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. 2009 Aug;20(15):3543-51.
doi: 10.1091/mbc.e09-02-0114. Epub 2009 May 28.

Induction of HoxB transcription by retinoic acid requires actin polymerization

Carmelo Ferrai  1 , Gabriela Naum-Onganía, Elena Longobardi, Martina Palazzolo, Andrea Disanza, Victor M Diaz, Massimo P Crippa, Giorgio Scita, Francesco Blasi
Affiliations

Induction of HoxB transcription by retinoic acid requires actin polymerization

Carmelo Ferrai et al. Mol Biol Cell. 2009 Aug.

Abstract

We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of "elongating" RNAPII, Prep1, beta-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes.

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Figures

Figure 1.
Figure 1.
RA increases the polymerization rate in NT2-D1 nuclear extracts, in vitro. Nuclear extracts from NT2-D1 cells were prepared as described in Materials and Methods. Left, equal amounts of extracts were mixed with GST or GST-VCA (10 μg), as indicated, and subjected to the pyrene actin polymerization assay. The lines represent the initial rate of polymerization. Right, purified Arp2/3 complex (10 nM) was used as an internal control for the reaction. N, nuclear extract; RA, retinoic acid; CD, cytochalasin D.
Figure 2.
Figure 2.
Down-regulation of N-WASP prevents HoxB1 and HoxB2 induction by RA. (A) Immunoblotting analysis to assess the down-regulation of N-WASP in NT2-D1 cells. Cells were transfected with an h-N-WASP–interfering oligonucleotide (siRNA) or with a scrambled oligonucleotide (see Materials and Methods). After 24 h, 1 μM RA was added, and 16 h thereafter proteins were isolated and immunoblotted with anti N-WASP and Histone 2b antibodies. (B) N-WASP down-regulation prevents HoxB1 induction. RNA isolated in parallel from the same experiment of A, was subjected to semiquantitative RT-PCR with primers specific for the HoxB1 and GADPH mRNA (see Materials and Methods). Lane 1, control, N-WASP+ cells; lane 2, RA-treated N-WASP+ cells (16 h) in which HoxB1 is induced; lane 3, control N-WASP− cells; lane 4, RA-treated N-WASP− cells; lane 5, control transcription of the sample used in lane 2 performed in the absence of reverse transcriptase. At the bottom, the same RNA samples used for lanes 1–4 were used to measure HoxB2 (with primers specific for HoxB2) and GAPDH RNA (see Materials and Methods). This experiment was carried out in duplicate with identical results.
Figure 3.
Figure 3.
Effect of a dominant-negative actin mutant on the RA-induced HoxB2 transcription. NT2-D1 cells were transfected with 1.6, 16, or 50 μg of DNA encoding either of two variants of actin containing a nuclear localization signal, the G13R mutant that is polymerization-defective (dominant-negative) and the S14C mutant, which is facilitated in polymerization. Cells were treated with RA for 24 h, after which RNA was extracted and HoxB2 mRNA measured by quantitative PCR. The data are the average of at least two independent experiments performed in triplicate. Error bars, SD.
Figure 4.
Figure 4.
Prep1 interaction with actin and N-WASP. (A) Nuclear extracts of untreated, 1 μM RA- and RA + CytD-(100 nM) treated NT2-D1 cells were immunoprecipitated with an anti-Prep1 antibody and blotted against a β-actin antibody. Right panel, the results with an irrelevant antibody. (B) RA and CytD do not modify the nuclear localization of Prep1. Nuclear or cytoplasmic extracts of NT2-D1 cells treated as indicated were immunoblotted with the indicated antibodies. An anti-histone and an anti-β-actin 2B antibody was used to assess equal loading of extracts. (C) RA-dependent interaction of Prep1 with N-WASP. Nuclear extracts from untreated or RA-treated (24 h) cells were immunoprecipitated with anti-Prep1 (IP) or control (Ctr) antibodies and blotted with N-WASP and actin antibodies.
Figure 5.
Figure 5.
DNA-binding Prep1 is associated with p54Nrb and PSF. (A) Nuclear extracts of untreated NT2-D1 cells were immunoprecipitated with a monoclonal anti-Prep1 or a control (C) antibody and blotted using polyclonal PSF, Prep1, or p54Nrb antibodies. (B) P54Nrb is present on the DNA-binding Prep1–Pbx complexes. EMSA analysis of HeLa nuclear extracts with the specific Prep1–Pbx-binding oligonucleotide (see Materials and Methods). n.s., nonspecific band. The nuclear extract from HeLa cells (lanes 1 and 6) forms two specifically retarded bands. Prep1 antibodies prevent the formation of both bands (lane 2). The lower band is inhibited by the anti-Pbx1 antibodies (lane 4), whereas the upper band is inhibited by the anti-Pbx2 (lane 5) antibodies. Therefore the two bands are identified as Prep1–Pbx1b (lower band) and Prep1–Pbx2 (upper band) as previously reported (Berthelsen et al., 1998). Formation of both bands is inhibited by anti-p54Nrb antiserum (lane 3).
Figure 6.
Figure 6.
RA treatment induces the recruitment of Prep1, actin, N-WASP, p54Nrb, PSF, and the elongating form of RNAPII on the enhancer of the HoxB2 gene, but this recruitment is prevented by CytD. Chromatin immunoprecipitation analysis of NT2-D1 cells (untreated, 24-h RA-treated and 24-h RA + CytD-treated). Cross-linked chromatin was immunoprecipitated with the indicated antibodies and the precipitated DNA amplified by quantitative PCR with oligonucleotides specifically identifying the HoxB2 enhancer or the uPA intergenic region (see Materials and Methods). The antibodies used are indicated on top. Antibodies to RNPII-S2p recognize the elongating form of the RNAPII enzyme. Results and error bars shown are the average and SD of quantitative PCRs performed in triplicate from three independent chromatin immunoprecipitation experiments. Values are reported as fold enrichment relative to input DNA.
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