SET-domain bacterial effectors target heterochromatin protein 1 to activate host rDNA transcription

Abstract

Transcription of rRNA genes (rDNAs) in the nucleolus is regulated by epigenetic chromatin modifications including histone H3 lysine (de)methylation. Here we show that LegAS4, a Legionella pneumophila type IV secretion system (TFSS) effector, is targeted to specific rDNA chromatin regions in the host nucleolus. LegAS4 promotes rDNA transcription, through its SET-domain (named after Drosophila Su(var)3-9, enhancer of zeste [E(z)], and trithorax [trx]) histone lysine methyltransferase (HKMTase) activity. LegAS4's association with rDNA chromatin is mediated by interaction with host HP1α/γ. L. pneumophila infection potently activates rDNA transcription in a TFSS-dependent manner. Other bacteria, including Bordetella bronchiseptica and Burkholderia thailandensis, also harbour nucleolus-localized LegAS4-like HKMTase effectors. The B. thailandensis type III effector BtSET promotes H3K4 methylation of rDNA chromatin, contributing to infection-induced rDNA transcription and bacterial intracellular replication. Thus, activation of host rDNA transcription might be a general bacterial virulence strategy.

Figure 1

LegAS4 is a nucleolar protein and activates host rDNA transcription. (A) Diagram of LegAS4 domain structure. Pink, the two nuclear localization sequences. (B) Nucleolar localization of GFP-LegAS4 in HeLa cells. B23 antibody and 4′,6-diamidino-2-phenylindole stains the nucleolus and DNA, respectively. Scale bar, 2 μm. (C) Schematic drawing of a single human rDNA repeat. (D,E) ChIP assay of LegAS4 binding to rDNA locus. 293T cells were transfected with an EV or Flag-LegAS4. Quantification of DNA binding was determined by qPCR with primer sets targeting indicated rDNA regions (D) or β-actin (ACTB) coding/promoter regions (E). Error bars indicate the s.d. of three experiments. (F) LegAS4 stimulates rDNA transcription. GFP-LegAS4 (WT or the Y172A/R191D MU) was expressed in 293T cells. Shown are qRT–PCR measurements of the relative pre-rRNA levels normalized to those of β-actin. The western blot shows the expression of GFP-LegAS4. Error bars indicate s.d. of three experiments. (G) LegAS4 increases Pol I occupancy at rDNA locus. 293T cells were transfected with GFP-LegAS4 (white bars) or the Y172A/R191D mutant (black bars) or an empty vector control (grey bars). Shown are ChIP analyses of the relative occupancy of Pol I at the rDNA promoter and the 18S rRNA coding region. Error bars indicate s.d. of three experiments. ANK, ankyrin; ChIP, chromatin immunoprecipitation; DAPI, 4′,6-diamidino-2-phenylindole; EV, empty vector; GFP, green fluorescent protein; IgG, immunoglobulin G; IGS, intergenic spacer; MU, mutant; qRT–PCR, quantitative real-time polymerase chain reaction; rRNA, ribosomal RNA.

Figure 2

HP1 is required for LegAS4 association with the rDNA. (A) Identification of HP1α/γ as the binding factors of LegAS4 by yeast two-hybrid screen. (B) Co-immunoprecipitation of LegAS4 with HP1 in HEK293T cells. (C) Domain structures of HP1α and its mutants subjected to LegAS4 binding assay in (D,E). (D,E) In vitro pulldown assay of LegAS4 binding to various HP1α mutants. (F) Mapping the HP1 binding region in LegAS4 by in vitro pulldown assay. GST-OspG protein (a type III effector from Shigella flexneri) was included as a negative control. (G) Effects of HP1α and HP1γ double knockdown on LegAS4 binding onto the rDNA. HP1α-targeting siRNA was transfected into the HP1γ shRNA stable knockdown 293T cells. Knockdown cells were transfected with Flag-LegAS4 for another 48 h followed by anti-Flag ChIP assay using primer sets targeting indicated rDNA regions. Error bars indicate s.d. of three experiments. ANK, ankyrin; CD, chromodomain; CSD, chromo shadow domain; ChIP, chromatin immunoprecipitation; GST, glutathione S-transferase; HEK, human embryonic kidney; HP1, heterochromatin protein 1; HP1^CD, HP1α CD domain; HP1^CSD, HP1α CSD domain; L4, GST-LegAS4; IgG, immunoglobulin G; shRNA, short hairpin RNA; siRNA, short interfering RNA; UBF, upstream binding factor.

