Btg2 enhances retinoic acid-induced differentiation by modulating histone H4 methylation and acetylation

Abstract

Retinoic acid controls hematopoietic differentiation through the transcription factor activity of its receptors. They act on specific target genes by recruiting protein complexes that deacetylate or acetylate histones and modify chromatin status. The regulation of this process is affected by histone methyltransferases, which can inhibit or activate transcription depending on their amino acid target. We show here that retinoic acid treatment of hematopoietic cells induces the expression of BTG2. Overexpression of this protein increases RARalpha transcriptional activity and the differentiation response to retinoic acid of myeloid leukemia cells and CD34+ hematopoietic progenitors. In the absence of retinoic acid, BTG2 is present in the RARalpha transcriptional complex, together with the arginine methyltransferase PRMT1 and Sin3A. Overexpressed BTG2 increases PRMT1 participation in the RARalpha protein complex on the RARbeta promoter, a target gene model, and enhances gene-specific histone H4 arginine methylation. Upon RA treatment Sin3A, BTG2, and PRMT1 detach from RARalpha and thereafter BGT2 and PRMT1 are driven to the cytoplasm. These events prime histone H4 demethylation and acetylation. Overall, our data show that BTG2 contributes to retinoic acid activity by favoring differentiation through a gene-specific modification of histone H4 arginine methylation and acetylation levels.

FIG. 1.

BTG2 expression is induced by RA treatment. (A) Northern blot analysis of BTG2 expression in the indicated cell lines, untreated or treated with RA for the indicated times. Blots were hybridized with radiolabeled BTG2 and control GAPDH probes. (B) Real-time PCR analysis of BTG2 expression in the indicated cell lines, untreated or treated with RA for the indicated times. (C) Western blot analysis of BTG2 expression in lysates from the indicated cells. C+ is a lysate from 293T cells transfected with a BTG2 expression vector. Blots were stripped and rehybridized with an anti-α-tubulin antibody as a loading control.

FIG. 2.

BTG2 expression increases RARα transcriptional activity. (A) Transcriptional activity of a luciferase-linked β-RARE responsive element cotransfected in HL60 cells with the indicated amounts of vector DNA (V) or a BTG2 expression vector. (B) Transcriptional activity of a luciferase-linked β-RARE responsive element cotransfected in HL60 cells with vector DNA (V) or a BTG2 expression vector with or without a PRMT1 expression vector. RA indicates an 8-h treatment with 10⁻⁷ M RA. The results are expressed as the transcriptional activity relative to that measured in cells transfected with the responsive element and empty vector alone. The means and standard deviations from three triplicate experiments are given.

FIG. 3.

BTG2 expression increases RA-induced differentiation of hematopoietic cells. (A) Western blot showing BTG2 overexpression in the indicated retrovirally infected cells. C+, lysates from 293T cell transiently transfected with a BTG2 expression vector; C, control uninfected cells; B, cells infected with a BTG2 expression vector. Blots were stripped and rehybridized with an anti-α-tubulin antibody as a loading control. (B) RA-induced differentiation of HL60 and U937 cells with or without BTG2 overexpression measured as the percentage of NBT- or CD11b-positive cells. Cells were treated with 10⁻⁷ M RA for the indicated times. C, cells infected with a control “empty” retroviral vector; BTG2, cells infected with a BTG2 retroviral vector. (C) Differentiation of CD34⁺ human hematopoietic progenitor cells with 10⁻⁷ M RA and growth factors cocktails inducing monocytic or granulocytic differentiation (see Materials and Methods). C, BTG2, as described above. The means and standard deviations from three independent experiments are given.

FIG. 4.

