micro-array analysis for genome-wide identification
ABSTRACT 
Nanobodies are single-domain antibody fragments derived from camelid heavy-chain antibodies . 
Because of their small size , straightforward production in Escherichia coli , easy tailoring , high affinity , specificity , stability and solubility , nanobodies have been exploited in various biotechnological applications . 
A major challenge in the post-genomics and post-proteomics era is the identification of regulatory networks involving nucleic acid -- protein and protein -- protein interactions . 
Here , we apply a nanobody in chromatin immunoprecipitation followed by DNA microarray hybridization ( ChIP-chip ) for genome-wide identification of DNA -- protein interactions . 
The Lrp-like regulator Ss-LrpB , arguably one of the best-studied specific transcription factors of the hyperthermophilic archaeon Sulfolobus solfataricus , was chosen for this proof-of-principle nanobody - assisted ChIP . 
Three distinct Ss-LrpB-specific nanobodies , each interacting with a different epitope , were generated for ChIP . 
Genome-wide ChIP-chip with one of these nanobodies identified the well-established Ss-LrpB binding sites and revealed several unknown target sequences . 
Furthermore , these ChIP-chip profiles revealed auxiliary operator sites in the open reading frame of Ss-lrpB . 
Our work introduces nanobodies as a novel class of affinity reagents for ChIP . 
Taking into account the unique characteristics of nanobodies , in particular , their short generation time , nanobody - based ChIP is expected to further streamline ChIP-chip and ChIP-Seq experiments , especially in organisms with no ( or limited ) possibility of genetic manipulation . 
INTRODUCTION
Chromatin immunoprecipitation ( ChIP ) is a widely used technique to measure DNA-binding events of transcription factors in vivo . 
ChIP , combined with DNA micro-array analysis ( ChIP-chip ) or high-throughput sequencing ( ChIP-Seq ) , allows genome-wide mapping of all locations where a factor is associated , through protein -- DNA or protein -- protein interactions ( 1,2 ) . 
In contrast to transcriptomics and proteomics that measure the consequences of the regulatory interactions ( changes in RNA or protein levels ) , which may be because of either direct or indirect ( cascade ) effects , ChIP-chip and ChIP-Seq provide information on the regulatory interactions themselves and are , therefore , the most direct ways to define regulons . 
An additional advantage of ChIP-chip and ChIP-Seq is that the analysis can be performed in a wild-type strain ; there is no need for a gene disruption mutant or a strain that overexpresses a tagged regulatory DNA-binding protein . 
The ChIP-chip procedures have been established for different organisms , ranging from prokaryotes and yeasts to higher eukaryotes , including mammals ( 3 -- 9 ) . 
In bacteria , ChIP-chip has been applied mainly to Escherichia coli ( 10,11 ) . 
The use of ChIP in archaea has been lagging behind and , to our knowledge , has only been applied to Halobacterium salinarum NRC-1 ( 12 -- 14 ) . 
A ChIP-chip assay consists of multiple sequential experimental steps . 
Living cells are first treated with formaldehyde , resulting in covalent cross-linking of DNA-associated proteins to DNA . 
Subsequently , nucleoprotein is extracted and sheared into shorter DNA fragments , usually by sonication . 
This preparation is then subjected to immunoprecipitation using an antibody specific for the protein of interest . 
After ChIP , the enriched nucleoprotein complexes are treated to hydrolyse the cross-linked complexes , and DNA is purified . 
Generally , the yield of ChIP DNA is too low and needs to be amplified before array hybridization . 
Given the large number of experimental parameters in a ChIP-chip experiment , it is not surprising that there is a wide variation in the design of different studies . 
One of the most critical determinants of a successful ChIP-based approach is the antibody ( 5,11,15,16 ) . 
ChIP antibodies should be capable of capturing specifically one single protein of a vast pool of DNA-binding proteins . 
It should also be considered that DNA binding and DNA -- protein cross-linking might provoke conformational changes in the nucleoprotein complexes that lead to epitope masking , causing false-negative outcomes , whereas cross-reactivity of the antibodies to non-cognate targets could generate falsepositive outcomes . 
Effects of epitope masking can be minimized by using polyclonal antibodies ( pAbs ) ( 17 ) . 
However , pAbs increase the frequency of false-positive outcomes , their production requires regular immunization and they exhibit batch to batch variability ( 18,19 ) . 
In comparison with pAbs , monoclonal antibodies ( mAbs ) suffer less from the aforementioned problems . 
However , the availability of high-quality ChIP-grade mAbs is apparently limited ( 11,20 ) . 
Epitope tagging , by homologous recombination-mediated knock-in of the tagged genes , could circumvent the lack of ChIP-grade mAbs . 
Although this technology is relatively straightforward for some well-established model organisms , such as Saccharomyces cerevisiae and E. coli ( 7,8,14,21 -- 23 ) , genetic tools to achieve this in many organisms such as Sulfolobus , one of the archaeal model organisms , are still limited ( 24,25 ) . 
Moreover , it is not excluded that the characteristics ( e.g. stability , folding efficiency , hydrophobi-city ) of a tagged protein may differ from those of the wild-type . 
Evidently , such potential differences can affect the outcome of the ChIP experiment . 
Monospecific antigen-binding domains can also be produced by microorganisms at a fraction of the cost of mAbs , and they might constitute a novel and valuable resource of ChIP-grade antibodies . 
Especially the recombinant single-domain antigen-binding fragments , such as Nanobodies , seem to be attractive for ChIP . 
Remarkably , the antibody repertoire of camelids contains , in addition to conventional antibodies , a novel class of antibodies comprising heavy chains only ( 26 ) . 
These antibodies , referred to as heavy-chain antibodies , bind their cognate antigen by virtue of one singlevariable domain , termed VHH or nanobody . 
In contrast , the antigen binding by conventional antibodies relies on variable regions of both heavy and light chains ( VH and VL , respectively ) . 
Therefore , construction of libraries of antigen-binding domains of conventional antibodies involves random association of VHs and VLs . 
Consequently , large libraries are required to restore all possible VH -- VL combinations , of which some may represent the original VH -- VL pairing as it was affinity matured in vivo during immunization with antigen . 
As camelid heavy-chain antibodies bind their target antigens by only one single domain , construction of large immune libraries to trap antigen-specific nanobodies has proven unnecessary ( 27,28 ) . 
Construction of libraries of antigen-binding repertoire of conventional antibodies is also complicated by the existence of multiple VH and VL gene families , whereas the vast majority of VHHs belong to one single sub-family ( 28 ) . 
The aforementioned technological advantages of constructing ` immune ' nanobody libraries , together with small size , recognition of unique epitopes , high affinity , high solubility , high expression yield in heterologous expression systems and easy tailoring , make nanobodies an interesting class of affinity reagents for various applications ( 27,29,30 ) . 
