BACKGROUND
photosynthetic plant life is exposed to potentially damaging uv-b radiation  which is intrinsic to sunlight.
uv-b photons are specifically and sensitively detected in plants by the uv-b photoreceptor uv resistance locus  <dig>  .
uvr8-mediated signalling is known to culminate in altered expression of a broad range of genes  <cit>  which ultimately leads to uv-b acclimation and uv-b tolerance.
uvr <dig> uv-b signalling involves the interaction of uvr <dig> with the e <dig> ubiquitin ligase constitutively photomorphogenic  <dig>  and associated stabilization of the bzip transcription factor elongated hypocotyl  <dig>  .
hy <dig> then influences expression of a number of uv-b regulated genes  <cit> , including expression of hy <dig> itself  <cit> .
indeed, a t/g-box cis-regulatory element within the hy <dig> promoter functions as an hy <dig> binding site and is required for uv-b-induced hy <dig> expression  <cit> .
apart from hy <dig> stabilization, past reports have proposed a more direct mechanism of uvr <dig> regulation of uv-b-dependent transcription.
uvr <dig> has sequence similarity to the eukaryotic guanine nucleotide exchange factor regulator of chromatin condensation , which interacts with chromatin and specifically with histones  <cit> .
rcc <dig> proteins act as guanine nucleotide exchange factors  and regulate the small gtpase ran.
rcc <dig> is essential to critical cellular processes in eukaryotes such as nucleocytoplasmic transport, nuclear envelope formation, and spindle assembly during mitosis .
although uvr <dig> is not otherwise functionally related to rcc <dig>  chromatin immunoprecipitation  assays have suggested that uvr <dig> binds chromatin in vivo .
it is tempting to assume that uvr <dig> chromatin association is due to structural similarity with rcc <dig> proteins.
recent studies of rcc <dig> structure illustrate how this protein interacts with histone and dna components of the nucleosome  <cit> .
rcc <dig> is a β-propeller protein that interacts with histones and nucleosomal dna through a ‘switchback loop’ region and its n-terminal tail  <cit> .
rcc <dig> and histones make contact through the h2a-h2b histone dimer surface of the nucleosome core particle whilst contacts between rcc <dig> and dna are made through the dna phosphate backbone.
this indicates that rcc <dig> interacts with chromatin by binding non-dna-sequence specific areas  <cit> .
consistently, the yeast rcc1-orthologue srm1/prp <dig> was found to bind most nucleosomes in the genome with no apparent sequence specificity  <cit> .
similar unspecific binding was indicated for arabidopsis uvr <dig> by reports of its association with a region larger than 3 kb around the hy <dig> genomic locus and its chromatin association via histones, preferentially histone h2b  <cit> .
however, in contrast to the yeast rcc <dig>  the proposed uvr <dig> chromatin association was confined to specific genes.
of the promoter regions tested, uvr <dig> was found to interact with chromatin of some  but not all  uvr8-regulated genes  <cit> .
however, both the specificity of uvr <dig> for particular target genes and the mechanism conferring specificity need to be more firmly established.
association of arabidopsis uvr <dig> with chromatin of uvr <dig> target genes was reported to be constitutive and not uv-b-responsive  <cit> , despite the fact that uvr <dig> accumulates in the nucleus upon uv-b irradiation  <cit> .
thus, independent of the ambiguities surrounding target gene specificity, it is also a valid question whether uvr <dig> chromatin association indeed underlies uvr <dig> involvement in transcription regulation and, if so, how uvr <dig> influences gene expression mechanistically.
it has been suggested that uvr <dig> is involved in the recruitment of transcriptional regulators and/or in chromatin remodelling  <cit> .
here, we have experimentally revisited chromatin association of uvr <dig> and suggest that a mode-of-action involving direct uvr <dig> chromatin association should be considered with caution and alternative mechanisms should not be dismissed.
RESULTS
arabidopsis uvr <dig> does not associate with promoter regions of hy <dig> and myb <dig> target genes in quantitative chip qrt-pcr assays
to better understand the dynamics and role of the proposed uvr <dig> chromatin association, we set out to establish whether uvr <dig> physically interacted with chromatin at previously described target genes using quantitative chip qrt-pcr.
we first tested association of endogenous uvr <dig> with the hy <dig> genomic locus using several target regions for which positive qualitative gel-based chip has been described  <cit>  .
our anti-uvr <dig> antibody was raised against the same peptide  used in previous chip experiments  <cit> .
this antibody is highly efficient and specific both in western blotting and co-immunoprecipitation of uvr <dig> with endogenous cop <dig>  <cit> .
to ensure adequate crosslinking conditions and a functional chip protocol, we used the same cross-linked chromatin pool to perform chip of hy <dig> using an anti-hy <dig> antibody and processed the samples in parallel to those arising from uvr <dig> chip.fig.