BTG2 interacts with the RARα transcriptional complex. Coimmunoprecipitation experiments from lysates of HL60 cells. Cells were treated with 10⁻⁶ M RA for the indicated times. Lysates were immunoprecipitated with the antibodies shown on the right. Samples were analyzed by Western blotting with the indicated antibodies. The positions of molecular mass markers expressed in kilodaltons are indicated on the right of each panel. C+, lysates from HL60; C−, immunoprecipitation with an anti-CD40 antibody from lysates of untreated HL60 cells. Ig, position of the immunoglobulin band when visible in the frame.

FIG. 5.

BTG2 takes part in the RARα transcriptional complex on the RARβ promoter. (A) Anti-PRMT1, anti-BTG2, anti-Sin3A, and anti-RARα ChIP experiments on HL60 cells infected with a control empty vector (C) or with a BTG2 vector (B) treated with RA for the indicated times. Input, PCR with RARβ promoter oligonucleotides on the cell lysates used for ChIP; Actin, control amplification of a β-actin gene sequence; RARβ, amplification with RARβ promoter oligonucleotides. In all of the experiments shown in panel A, PCR amplification with RARβ promoter oligonucleotides on chromatin precipitated with an anti-CD40 antibody, as a negative control, gave no signal; a representative panel is shown. (B) Western blot showing persistent RARα expression in HL60 or HL60 BTG2 cells treated with 10⁻⁶ M RA for the indicated times.

FIG. 6.

(A) Western blot of nuclear and cytoplasmic fractions extracted from HL60 cells treated with 10⁻⁶ M RA for the indicated times. Filters were transversely cut at a molecular mass of 31 kDa on the basis of stained molecular mass standards. The upper half was hybridized with an anti-PRMT1 antibody, and the lower half was stained with an anti-BTG2 antibody. The loading amounts of the nuclear fractions were controlled by staining the filters after the electrotransfer with Ponceau S. The staining identifies the core histone bands. The loading amounts of the cytoplasmic fractions were evaluated by stripping the filters and rehybridizing them with an anti-α-tubulin antibody. (B) Immunofluorescence experiments on cytospin slides showing HL60 cells before (−RA) and after (+RA) a 6-h treatment with 10⁻⁶ M RA. The primary antibodies are indicated on the left. The secondary antibodies were labeled with TRITC, tetramethyl rhodamine 5 (and 6)-isothiocyanate. DAPI is a nuclear stain used to identify the nuclei.

FIG. 7.

BTG2 expression increases histone H4 arginine methylation and lysine acetylation in response to RA. (A) Anti-methyl-arginine 3 histone H4 ChIP experiments on control (C) or BTG2- overexpressing (B) HL60 cells treated with RA for the indicated times. Input, Actin, and RARβ are as described for Fig. 5. (B) In vitro methylation of core histones extracted from HL60 cells by the indicated GST or GST fusion proteins. (C) Anti-acetyl-H4 ChIP experiments on control (lanes C) or BTG2-overexpressing (lanes B) HL60 cells treated with RA for the indicated times. Input, Actin, and RAR-β are as described for Fig. 5. (D) Anti-methyl-arginine 3 histone H4 and anti-acetyl-histone H4 ChIP experiments on lysates of control (lanes C) 293T cells or 293T cells transiently transfected with a BTG2 expression vector (lanes BTG2). For anti-methyl-arginine 3 histone H4 ChIP cells were not treated with RA to prevent demethylation (see Fig. 5). For anti-acetyl-H4 ChIP experiments, cells were studied before and after a 24-h RA treatment. Input, Actin, and RARβ are as described for Fig. 5. P, positive PCR control on genomic DNA; N, negative PCR control without DNA. In the experiments shown in panels A, C, and D PCR amplification with the RARβ promoter oligonucleotides on chromatin precipitated with an anti-CD40 antibody, as a negative control, gave no signal. (E) Real-time PCR showing the induction of RARβ expression by 10⁻⁶ M RA treatment of HL60 and HL60 BTG2 cells for the indicated times. (F) Western blot showing RARβ expression in HL60 or HL60 BTG2 cells treated with 10⁻⁶ M RA for the indicated times.