while the presence 
 of g - quadruplexes in telomeric sequences , promoter 
 elements , and 5utrs is well documented , and in some cases 
 with a biological role proposed , similar research is lacking for the 
 complementary dna secondary structure , the i - motif , although such 
 roles have been suggested . in promoter 
 elements where duplex dna is found
, the possibility exists that the 
 g - quadruplex and the i - motif form on opposite strands , but whether 
 they can coexist or are mutually exclusive remains unresolved , except 
 in the case of the insulin promoter where the formation of the two 
 structures is mutually exclusive . if the 
 latter were the case more generally , then one might imagine that the 
 g - quadruplex could act as a signal to silence gene expression , as 
 is the case with the myc promoter , and the i - motif as an activator signal . in support of this
, 
 the activating transcriptional factor hnrnp k binds to the ct boxes 
 on the c - rich strand in the myc promoter and induces myc expression .
recent 
 findings in our companion paper ( doi : 10.021/ja410934b ) further support the idea of dna secondary structures serving as 
 switches to turn gene transcription on or off .
we observed two different small molecules that bound to different 
 topological forms of the c - rich strand of the bcl2 cis - regulatory element and either repressed or activated transcription .
the compound ( imc-48 ) that bound to the i - motif 
 species to populate this species relative to the flexible hairpin 
 increased bcl2 gene expression .
in contrast , the 
 other compound ( imc-76 ) , which selected for the flexible hairpin species , 
 decreased gene expression .
antagonism between the two molecules was 
 found to occur with the dna species in solution as well as within 
 a cellular system . on the basis of these 
 results
, we postulated the presence of transcriptional factors that 
 would similarly bind to the two different dna structures , thereby 
 mimicking the effect of the two compounds on bcl2 gene expression . here
we identify hnrnp ll as a transcriptional 
 factor that recognizes the bcl2 i - motif and subsequently 
 unfolds it to activate transcription .
furthermore , hnrnp ll belongs 
 to the same protein family as hnrnp k , which previously was shown 
 to activate myc transcription by binding to the c - rich 
 strand of the myc promoter . following the identification of hnrnp ll as an activating transcriptional 
 factor for bcl2
, we then demonstrate that the two 
 small molecules that bind exclusively to one or the other of the two 
 equilibrating species of the bcl2 c - rich strand exert 
 their activity by modulating the amount of the i - motif available for 
 binding to hnrnp ll .
importantly , this principle was shown at both 
 the level of the dna species bound to hnrnp ll in solution and the 
 cellular level .
these results suggest that the bcl2 i - motif can be considered as a molecular switch similar in principle 
 to a riboswitch found in rna .
directly upstream ( 25 bases ) from the bcl2 p1 promoter is a gc - rich element known to form g - quadruplex and 
 i - motif structures ( figure 1a ) . under negative 
 superhelicity induced by transcriptional activity
it can be expected 
 that either the i - motif or the g - quadruplex will exist in the promoter 
 element .
previous in vitro studies using synthetic oligomers demonstrated 
 that the bcl2 g - rich promoter element forms three 
 different g - quadruplexes ; the major one exhibits a mixed parallel / antiparallel 
 structure .
the opposite strand is highly dynamic , existing 
 as a mixed population of two molecules at a ph of 6.6 , an i - motif 
 and a flexible hairpin ( figure 1b , c ) .
the relationship 
 between these two dna secondary structures , the interaction of imc-48 
 and imc-76 , and the subsequent effect on bcl2 gene 
 expression are also shown in figure 1c .
diagram of 
 the bcl2 gene promoter region with 
 the gc - rich element located directly upstream of the p1 promoter and 
 targeting with imc-48 and imc-76 .
three and one - half sets of two intercalated hemiprotonated 
 cytosine  cytosine base pairs form the i - motif structure . 
 the bases in bold correspond to the bases involved in base pairing 
 within each of the structures .
here and in subsequent figures , the 
 yellow , green , red , and blue circles represent the deoxynucleotides 
 cytosine , adenine , guanine , and thymine , respectively . in the lower 
 portion of the figure
we also show the proposed partial hairpin that 
 is in equilibrium with the i - motif and the proposed binding of imc-48 
 and imc-76 to the i - motif and partial hairpin , respectively , along 
 with the proposed transcriptional consequences of targeting with imc-48 
 and imc-76 .
specific proteins such as nucleolin and 
 nm23-h2 recognize and bind to g - quadruplexes 
 in promoter elements .
g - quadruplex - binding agents can interfere with 
 protein  dna complex formation , potentially modulating gene 
 expression .
we sought to identify nuclear proteins that could bind 
 to the i - motif or an unfolded form and might also be involved in bcl2 transcriptional modulation .
since the i - motif is highly 
 dynamic , any identified i - motif - binding protein may take advantage 
 of this property and form a stable dna complex by i - motif remodeling . 

