Differential chromatin structure within a tandem array 100 kb upstream of the maize b1 locus is associated with paramutation

Abstract

Recombination mapping defined a 6-kb region, 100 kb upstream of the transcription start site, that is required for B-I enhancer activity and paramutation-a stable, heritable change in transcription caused by allele interactions in maize (Zea mays). In this region, B-I and B' (the only b1 alleles that participate in paramutation) have seven tandem repeats of an 853-bp sequence otherwise unique in the genome; other alleles have one. Examination of recombinant alleles with different numbers of tandem repeats indicates that the repeats are required for both paramutation and enhancer function. The 6-kb region is identical in B-I and B', showing that epigenetic mechanisms mediate the stable silencing associated with paramutation. This is the first endogenous gene for which sequences required for paramutation have been defined and examined for methylation and chromatin structure. The tandem repeat sequences are more methylated in B-I (high expressing) relative to B' (low expressing), opposite of the typical correlation. Furthermore, the change in repeat methylation follows establishment of the B' epigenetic state. B-I has a more open chromatin structure in the repeats relative to B'. The nuclease hypersensitivity differences developmentally precede transcription, suggesting that the repeat chromatin structure could be the heritable imprint distinguishing the two transcription states.

Figure 1

Recombination experiments with B-I and B-Peru (B-P) to map enhancer sequences. The gray oval (E) contains the hypothetical enhancer sequences. The phenotypic marker glossy2 (gl2) and the molecular marker tmp were used to identify recombinants; polymorphisms are indicated with different symbols. Three recombination intervals between gl2 and the b1 coding region are indicated. The B-Peru promoter-proximal region contains sequences directing purple seed color (sc). B-I lacks these sequences. Vegetative and seed pigment phenotypes of B-I, B-Peru, and BI∥BP-D1 (D, dark pigment; L, light pigment; ∥, recombinant allele with the parental allele contributing sequences upstream of the recombination breakpoint indicated on the left of the vertical bars and the parental allele contributing the coding and promoter proximal region indicated on the right) are shown.

Figure 2

Sequence and structure of B‘, B-I, B-Peru, b-K55, and recombinants. (A) Percentage identity between B-I, B-Peru (B-P), and b-K55 as compared with B‘ is indicated by bar color. (B) Restriction maps of recombinant alleles defining 5′ and 3′ boundaries of B-I sequences required for enhancer and paramutation activities. The white ovals indicate the region in which recombination occurred. The arrows represent the tandem repeats (B-I is white, B-Peru and b-K55 are shaded). Restriction sites are (B) BamHI; (E) EcoRI; (N) NcoI; and (S) SwaI.

Figure 3

Sequence features of the tandem repeat in B‘ and B-I. Alignment of the B-Peru sequence with the consensus of the seven repeats in B‘ and B-I. Polymorphisms among the seven repeats are indicated below the consensus, the number in parentheses indicating in which repeat the polymorphism occurred (1 is most upstream, 7 is most downstream). Black highlights show positions of sequence divergence unique to the B-Peru repeat. Gray highlights show regions of similarity to yeast ARS, with one or two mismatches allowed from the 11-bp consensus, (A/T)TTTAT(A/G)TTT(A/T). The region of potential MAR similarity is underlined.

Figure 4

B-I DNA is more methylated within the tandem repeats than B‘ DNA. DNA blot analyses were done on B‘, new B‘ (nB‘), B-I, and B-Peru genomic DNA cut with EcoRI and Sau3AI (methylation-sensitive) or MboI (methylation-insensitive). Blots were hybridized with probes indicated on the B-I, B‘ map; the stippled bar indicates the region recognized by probe C. Arrows indicate tandem repeats. An enlargement of one repeat is shown. The restriction sites are (E) EcoRI; (S) SwaI. Vertical lines on the map indicate Sau3AI/MboI sites in regions recognized by the probes. Various symbols indicate fragments visible on blots; size is in base pairs. One blot hybridized with each indicated probe is shown.

Figure 5

The tandem repeat region shows DNase I hypersensitivity in B-I chromatin. Nuclei derived from B‘ and B-I sheaths were treated with increasing amounts of DNase I (0.05, 0.2, 0.5, and 8 units of DNase I/mL; indicated by wedge); naked DNA was treated with no DNase I or 0.003 units of DNase I/mL. No DNase I was added to the nuclei followed by no incubation (lane θ) or incubation (lane 0). All incubations were for 5 min. DNA was isolated, cut with EcoRV and BamHI, and blotted. The restriction map shows the region analyzed and the probes used (B, C, and E); the stippled bar shows the region recognized by probe C. Arrows indicate the EcoRV–BamHI fragments expected if the fragment is not cut by DNase I. The marker lane (M) contains a pool of B-I genomic DNA cut with EcoRV and one of the following enzymes: BamHI, PstI, XbaI, and BstEII. The restriction sites are (B) BamHI; (E) EcoRI; and (EV) EcoRV.

Figure 6

The tandem repeats in new B‘ chromatin show an intermediate chromatin structure relative to B-I and B‘. Nuclei from B‘, new B‘, and B-I tissue were treated with increasing amounts of DNase I (0, 0.05, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 units of DNase I/mL; indicated by wedge). Naked DNA was treated with 0 or 0.005 units of DNase I/mL. All incubations were for 6 min. The lack of digestion of naked DNA and cutting of DNA in nuclei only at the highest concentrations used indicated that the DNase I was not very active. The 0 DNase I control shows that most digestion was by endogenous nucleases. DNA was isolated, cut with EcoRI and BamHI, and blotted. The maps show the regions analyzed and the probes used (A, B1, D1, D2, and F). Arrows indicate the EcoRI–BamHI fragments expected if the fragments are uncut by nucleases. The probes monitor the same general regions as in Fig. 5, but are at the end of each restriction fragment to map hypersensitive sites. Endogenous nuclease-hypersensitive sites are indicated with vertical arrows beneath the restriction map. The restriction sites are (B) BamHI and (E) EcoRI.