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Figure 5

Figure 5. From: Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers.

NOEsY spectra demonstrate “peptide dissection” of intact DM motif (A) into major and minor fragments: Zn-binding module (DMΔ; B) and carboxy-terminal peptide (peptide DM-p; C). Shown are respective spectra in H2O at 25°C and 600 MHz; mixing times were in each case 175 msec. Cross peaks from amide and aromatic protons (vertical scale, θ2) and aliphatic protons (horizontal scale, θ1) are shown. Boxed regions highlight unresolved envelope in intact DM domain (A) assigned to nascent carboxy-terminal helix (C); this feature is absent in spectrum of DMΔ (B). Peptides were made 1.5 mm in 50 mm deuterated Tris-HCl (pH 6.5) in 90% H2O and 10% D2O. Spectra A and B also were obtained in presence of 4 mm ZnCl2.

Lingyang Zhu, et al. Genes Dev. 2000 July 15;14(14):1750-1764.
Figure 6

Figure 6. From: Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers.

(A) Stereo ribbon representation of the solution structure of the DM domain (DSX residues 41–81). An ensemble of side chains of the coordinating cysteate (yellow) and histidine (blue) side chains and two bound Zn2+ atoms (red) are shown. The two intertwined zinc-binding sites are designated sites I and II (boxes at right). (B) Ensemble of main chain structures (stereo pair) aligned according to the main chain atoms of residues 41–81. The positions of Zn2+ atoms are shown by red spheres (50% of van der Waal radius). (C) Structural relationships among side chains of conserved (green), otherwise ordered (white), or invariant ligand-binding (green) residues. Ligand-binding residues are C44, C47, H59, C63 and H50, C68, C70, C73 (see Fig. B). Conserved residues N49 and R79 are shown in green whereas side chains of R46 and F65 are shown in gold to highlight structural environments (see text). Otherwise well-order residues include side chains of N43, A45, R48, L52, K53, T55, L56, Y67, T69, L75, T76, A77, and D78; criterion for inclusion is an ensemble side chain RMSD of <1.0 Å.

Lingyang Zhu, et al. Genes Dev. 2000 July 15;14(14):1750-1764.
Figure 4

Figure 4. From: Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers.

(A,B) Analysis of protein-directed DNA bending by permutation gel electrophoresis. (A) Probes (each 140 bp) contain fbe site at positions shown; probes 3 and 4 contain SRY target site 5′-TTTGTT-3′. GMSA in B demonstrates very weak dependence of DM–fbe complexes c1 and c2 on fbe position in probe (lanes 14). Lanes 6 and 7 illustrate SRY control using probes 3 and 4 (). The weak band under major SRY–DNA complex arises from proteolytic fragment; more slowly migrating species represent higher order complexes. Free probe 3 is shown in lane 5. (C) Tail mutant R91Q impairs specific DNA binding but not cooperativity. GMSA used 33P-labeled 33-bp fbe probe (1.5 nm). (Lanes 27) Native DSX domain (respective protein concentrations 4, 8, 12, 18, 24, and 48 nm); (lanes 816) R91Q variant domain (respective protein concentrations 12, 24, 48, 96, 120, 156, 240, 480, and 560 nm); the free probe is shown in lane 1. At 48 nm concentrations the native domain is 52% shifted, whereas shift of variant domain is negligible (<1%). Complexes c1 and c2 represent 1:1 and 2:1 binding of domain to dsxA; c3 represents a higher order complex observed at high protein concentrations (>100 nm).

Lingyang Zhu, et al. Genes Dev. 2000 July 15;14(14):1750-1764.
Figure 2

Figure 2. From: Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers.

(A) Domain organization of DSXM, DSXF, and MAB-3. Amino-terminal black rectangles indicate DM domains; the carboxy-terminal DSX dimerization domain and sex-specific extension (cross-hatched or gray; not present in MAB-3) are also indicated. (B,top) Three transcription factors (AEF1, DSX, and bZIP1) bind fbe enhancer (,) at overlapping target sites (aef1 is shown in black, dsxA in red, and bzip1 in blue); (bottom) consensus sequences. Boxed sites are based on DNase protection; DSX footprint spans 21 bp, whereas DmC/EBP footprint spans 19 bp. DNase footprints overestimate DNA target sites due to steric occlusion between enzyme and protein–DNA complex. (C) Model of sex- and tissue-specific regulation of yolk–protein expression by fbe (,). DSX binds as dimer; only monomer is shown (ribbon model). In model dsxA and bzip1 are occupied simultaneously to activate promoter in female. Binding of either DSXM or DSXF displaces AEF1 from its target site aef1. In female, fat body expression of DSXF is higher than that of AEF1, and therefore in the presence of bZIP1, yolk proteins are expressed. Expression is repressed in ovary with higher levels of AEF1, which displace DSXF. Male-specific repression of yolk proteins occurs as binding of DSXM, which is 122 residues longer than DSXF (), occludes bzip1 or inactivates bound bZIP1, and therefore is repressed in male (C,bottom; ). It is not known which Drosophila bZIP family member is active in fat body. (D) Overlapping DSX DM and bZIP phosphate contacts are inferred based on methylphosphonate interference studies (red circles); sites of bZIP contact are as predicted by GCN4 cocrystal structure (blue circles; ). Filled or half-filled red circles indicate sites of strong or weak DM interference, respectively.

