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Regulation of transcriptional elongation in pluripotency and cell differentiation by the PHD-finger protein Phf5a

Nature Cell Biology volume 18, pages 1127–1138 (2016)Cite this article

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Abstract

Pluripotent embryonic stem cells (ESCs) self-renew or differentiate into all tissues of the developing embryo and cell-specification factors are necessary to balance gene expression. Here we delineate the function of the PHD-finger protein 5a (Phf5a) in ESC self-renewal and ascribe its role in regulating pluripotency, cellular reprogramming and myoblast specification. We demonstrate that Phf5a is essential for maintaining pluripotency, since depleted ESCs exhibit hallmarks of differentiation. Mechanistically, we attribute Phf5a function to the stabilization of the Paf1 transcriptional complex and control of RNA polymerase II elongation on pluripotency loci. Apart from an ESC-specific factor, we demonstrate that Phf5a controls differentiation of adult myoblasts. Our findings suggest a potent mode of regulation by Phf5a in stem cells, which directs their transcriptional programme, ultimately regulating maintenance of pluripotency and cellular reprogramming.

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Figure 1: Phf5a is required for maintenance of ESC self-renewal.
Figure 2: Phf5a regulates ESC pluripotency and cellular reprogramming.
Figure 3: Phf5a physically associates with the Paf1 complex.
Figure 4: Phf5a controls interactions among Paf1C subunits and its silencing abrogates Paf1C recruitment on pluripotency genes in ESCs.
Figure 5: Phf5a controls transcriptional elongation and RNA-PolII pause release of pluripotency genes in ESCs.
Figure 6: Phf5a regulates the deposition of histone marks characteristic of transcriptional elongation in pluripotency gene loci.
Figure 7: Phf5a loss leads to Paf1C destabilization and inhibits myogenic differentiation.

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Acknowledgements

We thank all members of the Aifantis laboratory for useful comments and discussions throughout the duration of this project; Z. Gao and P. Voigt for experimental help with glycerol gradients and helpful discussions; K. J. Armache and P. De Ioannes Fernandez for help with protein purification, helpful discussions and manuscript preparation; H.-J. Fehling for the Dppa4-RFP/Brachyury-GFP reporter ESC line; A. Heguy and the NYU Genome Technology Center (supported in part by National Institutes of Health (NIH)/National Cancer Institute (NCI) grant P30CA016087-30) for expertize with sequencing experiments; the NYU Histology Core (5P30CA16087-31) for assistance; C. Loomis and L. Chiriboga for immunohistochemistry experiments; and H. Li and T. Liu at the Center for Advanced Proteomics Research, New Jersey School of Medicine for mass spectrometry. This work has used computing resources at the High Performance Computing Facility of the Center of Health Informatics and Bioinformatics at the NYU Medical Center. A.S. is supported by the NYSTEM institutional NYU Stem Cell Training Grant (C026880). I.A. is supported by the NIH (RO1CA133379, RO1CA105129, RO1CA149655, 5RO1CA173636, 1RO1CA194923) and the NYSTEM programme of the New York State Health Department (NYSTEM-N11G-255).

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Author notes

  1. Panagiotis Ntziachristos & Shannon Buckley

    Present address: Present addresses: Department of Biochemistry and Molecular Genetics, Northwestern School of Medicine, Chicago, Illinois 60611, USA (P.N.); Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA (S.B.).,

Authors and Affiliations

  1. Department of Pathology, NYU School of Medicine, New York, New York 10016, USA

    Alexandros Strikoudis, Charalampos Lazaris, Thomas Trimarchi, Antonio L. Galvao Neto, Yan Yang, Panagiotis Ntziachristos, Shannon Buckley, Igor Dolgalev, Brian D. Dynlacht, Aristotelis Tsirigos & Iannis Aifantis

  2. Laura & Isaac Perlmutter Cancer Center, NYU School of Medicine, New York, New York 10016, USA

    Alexandros Strikoudis, Charalampos Lazaris, Thomas Trimarchi, Yan Yang, Panagiotis Ntziachristos, Shannon Buckley, Igor Dolgalev, Brian D. Dynlacht, Aristotelis Tsirigos & Iannis Aifantis

  3. Helen L. & Martin S. Kimmel Center for Stem Cell Biology, NYU School of Medicine, New York, New York 10016, USA

    Alexandros Strikoudis, Charalampos Lazaris, Thomas Trimarchi, Panagiotis Ntziachristos, Shannon Buckley, Igor Dolgalev, Matthias Stadtfeld & Iannis Aifantis