<dig> uvr <dig> does not associate with the hy <dig> genomic region.
a schematic representation of the hy <dig> genomic locus and surrounding region.
black and grey boxes depict the hy <dig> transcribed region  and portions of the transcribed regions of the neighbouring genes, respectively.
upper grey bars indicate qrt-pcr amplicons at various positions at and surrounding the hy <dig> genomic locus with numbers indicating the position of the 5’ end of the amplicon relative to the hy <dig> translation start site .
an arrow indicates the position of the t/g-box cis-element bound by hy <dig>  <cit> .
open bars are placed at genomic regions previously reported to be uvr <dig> binding targets in gel-based chip assays  <cit> .
b, c chip-qpcr of dna associated with hy <dig>  and uvr <dig>  presented as the percentage of total input dna recovered by immunoprecipitation .
the same chromatin pool was used for immunoprecipitation with both hy <dig> and uvr <dig> antibodies.
wt = ws/pro
hy5
:luc+, uvr <dig> ox = ws/pro
hy5
:luc + pro
35s
:uvr <dig>  arabidopsis mutant lines with corresponding genetic backgrounds  were included as negative controls.
d, e as in , but of dna associated with hy5-yfp  and yfp-uvr <dig> , performed using the same anti-gfp antibody for immunoprecipitation.
hy5-yfp = hy5-1/pro
35s
:hy5-yfp, yfp-uvr8 = uvr8-7/pro
35s
:yfp-uvr <dig>  arabidopsis mutant lines with corresponding genetic backgrounds  were included as negative controls.
b, c, d, e the number labelling indicates the location of each analysed dna fragment, specifically the position of the 5’ end of the amplicon relative to the translation start site .
experimental material was sourced from ten-day-old seedlings, either processed with no uv-b exposure  or following 4 h uv-b .
error bars represent sds of three technical replicates.
f input and eluate of d/e chip samples were analysed on western blots with an antibody against gfp.
bands resulting from degradation products  and unspecific background signal  are marked .
molecular weight marker positions representing  <dig> and 75 kda are shown
hy <dig> chip clearly confirmed binding of hy <dig> to its own promoter , as has been previously documented  <cit> .
however, in contrast to previous reports , we did not detect uvr <dig> chromatin association with hy <dig> using the anti-uvr <dig> antibody in conjuction with several different probes covering a large portion of the hy <dig> genomic region , including those regions previously reported to be bound by uvr <dig>  similar to wild type, we also did not detect uvr <dig> chromatin association with hy <dig> in a transgenic line overexpressing uvr <dig> .
to ensure comparable antibody functionality during immunoprecipitation, we performed further chip qrt-pcr assays and used the exact same antibody for both hy <dig> and uvr <dig>  for this, we resorted to the pro35s:yfp-uvr <dig>  pro35s:cfp-uvr <dig> and pro35s:hy5-yfp transgenic lines and used a polyclonal gfp  antibody to perform chips of hy5-yfp and yfp-uvr <dig> or cfp-uvr <dig> in parallel .
in agreement with our hy <dig> chip data, we obtained a clear signal for hy5-yfp binding to the endogenous hy <dig> promoter .
however, as for uvr <dig> chip, we did not detect any specific hy <dig> signal above background in yfp-uvr <dig> and cfp-uvr <dig> chip assays, a result that was mirrored in the non-transgenic negative controls  .
to verify that yfp-uvr <dig>  cfp-uvr <dig> and hy5-yfp were efficiently immunoprecipitated with the anti-gfp antibody during the chip procedure, we analysed the protein-chromatin eluate by western blotting.
following the immunoprecipitation step, we found yfp-uvr <dig> and cfp-uvr <dig> in the eluate and at positions in agreement with their expected size of 74 kda  .
hy5-yfp was detectable in the input and ip fractions  although it ran slightly higher than its predicted molecular weight of 45 kda .
however, a similar size discrepancy has been observed previously   <cit> .
it is of note that tagged uvr <dig> proteins were expressed and immunoprecipitated at higher levels than hy5-yfp, and yet, unlike hy5-yfp, yfp-uvr <dig> and cfp-uvr <dig> chip assays did not result in hy <dig> chip signal .
we further tested uvr <dig> binding to the promoter of myb <dig>  which is also a reported target gene of uvr <dig>  <cit>  and compared genomic association to that of hy <dig>  <cit> .
the primer pair used to detect the myb <dig> promoter amplifies a region that is included in the region amplified with a primer pair used previously  <cit> .
however, as for the hy <dig> target gene, we did not detect binding of uvr <dig>  yfp-uvr <dig> or cfp-uvr <dig> to the myb <dig> promoter region above background chip signal for a non-uv-b regulated gene  and a control intergenic region .
in contrast, clear myb <dig> chip signal resulted from both chip of hy <dig>  performed in parallel using an anti-hy <dig> antibody on the same cross-linked wild-type chromatin pool , and chip of hy5-yfp, performed with the same gfp antibody in transgenic lines .
indeed, uv-b enhanced the association of hy <dig> with the myb <dig> promoter region , as described before  <cit> .fig.
<dig> uvr <dig> does not bind to the myb <dig> promoter.
a uvr <dig> chromatin association in wild-type plants , a uvr <dig> mutant  and a uvr8-overexpression line .
chip-qpcr was performed using an anti-uvr <dig> antibody for immunoprecipitation.
b chromatin association in hy5-yfp expressing plants , a yfp-uvr <dig> line  and hy5- <dig> and uvr8- <dig> mutant controls.
chip-qpcr was performed using an anti-gfp antibody for immunoprecipitation.
c hy <dig> chromatin association in wild-type plants , a uvr <dig> overexpression line  and hy <dig> hyh mutant control .
chip-qpcr was performed using an anti-hy <dig> antibody.
a, b, c chromatin association was analysed by chip-qpcr for the myb <dig> promoter , actin <dig> and an intergenic control region between the at4g <dig> and at4g <dig> genes.
the analysed chromatin was the same as in the experiments presented in fig.
