And Histone H1-Like Domains in a Bacterial Transcriptional Factor

Functional equivalence of HMGA- and histone H1-like domains in a bacterial transcriptional factor

Francisco García-Herasa, S. Padmanabhanb,1, Francisco J. Murilloa, and Montserrat Elías-Arnanza,1

aDepartamento de Gene´tica y Microbiología, A´ rea de Gene´tica (Unidad Asociada al IQFR-CSIC), Facultad de Biología, Universidad de Murcia, Murcia 30100, Spain; and bInstituto de Química Física ‘‘Rocasolano’’, Consejo Superior de Investigaciones Cientíﬁcas, Serrano 119, 28006 Madrid, Spain

Edited by A. Dale Kaiser, Stanford University School of Medicine, Stanford, CA, and approved May 22, 2009 (received for review February 27, 2009) Histone H1 and high-mobility group A (HMGA) proteins compete AT-rich sequences occurring in at least 2 appropriately spaced dynamically to modulate chromatin structure and regulate DNA tracts of 4 to 8 bp in length (8–11). transactions in eukaryotes. In prokaryotes, HMGA-like domains are H1 and, more so, HMGA-like domains are rare in prokaryotes. known only in Myxococcus xanthus CarD and its Stigmatella auran- The only known examples of bacterial proteins with HMGA-like tiaca ortholog. These have an N-terminal module absent in HMGA domains occur in myxobacteria: Myxococcus xanthus CarD and its that interacts with CarG (a zinc-associated factor that does not bind Stigmatella aurantiaca ortholog, CarDSa [(12, 13); supporting infor- DNA) to form a stable complex essential in regulating multicellular mation (SI)Fig.S1A]. Myxobacteria are unique among prokaryotes development, light-induced carotenogenesis, and other cellular pro- in carrying out a programmed multicellular developmental process cesses. An analogous pair, CarDAd and CarGAd, exists in another on starvation (14–16). Temporal and spatial control of gene myxobacterium, Anaeromyxobacter dehalogenans. Intriguingly, the expression during M. xanthus development depends on eukaryotic- CarDAd C terminus lacks the hallmark HMGA DNA-binding AT-hooks like signal transduction proteins and transcription factors of which and instead resembles the C-terminal region (CTR) of histone H1. We CarD is one (17–19). Additionally, CarD is involved in regulating find that CarDAd alone could not replace CarD in M. xanthus.By other processes, such as light-induced carotenogenesis (19, 20). Its 136-residue C-terminal region, with a highly acidic stretch preced- contrast, when introduced with CarGAd, CarDAd functionally replaced CarD in regulating not just 1 but 3 distinct processes in M. xanthus, ing 4 AT-hooks, resembles human HMGA1a in its physical, structural, and DNA-binding properties (21). This HMGA-like segment despite the lower DNA-binding affinity of CarDAd versus CarD in vitro. is linked to a 180-residue N-terminal domain, CarDNter, essential MICROBIOLOGY The ability of the cognate CarDAd–CarGAd pair to interact, but not the noncognate CarD –CarG, rationalizes these data. Thus, in chimeras for function and absent in eukaryotic HMGA (13). Every known Ad CarD-regulated activity requires physical association of CarDNter that conserve CarD–CarG interactions, the H1-like CTR of CarDAd could replace the CarD HMGA AT-hooks with no loss of function in vivo. to CarG; the latter binds zinc via a H/C-rich motif of the type found in archaemetzincins (a class of metalloproteases) but lacks protease More tellingly, even chimeras with the CarD AT-hook region substi- or DNA-binding activity (22). The myxobacterium Anaeromyx- tuted by human histone H1 CTR or full-length H1 functioned in M. obacter dehalogenans also expresses sequence analogs to both CarD xanthus. Our domain-swap analyses showing functional equivalence (CarD ) and CarG (CarG ). Strikingly, whereas CarD has an of HMGA AT-hooks and H1 CTR in prokaryotic transcriptional regu- Ad Ad Ad N-terminal domain very similar to that in CarD, its C-terminal end lation provide molecular insights into possible modes of action un- lacks the hallmark AT-hooks. Instead, its K/A/P content is typical derlying their biological roles. of H1 CTRs. Whether an H1 domain can functionally replace the HMGA one ͉ ͉ Anaeromyxobacter dehalogenans CarD Myxococcus xanthus in CarD is studied here. Remarkably, we find that an H1-CTR like domain, whether from CarDAd or, even more notably, from human n eukaryotes, the DNA architectural factors histone H1 and histone H1.2, can supply the DNA-binding activity essential for Ihigh-mobility group (HMG) proteins remodel chromatin struc- CarD function in 3 distinct processes in M. xanthus just as well as ture and modulate the action of other regulatory factors in various the naturally occurring AT-hooks. Our study provides insights into DNA transactions (1). H1 (or linker histone) interacts with linker the modular design of this rather singular bacterial transcriptional DNA at or near the nucleosomal dyad axis, stabilizes higher-order factor. More importantly, it reveals that