Here , we demonstrate the use of target-specific nanobodies in ChIP experiments . 
As a model system , we chose the well-characterized transcription regulator Ss-LrpB from the hyperthermoacidophilic archaeon Sulfolobus solfataricus ( 31 ) . 
Ss-LrpB belongs to the leucine-responsive regulatory protein ( Lrp ) family , a wide-spread and abundant family of regulators in prokaryotes , both bacteria and archaea ( 32,33 ) . 
Several regulatory targets of Ss-LrpB have already been identified by in vitro binding experiments and by in vivo gene expression analysis ( 34 ) . 
These targets include the regulator gene itself and a gene cluster juxtaposed to it , encoding a putative ferredoxin oxidoreductase and two permeases . 
In this work , different Ss-LrpB-specific nanobodies were generated and assessed for their capacity to capture spe-cifically the regulator , either free or bound to DNA . 
We then developed a nanobody - based ChIP protocol for S. solfataricus . 
The genome-wide application of nanobody - based ChIP for Ss-LrpB is demonstrated by implementation of the Roche NimbleGen microarray TM platform . 
The results presented here demonstrate the utility and specificity of nanobodies as a novel class of affinity reagents for ChIP . 
MATERIALS AND METHODS Protein purifications
Full-length non-tagged Ss-LrpB protein was produced recombinantly in E. coli and was purified by heat treatment and ion exchange chromatography , as previously described ( 35 ) . 
The His-tagged C-terminal 2 + domain of Ss-LrpB was purified by Ni affinity chromatography ( 36 ) . 
LysM and Ss-Lrp proteins were produced and purified by the same procedure as the Ss-LrpB purification . 
For LysM , E. coli BL21 ( DE3 ) was first transformed with construct pLUW632 ( 37 ) . 
After purification , the Ss-LrpB and Ss-Lrp preparations were dialysed against 20 mM of Tris -- HCl ( pH 8.0 ) , 50 mM of NaCl , 0.4 mM of ethylenediaminetetraacetic acid ( EDTA ) , 0.1 mM of DTT , 12.5 % of glycerol and the LysM preparation against 20 mM of Tris -- HCl ( pH 8.0 ) and 20 % of glycerol 
After identification as described later in the text , the Ss-LrpB-specific VHH ( nanobody ) genes were cloned into the pHEN6c vector , which allows expression of nanobodies in fusion with His6 tag ( 38 ) . 
Expression and purification of nanobodies were performed as previously described ( 39 ) . 
Protein concentrations in the case of Ss-LrpB expressed in monomeric units were determined by ultraviolet absorption at 280 nm and by densitometric analysis of Coomassie stained sodium dodecyl sulphate ( SDS ) -- polyacrylamide gel ( PAG ) . 
Generation of Ss-LrpB-specific nanobodies
Ss-LrpB-specific nanobodies were generated by immunizing an alpaca ( Vicugna pacos ) with purified fulllength Ss-LrpB . 
Using peripheral blood lymphocytes of the animal , a VHH library was constructed , and specific nanobodies were selected according to published methods ( 38 ) . 
Surface plasmon resonance
Surface plasmon resonance ( SPR ) measurements of the interactions of Ss-LrpB-specific nanobodies with their antigen were performed with a Biacore T200 instrument . 
All measurements were performed in phosphate-buffered saline ( PBS ) at 25 C. CM5 chips ( GE Healthcare ) were used to covalently couple Ss-LrpB via its primary amines of lysine residues . 
Ss-LrpB was immobilized onto the chip until the signal reached 500 resonance units ( RUs ) . 
Measurements were performed by applying various concentrations of nanobodies ( between 3 and 500 nM ) as analyte to the chip , at a flow rate of 30 ml/min . 
An association phase of 150 s was followed by a dissociation phase of 600 s. Regeneration was achieved by washing the chip with 10 mM of glycine hydrochloride ( pH 2.0 ) for 20 s , at a flow rate of 60 ml/min . 
The association and dissociation curves of the sensorgrams were analysed with the Biacore Evaluation software , version 2.0 , yielding kinetic and equilibrium binding constants . 
Epitope analysis was done with 250 , 500 or 750 nM of each nanobody , either alone or combined , also at a flow rate of 30 ml/min . 
Each association phase lasted 200 s. 
Immunoprecipitation (pull-down) assays with crude cell extracts
Escherichia coli BL21 ( DE3 ) crude cell extracts containing one of the three Lrp-like transcription factors from S. solfataricus ( Ss-LrpB , LysM or Ss-Lrp ) , expressed from recombinant pET24 vectors , were used for these experiments . 
Crude extracts from BL21 ( DE3 ) containing an empty pET24 vector served as negative control . 
Cell pellets from 20 ml cultures were resuspended in 1 ml of IP buffer [ 150 mM of NaCl , 50 mM of Tris -- HCl ( pH 8.0 ) , 1 % of Triton X-100 , 0.5 % of NP-40 , 1 % of deoxycholate ] , sonicated and centrifuged . 
Aliquots of 200 ml of the supernatants were incubated with different amounts of His-tagged nanobodies for 20 min at room temperature . 
Subsequently , the pull-down was performed using Nickel-NTA magnetic particles ( Bio-Nobile ) following supplier 's recommendations . 
The nanobody -- antigen complexes were eluted with 100 ml of PBS containing 250 mM of imidazole , and the eluted proteins were analysed using 12 % of SDS -- PAG electrophoresis ( PAGE ) . 
Native protein polyacrylamide gel electrophoresis
Native protein gel electrophoresis was performed with 10 % of Tris -- glycine gel ( Invitrogen ) . 
Each reaction mixture , with a total volume of 20 ml , contained 20 mg of Nb9 and/or 10 mg of the respective Lrp-like protein . 
Reaction mixtures were incubated for 30 min at room temperature before gel analysis . 
The electrophoresis was performed in Tris -- glycine ( pH 8.5 ) electrophoresis buffer at 125 V for 4 -- 5 h . 
The gel was stained with Coomassie blue . 
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays ( EMSAs ) were performed with labelled DNA prepared by polymerase chain reaction ( PCR ) . 
One of the two oligonucleotides was 50-end labelled with 32P using [ g-32P ] - adenosine triphosphate ( Perkin Elmer ) and T4 polynucleotide kinase ( Fermentas ) . 
The PCR mixtures contained Taq DNA polymerase ( Ready Mix , Sigma-Aldrich ) , the labelled primer , a second non-labelled primer and the recombinant vector pUC18 / o Ss-lrpB or pBendBox1 as p template ( 31 ) . 
Primer sequences are given in Supplementary Table S1 . 
Labelled DNA fragments were purified from PAG . 
