Nucleic Acids Research

INTRODUCTION

In eukaryotes, DNA is packaged into a complex multilevel assembly by a range of DNA-binding proteins which includes the histones and the non-histone chromosomal proteins such as the high mobility group (HMG) proteins. There are three distinct classes of DNA-binding motifs found in the HMG proteins. The HMG-1 box family of proteins is at least 75 amino acids in length and is found in >100 eukaryotic proteins in the sequence databases (1). The HMG-14/-17 family of proteins bind to nucleosomes between the histones and the DNA and have so far only been isolated from birds, fish and mammals (2). This family binds DNA by virtue of a conserved stretch with arginines and lysines which probably form a rigid basic surface. The third family of HMG proteins comprise the HMG-I(Y) group which bind to the minor groove of DNA via a conserved nine amino acid peptide called an AT-hook (3). Many of these proteins have been shown to have an effect on the architecture of chromatin at levels beyond the action of the basic histones and also play a role in transcription regulation by acting as accessory factors which influence the association of transcription factors with chromatin (4,5). In particular, a number of experiments have demonstrated that AT-hook-containing proteins like HMG-I(Y) play important roles in chromatin structure and act as transcription factor cofactors (6-8).

Unlike several of the other well-characterized DNA-binding motifs, the AT-hook is a small motif which has a typical sequence pattern centered around a glycine-arginine-proline (GRP) tripeptide (3). The importance of this short conserved sequence is stressed by the observation that it is necessary and sufficient to bind DNA (3,9). The archetypal AT-hook protein, HMG-I(Y), has three copies of this motif and the motif has been found in several other proteins with from a single to 15 copies in the same peptide. Given the experimental evidence that this motif is central to the DNA binding and chromatin interaction of the HMG-I(Y) proteins, we have systematically investigated its distribution in the protein sequence databases. We present here a description of all detectable AT-hook motifs in the non-redundant protein database (NR) at the National Center for Biotechnology Information (NCBI) and suggest possible functional implications of their unexpected widespread presence in several chromosomal proteins. We also describe novel proteins containing AT-hook motifs and discuss the possibility that these may serve as accessory DNA-binding domains for several transcription factors, presumably to anchor them to particular DNA structures (e.g. minor grooves and four-way junctions). These AT-hook motifs seem to be auxiliary elements necessary for cooperation with other DNA-binding activities in the same or different proteins. We also propose that the AT-hook and other DNA-binding motifs, like the HMG-1 box and the novel TAM motif which we characterize here, may play a role in translocating regulatory chromosomal proteins to scaffold-associated regions (SARs) (10) or matrix-associated regions (MARs) (11).

MATERIALS AND METHODS

The non-redundant database (NCBI) was searched using the utilities in the SEALS package (12) which allow multiple file manipulations, pattern searches, local alignment searches like gapped BLAST (13) and iterative database searches. For the searches performed in this manuscript, the most effective tool in searching the database for short but highly significant matches was the flexible pattern search method GREF in the SEALS package. The proteins detected in the pattern search process were further evaluated for true positives using weighted matrices constructed using the average score method with the help of the MoST and BLOCK programs (14) and motif sampling using the information theoretic Gibbs sampling method. Two programs implementing the Gibbs sampling procedure were used, namely MACAW (15) and PROBE (16). Sequences were masked for compositional bias using the SEG program (17). Sequence logos were constructed using the WebLogo program (http://www.bio.cam.ac.uk/seqlogo ), an implementation of the MAKELOGO program (18). Three-dimensional structural visualizations and manipulations were carried out using the SwissPDB-viewer program (19).

RESULTS AND DISCUSSION

The AT-hook is a short stretch of sequence similarity which makes it difficult to detect in conventional searches and discern scores which are statistically significant. Two main approaches were used to detect the AT-hook motifs in the databases. The AT-hook proteins known to bind to DNA, namely HMG-I(Y) and its homologs from animals and plants, and plant AT-rich DNA-binding proteins were extracted from the database and repeats of the AT-hook motifs were identified and aligned using the Gibbs sampling methods in MACAW and PROBE (Materials and Methods). Based on these alignments, regular expression patterns were developed and used to search the NR database (i.e. the database of non-redundant protein sequences at the NCBI) using the GREF program of the SEALS package. With the same starting alignment we constructed a position-specific weight matrix using the MoST and BLOCK programs and searched the NR database for hits above different cut-offs (matrix available at ftp://ncbi.nlm.nih.gov/pub/landsman/hmg-i/weight_matrix ). The proteins recovered from these analyses were subjected to Gibbs sampling. Motifs identified by this procedure had a probability of chance occurrence <10^-5. In addition, the gapped BLAST program was used to query the database with AT-hooks after masking out adjacent, compositionally biased regions. Random expectation values of 0.01-0.001 were obtained for AT-hook hits, supporting the statistical significance of these motifs.

Figure 1A shows sequence logos generated from multiple alignments of copies of this motif. The central typical GRP motif (positions 11-13 of Fig. 1A) and the surrounding pattern of characteristic basic residues show a total information content >1.3 bits at each position. The striking feature of these search results was the central GRP motif with the flanking conserved residues which occurred in only a set of proteins with the notable characteristic of nuclear localization or a DNA-binding function. Where the proteins were uncharacterized, they frequently had recognizable sequence motifs found only in other nuclear proteins. A list of these proteins with the number of AT-hooks they have and the various additional domains they possess is shown in Table 1 (a complete alignment of the of the AT-hook motifs from all these proteins can be accessed at ftp://ncbi.nlm.nih.gov/pub/landsman/hmg-i/classI, classII, and classIII ). The specific occurrence of the AT-hook domain in known and predicted nuclear or DNA-binding proteins, together with evidence that it is capable of binding DNA on its own, make it a good predictor of nuclear function.

