Analysis of the Methylation Pattern of the Maize Opaque-2 (O2) Promoter and in Vitro Binding Studies Indicate That the O2 B-Zip Protein and Other Endosperm Factors Can Bind to Methylated Target Sequences

Abstract

The maize opaque-2 locus (o2) has an endosperm-specific expression and is positively autoregulated by its gene product, a b-Zip protein, to a TGACGTTG motif. The genomic sequencing method was used here to describe, in leaf and endosperm, the methylation pattern of a 390-base pair region of the o2 promoter. In leaf, 96% of the C residues are methylated, whereas in endosperm the 5-methylcytosine content is 84%. Comparison of these methylation patterns indicates that the o2 tissuespecific expression does not result from the demethylation of any specific C residue and that, in vivo, O2 interacts with a methylated target sequence. Consistently, gel-shift experiments using a CpG-methylated, partially methylated, and hemimethylated o2 promoter fragments showed that, in vitro, the O2 protein binds to the major groove of a methylated target sequence, although its binding activity decreases at increasing levels of Cmethylation and is more effectively reduced by methylation of the lower strand than of the upper strand of the DNA. Using partially purified endosperm cell extracts, we also show that, besides the O2 protein, other proteins specifically bind to a partially methylated o2 promoter fragment, therefore indicating that in plants a subset of different proteins may mediate the expression of a naturally occurring methylated o2 promoter.

Genome-Wide Characterization ofcis-Acting DNA Targets Reveals the Transcriptional Regulatory Framework ofOpaque2in Maize

“…The LKR/SDH mRNA is reduced by >90% in o2; consequently, this gene was predicted to be directly regulated by O2 (Kemper et al, 1999;Prioul et al, 2008). O2 itself is believed to be positively autoregulated by its gene product, based on a B5-like box in its promoter region (Lohmer et al, 1991;Rossi et al, 1997). (A) The specific sequence and origin of 13 previously reported O2 binding sites.…”

“…The cis-elements studied included the Z1 (TCACATGTGT), Z2 (TCATGCATGT) and Z3 (TCCACGTAGAT) sites from the 22-kD a-zein gene promoter (Muth et al, 1996); the B1 (GATGACATGG), B2 (GATGATATGG), B3 (GATGATGTGG), B4 (GATGAGATGA), and B5 (GTTGACGTGA) from the b-32 gene (Lohmer et al, 1991); the O2 BS-1 (GTTGATCTGA) and O2 BS-2 (CATGACGTGT) from the cyPPDK1 gene (Maddaloni et al, 1996); the O2-like-box (TTTACGTAGAT) from the 27-kD g-zein gene promoter (Wu and Messing, 2012); the C-box and odd C-box (ATGACGTCAT or ATGAGTCAT) from the pea (Pisum sativum) lectin (ps1) gene (de Pater et al, 1994;Cord Neto et al, 1995); and the B5-like-box (GTTGACGTTG) from the O2 gene (Lohmer et al, 1991;Rossi et al, 1997). As shown in Figure 4B, without the O2 protein no bands were observed, except for the free probe.…”

Self-regulated Cleavage of the Mitochondrial Intramembrane-cleaving Protease PARL Yields Pβ, a Nuclear-targeted Peptide

“…Samples were heated at 85°C for 2 min and separated on a 4 -12% polyacrylamide-SDS gel (Invitrogen). Proteins were blotted onto nitrocellulose membranes and probed with the specified antibodies as described (18,19). Immunoblots were developed using the SuperSignal system (Pierce).…”

Chromatin and DNA Modifications in theOpaque2-Mediated Regulation of Gene Transcription during Maize Endosperm Development

“…Subsequently, it was shown that, in specific genetic backgrounds, the demethylation of selected maternally inherited azs22 loci correlates with allele-specific expression (Lund et al, 1995a). Furthermore, the in vitro binding of O2 at the O2-box sequence is impaired by high cytosine methylation ( m C) levels that characterize sporophytic tissues, where O2 and its targets are not expressed (Rossi et al, 1997;Sturaro and Viotti, 2001). Finally, it was reported that O2 protein interacts with the maize homologs of the yeast transcriptional coactivator ADA2 (Ada2) and of the histone acetyltransferase GCN5 (Gcn5) and that the three proteins cooperate in driving the transient expression of O2 targets in a heterologous system, thus suggesting that O2 is implicated in modulating histone modifications (Bhat et al, 2003(Bhat et al, , 2004.…”