  4. Center for Health Informatics and Bioinformatics, NYU School of Medicine, New York, New York 10016, USA

    Charalampos Lazaris, Igor Dolgalev & Aristotelis Tsirigos

  5. Ronald O. Perelman Department of Dermatology, NYU School of Medicine, New York, New York 10016, USA

    Antonio L. Galvao Neto

  6. Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599, USA

    Scott Rothbart & Brian D. Strahl

  7. Genome Technology Center, Office of Collaborative Science, NYU School of Medicine, New York, New York 10016, USA

    Igor Dolgalev

  8. Department of Cell Biology, NYU School of Medicine, New York, New York 10016, USA

    Matthias Stadtfeld

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  1. Alexandros Strikoudis
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Contributions

I.A. and A.S. designed the experiments and wrote the manuscript. A.S. performed the experiments. A.T., C.L., T.T. and I.D. designed and performed the analysis of genome-wide data. P.N. provided expertize in sequencing experiments and contributed in manuscript preparation. A.L.G.N. performed histological examination of teratomas. S.B. contributed ideas. M.S., Y.Y., B.D.D., S.R. and B.D.S. provided materials and tips related to this study, helped with ideas and concepts and contributed to manuscript preparation.

Corresponding authors

Correspondence to Aristotelis Tsirigos or Iannis Aifantis.

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Integrated supplementary information

Supplementary Figure 1 Phf5a depletion results in aberrant ESC differentiation.

(a) Alignment of Phf5a protein sequence from different organisms. Green: Conserved cysteine residues, which coordinate Zn2+. Orange: Nuclear localization signal (NLS). (b) Relative expression of Phf5a transcript by qRT-PCR in ESC differentiation. n = 6 biologically independent replicates (see Supplementary Table 5). ∗∗p = 0.0001, respectively, two-sided Student’s t-test, values represent the mean ± s.d. (c) Nanog-GFP ESC colonies following knockdown with shControl or shPhf5a respectively. Scale bars, 100 μm. (d) FACS plots showing levels of apoptosis and cell death following shPhf5a knockdown in ESCs. Proteasome inhibition by MG132 is used as positive control. (e) Western blot analysis of total and cleaved Caspase-3 following shPhf5a knockdown in ESCs and differentiation. Proteasome inhibition by MG132 is used as positive control. (f) Gene Ontology (GO) analysis of significantly downregulated or upregulated genes, respectively, after RNA-sequencing in ESCs using shControl or shPhf5a silencing (g) Schematic of Phf5a mRNA indicating distinct target regions for shRNAs, gRNAs and siRNA used in the study. (h) Western blot analysis of Oct4 levels following siPhf5a knockdown in ESCs. (i) Alkaline phosphatase (AP) staining of ESCs following knockdown using siRNA transfections. Scale bars, 100 μm. (j) Western blot analysis of pluripotency factors following shPhf5a knockdown in ESCs. (k) Bar graphs showing expression levels of Phf5a in ESCs of different genetic backgrounds. n = 6 biologically independent replicates (see Supplementary Table 5). MK6 and CCE: D2 and D4 ∗∗p = 0.0001, respectively. KH2: D2 and D4 p = ∗∗0.0155 and 0.0052, respectively, two-sided Student’s t-test, values represent the mean ± s.d. (l) Western blot analysis of pluripotency factors following shPhf5a knockdown in MK6 ESCs (C57BL/6).

Supplementary Figure 2 Silencing of Phf5a affects ESC pluripotency and iPS generation.