Determination of the solution structure of AT-hooks 2 and 3 of the HMG-I(Y)-DNA complex by NMR has helped in understanding the structural basis for the DNA-protein interactions of this motif (9). The AT-hook forms a C-shaped structure with the `rear' of the concave surface inserted into the minor groove of the DNA (yellow part of ribbon in Fig. 2). The proline residues in the AT-hook are probably responsible for maintenance of the rigid structure of this domain in the presence of DNA while the characteristic RGR sequence motif of the AT-hook (positions 10-12 of Fig. 1A-D) adopts an extended structure and participates in DNA-protein interactions (Fig. 2). The arginine residues are seen to penetrate deep into the B-DNA helix and interact with the bases of DNA. Furthermore, the affinity of the AT-hooks has been shown to be influenced by DNA contacts outside these core residues. Based on the extended sequence conservation and binding affinities, the AT-hooks were divided into two types (9). Type I is characterized by an additional module, C-terminal of the core GRP (positions 11-13 in Fig. 1A-D), which includes basic residues forming a supporting polar network and additional contacts with DNA (Fig. 1B; note positions 16-19). The type I motifs also have a greater probability of having a glycine at the second position C-terminal of the GRP (i.e. position 15 in Fig. 1B). In contrast, the type II AT-hooks comprise only the basic similarity centered around the core GRP motif (positions 7-10 and 14-15 in Fig. 1D). These have a high probability of possessing a lysine in place of a glycine two residues downstream of the GRP. In addition, we recognized a third class of AT-hook motifs which possesses some features of both type I and type II AT-hooks. Single linkage clustering (using the GROUPER program in the SEALS package; 12) using serial BLAST hit score cut-offs (BLOSUM80 matrix) recognized not only the original two classes, but also a third class (type III). In the type III AT-hooks, a highly conserved basic position (lysine) and the presence of some polar residues downstream of the core motif are similar to type I AT-hooks. However, the type III AT-hooks, like the type II AT-hooks, have a predominant lysine at the second position downstream of the GRP (position 15 in Fig. 1C). In addition, a conserved lysine in the type III AT-hooks is four residues C-terminal of the core GRP motif (position 17 in Fig. 1C), whereas in type I AT-hooks there is a conserved lysine six residues downstream of the conserved GRP motif (position 19 in Fig. 1B). The type I AT-hooks with the extended basic motif have been shown to be high affinity DNA-binding modules, whereas the type II AT-hooks, which lack this feature, have been shown to have a low affinity for DNA (9). This correlates well with the additional DNA contacts seen in the type I AT-hooks due to this C-terminal conserved region. In addition, the type II AT-hooks bind at much lower affinities but with higher affinities than synthetic PRGRP peptides (9), suggesting that the additional basic residues beyond the PRGRP sequence is important for DNA binding. The type III AT-hooks, with an additional conserved C-terminal region and the characteristic downstream lysine, are predicted to have greater affinity for DNA than the type II AT-hooks, but possibly less than type I AT-hooks, which have a more extensive polar surface.

