Cis-regulatory sequences in plants: Their importance, discovery, and future challenges

Abstract

The identification and characterization of cis-regulatory DNA sequences and how they function to coordinate responses to developmental and environmental cues is of paramount importance to plant biology. Key to these regulatory processes are cis-regulatory modules (CRMs), which include enhancers and silencers. Despite the extraordinary advances in high-quality sequence assemblies and genome annotations, the identification and understanding of CRMs, and how they regulate gene expression, lag significantly behind. This is especially true for their distinguishing characteristics and activity states. Here, we review the current knowledge on CRMs and breakthrough technologies enabling identification, characterization, and validation of CRMs; we compare the genomic distributions of CRMs with respect to their target genes between different plant species, and discuss the role of transposable elements harboring CRMs in the evolution of gene expression. This is an exciting time to study cis-regulomes in plants; however, significant existing challenges need to be overcome to fully understand and appreciate the role of CRMs in plant biology and in crop improvement.

Figure 1

Combinatorial CRM actions elicit diverse transcriptional responses in distinct cell types. The activity of each CRM depends to a large extent on the expression levels of TFs that can bind the CRM. A, In cell type 1, silencer element 1 represses an enhancer, while a multifunctional sequence element and silencer element 2 repress promoter activity. B, In cell type 2, enhancer element 1 works cooperatively with a multifunctional sequence element to activate gene expression. C, In cell type 3, silencer element 1 represses the upstream enhancer 1, whereas the multifunctional sequence element activates the gene in concert with the promoter proximal enhancer (enhancer 2). S, silencer; E, enhancer; M, multifunctional sequence element; P, promoter.

Figure 2

Models for enhancer organization. A, Example of alternative complex formation. TFs can function both as transcriptional activators and repressors, depending on the proteins (co-activators or co-repressors) they interact with. B, Examples of TF collective enhancers. This model is characterized by cooperative DNA-binding, which can be achieved by many different mechanisms, and by proteins as well as DNA serving as a scaffold for the binding of TFs. It allows for flexible CRE arrangements, resulting in distinct regulatory outputs (exemplified by the two enhancers shown). C, Example of an enhanceosome enhancer. In the enhanceosome model, the binding of the various TFs to the respective CREs must occur in a specific order and orientation, following a particular grammar. D, Example of billboard enhancers. In the billboard model, the composition as well as the position and orientation of CREs within an enhancer is preserved. The regulatory output differs depending on the expression and activity level of TFs that can bind the CREs. Key: The thick gray line represents DNA, with the colored rectangles indicating CREs. Each different color indicates a different DNA motif. TFs are depicted in various shapes and colors, with each color denoting a different TF that recognizes a specific DNA sequence (matching colors).

Figure 3

Chromatin accessibility and modifications associated with different CRM activities. A, Example of a repressed CRM. CREs are occluded by nucleosomes due to inaccessible chromatin imparted by PcG protein complexes and H3K27me3. This is associated with transcriptional silencing of the target gene. B, Example of a poised CRM, where nucleosomes flanking CREs have low histone acetylation levels and only a few CREs are accessible for TF binding. PcG proteins and H3K27me3 are still present. The promoter is engaged by RNAPII, and flanking nucleosomes have low histone acetylation levels. PcG proteins and H3K27me3 hamper RNAPII elongation and transcript production. C, Example of an active enhancer, whereby multiple TFs and cofactors interact with a CRM, and target the promoter to activate transcription of the target gene. Nucleosomes are enriched for histone acetylation. D, Example of an active silencer where TFs and cofactors bind to the CRM to recruit PcG proteins to catalyze H3K27me3 and silencing of the target gene. Purple ovals, PCG protein complexes; P, promoter; RNAPII, RNA polymerase II; Purple circles, H3K27me3; orange circles, histone acetylation; red circles, DNA methylation.