the c - rich strand that gives rise to the folded i - motif has features 
 more commonly associated with secondary rna structures than dna ; therefore , 
 rna - binding proteins were considered .
candidates included rna recognition 
 proteins belonging to the hnrnp class normally associated with rna 
 splicing .
although not as yet reported to bind to an i - motif structure , 
 an example is hnrnp k , which binds to the ct element of the myc promoter to activate transcription .
nuclear proteins from standard and commercially available 
 hela nuclear extract that putatively bind to the bcl2 i - motif were purified using a biotinylated oligomer  streptavidin 
 bead complex pull - down assay and identified by liquid lc / ms / ms sequencing . 

bead complexes were used consisting 
 of either the wild - type bcl2 i - motif  forming 
 sequence or a mutant oligomer ( which can not form a stable i - motif ) 
 for nonspecific protein binding .
ninety - five proteins were identified 
 that bound either to the wild - type bcl2 i - motif  forming 
 sequence ( 35 proteins , supplemental table 1 ) , a mutant ( 20 proteins , supplemental table 
 2 ) , or both sequences ( 40 proteins , supplemental 
 table 3 ) .
proteins that bound uniquely to the bcl2 i - motif  forming promoter element were classified into functional 
 groups : ( 1 ) transcription , ( 2 ) translation or protein - folding , ( 3 ) 
 energy metabolism or other enzymatic processes , and ( 4 ) cell adhesion 
 or migration functions , mostly related to the cytoskeleton ( supplemental tables 13 ) .
of interest 
 were proteins having documented function related to transcription 
 ( table 1 ) , particularly hnrnp ll . while hnrnp 
 ll has not been extensively studied , the related protein hnrnp l is 
 a pre - mrna splicing factor that binds to and stabilizes bcl2 mrna .
the hnrnp ll protein is a paralog 
 of hnrnp l , shows tissue - specific distribution , and activates t - cells by shifting transcriptomes for cellular 
 proliferation and inhibition of cell death . to investigate hnrnp ll for its possible involvement 
 in bcl2 transcriptional modulation
, we studied the 
 effects 
 of sirna knockdown in mcf-7 breast cancer cells .
relative mrna levels 
 of hnrnp ll and bcl2 were determined by qpcr after 
 treatment with hnrnp ll sirna . significantly decreased bcl2 mrna levels resulted from hnrnp ll knockdown ( figure 2a ) . to demonstrate that this effect is specific and not caused 
 by an off - target sirna effect , we examined mrna levels of two other 
 genes ( pdgfr- and kras ) following 
 hnrnp ll knockdown .
the pdgfr- and kras promoter regions also contain the consensus sequences 
 ccgc for hnrnp ll binding , and preliminary data suggest that hnrnp 
 ll binds to the i - motif - forming oligomers ( data not shown ) with comparable 
 binding to bcl2 .
however , the pdgfr- and kras transcripts are not lowered by hnrnp ll 
 sirna , demonstrating selectivity for the bcl2 i - motif 
 at least among these promoter i - motifs ( figure 2a ) .
( a ) the effects of sirna knockdown of hnrnp ll on the bcl2 , kras , and pdgfr- mrna level in mcf-7 cells .
50 nm of hnrnp ll sirna was added to 
 mcf-7 cells for 72 h. gapdh was used as an internal control ( * * p < 0.01 ) .
( b ) effect on concentration - dependent binding 
 of hnrnp ll on the bcl2 i - motif  forming oligomer 
 ( py39wt ) by emsa at ph 6.8 .
( c ) competition emsa showing bcl2 i - motif  specific binding of hnrnp ll at ph 6.8 .
nonlabeled 
 ( cold ) oligomers were incubated with hnrnp ll on ice for 20 min and 
 end - labeled py39wt was added for 5 min .
( d ) comparative kd values for hnrnp ll binding to the biotin - py39wt and 
 biotin - py39mutt at two different ph levels determined by spr analysis .
mobility shift assays 
 ( figure 2b , c ) were used to determine whether 
 hnrnp ll bound to the bcl2 i - motif specifically . 