Table 1. Proteins in the databases which contain AT-hook motifs

Protein name	Species name +protein gi number	n	Additional domains	Proposed function
AT1 (21)	Os_1362172	15	Histone H1 globular domain
Chromosomal protein D1	Dm_117239	10		Binds to AT-rich D1 satellite DNA
pabf	Nt_555655	8	2 histone H1 globular domains	Tissue-specific transcription regulator
ENBP1 (44)	Vs_1360637	6	JOR domain (modified [beta]-helix domain) + cysteine-rich domain	DNA binding protein binding to the AT-rich elements in the promoter of the modulation-specific ENOD gene
ORF T05A7.4	Ce_1055146	4
AAC-rich mRNA	Dd_112945	4
HMG-Y-related A	Gm_1708261	4	Histone H1 globular domain
HMG-Y-related B	Gm_1708262	4	Histone H1 globular domain
PF1	Os_453692	4	Histone H1 globular domain	Binds to the AT-rich elements present in the rice phytochrome A3 promoter
HMG-I(Y)	At_1808590	4	Histone H1 globular domain
HMG-I(Y)	Ca_2129885	4	Histone H1 globular domain
CarD (32)	Mx_1022328	4	C-Terminal globular domain found in other bacteria	Transcription regulator of light and starvation responsive genes
Pathogenesis-related homeodomain	Pc_1346791	4	PHD finger + homeodomain	Specifically binds to fungal elicitor-responsive in response to fungal pathogens
HMG-I(C)	Hs_1708263	3		Chromatin architectural factor and cofactor for different DNA-binding proteins
HMG-I(Y)	Hs_123377	3		Chromatin architectural factor and cofactor for different DNA-binding proteins
HMG-I(Y)	Hs_123383	3		Chromatin architectural factor and cofactor for different DNA-binding proteins
ALL-1 (36)	Hs_1490271	3	SET domain + methylase-type Zn finger	Trithorax ortholog and positive regulator of chromatin decondensation and expression of the Hox cluster genes
F1N21.16	At_2760331	3	Mudra family transposase with C2HC finger and HTH
ORF T18B16.30	At_2828281	3	AP ATPase domain
Sum1p (45)	Sc_1711597	2		Involved in the regulation of transcription silencing by Sir genes; dominant mutations in this gene result in bypass os Sir dependence of silencing
Swi2p/Snf2p (46)	Sc_134589	2	Swi/Snf-like superfamily 2 helicase +bromodomain	ATPase component of the SWI chromatin remodeling complex
Lin-15B (42)	Ce_1078881	2	Modified Hermit type transposon	Negative regulator of the ras/tyrosine kinase pathway in vuval development
C11G6.1	Ce_1229035	2	Prodos/ZK1320.7-type histone fold protein	Predicted novel chromosomal protein
TAFII250 (47)	Dm_1705691	2	2 bromodomains	Histone acetyltransferase, basal transcription factor of RNA polymerase II (some fly strains have just 1 AT-hook)
ASH1 (48)	Dm_1335892	2	PHD fingers, SET domain and BAM domain	Member of the Trithorax group of chromatin proteins, proposed action in positively regulating chromatin decondensation.
zhx-1 (49)	Mm_2137401	2	2 C2H2 zinc fingers + 5 homeodomains	Transcription regulator
Nuclear protein SA-1 (50)	Mm_2204230	2	SA1/2-rec11 domain	Nuclear protein expressed in hematopoetic organs
ORF KIAA0437	Hs_2879925	2
TTF-I interacting peptide 5 (51)	Hs_2183083	2	Bromodomain + TAM DNA-binding domain	Forms a complex with the RNA polymerase I termination factor I; predicted architectural factor
DNA-binding protein PD1	At_2213536	2	PD1-type globular domain seen in archaean proteins MTH1230, AF0104	DNA-binding protein
SC1 (52)	Hs_809489	1	FHA domain	Proposed regulator of cell cycle progression
X box-binding regulatory factor (53)	Hs_1350587	1	RFX domain	Regulator of B cell development
LIM/homeodomain protein LH-2 (54)	Hs_423900	1	LIM domain and homeodomain	Neural and lymphocyte development regulator in human
Retinoblastoma-binding protein 1, RBP1	Hs_1710030	1	Chromodomain + JOR domain (modified [beta]-helix domain + Bright DNA-binding domain	Retinoblastoma protein binding phosphorylated protein; predicted chromatin architectural factor
ESX (epithelial-restricted with serine box) (55	Hs_1754538	1	ETS domain	Transcription factor regulates expression of TGF[beta] receptor II
DFS autoantigen	Hs_1945445	1		Autoantigen in atopic dermatitis
Putative transcription factor CR53	Hs_2407245	1	SCAN domain + zinc fingers?	Predicted transcriptional regulator
ORF HH0601	Hs_2662163	1	POZ domain + C2H2 zinc fingers
ORF R29381	Hs_2995577	1	Unique methyltransferase domain also seen in C.elegans
Peregrin (56)	Hs_1705500	1	Bromodomain + PHD fingers + C-rich region	Predicted transcription regulator/ chromatin architectural factor
Methyl-CpG-binding protein 2 (57)	Hs_420279	1	TAM DNA-binding domain	Avian ortholog has been shown to be a SAR-binding protein
AF-10 (58)	Hs_1703190	1	2 PHD fingers	Predicted chromosomal protein, forms fusion proteins with AT-hooks of the HRX (ALL-1) protein in leukemias
AF-17 (59)	Hs_2134713	1	2 PHD fingers	Predicted chromosomal protein, forms fusion proteins with AT-hooks of the HRX (ALL-1) protein in leukemias
Barx1 (60)	Mm_1550783	1	Homeodomain	Expressed in developing neural and cranial precursors
CTCF1 (61)	Mm_1256410	1	11 C2H2 zinc fingers	Neural development regulator
M33 polycomb-like protein (62)	Mm_2266988	1	Chromodomain	Involved in chromatin condensation
ORF epitope	Mm_2384711	1
Castor protein (63)	Dm_419965	1	Peculiar modified C2H2 fingers	Transcription regulator in neural development
Alternatively spliced tramtrack (64)	Dm_578319	1	POZ domain + 2 zinc fingers	Repressor in neural development
Doom-modifier of mdg4 (25)	Dm_942610	1	POZ domain	Insulator element interacting protein/enhancer of variegation with imprinting; an isoform, Doom is a pro-apoptotic regulator
ISWI (65)	Dm_1362615	1	SWI/SNF-like superfamily 2 helicase + SANT domain	ATPase component of the chromatin remodeling complex NURF
Apterous protein (66)	Dm_ 231559	1	LIM domains + homeodomain	Neural development and wing dorsoventral polarity regulator
Zinc finger 30C	Dm_2653645	1	12 C2H2 zinc fingers
Zinc finger AT-hook protein	Dm_2982495	1	11 C2H2 zinc fingers
ORF YN06	Ce_465974	1
ORF C27B7.4	Ce_1000467	1	SWI/SNF-type superfamily 2 helicase	Ortholog of the human ATR-X gene
ORF F40F9.7	Ce_1262959	1	Histone fold domain	Predicted chromosomal protein
ORF C44F1.2	Ce_780182	1	KDWK domain	Predicted DNA-binding protein
ORF C25G4.4	Ce_1256454	1	KDWK domain	Predicted DNA-binding protein
ORF T11A5.1	Ce_1301722	1
ORF YG23	Sc_1723670	1	2 bromodomains and 1 BAM domain
ORF F18E2.3	Ce_1418507	1	SA1/2-rec11 domain
ORF F16B12.6	Ce_1673468	1	Globular domain restricted to C.elegans
ORF C43E11.3	Ce_1703572	1	SET domain
ORF C34F6.9	Ce_264801	1	Ubiquitin C-terminal hydrolase
ORF F15E6.1	Ce_2702442	1	PHD finger + modified SET domain
Orc2p (67)	Sc_464317	1		Component of the replication origin recognition complex
Swi5p (68)	Sc_135077	1	3 C2H2 zinc fingers (1 modified finger)	Regulator of cell cycle-specific transcription in yeast
Mif2p (69)	Sc_462601	1	AraC type [beta]-helix motif	Centromere DNA-binding protein required for integrity of the mitotic spindle
ORF YDR36	Sc_2131446	1
ARS-binding protein 2	Sp_1724085	1		ARS-binding protein in S.pombe
Orc1+p (70)	Sp_1163108	1	BAM domain + AAA ATPase domain	Origin recognition complex ATP hydrolizing subunit
AMT1 (71)	Cg_1168451	1	Fungal-type metal-binding cluster	DNA-binding protein which regulates heavy metal response
Manx (72)	Mo_308967	1	Tudor domain + C-terminal C-rich domain	Master regulator of tail development in ascidians
ICP4 (73)	GHV_643430	1		Marek disease herpesvirus trans acting transcription activator
RF1	SHV_279827	1		Transcription activator of delayed early genes
Tuple1/HirA	Fr_2352031	1	WD40 repeats	Regulator of histone gene expression
MADS box homolog Umc1 (74)	Um_2708784	1	MADS box	Transcription regulator of several pheromone-responsive genes in Ustilago
C	To_2894102	1
Tc1(75)	An_1911486	1	Transposase + HTH	Tc1 family of transposons with HTH and integrase domains

The proteins which contain an AT-hook motif are listed in the column labeled Protein name. The column labeled Species name + protein gi number lists a two letter abbreviation of the species name (see below) associated with the database entry, followed by the GenBank gi number for the protein sequence (use these numbers to extract the sequences from GenBank at http://www.ncbi.nlm.nih.gov/Entrez/protein.html ). The column labeled n lists the number of AT-hook motifs identified in each protein sequence. The column labeled Accessory domains lists other protein domains or motifs found in each protein; if there is no entry there are none identified at this time. Some of these are as yet undescribed motifs found in other proteins. The column labeled Comments lists comments about a protein.
An, Aspergillus nidulans; At, Arabidopsis thaliana; Ca, Carassius auratus; Ce, Caenorhabditis elegans; Cg, Candida glabrata; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Fr, Fugu rubripes; GHV, Gallid herpesvirus 1; Gm, Glycine max; Hs, Homo sapiens; Mm, Mus musculus; Mo, Molgula oculata; Mx, Myxococcus xanthus; Nt, Nicotiana tabacum; Os, Oryza sativa; Pc, Petrostelium crispum; Sc, Saccharomyces cerevisiae; SHV, Saimiriine herpesvirus 1; Sp, Schizosaccharomyces pombe; To, Torulaspora delbrueckii; Um, Ustilago maydis; Vs, Vicia sativa.

