Identification and functional validation of super-enhancers in Arabidopsis thaliana.

Super-enhancers (SEs) are exceptionally large enhancers and are recognized to play prominent roles in cell identity in mammalian species. We surveyed the genomic regions containing large clusters of accessible chromatin regions (ACRs) marked by deoxyribonuclease (DNase) I hypersensitivity in Arabidopsis thaliana. We identified a set of 749 putative SEs, which have a minimum length of 1.5 kilobases and represent the top 2.5% of the largest ACR clusters. We demonstrate that the genomic regions associating with these SEs were more sensitive to DNase I than other nonpromoter ACRs. The SEs were preferentially associated with topologically associating domains. Furthermore, the SEs and their predicted cognate genes were frequently associated with organ development and tissue identity in A. thaliana. Therefore, the A. thaliana SEs and their cognate genes mirror the functional characteristics of those reported in mammalian species. We developed CRISPR/Cas-mediated deletion lines of a 3,578-bp SE associated with the thalianol biosynthetic gene cluster (BGC). Small deletions (131-157 bp) within the SE resulted in distinct phenotypic changes and transcriptional repression of all five thalianol genes. In addition, T-DNA insertions in the SE region resulted in transcriptional alteration of all five thalianol genes. Thus, this SE appears to play a central role in coordinating the operon-like expression pattern of the thalianol BGC.

Rearrangement with the nkd2 promoter contributed to allelic diversity of the r1 gene in maize (Zea mays).

The maize red1 (r1) locus regulates anthocyanin accumulation and is a classic model for allelic diversity; changes in regulatory regions are responsible for most of the variation in gene expression patterns. Here, an intrachromosomal rearrangement between the distal upstream region of r1 and the region of naked endosperm 2 (nkd2) upstream to the third exon generated a nkd2 null allele lacking the first three exons, and the R1-st (stippled) allele with a novel r1 5' promoter region homologous to 5' regions from nkd2-B73. R1-sc:124 (an R1-st derivative) shows increased and earlier expression than a standard R1-g allele, as well as ectopic expression in the starchy endosperm compartment. Laser capture microdissection and RNA sequencing indicated that ectopic R1-sc:124 expression impacted expression of genes associated with RNA modification. The expression of R1-sc:124 resembled nkd2-W22 expression, suggesting that nkd2 regulatory sequences may influence the expression of R1-sc:124. The r1-sc:m3 allele is derived from R1-sc:124 by an insertion of a Ds6 transposon in intron 4. This insertion blocks anthocyanin regulation by causing mis-splicing that eliminates exon 5 from the mRNA. This allele serves as an important launch site for Ac/Ds mutagenesis studies, and two Ds6 insertions believed to be associated with nkd2 mutant alleles were actually located in the r1 5' region. Among annotated genomes of teosinte and maize varieties, the nkd2 and r1 loci showed conserved overall gene structures, similar to the B73 reference genome, suggesting that the nkd2-r1 rearrangement may be a recent event.

The thick aleurone1 Gene Encodes a NOT1 Subunit of the CCR4-NOT Complex and Regulates Cell Patterning in Endosperm.

Maize (Zea mays) thick aleurone1 (thk1-R) mutants form multiple aleurone layers in the endosperm and have arrested embryogenesis. Prior studies suggest that thk1 functions downstream of defective kernel1 (dek1) in a regulatory pathway that controls aleurone cell fate and other endosperm traits. The original thk1-R mutant contained an ∼2-Mb multigene deletion, which precluded identification of the causal gene. Here, ethyl methanesulfonate mutagenesis produced additional alleles, and RNA sequencing from developing endosperm was used to identify a candidate gene based on differential expression compared with the wild-type progenitor. Gene editing confirmed the gene identity by producing mutant alleles that failed to complement existing thk1 mutants and that produced multiple-aleurone homozygous phenotypes. Thk1 encodes a homolog of NEGATIVE ON TATA-LESS1, a protein that acts as a scaffold for the CARBON CATABOLITE REPRESSION4-NEGATIVE ON TATA-LESS complex. This complex is highly conserved and essential in all eukaryotes for regulating a wide array of gene expression and cellular activities. Maize also harbors a duplicate locus, thick aleurone-like1, which likely accounts for the ability of thk1 mutants to form viable cells. Transcriptomic analysis indicated that THK1 regulates activities involving cell division, signaling, differentiation, and metabolism. Identification of thk1 provides an important new component of the DEK1 regulatory system that patterns cell fate in endosperm.