Mammalian Chromatin Remodeling Complex SWI/SNF Is Essential for Enhanced Expression of the Albumin Gene during Liver Development

Abstract

The chromatin remodeling complex SWI/SNF is known to regulate the transcription of several genes by controlling chromatin structure in an ATP-dependent manner. SWI/SNF contains the Swi2p/Snf2p like ATPases BRG1 or BRM exclusively. We found that the expression of BRM gradually increases and that of BRG1 decreases as liver cells differentiate. Chromatin immunoprecipitation assays revealed that the ATPase subunits of SWI/SNF and tumor suppressor retinoblastoma (RB) family proteins bind to the promoter region of the albumin gene in hepatocytes, and that the replacement of BRG1 with BRM and pRB with p130 at this site occurs over the course of differentiation. Small interfering RNA experiments showed that blocking the expression of BRG1 and BRM in fetal and adult hepatocytes, respectively, causes a reduction in albumin expression. In luciferase reporter assays with a pREP4-based reporter plasmid that forms a chromatin structure, BRG1 showed activity stimulating the expression of the albumin promoter mediated by CCAAT/enhancer-binding protein alpha (C/EBPalpha). This enhancement was facilitated by the RB family members pRB and p130. ATPase assays showed that both pRB and C/EBPalpha proteins directly stimulate the ATPase activity of BRG1. Our findings suggest that the mechanism by which the activity of transcription factors is enhanced by RB family members and SWI/SNF includes an increase in the ATPase activity of the chromatin remodeling complex.

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

“…We and other groups have shown that C/EBP␣ interacts with BRG1, an ATPase subunit of SWI/SNF chromatin remodeling complex (14) and C/EBP␣-mediated transactivation of the albumin gene is enhanced by the SWI/ SNF complex in hepatocytes (25). Because the binding site for BRG1 in C/EBP␣ overlaps with the SUMO-1 conjugation site (Fig.…”

“…The mammalian expression vector pSPORT6/mPIASy (MGC 35863) was purchased from Invitrogen. A pREP4-based luciferase reporter plasmid in which the luciferase gene is under the control of the albumin promoter, pcDNA4/C/EBP␣, pcDNA4/BRG1, pcDNA4/mutBRG1 (ATPase mutant) and pcDNA4/BRM, was described previously (25). Mutant C/EBP␣ expression vectors were generated by site-directed mutagenesis by replacing Lys-159 with Ala or Arg.…”

“…For the expression of GST-fused proteins, the SUMO-1 and C/EBP␣ TEI-III regions (amino acids 1-200) were cloned in-frame into the GST expression vector pGEX-5X-2 (Amersham Biosciences). For the expression of HSV-tagged BRG1, the BRG1 helicase domain (amino acids 757-1262) and BRG1 ⌬E7 (amino acids 714 -1308) were also cloned in-frame into pETBlue2 (Novagen) (25). pTS-1, which expresses SUMO-E1 and -E2 ligases and SUMO-1 itself (26), was purchased from Nippon Flour Mills.…”

“…Immunoprecipitation and GST Pulldown Assay-Immunoprecipitation and the GST pulldown assay were performed as described previously (25). The GST-C/EBP␣ TEI-III, sumoylated GST-C/EBP␣ TEI-III, or GST-SUMO-1 fusion protein was expressed in Escherichia coli BL21 (DE3) and purified using a glutathione-Sepharose column (Amersham Biosciences).…”

Mechanisms of ATP dependent chromatin remodeling

“…The role of SWI/SNF by all indications is far reaching. In mammals it is involved in many developmental programs such as muscle [19][20][21][22], heart [23], blood [24], skeletal [25], neuron [26][27][28][29], adipocyte [30], liver [31] and immune system/Tcell development [32,33]. Yeast SWI/SNF has been shown to be involved in an early step in homologous recombination (HR) while RSC promotes HR at the stage of strand invasion [34,35].…”