EMSA experiments were performed as previously described ( 40 ) . 
Binding reactions were allowed to equilibrate for 20 min at 37 C in Lrp-binding buffer [ 20 mM of Tris -- HCl ( pH 8.0 ) , 1 mM of MgCl2 , 50 mM of NaCl , 0.4 mM of EDTA , 0.1 mM of DTT , 12.5 % of glycerol ] . 
For binding reactions in which Ss-LrpB and nanobodies were combined , Ss-LrpB was pre-incubated with DNA for 20 min before addition of nanobody . 
Cell culture and formaldehyde cross-linking
Sulfolobus solfataricus P2 ( DSMZ 1617 ) was cultured aerobically by shaking at 80 C in Brock medium ( 41 ) supplemented with 0.1 % of tryptone as carbon and nitrogen source . 
Depending on the downstream application , 50 ml or 200 ml cultures were grown . 
When cells were in mid-exponential growth phase [ at optical density ( OD ) 600 nm of 0.5 ] , the cultures were cooled to 37 C , and formaldehyde was added to a final concentration of 1 % , while shaking for 5 min , unless otherwise noted . 
The cross-linking reaction was quenched by adding glycine to a final concentration of 125 mM , followed by an additional incubation of 5 min at 37 C. To find the optimal cross-linking time , 50 ml cultures were formaldehyde-treated for different periods , centrifuged and sonicated for 5 min ( see later in the text for sonication details ) . 
After this treatment , 200 ml aliquots were subjected to phenol extraction to separate proteinfree from complexed DNA . 
This extraction was done by mixing with 2 volumes of phenol/chloroform/isoamyl alcohol ( 25:24:1 ) followed by 5 min of centrifugation a 
20 817g . 
The protein-free DNA fractions were recovered from the upper aqueous phase by ethanol precipitation . 
Finally , the extracted DNA was treated with 16.5 ng RNase ( Invitrogen ) for 2 h at 37 C and column-purified . 
The degree of cross-linking was assessed by quantitative PCR ( qPCR ) analysis of the protein-free DNA samples relative to DNA from a non -- cross-linked sample , which represented total DNA , and was prepared by extraction and purification as described for cross-linked samples . 
Sonication
Cross-linked cells from either 50 ml or 200 ml cultures were centrifuged at 3220g for 10 min ; cell pellets were washed twice with PBS and resuspended in 3 ml IP buffer . 
Sonication was performed with a Bioblock Scientific-Vibracell sonicator at 20 % of the maximal amplitude , in a pulsed operating mode with 9 s rest in between each 3 s of operation . 
The total operating time was 9 min , unless otherwise stated . 
Cells were continuously cooled during sonication . 
After sonication , the samples were centrifuged at 21 000g for 15 min , and the supernatants were used for ChIP as described later in the text . 
For small-scale sonication tests , samples from 50 ml cultures were cross-linked for 5 min and sonicated with total operation times between 3 and 30 min . 
For each sample , a 200 ml aliquot was purified by phenol extraction , and a 200 ml aliquot was de-cross-linked , as described later in the text . 
These two samples , corresponding to proteinfree DNA and total genomic DNA , respectively , were analysed by qPCR to calculate the ratio of cross-linked protein -- DNA complexes versus total DNA . 
The DNA size distribution was analysed by 1 % agarose gel electrophoresis of de-cross-linked samples . 
Chromatin immunoprecipitation
For each ChIP assay , 0.5 mg of purified nanobody was added to 3 ml cross-linked sonicated sample obtained as previously described after centrifugation . 
The mixtures were incubated overnight at 4 C . 
In parallel , 1 ml of His-Select Nickel Affinity Gel suspension ( Sigma-TM Aldrich ) was blocked overnight at 4 C with IP buffer containing 0.5 % of bovine serum albumin , and it was added the next day to the mixtures of the nanobody -- cross-linked/sonicated sample . 
After 2 h incubation at room temperature , the gel pellets were washed three times with 4 ml of IP buffer each . 
The ChIP-enriched fractions were then eluted from the gel pellet by the addition of 400 ml of elution buffer [ 50 mM of Tris -- HCl ( pH 8.0 ) , 1 % of Triton X-100 , 0.5 % of NP-40 , 1 % of deoxycholate , 1 % of SDS , 300 mM of NaCl , 250 mM of imidazole ] and a further incubation of the gel mixture at room temperature for 1 h. Subsequently , 400 ml samples were subjected to de-cross-linking by incubation at 55 C for 16 h , followed by addition of 1 volume of protein lysis buffer [ 10 mM of Tris -- HCl ( pH 8.0 ) , 1 mM of EDTA , 31 nM of proteinase K , 0.9 mg/ml of glycogen ] and incubation at 37 C for 2 h. DNA was recovered from the mixture by phenol extraction , followed by a treatment with 50 ml of RNase A solution ( 33 ng/ml ) at 37 C for 2 h and by column purification ( Qiagen ) . 
Input DNA , sampled after sonication , was also de-cross-linked and purified as aforementioned . 
Finally , all ChIP samples were amplified by whole-genome amplification using the WGA-2 Kit ( Sigma-Aldrich ) following the manufacturer 's instructions for ChIP-chip samples in which the heat-induced fragmentation step is omitted . 
Mock immunoprecipitations were performed with a BcII b-lactamase-specific nanobody ( here referred to as NbX ) ( 38 ) . 
For the spiking immunoprecipitation experiment , covalently cross-linked Ss-LrpB -- DNA complexes were prepared as follows : formaldehyde was added ( 1 % , final concentration ) to a mixture of 7.7 pM of pUC18p/oSs-lrpB plasmid DNA ( 31 ) and 1.5 nM Ss-LrpB protein , and it was incubated at room temperature for 10 min . 
The reaction was then quenched by adding glycine ( final concentration 125 mM ) and by incubating for 5 min at 37 C . 
The mixture was sonicated as previously described . 
For all ChIP samples , enrichment was evaluated by qPCR relative to input DNA . 
Microarray design and data analysis
For DNA microarray analysis , a custom 385 K highdensity tiling array was designed and manufactured by NimbleGen ( Madison , WI , USA ; www.nimblegen . 
TM com ) . 
The probes ( 50 -- 75 bases , with an average tiling interval of 14 bases ) were designed based on the S. solfataricus P2 genome sequence ( 42 ) . 
Each probe occurred twice on each array . 
Sample labelling , hybridization and array processing were performed at NimbleGen . 
The ChIP input and output samples TM were labelled with Cy3 and Cy5 , respectively . 
The Ringo package ( 43 ) was applied to analyse the raw data sets , including removal of unreliable signals , normalization and smoothing of the data and assignment of ChIP-enriched regions ( chers ) . 
Venn diagrams were generated using ChIPpeakAnno ( 44 ) . 
