Mouse Î³-satellite is not homologous to human Î³-satellite, but human-mouse homologous sequences exist
Overall, absolute levels of cassini in cells and tissues that expressed it varied significantly, but were very high as compared to that of the reference gene gapdh or 18 s rRNA, and in some samples exceeded those of the housekeeping transcripts. Therefore, it was remarkable that this family had not been characterized. Further database searches revealed that cassini and sequences related to it belong to a class of DNA that has been collectively named γ-satellite DNA in the murine genome. Satellite DNA was named as such due to its buoyant density on CsCl gradients, differentiating it from that of the bulk of eukaryotic DNA. This is a consequence of its different overall base pair composition. When we evaluated the first DNA sequence reported for a mouse major satellite DNA from 1981, we found it to be a cassini-like sequence. Vissel and Choo coined the term γ-satellite DNA for these sequences to distinguish this mouse satellite DNA from other satellite families, such as the mouse “minor” satellite, the human centromeric N satellite and a new p-satellite family. However, the term γ-satellite, as proposed by Vissel and Choo, remains confusing in that it suggests relatedness between such sequences from different species. Mouse γ-satellites were regarded as non-conserved in evolution, and indeed we found that the sequences described as human γ-satellite DNA repeats bear no sequence homology to the mouse γ-satellite DNA (not shown). In addition, mouse γ-satellite DNA is reported to be pericentromeric, but we were unable to detect any homology between the cassini-type sequences and the different human pericentromeric repeats reported in Eymery et al.. We did find numerous human cassini-like EST sequences and two non-assigned human genomic clones from chromosome 7 and 18. However, in contrast to mouse, none of these sequences have been assigned to a chromosomal sublocation and therefore it is unknown whether the human CASSINI loci are pericentromeric.
Similar to some classes of human pericentromeric repetitive DNA, cassini mouse γ-satellite is transcriptionally regulated
Interestingly, as reviewed in Ugarkovic, some of the other satellite DNAs are also highly conserved. To the extent that the conservation of a sequence in evolution, such as that of an exon versus an intron, signifies an evolutionary constraint on divergence because of loss of function, this would imply that sequences such as cassini have a function. Vourc’h and Biamonti reviewed possible functions of satellite DNA, in particular human pericentromeric satellite DNA, which has been more extensively studied. Although the human pericentromeric satellites show no homology to the mouse γ-satellite DNA, those located in the human 9q12 region show increased expression in HeLa cells that are heat shocked or stressed. Our results show that cassini expression is regulated by heat shock suggesting that the human pericentromeric satellites and cassini share inducibility.
Mouse Î³-satellite could be part of one large transcriptional unit
Vissel and Choo described the basic repeating unit of the mouse γ-satellite DNA to be 234 bp, and the majority of monomers were reported to be organized into largely uninterrupted arrays that vary from a minimum of 240 kb to greater than 2000 kb in length. However, an inspection of the only segment of the mouse genome wherein the organization of the γ-satellite is precisely reported shows that the 234 bp sequences are in fact organized as exons within typical genes. The chromosome 9 cluster contains 12 blocks of these sequences (Additional file1: Figure S1A), organized in a tandem head-to-tail configuration in the same transcriptional orientation. The 12 blocks each have a possible intron-exon structure, with the γ-satellite sequences interspersed by non-γ-satellite DNA sequences. In fact, the automated DNA analysis software has annotated this region as containing genes.
It is currently unclear whether this area would be transcribed as 12 units or whether some of the units could be spliced together to form a larger transcript. Murine cDNAs corresponding in size to the predicted transcripts of these units are listed in the databases. The only indication that a very large spliced transcript could exist is the fact that the Plasmodium genome also contains homologous sequences, organized in a typical exon-intron structure; the largest virtual cDNA that was generated by automated annotation would be composed of 26 exons containing multiple “γ-satellite” units.
Cassini shows some of the features of a typical gene
Whereas in earlier years repetitive DNA, including γ-satellite DNA, was regarded as functionless, in the course of twenty years, views on mouse γ-satellite DNA appear to have evolved. One paper reported detecting a transcript in senescent cardiac muscle, and a different study linked mouse γ-satellite transcription to the cell cycle suggesting that some of these sequences may be transcribed.
Our current study, using real time RT/PCR, is the first to extensively analyze and quantify transcription of one such sequence, which we have named after the celestial satellite cassini. The primer pair that we selected detects a single product in the databases, and the melting curves obtained from both mouse and human samples indicate the primers amplify a single product. However, we cannot definitively exclude the possibility that we are detecting transcripts from more than one locus because we presume that not all of these sequences are reported in the databases. Nonetheless, individual γ-satellite sequences that are reported as physical cDNAs have distinct nucleotide sequences. Thus, they represent distinct loci, and the cassini primers detect transcription from only a subset of these loci.
Furthermore, the detection of large differences in basal and induced transcription of cassini in different mouse tissues supports the specificity of its expression. If the cassini transcript is the product from a single gene, then specific stimuli, including endotoxin exposure and drug treatment of pro-B ALL cells, induce extremely high levels of this specific RNA. Our experiments standardized cassini levels to those of the abundant gapdh and, in some experiments, levels were measured that vastly exceeded those of gapdh. Because the highest basal levels of cassini were detected in thymus and spleen, its expression may be regulated in hematopoietic cell types, and we speculate that this could be promoted through transcription factors that were reported to bind mouse γ-satellite DNA including Ikaros, a B-lineage specific transcription factor that regulates the early development of hematopoiesis, Glfi1b, which is an important regulator of hematopoiesis[19–22] or YinYang1, which is needed for differentiation of proB to preB cells.
It is currently unclear if cassini or other mouse γ-satellite RNA is translated into a protein (see Additional file5: Figure S5 and Additional file6) as we were unable to demonstrate the existence of a protein that corresponds in expression pattern to that of the cassini RNA. Although our data clearly show that a Cassini protein can be made, the product, if it exists, is likely to have unusual characteristics as is demonstrated by the migration abnormalities and aggregation of EGFP-Cassini after heat treatment in SDS-SB lysates (Additional file5: Figure S5).
Effect of cassini expression
Our experiments showed that induction of cassini RNA in cells correlates with specific stress stimuli, but our data do not provide information on whether the increased levels are detrimental to the cells or contribute a survival advantage. Because of the abundance of this RNA upon stress of cells, and the possibility that other transcribed loci exist in the genome with identical sequence, ablation of the RNA using siRNA does not appear technically feasible. We therefore used transient transfection in 293 cells to attempt to achieve expression levels that would significantly add to that already induced endogenously by stress, and also subjected the cells to a short-term treatment with chemotherapeutic drugs. The results of these experiments rule out the possibility that cassini induction correlates with or contributes to cell death, and suggest that high levels of this RNA provide some form of protection against the cytostatic activity of such drugs (Additional file7: Figure S6).