Figure 7: Rv1988 is required for H3 methylation during M. tuberculosis infection. | Nature Communications

Figure 7: Rv1988 is required for H3 methylation during M. tuberculosis infection.

From: Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3

Figure 7

(a) Generation of M. tuberculosis Rv1988 gene replacement mutant. Schematic representation of the strategy used for the generation of Rv1988 gene replacement mutant. (b) PCR amplifications were performed using specific primers (depicted with red arrows in a) as indicated below the agarose gel, using genomic DNA of M. tuberculosis H37Rv and ΔRv1988 mutant. Left panel shows the PCR amplification with F1-R2 pair which gives a product of 2.2 kb for H37Rv and 2.9 kb for ΔRv1988. Right panel shows PCR amplification with F1-R1 and F2-R2, which do not give any product with Rv genomic but yield a 2.2 kb product ΔRv1988 mutant. Mr: Marker, Rv: M. tuberculosis H37Rv, Δ1988: ΔRv1988 mutant. (c) Absence of Rv1988 protein from ΔRv1988 mutant. Western blot performed with cell lysate of M. tuberculosis H37Rv (lane 1) and ΔRv1988 mutant (lane 2) was probed with Rv1988 and GroEL1 (control) antibodies. (d) Rv1988 represses specific host genes. Expression levels of the indicated genes measured by real-time qRT-PCR in THP1 macrophages infected with M. tuberculosis H37Rv (white bars, WT) or M. tuberculosis H37Rv-ΔRv1988 mutant (black bars, ΔRv1988). Levels were normalized against GAPDH. ncRNA: lincRNA ENSG00000250584. (e) Histone H3-arginine methylation by Rv1988. Double ChIP was performed, using histone H3 and dimethyl arginine antibodies (Methods), on THP1 macrophages infected with M. tuberculosis H37Rv and ΔRv1988 mutant. Association of histone H3, dimethylated for arginine residue, with the promoters of the indicated genes was analysed by quantitative real-time PCR. (f) Western blot to compare the H3R42me2 levels between THP1 macrophages infected with M. tuberculosis H37Rv (lane 1) or ΔRv1988 mutant (lane 2). As a control the blot was also probed with H3R2me2 and H3 antibodies. (g) Histone H3R42 dimethylation by Rv1988. ChIP was performed using histone H3R42me2 antibody on THP1 macrophages infected with M. tuberculosis H37Rv and ΔRv1988 mutant. Association of histone H3, dimethylated at R42, with the promoters of the indicated genes was analysed by quantitative real-time PCR. The error bars represent s.d. Student’s t-test, *P<0.05, **P<0.01.

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