AT-hooks and H1 CTR can chromatin structure, and lowers nucleosomal access to transcrip- function equivalently in this system, and provides molecular insights tional factors so as to up- or downregulate their action; HMG that may be relevant in understanding their modes of action. proteins compete with H1 for overlapping binding sites in chromatin to decrease nucleosomal compactness and provide access to Results regulatory factors (1). The typical H1 structure consists of a central CarDAd Alone Cannot Replace CarD in M. xanthus. CarDAd and CarD globular winged-helix domain flanked by 2 basic, randomly struc- share 58% sequence identity (70% similarity) in their N-terminal tured segments: a short N-terminal stretch and an Ϸ100-residue region and 37% (45%) in their C-terminal part (Fig. S1). Further- C-terminal region (CTR) (1). The primary sequence of CTR, more, CarDAd, like CarD, has a C-terminal region with a highly crucial for DNA binding and chromatin condensation, diverges acidic stretch preceding a very basic one, with the most abundant among different species and isoforms but its amino acid composi- amino acids in the latter region being K, A, and P in both proteins tion is fairly constant in most of the higher eukaryotes: Ϸ40% K, (Fig. S1D). However, the basic region of CarDAd stands out from 20–35% A, Ϸ15% P, few S, T, G, or V, and virtually no aromatic that of CarD in lacking the hallmark AT-hooks, besides having residues (1–5). Proteins of the HMG superfamily are the second- most abundant components of chromatin after histones. Of these, Յ Author contributions: F.G.-H., S.P., and M.E.-A. designed research; F.G.-H. and S.P. per- high-mobility group A (HMGA) are small ( 107 residues), intrin- formed research; F.G.-H., S.P., F.J.M., and M.E.-A. analyzed data; and S.P. and M.E.-A. wrote sically disordered proteins characterized by multiple repeats of the the paper. conserved RGRP or ‘‘AT-hook’’ DNA-binding motif embedded in The authors declare no conflict of interest. a less conserved cluster of basic and proline residues, and flanked This article is a PNAS Direct Submission. by an acidic region that modulates protein stability and DNA 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. binding (6, 7). The AT-hooks, 3 of which occur in mammalian This article contains supporting information online at www.pnas.org/cgi/content/full/ HMGA, bind in a defined conformation to the minor groove of 0902233106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902233106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 26, 2021 Fig. 2. DNA binding of CarD and CarDAd in vitro. (A) Gel mobility shift assay with Fig. 1. Functional replacement of M. xanthus carD by carDAd in the absence or 32P-labeled DNA probes of 37 bp (Top gel) and 169 bp (Bottom gel) containing the presence of carGAd.(A) Schematic representation of CarD, human HMGA1a, CarD-binding site at the PQRS promoter region. Lane 1: no protein; lanes 2 to 4: CarDAd, and human H1 (H1.2 subtype). The AT-hooks, the acidic region, and the 0.19, 0.75 and 1.5 ␮M CarD; lanes 5 to 10: 0.19, 0.38, 0.75, 1.5, 3, and 6 ␮M CarD . ϩ Ϫ Ad H1 CTR are shown as boxes with ‘‘ ,’’ ‘‘ ,’’ and ‘‘X,’’ respectively. Black boxes in The 37-bp and 169-bp probes (coding strand) are shown schematically on Top, the CarD and CarDAd, and the line in human HMGA represent N-terminal domains. In numbers indicating positions relative to the transcription start site of the carQRS human H1, the short N-terminal domain is shown by the unfilled box and the operon. The CarD-binding sequence between positions Ϫ77 and Ϫ65 is also globular domain by the gray box. Residue numbers demarcate the domains. (B) shown with the 2 AT-rich tracts in uppercase. (B) DNase I footprint of the 169-bp Color phenotypes (Top) of cell spots after overnight growth on CTT plates in the probe with the coding strand labeled. Protein concentrations used were: 0, 3.75, dark (‘‘D’’) or in the light (‘‘L’’) vertically aligned with the corresponding expres- 7.5, 11.25, and 15 ␮M of CarD (lanes 1 to 5) or CarD (lanes 6 to 10). The Ј Ad sion levels of carQ ::lacZ in strains bearing the indicated carD and carG alleles CarD-binding site is marked on the Left. (Bottom). Cell cultures were grown to early exponential phase in the dark, divided into 2, grown for a further 14 h, one in the dark (filled bars) and the other in the light (empty bars), and specific ␤-galactosidase activities were estimated. ␮ 1C). In agreement with this, the strain with carDAd failed to express (C) Developmental phenotype (Top) after 5-day incubation of 15- L cell droplets ⍀ (1.25 ϫ 108 cells mlϪ1) spotted on TPM agar vertically aligned with the corre- the 4435 Tn5lac transposon insertion, which is one of the devel- sponding expression levels of the ⍀4435 developmental marker in strains bearing opmental markers known to depend on CarD (19). Finally, a the indicated carD and carG alleles (Bottom). Specific ␤-galactosidase activities reporter lacZ transposon probe at the ddvA locus (ddvA::Tn5lac) were determined for samples collected after 24-h development on TPM agar. (D) was expressed in the strain with carDAd at levels far lower than in ϭ Expression of ddvA::Tn5-lac in exponentially growing cultures (OD550 0.7) of wild-type and similar to those in the ⌬carD ⌬carG strain (Fig. 1D). strains containing the indicated carD and carG alleles. In B, C, and D, ␤-galacto- Thus, CarDAd on its own cannot compensate for the lack of CarD sidase activities (in nanomoles of o-nitrophenyl ␤-D-galactoside hydrolyzed/ in M. xanthus. This is unlikely to be the result of low levels of carD Ϯ Ad min/mg protein), shown as a percentage of the wild-type values (128 20 in B, expression in M. xanthus, because it maintains both the high GC 240 Ϯ 30 in C, and 2900 Ϯ 300 in D), are from 2 or more independent ϩ measurements. content and the codon third-position G CbiasofcarD. Other possible causes for why CarDAd differs from CarD in vivo might be in its binding to DNA and/or to CarG. These were therefore K/A/P contents closer to those observed in H1 CTRs (Fig. 1A; Fig. examined next. S1D). This and the reported competition between H1 and HMGA Human HMGA binds to the minor groove of appropriately for binding to DNA in eukaryotes (1) led us to examine whether spaced AT-rich tracts, as noted above (8–11). We have demon- CarDAd could replace CarD in M. xanthus. For this, a plasmid strated that the CarD HMGA-like domain resembles human bearing carDAd was electroporated into a ⌬carD M. xanthus strain, HMGA1a not only physically and structurally, but also in binding where it integrates into the chromosome at the carD locus by to specific human HMGA DNA sites, such as the IFN response homologous recombination resulting in merodiploids. Haploid element (21). In M. xanthus, CarD binding to DNA has been well strains with the carDAd allele replacing carD were then isolated as characterized only at the light-inducible PQRS promoter, where its described (see SI Materials and Methods) to test how CarDAd affects HMGA-like domain binds to 2 appropriately spaced AT-rich tracts the following distinct processes dependent on CarD (and CarG): (i) located in the segment between Ϫ65 and Ϫ77 upstream of the activation of PQRS, the promoter that on illumination drives tran- transcription start site (12, 21–23). Human HMGA1a also exhibits scription of the regulatory carQRS operon, thereby leading to preferential binding to this site in vitro (21) and yields a DNase I expression of the structural genes for carotenoid synthesis; (ii) footprint that resembles that of CarD (Fig. S2), showing that starvation-induced fruiting-body formation; and (iii) expression of HMGA1a and CarD bind at PQRS similarly. Two DNA probes, of the ddvA locus, unrelated to the light- or starvation-induced re- lengths 37 bp and 169 bp, containing the AT-rich tracts at PQRS, sponses, whose function remains to be defined (20, 22, 23). bind CarD in gel mobility shift assays (lanes 2–4, Top and Bottom In contrast to wild-type M. xanthus, which is yellow in the dark gels, respectively, in Fig. 2A). CarDAd also bound to these probes, and turns red in the light due to carotenogenesis (the Carϩ but at over a 10-fold higher concentration (lanes 5–10, Top and phenotype), a strain with carD replaced by carDAd remained yellow Bottom gelsinFig.2A). In DNase I footprinting assays with the in the light (CarϪ), like those with deletions of carD, carG, or both 169-bp probe, the segment between Ϫ65 to Ϫ77 containing the (Fig. 1B; ref. 22). Consistent with this, expression levels of the CarD-binding site was protected by CarD (lanes 2–5, Fig. 2B) but carQЈ::lacZ reporter probe, where lacZ is under PQRS control, were not by CarDAd at comparable concentrations (lanes 7–10, Fig. 2B), close to those observed for strains with carD, carG, or both deleted consistent with the lower affinity of CarDAd inferred from gel-shift (Fig. 1B). Furthermore, the strain with carDAd in place of carD did data. Higher CarDAd levels yielded protection extending through- not produce normal, mature spore-filled fruiting bodies on starva- out the DNA probe, indicative of nonspecific binding (data not Ϫ ϩ tion (Fru phenotype), in contrast to the wild-type Fru cells (Fig. shown). Thus, CarD binds to its site at PQRS with a higher affinity

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902233106 García-Heras et al. Downloaded by guest on September 26, 2021 in vitro than CarDAd, reminiscent of eukaryotic HMGA1a binding to a variety of DNA substrates such as AT-rich B-form DNA, 4-way junctions, or kinked, distorted structures with significantly greater affinities than H1 (24, 25). This could rationalize, at least in part, why CarDAd does not effectively replace CarD in vivo. However, as described next, a more critical determinant appears to be the lack of its cognate CarGAd partner.