All microarray data are available in Supplementary Material . 
Quantitative real-time PCR
qPCR reactions were performed with a My-iQ Single TM Colour Real-time PCR system ( Bio-Rad ) . 
Amplification and detection were achieved using SYBR Green Master Mix ( Bio-Rad ) . 
Each 25 ml of PCR reaction contained 10 ng of template DNA and 200 nM of each primer . 
Cycling conditions ( 10 min at 94 C and 40 cycles of 30 s at 94 C , 30 s at 60 C ) were followed by melt curve analysis . 
Amplicon sizes were between 100 and 250 bp ; all primers are listed in Supplementary Table S1 . 
Quantification cycles ( Cq ) were determined by My-iQ software ( Bio-Rad ) , and relative quantitative analysis was done using the 2 method ( 45 ) . 
All measurements Ct were normalized to reference DNA , a non-related sequence fragment amplified by PCR from E. coli gDNA , and spiked at 30 ng/sample before sonication . 
Experiments were performed at least in duplicate 
RESULTS
Generation , affinity determination and epitope mapping of Ss-LrpB-specific nanobodies 
An alpaca was immunized by six injections at weekly intervals , each time with 200 mg of purified full-length recombinant Ss-LrpB . 
The plasma obtained 4 days after the last injection showed an end-titre of 10 [ the plasma 5 dilution which still gives an antigen-specific enzyme-linked immunosorbent assay ( ELISA ) signal , which is 3-fold above background ] . 
Subsequently , an immune VHH library comprising 10 independent transformants was 8 constructed from the peripheral blood lymphocytes taken from the immunized animal 4 days after the last immunization . 
The library was subjected to two distinct bio-panning experiments , against either full-length Ss-LrpB or the C-terminal domain of Ss-LrpB ( ` Ss-LrpB CTerm ' ) ( 36 ) . 
We included the selection against Ss-LrpB C-Term because the N-terminal domain of Lrp-like transcription regulators contains the DNA-binding helix-turn-helix motif . 
By selecting binders that recognize epitopes located in the C-terminal oligomerization and effector binding domain , we aimed to increase the chances of obtaining nanobodies interacting with regions of the regulator that remain accessible on DNA binding . 
After three rounds of selection , > 200 clones were randomly chosen to assess their nanobody to recognize Ss-LrpB in an ELISA . 
The nanobody nucleotide sequence of 75 ELISA positive clones ( 19 and 56 clones from panning against full-length Ss-LrpB and Ss-LrpB CTerm , respectively ) was determined , resulting in 47 different genes ( 10 and 37 against full-length Ss-LrpB and Ss-LrpB C-Term , respectively ) , encoding proteins that differ from each other in at least 1 amino acid . 
Several of the nanobody sequences possess a nearly identical CDR3 ( the third hypervariable antigen-binding loop ) . 
Of these binders , it is known that they interact with the same epitope , although sequence differences in other antigen-binding loops ( CDR1 and CDR2 ) might affect the affinity for the antigen ( 46 ) . 
Remarkably , 24 different CDR3 sequences could be discerned for the Ss-LrpB C-Term target , and only two different CDR3 sequences were obtained for the full-length target . 
Twelve nanobody genes ( 2 for the full-length and 10 for the C-Term ) were re-cloned in an expression vector in fusion with a His6 tag , expressed and the recombinant protein purified to homogeneity to determine the affinity and to identify binders that recognize a unique epitope by both , surface plasmon resonance ( SPR ) and ELISA . 
Based on these criteria , it was decided to continue with three nanobodies , designated Nb1 , Nb11 and Nb9 ( Figure 1A ) . 
The first two nanobodies originated from the pannings on the full-length Ss-LrpB , the Nb9 was retrieved from the C-Term selections . 
The affinity ( KD ) as obtained from SPR ranged from 40 ( Nb11 ) to 1 nM ( Nb9 ) ( Table 1 ) . 
The high affinity of Nb9 is attributed to a high association rate constant ( kon ) , which is 6-and 64-fold higher than the kon of Nb1 and Nb11 , respectively ( Table 1 ) . 
The koff rate ( 10 s ) is similar 3 1 for all three nanobodies . 
Therefore , Nb9 is the best candidate nanobody for ChIP in terms of affinity . 
SPR experiments , involving the sequential injection of two nanobodies at target saturating concentrations on the immobilized Ss-LrpB , further demonstrated that Nb1 , Nb11 and Nb9 indeed bind to independent sites on the regulator ( Figure 1B ) . 
This figure shows that Nb1 and Nb9 bind concomitantly to the immobilized Ss-LrpB protein : a second injection of the same nanobody did not result in a significant RU change , indicating saturation of the first occupied epitope . 
However , a second injection of the counterpart nanobody resulted in a similar RU change as observed after the first injection . 
Similar results were obtained for Nb1/Nb11 and Nb9/Nb11 combinations ( data not shown ) . 
The same epitope grouping whereby the three nanobodies recognize three independent epitopes on the same antigen was further confirmed by ELISA ( data not shown ) . 
Specificity of the Ss-LrpB–nanobody interaction
Thus far , all interaction analyses were performed with purified immobilized Ss-LrpB and , therefore , do not address the specificity of the nanobodies for their cognate target in a complex mixture . 
Moreover , immobilization of antigen can lead to ( partial ) denaturation , thereby exposing epitopes that are not present or accessible in the native soluble protein . 
To evaluate the capacity of nanobodies to capture Ss-LrpB in solution and to provide further information on their specificity , we performed pull-down assays with the three selected nanobodies , using total protein extracts from E. coli cells expressing recombinant Ss-LrpB ( Figure 2A ) . 
It is clear that different amounts of Nb1 ( from 6.5 to 25 mg ) capture specifically Ss-LrpB from crude cell extract , and that E. coli endogenous proteins are not observed after pull-down . 
Similar results were obtained in pull-down assays with Nb9 and Nb11 ( data not shown ) . 
The control nanobody , NbX with specificity for an antigen that is not expressed by E. coli , fails to capture Ss-LrpB or any other protein , thereby demonstrating its suitability as a negative control in ChIP ( Figure 2A , lower panel ) . 
However , the lack of cross-reactive antigens in E. coli does not guarantee that proteins displaying homology with Ss-LrpB are absent in S. solfataricus . 
In particular , S. solfataricus Lrp family members , other than Ss-LrpB , may cross-react with Ss-LrpB-specific nanobodies . 
Two such Lrp family members , Ss-Lrp ( encoded by Sso0606 ) and LysM ( encoded by Sso0157 ) , have already been reported to exhibit 31 and 25 % sequence identity and 60 and 52 % sequence homology to Ss-LrpB , respectively ( 32,47 ) . 
