title: 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.
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 3.4kb 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.
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Misuzu
authors.initials: M
authors.lastname: Kurihara
authors.affiliation: Suntory Foundation for Life Sciences, Bioorganic Research Institute, Osaka 618-8503, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Shiraishi
authors.affiliation: Suntory Foundation for Life Sciences, Bioorganic Research Institute, Osaka 618-8503, Japan.
authors.forename: Honoo
authors.initials: H
authors.lastname: Satake
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan; Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan. Electronic address: akimura@sci.hokudai.ac.jp.
authors.forename: Atsushi P
authors.initials: AP
authors.lastname: Kimura
mesh_terms: D000818:Animals; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics; D017124:Conserved Sequence; D005786:Gene Expression Regulation; D006657:Histones / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics; D041681:NIH 3T3 Cells; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D062085:RNA, Long Noncoding / Q000235:genetics* / Q000378:metabolism; D013090:Spermatocytes / Q000378:metabolism
chemical_list: D015815:Cell Adhesion Molecules; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D009124:Muscle Proteins; D062085:RNA, Long Noncoding; C494056:Smarcd2 protein, mouse; C485520:Tcam1 protein, mouse
keywords: GC-2spd(ts); Tcam1; conserved noncoding sequence; enhancer; long noncoding RNA