<dig>  error bars represent sds of three technical replicates
taken together, the results of our quantitative chip qrt-pcr assays of endogenous uvr <dig> and transgenically expressed yfp-uvr <dig> and cfp-uvr <dig> do not support an association of uvr <dig> with previously reported chromatin regions.
however, it cannot be excluded that differences in plant growth conditions or chip procedure may explain the discrepancy between our and previously published results.
independent of this, a functional role of uvr <dig> at the chromatin level needs to be reconsidered.
vp16-uvr <dig> does not enhance expression of hy <dig> and myb <dig> in transient protoplast assays
as an alternative test of promoter association, we generated synthetic transcriptional activators by creating fusion proteins with the herpes simplex virus vp <dig> acidic activation domain  <cit>  and analysed the effect on target gene expression in a transient protoplast assay.
to have a comparative means to guage the effect of uvr <dig> , we used the established dna-binding protein hy <dig>  <cit>  as a positive control  and the non-dna-binding hfr <dig>  <cit>  as a negative control .
transient expression of vp16-hy <dig> in arabidopsis protoplasts strongly enhanced expression of its target genes  but not of others  .
this is in agreement with previous experiments showing strong activation of chs and myb <dig> in stable transgenic arabidopsis lines expressing vp16-hy <dig> fusion proteins  <cit> .
in contrast, vp16-uvr <dig> did not activate the expression of the suggested direct uvr <dig> target genes , either in white light  or in response to uv-b .
it is, however, noteworthy that uvr <dig> can act as a uv-b photoreceptor in arabidopsis protoplasts, as was evident by expression induction of marker genes hy <dig> and myb <dig> in response to uv-b in protoplasts derived from wild-type seedlings but not from uvr8- <dig> null mutants .
moreover, in uvr8-7-derived protoplasts, transient expression of wild-type uvr <dig> was seen to complement the mutant phenotype .
in agreement with protoplast uv-b-responsiveness, enhanced target gene expression associated with vp16-hy <dig> was reduced when protoplasts were treated with uv-b , reflecting the enhanced basal expression levels of the elip <dig>  chs and myb <dig> target genes resulting from a typical transcriptional response to uv-b.
conversely, vp16-hfr <dig> expression had no effect on chs, myb <dig>  elip <dig>  hy <dig> or uvr <dig> mrna levels , in agreement with the absence of dna-binding activity of hfr <dig>  <cit> .fig.
<dig> fusion of the vp <dig> activator domain to uvr <dig> does not affect expression of suggested target genes.
a, b, c, d, e, f the effect of fusing the vp <dig> activator domain to hy <dig> , uvr <dig>  and hfr <dig>  as measured by expression changes of target and negative control genes.
mutant protoplasts prepared from hy5-ks <dig>  uvr8- <dig> and hfr1- <dig> seedlings were transfected with pro
35s
:3xha-vp16ad-hy5/pro
35s
:hy <dig>  pro
35s
:3xha-vp16ad-uvr8/pro
35s
:uvr <dig> and pro
35s
:3xha-vp16ad-hfr1/pro
35s
:hfr <dig> respectively.
gene expression was analysed 19–24 h post-transfection with protoplasts maintained under weak white light  and weak white light plus a final 3 h uv-b irradiation .
gene expression levels were normalized with the expression levels of the respective transfected effectors .
data is presented as relative expression in the presence of the effectors fused to vp <dig> to the respective control effectors without the vp <dig> domain .
nd = non-determinable.
data are shown as the means of four  or three  biological replicates ± standard errors
drosophila rcc <dig> chromatin and dna binding residues are not conserved in arabidopsis uvr8
the initial investigation of uvr <dig> chromatin association was inspired by the sequence similarity of uvr <dig> with eukaryotic rcc <dig> proteins  <cit> , prior to publication of the uvr <dig> crystal structure  <cit> .
rcc <dig> binds chromatin as part of its biological activity in establishing a rangtp/rangdp gradient, with an increasing ratio in decreasing distance to chromatin  <cit> .
the crystal structure of drosophila melanogaster rcc <dig>  in association with a nucleosome core particle  revealed two distinct binding sites, a histone- and a dna-binding loop  <cit> .
the dmrcc <dig> histone-binding loop is made up of two key arginine residues  supported by a serine residue  that mediates the interaction with an acidic histone patch  <cit> .
on the other hand, the dna-binding activity of dmrcc <dig> is mediated by gln- <dig>  lys- <dig> and arg- <dig> through sequence-unspecific interaction with the dna phosphate backbone  <cit> .
although crystal structures of saccharomyces cerevisiae  and human  rcc <dig> proteins have been described and both proteins shown to bind nucleosomes, the molecular details of the chromatin interaction are less well understood .
structure comparison showed that critical arginine residues of the histone-binding loop are conserved, arg- <dig> in scsmr1/prp20p and arg- <dig> in hsrcc <dig>  corresponding to dmrcc <dig> arg- <dig>  <cit> .
furthermore, hsrcc <dig> arg- <dig> was shown experimentally to be necessary for nucleosome interaction  <cit>  and the mutation of basic residues in the dna-binding loop of hsrcc <dig> showed lys- <dig>  arg- <dig> and arg- <dig> to be important for dna binding  <cit> .fig.