the hnrnp ll protein bound with high affinity to the bcl2 i - motif at ph 6.8 ( figure 2b ) .
the cold bcl2 i - motif ( py39wt ) competed with the p - labeled bcl2 i - motif ; as expected , the cold mutant i - motif oligomer 
 ( py39mutt ) did not compete for hnrnp ll binding ( figure 2c ) .
also , hnrnp ll did not bind to duplex dna formed with 
 the cold complementary g - rich strand ( pu39wt ) annealed to end - labeled 
 py39wt . using surface plasmon resonance ( spr ) analysis ( figure 2d and supplemental figure 1 ) , hnrnp ll bound strongly to py39wt with a kd value of 19.4 pm at ph 6.5 .
for the py39mutt , the kd value was 2.5-fold higher at each ph compared 
 to the wild - type .
the hnrnp ll protein shares 57% sequence identity to 
 hnrnp l. both proteins have four rna 
 recognition motifs ( rrms ) , and at least two are required for stable 
 binding to single - stranded rna or dna .
two consensus sequences for 
 binding these rrms are found in the bcl2 i - motif , 
 and both are located in the lateral loops ( cccgc and cgccc ) ( figure 3a ) . to determine the importance of these loops in 
 comparison to the central loop ( having a ttcct sequence ) , cold mutant 
 py39 sequences were designed having one or more of these loops mutated 
 but still maintaining the basic i - motif core structure . of these four 
 mutated i - motif sequences , the one having both lateral loops mutated 
 ( mut5,3l ; 35% ) was the least effective competitor , 
 whereas the one having the central loop mutated ( mutcl ; 73% ) was the 
 most effective ( figure 3b ) .
the histograms 
 shown to the right of the emsas in figure 3b depict the quantification of band intensity . while the two individually 
 mutated loop oligomers ( mut5l and mut3l ) were of intermediate 
 potency in competing with the wild - type for hnrnp ll binding to the bcl2 i - motif , mut5l ( 48% ) appeared less effective 
 than mut3l ( 64% ) . to complement these experiments
, we also 
 determined the biological significance of the lateral loops by investigating 
 whether hnrnp ll knockdown depended on the wild - type loop sequences . 

two mutant luciferase constructs were prepared , one in which both 
 lateral loops were mutated ( mut5,3l ) and a second 
 in which the central loop was mutated ( mutcl ) . as anticipated , while 
 knockdown of the hnrnp ll still had an inhibitory effect on luciferase 
 activity with the wild - type and mutcl promoter constructs , there was 
 no significant effect on reporter activity with the mut5,3l 
 ( figure 3c ) .
the knockdown effect of hnrnp 
 ll sirna on reporter activity ( 25% ) could be considered modest ; 
 however , this is probably due to the limited ( 5060% ) knockdown 
 of hnrnp ll and suggests that activation of bcl2 expression 
 may involve other transcriptional factors . to determine the relative 
 importance of the sequence of each of the lateral loops ,
a similar 
 emsa competition experiment was carried out in which either the 5 
 or 3 sequence was swapped out ( mut6 ) or both lateral loops 
 carried either the 5 ( mut6 - 1 ) or 3 ( mut6 - 2 ) loop sequences 
 ( supplemental figure 2 , left ) .
the histograms 
 of band quantification did not reveal significant differences for 
 any of the sequences ( 64% for mut6 , 65% for mut6 - 1 , and 70% for mut6 - 2 ) , 
 indicating that the variance observed between mut6 - 1 and mut6 - 2 may 
 be related to the 5 or 3 positions of the lateral 
 loop and the associated rrms rather than sequences ( supplemental figure 2 , right ) .
emsa and sirna knockdown 
 experiments demonstrate that hnrnp ll 
 recognizes the i - motif through the lateral loops .
( a ) folding pattern 
 of the bcl2 i - motif showing the 5 and 3 lateral loops 
 ( ii and v , respectively ) and central loop ( cl ) .
( b ) competition emsa 
 showing selective binding of hnrnp ll to the two lateral loops of 
 py39wt with 8:5:7 loop folding pattern .
mut5,3l has mutations in two lateral loops , 
 and mut5l and mut3l have mutations in the 5 
 and 3 loops , respectively ( binding sequences are color coded 
 to match i - motif folding pattern in a ) .
the percents above the sets of histograms for mut5,3l , 
 mut5l , mut3l , and mutcl indicates the addition of 
 free dna for each concentration of cold oligomers divided by three . 

