H2B Tyr37 phosphorylation suppresses expression of replication-dependent core histone genes

Abstract

Histone gene transcription is actively downregulated after completion of DNA synthesis to avoid overproduction. However, the precise mechanistic details of the cessation of histone mRNA synthesis are not clear. We found that histone H2B phosphorylation at Tyr37 occurs upstream of histone cluster 1, Hist1, during the late S phase. We identified WEE1 as the kinase that phosphorylates H2B at Tyr37. Loss of expression or inhibition of WEE1 kinase abrogated H2B Tyr37 phosphorylation with a concomitant increase in histone transcription in yeast and mammalian cells. H2B Tyr37 phosphorylation excluded binding of the transcriptional coactivator NPAT and RNA polymerase II and recruited the histone chaperone HIRA upstream of the Hist1 cluster. Taken together, our data show a previously unknown and evolutionarily conserved function for WEE1 kinase as an epigenetic modulator that marks chromatin with H2B Tyr37 phosphorylation, thereby inhibiting the transcription of multiple histone genes to lower the burden on the histone mRNA turnover machinery.

Figure 1

WEE1 phosphorylates histone H2B at Tyr37. (a) Synchronized MEF lysates immunoprecipitated with pTyr37 H2B antibodies and immunoblotted with H2B antibody (top). Total levels of histone H2B are shown in the bottom blot. Unsyn, unsynchronized. (b) H1975 cells treated with the WEE1 inhibitor MK-1775 (0.31, 0.62 and 1.25 µM) had loss of H2B Tyr37 phosphorylation. (c) MEFs transfected with WEE1 siRNA had loss of H2B Tyr37 phosphorylation compared to cells transfected with control siRNAs. (d) In vitro kinase assay with purified proteins showing direct phosphorylation of H2B by WEE1 kinase. (e) In vitro kinase assay with purified proteins indicating that WEE1 kinase phosphorylates H2B but not other core histones. (f) Coimmunoprecipitation revealing endogenous WEE1–pTyr37 H2B complexes in MEFs. (g) MEFs coexpressing Myc-tagged WEE1 (WT-WEE1) or kinase-dead mutant WEE1 (KD-WEE1) and empty vector, Flag-tagged H2B or Y37F mutant H2B were immunoprecipitated with pTyr37 H2B antibodies and then immunoblotted with Flag antibody, revealing that WEE1 specifically phosphorylated H2B at Tyr37.

Figure 2

H2BpY37 occurs upstream of the histone gene cluster Hist1. (a) Shown are the positions of histone genes in the mouse Hist1 subcluster 1 and two pTyr37 H2B binding sites. The starting nucleotide position is shown above. Subcluster 1 is conserved between human and mouse. (b–d) Native ChIP with pTyr37 H2B or IgG antibodies followed by qPCR using primers corresponding to site 1 (b), site 2 (c) or the control site (d) validated the presence of H2BpY37 at sites 1 and 2, upstream of the Hist1 cluster. Data shown in the bar graphs are the mean ± s.d. from three independent experiments. *P = 0.001, **P = 0.006. (e,f) ChIP using WEE1 or IgG antibodies followed by qPCR for site 2 (e) or the control site (f) revealing recruitment of WEE1 to site 2. Data shown in the bar graphs are the mean ± s.d. from three independent experiments. ***P = 0.02. (g,h) ChIP performed in cells transfected with WEE1 siRNAs showed loss of H2B Tyr37 phosphorylation at site 2. (g) but not at the control site (h). Data shown in the bar graphs are the mean ± s.d. from three independent experiments. ****P = 0.001. Student’s t test was used to calculate statistical significance throughout the paper.

Figure 3

H2BpY37 suppresses the transcription of histone genes located in the Hist1 cluster. (a) qRT-PCR of RNA prepared from synchronized MEFs indicating a rapid decrease in histone mRNA levels after 6.5 h after thymidine release. The y axis label “histone/actin” indicates the amount of histone transcript compared to that of actin. Error bars indicate the standard error. (b) Synchronized MEFs were harvested, stained with propidium iodide and then subjected to flow cytometry, which indicated that cells were in the late S/G2 phase at 6–8 h after thymidine release. (c–m) qRT-PCR of RNA prepared from synchronized MEFs indicating an increase in histone mRNA levels at 8.5 h in samples treated with WEE1 inhibitor compared to untreated samples. The histograms include data from two independent biological experiments, each done in triplicates. (n) The nuclei were prepared from synchronized MEFs treated with the WEE1 inhibitor MK-1775 (0.625 µM, 14 h) or left untreated. Run-on assay was performed, revealing that WEE1 regulates histone transcription. Data in all the bar graphs are the mean ± s.d. Data are representative of two independent biological experiments.

Figure 4

Histone tyrosine phosphorylation by SWE1 is required for the transcriptional suppression of histones in yeast. (a–i) WT and Y40A mutant yeast cells (a–d) and WT and swe1Δ mutant yeast cells (e–i) were synchronized followed by RNA preparation and qRT-PCR. The mutants Y40A and swe1Δ showed marked increases in histone transcript abundances. The data shown are derived from individual experiments that are representative of at least three independent replicates. All data are the mean ± s.d.

Figure 5

H2BpY37 differentially regulates NPAT and HIRA recruitment. (a) ChIP performed using NPAT and IgG antibodies followed by qPCR revealed loss of NPAT recruitment at site 2 after deposition of H2BpY37 marks. *P = 0.0018. (b) Immunoblotting revealed that NPAT protein amounts were not altered in MEFs treated with WEE1 inhibitor. (c) ChIP performed using RNA polymerase II (Pol) and control IgG antibodies followed by qPCR revealed loss of RNA polymerase II recruitment at site 2 in presence of H2BpY37. ^#P = 0.01. (d) Immobilized unmodified H2B and Tyr37-phosphorylated H2B_25–49 peptides were incubated with HEK 293 cell lysates followed by immunoblotting with NPAT antibodies, indicating that NPAT binds unmodified H2B. (e) A filter-binding assay confirming the selectivity of NPAT binding to unmodified H2B. (f) Synchronized MEFs were harvested and ChIP was performed using NPAT antibodies followed by qPCR, revealing decreased NPAT binding to site 2 after 6.5 h. (g) Recruitment of HIRA at site 2 is dependent on H2B Tyr37 phosphorylation. Data shown in the bar graphs are the mean ± s.d. from three independent experiments. **P = 0.017. (h) Pull-down assay with immobilized unmodified and Tyr37-phosphorylated H2B_25–49 peptides revealing selective binding of HIRA to Tyr37-phosphorylated H2B_25–49 peptide.

Figure 6

A model of H2B Tyr37 phosphorylation upstream of the major histone gene cluster Hist1 suppresses histone mRNA transcription. WEE1 phosphorylates H2B at Tyr37 upstream of histone cluster 1, Hist1. NPAT is excluded from binding to the Hist1 cluster because of its inability to recognize Tyr37-phosphorylated H2B, which in turn inhibits RNA polymerase II (Pol II) recruitment. Subsequently, HIRA is recruited to Tyr37-phosphorylated H2B, preventing NPAT rebinding and effectively resulting in the suppression of histone mRNA synthesis.