(a) Schematic of Tet-inducible mir-30 shRNA expression cassette at the targeted Col1a1 locus in ESCs. (b) Brightfield and fluorescent images of ESCs targeted with shControl and shPhf5a mir-30 shRNA expression cassettes at the Col1a1 locus following the addition of doxyxycline. Scale bars, 100 μm. (c) Comparison of Phf5a expression levels by qRT-PCR in ESCs targeted with shControl and shPhf5a mir-30 shRNA expression cassettes at the Col1a1 locus following the addition of doxycycline. n = 3 biologically independent replicates (see Supplementary Table 5). shPhf5a-2 hairpin: ∗∗p = 0.0072 and shPhf5a-3 hairpin ∗∗p = 0.008, respectively, two-sided Student’s t-test, values represent the mean ± s.d. (d) Western blot analysis of Phf5a protein levels in ESCs targeted with shControl and shPhf5a mir-30 shRNA expression cassettes at the Col1a1 locus following the addition of doxycycline. (e–g) Histology and comparison of shControl and shPhf5a teratomas using H&E or PAS stain (e and f) and desmin, nestin or cytokeratin immunohistochemistry (g), respectively. Teratomas tissue formations include respiratory cilia, keratinized skin, cartilage and goblet cells, representative of all germ layers. Arrows indicate ciliated respiratory epithelium; stars indicate goblet cells stained positive for PAS stain. Scale bars, 100 μm. (h) FACS plots showing levels of Dppa4-RFP and Brachyury-GFP following induction of mesoderm differentiation in the presence or absence of Phf5a. (i) Western blot analysis of Brachyury protein levels following induction of mesoderm differentiation in the presence or absence of Phf5a. (j) Levels of several mesoderm markers following shPhf5a depletion. n = 3 biologically independent replicates (see Supplementary Table 5). Brachyury: ∗∗p = 0.0078, Msgn: n.s: non-significant p = 0.0694, Nkx2-5: ∗∗p = 0.0010, n.s: non-significant Isl1: p = 0.2223, two-sided Student’s t-test, values represent the mean ± s.d. (k and l) Proliferation assays of ESCs (k) or MEFs (l), respectively, following shControl or shPhf5a depletion. n = 3 biologically independent replicates (see Supplementary Table 5). ESCs Day2 and Day4: ∗∗p = 0.0001, respectively. MEFs Day2 and Day4: n.s: non-signifficant, p = 0.4169 and p = 0.8769, respectively, two-sided Student’s t-test, values represent the mean ± s.d. (m and n) Expression levels of pluripotency factors (m) and western blot for Nanog protein levels (n), respectively, during ESC differentiation, in the presence or absence of ectopic expression of Phf5a. n = 3 biologically independent replicates (see Supplementary Table 5). Phf5a: ∗∗p = 0.0016, Nanog: ∗∗p = 0.0451, Pou5f1: ∗∗p = 0.0335, Sox2: p = 0.0485, Zfp42: n.s: non-signifficant p = 0.0908, Nr0b1: p = 0.0209, two-sided Student’s t-test, values represent the mean ± s.d. (o) Representative iPSC colony morphology and AP-staining following shPhf5a knockdown. Scale bars, 100 μm. (p) Western blot analysis of reprogramming markers in OKSM MEFs following shPhf5a knockdown, on day 14 post-initial doxycycline induction.

Supplementary Figure 3 Phf5a interacts with the Paf1C.

(a) Western blot analysis of Phf5a and control tagged-proteins following doxycycline induction and cytoplasmic or nuclear fractionation in ESCs. (b) Flag immunofluorescence showing nuclear localization of tagged-Phf5a following doxycycline induction in ESCs. Scale bars, 100 μm. (c) Western blot analysis of Paf1C subunits in ESC differentiation. (d) Western blot analysis of interacting proteins following doxycycline induction and streptactin purification of Paf1C subunits or GFP control in ESCs. (e) Western blot analysis of Phf5a- or control- interacting proteins following doxycycline induction and tandem affinity purification using strep-tagII and flag tags in ESCs. (f) Representative time point analysis for presence of nucleic acids following lysis of ESCs in the presence of benzonase nuclease prior to immunoprecipitation in ESCs.

Supplementary Figure 4 Phf5a regulates Paf1C subunit composition.

(a) Venn diagrams showing number of all significantly differentially expressed genes, as well as significantly downregulated and upregulated genes using RNA-sequencing after shPhf5a and shPaf1 depletion in ESCs. (b) Western blot of fractions following glycerol gradient sedimentation analysis of Paf1C subunits using one-step purification from ESCs in the presence or absence of shPhf5a knockdown. (c–e) Graphs showing quantification of percent distribution for the Paf1C subunits Paf1, Wdr61 and Cdc73 shown in (c), in the presence or absence of Phf5a, respectively, in ESCs. (f) Western blot of fractions following glycerol gradient sedimentation analysis of Swi/Snf complex subunit Smarca4 and NELF complex subunit NELF-A using one-step purification from ESCs in the presence or absence of shPhf5a knockdown. (g) Binding profiles for genomic distribution of Leo1 peaks (upstream, promoter, coding region, 5’UTR, 3’UTR, downstream and intergenic) in ESCs, showing preferential binding (42%) within gene bodies of downregulated genes, but preferential binding (31%) within promoters of upregulated genes. (h) A Tet-inducible “knock-in” tagged Phf5a ESC line was used to perform ChIP-sequencing of Phf5a using a HA-epitope in the presence of absence of doxycycline. Snapshots of Phf5a binding on representative pluripotency gene targets are shown (Pou5f1, Sall4, Prdm14, Esrrb) in the presence (blue) of absence (gray) of doxycycline.

