Two mammalian MOF complexes regulate transcription activation by distinct mechanisms

Abstract

In mammals, MYST family histone acetyltransferase MOF plays important roles in transcription activation by acetylating histone H4 on K16, a prevalent mark associated with chromatin decondensation, and transcription factor p53 on K120, which is important for activation of proapoptotic genes. However, little is known about MOF regulation in higher eukaryotes. Here, we report that the acetyltransferase activity of MOF is tightly regulated in two different but evolutionarily conserved complexes, MSL and MOF-MSL1v1. Importantly, we demonstrate that while the two MOF complexes have indistinguishable activity on histone H4 K16, they differ dramatically in acetylating nonhistone substrate p53. We further demonstrate that MOF-MSL1v1 is specifically required for optimal transcription activation of p53 target genes both in vitro and in vivo. Our results support a model that these two MOF complexes regulate distinct stages of transcription activation in cooperation with other histone modifying activities.

Figure 1. MSL1v1 is essential for regulating the nucleosomal activity of the MOF-MSL1v1 Complex

(A) Immunoblots of proteins co-purified with Myc-MSL1v1. Antibodies used are indicated on right. (B) Immunoblot analysis of MOF complexes following fractionation on the superose 6 sizing column. Positions of molecular weight markers are indicated on top. (C) Schematic for comparing MSL1 and MSL1v1 proteins. Both the coiled-coil domain (red) and the MOF interacting domain (PEHE, blue) are conserved in the two proteins. However, only MSL1 contains a MSL3 interacting domain (yellow) C-terminal to the PEHE domain. (D) Coomassie stain of the recombinant MOF or MOF complexes (as indicated on right) purified from baculovirus infected insect cells. The MOF-MSL1v1 and the MSL complexes were purified through a FLAG-epitope on either MSL1v1 or MSL3 proteins. (E) In vitro HAT assays for MOF, MOF-MSL1v1 or the MSL complex. Both HeLa nucleosomes (top) and free recombinant histone octamers (bottom) were used as substrates. [³H]-acetyl-CoA was used as the cofactor.

Figure 2. MOF-MSL1v1 is specifically required to acetylate p53 K120 in vitro

(A) Coomassie stain of the purified recombinant p53 and p53 K120A proteins. (B) In vitro HAT assay using wild-type p53 or p53 K120A as the substrate. Proteins present in the assay are indicated on top. [³H]-acetyl-CoA was used as the cofactor.

Figure 3. Distinct domains of MSL1v1 are required for MOF-MSL1v1 mediated acetylation of nucleosomal H4 and p53

(A, C) Schematic for MSL1v1 deletion mutants used in the HAT assay. (B, D) In vitro HAT assay using HeLa nucleosomes or p53 as substrates. Different MOF-containing complexes used for the assay are indicated on top. Equivalent amounts of MOF were used in all cases. [³H]-Acetyl-CoA was used for detection. (E) MSL1v1 705-982 but not MSL1v1 Δ800 or MSL1 binds to GST-p53. Bound proteins were analyzed by immunoblot using anti-Flag antibody. 1% Input was loaded on the gel as controls.

Figure 4. MOF-MSL1v1 mediated p53 K120 acetylation is important for transcription activation in vitro

(A) Schematic of in vitro transcription assay using DNA templates (An and Roeder, 2004). (B) DNA-templated transcription assays with no p53, p53 K120A and wild-type p53 as indicated. (C) In vitro transcription assay using purified p53, MOF and the MOF-MSL1v1 complex with or without acetyl-CoA as indicated. (D) In vitro transcription assay using purified p53, MOF, the full length MOF-MSL1v1, MOF-MSL1v1 705-982 and MOF-MSL1v1 Δ800 as indicated. The transcription reactions in (C) and (D) were performed according to the scheme in (A).

Figure 5. MSL1v1 is important for global H4 K16 acetylation and p53 K120 acetylation in vivo

(A) SiRNAs or shRNA against MOF, MSL1v1 or MSL1 were used to transfect 293T cells, as indicated on top. The protein level after knockdown was demonstrated by immunoblot using antibodies against respective proteins. Immunoblot for H3 was used as the loading control. (B) SiRNA or shRNA treatments for MOF, MSL1v1 or MSL1 reduced global H4 K16 acetylation. The immunoblot of whole cell lysate using anti-H4 K16 antibody is shown on top. Immunoblot using anti-H3 antibody was used as the loading control. (C) SiRNA or shRNA treatments for MOF, MSL1v1 affects the global p53 K120 acetylation. Acetylated p53 in the knockdown cells was immunoprecipitated with anti-p53 K120ac antibody and detected by anti-p53 antibody (bottom). 2% input of each sample was included on top.

Figure 6. MOF-MSL1v1 activates PUMA and BAX expression by acetylating p53 K120 in vivo

(A) RT-PCRs were performed using RNAs recovered from H1299 cells after transfecting with different expression vectors as indicated on bottom. The gene tested is indicated on top of each panel. For each sample, gene expression level after normalizing against 5S rRNA was compared with that of the control cells, which is arbitrarily set at 1. (B) RT-PCRs were performed as in (A) except that MSL1v1 Δ800 was used. (C) RT-PCRs were performed as in (A) for 293T cells after knocking down MOF, MSL1v1 or MSL1 respectively. (D) Top, ChIP experiments using anti-p53 K120ac antibody were performed in cells co-transfected with various expression vectors as indicated. Bottom, ChIP experiment using anti-H4 K16ac antibody was performed in cells transfected with different expression vectors as indicated. Primers for either transcription start site (P) or around stop codon (3′-UTR) were used for quantitative PCR. (E) ChIP experiments were performed as in (D) in 293T cells treated with MOF, MSL1v1, MSL1 and control siRNA or shRNA. Both p53 K120 acetylation and K16 acetylation levels in (D) and (E) are normalized to 2% input. Means and standard deviations from at least three independent experiments were presented in (A)–(E). Standard deviations are indicated by error bars.

Figure 7. Model for the roles of two MOF complexes in transcription activation

(A) ChIP experiments using anti-Myc antibody were performed in cells transfected with MOF, Myc-MSL1v1 or MOF, Myc-MSL1/3 with or without p53. ChIP was quantified using four primer pairs as indicated in gene schematics. Signals in all cases were presented as % input. Standard deviations are indicated by error bars. (B) The MOF-MSL1v1 complex is recruited to the promoter of target genes through coordinated interactions with the histone methyltransferase MLL as well as the transcription factor p53. Once recruited, MOF-MSL1v1 acetylates both p53 and H4 K16 to facilitate transcription initiation. The recruitment of the MSL complex by tri-methylated H3K36 (Larschan et al., 2007) plays important roles in acetylating H4 K16 at downstream of the genes, which facilitates transcription elongation (Bell et al., 2007; Kind et al., 2008).