In addition , these Lrp-like transcription factors exhibit large structural homologies ( 33 ) . 
Nevertheless , physical interactions between Nb9 and LysM could be excluded from the results of a native protein PAGE with mixtures of the two proteins ( Figure 2B ) . 
The Lrp-like regulators migrate out of the gel towards the cathode because of their high-isoelectric point , whereas the nanobodies migrate into the gel because of their overall negative charge at pH 8.5 . 
Complexes between Lrp proteins and nanobodies enter into the gel , but with a slower migration velocity than the nanobodies alone . 
Nb9 forms a stable complex with Ss-LrpB , whereas this type of complex is not observed with LysM ( Figure 2B ) . 
Pull-down assays with total protein extracts from E. coli cells overexpressing Ss-Lrp or LysM further demonstrate the inability of Nb9 to capture these proteins ( Figure 2C ) . 
Similar results were obtained with Nb1 and Nb11 ( data not shown ) . 
Interaction between nanobodies and DNA-bound Ss-LrpB
DNA binding might influence the interaction between antibody and transcription factor because of epitope masking and/or conformational changes . 
Here , we used EMSAs ( Figure 3 ) to investigate the possible occurrence of supershifts as readout for nanobodies associating in vitro with Ss-LrpB in complex with its cognate target DNA , and as an indicator for the suitability of nanobodies for ChIP . 
Using a DNA fragment containing a single binding site for SsLrpB , stable complexes are formed , in which the semi-palindromic binding site is bound by an Ss-LrpB dimer ( Figure 3A ) . 
It is shown that the nanobodies do not provoke a band shift of the free DNA in an EMSA ( last lane ) . 
However , the addition of Nb1 or Nb11 to pre-equilibrated Ss-LrpB -- DNA complexes shifts the protein -- DNA equilibrium towards dissociation of the preformed complexes . 
The Nb1 induced dissociation occurs at lower nanobody concentration than with Nb11 , indicating that the dissociation is proportional to the affinity of the nanobody -- Ss-LrpB interaction . 
This dissociation can be explained as resulting either from a direct association of the nanobody with the DNA-binding face o the Ss-LrpB , so that the nanobody competes effectively with the DNA for the Ss-LrpB protein , or from a nanobody - induced conformational change in Ss-LrpB that affects its DNA binding allosterically in a negative fashion . 
The former explanation would discourage the use of Nb1 and Nb11 in ChIP , as their Ss-LrpB epitopes are probably unavailable in cross-linked nucleoprotein . 
Conversely , Nb9 , with specificity to the C-terminal domain of Ss-LrpB , binds to the Ss-LrpB -- DNA complex as evidenced by supershifting ( Figure 3A , middle panel ) . 
This suggests that Nb9 recognizes an epitope that is not directly involved in , or affected by , DNA binding . 
Therefore , Nb9 is definitely the best candidate for ChIP . 
Ss-LrpB interacts with its main DNA targets ( control region of own gene and of the neighbouring pyruvate ferredoxin oxidoreductase ( porDAB ) operon ) by binding cooperatively to three regularly spaced semi-palindromic binding sites ( 31,34 ) . 
On binding , all three sites of the Ss-lrpB control region , the three protein dimers closely interact and are assumed to wrap the DNA , causing large conformational changes with respect to the nucleoprotein complex with a single binding site ( 35 ) . 
Besides the formation of three specific complexes , nonspecific binding is observed on adding larger amounts of Ss-LrpB , visible as a complex ( annotated ` NS ' ) of variable relative mobility in gel and dependent on the protein concentration ( 31 ) . 
To analyse Nb9 interaction with nucleoprotein complexes involving three Ss-LrpB binding sites , which are expected to be prevalent in vivo , an EMSA was performed with a tripartite operatorcontaining DNA fragment ( Figure 3B ) . 
At the highest Ss-LrpB concentration used , the addition of Nb9 causes supershifting and the disappearance of the triple bound Ss-LrpB -- DNA complex ( C3 ) , indicating the recognition of these complexes by Nb9 . 
At the lowest Ss-LrpB concentration used , with all three distinct complexes ( C1 , C2 and C3 ) being present , Nb9 interacts only with complexes having two and three Ss-LrpB dimers bound ( C2 and C3 ) . 
These data , in conjuncture with those presented in Figure 3A ( middle panel ) , suggest that although Nb9 binds all three complexes , it preferentially interacts with complexes involving two and three Ss-LrpB dimers . 
Optimization of the nanobody -assisted ChIP assay for S. solfataricus
Cross-linking conditions
The formaldehyde cross-linking of DNA -- protein complexes is a crucial step in ChIP ( 17 ) . 
As this process is temperature-dependent and is reversed at high temperatures , it is impossible to perform cross-linking at physiological temperatures of hyperthermophilic organisms . 
Although formaldehyde-induced fixation of hyperthermophilic archaea chromatin works sufficiently well at room temperature ( 48,49 ) , we performed formaldehyde cross-linking at 37 C which is , as compared with room temperature , closer to hyperthermophiles ' physiological temperature . 
Cross-linking time is also an important parameter : a time that is too short might lead to insufficient cross-linking , and as a consequence to inability to detect an interaction , whereas excessive cross-linking might lead to epitope unavailability because of epitope masking or aggregation ( 11,17,50 ) . 
To optimize the cross-linking time , we performed a time-course experiment in which the efficiency of cross-linking was evaluated by separating cross-linked from non -- cross-linked DNA with phenol extraction followed either by gel electrophoresis analysis ( see Supplementary Figure S1 ) or by qPCR quantification of SsLrpB-target and non-target genomic regions ( Figure 4A ) . 
For all genomic regions tested , of which two are shown in Figure 4A , the fraction of protein -- cross-linked DNA ( over total DNA ) reached values between 84 and 99 % after 1 min cross-linking . 
Moreover , these values did not change significantly on increasing the cross-linking time . 
Cross-linking efficiencies varied somewhat depending on the genomic region , which can be explained by differences in the abundance of genome-associated proteins ( 51 ) . 
In conclusion , formaldehyde treatment for 1 min at 37 C is sufficient to cross-link S. solfataricus chromatin . 
This time is considerably shorter than the cross-linking time reported previously in eukaryotic or bacterial ChIP protocols , which varies from 10 min to several hours ( 7,50,52 ) . 
Note that we analysed cross-linking globally while individual proteins can have varying cross-linking efficiencies . 
To ensure successful cross-linking of Ss-LrpB , we decided to perform the cross-linking for 5 min for all further experiments . 
Sonication conditions
Sonication , to fragment the DNA in appropriate sizes and to solubilize the chromatin , is one of the most variable and critical steps in ChIP , and optimal conditions depend on cell type , cell quantity , chromatin structure and so forth ( 5 ) . 
