A Haploid Expressed Gene Cluster Exists as a Single Chromatin Domain in Human Sperm

Abstract

Mammalian spermiogenesis is marked by the initial disruption of the nuclear-histone-DNA complex by the transition proteins for ultimate replacement with protamines. The genes for three of these low molecular weight basic nuclear proteins exist as a single linear array of PRM1, PRM2, and TNP2 on human chromosome 16p13.2. To begin to address the mechanism governing their transcriptional potentiation, a region of approximately 40 kilo-bases of the human genome encompassing these genes was introduced into the germ line of mice. Fluorescence in situ hybridization and Southern analysis showed that this segment of the human genome integrated into independent chromosomal sites while maintaining its fidelity. Transcript analysis demonstrated that the expression of the endogenous mouse protamine Prm1 and Prm2 genes as well as the mouse transition protein Tnp2 gene were expressed along with their human transgene counterparts. The pattern of expression of these transgenic human genes within this multigenic cluster faithfully represented that observed in vivo. In addition, all members of this transgenic gene cluster were expressed in proportions similar to those in human testis. Copy number-dependent and position-independent expression of the transgenic construct demonstrated that the corresponding biological locus was contained within this segment of the human genome. Furthermore, DNase I sensitivity established that in sperm the human PRM1-->PRM2-->TNP2 genic domain was contained as an approximately 28.5-kilobase contiguous segment bounded by an array of nuclear matrix associated topoisomerase II consensus sites. This is the first description of a multigenic male gamete-specific domain as a fundamental gene regulatory unit. A model of haploid-specific gene determination is presented.

Chromatin loops are selectively anchored using scaffold/matrix-attachment regions

“…The 3′ end of the domain was further flanked by a single somatic S/MAR of an independently linked constitutively expressed domain. The head-to-tail array provided at least nine 40 kb regions that were bounded on each side by a somatic S/MAR (Choudhary et al, 1995;Kramer and Krawetz, 1996). Thus, if each of the somatic S/MARs served as an anchor, then every copy of this 40 kb segment should form a 40 kb loop, bounded by a pair of regions attached to the nuclear matrix.…”

“…We have effectively tested this premise by analyzing the distribution of human S/MARs with respect to the nuclear matrix that we have inserted into the mouse genome. Several varied-copy-number lines of a head-to-tail tandemly arrayed transgene of a 40 kb segment of human chromosome 16 containing the multigenic PRM1→PRM2→TNP2 locus were created (Choudhary et al, 1995;Stewart et al, 1999). Each 40 kb segment contained two haploid specific S/MARs bounding the ends of a haploid expressed multigenic domain.…”

“…Several varied copy number transgenic lines were created. The 40 kb transgenes were arranged in a head-to-tail tandem array in a single locus (Choudhary et al, 1995;Kramer and Krawetz, 1996;Stewart et al, 1999).…”

Prolactin Induces Protamine 2 mRNA Expression in Rat Testis.

“…Rat PRM2 mRNAs were 830 and 700 bps in size, the smallest fragment (approximately 0.9 kbps) appears to be PRM 2 mRNA including the 5' and 3' untranslated region [29,30]. This expression pattern has also been shown in human sperm [31]. The intensity of this band was influenced by PRL administration.…”

Protamine 3 expressions in crossbred bull spermatozoa may not be a prognostic marker for differentiating good and poor quality semen

“…The expression of the PRM3 gene sequence was first reported in rat testis (Singh and Rao, 1988), located in the cytoplasm instead of the nucleus (Kleene, 1989). The genomic sequences of the PRM1 and PRM2 genes are organized in the form of a loop domain together with the transition protein 2 gene (TNP2) and a sequence called PRM3 (Choudhary et al, 1995;Schluter et al, 1996;Singh and Rao, 1988;Krawetz and Dixon, 1988;Wykes and Krawetz, 2003;Nelson and Krawetz, 1993). Due to their spatial organization, they may allow a coordinated expression of these genes during spermiogenesis.…”