G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis

Abstract

Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes.

Figure 1

Targeting and genotyping of G9a-mutant mice. (A) Schematic representation of the mouse G9a gene locus, the G9a targeting construct, and the targeted allele. Exons are numbered and indicated by black boxes. Two splicing variants of the murine G9a (mG9a-S and mG9a-L) are presented at the top. The G9a targeting construct was designed to delete exons 5 to HindIII site of 21, which correspond to the glutamic acid stretch to ankyrin (ANK) repeats. R, EcoRI; D, DraI (not unique in the 14-kb EcoRI fragment); N, NotI; H, HindIII; B, BamHI. (B) Southern blot of EcoRI-digested DNA isolated from wild-type, G9a^+/−, and G9a^−/− ES cells. (C) Northern blot analyses of G9a^−/− ES cells. ANK and SET probes correspond to the region encoding for the ankyrin-repeats and the SET-domain, respectively. (D) Western blot analyses of G9a expression in wild-type and G9a^−/− ES cells and G9a^−/− ES cells stably expressing cDNA for the G9a-S or G9a-L isoform.

Figure 2

G9a^−/− embryos were delayed in their development. (A) The wild-type littermate at E9.5. (D) The lateral view of G9a^−/− embryo at E9.5. The approximate levels of the section presented in B, C, E, and F are indicated as dashed lines. Allantois (Al) develops normally. (B,C) Section through hindbrain (HB in B) to tail region (C). (E,F) Section through forebrain to tail region. S, somites; NT, neural tube; G, gut; VA, vitelline artery; A, amnion; NP, neural plate. (G) TUNEL analysis and α-phospho H3-S10 staining of E9.5 G9a^−/− (bottom) and the E8.5 wild-type embryos (top) on transversal sections. (H) Growth characteristics of G9a^−/− PEFs. Here, 10⁴ cells of PEFs derived from individual E9.0 embryos were cultured, and population doublings (PDL) were monitored. (I) Measurement of DNA content of G9a^−/− PEFs. The wild-type and G9a^−/− PEFs prepared from E9.5 embryos were cultured for 16 h and the DNA content was subsequently visualized by PI staining. Calculated populations (%) at each cell cycle stage are represented below. (J) Growth defect of the G9a-mutant ES cells in differentiation conditions. Here, 10⁵ cells of each ES cell line were cultured in ES medium (upper) or RA (1 μM), no LIF-containing differentiation medium (lower). Vertical bars represent the cell numbers after 4 d of culture. Typical data were picked up from triplicate independent reproducible examinations.

Figure 2

G9a^−/− embryos were delayed in their development. (A) The wild-type littermate at E9.5. (D) The lateral view of G9a^−/− embryo at E9.5. The approximate levels of the section presented in B, C, E, and F are indicated as dashed lines. Allantois (Al) develops normally. (B,C) Section through hindbrain (HB in B) to tail region (C). (E,F) Section through forebrain to tail region. S, somites; NT, neural tube; G, gut; VA, vitelline artery; A, amnion; NP, neural plate. (G) TUNEL analysis and α-phospho H3-S10 staining of E9.5 G9a^−/− (bottom) and the E8.5 wild-type embryos (top) on transversal sections. (H) Growth characteristics of G9a^−/− PEFs. Here, 10⁴ cells of PEFs derived from individual E9.0 embryos were cultured, and population doublings (PDL) were monitored. (I) Measurement of DNA content of G9a^−/− PEFs. The wild-type and G9a^−/− PEFs prepared from E9.5 embryos were cultured for 16 h and the DNA content was subsequently visualized by PI staining. Calculated populations (%) at each cell cycle stage are represented below. (J) Growth defect of the G9a-mutant ES cells in differentiation conditions. Here, 10⁵ cells of each ES cell line were cultured in ES medium (upper) or RA (1 μM), no LIF-containing differentiation medium (lower). Vertical bars represent the cell numbers after 4 d of culture. Typical data were picked up from triplicate independent reproducible examinations.

Figure 2

G9a^−/− embryos were delayed in their development. (A) The wild-type littermate at E9.5. (D) The lateral view of G9a^−/− embryo at E9.5. The approximate levels of the section presented in B, C, E, and F are indicated as dashed lines. Allantois (Al) develops normally. (B,C) Section through hindbrain (HB in B) to tail region (C). (E,F) Section through forebrain to tail region. S, somites; NT, neural tube; G, gut; VA, vitelline artery; A, amnion; NP, neural plate. (G) TUNEL analysis and α-phospho H3-S10 staining of E9.5 G9a^−/− (bottom) and the E8.5 wild-type embryos (top) on transversal sections. (H) Growth characteristics of G9a^−/− PEFs. Here, 10⁴ cells of PEFs derived from individual E9.0 embryos were cultured, and population doublings (PDL) were monitored. (I) Measurement of DNA content of G9a^−/− PEFs. The wild-type and G9a^−/− PEFs prepared from E9.5 embryos were cultured for 16 h and the DNA content was subsequently visualized by PI staining. Calculated populations (%) at each cell cycle stage are represented below. (J) Growth defect of the G9a-mutant ES cells in differentiation conditions. Here, 10⁵ cells of each ES cell line were cultured in ES medium (upper) or RA (1 μM), no LIF-containing differentiation medium (lower). Vertical bars represent the cell numbers after 4 d of culture. Typical data were picked up from triplicate independent reproducible examinations.