Insufficient sonication might lead to loss of resolution of binding events , whereas over-sonication can result in the disruption of cross-linked protein -- DNA complexes and introduction of noise in the microarray data . 
To determine the optimal sonication conditions , we performed small-scale tests for different periods of time ranging from 3 to 30 min and analysed both fragment size distribution and dissociation of protein -- DNA complexes ( Figure 4B and C ) . 
Sonication for 6 , 9 and 18 min yielded similar results with DNA size distribution of 0.2 -- 0.6 kb . 
On longer sonication ( 18 min ) , cross-linked protein -- DNA complexes tend to dissociate ( Figure 4C ) . 
The extent of dissociation was somewhat variable , depending on the genomic region under study . 
To ensure both the stability of cross-linked protein -- DNA complexes and an optimal fragment size distribution , we chose to sonicate for 9 min in further experiments . 
Minimal amount of cells
The number of cells subjected to ChIP is also an important element for a successful assay . 
It needs to be sufficiently high to obtain robust results ( 5 ) , whereas it also affects the concentration of targets , so that i combination with the antibody affinity ( and specificity ) it might influence the outcome as well . 
Cell counting by plating and by microscopy indicated a cell density of 2 10 cells/ml for a cell suspension with 7 OD600 nm of 0.5 ( exponential growth phase ; data not shown ) . 
Taking into account that S. solfataricus is a haploid species characterized by a long G2 cell cycle phase ( 53,54 ) , most cells are expected to have two chromosomal copies , and a 50 ml culture at an OD600 nm of 0.5 is expected to harbour 1 -- 2 10 copies of each 9 genomic binding site . 
To determine the minimal amount of cells required for ChIP-based DNA enrichment with Ss-LrpB-specific nanobodies , a preliminary immunoprecipitation assay was performed with Nb9 ( Figure 4D ) . 
Here , different amounts of in vitro prepared cross-linked Ss-LrpB -- DNA complexes , containing the Ss-lrpB operator , were added to a constant amount of cross-linked S. solfataricus cells . 
The mixture was subjected to ChIP by Nb9 . 
The enrichment of the Ss-lrpB operator , compared with input DNA and normalized to E. coli reference DNA that was added to all samples before sonication , in the immunoprecipitated DNA was analysed by qPCR . 
Likewise , an unrelated genomic region , not bound by Ss-LrpB , was analysed as negative control . 
No ChIP enrichment was observed using cells from 50 ml culture ( Figure 4D ) . 
In contrast , after spiking , the samples with different amounts of cross-linked Ss-LrpB -- DNA complexes , enrichments exceeding a 4-fold ratio ( log2 value of 2 ) , were observed . 
Parallel ChIP with control nanobody NbX showed no enrichment ( data not shown ) . 
These experiments suggest 9 that at least 2 -- 3 10 specific complexes need to be present for detection by qPCR . 
Based on this result , 200 ml cultures , corresponding to 4 10 cells , were 9 used in subsequent ChIP-chip experiments . 
Comparative analysis of ChIP performance of nanobodies using predefined DNA targets
Although Nb9 is the most promising nanobody for ChIP in terms of affinity and epitope location , we compared the performance of the three Ss-LrpB-specific nanobodies by evaluating nanobody - mediated enrichment of known Ss-LrpB binding sites by qPCR and ChIP-chip . 
To avoid variability introduced by ChIP , the same ChIP DNA was used for both qPCR and ChIP-chip . 
In the first approach , enrichment of known target regions wa analysed as the evaluation criterion ( Figure 5 ) . 
A nontarget genomic region and mock immunoprecipitation with the nanobody NbX were used as negative controls . 
By using qPCR as readout , significant enrichment was observed with negative controls in ChIP DNA , as compared with the input DNA ( log2 values between 1 and 3 ) . 
Although the whole-genome ampli-fication protocol might result in a minimal amplification bias ( 16 ) , we observed a bias towards more efficient amplification of longer DNA molecules ( data not shown ) , probably because longer DNA fragments might anneal to more primers yielding a larger number of amplification products . 
This bias possibly explains the observed background enrichment , as the molecular weight of the reference E. coli DNA is significantly lower than the average molecular weight of the chromatin DNA . 
Therefore , amplification could cause a higher ratio of chromatin DNA/reference DNA in the ChIP DNA as compared with the unamplified input DNA , irrespective of immuno-enrichment ( Figure 5A ) . 
Nevertheless , this bias does not affect the assessment of the ChIP enrichments , as they were calculated based on the relative fold enrichment and were compared with the ChIP enrichment of the negative control NbX . 
The experiment with the Nb9 resulted in 500 - and 33-fold enrichment of the Ss-lrpB and porDAB operators DNAs , respectively ( Figure 5A ) . 
This difference might reflect the difference in the binding affinities of Ss-LrpB for the two operators . 
The use of Nb1 and Nb11 enriched the target Ss-lrpB operator 47 - and 62-fold , respectively . 
Furthermore , the Nb1 and Nb11 enrichment of porDAB operator DNA failed to exceed the background levels . 
Next , genome-wide ChIP-chip experiments were performed with DNA prepared with each of the nanobodies , and raw log2 fold-enrichment values were compared for the genomic regions known to bind Ss-LrpB ( Figure 5B ) . 
Although the sensitivity of this assay is lower than qPCR , the trends of the peaks confirm th relative enrichment ratios observed with qPCR . 
For the porDAB operator region , absolute log2 values were somewhat higher , but the ChIP curve obtained with control nanobody NbX coincided with those of Nb1 and Nb11 ( data not shown ) . 
The ChIP-chip derived binding peaks for the known autoregulatory binding sites exhibit an unexpected shape , centred over the coding part of the gene rather than over the operator region ( Figure 5B ) . 
This observation prompted us to re-investigate autoregulatory binding of Ss-LrpB , and indeed , two additional potential binding sites were predicted in silico in the open reading frame ( ORF ) sequence ( Figure 6A ) . 
These sites , tentatively called Box4 and Box5 , are located at the 30-end of the ORF with a spacing of 26 bp . 
Based on sequence similarity with the Ss-LrpB consensus sequence ( 56 ) , both sites are expected to be low-affinity sites ( Figure 6B ) . 
To further investigate possible Ss-LrpB binding within the ORF , EMSAs were performed with a fragment encompassing the promoter region only , both the promoter region and the coding region and with a fragment spanning the coding region only ( Figure 6C and D ) . 
In the former case , three complexes ( C1 -- C3 ) are formed , whereas a fourth complex ( C4 ) that migrates slower than the other three complexes ( C1 -- C3 ) is clearly present when the DNA fragment comprises both the promoter and the ORF . 
In contrast to Ss-LrpB complexes with the fragment containing the promoter region-only , the presence of the third complex ( C3 ) is seriously reduced , obviously in favour of forming a new complex , C4 . 