CarDAd Can Replace CarD in M. xanthus if CarGAd Is also Present. All known CarD-dependent processes require CarG, a zinc-bound protein with no DNA-binding activity, which interacts physically with CarDNter to form a stable regulatory complex (22). In M. xanthus, carD and carG are contiguous genes in an operon, as are carDAd and carGAd in A. dehalogenans, where overlap of the carDAd stop codon and the carGAd start codon suggests translational coupling. CarGAd is 46% identical (62% similar) to CarG and conserves the structurally critical Zn-binding motif (Fig. S1). To test if it is the presence of CarG rather than the cognate CarGAd that makes CarDAd unable to replace CarD in M. xanthus, a plasmid bearing the contiguous carDAd and carGAd genes was constructed and electroporated into the ⌬carD ⌬carG strain. Chromosomal integration of the plasmid by homologous recombination resulted in merodiploids that were Carϩ, suggesting that the simultaneous presence of carDAd and carGAd can complement the lack of carD and carG. Consistent with this, the haploid strain with carDAd and carGAd (generated as described in SI Materials and Methods) was also Carϩ, in marked contrast to the CarϪ phenotype observed on

replacing only carD by carDAd. Accordingly, equivalent levels of MICROBIOLOGY reporter carQЈ::lacZ expression were observed in the light for the wild-type strain and for that with the carDAd–carGAd pair (Fig. 1B). Furthermore, the latter strain was Fruϩ, with normal expression of the ⍀4435 developmental marker (Fig. 1C), and of the ddvA::Tn5lac probe (Fig. 1D). Thus, despite the apparently lower Fig. 3. Physical interactions between cognate and noncognate CarD–CarG DNA-binding affinity of CarDAd relative to CarD in vitro, CarDAd pairs. (A) Two-hybrid analysis in E. coli, showing reporter lacZ expression for can functionally replace CarD in regulating not just 1 but several the different pairs as indicated. ‘‘CϪ’’ is the negative control, where cells distinct processes in M. xanthus, so long as the cognate CarGAd contain the vectors without fusions. Values are from 2 or more independent partner is also present. measurements. (B) Analytical gel ﬁltration using a Superdex200 column. Protein concentrations were Ն10 ␮M. Elution proﬁles as tracked by absor- bance at 220 nm in arbitrary units (a.u.) are shown. Bottom panel: pure CarD CarDAd Forms a Stable Complex with CarGAd but Not with CarG. Why (black solid line), CarG (black dashed line), CarDAd (gray solid line displaced the CarDAd–CarGAd pair, but not the mixed CarDAd–CarG pair, vertically for easy viewing), and CarGAd (gray dashed line). Top panel: a functions in M. xanthus may be the result of differences in inter- mixture of CarD and CarG (black solid line), of CarDAd and CarGAd (gray solid actions between cognate and noncognate CarD–CarG pairs. This line), and of CarDAd and CarG (black dashed line). On Top is shown Mr (in kDa) was checked next. A bacterial 2-hybrid system in which interaction aligned with the corresponding elution volume. between 2 test proteins leads to functional complementation between the T25 and T18 fragments of the catalytic domain of Ϸ Bordetella pertussis adenylate cyclase (26) demonstrated that CarD with excess CarDAd, and both proteins coeluted with Mr 200 or CarDNter interacts with CarG (22). The same analysis showed kDa (Fig. 3B, Top panel). In contrast, CarG with excess CarDAd this to be true for CarDAd and CarGAd as well (Fig. 3A). By contrast, yielded only the pure protein peaks (Fig. 3B, Top panel). CarG did not interact with the CarDAd N-terminal domain (Fig. Therefore, the cognate CarDAd–CarGAd pair forms a stable 3A) or CarDAd (results not shown). complex (like CarD and CarG) but the mixed CarDAd–CarG pair Analytical gel filtration using purified proteins further con- does not. firmed these findings in vitro. We have shown elsewhere that native CarD is an elongated dimer with a globular N-terminal CarD Chimeras Containing the CarDAd C-Terminal Segment Function in dimerization domain and an extended C-terminal domain, and M. xanthus. CarDAd, despite its lower DNA-binding affinity in vitro, although its calculated molecular weight (MW) is 34 kDa it could replace CarD in vivo if CarGAd was also present. This led us elutes off a Superdex200 column with an apparent MW, Mr,of to infer that so long as CarD–CarG interactions are maintained (118 Ϯ 7) kDa (21). CarG (calculated MW ϭ 19 kDa) elutes as (which occurs only with the cognate pairs), differences resulting a compact monomer with Mr of (17 Ϯ 1) kDa but, when mixed from the HMGA-like domain of CarD versus the H1-like one of with Ն2-fold excess of CarD, it coelutes with the latter as a stable CarDAd do not significantly affect function. In other words, the complex with Mr of Ϸ129 kDa, with the pure CarG peak CarD HMGA-like domain and the CarDAd H1-like domain