<dig> structural comparison of uvr <dig> with the nucleosome binding interface of rcc <dig> shows that uvr <dig> lacks positive surfaces to interact with nucleosomes.
a overview of the drosophila rcc1-nucleosome complex determined by x-ray crystallography  <cit> .
both rcc <dig> molecules bind identically to the nucleosome.
b dmrcc <dig> viewed through the nucleosome shows the nucleosome interaction interface.
left: for rcc <dig>  the solvent accessible surface is shown coloured in terms of electrostatic potential  <cit> .
the nucleosome is depicted as semi-transparent.
right: the same representation of rcc <dig> with the nucleosome contact surface outlined in pink.
dna and histone-binding regions are indicated.
c, d, e comparison of electrostatic surface potential of hsrcc <dig>  , scsmr1/prp <dig>   and uvr <dig>   with an orientation identical to that of dmrcc <dig> in .
the electrostatic surface potential for the opposite side of uvr <dig> is also shown by rotation of the molecule .
pymol  and apbs plugin for pymol  was used for visualization of these structures.
f structure-based multiple sequence alignment of rcc <dig> homologs with uvr <dig>  shaded residues indicate those confirmed or proposed to be critical for rcc <dig> binding to the nucleosome
the recent publication of the crystal structure of arabidopsis uvr <dig>  <cit>  allowed us to carry out a structural comparison of arabidopsis uvr <dig> with dmrcc <dig> to check for the presence in uvr <dig> of a conserved nucleosome-binding surface that is critical for histone and dna interaction in dmrcc <dig>  in addition, we compared dmrcc <dig> and scsmr1/prp20p as well as dmrcc <dig> and hsrcc <dig> .
as the free structures of hsrcc <dig>  scsmr1/prp20p, and uvr <dig> are likely to undergo structural rearrangements in the switch-back loop and the dna-binding surface upon binding to nucleosomes, it is difficult to compare individual side-chain positions.
thus, we chose to compare the electrostatic surface potentials as a measure of histone and dna interaction potential.
since both dna and the negative patch on the nucleosome surface are negatively charged, dmrcc <dig> shows strong accumulation of positive charge on both the histone- and dna-interacting surfaces .
aligning the structures of hsrcc <dig> and scsmr1/prp <dig> revealed similar positive surface patches .
however, uvr <dig> showed a rather negatively charged surface across the entire predicted nucleosome interaction surface, which argues strongly against an rcc1-like binding mode to nucleosomes.
in addition, the uvr <dig> surface is generally devoid of strongly positively charged patches  and, thus, does not display the characteristics of a bona fide dna-binding protein.
comparing structure-based sequence alignments, it became evident that the overall structural similarity between the uvr <dig> and dmrcc <dig> 7-bladed β-propeller structures is striking but the histone-binding loop in dmrcc <dig> is not conserved in uvr <dig> .
similarly, we did not find a potential dna-binding loop in uvr <dig> through structural comparison with dmrcc <dig>  we conclude that the presence of histone- and dna-binding surfaces is a general feature of the chromatin association of eukaryotic rcc <dig> proteins but these are apparently absent in arabidopsis uvr <dig>  this suggests that uvr <dig> may have lost the ability to bind to chromatin, corresponding to the differences in activity between the uvr <dig> and rcc <dig> proteins.
as suggested by the structural analysis, recombinant uvr <dig> did not bind to reconstituted nucleosomes in vitro, either in its ground-state as homodimer or as uv-b-activated monomer .
in contrast, recombinant rcc <dig> showed a clear electrophoretic mobility gel shift of the nucleosomes, demonstrating nucleosome binding .fig.
<dig> uvr <dig> does not bind to nucleosomes in vitro.
a purified recombinant rcc <dig> and uvr <dig>  were electrophoretically separated on sds-page together or in the absence of reconstituted nucleosomes  and stained by coomassie blue.
samples were boiled or non-boiled before loading the gel.
b electrop horetic mobility gel shift assay of nucleosome binding by rcc <dig> and uvr <dig>  between  <dig>  - 10 μm rcc <dig> and  <dig>  - 40 μm uvr <dig>  were added to reconstituted mononucleosomes.
binding was assayed by emsa and visualized by ethidium bromide staining
CONCLUSIONS
contrary to the current models of uvr <dig> signalling, our data do not support binding of uvr <dig> to chromatin at target genes.
moreover, no chromatin-based activity of uvr <dig> could be demonstrated experimentally.
however, it has been rather well established that uvr <dig> activity requires uv-b-dependent interaction with cop <dig>  which leads to hy <dig> stabilization .
hy <dig> associates with target gene promoters and is required for uv-b-induction of a large portion of the uv-b-induced genes  <cit> .
thus, we propose that the effect of uvr8-cop <dig> on gene expression is through hy <dig> and its homolog hyh, without involving direct uvr <dig> chromatin association at the same target genes.
however, we cannot exclude the existence under uv-b of short-lived and/or limited amounts of a possible uvr8-cop1-hy <dig> protein complex that was not detected by our qrt-pcr-based chip assays.