( c ) a luciferase assay shows that knockdown with hnrnp ll sidna was 
 dependent on the wild - type sequence in the lateral loops of the i - motif . 

three pgl3 constructs of wild - type , mut5,3l , and mutcl 
 were co - transfected with prl - tk for normalization and sirna to hnrnp 
 ll for 72 h. final relative luciferase activities were obtained by 
 normalization of the ratio of firefly to renilla to sirna - untreated 
 control of each construct p values ( * * * * p < 0.0001 , * * * p < 0.001 ,
there was a significant decrease 
 in the ellipticity of py39wt at 286 nm in the presence of hnrnp ll 
 in a concentration - dependent manner ( figure 4a , left ) and a shift to a lower wavelength by about 2.2 nm at 2 equiv 
 as detected by cd analysis .
this suggests a partial unfolding of the 
 i - motif or conversion to another topological form by hnrnp ll , which 
 has been supported by single - molecule experiments in a recently published 
 paper . to a lesser extent
, the cd signal 
 of py39mutt also decreased upon binding of hnrnp ll ( figure 4a , right ) and shifted to a higher wavelength by 
 about 12 nm at 2 equiv . to determine the optimum distance 
 between the two lateral - loop binding sites for competition with py39wt
, 
 a series of oligomers containing the two consensus sequence binding 
 sites connected by variable ( 217 nucleotides ) spacers was 
 used .
a 13-nucleotide spacer was found to be optimal for competition 
 with the end - labeled py39wt ( figure 4b ) , which 
 is the exact nucleotide distance between the two lateral loops in 
 the wild - type sequence .
it is important to note that the molar ratio 
 of the unlabeled , unstructured 39-mers to the labeled 39-mer i - motif 
 was 150:1 .
pre - organization of the consensus binding sequences into 
 the lateral loops of the folded i - motif provides a significant entropic 
 and kinetic binding advantage .
we conclude that a key function of 
 the i - motif folded structure is to provide a rigid chemical scaffold 
 upon which to display the preorganized lateral loops for optimum kinetic 
 advantage for binding of hnrnp ll .
in addition , the sequential recognition 
 and binding of not just one but both lateral loops also provide a 
 significant kinetic advantage . on the basis of these emsa experiments 

we propose that the rrms of hnrnp ll recognize the mixed cytosine / guanine 
 sequences in the lateral loops by binding to one or both of the lateral 
 loops ( the 5 lateral loop is the favored one ) .
then , after 
 subsequent protein - facilitated i - motif unfolding , hnrnp ll binds more 
 stably to an unfolded i - motif species not present initially .
emsa , br2 footprinting , and fret show that hnrnp ll 
 unfolds the bcl2 i - motif after binding .
( a ) cd analysis shows that 
 binding of hnrnp ll produces a conformational change in the i - motif . 

hnrnp ll was preincubated with py39wt or py39mutt at ph 6.5 for 5 
 min at room temperature before measuring the cd .
( b ) competition emsa 
 showing that 13 nt is the optimal length between two hnrnp ll binding 
 sites for the binding of hnrnp ll .
competition 
 emsa experiments were conducted in a binding buffer ( ph 6.8 ) for 20 
 min of preincubation of 250 nm of cold oligomers with hnrnp ll and 
 subsequent 5 min incubation of end - labeled py39wt .
this represents 
 about a 150 molar excess of cold dna to labeled i - motif .
the histogram 
 below the gel shows the relative binding intensity from the emsa gel . 

( c ) bromine footprinting of the bcl2 i - motif and 
 hnrnp ll complex showing the conformational change of py39wt induced 
 by hnrnp ll .
py39wt and hnrnp ll were incubated for 5 min at room 
 temperature , and bromine generated in situ was added for 30 min .
black 
 and red plots are 0 and 10 g of hnrnp ll , respectively .
the 
 peaks with the black dots correspond to those where maximum inhibition 
 occurs and include c runs ii and iv and the central loop .
the right 
 panel shows the folding pattern of the bcl2 i - motif with that region 
 protected from br2 cleavage shown in the blue shading . 

( d ) fret experiments showing i - motif - specific unfolding 
 activity by hnrnp ll .
fam / tamra dual - labeled probes were incubated 
 at ph 6.5 or 7.9 with hnrnp ll at room temperature for 5 min , and 
 then fluorescence intensity was measured at 495 nm ( ex.)/528 ( em . ) . 

right panel shows the unfolding of the i - motif consistent with the 
 fluorescence enhancement seen in the left panel ( wt at ph 6.5 ) .
p values ( * * p < 0.01 , * * * p < 0.001 ) were determined by t - test analysis .
the structure of the py39wt oligomer after hnrnp ll binding 
 was 
 examined by bromine footprinting ( figure 4c ) . 