Lingyang Zhu, et al. Genes Dev. 2000 July 15;14(14):1750-1764.

Figure 7. From: Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers.

The DM tail is a nascent α-helix that folds on DNA binding. (A,B) CD studies of free (A) and bound (B) DSX domain. (A,a) Spectra of intact domain at 4°C (solid line) and 37°C (dotted line); (A,b) corresponding spectra of DMΔ. (A,c) Difference spectra: (solid line a), between DM spectra at 4° and 37°C; (dashed dotted line Δ), between DM and DMΔ at 4°C; (dotted line b), baseline attenuation of stable helices in DMΔ between 4° and 37°C. That difference spectra a and Δ are similar suggests that the labile segment in DM corresponds to the missing element in DMΔ, i.e., a nascent carboxy-terminal helix. (A,d). Difference spectrum [Δ] (dashed dotted line), obtained from Δ normalized relative to 19 deleted residues, exhibits a helical signature. For comparison, spectra of isolated carboxy-terminal peptide DM-p 19 residues; underlined in Fig. B) are shown in d at 4° and 37°C (solid and dotted lines). Magnitude of [θ] at 222 nm (P[θ]222) is similar to that of difference spectra Δ and a in A,c. (B) Comparison of spectra of protein–DNA complexes (solid line), free domain (dashed dotted line), and free DNA (dotted or dashed line) at 4°C (B,a) and 37°C (B,b). (B,c) CD melting curves of free DM domain (dashed dotted line) and specific 2:1 complex (solid line) as monitored at 222 nm from 4° to 95°C. Whereas free domain exhibits steep loss in mean residue ellipticity with increasing temperature, the complex exhibits an attenuation rate (d[θ]222/dT) typical of pretransition regime (arrows). (B,d) CD difference spectra at 37°C: (solid line Δ1) between complex and the free DNA; (solid line Δ2), between the spectrum of a complex and the sum of the spectra of its isolated parts. Induced helical feature in far UV is labeled. Arrow in near-UV indicates difference feature arising from change in DNA structure on protein binding. Peptide and DNA concentrations were 10 and 5 μm, respectively. For reference, the spectrum of free DM domain is also shown (dashed dotted line).

Lingyang Zhu, et al. Genes Dev. 2000 July 15;14(14):1750-1764.
Figure 3

Figure 3. From: Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers.

DNA-binding studies of DSX domain demonstrate minor groove binding mode. (A,top) Consensus sequence of DSX-binding sites, an imperfect palindrome containing an odd number of base pairs. (Second from top) DSX target site in fbe11–13. (Third from top) Analog containing multiple AT → IC substitutions (boldface type). (Bottom two sequences) Variants containing additional AT or TA base pairs in center. Labels b, c, d, e, f (circled) refer to lanes of gels in B. (B) 33P-detected GMSA at 4°C of DSX domain binding to sites defined in A. Bands c1 and c2 designate 1:1 and 2:1 protein–DNA complexes. Protein binds equally well to native and IC-containing sites. Insertion of central AT or TA base pairs reduces protein binding by about 100-fold. Protein concentration was 8 nm and DNA concentration was 0.5 nm. (C) Cooperative low-affinity binding of the DSX DM domain to a palindromic (sym) DNA site (5′-ACTACAATTGTTGCA-3′; central base pair in boldface type) using fluorescein (at 5′ of top strand) as probe. DNA concentration was 500 nm in lanes ai, with respective protein concentrations 0, 250, 500, 600, 700, 800, 900, and 1000 nm. Lane j contains an equimolar solution in which concentrations of DNA and protein were each 5 μm; predominance of 2:1 complex and free DNA indicates cooperativity is retained. (D,left) Structures of AT and variant nebularine NT base pair. (D,right) Structures of GC and inosine IC base pair. Positions of major and minor grooves are indicated. (E,right gel) Major groove base modifications do not perturb binding of DSX domain to 15 base-pair DNA probe (5′-CACTACAATGTTGCA-3′ and complement): (lanes eg) single nebularine substitutions at positions 5, 7, and 8 of upper strand and (lanes hj) single 5-methylcytosine or uridine substituitions at positions 6, 9, and 11 of upper strand. (E,left gel) DMRT1 DM domain (25 nm) recognizes DSX target site with weaker apparent affinity; cooperative 2:1 binding is maintained. (F) 33P-detected GMSA of fbe analog in which the 8 AT bp in the DSX-binding site (boxed in Fig. A, sequence c,d) are substituted by 2,6-diaminopurine (structure at right). No binding is observed to λ operator site OL1 (lanes c,d) or to the diaminopurine fbe analog (lanes en) at successive protein concentrations of 0, 1, 2, 4, 8, 16, 32, 64, and 128 nm. DNA concentration was ∼1 nm. Protein concentrations were 8 nm for lanes b and d. Sequence of λ probe is 5′-TACCACTGGCGGTCATA-3′ and complement.