Experiments with HMG-I(Y) have shown that this protein is capable of reversing inherent bends present in DNA, presumably to allow effective binding of transcription factors (7). Similarly, synthetic proteins with several AT-hook motifs have been shown to have dramatic effects on chromatin organization when added to sperm chromatin (5) or on position effect variegation in Drosophila when expressed in vivo (8). Single or double AT-hooks do not appear to alter the structure of B-DNA dramatically because, unlike other minor groove binding proteins, they do not insert a hydrophobic residue into the DNA helix. However, the above experiments suggest that the cooperative action of several of these AT-hook motifs could introduce significant changes in DNA conformation over a long range. This is probably dependent on the spacing between successively interacting AT-hooks and their binding sites may be crucial for conformational changes of the DNA.

Figure 1. Sequence logos of AT-hook motifs. The classification of AT-hook motifs is similar to that used in Huth et al. (9). For all logos, the information content (in bits) is proportional to the size of the letter at each position in the logo. This is related to the frequency of occurrence of each amino acid at each position of each multiple sequence alignment (18). (A) The sequence logo was prepared from the sequences of all AT-hook sequences detected in this study. The conserved core defining an AT-hook is clearly indicated by the larger collections of letters at positions 7-15. (B) Sequence logo of the type I sequences (29 sequences). Note the characteristic, frequently observed G residue in the second position (position 15) downstream of the conserved GRP (positions 11-13) and the predominance of polar and charged residues in the C-terminal high information region (positions 16-19) which form an additional DNA contact in the 3D structure (9). (C) Sequence logo of the newly defined type III AT-hook sequences (34 sequences). Note the high information positions (positions 14-15) downstream of the GRP conserved core and a characteristic K in the fourth position downstream (position 17). (D) Sequence logo of the type II AT-hook sequences (122 sequences). The high information region corresponds to the minimal universal conserved region for all AT-hooks [compare positions 7-15 in (A) and (D)]. Note the presence of a prominent K residue two positions downstream of the GRP conserved core (position 15). A complete list of proteins with each of these types of motif and the alignments used to generate the logos are available at ftp://ncbi.nlm.nih.gov/pub/landsman/hmg-i/

In this study, we have identified AT-hook motifs in many proteins, several of which have not been reported before. The AT-hook motifs in these proteins may be the sole motifs in the protein, similar to canonical HMG-I(Y), or they may be part of a polypeptide with other recognizable motifs. Several proteins have more than one AT-hook motif (Table 1). The best studied of these multiple AT-hook proteins are the vertebrate HMG-I(Y) family members, which have three AT-hooks (20). Related proteins with three or four AT-hook motifs are detectable in Caenorhabditis elegans and Dictyostelium discoideum, suggesting that HMG-I(Y)-like architectural factors were functional from early in evolution. Plants are characterized by a set of AT-hook proteins which have an N-terminal histone H1 globular domain motif followed by multiple (4-15) AT-hook motifs (21). The combination of the H1 globular domain with the AT-hook motifs suggests that they could function as a special class of linker histone involved in chromatin organization. This is consistent with detection of the AT-hook in two distinct C.elegans proteins in combination with the histone fold motif, implying that in animals too, these AT-hook-containing histone-like proteins could function in specific atypical nucleosome-like structures involved in transcriptional or architectural regulation.

Multiple or single AT-hook motifs are also found in several multi-domain proteins which associate with chromatin (Table 1). Striking examples include the human HRX (ALL-1) protein, TAF_II250, Saccharomyces cerevisiae ASH1, S.cerevisiae SWI2, Drosophila melanogaster ISWI, ENBP1, M33 (vertebrate polycomb), doom/Mod(Mdg4) and tramtrack. Several of these proteins are involved in the organization of chromatin into specific states. HRX (ALL-1) and ASH1 are members of the trithorax group of regulatory genes which are involved in chromatin decondensation (22). SWI2 and ISWI are ATP-dependent chromatin remodeling proteins (23). TAF_II250 is a basal transcription factor which is involved in transcription regulation and also contains histone acetyltransferase activity (24). In contrast, the POZ domain proteins Mod(Mdg4) (25) and tramtrack (26) act as regulators of transcriptional insulation and mediators of transcription repression, respectively. The presence of AT-hooks in these proteins suggests that these chromatin architecture regulators probably use DNA sequence information in the form of AT-rich tracts, to target specific regions on the chromosomes. The AT-richness of SARs and their presumptive involvement in transcription activation suggest that the AT-hooks may indeed play a role in tethering these proteins to specific chromosomal regions.

Figure 2. Three-dimensional model of an AT-hook DNA complex. The image was constructed from the coordinates of the solution structure of a HMG-I(Y) peptide complexed with a DNA dodecamer derived from the interferon-[beta] promoter (PDB, 2EZD) (9). The peptide backbone is represented as a pink ribbon with the AT-hook central region highlighted in yellow. The side chains of the conserved RGR amino acids, which are inserted into the minor groove of the DNA in an extended conformation, are included, as well as the flanking prolines. This is the second AT-hook of the HMG-I(Y) protein and belongs to type II (Fig. 1). Note the typical long C-terminal extension which forms a surface for additional DNA contacts on either side of the helix (9).

