A Novel CpG-free Vertebrate Insulator Silences the Testis-specific SP-10 Gene in Somatic Tissues

Abstract

Regulation of cell type-specific gene transcription is central to cellular differentiation and development. During spermatogenesis, a number of testis-specific genes are expressed in a precise spatiotemporal order. How these genes remain silent in the somatic tissues is not well understood. Our previous studies using the round spermatid-specific mouse SP-10 gene, which codes for an acrosomal protein, revealed that its proximal promoter acts as an insulator and prevents expression in the somatic tissues. Here we report that the insulator tethers the SP-10 gene to the nuclear matrix in somatic tissues, sequestering the core promoter in the process, thus preventing transcription. In round spermatids where the SP-10 gene is expressed, this tethering is released. TAR DNA-binding protein of 43 kDa (TDP-43), previously shown to interact with the SP-10 insulator, was found to be in the 2 M NaCl-insoluble nuclear matrix fraction. TDP-43 prevented enhancer-promoter interactions when artificially recruited between the two by Gal4 strategy. Knockdown of TDP-43 using small interfering RNA released the enhancer-blocking effect of the SP-10 insulator in a stable cell culture model. Mutation of TDP-43 binding sites abolished this effect. Finally, a 50-bp subfragment of the SP-10 insulator, which includes TDP-43 binding sites, functioned as a minimal insulator in transgenic mice and silenced an otherwise ectopically expressed transgene in somatic tissues. The SP-10 insulator lacks CpG dinucleotides or CTCF binding sites. Thus, the present study characterized a novel vertebrate insulator in a physiological context and showed for the first time how a testisspecific gene is silenced in the somatic tissues by an insulator.Regulation of transcription of a tissue-specific gene is central to cellular differentiation and development. Mechanisms must be in place not only to activate the gene in the correct cell type but also to keep the gene silenced in all other tissues. We study this problem using the mouse testis-specific SP-10 gene as a model. The SP-10 gene codes for an acrosomal protein conserved in mammals and is exclusively expressed in round spermatids (1). In this report, we address the mechanism of transcriptional silencing of the SP-10 gene in the somatic tissues. Our previous work in transgenic mice showed that the SP-10 proximal promoter performs dual functions; it activates testisspecific transcription and also acts as an insulator preventing the possibility of transcription in the somatic tissues (2).Insulators are DNA sequences located at gene boundaries (3). Their action prevents ectopic expression of genes they flank. Insulators are operationally defined as enhancer blockers and barrier elements. Enhancer-blocking insulators prevent a foreign enhancer from inappropriately encroaching into the promoter of the neighboring gene. The barrier insulators prevent the spread of heterochromatin and thus prevent untimely shut down of gene expression. Insulators may possess only one or both of the above properties (4).The chicken...

TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis

“…35 In addition, the C-terminal domain functions in the transcriptional repression of the mouse testis-specific SP-10 gene, regulating the tissuespecific expression of this gene. 36 Finally, the glycine-rich C-terminal domain binds several heterogeneous nuclear ribonucleoproteins involved in the biogenesis of mRNA. 37,38 Thus, while the physiological functions of TDP-43 are as yet incompletely characterized, TDP-43, and in particular the C-terminal domain, appears to function in the regulation of gene expression.…”

Implications of the prion-related Q/N domains in TDP-43 and FUS

“…31,33,34 Instead, the C-terminus of TDP-43 is required for it to function as a suppressor at several splicing targets 35,36 and for TDP-43 to act as a transcriptional insulator for the mouse sp-10 gene. 37 Importantly, all but one of the ALS associated mutations in TDP-43 occur in the C-terminal domain.…”

Structural determinants of the cellular localization and shuttling of TDP-43

“…A recent report has shown that TDP-43, acting as a transcriptional insulator at the promoter of the spermatid-specific SP-10 gene, is predominantly present in the nuclear matrix fraction (Abhyankar et al, 2007). We wanted to test whether removal of TDP-43 disrupted chromatin organization.…”

“…hnRNP A1 and hnRNP A2/B1) through this region specifically. The functional importance of this domain in additional biological properties is highlighted by studies on the regulation of the spermatid-specific SP-10 gene (also known as ACRV1) (Abhyankar et al, 2007). Transcriptional inhibition of SP-10 by TDP-43 depends on the presence of the carboxyl domain and RRM-1 of the protein.…”