binding of hnrnp ll to the bcl2 i - motif changed the 
 cleavage pattern of py39wt following bromination / piperidine treatment 
 ( compare lanes 2 and 4 in figure 4c )
. cytosines 
 in run i were more cleaved , while other cytosines in runs iii to iv 
 were less cleaved with increasing concentrations of hnrnp ll .
bromine 
 footprinting of the same sequence in the presence of imc-76 indicated 
 the opposite effect on runs iii and iv .
this suggests that the unfolded form induced by hnrnp ll is not 
 the partial hairpin .
since runs iii to iv are positioned between the 
 5 and 3 lateral loops , which the spacer experiment 
 demonstrated must be unfolded in the hnrnp ll  bound species , 
 bromine footprinting inhibition suggests that they are more protected 
 by close association with hnrnp ll than even the lateral loops ( figure 4c ) .
the i - motif unfolding activity of hnrnp 
 ll was further confirmed 
 by fret assay ( figure 4d ) . in this experiment 

hnrnp ll increased the fluorescence intensity by 1.8-fold at ph 6.5 , 
 where the i - motif is expected to be initially present but had little 
 effect at ph 7.9 where the i - motif is absent ( figure 4d , left ) .
in addition , hnrnp ll selectively increased the 
 fluorescence signal of the wild - type sequence compared to the lateral 
 loop mutant ( mut5,3l ) at ph 6.5 ( figure 4d , right ) .
this result strongly suggests that the binding 
 and associated unfolding activity of hnrnp ll is restricted to the 
 i - motif structure with the wild - type sequence in the lateral loop .
the binding of hnrnp ll to two similar consensus sequences in the 
 c - rich strand of the bcl2 promoter that results in 
 transcriptional activation is quite analogous to hnrnp k binding to 
 the ct elements in myc nhe iii1 .
the hnrnp 
 k protein contains three kh domains that are spaced apart in a similar 
 manner to hnrnp ll but recognize tccc sequences .
significantly , tccc elements are found in the lateral loops 
 of the myc i - motif and are spaced the same distance 
 apart in the unfolded structure as those found in the bcl2 i - motif ( unpublished results ) .
thus , 
 hnrnp k and hnrnp ll may have similar roles in transcriptional activation 
 of myc and bcl2 : they recognize 
 similar single - stranded elements in the lateral loops of their respective 
 i - motifs , and both presumably remodel the i - motif to form a thermodynamically 
 stable species prior to transcriptional activation .
the partitioning 
 of biological molecules between two equilibrating species in which 
 only one is biologically active is well - known in the rna world .
these can act as switches if the chemical equilibrium 
 can be changed by the preferential sequestration of one of the forms 
 by a small molecule . to determine whether a similar mechanism might 
 operate in a system consisting of two equilibrating dna species
, we 
 explored the biological outcome ( transcriptional silencing or activation ) 
 of using a small molecule that bound preferentially to each of the 
 dna forms .
characterization of the ternary interactions between the 
 dna , protein , and each small molecule in a cell - free system permitted 
 extension into a cellular system .
we have demonstrated that 
 imc-76 can change the dynamic chemical populations of equilibrating 
 c - rich strand species in solution by sequestering the flexible hairpin .
we have also shown that the rrms of hnrnp ll 
 require the presence of the cgccc and cccgc sequences in the lateral 
 loops of the i - motif for optimum binding and subsequent unfolding 
 leading to transcriptional activation . taken together , this suggests 
 a competition between imc-76 and hnrnp ll for binding to the equilibrating 
 populations of the flexible hairpin and i - motif . binding of imc-76 
 to the flexible hairpin should increase the population of this species 
 and deplete the population of the hnrnp ll  bound i - motif species . 
 in cells , imc-76 is expected to decrease the i - motif population in 
 the promoter element and thus reduce hnrnp ll promoter occupancy . 

