All eukaryotes have three nuclear DNA-dependent RNA polymerases, namely, Pol I, II, and III. Interestingly, plants have catalytic subunits for a fourth nuclear polymerase, Pol IV. Genetic and biochemical evidence indicates that Pol IV does not functionally overlap with Pol I, II, or III and is nonessential for viability. However, disruption of the Pol IV catalytic subunit genes NRPD1 or NRPD2 inhibits heterochromatin association into chromocenters, coincident with losses in cytosine methylation at pericentromeric 5S gene clusters and AtSN1 retroelements. Loss of CG, CNG, and CNN methylation in Pol IV mutants implicates a partnership between Pol IV and the methyltransferase responsible for RNA-directed de novo methylation. Consistent with this hypothesis, 5S gene and AtSN1 siRNAs are essentially eliminated in Pol IV mutants. The data suggest that Pol IV helps produce siRNAs that target de novo cytosine methylation events required for facultative heterochromatin formation and higher-order heterochromatin associations.

The Dof (DNA binding with one finger) transcriptional activator rice (Oryza sativa) prolamin box binding factor (RPBF), which is involved in gene regulation of rice seed storage proteins, has been isolated from rice cDNA expressed sequence tag clones containing the conserved Dof. RPBF is found as a single gene per haploid genome. Comparison of RPBF genomic and cDNA sequences revealed that the genomic copy is interrupted by one long intron of 1,892 bp in the 5# noncoding region. We demonstrated by transient expression in rice callus protoplasts that the isolated RPBF trans-activated several storage protein genes via an AAAG target sequence located within their promoters, and with methylation interference experiments the additional AAAG-like sequences in promoters of genes expressed in maturing seeds were recognized by the RPBF protein.Binding was sequence specific, since mutation of the AAAG motif or its derivatives decreased both binding and transactivation by RPBF. Synergism between RPBF and RISBZ1 recognizing the GCN4 motif [TGA(G/C)TCA] was observed in the expression of many storage protein genes. Overexpression of both transcription factors gave rise to much higher levels of expression than the sum of individual activities elicited by either RPBF or RISBZ1 alone. Furthermore, mutation of recognition sites suppressed reciprocal trans-activation ability, indicating that there are mutual interactions between RISBZ1 and RPBF. The RPBF gene is predominantly expressed in maturing endosperm and coordinately expressed with seed storage protein genes, and is involved in the quantitative regulation of genes expressed in the endosperm in cooperation with RISBZ1.