The above observation offers a generalization: several chromatin proteins may use different DNA-binding motifs to target AT-rich tracts and SARs. Along with the AT-hook motif, the HMG-1 box and the Bright domain (L.Aravind, unpublished data) are also prevalent in chromatin proteins. In this context, we note that the ARBP protein from chicken, which contains AT-hook motifs, has been demonstrated to be a SAR-binding protein (27). Both ARBP and its ortholog, the mammalian methylated DNA binding protein MeCP2, contain both an AT-hook motif and a methylated CpG DNA binding domain (28). Further analysis of the latter domain allowed us to characterize it in several predicted chromatin proteins from plants and C.elegans (Fig. 3). As this domain is found in organisms not known to contain significant amounts of methylated DNA, we suggest that it is a more generalized DNA binding motif which, like the AT-hook, recognizes specific features in or associated with SAR DNA. Although this domain has been called a methyl-CpG-binding domain (MBD) (28), we would prefer to give it a more generalized name. We propose the name TAM for this domain after the three proteins in which it is found, namely TTF-IIP5, ARBP and MeCP1. It is quite probable that in organisms with methylation, it was additionally recruited to recognize methylation which occurs in close proximity to the SARs (29).

There are several examples where a single AT-hook motif was detected in proteins which have additional larger DNA-binding domains (Table 1). In addition to the AT-hook: D.melanogaster apterous, mammalian LH2 and Barx1 also contain homeodomains; human RFX5 protein also contains the DNA-binding RFX box; human ESE-1 protein also contains an ETS domain; S.cerevisiae SWI5, Drosophila castor and mouse CTCF1 also contain zinc fingers; the fungal metal binding transcription factors of the AMT1 family of proteins contain a zinc cluster (30). This suggests that in several cases, the AT-hook may serve as an additional peptide contact in the minor groove even as the `principal or larger' DNA-binding protein contacts the major groove. It is tempting to speculate that in these cases the AT-hook may serve as a `built-in' cofactor like the HMG-I(Y) protein and subtly alter the affinity and specificity of the `principal or larger' DNA-binding domain by recognizing sites in the vicinity of the principle DNA binding site. This may again be a general feature of eukaryotic DNA-binding proteins: small basic patches such as the N-terminus of homeodomains and the recently described GRIP box of nuclear hormone receptors which contact the minor groove in addition to the main DNA-binding activity in the major groove (31).

Outside the eukaryotic world, the AT-hook motif is present infrequently and is undetectable in the archaea, while it is found in some bacterial species. The only studied bacterial protein with an identifiable AT-hook motif is CarD from Myxococcus xanthus, which has been demonstrated to bind AT-rich DNA (32). It is interesting to note that several mariner family transposons, both bacterial and eukaryotic, have AT-hook motifs in their transposase proteins (Table 1), suggesting that it may act with the major groove interacting helix-turn-helix motif (33) of these proteins to additionally contact the DNA minor groove. The predominant presence of the AT-hook motif in eukaryotes suggests that this motif was actively selected for only after eukaryotic chromatin had evolved, resulting in its major proliferation.

The AT-hook domains of human HRX (ALL-1) and related sequences in the AF-10 and AF-17 proteins have been implicated in several chromosomal translocations resulting in different lymphoid leukemias (34-36). Similarly, translocations of the AT-hook motifs of HMG-I(Y) result in lipomas and atypical lipomatous tumors (37). The participation of AT-hook motifs in many different translocations is surprisingly correlated with their evolutionary mobility. It is interesting to note that pairs of orthologous or closely related paralogous genes can be quite clearly distinguished by the conspicuous presence or absence of the AT-hook motif. For example, HRX (ALL-1) has AT-hook motifs while the D.melanogaster ortholog, trithorax, lacks them. Similarly, the plant pathogenesis-related homeodomain protein (PRHP) from Petroselium crispum contains both a homeodomain and a PHD finger as well as four AT-hook motifs while the Arabidopsis thaliana ortholog of PRHP appears to lack these AT-hook motifs (38). Similar cases have been noted when the TAF_II250, polycomb and SWI2 proteins are compared between different taxa. This suggests that the AT-hook motif is an evolutionarily mobile module which may have been dispersed in events similar to the translocations occurring in the lymphoid leukemias and lipomas. Each of the AT-hook motifs in the HMG-I(Y) gene family is encoded by a separate exon (39), which suggests that such an organization possibly favors the translocation of complete and contiguous units of this motif.

A survey of the uncharacterized proteins with AT-hook motifs (Table 1) reveals several potentially interesting candidates for experimental study. One particularly interesting example is a human protein (gi 2995577) which combines an AT-hook motif with a methyltransferase domain, which suggests that it could be a candidate for a novel chromatin modifying methylase. Likewise, other proteins which combine the AT-hook motif with chromatin-specific domains, like PHD fingers (40) and SET domains (41), could be potential chromatin architecture and/or regulatory proteins. The presence of AT-hooks in the C.elegans tyrosine kinase pathway regulator lin-15 protein (42) suggests that it is a DNA-binding protein probably acting during transcription. Furthermore, it has a divergent transposase domain typical of the hermit family of transposons (L.Aravind, unpublished data). This suggests that the protein probably serves as a novel transcription regulator or chromatin architecture factor which regulates development during transcription by direct interaction with DNA.

Figure 3. A sequence alignment of the TAM domain. The TAM domain is found in several proteins other than MeCPs. TTF-IIP5 is a protein which interacts with the transcription termination factor TTFI. Caenorhabditis elegans Topoisomerase II apparently has the TAM domain along with the SET domain found in other chromatin proteins. Colors are assigned according to the 90% consensus rule with the following convention: yellow background, hydrophobic residues (L, W, Y, I, M, F, V, A and C); purple, charged residues (R, K, H, E and D); turquoise background, small residues (A, G, S, T, D, N, V, P and H); blue, hydroxy residues (S and T); green background, tiny residues (A, G and S); brown, polar residues (D, E, N, Q, R, K, H, S and T). The species abbreviations are as indicated in the legend to Table 1 and the GenBank gi numbers are attached to the extreme right of the protein name. The PHD secondary structure prediction (43) is shown above (E, [beta]-strands; H, [alpha]-helices).

