Journal of Molecular Biology
Volume 426, Issue 17, 26 August 2014, Pages 3069-3093
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A Conserved Noncoding Sequence Can Function as a Spermatocyte-Specific Enhancer and a Bidirectional Promoter for a Ubiquitously Expressed Gene and a Testis-Specific Long Noncoding RNA

https://doi.org/10.1016/j.jmb.2014.06.018Get rights and content

Highlights

  • CNS1 may be a spermatocyte-specific enhancer for the mouse Tcam1 gene.

  • CNS1 is a bidirectional promoter for Smarcd2 and lncRNA-Tcam1 in spermatocytes.

  • lncRNA-Tcam1 is germ cell specific but unlikely to control the Tcam1 expression.

  • Bidirectional promoter activity of CNS1 may depend on the epigenetic state.

  • This is the first report for a dual promoter–enhancer in mammals.

Abstract

Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4 kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5′-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and lncRNA, designated lncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, lncRNA-Tcam1 expression was restricted to testicular germ cells, although this lncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for lncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and lncRNA-Tcam1.

Introduction

Tissue-specific gene activation is controlled by complicated mechanisms that involve the activity of promoters and enhancers, epigenetic modification, and noncoding transcription [1], [2], [3]. In mammals, many tissue-specific genes require distal enhancers and a proximal promoter for full activation [4], [5]. The enhancer is a sequence to which transcription factors bind for increasing the rate of target gene transcription [4], [5]. Recent studies have demonstrated that enhancers physically interact with the target gene promoter by looping out the intervening sequences and are associated with many transcription factors [4], [5]. Genome-wide analyses have also revealed that a large number of enhancers are occupied by RNA polymerase II [6], [7], [8], [9], [10], [11], and consistent with this, many enhancers are actually transcribed into long noncoding RNAs (lncRNAs) that are often essential for target gene activation [11], [12]. However, the relationship between lncRNAs and enhancers seems to be diverse and is not completely understood [11], [12], [13].

To understand the regulatory mechanism for tissue-specific gene activation, various genes have been investigated as model genes [14], [15], [16], [17], [18], [19], [20]. The human growth hormone (hGH) gene cluster is one such model. This cluster is located on chromosome 17q22-24 and encompasses five paralogous growth hormone genes. Although the primary structure of the five genes is well conserved, hGH-N is specifically expressed in the pituitary and the other four genes are placenta specific [21], [22]. The tissue-specific activation of the hGH cluster is dependent on the 5′-distal locus control region (LCR) [23], [24], and epigenetic regulation and noncoding transcription are known to play crucial roles in activation by the hGH LCR [25], [26], [27], [28], [29]. Interestingly, the hGH cluster is linked to two other tissue-specific genes: the B-cell-specific CD79b gene, which is located between the cluster and LCR, and the testis-specific testicular cell adhesion molecule (TCAM1P) gene in the 3′-region of the cluster. Therefore, this locus is an excellent model for tissue-specific gene activation; however, the TCAM1P gene regulation has not been studied in detail.

TCAM1P is a highly conserved gene among placental mammals such as the cow, rat, mouse, and rhesus monkey. Although the human TCAM1P gene does not seem to encode a protein, the orthologous gene in other species is translated to a protein related to cell adhesion [30]. The testis-specific expression of this gene has been confirmed in the rat and mouse [30], [31], and the mouse Tcam1 gene has been found to be expressed in the 17-day-old testis, when germ cell meiosis reaches the late pachytene spermatocyte stage [32]. Consistent with this, mouse Tcam1 mRNA has been found to be localized in such spermatocytes by in situ hybridization [30]. With regard to the regulation of this gene, a DNase I hypersensitive site has been detected in the rat Tcam1 promoter, and a high level of histone acetylation has been observed at DNase I hypersensitive site in rat plasmacytoma-derived Y3-Ag1.2.3 cells [33], [34]. However, no cis-elements have been identified for Tcam1 regulation and the regulatory mechanism in native testicular germ cells has not been investigated.

In the present study, we focused on conserved noncoding sequences (CNSs) to examine the regulation of the mouse Tcam1 gene. There were six CNSs at the Tcam1 locus, among which CNS1 was identified as a potential spermatocyte-specific enhancer. Interestingly, CNS1 also contained bidirectional promoter activity, and the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 2 (Smarcd2) gene and a novel testis-specific lncRNA (designated lncRNA-Tcam1) were actually expressed from the upstream and downstream of CNS1, respectively. The results indicated that CNS1 may work as a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of the ubiquitously expressed Smarcd2 gene and the testicular germ cell-specific lncRNA-Tcam1. This is the first indication of a dual promoter–enhancer in mammals.

Section snippets

Tcam1 is a spermatocyte-specific gene

The mouse Tcam1 gene has been reported to be specifically expressed in the testis, particularly in germ cells at stages from pachytene spermatocytes to secondary spermatocytes [30]. We first attempted to confirm this expression pattern of Tcam1 mRNA. By Northern blot analysis using total RNAs from 15 mouse tissues, we detected a specific signal for Tcam1 at the 3.0-kb position only in the testis (Fig. 1a). This was also confirmed by the transcriptomic data (GSE9954), which demonstrated

CNS1 may be a spermatocyte-specific enhancer for the Tcam1 gene

An enhancer is a sequence that increases the transcription rate of its target gene and is usually located in a remote upstream or downstream region [4], [5]. It enhances gene transcription when it physically interacts with the target gene promoter in the nucleus [4], [5]. In the present study, CNS1 increased Tcam1 promoter activity in vitro in GC-2spd(ts) cells (Fig. 4a and Supplementary Fig. 2), which indicates that CNS1 is a potential enhancer in these cells. To assess the enhancer activity

Animals

The mice (C57/BL6) were maintained at 25 °C with a photoperiod of 14:10 h light:dark with free access to food and water. Experimental procedures used in this study were approved by the Institutional Animal Use and Care Committee at Hokkaido University.

RNA analyses

Northern blot, in situ hybridization, RT-PCR, and qRT-PCR were performed as described previously [92]. qRT-PCR was also performed using the 7300 real-time PCR system and KOD SYBR qPCR Mix (Toyobo, Osaka, Japan) in a total volume of 10 μl per well.

Acknowledgements

We thank Dr. Shin Matsubara for his valuable comments on this manuscript. This work was supported by Grants-in-Aid for Scientific Research 19770048 and 21770068 to A.P.K. from the Ministry of Education, Culture, Sports, Science and Technology, Japan. M.K. was supported by a research fellowship of the Japan Society of the Promotion of Science (24-6230).

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