Assaying the regulatory potential of mammalian conserved non-coding sequences in human cells

Abstract

Background: Conserved non-coding sequences in the human genome are approximately tenfold more abundant than known genes, and have been hypothesized to mark the locations of cis-regulatory elements. However, the global contribution of conserved non-coding sequences to the transcriptional regulation of human genes is currently unknown. Deeply conserved elements shared between humans and teleost fish predominantly flank genes active during morphogenesis and are enriched for positive transcriptional regulatory elements. However, such deeply conserved elements account for <1% of the conserved non-coding sequences in the human genome, which are predominantly mammalian.

Results: We explored the regulatory potential of a large sample of these 'common' conserved non-coding sequences using a variety of classic assays, including chromatin remodeling, and enhancer/repressor and promoter activity. When tested across diverse human model cell types, we find that the fraction of experimentally active conserved non-coding sequences within any given cell type is low (approximately 5%), and that this proportion increases only modestly when considered collectively across cell types.

Conclusions: The results suggest that classic assays of cis-regulatory potential are unlikely to expose the functional potential of the substantial majority of mammalian conserved non-coding sequences in the human genome.

Figure 1

Multi-tissue DNaseI hypersensitivity patterns of CNCSs. Shown are the locations of Chr21 CNCSs (top row, black vertical marks), 192 CNCSs tested for DHSs potential (second row, black vertical marks), and CNCSs encoding DHSs in one or more cell types (colored vertical marks). Absence of a colored vertical mark beneath a CNCSs from row 2 indicates lack of DHS potential in the tissue tested.

Figure 2

Unbiased mapping of DHSs and DHS CNCS overlaps. (a) Shown for a 1.7 Mb region of Chr21 are locations of CNCSs (top row, vertical red marks), locations of known genes and annotated transcripts, and maps of DNaseI hypersensitivity in intestinal (CACO2), lymphoid (GM06990), cervical (HeLa), and neural (SKnSH) cell types. A total of 416 distinct DHSs map to this region. (b) Results from 1,000 random trials of sample size 416 and corresponding overlap with CNCSs. The vertical arrow indicates actual result, which is within random expectation.

Figure 3

Chr21 CNCSs and control sequences. Shown are the mapping locations of the human chromosome 21 CNCSs and control non-genic non-transcribed sequences used in this study relative to known Chr21 genes: a) 2262 CNCSs described in Dermitzakis et al. [18]; b) 71 CNCSs randomly selected; c) 21 control single-copy sequences chosen randomly along Chr21; d) 23 CNCSs from Dermitzakis et al. coinciding with Sp1/Myc/p53 binding sites determined by Cawley et al. [30]; e) 44 putative promoter CNCSs.

Figure 4

Enhancer/repressor assay of CNCSs. (a, b) Boxplots showing the distribution of the luciferase activity for each subset of sequences in 293T (a) and Huh7 (b) cell lines. The proportion of putative regulatory elements of each subgroup is indicated at the bottom of both graphs. (c-f) Bar graphs showing the fold change of luciferase activity compared to the control sequence set for 71 selected CNCSs (c, d), 23 CNCS overlapping transcription factor binding sites (TFBSs) (e, f), in 293T and Huh7 cell lines, respectively. Red lines show ± 2-fold change threshold. Asterisks denote statistically significant change (one-sample t-test).

Figure 5

Assay of putative CNCS promoters. (a) Bar graph showing the normalized luciferase activity of putative promoter CNCSs in an episomal vector without minimal promoter. Bidirectionality was tested by cloning the sequences in the native or reverse orientation. Broken bars show values that are off scale. All CNCSs overlapping DHSs are included. (b) Pie chart showing the proportion of random CNCSs with enhancer, silencing, promoter or no activity.