may be undetected (22; Fig. 3B). Purified CarDAd, whose calculated MW interchangeable in vivo. We therefore tested if CarD chimeras that is Ϸ42 kDa, eluted off Superdex200 with a 4-fold higher Mr of retained the N-terminal part (and thus, interactions with CarG), but (166 Ϯ 8) kDa. This behavior, which parallels that of CarD, in which the HMGA domain was replaced by the CarDAd H1 suggests that CarDAd is also elongated with a globular N- CTR-like segment, could function in M. xanthus. Two such chime- terminal dimerization domain and an extended C-terminal do- ras were examined: in 1 (C1), the CarDAd segment spanning the main; CarGAd (calculated MW Ϸ 20 kDa) eluted with Mr of H1-like basic region and the preceding highly acidic region replaced (18 Ϯ 1) kDa and so appears to be compact like CarG (Fig. 3B, the entire CarD HMGA-like domain; in the other (C2), the basic Bottom panel). The pure CarGAd peak disappeared on mixing H1-like domain in CarDAd substituted the basic AT-hooks of CarD

García-Heras et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 26, 2021 Fig. 4. CarD chimeras with a CarDAd C-terminal domain are functional in M. xanthus.(A) Schematic representation of CarD chimeras C1 and C2. CarD and CarDAd segments in each chimera are indicated with numbers and boxes are coded as in Fig. 1A.(B) Expression of carQЈ::lacZ in the dark (ﬁlled bars) and in the light (empty bars) for strains bearing the wild-type carD (Cϩ), ⌬carD (CϪ), C1, or C2 alleles. (C) Developmental phenotype on TPM agar for strains as in A, vertically aligned with the corresponding expression levels of ⍀4435. (D) Expression of ddvA::Tn5-lac for strains as in A. ␤-galactosidase activities in B, C, and D are shown Fig. 5. CarD chimeras with human HMGA1a or histone H1.2, but not and estimated as in Fig. 1. protamine 2 or cytochrome C, function in M. xanthus.(A) Schematic representation of CarD chimeras C3 to C8. Segments corresponding to CarD, HMGA1a, or H1.2 in each chimera are indicated with numbers and boxes and (Fig. 4A). An acidic region therefore exists in both C1 and C2, since are coded as in Fig. 1A.(B) Expression of carQЈ::lacZ in the dark (ﬁlled bars) and ϩ Ϫ lack of the acidic region decreases CarD stability (21) and impairs in the light (empty bars) for strains bearing the wild-type carD (C ), ⌬carD (C ), function in vivo (Fig. S3). To test if C1 and C2 function in M. or the indicated chimeric alleles. (C) Developmental phenotype on TPM agar for strains as in A, vertically aligned with the corresponding expression levels xanthus, plasmid vectors bearing the corresponding genes were of ⍀4435. (D) Expression of ddvA::Tn5-lac in strains as in A.InB, C, and D, independently introduced into the ⌬carD strain, where they incor- ␤-galactosidase activities are shown and estimated as in Fig. 1. porate into the chromosome by homologous recombination to generate merodiploids. Both types of merodiploids turned red in the light, suggesting that C1 and C2 can complement the CarϪ fused to HMGA1a in chimera C3, while in C4, C5, and C6, the phenotype provoked by the ⌬carD allele. Haploid strains expressing CarD stretch from residues 1 to 226 was linked to the HMGA1a C1 or C2 were isolated as described (see SI Materials and Methods) AT-hook segment between residues 2 and 90, the entire H1.2, or the and confirmed to be Carϩ (Fig. S4A). Both strains also recovered H1.2 CTR part (residues 110–213), respectively. C4, C5, and C6 the Fruϩ developmental phenotype (Fig. 4C). Furthermore, the thus retain the CarD acidic segment (residues 180–226) while C3 strains with C1 or C2 showed levels of reporter lacZ expression has the HMGA1a acidic segment that, unlike in CarD, is at the within Ϯ20% of the wild-type for the carQЈ::lacZ (Fig. 4B), ⍀4435 C-terminal end and follows the AT-hook segment. Plasmid vectors (Fig. 4C), and ddvA::Tn5lac (Fig. 4D) probes, confirming that the bearing the coding sequences for each chimera were constructed CarDAd H1-like domain can replace the CarD HMGA domain using the natural coding sequence for human HMGA1a and a in vivo. synthetic coding sequence for H1.2, which was optimized for high GC content and G ϩ C bias at the third codon position. These were CarD Chimeras with Human HMGA or Histone H1 Are Functional in M. then introduced into the ⌬carD M. xanthus strain by electropora- xanthus. The above results were sufficiently intriguing to prompt us tion, where each construct incorporates into the chromosome by to examine whether other variants of HMGA or H1 domains in homologous recombination at the carD locus, as described earlier. CarD are equally capable of functioning in M. xanthus. We checked Unlike the CarϪ ⌬carD recipient strain, the merodiploids with each this using human HMGA1a or histone H1 (its H1.2 subtype) in of these chimeras turned red on illumination with blue light. chimeric CarD