A single high-zinc activation enhancer can control two genes orientated head-to-head in C. elegans

Abstract

Enhancers play critical roles in gene expression, but a full understanding of their complex functions has yet to be defined. The cellular response to excess zinc levels in C. elegans requires the HIZR-1 transcription factor, which binds the high-zinc activation (HZA) enhancer in the promoters of multiple target genes. Cadmium hijacks the excess zinc response by binding and activating HIZR-1. By analyzing the genome-wide transcriptional response to excess zinc and cadmium, we identified two positions in the genome where head-to-head oriented genes are both induced by metals. In both examples, a single predicted HZA enhancer is positioned between the two translational start sites. We hypothesized that a single enhancer can control both head-to-head genes, an arrangement that has not been extensively characterized. To test this hypothesis, we used CRISPR genome editing to precisely delete the HZA^mT enhancer positioned between mtl-2 and T08G5.1; in this mutant, both head-to-head genes display severely reduced zinc-activated transcription, whereas zinc-activated transcription of more distant genes was not strongly affected. Deleting the HZA^cF enhancer positioned between cdr-1 and F35E8.10 caused both head-to-head genes to display reduced cadmium-activated transcription, whereas cadmium-activated transcription of more distant genes was not strongly affected. These studies rigorously document that a single HZA enhancer can control two head-to-head genes, advancing our understanding of the diverse functions of enhancers.

Keywords: C. elegans; HZA; Zinc metabolism; divergent transcription; enhancer; high-zinc activation element.

Figure 1.

Two regions of Chromosome V have genes that are oriented head-to-head, activated by cadmium, and require hizr-1 for activation. A, E) Schematics of regions of Chromosome V are drawn to scale in base pairs (bp). Numeric chromosome location indicates the first base pair of the HZA. Thick black line represents DNA; yellow box indicates HZA enhancer; numbers are intervals from HZA enhancer to ATG start codon and end of transcript; arrows above represent pre-mRNA for indicated genes. Scale bar as shown. B, C, F, G) Wild type (WT) and hizr-1(am286) mutants at the L4 stage were cultured with or without 100μM cadmium for 16 hours and analyzed by qPCR. Values for WT and hizr-1(am286) with no cadmium were set equal to 0, and values with cadmium represent log2 fold change. N = 4 initial biological replicates but may vary in panels due to outlier removal. Circles are technical replicates (repeated measurements of the same sample), and triangles are biological replicates (different samples from different days). Same color denotes the same experiment trial. Statistical analysis by pairwise one-way ANOVA. Non-significant p-values are listed for p<0.3; otherwise, “ns”. For significant p-values: *<0.05; **<0.01; ***<0.001; ****<0.0001. Error bars represent mean ± 95% confidence intervals. Mean values are listed. All fold changes are in base 2 logarithm (e.g. 3 folds = 8x). The same approach is used to represent qPCR data in Figures 2–4, S1-S3. D, H) Distances from the HZA to the start codons (shown in panels A and E) plotted against fold change for WT plus cadmium (shown in B, C, F, and G).

Figure 2.

The HZA^mT element is necessary for zinc-induced transcription of both mtl-2 and T08G5.1. A) Schematic of a region of Chromosome V drawn to scale in base pairs (bp). Thick black line represents DNA; yellow box indicates HZA enhancer. Arrows above represent pre-mRNA for indicated genes. Red line below indicates mutation syb4265, a 15-bp deletion of the HZA^mT (named HZA^mTΔ). Scale bar: 100bp. B) Alignment of DNA sequences from the 5’ untranslated regions of mtl-2 genes, containing the conserved HZA^mT element and six flanking base pairs. Sequences are from four WT Caenorhabditis species and the C. elegans syb4265 deletion strain. Location indicates the number of base pairs upstream from the ATG start codon and the strand orientation. C-E) Wild type (WT) and syb4265 mutants at the L4 stage were cultured with replete (-) or 200μM supplemental zinc (+) for 16 hours and analyzed by qPCR. N = 15 initial biological replicates but may vary in panels due to outlier removal.

