TE2 and TE1 sub‐elements of the testis‐specific histone H1t promoter are functionally different

Abstract

The testis-specific linker histone H1t gene is transcribed exclusively in pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in part by transcriptional factors that bind elements located within the proximal and distal promoter. A 40 bp promoter element, designated H1t/TE, that is located within the proximal promoter between the CCAAT-box and AC-box, is known to be essential for H1t gene transcription in transgenic animals. In the present study, we show by SDS-PAGE analysis of UV crosslinked protein and DNA and by electrophoretic mobility shift assays (EMSA) of testis nuclear proteins separated on a non-denaturing glycerol gradient that the TE1 sub-element is bound by a protein complex. Mutation of TE1 leads to a drop in H1t promoter activity in germinal GC-2spd cells as well as in nongerminal Leydig, NIH3T3, and C127I cell lines. Although TE1 and TE2 sub-elements have similar sequences, mutation of the TE2 sub-element causes an increase in promoter activity in C127I and Leydig cells. The rat TE1 but not TE2 contains a CpG dinucleotide and this cytosine is methylated in liver but not in primary spermatocytes. Methylation of the cytosine at this site almost eliminates nuclear protein binding. Thus, there are significant functional differences in the TE2 and TE1 sub-elements of the H1t promoter with TE1 serving as a transcriptional activator binding site and TE2 serving as a repressor binding site in some cell lines.

RFX2 Is a Potential Transcriptional Regulatory Factor for Histone H1t and Other Genes Expressed During the Meiotic Phase of Spermatogenesis1

“…As a first step to test if the binding proteins detected by Clare et al [24] are related to the RFX family [25], we compared testis and liver NE by band-shift analysis, using labeled oligos containing either the H1t palindrome or the strong, interleukin-5 receptor ␣ (IL-5R␣) RFX site [34] ( Fig. 1, lanes 1-4).…”

“…Clare et al [24] confirmed their observation but found that an additional set of complexes of similar mobilities was also formed from all somatic nuclear extracts. The identities of these bands have remained obscure, although Grimes recently suggested that the Te1 palindrome motif was related to the so-called X box in the promoters of the major histocompatibility complex II (MHCII) genes [25].…”

Testis-specific transcriptional control

“…However, the GC-box overlaps TE1 and TE2, and it is not yet known if it is functional in activating transcription in spermatocytes in vivo. Mutations within TE1, TE2 or GC-box sub-elements leads to partial inactivation of the H1t promoter in transient expression assays (Wilkerson et al, 2002a;Wilkerson et al, 2003;Wolfe et al, 2004). Mutagenesis of any of these sites causes a decrease in reporter gene expression in the germinal derived GC-2spd cell line as well as in transfected nongerminal cells.…”

“…2). The TE1 and TE2 subelements are imperfect inverted repeats, and TE1 serves as a binding site for a transcriptional activator (Grimes et al, 1992b;Grimes et al, 1992a;Wilkerson et al, 2002b;Wilkerson et al, 2002a;Wilkerson et al, 2003). The GC-box, located between TE1 and TE2, contribute to transcriptional activation in transient expression assays in some cell lines (Wilkerson et al, 2002b;Wilkerson et al, 2002a).…”

Binding of RFX2 and NF‐Y to the testis‐specific histone H1t promoter may be required for transcriptional activation in primary spermatocytes

“…Unlike other histone promoters, the H1t promoter contains an imperfect inverted repeat called the testis element (TE) shown to contain the two elements of the X‐box initially called TE1 and TE2, now called X‐box 1 and X‐box 2 [Grimes et al, 2003; Wilkerson et al, 2003]. Studies in transgenic mice showed that mutation of X‐box 1 and X‐box 2 in the rat H1t promoter abolished expression of the transgene [vanWert et al, 1998].…”