIP60 complex and H3K9acS10P in the activated neuronal nucleus.
- Figure 6
Knockdown of PHF8 impairs activity-dependent induction of H3K9acS10P and Arc and Fos expression. A, Neurons transfected with PHF8 shRNA1 and subsequently treated with chemLTP activation for 3 h (DAPI-stained nuclei are outlined in magenta) were immunostained for H3K9acS10P and ARC. The right panel shows a corresponding quantification of the staining density (intensity/area/nucleus) normalized to the mean density for each condition, showing a significant decrease in H3K9acS10P induction as well as Arc gene expression (p = 0.0052). B, C, Representative microscopic fields showing neurons transfected with PHF8 shRNA1 (B) and PHF8 shRNA2 (C) and subsequently treated with chemLTP activation for 3 h (DAPI-stained nuclei are outlined in magenta), immunostained for products of immediate-early genes Arc and Fos. The right panel shows a corresponding quantification of the staining density (intensity/area/nucleus) normalized to the mean density for each condition, showing that shRNA knockdown by two individual PHF8 shRNAs succesfully inhibited Arc and Fos induction (B: p = 0.0301, 0.0408; C: p = 0.0002, 0.0452; asterisks indicate the level of significance: * indicate p < 0.05; *** p < 0.001).
- Figure 7
PHF8 and TIP60 are actively recruited to specific neuronal gene promoters. A, B, Within minutes of synaptic activation (x-axis: time of increased network activity, in minutes), time-course ChIP shows an early detectable decrease in the chromatin mark H3K9me2 at the Arc TSS (A), which is mirrored by a concomitant, highly transient increase in the levels of H3K9acS10P at the Arc TSS (B). This increase in H3K9acS10P was specific to the Arc promoter as analyses of Rpl19, JunB, Synaptophysin, Homer1A, BDNF promoter 1, Txnip, Gapdh, and Fos intergenic region (Negative Ctrl) did not show an activity-dependent increase. C, D, Time-course ChIP followed by qRT-PCR using primers against the transcriptional start site regions of the Arc gene, arc synaptic response element, ribosomal protein L19 (Rpl19), neuronal PAS domain protein 4 (Npas4), and synaptophysin. Both TIP60 (C) and PHF8 (D) are recruited to the Arc TSS within minutes of activation of the neuronal network, but not to the Rpl19, Npas4, or Synaptophysin transcriptional start sites.
- Figure 8
Common interacting partners between PHF8 and TIP60 function primarily in transcription and mRNA processing. Top, A Venn diagram showing several interacting partners of PHF8 and TIP60 as identified by immunoprecipitation followed by mass spectrometry. The overlapped region in the middle represents common partners that interact with PHF8 and TIP60, which include the splicing factor SFPQ (PSF) and its partner NONO, as well as several ATP-dependent RNA helicases, and the histone chaperone nucleolin. Proteins that have known acetylation sites are marked by a triangle (Choudhary et al., 2009). Arrows indicate known functional interactions between identified proteins. Font size indicates the percentage of the total protein that the identified MS/MS peptides covered (large font: >25% coverage; medium font: 5 − 25% coverage; small font: <5% coverage). Histone proteins identified in the IP−MS are in bold. Bottom, A listing of the top eight biological functions attributed to the proteins identified in the IP−MS of both PHF8 and TIP60 in order of abundance, as computed by the software DAVID (http://david.abcc.ncifcrf.gov/home.jsp), with the associated p value and Benjamini factor, showing that interactors of PHF8 and TIP60 are enriched in the functions of RNA processing, RNA splicing, and mRNA processing.
- Figure 9
Endogenous TIP60 is located within 30 nm of PHF8 in the activated hippocampal neuronal nucleus. A, A maximum intensity projection of a dual color 3D STORM image of a hippocampal neuronal nucleus that has undergone 1 h of chemLTP. The neuron has been labeled for endogenous TIP60 (red) and endogenous PHF8 (green), showing that the two molecules closely interact in various localized puncta in the nucleus. Scale bar, 1 μm. The insets on the right show six representative complexes at higher magnification (scale bar, 50 nm). B, A highly magnified view of two endogenous PHF8−TIP60 complexes shown in the outlined area in A. The insets on the right show three projections of the single-molecule interaction between PHF8 and TIP60 viewing down the x-, y-, and z-axes, demonstrating that the complexes formed between these two chromatin-modifying enzymes have well-defined spatial relationship. Each dot corresponds to the localization of a single molecule. Scale bars, 50 nm.
- Figure 10
PHF8 and TIP60 form a tripartite complex with the splicing factor PSF and associates with newly transcribed nascent RNA. A, A maximum intensity projection of a 3D STORM image of an activated hippocampal neuronal nucleus. Single-molecule imaging of endogenous PHF8 (green), endogenous TIP60 (red), and PTB-associated splicing factor (blue), with the corresponding single-channel views. Each dot corresponds to the localization of a single molecule. Scale bar, 500 nm. B, C, Four representative higher magnification views of the neuronal nucleus depicted in A, showing that PSF (blue) forms a tailing structure within the interface between PHF8 (green) and TIP60 (red) viewed axially B or longitudinally C as a recognizable tripartite complex. Scale bar, 50 nm.
Serum stimulation induces proviral integration site 1 (PIM1)-mediated phosphorylation (P) of pre-acetylated (Ac) H3K9S10 (serine 10 of histone H3 lysine 9) at the FOS-like antigen 1 (FOSL1) enhancer. PIM1 is a proto-oncogene-encoded serine/threonine protein kinase. Binding of 14-3-3 to nucleosomes that carry histone H3 acetylation at lysine 9 and phosphorylation at serine 10 (H3K9acS10ph) recruits MOF, a histone H4 lysine 16-specific acetyltransferase, which in turn acetylates H4K16 (H4K16ac). In Drosophila melanogaster, elongator complex protein 3 (ELP3) and JIL1 (a serine/threonine kinase that is related to nuclear mitogen- and stress-activated protein kinase 1 (MSK1 in humans)) respectively acetylate and phosphorylate H3K9S10 at the promoter. The modified nucleosomes act as a platform for the sequential recruitment of bromodomain-containing 4 (BRD4) and positive transcription elongation factor b (PTEFb). PTEFb then facilitates the release of the promoter-proximal paused RNA polymerase II (RNAPII). The type of Mediator (shown by a purple diamond) and cofactors (shown by yellow circles) involved remains to be determined.
Nature Reviews Genetics 12, 283-293 (April 2011)
Movies
- Movie 1
Stochastic optical reconstruction microscopy of a tripartite complex formed by PHF8, TIP60, and the splicing factor PSF, rotated around the y-axis, showing the configuration of the epigenetic enzymes around a tail-like structure composed of the neuronal splicing factor PSF.
In this issue
