Characterization of Asxl1, a murine homolog of Additional sex combs, and analysis of the Asx-like gene family
Introduction
Epigenetic regulators control cell memory and/or inheritance in ways that do not result in changes to DNA sequence and/or do not follow Mendelian inheritance patterns; disruption of epigenetic regulation leads to cancer and other forms of disease in humans (Lund and van Lohuizen, 2004). Polycomb Group (PcG) and trithorax Group (trxG) proteins are epigenetic regulators, initially discovered due to their requirement for maintaining proper Hox homeotic gene expression patterns during Drosophila embryogenesis once correctly initiated. Hence, PcG and trxG proteins are collectively referred to as maintenance proteins. There are estimated to be 30–40 PcG genes in Drosophila, of which 15 have been cloned and characterized. Each maintenance protein characterized in Drosophila has at least one homolog in mammals that regulates Hox gene expression. PcG and trxG proteins regulate other target loci in addition to the Hox genes, and possess additional functions including random X inactivation, genomic and autosomal locus imprinting, RNA interference, cell cycle regulation, oncogenesis, and stem cell maintenance (Brock and Fisher, 2005).
Maintenance proteins have recently been further categorically sub-divided. The Enhancer of trithorax and Polycomb (ETP) Group was created on the basis of a Drosophila genetic screen for enhancers and suppressors of trxG mutations (Gildea et al., 2000). Mutations in six genes formerly classified in the PcG, namely Additional sex combs (Asx), Enhancer of zeste (E(z)), Enhancer of Polycomb (E(Pc)), Posterior sex combs (Psc), Suppresor of zeste 2 (Su(z)2), and Sex combs on midleg (Scm) were found to enhance trxG mutations, thereby leading to the reclassification of those genes to the ETP Group, defined as genes whose mutations enhance the phenotypes of both PcG and trxG gene mutants (LaJeunesse and Shearn, 1996, Milne et al., 1999, Gildea et al., 2000). The phenotypes of ETP group gene mutants imply these genes function in both repression and activation of Hox target genes (Brock and Fisher, 2005).
Additional sex combs (Asx) is an ETP Group gene because mutations in Asx enhance both Polycomb and trithorax Group gene mutations (Milne et al., 1999, Gildea et al., 2000). Consistent with this classification, Asx mutants exhibit bidirectional transformations along the anterior–posterior body axis, and Asx is required to maintain repression and activation of Hox loci (Breen and Duncan, 1986, Sinclair et al., 1992, Milne et al., 1999). PcG and trxG proteins are now thought to have interconnected functions in maintaining the appropriate transcriptional state of a target locus, depending on the context, and ETP proteins may provide insight into the bridge between these functions (Brock and Fisher, 2005). We have undertaken the study of Asx and its homologs as a model for ETP gene function. Here we report the cloning and characterization of a murine Asx homolog, called Additonal sex combs-like 1, and compare it to Asx and two other known Asx homologs in mice, Asxl2 and Asxl3, in an attempt to understand the structural basis for Asx function in Drosophila and mammals.
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cDNA library screening, cDNA and genomic cloning and sequencing
A 5′ region 1.8 kb cDNA clone, and a 3′ region 4.8 kb cDNA clone, each containing partial mouse Asxl1 sequence, were obtained by screening a mixed random- and oligo(dT)-primed mouse neonatal brain cDNA library (Stratagene) with the 0.45 kb NotI-EcoRI fragment from EST W41911 obtained from the IMAGE consortium. Sequencing was done by Filippo Randazzo (Chiron Corp., Palo Alto CA) and the UBC NAPS unit service (Vancouver, Canada).
3′ RACE experiments for mouse Asxl1 were performed using a mouse day
Characterization of Asxl1 cDNA and genomic clones
As described in the Material and methods, sequencing and alignment of two embryonic cDNA clones, a 4.8 kb clone mAsx7A and a 1.8 kb clone mAsx9A revealed a putative open reading frame of 4541 bp. This partial mouse Asxl1 cDNA sequence was submitted to GenBank under Accession number AR072722. Following alignment of overlapping ESTs, cDNA and genomic clones from public databases with the partial Asxl1 cDNA sequence, we generated a putative full length cDNA contig of 6674 bp that was named Asxl1
Conclusions
- 1.
A murine homolog of Asx has been cloned and characterized and named Asxl1. Asxl1 predicted protein is highly (81%) similar to human ASXL1, but shows similarity to Asx only within the C-terminal PHD finger and the N-terminal region containing the ASXH domain.
- 2.
Asxl1 consists of 13 exons spanning 58 kb. The intron–exon structure is highly similar to that of human ASXL1, although the locus size is smaller.
- 3.
There are two other murine homologs to Asx, termed Asxl2 and Asxl3. Each of the three murine
Acknowledgements
This research was supported by a grant from the Canadian Institutes of Health Research to H.W.B., and by a Medical Research Council of Canada Studentship to C.L.F. We thank Patty Rosten for technical assistance, and Jacquie Damen for the GAPDH control probe.
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