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Tyrosine phosphorylation of histone H2A by CK2 regulates transcriptional elongation

Abstract

Post-translational histone modifications have a critical role in regulating transcription, the cell cycle, DNA replication and DNA damage repair1. The identification of new histone modifications critical for transcriptional regulation at initiation, elongation or termination is of particular interest. Here we report a new layer of regulation in transcriptional elongation that is conserved from yeast to mammals. This regulation is based on the phosphorylation of a highly conserved tyrosine residue, Tyr 57, in histone H2A and is mediated by the unsuspected tyrosine kinase activity of casein kinase 2 (CK2). Mutation of Tyr 57 in H2A in yeast or inhibition of CK2 activity impairs transcriptional elongation in yeast as well as in mammalian cells. Genome-wide binding analysis reveals that CK2α, the catalytic subunit of CK2, binds across RNA-polymerase-II-transcribed coding genes and active enhancers. Mutation of Tyr 57 causes a loss of H2B mono-ubiquitination as well as H3K4me3 and H3K79me3, histone marks associated with active transcription. Mechanistically, both CK2 inhibition and the H2A(Y57F) mutation enhance H2B deubiquitination activity of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, suggesting a critical role of this phosphorylation in coordinating the activity of the SAGA complex during transcription. Together, these results identify a new component of regulation in transcriptional elongation based on CK2-dependent tyrosine phosphorylation of the globular domain of H2A.

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Figure 1: The conserved Tyr 57 residue in H2A is phosphorylated.
Figure 2: CK2 phosphorylates Tyr 57 in H2A.
Figure 3: The H2A(Y58F) mutation enhances H2B deubiquitination, and impairs transcriptional elongation in yeast.
Figure 4: CK2 regulates transcriptional elongation.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

ChIP-seq data has been deposited in the Gene Expression Omnibus database under accession number GSE58607.

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Acknowledgements

We acknowledge M. Ghassemian for assistance in mass spectrometry analysis. We also acknowledge K. Arndt for providing the UBP8 null yeast strain. CK2 constructs were provided by D. Litchfield. We acknowledge J. Hightower for assistance in figure preparation. We acknowledge K. Tumaneng and I. Bassets for critical reading of the manuscript and D. J. Forbes for discussion. This work was supported by grants NS034934, DK039949, DK018477, HL065445 and CA173903 from NIH to M.G.R., UC-CRCC to L.P., NIH-GM033279 support for X.B.S. and NCI CA82683 grant to T.H. M.G.R. is an Investigator with the HHMI. T.H. is a Frank and Else Schilling American Cancer Society Professor and holds the Renato Dulbecco Chair in Cancer Research.

Author information

Authors and Affiliations

Authors

Contributions

M.G.R. and H.B. conceived the idea and wrote the manuscript with contributions from L.P., X.B.S. and T.H. M.G.R. and H.B. designed the experiments with mammalian cells, and H.B. performed the experiments. L.P., X.B.S. and H.B. designed the yeast experiments, and X.B.S. and H.B. performed the experiments. Y.T. did the bioinformatics analysis. D.M. aligned the ChIP-seq data. K.A.O. prepared the ChIP-seq library, and conducted the high-throughput sequencing. T.H. designed the PAA experiments, and J.M. performed the experiments. All authors read the manuscript, and approve the content.

Corresponding authors

Correspondence to Lorraine Pillus or Michael G. Rosenfeld.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The conserved Tyr 57 residue in H2A is phosphorylated.