in contrast , imc-48 , like hnrnp ll , binds exclusively to the bcl2 i - motif ; thus imc-48 should increase the i - motif population 
 and thereby increase the amount of hnrnp ll  bound i - motif species  assuming 
 that hnrnp ll binds to the i - motif tightly enough to displace imc-48and 
 increase the promoter occupancy in cells .
experiments were designed 
 using emsa , hnrnp ll knockdown , and chip analysis to test these hypotheses .
the results from the emsa experiment in which different amounts 
 of imc-76 were incubated at ph 6.5 with the bcl2 i - motif 
 and its equilibrating species for 3 h prior to the addition of hnrnp 
 ll are shown in figure 5a . in the absence of 
 imc-76 and hnrnp ll , there are two conformationally different oligomer 
 species separated in the gel .
we propose that species 1 , the predominant 
 form , is most likely the i - motif , which leaves species 2 as the flexible 
 hairpin .
upon addition of hnrnp ll , species 1 is depleted preferentially 
 to form the hnrnp ll high - mobility - shifted complex . however , as imc-76 
 concentration is increased , the amount of the hnrnp ll 
bcl2 i - motif complex is decreased , and species 2 , putatively 
 the flexible hairpin , is increased at the expense of species 1 , the 
 presumed i - motif .
this is in accord with the idea that imc-76 and 
 hnrnp ll compete for the pool of equilibrating species to trap ( imc-76 ) 
 or remodel ( hnrnp ll ) the i - motif to an unfolded species . in a parallel 
 experiment , the effect of imc-48 on the distribution of the three 
 species was determined ( figure 5b ) .
as the 
 concentration of imc-48 increased , there was a depletion of species 
 1 and 2 and an increased band intensity of the hnrnp ll 
this supports the hypothesis that 
 the increase of i - motif population by imc-48 facilitates the binding 
 of hnrnp ll to the i - motif structure .
the consequences of sequestration of the 
 flexible hairpin or the 
 bcl2 i - motif by imc-76 ( a ) and imc-48 ( b ) , respectively , on the binding 
 of hnrnp ll to the i - motif .
( a ) emsa analysis of the competition between 
 imc-76 and hnrnp ll for the i - motif ( left ) and densitometric analysis 
 ( right ) .
( b ) emsa analysis of the cooperativity between imc-48 and 
 hnrnp ll for the i - motif ( left ) and densitometric analysis ( right ) . 

imc-76 or imc-48 was incubated with py39wt for 3 h , and hnrnp ll was 
 added for 10 min at ph 6.5 before running the 6% native page .
relative 
 band intensities are plotted against imc-76 or imc-48 concentrations 
 ( right ) .
species 1 and 2 are proposed to be the i - motif and flexible 
 hairpin , respectively . to investigate the imc-76 and imc-48 cellular effects , which 
 are 
 known to affect bcl2 transcription , sirna and chip 
 experiments were performed .
first , the potentially additive or subtractive 
 inhibitory effects of hnrnp ll sirna together with imc-76 or imc-48 
 were determined on bcl2 mrna transcription , following 
 knockdown of hnrnp ll .
second , to determine the effects of imc-76 
 or imc-48 on promoter occupancy by sp1 and hnrnp ll , chip analysis 
 was performed using mcf-7 cells . while the treatment of 50 nm hnrnp 
 ll sirna alone significantly decreased bcl2 mrna 
 levels by 24% , addition of either 0.5 or 2 m of imc-76 further 
 decreased the mrna levels to a total of 33% and 47% , respectively 
 ( figure 6a ) .
in contrast , imc-48 reversed the 
 inhibitory effects of the hnrnp ll sirna ( figure 6b ) .
this is expected because both knockdown of hnrnp ll and 
 depletion of the i - motif population by imc-76 should be additive in 
 lowering transcription , although they act on different targets . in 
 contrast
, the effect of imc-48 in cells should antagonize the inhibitory 
 effects of bcl2 mrna expression knockdown .
effect of imc-76 
 and imc-48 combined with knockdown of hnrnp ll 
 on bcl2 mrna levels .
( a ) enhanced effect of imc-76 treatment on the bcl2 mrna levels after knockdown of hnrnp ll .
( b ) restoration 
 of bcl2 mrna levels after treatment with imc-48 following knockdown 
 of hnrnp ll .
after transfection of 50 nm of hnrnp ll sirna into mcf-7 
 cells for 48 h , imc-76 or imc-48 was incubated for a further 24 h 
 ( * * * p < 0.001 , * * p < 0.01 ) . 

the p value ( * * p < 0.01 ) was 
 determined by one - way anova analysis . to directly assess the effect of imc-76 and imc-48 on recruitment 
 of transcriptional factors to the bcl2 promoter , 
 which is proposed to contain the i - motif - forming element , a chip assay 
 was performed on sp1 and hnrnp ll using mcf-7 and bjab cells , respectively . 