proteins. Human HMGA1a, as noted previously, Haploid strains expressing each of these chimeras were isolated as resembles the CarD C-terminal HMGA-like domain in its physical, described (see SI Materials and Methods) and confirmed to be Carϩ structural, and DNA-binding properties (21). However, human (Fig. S4A). Moreover, they were all Fruϩ (Fig. 5C). Expression of HMGA1a has 1 less AT-hook and its acidic region (significantly the reporter lacZ probes carQЈ::lacZ (Fig. 5B), ⍀4435 (Fig. 5C), and shorter than in CarD) flanks the C- rather than the N-terminal side ddvA::Tn5lac (Fig. 5D) in the strains expressing any 1 of the 4 of the AT-hook segment (Fig. 1A). Likewise, although the C- chimeras was within Ϯ20% of the wild type. Thus, in all 3 terminal region of CarDAd is rich in K/A/P like H1 CTRs, its CarD-dependent processes examined, human HMGA1a or H1 sequence and overall amino acid composition vary from that in could replace the CarD HMGA domain in vivo. The results human H1.2, and the latter’s globular and N-terminal domains do demonstrate that CarD remains functional despite 1 less AT-hook not occur in CarDAd. The globular domain has been implicated in or a different juxtaposition of the acidic and AT-hook segments. positioning H1 to the nucleosome after the initial binding of CTR Moreover, given that H1 CTR alone appears to function as well as to linker DNA, while the N-terminal region may modulate the whole H1.2, we can conclude that the H1 globular domain together binding affinity of H1 to chromatin (1). with its N-terminal region does not contribute to CarD activity in Fig. 5A depicts schematically the CarD chimeras with segments vivo, consistent with the roles of these 2 domains being more corresponding to human HMGA1a or histone H1.2 subtype that pertinent in the context of eukaryotic chromatin. were examined. The CarD segment spanning residues 1 to 179 was We next checked if any basic polypeptide could function as the

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902233106 García-Heras et al. Downloaded by guest on September 26, 2021 CarD AT-hook region by constructing 2 additional chimeras, C7 Interestingly, a naturally existing domain swap between AT-hooks and C8 (Fig. 5A). In C7, the AT-hooks are replaced by the and H1 CTR occurs in plants, where a globular domain like the one 102-residue, R-rich human protamine 2 (Prt2; theoretical pI ϭ linked to H1 CTR is also found associated with the AT-hook region 11.9), which lacks a defined structure and binds DNA in vitro like of plant HMGA (31). H1 CTR but with higher affinity (27, 28). In C8, the AT-hooks are A bacterial origin for H1 CTR was based on similar domains replaced by the 105-residue human cytochrome C (CytC; theoret- existing in various bacterial species but not in archaea (2). These ical pI ϭ 9.58), which has defined structure and can bind DNA bacterial H1 CTR-like domains have been implicated in DNA because of its positive charge although it is not a proper DNA- compaction, gene regulation, and other DNA transactions but binding protein (29). Plasmid vectors bearing DNA coding for molecular details on their action or interaction with other proteins CarD residues 1 to 226 plus synthetic sequences coding for Prt2 or are scarce. They all differ from CarDAd in lacking the characteristic CytC (both optimized for the high GC content and codon usage in N-terminal domain similar to CarDNter. Accordingly, the corre- M. xanthus, as with H1.2 above) were introduced into the ⌬carD M. sponding bacteria also lack a CarG analog. Specific processes in A. xanthus strain, and merodiploids and haploids expressing chimeras dehalogenans subject to CarDAd–CarGAd control remain an open C7 or C8 were isolated as before. Unlike strains bearing chimeras question, as this bacterium lacks genes for fruiting body develop- with H1 CTR, those with C7 or C8 were CarϪ FruϪ (Fig. 5C, Fig. ment or carotenogenesis that are CarD–CarG dependent in M. S4B). In agreement with this, expression of the carQЈ::lacZ and the xanthus (30). However, our study indicates that CarD–CarG pairs ⍀4435 reporter probes in strains with C7 or C8 was low or negligible in M. xanthus and A. dehalogenans share the same molecular relative to wild type (similar to the ⌬carD strain for C7, and architecture and interactions and thus, very likely, employ con- Ϸ10–20% for C8; Fig. 5 B and C). Expression of the ddvA::Tn5lac served molecular mechanisms of action. probe in strains with C7 or C8 was also significantly lower than in The role of CTR in histone H1 function has long been an enigma those with C5 or C6 (Ͻ20% for C7 and Ϸ35% for C8, relative to given its high sequence diversity and intrinsically disordered struc- wild type; Fig. 5D). We checked whether lack of complementation ture. It was supposed that this very basic region functions simply as by C7 and C8 could be the result of their instability or deficient a polycation that condenses DNA (5, 32). The winged-helix globular expression (despite optimizing codon usage in M. xanthus)ofthe domain of H1, which is its most highly conserved part, has been corresponding cell extracts in Western blots using a monoclonal more amenable for structure–function studies (33–36). Recently, antibody that specifically recognizes the CarD region present in all however, CTR is emerging as a critical determinant of H1 function, chimeras. C8, but not C7, could be detected at levels comparable and specific subdomains and length differences in CTR have been