a, The Tyr 57 residue is conserved in all variants of H2A. Sequences of H2A variants surrounding the Tyr 57 residue (arrow) in mammals is shown, particular variant residues are highlighted in blue. b, The Y57F mutation in H2A does not affect the structural integrity of nucleosomes. Mononucleosomes containing Flag-tagged wild-type H2AX or H2AX(Y57F) were immunoprecipitated, and histones and DNA were visualized by Ponceau staining (right) and by ultraviolet light (left), respectively. c, The H2A Tyr 58 residue has overlapping functions with the H2AZ Tyr 65 residue in yeast. Fivefold serial dilutions of the indicated transformants were plated on SC–His–Ura–Trp for growth and 5-FOA for the loss of pJH33. d, e, H2A Tyr 57 is phosphorylated in 293T cells. d, Nuclear extracts from 293T cells were immunoblotted (IB) with anti-pTyr 57 H2A pre-incubated with indicated peptides, and re-probed with anti-H2A. e, Histone extracts from 293T cells were treated with calf intestinal phosphatase (CIP) for 1 h at 37 °C, and immunoblotted. f, The anti-pTyr 57 H2A antibody specifically recognizes the H2A peptide phosphorylated at Tyr 57 but not the non-phosphorylated peptide. Indicated peptides were spotted on nitrocellulose, and probed with anti-pTyr 57 H2A. Data represent three independent experiments.

Extended Data Figure 2 CK2α phosphorylates Tyr 57 in H2A.

An in vitro kinase reaction was performed using recombinant GST-CK2α, 10 μCi of [γ-32P]ATP supplemented with 10 μM cold ATP, and nucleosomes containing Flag-tagged wild-type H2AX or H2AX(Y57F) from 293T cells, and phosphoamino acid analysis of the phosphorylated Flag-tagged H2AX was performed. The red circle indicates pSer, the blue circle indicates pThr and the green circle indicates pTyr. Data represent two independent experiments.

Extended Data Figure 3 H2A Tyr 57 phosphorylation regulates transcriptional elongation.

a, H2A(Y58F) mutation does not affect several other histone marks. Whole-cell extracts from wild-type (WT) or H2A(Y58F) yeast cells were immunoblotted (IB). b, c, H2A(Y57F) mutation affects H2A ubiquitination in 293T cells. b, Flag-tagged wild-type H2A/H2AX and Y57F mutants were expressed in 293T cells, and mono-ubiquitination was assessed by immunoblotting with Flag antibody. c, The Flag-tagged H2A mutants were expressed in 293T cells, immunoprecipitated (IP) under denaturing conditions, and immunoblotted. d, H2A(Y58F) mutant cells are defective in transcriptional elongation. Fivefold serial dilutions of wild-type and H2A(Y58F) cells were plated on SC supplemented with NH4OH (solvent) or 100 μg ml−1 6-azauracil (6-AU). e, Pol II protein level is comparable in wild-type and H2A(Y58F) yeast. Whole-cell extracts from wild-type or H2A(Y58F) yeast were immunoblotted. f, H2A(Y58F) mutation affects transcription. Wild-type H2A, H2A(Y58F), and H2A(Y58F) H2AZ(Y65F) yeast were grown at 30 °C or shifted to 37 °C for 10 min. RNA was extracted and transcript levels of the indicated genes were measured by reverse-transcription–qPCR, and normalized to SCR1, a Pol III transcript (n = 3, mean ± s.e.m., *P < 0.05, **P < 0.01). P values were calculated with two-tailed Student’s t-tests. g, H2AZ(Y65F) mutation alone in yeast does not affect transcription significantly. Wild type (HTZ1) transformed with vector, and htz1Δ strains transformed with vector (htz1Δ), HTZ1 (WT H2AZ) or HTZ1(Y65F) (H2AZ(Y65F)) were grown at 30 °C (blue bars) or shifted to 37 °C for 10 min. (orange bars), and transcript levels of the indicated genes were measured as in f (n = 3, mean ± s.e.m., *P < 0.05, **P < 0.01). P values were calculated with two-tailed Student’s t-tests. h, Tyr 57 in H2A is phosphorylated during transcriptional elongation. 293T cells were treated with vehicle (DMSO) or flavopiridol (FP) (1 μM) for 4.5 h, then flavopiridol was washed out (release). Cells were harvested at the indicated minute (,) after release, and the nuclear extracts were immunoblotted. Data represent two (a, d, h) or three (b, c, eg) independent experiments.