the mcf-7 cells , which overexpress bcl2 and have 
 detectable levels of hnrnp ll , were used to determine the inhibitory 
 effect of imc-76 on bcl2 transcription .
alternatively , 
 the bjab cells , which only express basal levels of bcl2 , were used to evaluate the activating effect of imc-48 on bcl2 transcription .
sp1 is a ubiquitous transcription factor 
 bound to the gc - rich region in gene promoters .
mcf-7 and bjab cells 
 were treated with imc-76 and imc-48 ( at 0.5 and 2 m ) for 24 
 h , respectively .
quantification of immunoprecipitated dna was performed 
 by sybr green i qpcr using two specific sets of primers , amplifying 
 either the closest upstream region ( 103 to 3 base 
 pairs ) or a far upstream region ( > 3000 base pairs ) from the i - motif / g - quadruplex - forming 
 site of the p1 promoter , the latter serving as a negative control 
 for normalization . as shown , imc-76 decreased the occupancy of both 
 sp1 and hnrnp ll bound to the bcl2 p1 promoter region 
 in a concentration - dependent manner in mcf-7 cells ( figure 7a ) .
in contrast , imc-48 increased the promoter occupancy 
 of both sp1 and hnrnp ll in bjab cells ( figure 7b ) . to ensure that the effect of imc-76 on promoter occupancy by 
 sp1 and hnrnp ll was not due to inhibition of transcription of these 
 proteins , qpcr was carried out . in a similar way , the effect of imc-48 
 on the transcription level of sp1 and hnrnp ll was tested with bjab 
 cells ( supplemental figure 3 ) .
in addition , 
 immunoprecipitation ( ip ) experiments were carried out for both sp1 
 and hnrnp ll to verify antibody specificity ( supplemental 
 figure 4 ) .
chip analysis of the effect of imc-76 and imc-48 given 
 either singly 
 ( a and b ) or in sequential order ( imc-76 followed by imc-48 ) ( c ) on 
 promoter occupancy of sp1 and hnrnp ll .
for single drug treatments 
 ( a and b ) , two concentrations ( 0.5 and 2 m ) of imc-76 or imc-48 
 with dmso as a control were incubated with mcf-7 and bjab cells , respectively , 
 for 24 h. for sequential treatments ( c ) , antagonism between imc-76 
 and imc-48 was shown through restoration of sp1 and hnrnp ll promoter 
 occupancy levels following administration of imc-48 after prior knockdown 
 with imc-76 in mcf-7 cells .
mcf-7 cells were treated with dmso or 
 2 m of imc-76 for 24 h. in a similar way , other mcf-7 cells 
 were treated first with 2 m of imc-76 and then with 2 or 4 
 m of imc-48 for a further 24 h. ip was performed with antibodies 
 to sp1 and hnrnp ll and igg as a negative control and acetyl - histone 
 h3 ( ach3 ) as a positive control
. the p values ( * * * p < 0.001 , * * p < 0.01 ) were determined by one - way 
 anova analysis . in the companion paper 
 ( doi : 10.021/ja410934b )
we have demonstrated 
 that imc-48 and imc-76 are antagonistic in 
 redistribution of the two populations of dna species in solution using 
 1d nmr studies as well as cellular studies by following the chemosensitization 
 to cyclophosphamide . to extend these studies 
 to examine what happens at the promoter level to the transcriptional 
 factors that bind to the bcl2 regulatory element
, 
 we carried out an experiment in mcf-7 cells in which we first depleted 
 sp1 and hnrnp ll from the promoter element by treatment with imc-76 . 

