The K-SPMM database [50
] describes the location of promoters, transcription factor binding sites and the location of transcription factor binding modules of genes expressed during spermatogenesis in the murine testis. We used TRANSFAC analysis to identify additional non-spermatogenic-specific features [51
]. The promoter sequences analysed in K-SPMM incorporated 1 kB upstream of the Transcriptional Start Site (TSS) of the most 5’ TSS of splice variants, whilst non-spermatogenic TRANSFAC was extended to 2 kB upstream and ~50 bp downstream of the TSS. The K-SPMM analysis additionally allowed for identification of promoter element modules that are associated with, or excluded from, specific testis cell types: Sertoli cells, spermatogonia, spermatocytes and spermatids.
The promoter modules identified using the K-SPMM/TRANSFAC analysis for a representative member of each of mIMP α subfamily (IMP αs 2, 4 and 6) are given in Figs. (-)). By way of example, the K-SPMM data obtained also provides some information on conservation within select module regions relative to four other vertebrate genomes (H. sapiens, R. norvegicus, G. gallus and C. familiaris) as presented in Figs. (-)). In addition, the testis cell types which express other genes with these same putative promoter modules as the IMP αs, are highlighted in Figs. (-)).
Fig. (5) Figs. (-).Identification of paired transcription factor binding sites that are likely to be functional in the putative promoter regions of mouse IMP αs.A representative member of each IMP α subfamily (more ...)
Fig. (7) Figs. (-).Identification of paired transcription factor binding sites that are likely to be functional in the putative promoter regions of mouse IMP αs.A representative member of each IMP α subfamily (more ...)
Amongst those motifs identified that are common to all mouse IMP α putative promoter regions were canonical TATA and GC boxes, as well as motifs for ubiquitously expressed TFs including SPI1, TCF11, cETS1 (Figs.
). In addition, all mouse IMP αs expressed a variety of binding sites for transcription factors that display restricted patterns of tissue expression, including GATA 1 and 2 and winged helix factor HFH3 [52
]. Potential binding sites specific to each of the IMPs were also identified, including FREAC-7 and HFH1 for IMP α2 (Fig.
) and FREAC-4 for IMP α6 (Fig.
). Variations in the promoter elements present within the various IMP αs may account for their differential expression patterns. Genes with similar promoter modules represent a class of genes that may be co-expressed at distinct stages of spermatogenesis when the appropriate transcription factors are present. In the human, FREAC-4 expression is restricted to the testis and kidney [54
]. Confirmation of expression of transcription factors in the testis that have potential binding sites within the IMP α promoters will be the first step in understanding the regulation of IMP α expression.
Fig. (6) Figs. (-).Identification of paired transcription factor binding sites that are likely to be functional in the putative promoter regions of mouse IMP αs.A representative member of each IMP α subfamily (more ...)
Putative SRY-binding modules were detected in all IMP α promoter regions. In the mouse, SRY expression is restricted to the testis within a very narrow period between 10.5 and 12.5 days post coitum (dpc), the time during which sexual differentiation occurs [55
]. Microarray analysis of IMP α levels indicates significant levels of IMP α2 and 3 mRNAs, in particular, are present at 11.5 and 12.5 dpc (Fig.
]. Moreover, IMP α2 levels drop dramatically after 12.5 dpc, corresponding to the time when SRY expression abruptly ceases. SRY is transported into the nucleus by IMP β1 and hence it could well play a central role in IMP α gene regulation subsequent to its synthesis [58
The testicular cell types in which mRNAs from genes which possess these promoter motifs are known to be produced are boxed in red in Figs. (
). Putative mIMP α2 promoter modules are present in other genes of all the spermatogenic cell types, whereas IMP α4 modules are found in all cells except spermatogonia and IMP α6 modules are detected in spermatids only. These in silico
data do show some correlation with the IMP mRNA expression levels determined via
microarray and in situ
hybridisation studies in each of the spermatogenic cells types, with mIMP α2 highly expressed in spermatocytes and spermatids and mIMP α6 predominantly expressed in round spermatids (Figs. ,
]). The IMP α4 data correlates with high expression of mRNA in spermatocytes and spermatids which are represented in the putative modules detected (Figs. ,
). However consideration must be given to the likelihood that not all TFBP regions are identified using KSPMM, and the putative modules detected may not actually be functional in a particular cellular context. These data illustrate that in silico
data may provide a useful tool for identifying potential transcription factor sites and promoter regions of spermatogenic genes, however they must be biologically validated.
The conservation scores of the selected promoter motifs illustrated in Figs. (-) indicates that there is a particularly high level of conservation at the base-pair level for certain promoter regions. For example the TCF11MafG/TCF11MafG, Yin Yang/ TCF11MafG and TCF11MafG/S8 motifs of IMP α4 are 99% percent conserved between the four mammalian species. Such information will aid in identifying the regions and transcription factors binding them that are conserved across evolution and hence likely to be of critical importance.
These in silico
data provide a useful basis from which to develop models on regulation of IMP α gene expression. In terms of understanding transcriptional control of IMP α genes, modulation of IMP α gene expression represents only one form of IMP α regulation. Other mechanisms include post-transcriptional and post-translational modifications of the IMPs that alter of affinity and accessibility to target NLSs (reviewed in [60
]) and must also be considered when unravelling regulation of IMP α activity and its role in developmental systems in general.