The HARE-HTH and associated domains: novel modules in the coordination of epigenetic DNA and protein modifications

Abstract

Human ASXL proteins, orthologs of Drosophila Additional Sex combs, have been implicated in conjunction with TET2 as a major target for mutations and translocations leading to a wide range of myeloid leukemias, related myelodysplastic conditions (ASXL1 and ASXL2) and the Bohring-Opitz syndrome, a developmental disorder (ASXL1). Using sensitive sequence and structure comparison methods, we show that most animal ASXL proteins contain a novel N-terminal domain that is also found in several other eukaryotic chromatin proteins, diverse restriction endonucleases and DNA glycosylases, the RNA polymerase delta subunit of Gram-positive bacteria and certain bacterial proteins that combine features of the RNA polymerase α-subunit and sigma factors. This domain adopts the winged helix-turn-helix fold and is predicted to bind DNA. Based on its domain architectural contexts, we present evidence that this domain might play an important role, both in eukaryotes and bacteria, in the recruitment of diverse effector activities, including the Polycomb repressive complexes, to DNA, depending on the state of epigenetic modifications such as 5-methylcytosine and its oxidized derivatives. In other eukaryotic chromatin proteins, this predicted DNA-binding domain is fused to a region with three conserved motifs that are also found in diverse eukaryotic chromatin proteins, such as the animal BAZ/WAL proteins, plant HB1 and MBD9, yeast Itc1p and Ioc3, RSF1, CECR2 and NURF1. Based on the crystal structure of Ioc3, we establish that these motifs in conjunction with the DDT motif constitute a structural determinant that is central to nucleosomal repositioning by the ISWI clade of SWI2/SNF2 ATPases. We also show that the central domain of the ASXL proteins (ASXH domain) is conserved outside of animals in fungi and plants, where it is combined with other domains, suggesting that it might be an ancient module mediating interactions between chromatin-linked protein complexes and transcription factors via its conserved LXLLL motif. We present evidence that the C-terminal PHD finger of ASXL protein has certain peculiar structural modifications that might allow it to recognize internal modified lysines other than those from the N terminus of histone H3, making it the mediator of previously unexpected interactions in chromatin.

Figure 1

Domain architectures, gene neighborhoods and contextual network graph of the HARE-HTH domain and WHIM motifs. Standard abbreviations are used for domain names. LF ZnR refers to the little finger-type zinc ribbon that binds Ub. X and Y, respectively, refer to a domain and protein of uncharacterized function. Gene neighborhoods are shown for prokaryotic HARE-HTH-containing genes. Genes are shown as boxed arrows with the arrowhead pointing from the 5′ to the 3′ gene. The data from domain architectures and gene neighborhoods are combined into a contextual network graph with nodes representing domains and edges representing either their physical connectivity (solid black lines) or gene neighborhoods (blue dashed lines). Temporary IDs are assigned for Cyanidioschyzon and Emiliania, whose completed genomes are not in the non-redundant protein database. The sequences for the represented proteins are available in the Supplemental Material. The arrowheads depict directionality; for domain architectures, they point from the N- to the C-terminal domain, whereas for gene neighborhoods, they point from the 5′ to the 3′ gene. Domains with similar functional roles are grouped together.

Figure 2

Multiple sequence alignment (A) and structure (B) of the HARE-HTH domain and multiple sequence alignment of the PHD finger in ASX proteins (C). For the multiple alignments, proteins are denoted by their gene names, species names and GenBank index (GI) numbers. The consensus abbreviations, coloring scheme and secondary structure representation are given in the key at the bottom of the figure. The crystal structure of the Bacillus subtilis RNA polymerase δ subunit (PDB: 2KRC) was used to generate the cartoon representation showing the equivalent residues corresponding to the conserved positions at the N terminus of helix-1 and in helix 3. The same positions are marked with an asterisk below the alignment. For the PHD finger, residues involved in binding modified lysine residues are marked with an asterisk below the alignment.

Figure 3

Multiple sequence alignment of the WHIM motifs (A) and their relative structural organization in ISWI1a complexed with DNA (B). For multiple alignments, proteins are denoted by their gene names, species names and GenBank index (GI) numbers. Conserved residues predicted to interact with DNA and with other protein domains are marked in the alignment with red and purple asterisks, respectively. The coloring scheme, consensus abbreviations and secondary structure representation are as in the Figure 2 key. The cartoon structure was derived from the crystal structure of ISWIa complexed with DNA (PDB: 2y9y). Only key domains such as the DDT and WHIM motifs of Ioc3, and SLIDE domain of Iswi1 are shown with key residues depicted as space-fill models.