Figure 3.

The HZA^cF element was necessary for full cadmium-induced transcript accumulation of both cdr-1 and F35E8.10. A) Schematic of a region of Chromosome V drawn to scale in base pairs (bp). Thick black line represents DNA; yellow box indicates HZA enhancer. Arrows above represent pre-mRNA for indicated genes. Red line below indicates the syb4134 mutation, a 14-bp deletion of the HZA^cF enhancer (named HZA^cFΔ). Scale bar: 200bp. B) Alignment of DNA sequences from the 5’ untranslated regions of cdr-1 that contain the conserved HZA^cF element and six flanking base pairs in WT and syb4134 mutant. Location indicates the number of base pairs upstream from the ATG start codon and the strand orientation. C-G) Wild type (WT) and syb4134 mutants at the L4 stage were cultured with (+) or without (−) 100μM cadmium for 16 hours and analyzed by qPCR. N = 6 initial biological replicates, but may vary in panels due to outlier removal.

Figure 3.

The HZA^cF element was necessary for full cadmium-induced transcript accumulation of both cdr-1 and F35E8.10. A) Schematic of a region of Chromosome V drawn to scale in base pairs (bp). Thick black line represents DNA; yellow box indicates HZA enhancer. Arrows above represent pre-mRNA for indicated genes. Red line below indicates the syb4134 mutation, a 14-bp deletion of the HZA^cF enhancer (named HZA^cFΔ). Scale bar: 200bp. B) Alignment of DNA sequences from the 5’ untranslated regions of cdr-1 that contain the conserved HZA^cF element and six flanking base pairs in WT and syb4134 mutant. Location indicates the number of base pairs upstream from the ATG start codon and the strand orientation. C-G) Wild type (WT) and syb4134 mutants at the L4 stage were cultured with (+) or without (−) 100μM cadmium for 16 hours and analyzed by qPCR. N = 6 initial biological replicates, but may vary in panels due to outlier removal.

Figure 4.

A deletion of mtl-2 did not influence zinc-activated transcription of T08G5.1. A) Schematic of a region of Chromosome V drawn to scale in base pairs (bp). Thick black line represents DNA; yellow box indicates HZA enhancer. Arrows above represent pre-mRNA for indicated genes. Red line below indicates the extent of the gk125 deletion that removes the mtl-2 open reading frame (labeled mtl-2Δ). Scale bar: 200bp. B) An enlarged region of panel A is indicated by dashed lines and shows the positions of predicted control elements: TATA box, GATA element 1, HZA^mT, and GATA element 2. The gk125 deletion removes the TATA box and GATA element 1. Scale bar: 100bp. C, D) Wild type (WT) and mtl-2(gk125) mutants at the L4 stage were cultured with or without 200μM zinc for 16 hours and analyzed by qPCR. Biological replicates: T08G5.1 N = 10, srh-308 N = 3. E) Model for the orientation-independent enhancer function of the HZA element. In intestinal cells, the ligand-binding domain of HIZR-1 (red) binds to zinc ions (cyan circles) during zinc excess conditions or cadmium ions (green circles) during cadmium stress (blue arrows). HIZR-1 then enters the nucleus (blue arrows), the DNA-binding domain binds the HZA element (yellow), and transcription of both flanking genes is activated (red arrows). Thick black lines in the nucleus represent chromosome V, and thin black arrows indicate transcription start sites of genes. Not drawn to scale. F) Different modes of enhancer function. Top, a single enhancer controls gene 1 and gene 2, which are encoded on the same DNA strand. Middle, a single enhancer controls gene 1 in condition 1 or gene 2 in condition 2, and these head-to-head genes are encoded on different DNA strands. Bottom, a single HZA enhancer controls gene 1 and gene 2 in the same condition, and these head-to-head genes are encoded on different DNA strands.