We describe the presence of a small DNA-binding motif in many proteins and propose that it shows a striking over-representation in eukaryotic proteins with a nuclear function. We propose that these motifs serve to anchor several chromatin modifying proteins to the minor groove of DNA thus allowing their translocation to AT-rich sites on the chromosome (e.g. SARs). We also propose that some of these proteins with histone globular domains could serve as non-canonical histones in an altered chromatin organization affecting the regulation of transcription. In general, it seems that the AT-hook motif is an auxiliary protein motif cooperating with other DNA-binding activities or facilitating changes in the structure of the DNA. It is interesting to note that these small motifs seem to be found exclusively in nuclear proteins known to interact with DNA.

ACKNOWLEDGEMENT

We would like to thank Dr Michael Bustin for critically reviewing this manuscript.

REFERENCES

1. Baxevanis,A.D. and Landsman,D. (1995) Nucleic Acids Res., 23, 1604-1613. MEDLINE Abstract

2. Bustin,M., Trieschmann,L. and Postnikov,Y.V. (1995) Semin. Cell Biol., 6, 247-255. MEDLINE Abstract

3. Reeves,R. and Nissen,M.S. (1990) J. Biol. Chem., 265, 8573-8582. MEDLINE Abstract

4. Thanos,D. and Maniatis,T. (1992) Cell, 71, 777-789. MEDLINE Abstract

5. Strick,R. and Laemmli,U.K. (1995) Cell, 83, 1137-1148. MEDLINE Abstract

6. Onate,S.A., Prendergast,P., Wagner,J.P., Nissen,M., Reeves,R., Pettijohn,D.E. and Edwards,D.P. (1994) Mol. Cell. Biol., 14, 3376-3391. MEDLINE Abstract

7. Falvo,J.V., Thanos,D. and Maniatis,T. (1995) Cell, 83, 1101-1111. MEDLINE Abstract

8. Girard,F., Bello,B., Laemmli,U.K. and Gehring,W.J. (1998) EMBO J., 17, 2079-2085. MEDLINE Abstract

9. Huth,J.R., Bewley,C.A., Nissen,M.S., Evans,J.N., Reeves,R.,Gronenborn,A.M. and Clore,G.M. (1997) Nature Struct. Biol., 4, 657-665.

10. Gasser,S.M. and Laemmli,U.K. (1986) Cell, 46, 521-530. MEDLINE Abstract

11. Cockerill,P.N. and Garrard,W.T. (1986) Cell, 44, 273-282. MEDLINE Abstract

12. Walker,D.R. and Koonin,E.V. (1997) ISMB, 5, 333-339. MEDLINE Abstract

13. Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Nucleic Acids Res., 25, 3389-3402. MEDLINE Abstract

14. Tatusov,R.L., Altschul,S.F. and Koonin,E.V. (1994) Proc. Natl Acad. Sci. USA, 91, 12091-12095. MEDLINE Abstract

15. Schuler,G.D., Altschul,S.F. and Lipman,D.J. (1991) Proteins, 9, 180-190. MEDLINE Abstract

16. Neuwald,A.F., Liu,J.S., Lipman,D.J. and Lawrence,C.E. (1997)Nucleic Acids Res., 25, 1665-1677. MEDLINE Abstract

17. Wootton,J.C. and Federhen,S. (1996) Methods Enzymol., 266, 554-571. MEDLINE Abstract

18. Schneider,T.D. and Stephens,R.M. (1990) Nucleic Acids Res., 18, 6097-6100. MEDLINE Abstract

19. Guex,N. and Peitsch,M.C. (1997) Electrophoresis, 18, 2714-2723. MEDLINE Abstract

20. Reeves,R. and Nissen,M.S. (1993) J. Biol. Chem., 268, 21137-21146. MEDLINE Abstract

21. Meijer,A.H., van Dijk,E.L. and Hoge,J.H. (1996) Plant Mol. Biol., 31, 607-618. MEDLINE Abstract

22. Pirrotta,V. (1998) Cell, 93, 333-336. MEDLINE Abstract

23. Cairns,B.R. (1998) Trends Biochem. Sci., 23, 20-25. MEDLINE Abstract

24. Imhof,A., Yang,X.J., Ogryzko,V.V., Nakatani,Y., Wolffe,A.P. and Ge,H. (1997) Curr. Biol., 7, 689-692. MEDLINE Abstract

25. Gerasimova,T.I., Gdula,D.A., Gerasimov,D.V., Simonova,O. and Corces,V.G. (1995) Cell, 82, 587-597. MEDLINE Abstract

26. Tang,A.H., Neufeld,T.P., Kwan,E. and Rubin,G.M. (1997) Cell, 90, 459-467. MEDLINE Abstract

27. Weitzel,J.M., Buhrmester,H. and Stratling,W.H. (1997) Mol. Cell. Biol., 17, 5656-5666. MEDLINE Abstract

28. Cross,S.H., Meehan,R.R., Nan,X. and Bird,A. (1997) Nature Genet., 16, 256-259. MEDLINE Abstract

29. Jenuwein,T., Forrester,W.C., Fernandez-Herrero,L.A., Laible,G., Dull,M. and Grosschedl,R. (1997) Nature, 385, 269-272. MEDLINE Abstract

30. Turner,R.B., Smith,D.L., Zawrotny,M.E., Summers,M.F., Posewitz,M.C. and Winge,R.D. (1998) Nature Struct. Biol., 5, 551-555.

31. Zhao,Q., Khorasanizadeh,S., Miyoshi,Y., Lazar,M.A. and Rastinejad,F. (1998) Mol. Cell, 1, 849-861.

32. Nicolas,F.J., Cayuela,M.L., Martinez-Argudo,I.M., Ruiz-Vazquez,R.M. and Murillo,F.J. (1996) Proc. Natl Acad. Sci. USA, 93, 6881-6885. MEDLINE Abstract

33. van Pouderoyen,G., Ketting,R.F., Perrakis,A., Plasterk,R.H. and Sixma,T.K. (1997) EMBO J., 16, 6044-6054. MEDLINE Abstract

