The MUT9p kinase phosphorylates histone H3 threonine 3 and is necessary for heritable epigenetic silencing in Chlamydomonas

Abstract

Changes in chromatin organization are emerging as key regulators in nearly every aspect of DNA-templated metabolism in eukaryotes. Histones undergo many, largely reversible, posttranslational modifications that affect chromatin structure. Some modifications, such as trimethylation of histone H3 on Lys 4 (H3K4me3), correlate with transcriptional activation, whereas others, such as methylation of histone H3 on Lys 27 (H3K27me), are associated with silent chromatin. Posttranslational histone modifications may also be involved in the inheritance of chromatin states. Histone phosphorylation has been implicated in a variety of cellular processes but, because of the dynamic nature of this modification, its potential role in long-term gene silencing has remained relatively unexplored. We report here that a Chlamydomonas reinhardtii mutant defective in a Ser/Thr protein kinase (MUT9p), which phosphorylates histones H3 and H2A, shows deficiencies in the heritable repression of transgenes and transposons. Moreover, based on chromatin immunoprecipitation analyses, phosphorylated H3T3 (H3T3ph) and monomethylated H3K4 (H3K4me1) are inversely correlated with di/trimethylated H3K4 and associate preferentially with silenced transcription units. Conversely, the loss of those marks in mutant strains correlates with the transcriptional reactivation of transgenes and transposons. Our results suggest that H3T3ph and H3K4me1 function as reinforcing epigenetic marks for the silencing of euchromatic loci in Chlamydomonas.

Fig. 1.

Effect of the mut9 mutation on transcriptional gene silencing, sensitivity to DNA-damaging agents, and meiotic heritability of the repressed states. (A) Growth and survival on Tris-acetate-phosphate (TAP) medium or on TAP medium containing spectinomycin of the wild-type untransformed strain (CC-124), the silenced parental strain (11-P[300]), the mutant strain (Mut-9), and two strains complemented with a MUT9 wild-type copy [Mut-9(MUT9)-20 and Mut-9(MUT9)-28]. (B) Northern blot analysis of the indicated strains. The levels of the wild-type MUT9 mRNA, the truncated transcript resulting from the insertion mutation (mut9), and the RbcS2:aadA:RbcS2 transgenic RNA (aadA) are shown. The RBCS2 transcript was used as a control for equivalent loading of the lanes. (C) Survival of 11-P[300] (filled squares), Mut-9 (filled diamonds), and Mut-9(MUT9)-20 (open triangles) on TAP medium containing increasing concentrations of bleomycin. (D) A meiotic tetrad product (Mut-9-9-1), containing exclusively the mut9 mutation, was crossed to 11-P[300], and reactivation of transgene expression was tested in the progeny by their ability to grow on spectinomycin-containing medium. The segregation of the mut9 mutation and the RbcS2:aadA:RbcS2 transgene (aadA) was assessed by Southern blotting (Lower). (E) Mut-9 was crossed to the untransformed wild-type strain of opposite mating type (CC-125), and transgene expression as well as meiotic segregation was evaluated as described above.

Fig. 2.

MUT9p is a Ser/Thr protein kinase that phosphorylates histone H3 Thr-3 and histone H2A. (A) Schematic representation of MUT9p and related Ser/Thr protein kinases. The catalytic domain and a nuclear localization signal (NLS) are indicated on the MUT9p diagram. The brown or pink regions in related proteins represent aligned sequences with >55% or >35% identity, respectively. At, Arabidopsis thaliana; Hs, Homo sapiens; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe. (B) Phosphorylation activity of MUT9p on calf thymus histones. An RNA helicase (MUT6p) and a MUT9p point mutant (MUT9p K174R) were used as negative controls. (C) Recombinant MUT9p (rM9) activity on itself and on Chlamydomonas (CrH3) or calf thymus (cH3) histone H3. (D) Phosphorylation activity of MUT9p on HPLC-purified Chlamydomonas histones, with the specific histones indicated on the left. (E) Cauliflower mononucleosomes as the substrate for MUT9p. (F) Mononucleosomes, predigested with trypsin, as the substrate for MUT9p. The globular histone cores are indicated by asterisks. (G) Identification of the MUT9p phosphorylation site on H3 by N-terminal sequencing of ³²P-labeled protein. The MUT9p activity on recombinant H3 was also examined by immunoblotting with an anti-H3T3ph antibody (Inset). (H) In vivo phosphorylation levels, detected with an anti-phospho-Thr antibody, in partly purified histones from the indicated strains. Calf thymus histones were used as a positive control (Histones). (I) Immunoblot analysis of in vivo H3T3ph amounts with the modification specific antibody.

Fig. 3.

Phosphorylated H3T3 and monomethylated H3K4 are preferentially associated with transcriptionally silent loci. (A) Immunoblot analysis of global H3T3ph and H3K4 methylation states in the indicated strains. The asterisk denotes a nonspecifically cross-reacting protein. Mut-11, mutant defective in a core subunit of H3K4 methyltransferase complexes; Mut-9, Mut-11, mut9 and mut11 double mutant. (B) Chromatin immunoprecipitation assay of H3T3ph levels (in relative units) associated with the promoter regions of the RbcS2:aadA:RbcS2 transgene (aadA), the TOC1 retrotransposon (TOC1-LTR), and the constitutively expressed RPS3 gene in the described strains. (C) ChIP analysis of H3T3ph and H3K4 methylation states associated with the transcription units described above and with the TOC1 coding region.