Lingyang Zhu, et al. Genes Dev. 2000 July 15;14(14):1750-1764.
Figure 1

Figure 1. From: Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers.

(A) Genetic pathways of sex determination in D. melanogaster (a) and C. elegans (b) are initiated by X:autosome (A) ratio but otherwise different (). (A,a) In the fly a high X:A ratio (2:2) activates sxl, which encodes a splicing factor. In turn, Sxl allows female-specific expression of the splicing factor tra, which together with tra-2, permits expression of female isoform DSXF. The pathway then ramifies. Intersex (ix), hermaphrodite (her), and fruitless (not shown) are other downstream elements. When the X:A ratio is low, male-specific isoform DSXM occurs by default; isoforms differ in carboxy-terminal domain (see Fig. A). (A,b) In nematode a high X:A ratio (2:2) is associated with absent her-1 expression, in turn enabling unrepressed expression of tra-2 and tra-3 (unrelated to Drosophila genes of similar nomenclature). Hermaphrodite-specific expression of tra-2 and tra-3 represses fem-1, fem-2, and fem-3. In their absence, tra-1 is expressed in hermaphrodite and turns off male-determinant mab-3 (encoding a DM transcription factor). TRA-1 has MAB-3 independent targets as pathway ramifies. When the X:A ratio is low (1:2), HER-1 (involved in cell–cell signaling) represses tra-2 and tra-3. Unrepressed expression of female genes represses tra-1 and therefore default expression of mab-3 directs male development and turns off hermaphroditic program. Nematode genes (b) that enumerate X:A ratio and control X dosage compensation (including xol-1 and sdc-2; ) are omitted for clarity. (B) Alignment of metazoan DM sequence motifs. Cysteines and histidines that coordinate Zn2+ are aligned as two intertwined binding sites (boxes): site I (red) and site II (green). Conserved residues in zinc module are otherwise shown in blue; conserved residues in tail are color coded (R, red; Q, green; A, magenta; D/E, violet; L/V/T/M, blue). Stable α-helical elements are highlighted by magenta ribbons above DSX sequence. Nascent carboxy-terminal α-helix is indicated by gray extension; it is not meant to convey a continuous α-helix (see text). (DSX) DM domain in D. melanogaster (accession no. M25292). (MAB-3a and MAB-3b) The first and the second DM domains in C. elegans protein (accession no. Z99278). Other C. elegans DM sequences: F10C1.5, cosmid F10C1 (accession no. U49831); C34D1.1, cosmid C34D1 (accession no. Z78060); K08B12, cosmid K08B12 (accession no. U97001); F13G11, cosmid F13G11 (accession no. Z83317); T22H9.4: cosmid T22H9 (accession no. AF101315); C27C12 (accession no. Z69883); and Y43F8C (accession no. AL032637.1). Vertebrate DM sequences: DMRT1 and DMRT2, human homologs on the short arm of chromosome 9 (DMRT1: accession no. AF130728 and DMRT2: accession no. AF130729). Other homologs: mDmrt1 (murine, accession no. AF202778), pDmrt1 (porcine, accession no. AF216651), and cDmrt1 (chicken, accession no. AF123456). TERRA: DM domain of zebrafish terra (accession no. AF080622). Sequences shown include genes (such as TERRA; ) not known to be involved in sex determination. Arrows without parentheses indicate sites of point mutations in dsx or mab-3 associated with intersex development; substitutions in parenthesis indicate variants characterized only by biochemical assays (). Three additional putative DM genes of unknown function have recently been found in the genome of D. melanogaster (accession nos. AAF56919, AAF55843, and AAF48261; sequences not shown).

Lingyang Zhu, et al. Genes Dev. 2000 July 15;14(14):1750-1764.

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