This suggests a cooperative binding to additional binding sites within the ORF . 
Supplementary DNA deformations ( looping ) and a higher protein stoichiometry may explain the significant reduction in the relative mobility of complex C4 . 
Furthermore , low-affinity binding to the ORF fragment is inferred ( Figure 6D ) , as two nucleoprotein complexes are detected in these EMSA ( C1 and C2 ) , although they result in smearing , reflecting binding instability . 
In conjunction , we provide strong evidence both in vivo and in vitro that Ss-LrpB binds two additional binding sites in the Ss-lrpB ORF , located 392 bp downstream of Box1 and oriented on the same side of the DNA helix ( with a centre-to-centre distance of four helical turns ) 
Thus far we analysed the ChIP enrichment of two highaffinity Ss-LrpB targets . 
Two other known Ss-LrpB targets , the operator regions of Sso2126 and Sso2127 , bind Ss-LrpB at a single site in vitro ( 34 ) , and Ss-LrpB indeed exerts a weak activation effect on gene expression of these targets . 
After inspection of the ChIP-chip binding profiles , recorded in the growth conditions in which the expression of Sso2126 and Sso2127 genes was analysed , none of the nanobodies enriched these sequences . 
Given the weak regulatory effect of Ss-LrpB on these targets under the growth conditions used , the Ss-LrpB binding affinity for these sequences might be low , or the Ss-LrpB is only binding to these recognition sites in a subpopulation of cells , possibly because of the effect of cofactors . 
Comparative analysis of ChIP performance of nanobodies using genome-wide data
In an alternative approach , genome-wide ChIP-chip binding profiles were evaluated to assess the performance of different nanobodies ( Figure 7 ) . 
Binding patterns obtained with Nb1 and Nb11 almost completely overlap with the patterns obtained with control NbX and , consequently , fail to reveal any novel potential Ss-LrpB binding sites ( Figure 7A , first and third panel ) . 
The two sites identified by Nb11 at cut-off of 1.0 and the site identified both by Nb11 and Nb1 at cut-off of 0.8 ( Figure 7B ) are considered as false-positive sites because the log2 enrichment of the negative control NbX corresponding to these sites are 0.93 , 0.82 and 0.79 , respectively . 
The mean log2 enrichment over the whole genome by NbX is 0.22 . 
In contrast , the binding profile obtained with Nb9 showed significant novel Ss-LrpB binding regions throughout the entire genome , besides the previously known Ss-LrpB target sites ( Figure 7A , middle panel ) . 
Depending on the significance threshold , ChIP-chip analysis using Nb9 revealed between 36 ( cutoff = 2-fold or 1.0 log2-fold enrichment ) and 181 ( cutoff = 1.5-fold or 0.6 log2-fold enrichment ) novel putative Ss-LrpB binding sites ( Figure 7B ) . 
The ChIP-chip signals of most of the newly discovered potential binding sites , called ChIP-enriched regions ( chers ) , were higher than that of the Ss-lrpB operator region . 
To further validate the validity of these chers to represent genuine novel Ss-LrpB genomic association sites , qPCR analysis was performed for a selection of 13 chers that scored a log2 ChIP-chip value between 1.0 and 2.0 ( Figure 8 ) . 
All these chers showed enrichment in qPCR , and for more than half of them , enrichment values far exceeded the background enrichment level . 
Therefore , the use of Nb9 leads to the discovery of novel potential targets with ChIP-chip , whereas the use of Nb1 and Nb11 does not , although qPCR analysis shows enrichment of the main target ( p/o Ss-lrpB ) by these latter Nbs ( Figure 5A ) . 
A statistically solid identification and further analysis of novel Ss-LrpB targets in the context of the physiological function of the transcription factor is beyond the scope of this work and will be published elsewhere . 
DISCUSSION
Chromatin immunoprecipitation is a valuable technique , especially in combination with deep sequencing or microarray analysis to decipher gene regulatory networks . 
However , its success is largely dependent on the quality of the antibodies ( i.e. specificity and affinity for its cognate antigen ) ( 5,11,15,16 ) . 
Cross-reactivity of the antibody with other non-cognate antigens is an important source of high background signals and false-positive outcomes in genome-wide ChIP assays . 
A study with antibodies directed against modified histones has demonstrated a high level of specificity problems , as > 20 % of a panel of tested antibodies , including those with a ` ChIP-grade ' label , were shown to fail in ChIP experiments ( 18 ) . 
As argued in the ` Introduction ' section , recombinant antibodies constitute an interesting and renewable source of monospecific antibodies for various applications including ChIP . 
What is the problem with pAbs and mAbs in ChIP ? 
The polyclonal antibody preparations consist of a mixture of different antibodies , each with a different epitope recognition mode . 
Hence , it can be argued that pAbs are to be preferred over mAbs because of lower incidences of epitope masking in the cross-linked chromatin ( 17 ) . 
However , the pAbs are obviously less suitable for ChIP in terms of specificity ( 18,19 ) , and their use increases the risk of association to non-cognate antigens , thus crossreaction . 
Furthermore , the specificity of pAbs varies from batch to batch , necessitating a specificity analysis for each preparation ( 19 ) . 
With mAbs , which is renewable antibody source , most of these problems of non-cognate antigen binding can be avoided , and the antibody that performs best in terms of specificity and ChIP-efficiency can be selected and used repeatedly and reproducibly . 
However , as the mAb recognizes , in principle , only one epitope structure , this epitope may be masked during DNA binding , or within the chromatin architecture when interaction occurs with other transcription factors , or by fixation during cross-linking . 
The problem of epitope masking can be avoided by careful design of the immunization and antibody selection protocols . 
For instance , the use of cross-linked DNA -- protein complexes to screen the mAbs from hybridomas should yield antibodies with greater chance of success in ChIP . 
However , this is rarely done . 
Finally , irrespective of whether polyclonal or monoclonal antibodies are used , antibodies are complex molecules comprising an Fc part that is recognized by multiple effector molecules , and thus forms a possible source of multiple unwanted binding events in ChIP . 
The latter complication is expected to be absent with antibody fragments , such as scFv and nanobodies , which lack the Fc part . 
Nanobodies are recombinant single-domain antigen-binding entities derived from unique heavy chain only antibodies naturally occurring in camelids ( 26 ) . 
Sharks also possess such heavy chain antibodies , referred to as IgNARs . 
However , IgNARs are more ancestral antibodies compared with the camel variant ( 57 ) , and the immunization of sharks might be rather complicated . 
The immunization of camelid 
( camel , dromedary , alpaca and llama ) is more practical : these animals are routinely vaccinated in farms with optimized adjuvants , and we shortened the immunization time to 6 weeks . 