to functional chimeras (Fig. S4C). The apparent lack of C7 could invoked to explain the functional heterogeneity observed among its MICROBIOLOGY therefore account for its inability to function in M. xanthus.Onthe subtypes (5, 27, 32). The importance of CTR is underscored by the other hand, CytC could not functionally replace the CarD AT-hook existence of single-domain H1 proteins in protists, which lack a region despite stable expression of the corresponding chimera. The globular domain and have compositions very similar to CTR, as is basic regions in CarD and its chimeras although comparable in size exemplified by the Tetrahymena thermophila macronuclear linker (90–105 residues) vary in net positive charge besides amino acid histone (2). Interestingly, although this H1 is indeed involved in composition (Fig. S1D). That different HMGA and H1-like do- chromatin condensation that could lead to global transcriptional mains, but not CytC, can be interchanged with no loss of CarD repression, it was shown to activate or repress specific genes, thus function in CarD suggests that net charge alone may not be linking H1 CTR to specific control of gene expression (37). sufficient to determine function. Overall, our data indicate that A key finding of our present study is that H1 CTR can substitute although CarD can tolerate a remarkable plasticity in the nature of the HMGA AT-hooks in CarD with no loss of function in vivo. the AT-hook/H1 region and its arrangement relative to the acidic Thus, we find that CarDAd can replace CarD in M. xanthus so long region, the AT-hook region cannot be replaced by just any basic as CarGAd, with which it interacts specifically, is also present. In line domain. with this, chimeras with the CarD AT-hooks replaced by the CarDAd H1-like domain or even human histone H1.2 CTR, but Discussion which retain CarDNter and thus specific interactions with CarG, The unique 2-domain architecture of CarD, consisting of an are functional in vivo. We argued elsewhere that the essential role N-terminal domain of exclusively bacterial origin and a C-terminal of CarG, which does not bind DNA directly, maybe as an adaptor domain similar to eukaryotic HMGA, led us to propose that it may that bridges CarD to the transcriptional machinery and/or other have evolved by lateral gene transfer (LGT) of the HMGA domain factors that remain to be identified (22). Such interactions involving followed by its fusion to a preexisting N-terminal bacterial module CarG could conceivably override differences in affinity between (13). That a CarD analog with a C-terminal domain akin to H1 CarD and CarDAd, as both cause similar expression of 3 distinct CTR exists in A. dehalogenans, another myxobacterium that is most reporter fusions, even though CarDAd binds DNA with a lower closely related to the suborder that includes M. xanthus and S. affinity in vitro, at least to the well-defined site at the light-inducible aurantiaca (30), is intriguing both from evolutionary and functional PQRS promoter. A CarD-binding site like the one at PQRS is not standpoints. It has been argued that the evolutionary origin of H1 discernible at other promoters that are also regulated by CarD and CTR can be traced to eubacteria and that eukaryotes subsequently CarG. Thus, a rather broad DNA-binding specificity may be acquired it by LGT, with the globular and N-terminal domains tolerated by CarD, which would be compatible with its ability to evolving much later (2). CarDAd could then have evolved by the accommodate HMGA to H1 domain swaps with no apparent loss fusion of 2 preexisting bacterial modules. Hence, given the relat- of function. Our findings on the functional equivalence of these 2 edness between A. dehalogenans and M. xanthus, and that both domains in a bacterial transcriptional factor are echoed in the case CarD proteins have a K/A/P-rich C-terminal region, the most of the human Epstein-Barr virus protein EBNA1, whose first 378 parsimonious explanation for how M. xanthus CarD acquired the residues could be replaced by HMGA1a (residues 1 to 90) or H1.2 AT-hooks would be convergent evolution from an ancestral H1-like to support replication and maintenance of oriP-containing plasmids domain (as in