Abstract
The human ATRX gene encodes hATRX, a chromatin-remodeling protein harboring an helicase/ATPase and ADD domains. The ADD domain has two zinc fingers that bind to histone tails and mediate hATRX binding to chromatin. dAtrx, the putative ATRX homolog in Drosophila melanogaster, has a conserved helicase/ATPase domain but lacks the ADD domain. A bioinformatic search of the Drosophila genome using the human ADD sequence allowed us to identify the CG8290 annotated gene, which encodes three ADD harboring- isoforms generated by alternative splicing. This Drosophila ADD domain is highly similar in structure and in the amino acids which mediate the histone tail contacts to the ADD domain of hATRX as shown by 3D modeling. Very recently the CG8290 annotated gene has been named dadd1. We show through pull-down and CoIP assays that the products of the dadd1 gene interact physically with dAtrxL and HP1a and all of them mainly co-localize in the chromocenter, although euchromatic localization can also be observed through the chromosome arms. We confirm through ChIP analyses that these proteins are present in vivo in the same heterochromatic regions. The three isoforms are expressed throughout development. Flies carrying transheterozygous combinations of the dadd1 and atrx alleles are semi-viable and have different phenotypes including the appearance of melanotic masses. Interestingly, the dAdd1-b and c isoforms have extra domains, such as MADF, which suggest newly acquired functions of these proteins. These results strongly support that, in Drosophila, the atrx gene diverged and that the dadd1-encoded proteins participate with dAtrx in some cellular functions such as heterochromatin maintenance.
Highlights
The human ATRX gene was described approximately 20 years ago as the main gene mutated in ATRX syndrome (Alpha-Thalassemia with mental Retardation X-related)
Performing in silico analyses, we demonstrate that the annotated gene CG8290, recently named dadd1 [11], encodes three proteins with a conserved ADD domain that physically interact with dAtrxL
The ADD domain of Atrx is highly conserved in Drosophila and other invertebrates We searched for genes encoding proteins with the ADD domain in the Drosophila genome using the ADD region of the human ATRX gene (hATRX) protein with the BLAST (Basic Local Alignment Search Tool) of the NCBI (National Center of Biotechnology Information)
Summary
The human ATRX gene (hATRX) was described approximately 20 years ago as the main gene mutated in ATRX syndrome (Alpha-Thalassemia with mental Retardation X-related). There are mainly two isoforms named hATRX (289 kDa) and hATRXt (t, from truncated, 200 kDa) that are encoded by this gene [2] Both proteins contain an amino-terminal domain which is composed of PHD and GATA-like zinc fingers, named ADD after the three proteins that contain this domain (ATRX, DNMT3b and DNMT3L). It was recently demonstrated through different in vitro and in vivo approaches that this domain recognizes the combination of K9me and unmethylated K4 residues of the histone H3 tail [3]. This particular histone variant is incorporated at different chromatin regions, such as promoters, enhancers and heterochromatic regions, and it has been proposed to have dual functions in promoting both an active chromatin state and the maintenance of heterochromatin [8]. hATRX ATPase activity is important for incorporation of the histone variant H3.3 by the chaperone DAXX into specific regions of the chromosomes, such as telomeres and pericentric heterochromatin [9]
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