arguing against this is the evidence from previous gel-based chip assays which firstly suggested that uvr <dig> associates with the hy <dig> promoter in the absence of uv-b and functional cop <dig> and secondly failed to detect chromatin association of gfp-cop <dig>  <cit> .
moreover, no evidence was found for hy <dig> being part of a uvr8-cop <dig> complex  <cit> , nor did we identify direct interaction of uvr <dig> with hy <dig> in yeast two-hybrid assays .
however, despite the prime role in uv-b signalling assigned to hy <dig>  hy <dig> lacks a transcriptional activation domain and cannot alone activate transcription  <cit> .
thus, to better understand how uvr <dig> regulates gene expression through hy <dig>  we now need to identify hy <dig> partner protein that provide a transcriptional activation domain and enable uv-b-responsive gene expression.

 photosynthetic plant life is exposed to potentially damaging uv-b radiation which is intrinsic to sunlight. uv-b photons are specifically and sensitively detected in plants by the uv-b photoreceptor uv resistance locus. uvr8-mediated signalling is known to culminate in altered expression of a broad range of genes which ultimately leads to uv-b acclimation and uv-b tolerance. uvr uv-b signalling involves the interaction of uvr with the e ubiquitin ligase constitutively photomorphogenic and associated stabilization of the bzip transcription factor elongated hypocotyl. hy then influences expression of a number of uv-b regulated genes, including expression of hy itself. indeed, a t/g-box cis-regulatory element within the hy promoter functions as an hy binding site and is required for uv-b-induced hy expression. apart from hy stabilization, past reports have proposed a more direct mechanism of uvr regulation of uv-b-dependent transcription. uvr has sequence similarity to the eukaryotic guanine nucleotide exchange factor regulator of chromatin condensation, which interacts with chromatin and specifically with histones. rcc proteins act as guanine nucleotide exchange factors and regulate the small gtpase ran. rcc is essential to critical cellular processes in eukaryotes such as nucleocytoplasmic transport, nuclear envelope formation, and spindle assembly during mitosis. although uvr is not otherwise functionally related to rcc chromatin immunoprecipitation assays have suggested that uvr binds chromatin in vivo. it is tempting to assume that uvr chromatin association is due to structural similarity with rcc proteins. recent studies of rcc structure illustrate how this protein interacts with histone and dna components of the nucleosome. rcc is a β-propeller protein that interacts with histones and nucleosomal dna through a ‘switchback loop’ region and its n-terminal tail. rcc and histones make contact through the h2a-h2b histone dimer surface of the nucleosome core particle whilst contacts between rcc and dna are made through the dna phosphate backbone. this indicates that rcc interacts with chromatin by binding non-dna-sequence specific areas. consistently, the yeast rcc1-orthologue srm1/prp was found to bind most nucleosomes in the genome with no apparent sequence specificity. similar unspecific binding was indicated for arabidopsis uvr by reports of its association with a region larger than 3 kb around the hy genomic locus and its chromatin association via histones, preferentially histone h2b. however, in contrast to the yeast rcc the proposed uvr chromatin association was confined to specific genes. of the promoter regions tested, uvr was found to interact with chromatin of some but not all uvr8-regulated genes. however, both the specificity of uvr for particular target genes and the mechanism conferring specificity need to be more firmly established. association of arabidopsis uvr with chromatin of uvr target genes was reported to be constitutive and not uv-b-responsive, despite the fact that uvr accumulates in the nucleus upon uv-b irradiation. thus, independent of the ambiguities surrounding target gene specificity, it is also a valid question whether uvr chromatin association indeed underlies uvr involvement in transcription regulation and, if so, how uvr influences gene expression mechanistically. it has been suggested that uvr is involved in the recruitment of transcriptional regulators and/or in chromatin remodelling. here, we have experimentally revisited chromatin association of uvr and suggest that a mode-of-action involving direct uvr chromatin association should be considered with caution and alternative mechanisms should not be dismissed.  arabidopsis uvr does not associate with promoter regions of hy and myb target genes in quantitative chip qrt-pcr assays to better understand the dynamics and role of the proposed uvr chromatin association, we set out to establish whether uvr physically interacted with chromatin at previously described target genes using quantitative chip qrt-pcr. we first tested association of endogenous uvr with the hy genomic locus using several target regions for which positive qualitative gel-based chip has been described. our anti-uvr antibody was raised against the same peptide used in previous chip experiments. this antibody is highly efficient and specific both in western blotting and co-immunoprecipitation of uvr with endogenous cop. to ensure adequate crosslinking conditions and a functional chip protocol, we used the same cross-linked chromatin pool to perform chip of hy using an anti-hy antibody and processed the samples in parallel to those arising from uvr chip.fig. uvr does not associate with the hy genomic region. a schematic representation of the hy genomic locus and surrounding region. black and grey boxes depict the hy transcribed region and portions of the transcribed regions of the neighbouring genes, respectively. upper grey bars indicate qrt-pcr amplicons at various positions at and surrounding the hy genomic locus with numbers indicating the position of the 5’ end of the amplicon relative to the hy translation start site. an arrow indicates the position of the t/g-box cis-element bound by hy. open