34. Slany,R.K., Lavau,C. and Cleary,M.L. (1998) Mol. Cell. Biol., 18, 122-129. MEDLINE Abstract

35. Zeleznik-Le,N.J., Harden,A.M. and Rowley,J.D. (1994) Proc. Natl Acad. Sci. USA, 91, 10610-10614. MEDLINE Abstract

36. Tkachuk,D.C., Kohler,S. and Cleary,M.L. (1992) Cell, 71, 691-700. MEDLINE Abstract

37. Ashar,H.R., Fejzo,M.S., Tkachenko,A., Zhou,X., Fletcher,J.A., Weremowicz,S., Morton,C.C. and Chada,K. (1995) Cell, 82, 57-65. MEDLINE Abstract

38. Korfhage,U., Trezzini,G.F., Meier,I., Hahlbrock,K. and Somssich,I.E. (1994) Plant Cell, 6, 695-708. MEDLINE Abstract

39. Friedmann,M., Holth,L.T., Zoghbi,H.Y. and Reeves,R. (1993)Nucleic Acids Res., 21, 4259-4267. MEDLINE Abstract

40. Aasland,R., Gibson,T.J. and Stewart,A.F. (1995) Trends Biochem. Sci., 20, 56-59. MEDLINE Abstract

41. Jenuwein,T., Laible,G., Dorn,R. and Reuter,G. (1998) Cell. Mol. Life Sci., 54, 80-93. MEDLINE Abstract

42. Clark,S.G., Lu,X. and Horvitz,H.R. (1994) Genetics, 137, 987-997. MEDLINE Abstract

43. Rost,B. and Sander,C. (1993) J. Mol. Biol., 232, 584-599. MEDLINE Abstract

44. Christiansen,H., Hansen,A.C., Vijn,I., Pallisgaard,N., Larsen,K., Yang,W.C., Bisseling,T., Marcker,K.A. and Jensen,E.O. (1996)Plant Mol. Biol., 32, 809-821. MEDLINE Abstract

45. Chi,M.H. and Shore,D. (1996) Mol. Cell. Biol., 16, 4281-4294. MEDLINE Abstract

46. Laurent,B.C., Treich,I. and Carlson,M. (1993) Genes Dev., 7, 583-591. MEDLINE Abstract

47. Weinzierl,R.O., Dynlacht,B.D. and Tjian,R. (1993) Nature, 362 (6420), 511-517. MEDLINE Abstract

48. Tripoulas,N., LaJeunesse,D., Gildea,J. and Shearn,A. (1996) Genetics, 143, 913-928. MEDLINE Abstract

49. Barthelemy,I., Carramolino,L., Gutierrez,J., Barbero,J.L., Marquez,G. and Zaballos,A. (1996) Biochem. Biophys. Res. Commun., 224, 870-876. MEDLINE Abstract

50. Carramolino,L., Lee,B.C., Zaballos,A., Peled,A., Barthelemy,I., Shav-Tal,Y., Prieto,I., Carmi,P., Gothelf,Y., Gonzalez de Buitrago,G., Aracil,M., Marquez,G., Barbero,J.L. and Zipori,D. (1997) Gene, 195, 151-159. MEDLINE Abstract

51. Langst,G., Blank,T.A., Becker,P.B. and Grummt,I. (1997) EMBO J., 16, 760-768. MEDLINE Abstract

52. Ku,D.H., Chang,C.D., Koniecki,J., Cannizzaro,L.A., Boghosian-Sell,L., Alder,H. and Baserga,R. (1991) Cell Growth Differentiat., 2, 179-186.

53. Durand,B., Sperisen,P., Emery,P., Barras,E., Zufferey,M., Mach,B. and Reith,W. (1997) EMBO J., 16, 1045-1055. MEDLINE Abstract

54. Xu,Y., Baldassare,M., Fisher,P., Rathbun,G., Oltz,E.M., Yancopoulos,G.D., Jessell,T.M. and Alt,F.W. (1993) Proc. Natl Acad. Sci. USA, 90, 227-231. MEDLINE Abstract

55. Oettgen,P., Alani,R.M., Barcinski,M.A., Brown,L., Akbarali,Y., Boltax,J., Kunsch,C., Munger,K. and Libermann,T.A. (1997) Mol. Cell. Biol., 17, 4419-4433. MEDLINE Abstract

56. Thompson,K.A., Wang,B., Argraves,W.S., Giancotti,F.G., Schranck,D.P. and Ruoslahti,E. (1994) Biochem. Biophys. Res. Commun., 198, 1143-1152. MEDLINE Abstract

57. Meehan,R.R., Lewis,J.D. and Bird,A.P. (1992) Nucleic Acids Res., 20, 5085-5092. MEDLINE Abstract

58. Chaplin,T., Ayton,P., Bernard,O.A., Saha,V., Della Valle,V., Hillion,J., Gregorini,A., Lillington,D., Berger,R. and Young,B.D. (1995) Blood, 85, 1435-1441. MEDLINE Abstract

59. Prasad,R., Leshkowitz,D., Gu,Y., Alder,H., Nakamura,T., Saito,H., Huebner,K., Berger,R., Croce,C.M. and Canaani,E. (1994)Proc. Natl Acad. Sci. USA, 91, 8107-8111. MEDLINE Abstract

60. Tissier-Seta,J.P., Mucchielli,M.L., Mark,M., Mattei,M.G., Goridis,C. and Brunet,J.F. (1995) Mech. Dev., 51, 3-15. MEDLINE Abstract

61. Filippova,G.N., Fagerlie,S., Klenova,E.M., Myers,C., Dehner,Y., Goodwin,G., Neiman,P.E., Collins,S.J. and Lobanenkov,V.V. (1996)Mol. Cell. Biol., 16, 2802-2813. MEDLINE Abstract