CarDAd), rather than LGT. This rapid evolution in metazoan cells (38). toward AT-hooks should have occurred before M. xanthus and S. Both in H1 CTR and in the analogous CarDAd domain, Ϸ76% aurantiaca branched out, as both species possess an HMGA-like of the residues are K, A, or P (Fig. S1D). However, whereas H1 domain. Less probable would be the alternative that, in 2 closely CTR has several S/TPXK motifs (X is any amino acid), which are related bacteria, 2 independent fusion events involving the N- subject to phosphorylation and occur within subdomains implicated terminal CarD module occurred, one to an H1-like domain (as in in DNA binding and chromatin condensation (1, 5, 32), these are CarDAd) and the other to an HMGA-like domain (as in CarD). absent from the CarDAd H1-like domain (or the CarD AT-hook

García-Heras et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 26, 2021 region). Given that different chimeras function essentially as well as H1 are functionally distinct in eukaryotes, where DNA is packed CarD in vivo despite the diversity in amino acid compositions (Fig. into nucleosomes. The functional equivalence of HMGA AT-hooks S1D) and primary sequences, defined functional subdomains in and H1 CTR that we find in bacterial transcriptional regulation CarD CTR, if they do exist, may not be readily discernible. may, nevertheless, provide insights that could be relevant in un- It is remarkable that 2 apparently unrelated DNA-binding do- derstanding the molecular basis underlying the modes of action of mains like the AT-hooks and CTR are functionally interchangeable. HMGA AT-hooks and H1 CTR. Both have regions of high content in basic amino acids and their intrinsically disordered structure has been hypothesized as provid- Materials and Methods ing the conformational malleability required in the biological need Strains, Plasmids, and Growth Conditions. Table S1 lists the plasmids and Esch- to interact with many different targets (4, 10, 21, 32, 39–43). Also, erichia coli/M. xanthus strains used in this study. Vegetative growth was carried H1 and HMGA both bind preferentially to DNA of scaffold- out in rich CTT medium at 33 °C, and fruiting body development was induced on associated regions or SARs (proposed to be implicated in delimiting TPM agar and examined with a Zeiss dissecting microscope as described before DNA loops and in chromosome dynamics) (25). The preferential (22). E. coli DH5␣ (for plasmid constructions; SI Materials and Methods) and binding of H1 has been mapped to its CTR domain, which appears BL21-(DE3) (for protein overexpression) were grown in Luria broth at 37 °C. to recognize the narrow minor groove of AT-rich tracts in SARs, Complementation Analyses. Complementation analyses were performed using as had been shown for the HMGA AT-hooks (8, 9, 25, 27). Thus, ⌬ ⌬ ⌬ the common features of a basic, intrinsically disordered region, the carD and carD carG strains as recipients for electroporation with a given plasmid construct bearing carD or the gene encoding a specific CarD coupled to their similar DNA-binding modes, could explain the Ad chimera, or carDAd and carGAd. These plasmids do not replicate in M. xanthus functional interchangeability that we observe between these 2 but integrate into the chromosome by homologous recombination. From the DNA-binding domains. A requirement for structural disorder resulting merodiploids haploid strains bearing the desired allele were gener- might account for why CytC, with a defined structure, could not ated and verified as detailed in SI Materials and Methods. replace the AT-hooks/H1 CTR in CarD function, despite its basic nature. In sum, our findings indicate that an AT-hook region or H1 Protein Purification, Protein–Protein, Protein–DNA Interactions, and Western Blots. CTR, but not just any basic domain, can supply the DNA-binding Procedures for protein purification and analysis are described in SI Materials activity for CarD function. and Methods. In eukaryotes, where they are implicated in multiple cellular functions, histone H1 and HMGA proteins co-exist and can be ACKNOWLEDGMENTS. We thank Prof. F. E. Loeffler (Georgia Institute of Tech- present as a variety of isoforms. Pinning down the specific functions nology) for A. dehalogenans strain 2CP-C genomic DNA, Prof. T. Maniatis (Har- vard University) for the pET15b-human HMGA1a, J. A. Madrid for technical and understanding the molecular basis for their modes of action assistance, and C. Flores for DNA sequencing. This work was supported by the remain among the most perplexing questions in chromosome Ministerio de Educacioń y Ciencia (Spain) grants BFU2006–14524 (to M.E.A.), biology. There is significant evidence, however, that HMGA and BFU2005–01040 and BFU2008–00911 (to S.P.), and a PhD fellowship (to F.G.H.).

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