Figure 4

Evaluation of CRM activity using reporter assays. A, Candidate cis-regulatory sequences (candidate) can be tested for enhancer activity by fusion with a minimal promoter and reporter gene such as GFP, β-glucuronidase, or luciferase (left). Constructs are either transiently transfected into protoplasts or stably integrated into a plant genome to evaluate reporter gene activity (right). Some assays, for example luciferase assays, provide quantitative read-outs. Activities need to be examined relative to negative control sequences. B, Candidate sequences can also be evaluated using genome-wide assays such as STARR-seq or MPRA (left). In these assays, fragmented genomic DNA is cloned (e.g. using Gateway Technology), into a reporter construct in the 3′-UTR (STARR-seq) or upstream of the gene (MPRA). With MPRA, barcodes are inserted into the open reading frame (pink vertical bar). The resulting reporter construct library is transiently transfected into protoplasts or infiltrated into N. benthamiana leaves. Activity of the candidate fragments (left) is evaluated by measuring reporter transcript abundance in comparison to the abundance of input plasmid (right). For MPRAs, next-generation sequencing of the plasmid library is used to pair the unique barcodes in the reporter gene with the inserted candidates.

Figure 5

Large genomes: distant CRMs and chromatin interactions. A, The number and distance of CRMs to their target gene can increase in larger plant genomes. Hypothetical regions of accessible chromatin (dark blue peaks) are shown in a region of synteny between B. distachyon and Z. mays. The shaded light purple region indicates a gene with expanded intergenic space. The region of accessible chromatin for this gene in B. distachyon possesses one CRM (A/B) containing several CREs within a single accessible chromatin region, whereas in Z. mays the CREs within CRM (A/B) are split into two CRMs, A and B, through insertion of TEs. CRM A and B together carry similar CREs as CRM (A/B) in B. distachyon, but now separated by 15 kb of intergenic sequence. B, Chromatin interactions between distal CRMs and their target gene affect gene expression levels. An example of chromatin interactions that positively correlate with expression levels. The maize Booster-Intense (B-I) allele contains the b1 hepta-repeat enhancer 100-kbp upstream of the b1 TSS, and other putative CRMs at ∼15-, 45-, and 107-kbp upstream of the TSS. In seedling tissue of B-I plants, b1 is lowly expressed, low H3K9ac and H3K27me2 levels are observed at the gene body, and low H3K27me2 levels at the enhancer. Upon transcriptional activation of b1 in husk tissue, nucleosomes and H3K27me2 are lost at the enhancer and gene, H3K9ac levels increased, and the repeat enhancer and additional CRMs upstream physically interact with each other and the TSS of b1, resulting in enhanced b1 expression. Orange triangles, histone acetylation; light purple octagonal shapes, H3K27me2; grey barrels, nucleosomes; green circles, TFs.

Figure 6

Examples of transposable elements acting as regulatory sequences. A, In Z. mays, a Hopscotch retrotransposon inserted ∼60-kbp upstream of tb1. This TE is absent in teosinte (Studer et al., 2011). The Hopscotch TE acts as an enhancer of tb1 expression and partially explains the increased apical dominance observed in Z. mays versus teosinte. B, In apple, a 4-kbp Gypsy-like retrotransposon, redTE, inserted upstream of the MdMYB1-1 gene, increases the expression of this gene, resulting in red-skinned apples (Zhang et al., 2019). RedTE contains the “GCCGACTT” CRE, a TFBS for a DREB/CBF TF that enhances the expression level of MdMYB1 at low ambient temperatures. Created using Biorender.com.

Figure 7

Conserved non-coding sequences underlying ACRs show distinct locations relative to target genes between species. An example of a syntenic region between maize and sorghum. The region depicts two orthologous pairs of genes (linked by gray shading). An ACR detected in both species contains conserved DNA sequences (linked by red shading). Although the ACR in sorghum is near the TSS of Sobic.009G225100, the homologous ACR in maize is located more than 102 kb away from the orthologous gene, Zm001d0009497.