In addition , the cloning of the nanobody genes form the peripheral blood B-cells of the immune animal and the subsequent identification of recombinant , antigen-specific nanobodies after phage display became indeed a fast and straightforward technology ( 27,30,58 ) . 
Moreover , nanobodies are well expressed in microbial systems , and with their small size ( < MW 15 000 ) , high robustness and high specificity for their cognate antigen they are versatile . 
Nanobodies seem to suffer minimally from non-specific antigen capturing in the context of complex proteomes as illustrated here ( Figure 2 ) 
This seems to be a general property of nanobodies , as they have already been used successfully as highly specific probes in antigen capturing and intracellular imaging ( 59,60 ) . 
Here , we have demonstrated the successful use of an Ss-LrpB-specific nanobody ( Nb9 ) in ChIP in S. solfataricus . 
A high-affinity interaction between the nanobody and its cognate antigen [ KD in the nM range as routinely observed ( 28 ) ] warrants a specific and efficient immunoprecipitation of the target nucleoprotein complex from the chromatin . 
However , it is clear that the exact epitope recognized by the antibody is also of crucial importance , and nanobodies are no exception to this rule . 
Indeed the nanobodies , like mAbs , need to be carefully screened . 
This is illustrated with two other Ss-LrpB specific nanobodies ( Nb1 and Nb11 ) with similar good affinity characteristics as the Nb9 ( i.e. KD in low nM range ) but targeting a different epitope . 
These two Ss-LrpB-specific nanobodies failed in ChIP , as their antigen binding provokes a clear dissociation of the SsLrpB from its DNA ( Figure 3 ) . 
Hence , the epitope should be preferentially located not only outside the DNA-binding domain of the protein but also outside the regions used to interact with ( other ) partner chromatin proteins , and these are not always known in advance . 
It is possible to increase the chances to retrieve ` ChIP-able ' nanobodies by selecting during phage display pannings on truncated protein constructs lacking the DNA-binding domain as done here or by selecting on cross-linked DNA -- protein complexes . 
Finally , the use of nanobodies has the advantage that the vast majority of them are directed to conformational epitopes , which increases the specificity and decreases the background and false-positive signal after immunoprecipitation . 
The chance is indeed higher that a binder to a linear epitope also interacts with a mimetic peptide . 
The weakness of the nanobody - based ChIP technology is that a specific nanobody needs to be identified for each target . 
This can be avoided by using an epitope-tagging approach , where a unique peptide tag ( e.g. GFP , hemagglutinin , GST , myc , FLAG ) for which ChIP-able antibodies are available is knocked-in in the target gene , preferentially by homologous recombination ( 14 ) . 
Such tagging workflow is available , for example , in model organisms Saccharomyces cerevisae or E. coli ( 61 ) and the halophilic archaeon Halobacterium salinarum ( 14 ) . 
For those systems where a GFP has been introduced as a tag , a GFP binding nanobody could be used for ChIP . 
This GFP-specific nanobody has an excellent track record for intracellular targeting and for immune precipitation from cells expressing fluorescent DNA-binding proteins as well ( 58,62,63 ) . 
Although the homologous recombination of tagged genes replacing the endogenous genes avoids the overexpression of recombinant proteins that are naturally of low abundance within the cell , the presence of an unnatural C - or Nterminal tag at the target protein might lead to complications , such as an induced loss of function by mislocalization , or multimerization and aggregation of the GFP-tagged protein . 
Therefore , it is probably safer and more relevant to avoid the strategy of tagged gene product . 
In addition , for higher eukaryotes and many ( extremophilic ) archaeal organisms for which genetic tools have not been developed yet or only work in the hands of specialists , homologous recombination may be less practicable . 
The advantage of the method propose here is that it is applicable to any organism . 
We , therefore , prefer the standard ChIP technology using dedicated antibodies , where mAbs have been substituted by nanobodies . 
As aforementioned , the generation of antigen-specific nanobodies is not a bottleneck for high-throughput ChIP experiments , as they are straightforward to generate in a short time . 
We use a fast immunization scheme with multiple antigens in one camel or llama . 
The following library construction and identification of antigen-specific nanobodies requires only 2 weeks each . 
Hence , antigen-specific nanobodies against > 100 different antigens can be isolated by one researcher per year . 
The subsequent microarray or deep sequencing and the interpretation of the data is much more time consuming . 
Thus , the work presented here paves the way for a more widespread use of nanobodies in ChIP-chip and ChIP-seq approaches to analyse genome-wide binding of any desired chromatin-associated protein in any organism . 
Interestingly , the shape of the ChIP-chip profiles for the autoregulatory binding of Ss-LrpB , for which three binding sites are present in the promoter region , has led to the identification of additional novel low-affinity operator sites in the Ss-lrpB ORF . 
Supplementary binding of transcription factors to coding regions is not uncommon and has been previously observed for another archaeal Lrp-like transcription factor from Methanocaldococcus jannaschii called Ptr2 ( 64 ) . 
In this case , the additional site was located at the promoterproximal side of the gene ( position +7 ) at a reasonable distance from the main operator sites . 
In contrast , the auxiliary Ss-LrpB sites are located almost 400-bp downstream of the promoter Box1 . 
This situation is reminiscent of the E. coli Lac repressor , which binds to a site 401-bp downstream of the main operator site ( 65 ) . 
Simultaneous binding of the main operator O1 and the auxiliary operator O2 by a single Lac repressor tetramer induces the formation of a DNA loop and contributes to transcriptional repression . 
Possibly , Ss-LrpB binding to both operator regions ( upstream of the ORF and at the 0 3 - end of the ORF ) also alters the local conformation of the DNA and might even cause DNA looping . 
In any case , Box4 and Box5 binding is expected to contribute to autoregulation by increasing the local concentration of the transcription factor . 
Thus , the discovery of the novel Ss-LrpB binding sites within the Ss-LrpB ORF is a nice illustration of the capacity of nanobodies in ChIP-chip to rapidly identify novel operator sites . 
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online : Supplementary Tables 1 and 2 and Supplementary Figure 1 . 
ACKNOWLEDGEMENTS
The authors are grateful to Dr John van der Oost for the gift of pLUW632 plasmid . 
They thank Ningning Song and 
Amelia Vassart for purification of Ss-LysM and Ss-Lrp protein , respectively . 
Liesbeth van Oeffelen is greatly acknowledged for assistance with the microarray data analysis . 
FUNDING
Fonds voor Wetenschappelijk Onderzoek-Vlaanderen ( to E.P. ) ; Vlaams Interuniversiair Instituut ( VIB ) ( to T.N.D. ) . 
Funding for open access charge : Onderzoeksfonds ( OZRGOA ) , Vrije Universiteit Brussel . 
Conflict of interest statement. None declared.