A functional N-terminal domain in C/EBPβ-LAP* is required for interacting with SWI/SNF and to repress Ric-8B gene transcription in osteoblasts

Abstract

The chromatin remodeling complex SWI/SNF and the transcription factor C/EBPβ play critical roles in osteoblastic cells as they jointly control transcription of a number of bone-related target genes. The largest C/EBPβ isoform, LAP*, possesses a short additional N-terminal domain that has been proposed to mediate the interaction of this factor with SWI/SNF in myeloid cells. Here we examine the requirement of a functional N-terminus in C/EBPβ-LAP* for binding SWI/SNF and for recruiting this complex to the Ric-8B gene to mediate transcriptional repression. We find that both C/EBPβ-LAP* and SWI/SNF simultaneously bind to the Ric-8B promoter in differentiating osteoblasts that repress Ric-8B expression. This decreased expression of Ric-8B is not accompanied by significant changes in histone acetylation at the Ric-8B gene promoter sequence. A single aminoacid change at the C/EBPβ-LAP* N-terminus (R3L) that inhibits C/EBPβ-LAP*-SWI/SNF interaction, also prevents SWI/SNF recruitment to the Ric-8B promoter as well as C/EBPβ-LAP*-dependent repression of the Ric-8B gene. Inducible expression of the C/EBPβ-LAP*R3L protein in stably transfected osteoblastic cells demonstrates that this mutant protein binds to C/EBPβ-LAP*-target promoters and competes with the endogenous C/EBPβ factor. Together our results indicate that a functional N-terminus in C/EBPβ-LAP* is required for interacting with SWI/SNF and for Ric-8B gene repression in osteoblasts.

FIGURE 1

Ric-8B repression during osteoblastic differentiation involves simultaneous recruitment of C/EBPβ and SWI/SNF at the Ric-8B gene promoter, but is independent of histone H3 acetylation. Mouse preosteoblastic MC3T3 cells were cultured between 3 and 13 DIV (days of differentiation in vitro) in the presence of 50 μg/mL ascorbic acid. At the indicated days, cells were crosslinked with 1% formaldehyde and sonicated chromatin fragments were immunoprecipitated with specific antibodies. A. Sequential chromatin immunoprecipitation (re-ChIP) studies were performed using antibodies against C/EBPβ and the SWI/SNF-catalytic subunit Brg-1. As a negative control a distal upstream sequence of the Ric-8b gene promoter (−4,636/−4,421) was also amplified. The results are expressed as fold change relative to the binding determined at 3 DIV on the same region. B. ChIP studies were performed using antibodies against the active elongation-associated epigenetic mark H3K36me3 and RNA polymerase II (RNA PolII). C. Additional ChIP studies were performed using antibodies against histone H3 (H3) and acetylated histone H3 (H3ac). The enrichment levels of Ric-8B gene sequences in the precipitated chromatin were determined by QPCR using specific primers. The DNA sequences analyzed at the Ric-8B gene locus are indicated at the top of each graph. The bars represent mean ± standard error of three independent experiments quantified by duplicate. Statistically significant differences were determined by the analysis of variance (ANOVA) test. ***, P<0.001; **, P<0.01; NS, non-significant.

FIGURE 2

3xFlag-C/EBPβ-LAP*R3L mutant protein is unable to interact with SWI/SNF in osteoblastic cells. Sub-confluent MC3T3 cells were transfected with vectors encoding for 3xFlag-C/EBPβ-LAP* (A) or its mutant variant 3xFlag-C/EBPβ-LAP*R3L (B). 24 hrs later, nuclear extracts were obtained and immunoprecipitation studies were performed using anti-Flag antibodies. Precipitation with normal mouse IgG was carried out as specificity control. Coimmunoprecipitated proteins were recovered and Western blot analyses performed using antibodies against the Brg-1 and Ini1 subunits of the SWI/SNF complex. Precipitation of each Flag-tagged variant of C/EBPβ-LAP* was controlled by re-blotting against Flag and C/EBPβ. The band corresponding to the IgG high chain (approx. 60 kDa) is also shown.

FIGURE 3

C/EBPβ-LAP*-mediated repression of the Ric-8B promoter in osteoblastic cells is impaired in the R3L mutant protein unable to interact with SWI/SNF. Subconfluent MC3T3 cells were cotransfected with p2.1Ric-8B-Luc together with increasing amounts (100–800 ng) of a vector coding for 3xFlag-C/EBPβ-LAP* or its mutant 3xFlag-C/EBPβ-LAP*R3L protein. p2.1Ric8B-Luc construct contains a 2,124 bp segment of the Ric-8B promoter fused to the Luciferase reporter gene. A. 5 ug of nuclear extracts were obtained from cells transfected for 24 hrs and overexpression of Flag-tagged C/EBPβ proteins confirmed by Western blot using anti-Flag antibodies. Reblotting against TFIIB was used to control for equal loading. B. Upon 24 hrs of transfection with the plasmids, luciferase activity was determined. The bars represent mean ± standard error of at least three independent experiments quantified by duplicate. Statistically significant differences were determined by the analysis of variance (ANOVA) test. **, P<0.01; *, P<0.05; NS, non-significant.

FIGURE 4

C/EBPβ-LAP* differentially regulates transcription of bone-related genes Ric-8B and Osteocalcin genes in osteoblastic cells. ROS-LAP* and ROS-LAP*R3L cells expressing 3xFlag-C/EBPβ-LAP* or its R3L mutant protein, respectively, under control of the tetracycline-inducible Tet-off system were cultured for 4 days in presence (+Tet) or absence (−Tet) of 10 μg/mL tetracycline. A. To confirm the expression of Flag-tagged C/EBPβ-LAP* proteins, cells were collected and nuclear extracts were analyzed by Western blot using specific antibodies against the Flag epitope. Re-blotting using an antiC/EBPβ antibody allowed simultaneous detection of the Flag-tagged and endogenous isoforms of C/EBPβ. B, C. To demonstrate the binding of the Flag-tagged proteins to the Osteocalcin (OC) and Ric-8B gene promoter in ROS-LAP* (B) or ROS-LAP*R3L (C) cells, ChIP experiments were performed and purified DNA analyzed by QPCR using specific primers. Binding of the SWI/SNF catalytic subunit Brg-1 to the Ric-8B promoter was also analyzed. The DNA sequences amplified during each quantitation are indicated at the top of each graph. C. OC and Ric-8B mRNA expression levels in each cell line were determined by QPCR using specific primers. The bars represent mean ± standard error of three independent experiments quantified by duplicate. Statistically significant differences were determined by the Student’s t-test. ***, P<0.001; **, P<0.01; *, P<0.05.