MicroRNA Binding Sites in Arabidopsis Class III HD-ZIP mRNAs Are Required for Methylation of the Template Chromosome  Ning Bao, Khar-Wai Lye, M.Kathryn Barton  Developmental Cell  Volume 7, Issue 5, Pages 653-662 (November 2004) DOI: 10.1016/j.devcel.2004.10.003

Figure 1 Map of Methylated Msp/HpaII Sites Downstream of the microRNA 165/166 Complementary Site of PHABULOSA and PHAVOLUTA Genes in Wild-Type and Mutant Plants (A) Alignment of microRNA complementary site in PHB or PHV mRNA with microRNAs 165 and 166. 18 of 21 bases of the small RNA mir165 are complementary to 18 bp of processed PHABULOSA mRNA that flanks the junction of exons 4 and 5. An additional base pair can be formed through wobble base pairing (marked with a w). Dominant PHB mutations fall within this predicted microRNA binding site. The phb-1d mutation is a G to an A transition at the first position of intron 4, resulting in the use of a cryptic downstream splice donor site that generates a 30 or 33 bp in-frame insertion. The phv-1d mutation is a G to A transition in the microRNA complementary site. (B) Map of PHB/ATHB14 (GenBank accession Y11122) genomic DNA showing coordinates and extent of methylation of Msp/HpaII sites. (C) Map of PHV/ATHB9 (GenBank accession Y10922) genomic DNA showing coordinates and extent of methylation of Msp/HpaII sites. Msp/HpaII sites in shaded boxes show greater than 95% methylation in wild-type plants. Exons appear as rectangles (some numbered) and introns as lines. The predicted mir165/166 binding site is at the ends of exons 4 and 5. The deduced location of fragments A–F from Southern blots in Figure 2 are shown below maps. Methylation status in mutant and wild-type is shown above each cluster of MspI/HpaII sites. The percent methylated (%M) is the percent of molecules in which all sites in a cluster are methylated (if just one site in a cluster is unmethylated, the cluster will be cut and appear unmethylated). %M values were determined from the set of experiments shown in Figure 4. Probe locations are: PHB probe 1 (719–1241), PHB probe 2 (1794–3456), PHB probe 3 (3697–4440), PHB probe 4 (3564–4438), PHV probe 5 (689–1917), PHV probe 6 (1939–2937), PHV probe 7 (3141–3864), and PHV probe 8 (4002–4522). Coordinates are relative to first base pair of exon 1 as determined by Sessa et al. (1998). Developmental Cell 2004 7, 653-662DOI: (10.1016/j.devcel.2004.10.003)

Figure 2 Southern Blot of DNA Extracted from wt, phb, and phv Mutant Plants Digested with the Methylcytosine-Sensitive Enzyme HpaII (A) Blot probed with a cDNA representing the entire PHB coding sequence. (B) Blot probed with PHB probe 1. (C) Blot probed with PHB probe 2. (D) Blot probed with PHB probe 3. (E) Blot probed with PHV probe 7. Experiment in (E) showed some cross-hybridization of probe to PHB. Coordinates of probes are shown in Figure 1. Size markers (in kb) are to the left of each blot. Lettered names of fragments are to the right of each blot. Deduced origin of each fragment is shown in Figure 1. Developmental Cell 2004 7, 653-662DOI: (10.1016/j.devcel.2004.10.003)

Figure 3 Extent of PHB Methylation in Various Tissues and Mutants Histogram showing percent of total for fragments A, B, and C. (A) PHB methylation in various tissue types in wild-type plants. The difference between values in siliques and whole wild-type plants is significant at p < 0.01 (t test). (B) Wild-type versus phb-1d heterozygotes. Difference between wt and phb-1d heterozygotes is significant at p < 0.01 (t test). (C) PHB methylation in shoots from various mutants relative to wild-type shoots. None of the values are significantly different from one another. “I” bars indicate ± one standard error of the mean. The values for each genotype/tissue type are based on at least 2 independently isolated DNA samples and typically several independent replicate determinations per DNA sample. The exception is the argonaute1-7(ago1-7) mutant, which is represented by only one DNA sample. Inf, inflorescence; dcl1, dicer-like1; pnh-2, pinhead-2. Developmental Cell 2004 7, 653-662DOI: (10.1016/j.devcel.2004.10.003)

Figure 4 Distribution of Methylation in PHB Exon 12 to 13 among Sequenced Clones Derived from Bisulfite-Treated DNA (A) DNA from wild-type Landsberg erecta inflorescences. The majority of chromosomes show high levels of methylation. (B) DNA from phb-1d/phb-1d inflorescences. Most chromosomes are unmethylated. (C) DNA from inflorescence meristems (cal; ap1 homozygotes). Both methylated and unmethylated chromosomes are present. H, any nucleotide except G. Developmental Cell 2004 7, 653-662DOI: (10.1016/j.devcel.2004.10.003)

Figure 5 Decrease in Methylation in phb-1d Heterozygotes Occurs on the Mutant Chromosome (A) Map of genomic region of PHB from 3031 to 4367. The PciI site at position 3707 (double asterisk) is present in Columbia DNA but missing in Landsberg erecta DNA. Methylation at HpaII-4097 is reduced in phb-1d/+ DNA relative to wild-type DNA; HpaII-3663 and HpaII-3701 are predominantly methylated in both wild-type and mutant DNA (see Figures 1 and 2). Predicted sizes of each expected fragment are shown below the map. (B) Southern blot of DNA (shoots) from wild-type Landsberg, wild-type Columbia, and wild-type Columbia/Landsberg hybrids from reciprocal crosses probed with PHB probe 4. Lanes 1–4, DNA digested with HpaII and PciI; lanes 5–8, DNA digested with HpaII. (PHV), cross-hybridizing band from PHV locus; Ler, m, methylated Landsberg band; Ler, u, unmethylated Landsberg band; Col, m, methylated Columbia band. (C) Southern blot of wild-type Landsberg, Columbia, and phb-1d (Ler)/PHB+ (Ler), phb-1d (Ler)/PHB+ (Col) probed with PHB probe 3. (D) RT-PCR of RNA isolated from wild-type and phb-1d heterozygotes. RNA was subjected to reverse transcriptase using primers flanking a polymorphic Alu1 site. (Amplified fragment size is 663 bp in both ecotypes. The AluI site is present at position 2111 in Col coding sequence and absent at this site in Ler coding sequence. This fragment does not include the phb-1d mutation.) In the wild-type Ler/Col hybrids (lanes 3 and 4), we estimate that 35% of PCR products have the Columbia polymorphism and 65% have the Landsberg erecta polymorphism. In the phb-1d heterozygotes (lane 6), all detectable cDNA was derived from the Landsberg chromosome. Developmental Cell 2004 7, 653-662DOI: (10.1016/j.devcel.2004.10.003)

Figure 6 Model for Negative Regulation of PHB by mir165/166 The microRNAs recognize newly synthesized and processed PHB mRNA, perhaps before the RNA is released from the template chromosome. This interaction results in the recruitment of a chromatin-modifying complex to the PHB locus. This recruitment may occur directly, for instance if the microRNA is a part of such a complex, or indirectly, for instance if the microRNA acts to modify or cleave the target mRNA, which in its turn recruits the complex. Developmental Cell 2004 7, 653-662DOI: (10.1016/j.devcel.2004.10.003)