bars are placed at genomic regions previously reported to be uvr binding targets in gel-based chip assays. b, c chip-qpcr of dna associated with hy and uvr presented as the percentage of total input dna recovered by immunoprecipitation. the same chromatin pool was used for immunoprecipitation with both hy and uvr antibodies. wt = ws/pro hy5:luc+, uvr ox = ws/pro hy5:luc + pro 35s:uvr arabidopsis mutant lines with corresponding genetic backgrounds were included as negative controls. d, e as in, but of dna associated with hy5-yfp and yfp-uvr, performed using the same anti-gfp antibody for immunoprecipitation. hy5-yfp = hy5-1/pro 35s:hy5-yfp, yfp-uvr8 = uvr8-7/pro 35s:yfp-uvr arabidopsis mutant lines with corresponding genetic backgrounds were included as negative controls. b, c, d, e the number labelling indicates the location of each analysed dna fragment, specifically the position of the 5’ end of the amplicon relative to the translation start site. experimental material was sourced from ten-day-old seedlings, either processed with no uv-b exposure or following 4 h uv-b. error bars represent sds of three technical replicates. f input and eluate of d/e chip samples were analysed on western blots with an antibody against gfp. bands resulting from degradation products and unspecific background signal are marked. molecular weight marker positions representing and 75 kda are shown hy chip clearly confirmed binding of hy to its own promoter, as has been previously documented. however, in contrast to previous reports, we did not detect uvr chromatin association with hy using the anti-uvr antibody in conjuction with several different probes covering a large portion of the hy genomic region, including those regions previously reported to be bound by uvr similar to wild type, we also did not detect uvr chromatin association with hy in a transgenic line overexpressing uvr. to ensure comparable antibody functionality during immunoprecipitation, we performed further chip qrt-pcr assays and used the exact same antibody for both hy and uvr for this, we resorted to the pro35s:yfp-uvr pro35s:cfp-uvr and pro35s:hy5-yfp transgenic lines and used a polyclonal gfp antibody to perform chips of hy5-yfp and yfp-uvr or cfp-uvr in parallel. in agreement with our hy chip data, we obtained a clear signal for hy5-yfp binding to the endogenous hy promoter. however, as for uvr chip, we did not detect any specific hy signal above background in yfp-uvr and cfp-uvr chip assays, a result that was mirrored in the non-transgenic negative controls. to verify that yfp-uvr cfp-uvr and hy5-yfp were efficiently immunoprecipitated with the anti-gfp antibody during the chip procedure, we analysed the protein-chromatin eluate by western blotting. following the immunoprecipitation step, we found yfp-uvr and cfp-uvr in the eluate and at positions in agreement with their expected size of 74 kda. hy5-yfp was detectable in the input and ip fractions although it ran slightly higher than its predicted molecular weight of 45 kda. however, a similar size discrepancy has been observed previously. it is of note that tagged uvr proteins were expressed and immunoprecipitated at higher levels than hy5-yfp, and yet, unlike hy5-yfp, yfp-uvr and cfp-uvr chip assays did not result in hy chip signal. we further tested uvr binding to the promoter of myb which is also a reported target gene of uvr and compared genomic association to that of hy. the primer pair used to detect the myb promoter amplifies a region that is included in the region amplified with a primer pair used previously. however, as for the hy target gene, we did not detect binding of uvr yfp-uvr or cfp-uvr to the myb promoter region above background chip signal for a non-uv-b regulated gene and a control intergenic region. in contrast, clear myb chip signal resulted from both chip of hy performed in parallel using an anti-hy antibody on the same cross-linked wild-type chromatin pool, and chip of hy5-yfp, performed with the same gfp antibody in transgenic lines. indeed, uv-b enhanced the association of hy with the myb promoter region, as described before.fig. uvr does not bind to the myb promoter. a uvr chromatin association in wild-type plants, a uvr mutant and a uvr8-overexpression line. chip-qpcr was performed using an anti-uvr antibody for immunoprecipitation. b chromatin association in hy5-yfp expressing plants, a yfp-uvr line and hy5- and uvr8- mutant controls. chip-qpcr was performed using an anti-gfp antibody for immunoprecipitation. c hy chromatin association in wild-type plants, a uvr overexpression line and hy hyh mutant control. chip-qpcr was performed using an anti-hy antibody. a, b, c chromatin association was analysed by chip-qpcr for the myb promoter, actin and an intergenic control region between the at4g and at4g genes. the analysed chromatin was the same as in the experiments presented in fig.  error bars represent sds of three technical replicates taken together, the results of our quantitative chip qrt-pcr assays of endogenous uvr and transgenically expressed yfp-uvr and cfp-uvr do not support an association of uvr with previously reported chromatin regions. however, it cannot be excluded that differences in plant growth conditions or chip procedure may explain the discrepancy between our and previously published results. independent of this, a functional role of uvr at the chromatin level needs to be reconsidered. vp16-uvr does not enhance expression of hy and myb in transient protoplast assays as an alternative test of promoter association, we generated synthetic transcriptional activators by creating fusion proteins with the herpes simplex virus vp acidic activation domain and analysed the effect on target gene expression in a transient protoplast assay. to have a comparative means to guage the effect of uvr, we used the established dna-binding protein hy as a positive control and the non-dna-binding hfr as a negative control. transient expression of vp16-hy in arabidopsis protoplasts strongly enhanced expression of its target genes but not of others. this is in agreement