Figure 3

(A–D) Alteration of G9a-dependent covalent H3 modifications detected by Western blot analyses. (A) Purified and precalibrated histones with α-H3 (upper panel) from the G9a^+/− and G9a^−/− embryos were stained with α-dimethyl H3-K9 antibodies. (B) α-dimethyl H3-K9 staining of H3 from wild-type and G9a^−/− ES cells and G9a^−/− ES cells expressing exogenous G9a-L. (C) Calibration of the dimethyl H3-K9 content in G9a^−/− ES cells. The ratio of H3 content between wild-type and mutant were precalibrated and are represented below. (D) Other covalent modification statuses of H3 between wild-type and G9a^−/− ES cells. All the presented data shown in Fig. 2A–D were reproducible at least twice. (E) HMTase activity of G9a-HMTase domain. Amino acid sequences of GST-fused recombinant H3 N terminus protein as substrates are at the top. Recombinant G9a-HMTase domain could add methyl groups to H3-K9 and H3-K27. (F) H3 HMTase activity in wild-type and G9a^−/− ES cells. HMTase activity and substrate specificity of nuclear extracts of wild-type and G9a^−/− ES cells were indistinguishable. HMTase activities of the nuclear extracts toward Lys 4, Lys 9, and Lys 27 were calibrated as Nx–NT.

Figure 4

(A) Nuclear localization profiles of transiently expressed EGFP-tagged mouse G9a molecules (G9a-L) and DsRed-tagged human HP-1β molecules in the murine fibroblast cell line C3H10T 1/2 . EGFP-G9a-L molecules exist broadly in interphase nuclei and mostly excluded from HP-1β-enriched pericentric heterochromatin (arrows). (B) Immunohistochemical analyses of G9a^−/− ES cells with α-dimethyl H3-K9. Nuclei of G9a^−/− ES cells contained dimethyl H3-K9 only in DAPI-dense regions, whereas these were broadly distributed to both euchromatic and heterochromatic loci in wild-type nuclei.

Figure 5

HMTase-dependent transcriptional repression mediated by G9a. (A) Schematic representation of the used plasmids for in vitro luciferase assays. (B) Protein expression analysis of the GAL4-DBD-fused constructs described in A. Fusion molecules were detected by α-GAL4-DBD. (C) Full-length G9a (PM-G9a-L) and a G9a HMTase domain (PM-HMT) suppress transcription, but dead HMTase full-length G9a (PM-G9a-SΔNHLC ) and a G9a dead HMTase domain (PM-ΔNHLC) cannot. All the transient luciferase assays were performed multiple times and the results presented were reproduced. (D) The G9a-mediated suppression was not relieved in the presence of TSA (100 ng/mL). Upper left panel shows an accumulation of H3-K9 acetylation by the TSA treatment.

Figure 6

Characterization of Mage-a genes. (A) RNA expression of Mage-a genes. Six micrograms of total RNA prepared from G9a^+/+ (TT2), G9a^+/− (#36), G9a^−/− (2–3 and 22–10), 2–3-expressing exogenous G9a-L (2–3-L), G9a-S (2–3-S), or a drug-selectable molecule alone (2–3+mock1 and 2) were separated, blotted to a nylon membrane, and hybridized with a ³²P-labeled Mage-a8 cDNA. Expression of Mage-a2, Mage-a6, and Mage-a8 genes was determined in G9a^−/− ES cells by nucleotide sequencing of the RT-PCR products. The lower panel shows 28S rRNA stained with ethidium bromide in the same gel before blotting. (B) ChIP analyses on the Mage-a2 promoter region. Formaldehyde cross-linked chromatin from TT2, 22–10, and 15–3 (2–3-S) ES cells was immunoprecipitated without (−) or with α-dimethyl H3-K9 (K9), α-dimethyl H3-K4 (K4), or α-acetyl-H3-K9+14 (right panel). An equal amount of Mage-a2 promoter sequence in TT2, 22–10, and 15–3 nucleosomal preparations was determined by PCR from 1.4% of the input ChIP/ PCR reaction (lanes 12–14). The linearity of this PCR was confirmed by serial dilution of the TT2 input DNA (lanes 12,15–17). The product size of the Mage-a2 PCR is 242 bp. Similar results were obtained with multiple independent experiments.