62. Pearce,J.J., Singh,P.B. and Gaunt,S.J. (1992) Development, 114, 921-929. MEDLINE Abstract

63. Mellerick,D.M., Kassis,J.A., Zhang,S.D. and Odenwald,W.F. (1992) Neuron, 9, 789-803. MEDLINE Abstract

64. Harrison,S.D. and Travers,A.A. (1990) EMBO J., 9, 207-216. MEDLINE Abstract

65. Tsukiyama,T., Daniel,C., Tamkun,J. and Wu,C. (1995) Cell, 83, 1021-1026. MEDLINE Abstract

66. Cohen,B., McGuffin,M.E., Pfeifle,C., Segal,D. and Cohen,S.M. (1992) Genes Dev., 6, 715-729. MEDLINE Abstract

67. Micklem,G., Rowley,A., Harwood,J., Nasmyth,K. and Diffley,J.F. (1993) Nature, 366 (6450), 87-89. MEDLINE Abstract

68. Nasmyth,K., Seddon,A. and Ammerer,G. (1987) Cell, 49, 549-558. MEDLINE Abstract

69. Meluh,P.B. and Koshland,D. (1995) Mol. Biol. Cell, 6, 793-807. MEDLINE Abstract

70. Gavin,K.A., Hidaka,M. and Stillman,B. (1995) Science, 270, 1667-1671. MEDLINE Abstract

71. Zhu,Z. and Thiele,D.J. (1996) Cell, 87, 459-470. MEDLINE Abstract

72. Swalla,B.J. and Jeffery,W.R. (1996) Science, 274, 1205-1208. MEDLINE Abstract

73. Anderson,A.S., Francesconi,A. and Morgan,R.W. (1992) Virology, 189, 657-667. MEDLINE Abstract

74. Kruger,J., Aichinger,C., Kahmann,R. and Bolker,M. (1997) Genetics, 147, 1643-1652. MEDLINE Abstract

75. Glayzer,D.C., Roberts,I.N., Archer,D.B. and Oliver,R.P. (1995)Mol. Gen. Genet., 249, 432-438. MEDLINE Abstract

---

*To whom correspondence should be addressed. Tel: +1 301 435 5981; Fax: +1 301 480 9241; Email: landsman@ncbi.nlm.nih.gov

---

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 19 Sep 1998
Copyright©Oxford University Press, 1998.
CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				X. F. Yang, M. S. Goldberg, M. He, H. Xu, J. S. Blevins, and M. V. Norgard Differential Expression of a Putative CarD-Like Transcriptional Regulator, LtpA, in Borrelia burgdorferi Infect. Immun., October 1, 2008; 76(10): 4439 - 4444. [Abstract] [Full Text] [PDF]

				R. L. Cohen, C. W. Espelin, P. De Wulf, P. K. Sorger, S. C. Harrison, and K. T. Simons Structural and Functional Dissection of Mif2p, a Conserved DNA-binding Kinetochore Protein Mol. Biol. Cell, October 1, 2008; 19(10): 4480 - 4491. [Abstract] [Full Text] [PDF]

				B. A. Rasala, C. Ramos, A. Harel, and D. J. Forbes Capture of AT-rich Chromatin by ELYS Recruits POM121 and NDC1 to Initiate Nuclear Pore Assembly Mol. Biol. Cell, September 1, 2008; 19(9): 3982 - 3996. [Abstract] [Full Text] [PDF]

				C.-C. Yuan, X. Zhao, L. Florens, S. K. Swanson, M. P. Washburn, and N. Hernandez CHD8 Associates with Human Staf and Contributes to Efficient U6 RNA Polymerase III Transcription Mol. Cell. Biol., December 15, 2007; 27(24): 8729 - 8738. [Abstract] [Full Text] [PDF]

				D. Palmeri, S. Spadavecchia, K. D. Carroll, and D. M. Lukac Promoter- and Cell-Specific Transcriptional Transactivation by the Kaposi's Sarcoma-Associated Herpesvirus ORF57/Mta Protein J. Virol., December 15, 2007; 81(24): 13299 - 13314. [Abstract] [Full Text] [PDF]

				J. Panee, Z. R. Stoytcheva, W. Liu, and M. J. Berry Selenoprotein H Is a Redox-sensing High Mobility Group Family DNA-binding Protein That Up-regulates Genes Involved in Glutathione Synthesis and Phase II Detoxification J. Biol. Chem., August 17, 2007; 282(33): 23759 - 23765. [Abstract] [Full Text] [PDF]

				T. Kubota, S. Maezawa, K. Koiwai, T. Hayano, and O. Koiwai Identification of functional domains in TdIF1 and its inhibitory mechanism for TdT activity Genes Cells, August 1, 2007; 12(8): 941 - 959. [Abstract] [Full Text] [PDF]

				D. Vom Endt, M. Soares e Silva, J. W. Kijne, G. Pasquali, and J. Memelink Identification of a Bipartite Jasmonate-Responsive Promoter Element in the Catharanthus roseus ORCA3 Transcription Factor Gene That Interacts Specifically with AT-Hook DNA-Binding Proteins Plant Physiology, July 1, 2007; 144(3): 1680 - 1689. [Abstract] [Full Text] [PDF]

				R. Griffiths and A. Whitehouse Herpesvirus Saimiri Episomal Persistence Is Maintained via Interaction between Open Reading Frame 73 and the Cellular Chromosome-Associated Protein MeCP2 J. Virol., April 15, 2007; 81(8): 4021 - 4032. [Abstract] [Full Text] [PDF]

				A. Matsushita, T. Furumoto, S. Ishida, and Y. Takahashi AGF1, an AT-Hook Protein, Is Necessary for the Negative Feedback of AtGA3ox1 Encoding GA 3-Oxidase Plant Physiology, March 1, 2007; 143(3): 1152 - 1162. [Abstract] [Full Text] [PDF]

				R. J. Katzenberger, M. S. Marengo, and D. A. Wassarman ATM and ATR Pathways Signal Alternative Splicing of Drosophila TAF1 Pre-mRNA in Response to DNA Damage Mol. Cell. Biol., December 15, 2006; 26(24): 9256 - 9267. [Abstract] [Full Text] [PDF]

				M. M. Babu, L. M. Iyer, S. Balaji, and L. Aravind The natural history of the WRKY-GCM1 zinc fingers and the relationship between transcription factors and transposons Nucleic Acids Res., December 2, 2006; 34(22): 6505 - 6520. [Abstract] [Full Text] [PDF]

				M. Drobni, T. Li, C. Kruger, V. Loimaranta, M. Kilian, L. Hammarstrom, H. Jornvall, T. Bergman, and N. Stromberg Host-Derived Pentapeptide Affecting Adhesion, Proliferation, and Local pH in Biofilm Communities Composed of Streptococcus and Actinomyces Species Infect. Immun., November 1, 2006; 74(11): 6293 - 6299. [Abstract] [Full Text] [PDF]

</