with previous experiments showing strong activation of chs and myb in stable transgenic arabidopsis lines expressing vp16-hy fusion proteins. in contrast, vp16-uvr did not activate the expression of the suggested direct uvr target genes, either in white light or in response to uv-b. it is, however, noteworthy that uvr can act as a uv-b photoreceptor in arabidopsis protoplasts, as was evident by expression induction of marker genes hy and myb in response to uv-b in protoplasts derived from wild-type seedlings but not from uvr8- null mutants. moreover, in uvr8-7-derived protoplasts, transient expression of wild-type uvr was seen to complement the mutant phenotype. in agreement with protoplast uv-b-responsiveness, enhanced target gene expression associated with vp16-hy was reduced when protoplasts were treated with uv-b, reflecting the enhanced basal expression levels of the elip chs and myb target genes resulting from a typical transcriptional response to uv-b. conversely, vp16-hfr expression had no effect on chs, myb elip hy or uvr mrna levels, in agreement with the absence of dna-binding activity of hfr.fig. fusion of the vp activator domain to uvr does not affect expression of suggested target genes. a, b, c, d, e, f the effect of fusing the vp activator domain to hy, uvr and hfr as measured by expression changes of target and negative control genes. mutant protoplasts prepared from hy5-ks uvr8- and hfr1- seedlings were transfected with pro 35s:3xha-vp16ad-hy5/pro 35s:hy pro 35s:3xha-vp16ad-uvr8/pro 35s:uvr and pro 35s:3xha-vp16ad-hfr1/pro 35s:hfr respectively. gene expression was analysed 19–24 h post-transfection with protoplasts maintained under weak white light and weak white light plus a final 3 h uv-b irradiation. gene expression levels were normalized with the expression levels of the respective transfected effectors. data is presented as relative expression in the presence of the effectors fused to vp to the respective control effectors without the vp domain. nd = non-determinable. data are shown as the means of four or three biological replicates ± standard errors drosophila rcc chromatin and dna binding residues are not conserved in arabidopsis uvr8 the initial investigation of uvr chromatin association was inspired by the sequence similarity of uvr with eukaryotic rcc proteins, prior to publication of the uvr crystal structure. rcc binds chromatin as part of its biological activity in establishing a rangtp/rangdp gradient, with an increasing ratio in decreasing distance to chromatin. the crystal structure of drosophila melanogaster rcc in association with a nucleosome core particle revealed two distinct binding sites, a histone- and a dna-binding loop. the dmrcc histone-binding loop is made up of two key arginine residues supported by a serine residue that mediates the interaction with an acidic histone patch. on the other hand, the dna-binding activity of dmrcc is mediated by gln- lys- and arg- through sequence-unspecific interaction with the dna phosphate backbone. although crystal structures of saccharomyces cerevisiae and human rcc proteins have been described and both proteins shown to bind nucleosomes, the molecular details of the chromatin interaction are less well understood. structure comparison showed that critical arginine residues of the histone-binding loop are conserved, arg- in scsmr1/prp20p and arg- in hsrcc corresponding to dmrcc arg-. furthermore, hsrcc arg- was shown experimentally to be necessary for nucleosome interaction and the mutation of basic residues in the dna-binding loop of hsrcc showed lys- arg- and arg- to be important for dna binding.fig. structural comparison of uvr with the nucleosome binding interface of rcc shows that uvr lacks positive surfaces to interact with nucleosomes. a overview of the drosophila rcc1-nucleosome complex determined by x-ray crystallography. both rcc molecules bind identically to the nucleosome. b dmrcc viewed through the nucleosome shows the nucleosome interaction interface. left: for rcc the solvent accessible surface is shown coloured in terms of electrostatic potential. the nucleosome is depicted as semi-transparent. right: the same representation of rcc with the nucleosome contact surface outlined in pink. dna and histone-binding regions are indicated. c, d, e comparison of electrostatic surface potential of hsrcc, scsmr1/prp and uvr with an orientation identical to that of dmrcc in. the electrostatic surface potential for the opposite side of uvr is also shown by rotation of the molecule. pymol and apbs plugin for pymol was used for visualization of these structures. f structure-based multiple sequence alignment of rcc homologs with uvr shaded residues indicate those confirmed or proposed to be critical for rcc binding to the nucleosome the recent publication of the crystal structure of arabidopsis uvr allowed us to carry out a structural comparison of arabidopsis uvr with dmrcc to check for the presence in uvr of a conserved nucleosome-binding surface that is critical for histone and dna interaction in dmrcc in addition, we compared dmrcc and scsmr1/prp20p as well as dmrcc and hsrcc. as the free structures of hsrcc scsmr1/prp20p, and uvr are likely to undergo structural rearrangements in the switch-back loop and the dna-binding surface upon binding to nucleosomes, it is difficult to compare individual side-chain positions. thus, we chose to compare the electrostatic surface potentials as a measure of histone and dna interaction potential. since both dna and the negative patch on the nucleosome surface are negatively charged, dmrcc shows strong accumulation of positive charge on both the histone- and dna-interacting surfaces. aligning the structures of hsrcc and scsmr1/prp revealed similar positive surface patches. however, uvr showed a rather negatively charged surface across the entire predicted nucleosome interaction surface, which argues strongly against an rcc1-like binding mode to nucleosomes. in addition, the uvr surface is generally devoid of strongly positively charged patches and, thus, does not display the