Supplementary Figure 5 Phf5a regulates Paf1C functions on transcriptional elongation in ESCs.

(a) Western blot analysis of Ser2-phospho- and Total RNA PolII in ESCs following CRISPR-Cas9 mediated Phf5a depletion. (b) Comparison of GRO-seq read density profiles of downregulated genes 72h following shControl or differentiated ESCs in the absence of LIF. RPKM: Reads Per Kilobase per Million total reads. (c) Comparison of GRO-seq read density profiles of downregulated genes 72h in all conditions tested (shControl, shPhf5a or differentiated ESCs in the absence of LIF). RPKM: Reads Per Kilobase per Million total reads. (d) Box plot showing comparison of log2 pausing index for downregulated genes, 72h following shControl, shPhf5a, ESC differentiated in the absence of LIF, or flavopiridol-treated ESCs, respectively, using GRO-seq analysis. Flavopiridol treatment is used as a positive control of pause-release block. n = 3 biologically independent replicates, Wilcoxon signed rank test non-parametric. (e) (Upper) Venn diagrams between Leo1 ChIP-sequencing targets in ESCs and either downregulated or upregulated genes using RNA-sequencing after shPhf5a knockdown. (Lower) Box plot showing pausing index ratios after GRO-seq analysis for the direct Leo1 targets shown above. Only downregulated targets exhibit significant promoter-proximal pausing. n = 3 biologically independent replicates, Wilcoxon signed rank test non-parametric. (f and g) Comparison of read density profiles for Ser5- (on TSSs) or Ser2- (on gene bodies) phosphorylated RNA PolII, respectively, for the specific Gene Ontology Terms chromatin organization (f) and positive regulation of transcription (g), respectively. n = 3 biologically independent replicates, lines represent the mean ± s.d. In box plots (d and e) the central mark is the median, and the edges of the box are the first and third quartiles. Whiskers extend to the most extreme non-outlier data points.

Supplementary Figure 6 Phf5a silencing inhibits myotube differentiation through Paf1C destabilization.

(a) Brightfield images of self-renewing myoblasts or 72h-differentiated myotubes. (b) Western blot analysis of self-renewal and differentiation markers in C2C12 cells following knockdown of Paf1C subunits and 72h differentiation. (c) Myocin heavy chain (MHC) immunofluorescence on differentiated myotubes for 72h following shControl or shPhf5a knockdown. Scale bars, 100 μm. (d) Quantification of myocin heavy chain (MHC) immunofluorescence intensity of Rosa26rtTACol1a1TREshPhf5a primary myotubes following addition of doxycycline. n = 3 biologically independent replicates (see Supplementary Table 5). ∗∗p = 0.0001, two-sided Student’s t-test, values represent the mean ± s.d. (e) Relative expression of Phf5a and multiple Paf1C subunits using qRT-PCR showing higher levels of most of Paf1C subunits in primary mouse myotubes. n = 3 biologically independent replicates (see Supplementary Table 5). Phf5a: p = 0.0193, Paf1: p = 0.0001, Cdc73: p = 0.0031, Leo1: p = 0.0378, Wdr61: p = 0.0238, Rtf1: n.s, non-significant, p = 0.1103, two-sided Student’s t-test, values represent the mean ± s.d. (f) Desmin immunofluorescence on 72h differentiated myotubes following CRISPR-Cas9 depletion of Phf5a. Scale bars, 100 μm. (g) Western blot analysis of the differentiation marker myocin heavy chain following CRISPR-Cas9 depletion of Phf5a. (h–j) Gene Ontology (GO) analysis of Leo1-bound genes after ChIP-sequencing in myoblasts (h) and myotubes (i) under shControl conditions, or myotubes (j) after shPhf5a depletion, respectively.

Supplementary Figure 7 Scans of unprocessed key blots.

(a) Scans from Figure 1a (b) Scans from Figure 1i (c) Scans from Figure 2d (d) Scans from Figure 3b (e) Scans from Figure 3c (f) Scans from Figure 3d (g) Scans from Figure 3e (h) Scans from Figure 4a (i) Scans from Figure 5a (j) Scans from Figure 7a (k) Scans from Figure 7e (l) Scans from Figure 7j.

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Strikoudis, A., Lazaris, C., Trimarchi, T. et al. Regulation of transcriptional elongation in pluripotency and cell differentiation by the PHD-finger protein Phf5a. Nat Cell Biol 18, 1127–1138 (2016). https://doi.org/10.1038/ncb3424

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