Figure 3

LegAS4 defines a family of rDNA-associated SET-domain bacterial effectors. (A) Multiple sequence alignments of the LegAS4 family of bacterial SET-domain proteins. The LegAS4 family contains YP_443833 from B. thailandensis E264 (renamed as BtSET), NP_879327 from Bordetella pertussis Tohama I, NP_891504 from B. bronchiseptica RB50 (renamed as BbSET), YP_525184 from Rhodoferax ferrireducens T118, YP_004158192 from Variovorax paradoxus EPS, YP_297593 from Ralstonia eutropha JMP134, YP_585678 from Cupriavidus metallidurans CH34, YP_002554768 from Acidovorax ebreus TPSY, YP_003280830 from Comamonas testosteroni CNB-2, YP_364808 from Xanthomonas campestris and YP_003952558 Stigmatella aurantiaca DW4 3-1. (B) Domain diagram and nucleolar localization of BbSET and BtSET. HeLa cells were transfected with GFP-BbSET or BtSET. Scale bar, 2 μm. (C) BtSET specifically binds to the rDNA locus. The experiments were performed and data are presented similarly as those in Fig 1D. Error bars indicate s.d. of three experiments. (D) In vitro HKMTase assay of BtSET using calf thymus-derived core histone substrate. MU, the Y33A/R78D mutant of purified GST-BtSET. (E,F) BtSET prefers to catalyse H3K4me1 and H3K4me2 in vitro. Immunoblotting of the reaction mixtures using indicated methylation site-specific antibodies is shown in (E). Quantification of H3K4me1 and H3K4me2 is shown in F as the average of two experiments (values from mock-treated histone were arbitrarily set as 1). GFP, green fluorescent protein; GST, glutathione S-transferase; HKMTase, histone lysine methyltransferase; MU, mutant.

Figure 4

BtSET activates host rDNA transcription and contributes of Burkholderia intracellular replication. (A) qRT–PCR assay of BtSET-induced rDNA transcription in 293T cells infected with indicated B. thailandensis E264 strains (MOI, 20). Actinomycin D (ActD, 40 ng/ml) is used to block Pol I activity. Error bars indicate s.d. of three experiments. Statistical analysis was done using the Student’s t-test. *P<0.5, **P<0.05, ***P<0.001, ^#P>0.5. (B) BtSET is translocated into host cells through the B. thailandensis type III secretion system. U937 cells were infected with B. thailandensis or the ΔbipB mutant strain harbouring BtSET-TEM or GST-TEM β-lactamase expression plasmids. Scale bar, 100 μm. (C) Effects of inhibiting rDNA transcription on B. thailandensis intracellular replication. Left, B. thailandensis infection of HeLa cells (MOI, 1) in the presence of actinomycin D (40 ng/ml) (Student’s t-test, **P<0.005). Right, B. thailandensis E264 strain was cultured in the DMEM medium in the presence of 40 ng/ml ActD and fold increase of the number of bacteria following replication is shown (Student’s t-test, P>0.05). Error bars indicate s.d. of three experiments. (D) Deletion of BtSET inhibits B. thailandensis intracellular growth. HeLa cells were infected with indicated B. thailandensis strains (MOI, 1). Error bars indicate s.d. of three experiments. (E–G) Inhibition of pre-rRNA synthesis suppresses B. thailandensis intracellular growth. HeLa cells were transfected with a non-targeting control siRNA (NC) or siRNA-targeting hTAF48. Shown in E and F are levels of hTAF48 mRNA (knockdown efficiency) and pre-rRNA synthesis, respectively. (Error bars indicate s.d. of three experiments.) In G, knockdown cells were infected with B. thailandensis (MOI, 1), and bacterial intracellular replication is shown as the average of two experiments. ActD, Actinomycin D; DMEM, Dulbecco’s modified Eagle’s medium; GST, glutathione S-transferase; MOI, multiplicity of infection; mRNA, messenger RNA; NC, non-targeting control; qRT–PCR, quantitative real-time polymerase chain reaction; rRNA, ribosomal RNA; siRNA, short interfering RNA; UI, uninfected.