characteristics of a bona fide dna-binding protein. comparing structure-based sequence alignments, it became evident that the overall structural similarity between the uvr and dmrcc 7-bladed β-propeller structures is striking but the histone-binding loop in dmrcc is not conserved in uvr. similarly, we did not find a potential dna-binding loop in uvr through structural comparison with dmrcc we conclude that the presence of histone- and dna-binding surfaces is a general feature of the chromatin association of eukaryotic rcc proteins but these are apparently absent in arabidopsis uvr this suggests that uvr may have lost the ability to bind to chromatin, corresponding to the differences in activity between the uvr and rcc proteins. as suggested by the structural analysis, recombinant uvr did not bind to reconstituted nucleosomes in vitro, either in its ground-state as homodimer or as uv-b-activated monomer. in contrast, recombinant rcc showed a clear electrophoretic mobility gel shift of the nucleosomes, demonstrating nucleosome binding.fig. uvr does not bind to nucleosomes in vitro. a purified recombinant rcc and uvr were electrophoretically separated on sds-page together or in the absence of reconstituted nucleosomes and stained by coomassie blue. samples were boiled or non-boiled before loading the gel. b electrop horetic mobility gel shift assay of nucleosome binding by rcc and uvr between - 10 μm rcc and - 40 μm uvr were added to reconstituted mononucleosomes. binding was assayed by emsa and visualized by ethidium bromide staining  contrary to the current models of uvr signalling, our data do not support binding of uvr to chromatin at target genes. moreover, no chromatin-based activity of uvr could be demonstrated experimentally. however, it has been rather well established that uvr activity requires uv-b-dependent interaction with cop which leads to hy stabilization. hy associates with target gene promoters and is required for uv-b-induction of a large portion of the uv-b-induced genes. thus, we propose that the effect of uvr8-cop on gene expression is through hy and its homolog hyh, without involving direct uvr chromatin association at the same target genes. however, we cannot exclude the existence under uv-b of short-lived and/or limited amounts of a possible uvr8-cop1-hy protein complex that was not detected by our qrt-pcr-based chip assays. arguing against this is the evidence from previous gel-based chip assays which firstly suggested that uvr associates with the hy promoter in the absence of uv-b and functional cop and secondly failed to detect chromatin association of gfp-cop. moreover, no evidence was found for hy being part of a uvr8-cop complex, nor did we identify direct interaction of uvr with hy in yeast two-hybrid assays. however, despite the prime role in uv-b signalling assigned to hy hy lacks a transcriptional activation domain and cannot alone activate transcription. thus, to better understand how uvr regulates gene expression through hy we now need to identify hy partner protein that provide a transcriptional activation domain and enable uv-b-responsive gene expression. 
these proteins act as guanine nucleotide exchange factors for ran gtpases and bind chromatin via histones. subsequent to the revelation of this sequence homology, evidence was presented showing that uvr activity involves interaction with chromatin at the loci of some target genes through histone binding. moreover, fusion of a vp activation domain to uvr did not alter expression of proposed uvr target genes in transient gene expression assays. this has led us to conclude that the cellular activity of uvr likely does not involve its specific binding to chromatin at target genes. plants perceive uv-b through the uv resistance locus photoreceptor and uvr activation leads to changes in gene expression such as those associated with uv-b acclimation and stress tolerance. in contrast to human rcc recombinant uvr also does not bind nucleosomes in vitro.
 plants perceive uv-b through the uv resistance locus photoreceptor and uvr activation leads to changes in gene expression such as those associated with uv-b acclimation and stress tolerance. albeit functionally unrelated, uvr shows some homology with rcc proteins from non-plant organisms at the sequence level. these proteins act as guanine nucleotide exchange factors for ran gtpases and bind chromatin via histones. subsequent to the revelation of this sequence homology, evidence was presented showing that uvr activity involves interaction with chromatin at the loci of some target genes through histone binding. this suggested a uvr mode-of-action intimately and directly linked with gene transcription. however, several aspects of uvr chromatin association remained undefined, namely the impact of uv-b on the process and how uvr chromatin association related to the transcription factor elongated hypocotyl, which is important for uv-b signalling and has overlapping chromatin targets. therefore, we have investigated uvr chromatin association in further detail.  unlike the claims of previous studies, our chromatin immunoprecipitation experiments do not confirm uvr chromatin association. in contrast to human rcc recombinant uvr also does not bind nucleosomes in vitro. moreover, fusion of a vp activation domain to uvr did not alter expression of proposed uvr target genes in transient gene expression assays. finally, comparison of the drosophila dmrcc and the arabidopsis uvr crystal structures revealed that critical histone- and dna-interaction residues apparent in dmrcc are not conserved in uvr  this has led us to conclude that the cellular activity of uvr likely does not involve its specific binding to chromatin at target genes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords arabidopsischipuv-buvr8chromatinrcc1photoreceptorhttp://dx.doi.org/ /501100001711schweizerischer nationalfonds zur förderung der wissenschaftlichen forschung31003a-153475ulm roman http://dx.doi.org/ /501100000781european research council310539ulm roman issue-copyright-statement© the author 2016 