Extended Data Figure 4 H2A(Y58F) mutation enhances H2B deubiquitination.

ac, The H2A Tyr 58 mutation has moderate to no effect on the recruitment of the H2B ubiquitination machinery. Binding of (a) Rtf1-HA, (b) Paf1-myc, and (c) Rad6-HA was measured by ChIP-qPCR in the indicated genes in wild-type and H2A(Y58F) yeast. Whole-cell extracts from the yeast strains were immunoblotted (IB) to compare the protein levels. ORF of the genes, and the region amplified by the primer pairs are shown (n = 2, mean ± s.e.m.). d, UBP8 deletion does not rescue Pol II binding in the H2A(Y58F) mutant. Pol II binding in the indicated strains was measured by ChIP-qPCR. (n = 3, mean ± s.e.m., *P < 0.05, **P < 0.01). P values were calculated with two-tailed Student’s t-tests. The ORF of the genes and the regions amplified by the primer pairs are shown. e, UBP8 deletion does not rescue the defect in transcriptional output in the H2A(Y58F) yeast. The mRNA levels of the indicated genes were determined by RT–qPCR and normalized to the SCR1 transcript. (n = 2, mean ± s.e.m.). f, UBP8 deletion does not rescue the growth defect in the H2A(Y58F) yeast. UBP8 and ubp8Δ strains expressing either wild-type (WT) H2A or H2A(Y58F) were plated at 2.5-fold serial dilutions on SC–His–Ura for growth and 5-FOA for the removal of pJH33. Data represent two (ac, e, f) or three (d) independent experiments.

Extended Data Figure 5 CK2 regulates transcriptional elongation.

a, CK2 kinase activity is necessary for normal gene expression. LNCaP cells were treated with vehicle (DMSO) or TBBz (25 μM) for 60 min, and then treated with vehicle (ethanol) or 100 nM DHT for 90 min, and induction of the indicated androgen receptor (AR) target genes was measured by RT-qPCR (n = 3, mean ± s.e.m., *P < 0.05, **P < 0.01). P values were calculated with two-tailed Student’s t-tests. b, Nuclear extracts from 293T cells were immunoprecipitated (IP) using CK2α antibody and immunoblotted (IB). c, Enrichment of CK2α, H3K4me1 (ref. 18), H3K27ac (ref. 18) and androgen receptor genes18 at a representative androgen receptor enhancer (KLK3) is shown. d, Pol II tag density in cells treated with vehicle or TBBz at a representative RHOU enhancer is shown. Data represent two (bd) or three (a) independent experiments; kb, kilobase.

Supplementary information

Supplementary Table 1

This file shows Identification of post-translational modifications in histones from 293T cells. Histones were extracted from 293T cells by acid extraction, precipitated by Trichloroacetic acid (TCA), and subjected to mass spectrometry as described in the Methods. (XLSX 631 kb)

Supplementary Table 2

This file shows Identification of proteins interacting with H2AX. Flag-tagged Wild type (WT) H2AX, H2AX-Y57F or vector were transfected in 293T cells, and nuclear extracts from the cells were immunoprecipitated with anti-Flag magnetic beads, washed three times with wash buffer (described in Methods), and the proteins bound to the beads were analyzed by Mass Spectrometry as described in the Methods. Sample C (sheet 1) shows vector interacting proteins, H2AX_minus IR (sheet 2) shows WT H2AX interacting proteins, 57H2AX_minus IR (sheet 3) shows H2AX-Y57F interacting proteins. (XLS 159 kb)

Supplementary Table 3

This file contains oligonucleotide sequences as primers. (XLS 43 kb)

Supplementary Table 4

This file contains genotypes of the yeast strains together with their sources. Yeast plasmids are also described. (XLSX 47 kb)

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Basnet, H., Su, X., Tan, Y. et al. Tyrosine phosphorylation of histone H2A by CK2 regulates transcriptional elongation. Nature 516, 267–271 (2014). https://doi.org/10.1038/nature13736

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