PMC

Multiple Repeats Are Required for a Frequent, Tissue-Specific Interaction between the Repeats and the TSS Region.
Relative cross-linking frequencies are shown between fragment I (TSS region) and the rest of the B-615 allele (A) and between fragment VII and the rest of the B-615 allele (B). The 1 on the y axes in (A) and (B) is the same value 1 as in , respectively. The dark-blue line represents B-615 husk tissue, and the light blue line represents B-615 inner tissue. The single 853-bp B-615 sequence is indicated with a black arrowhead. For comparison, the relative cross-linking frequencies observed for the B' allele are shown. See for further details.

Model of Tissue- and Expression Level–Specific Chromatin Looping at the b1 Locus.
In the cartoons a blow-up of part of the hepta-repeat is shown. The hepta-repeat tissue specifically interacts with the TSS region, and this interaction is mediated by a transcription factor (TF).
(A) Tissue-specific, multi-loop formation at the B-I epiallele. The enhancer function of the B-I hepta-repeat is tissue-specifically activated by binding of a transcription factor and proteins mediating enhancer activity (E). The activated enhancer interacts with the TSS region. Regulatory sequences ∼107, ∼47, and ∼15 kb upstream of the TSS interact with the TSS and hepta-repeat as well, resulting in the formation of a multiloop structure that mediates high b1 expression levels in B-I husk.
(B) Tissue-specific, single-loop formation at the B' epiallele. The chromatin state at the B' hepta-repeat is inactive. In husk tissue, binding of the TF occurs and mediates the interaction between the hepta-repeat and the TSS region. However, the inactive B' chromatin state prevents the formation of a multiloop structure and results in a single-loop structure. This single-loop structure is associated with a low expression level in B' husk.

FAIRE Enrichment at ∼110 kb Chromatin Domain in B-I over B' Husk Tissue.
(A) Schematic representation of the b1 and Sam locus, indicating the primer sets used for FAIRE with letters. See legend of for further details.
(B) Quantitative FAIRE analysis on B-I and B' husk tissue. FAIRE values obtained for b1 were normalized against those measured for Sam using amplicon q. Error bars indicate the se of six samples. Ex3, exon 3 of the b1 coding region.
(C) ChIP-qPCR analysis on B-I and B' husk tissue using an antibody against histone H3. The b1 ChIP data were normalized against the ChIP data obtained for the Sam q amplicon. Error bars indicate the se of three samples. The signals levels for the no-antibody immunoprecipitation control were negligible for all amplicons and not shown.
In (B) and (C), purple bars represent B-I husk tissue, and green bars represent B' husk tissue. Values that differ significantly between B-I and B' in a two-tailed Student's t test are indicated with one, two, or three asterisks, specifying a 90, 95, and 99% CI, respectively.

3C-qPCR Analyses Demonstrate Tissue-Specific and Expression Level–Dependent Chromatin Looping at the b1 Locus.
The b1 locus is shown at the top of each graph (see also ). The x axis shows the position in kilobases relative to the transcription start site (hooked arrow). The hepta-repeat is indicated with arrowheads. The position and size of the BglII fragments analyzed is indicated by vertical gray shading and Roman numerals; black shading represents the fixed fragment for each experiment. The y axis depicts relative cross-linking frequencies. Data were normalized against cross-linking frequencies measured for the Sam locus. The data for B-I inner and husk tissue are indicated in pink and purple, respectively. Dark green represents B' husk tissue, and light green represents B' inner tissue. Error bars indicate the standard error of mean of four to eight different samples. Relative cross-linking frequencies are shown between fixed fragment I (TSS region) and the rest of the b1 locus (A); fixed fragment X (containing the hepta-repeat) and the rest of the b1 locus (B); and fixed fragment VII (∼47 kb upstream of the TSS) and the rest of the b1 locus (C).

Features of the b1 and Sam Loci.
(A) Schematic representation of the b1 (top panel) and Sam (bottom panel) loci. The b1 TSS is indicated at 0 kb with a hooked arrow; the open box represents the b1 coding region, and the hepta-repeat is indicated with arrowheads. Distances are indicated in kilobases relative to the TSS. The BglII fragments analyzed by 3C are indicated with Roman numerals. Gray bars represent transposon and other repetitive sequences (). Dr1 and Dr2, direct repeats 1 and 2; IR, inverted repeat; Uni, Unigene Zm.5756. The Sam locus was used to normalize the 3C, FAIRE, and ChIP data obtained for b1. The hooked arrow indicates the TSS (BT042811.1). Black box, Sam probe used for RNA gel blot shown in (B); B, BglII sites; triangles, 3C primers.
(B) RNA gel blot analyses of RNA isolated from B-I, B', and B-615 inner tissue and husk. Pictures from each type of tissue are shown above the respective lane; inner tissue consists of young tissue present inside the stem (see Methods). The blots were hybridized with probes recognizing the coding region of b1 and Sam. The arrow indicates the full-length b1 transcript. The green color of B-615 plant tissue is due to the presence of a very weak or null Pl1 allele and does not reflect the b1 expression level (). The bottom panel shows the corresponding ethidium bromide–stained gel. I, inner tissue; H, husk tissue.
(C) The band intensities of the full-length b1 and Sam transcripts of two independent experiments (gray and black bars) were quantified, the background signals subtracted, and the b1/Sam ratio calculated and depicted in the bar graph. The gray bars correspond to the blot shown in (B).