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Small interfering RNAs (siRNAs) are widespread in various eukaryotes and are involved in maintenance of chromatin modifications, especially those for inert states represented by covalent modifications of cytosine and/or histones. In contrast to mammalian genomes, in which cytosine methylation is restricted mostly to CG dinucleotide sequences, inert chromatin in plants carries cytosine methylation in all sequence contexts, and siRNAs play a major role in directing cytosine methylation through the process of RNA-directed DNA methylation. Recent advances in this field have revealed that siRNA-mediated maintenance of inert chromatin has diverse roles in development as well as in plant responses to the environment. Various proteinaceous factors required for siRNA-mediated chromatin modification have been identified in Arabidopsis thaliana, and much effort has been invested in understanding their function and interaction, resulting in the assignment of many of these factors to specific biochemical activities and engagement with specific steps such as transcription of intergenic RNAs, RNA processing, and cytosine methylation. However, the precise functions of a number of factors remain undesignated, and interactions of distinct pathways for siRNA-mediated chromatin modification are largely unknown. In this review, we summarize the roles of siRNA-mediated chromatin modification in various biological processes of A. thaliana, and present some speculation on the functions and interactions of silencing factors that, while not yet assigned to defined biochemical activities, have been loosely assigned to specific events in siRNA-mediated chromatin modification pathways. Special Issue entitled: Epigenetic control of cellular and developmental processes in plants.

Summary The organ-specific and temporally regulated expression of the genes for Kunitz chymotrypsin inhibitor-3 in winged bean is known to be governed by its 5′ flanking region. Two nuclear factors that bind to AT-rich sequences in this region were detected in gel retardation assays. The binding activity of one of these factors, band 1-forming factor, was inhibited by phosphorylation, as is the AT-binding protein ATBP-1 of tobacco, which functions as an activator of transcription. In addition, the binding of band 1-forming factor was reduced in the presence of a low concentration of the minor groove-binding dye, Hoechst 33258, suggesting that band 1-forming factor recognizes the minor groove of the probe, as does ATBP-1. The binding of another factor, band 3-forming factor, was also reduced by Hoechst 33258, but the binding activity of this factor was not affected by phosphorylation. During partial purification by gel-filtration and affinity chromatography, the binding activity of band 3-forming factor was cofractionated with a 75-ku (kDa) DNA-binding protein. These results indicate that band 3-forming factor is a new DNA-binding factor that recognizes the minor groove of AT-rich DNA.

The human macrophage-like cell line U937 constitutively produces a lymphocyte blastogenesis inhibitory factor (LBIF). Using fast protein liquid chromatography (FPLC)-chromatofocusing, FPLC-anion-exchange chromatography and high-performance liquid chromatography-gel permeation chromatography, LBIF has been purified to homogeneity. It is a single polypeptide chain and has a molecular weight of 57,000 daltons according to sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions. LBIF shows pI 4.28-4.56 upon chromatofocusing on Mono P.