Changes in nucleosome position at transcriptional start sites of specific genes in Zea mays mediator of paramutation1mutants

Figures & data

Figure 1. Agarose gel fractionation of MNase digested DNA from leaf, immature tassel, ear shoot and seedlings of homozygous wild type (WT MOP1) and homozygous mutant mop1–1 (MUT mop1–1) plants. For each genotype and tissue type, separate reactions were conducted using 0.5, 0.75, and 1 unit of MNase respectively. Lanes with molecular weight markers are indicated (λ HindIII and Lad); the size of each band is indicated in base pairs.

Figure 2. Schematic representation of comparative genomic hybridization (CGH) on the 400 gene TSS microarray for 13 exemplar genes with their respective chromosomal locations. The magnitude of sequence differences (ratio of the log2 ratios of signal intensity for respective samples) between wild type and mutant mop1–1 is displayed on the y axis for each gene. A value of 1 in the bar chart indicates that there is no detectable difference in hybridization behavior between the genomic DNA from wild type and mutant mop1–1 individuals in the probed region for the gene.

Figure 3. Nucleosome distribution plots for 3 genes exhibiting differences between wild type and mop1–1 homozygous mutants. The x-axis shows the coordinates of the genes flavanone 3-hydroxylase1 ((A), liguleless1 (B), and yabby15 (C) on their respective chromosomes, while the y-axis represent the log 2 ratio of the signal intensity between the test (nucleosomal DNA) and reference (genomic DNA) samples. The black lines represent replicates of wild type MOP1, while the red lines represent replicates of mutant mop1–1. The location and direction of transcription is indicated with an arrow and label for each gene. Open arrows indicate regions where nucleosome occupancy appears to vary between wild type and mutant plants. For yabby15 and liguless1, the plots represent a comparison of wild type and mutant immature tassel samples. For flavanone 3-hydroxylase1, the plot represents a comparison of wild type and mutant ear shoot samples.

Table 1. Genes showing consistent differences in nucleosome distribution between wild type and mop1–1 mutants

Gene (B73 refgen2)^a	Tissue type^b	Average correlation^c WT:WT; Mu:Mu; WT:Mu
Flavanone 3-hydroxylase1 (GRMZM2G062396)	LF	0.936; 0.937; 0.85
	TSL	0.932; 0.952; 0.86
	ES^d	0.949; 0.913; 0.73
Liguleless1 (GRMZM2G036297)	LF^d	0.924; 0.928; 0.497
	TSL^d	0.926; 0.951; 0.599
	ES^d	0.949; 0.956; 0.265
Yabby15 (GRMZM2G529859)	LF	0.6; 0.883; 0.755
	TSL^d	0.849; 0.915; 0.802
	ES	0.91; 0.847; 0.906

^a Gene name and gene identification number from the reference genome, version 2, are indicated for each gene. ^bLF indicates leaf tissue, TSL indicates immature tassel, and ES indicates immature ear shoot. ^cCovariance (r-value) was calculated for each pair of wild type replicates (WT) and mutant replicates (Mu) and averaged to determine the average correlation between replicates of the same genotype (WT:WT and Mu:Mu). Each WT replicate was compared pairwise to each Mu replicate; these r-values were also averaged to determine the average correlation between the two distinct genotypes in each tissue (WT:Mu). ^dTissues where apparent differences in nucleosome position were noted between wild type and mutant genotypes.

Table 2. Comparison between observed nucleosome distribution and support vector machine predicted nucleosome-forming and -inhibitory sequences in the TSSs of three genes with altered nucleosome distribution in mop1–1 mutants

Gene	Tissue type^a	Average correlation^b NOL:WT; NOL:Mu; NOL:Total
Flavanone 3-hydroxylase1	LF	0.700; 0.710; 0.700
	TSL	0.710; 0.710; 0.710
	ES^c	0.583; 0.592; 0.588
Liguleless1	LF^c	0.190; 0.398; 0.294
	TSL^c	0.276; 0.412; 0.344
	ES^c	0.129; 0.378; 0.253
Yabby15	LF	0.113; 0.158; 0.240
	TSL^c	0.129; 0.060; 0.095
	ES	0.321; 0.158; 0.240

^a LF indicates leaf tissue, TSL indicates immature tassel, and ES indicates immature ear shoot. ^bCovariance (r-value) was calculated for each pair of wild type replicates (WT) and mutant replicates (Mu) compared with the NOL plot and averaged to determine the average correlation between the NOL and each genotype (NOL:WT and NOL:Mu respectively). The average r-value of all replicates and genotypes for each tissue compared with the NOL (NOL:Total) is also reported. In cases where the r-value was negative, the absolute value was utilized for averaging. ^cTissues where apparent differences in nucleosome position were noted between wild type and mutant genotypes.

Figure 4. Real time PCR results for flavanone 3-hydroxylase1 (A) liguleless1 (B) yabby15 (C), and colored plant1 (D). Transcript abundance was measured in leaf, tassel and ear shoot of mop1–1/mop1–1 mutant (MUT) and wild type MOP1/MOP1 (WT) plants using ubiquitin as an endogenous control. Relative quantification (Rq) values were averaged for three replicates of mop1–1 samples for each tissue type, and compared with the average value for three replicates of wild type samples (set at 1 for each tissue type). The averaged Rq values are plotted on the y-axis. Error bars represent standard error for the averaged Rq values.