A dual enhancer-silencer element, DES-K16, in mouse spermatocyte-derived GC-2spd(ts) cells
Introduction
The genome consists of different types of cis-regulatory elements such as transcriptional enhancers and silencers [1,2]. Transcriptional enhancers play a major role in activating gene expression and can be located upstream or downstream of transcriptional start sites of target genes or within gene bodies [2]. A silencer is a cis-regulatory element which negatively regulates the transcription of its target gene. Since its discovery in 1985, various silencers have been identified in different species [[3], [4], [5], [6], [7]], but compared to enhancers, detailed studies about silencers are limited.
Epigenomic analyses have revealed the association of epigenetic marks with cis-regulatory elements. Histone H3 lysine 4 monomethylation (H3K4me1) has been identified as an important and enriched mark at active and primed enhancers, and possibly finetunes, rather than tightly controls, the activity and function of enhancers [[8], [9], [10]]. H3 lysine 27 acetylation (H3K27ac) distinguishes active enhancers from inactive/poised enhancer elements that contain H3K4me1 alone [8,11]. H3K27 trimethylation (H3K27me3) was recently reported to be a mark of silencers when it coincides with DNase I hypersensitivity [12]. Thus, epigenetic marks are useful for exploration of the functional genomic regions.
In addition, the multifunctionality of genome and its association with epigenetic marks have been revealed by recent studies. A dual promoter-enhancer or epromoter is associated with H3K4me1, H3K27ac, and histone H3 lysine 4 trimethylation (H3K4me3), a typical mark of promoter, and possesses both promoter and enhancer activity to control the transcription of two or more genes at a time [[13], [14], [15], [16], [17], [18], [19], [20]]. A dual enhancer-silencer element possesses both enhancer and silencer activity [21,22], and a recent study with Drosophila indicated its association with H3K4me1, H3K27ac, and H3K27me3 [23]. However, while dual promoter-enhancers mostly function as both enhancer and promoter in a single cell type, enhancer and silencer activity of a dual enhancer-silencer is exerted in different tissues [[21], [22], [23], [24]]. This raises a question whether a dual enhancer-silencer element can function as both enhancer and silencer in a single cell type or not.
In this study, we identified a novel gene regulatory genomic element in mouse primary spermatocytes, DES-K16, in an intron of the Kctd16 gene. The present data demonstrate that DES-K16 exhibits unprecedented dual enhancer-silencer activity in a spermatocyte-derived GC-2spd(ts) cell line. To the best of our knowledge, this is the first demonstration of the simultaneous enhancer and silencer activity in a single cell type.
Section snippets
Animals
C57Bl/6 mice were maintained on 14 h light/10 h dark cycles at 25 °C and given food and water ad libitum. The experimental procedures in this study were approved by the Institutional Animal Use and Care Committee at Hokkaido University.
ChIP-sequencing data analysis
ChIP-sequencing (ChIP-seq) data were collected and analyzed as previously described [15].
Reporter constructs
The thymidine kinase (TK) promoter was obtained from a pEBMulti-Neo vector (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) by NheI and BglII restriction digestion,
Discovery of DES-K16 with H3K4me1, H3K27ac, and H3K27me3 marks in mouse spermatocytes
To find novel cis-regulatory elements during spermatogenesis, we analyzed publicly available ChIP-seq data indicating H3K4me1, H3K27ac, and H3K27me3 patterns (GEO accession numbers: GSM1202711, GSM1202714, and GSM1674011) in mouse primary spermatocytes [28,29]. We detected a high peak for all of the three histone marks overlapping with an intron region of the Kctd16 gene on chromosome 18 (Fig. 1). This approximately 350-bp region contained a 100-bp high peak and 250-bp small but clear peaks and
Discussion
Here, we identified the DES-K16 region based on histone modification patterns in mouse spermatocytes and assessed its transcriptional activity in cultured cells. DES-K16 showed significant enhancer activity in two cell types by reporter gene assay, and a deletion resulted in both increase and decrease in expression of linked genes in GC-2spd(ts) cells. These data suggest that DES-K16 functions as a dual enhancer-silencer in this cell type. Hitherto known dual enhancer-silencer elements act as
Funding
This work was supported by KAKENHI 15H04317 and 20H03285 from Japan Society for the Promotion of Science and Suhara Memorial foundation.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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