then we treated 24 h later with imc-48 , which should reverse these 
 effects relative to the control in which only imc-76 has been previously 
 added .
the results ( figure 7c ) showed that 
 the decreased promoter occupancy by both sp1 and hnrnp ll in mcf-7 
 cells produced by imc-76 was reversed by imc-48 in a concentration - dependent 
 manner , illustrating an antagonistic relationship between imc-76 and 
 imc-48 at the promoter level .
this result , together with the results 
 from the previous complementary antagonism experiments carried out 
 at the solution level using nmr and at the cellular level using chemosensitization 
 to cyclophosphamide , provide very strong 
 evidence for direct competition between imc-76 and imc-48 for the 
 two equilibrating populations of the bcl2 i - motif 
 and flexible hairpin , resulting in the cellular consequences mediated 
 via hnrnp ll . of the noncanonical 
 dna structures , the i - motif is perhaps the most dynamic at ph levels 
 that are either slightly acidic or even close to neutral .
because 
 the i - motif is formed from hemiprotonated c  ch base 
 pairs that have a pka of 4.58 for the 
 n3 of cytosine , their existence in cells 
 has not been generally anticipated . however , an important contributor 
 to their increased stability is favorable van der waals energies , 
 due to close contacts between deoxyribose sugars in the narrow groove 
 of the tetrad , and this is dependent upon the precise topology of 
 the phosphodiester backbone with intercalation of c  ch pairs .
significantly , i - motifs in rna 
 can not be formed , even at low ph , because of the steric hindrance 
 of the 2-hydroxyl group . since the 
 topology of the phosphodiester backbone appears to be critical in 
 stabilization of the i - motif through sugar
sugar interaction , 
 conditions such as molecular crowding , negative superhelicity , and 
 loop constraints may play important roles if they influence these 
 parameters .
what is critical for the proposed role of i - motifs as 
 molecular switches in transcriptional regulation is that their dynamic 
 nature is such that they can easily move between folded ( i - motif ) 
 and unfolded ( hairpin ) populations of molecules under physiological 
 conditions .
thus , factors such as those described above that influence 
 the topology of the deoxyribose backbone of the intercalated c  ch base pairs likely play critical roles in determining the 
 dynamic nature of promoter i - motifs .
recently , the function of i - motifs 
 as reversibly conformational switches for nanobiotechnology has been 
 reviewed . in cellular systems , 
 where both transcriptionally induced negative
superhelicity and molecular 
 crowding can occur , this molecular plasticity of i - motifs in promoter 
 elements is likely to be evident under physiological conditions , even 
 without considering the presence of transcriptional factors such as 
 hnrnp ll , which can recognize and then unfold the i - motif to form 
 a thermodynamically stable species .
thus we propose that promoter 
 i - motifs may have dynamic properties more like rna secondary structures 
 than what are typically associated with dna . in 
 the studies described here ,
we show that both small molecules ( imc-76 
 and imc-48 ) and a transcriptional factor ( hnrnp ll ) can either change 
 the relative population states of the i - motif and its equilibrating 
 conformers or , in the case of hnrnp ll , drive changes in the interhelical 
 conformation of the i - motif to bind most stably to an alternative 
 conformation not originally present . we now show that the competition 
 between these ligand- or protein - associated dynamic states has functional 
 consequences , leading to gene expression modulation .
this is analogous 
 to metabolite - sensing riboswitches that regulate gene expression in 
 response to small molecules by causing a redistribution of the conformational 
 states with functional consequences .
the 
 underlying common feature of the bcl2 i - motif and 
 the riboswitch is the ability of ligands and proteins to take advantage 
 of the intrinsic dynamic chemical behavior of dna or rna .
this i - motif 
 structure is found in dna but not in rna and thus is present in the genome at a very low number of copies , 
 implying that there are different drug - receptor characteristics present 
 than those found commonly in protein and rna or even duplex dna . beyond 
 the ability of the molecules such as imc-48 and imc-76 to recognize 
 and bind to the i - motif and its equilibrium partners , they must also 
 transport into the cell nucleus and
then bind to these structures 
 in open chromatin regions for them to be biologically active .
therefore 
 the chromatin state must also be favorable for transcriptional regulation 
 and involve chromatin - modifying proteins as well as other epigenetic 
 changes .
these factors , taken together with the critical need for 
 transcriptionally induced negative superhelicity , imply a complex 
 but inherently attractive new drug receptor class for exploitation 
 in drug combinations , such as with topoisomerase and hdac inhibitors . in the dynamic transitional system shown in figure 8a ,
we propose two predominant conformational states , of which 
 the flexible hairpin can coexist with either a single - stranded form 
 or the fully folded species ( i - motif ) . for i - motif - forming sequences 
 at neutral ph , both the partially folded and single - stranded states 
 coexist , and under molecular crowding 
 conditions , the i - motif conformation can exist even at neutral conditions . under negative superhelicity , the i - motif has 
 been observed in the myc promoter under physiological 
 conditions . for the bcl2 promoter sequence , at ph 6.6
the flexible hairpin and i - motif forms 
 can be observed both by h nmr and in an emsa 
 gel ( figure 5 ) .
incremental imc-76 addition 
 sequesters the flexible hairpin form , which contains five gc base 
 pairs . by analogy with the binding of a steroidal diamine to a poly(dadt ) 
 duplex , imc-76 most likely binds in the 
 non - watson  crick base pair regions where unstacked base pairs 
 exist capped at either side by gc base pairs ( figure 8b ) .
this redistribution of the conformation species 
 in box a results in a reduction in the amount of the hnrnp ll  shifted 
 band in figure 5a , which in a cellular context 
 would result in reduction of bcl2 transcriptional 
 activation and chemosensitization .
imc-48 does exactly the opposite 
 and increases the amount of the hnrnp ll  shifted band in figure 5b .
it does this by most likely binding to the central 
 loop of the bcl2 i - motif and further constrains the 
 lateral loops through which hnrnp ll recognizes and binds before unfolding 
 the structure .
this would further accelerate the kinetic step and 
 lead to enhanced transcriptional activation of bcl2 .
furthermore , it is likely that under varying extents of negative 
 superhelicity produced during transcriptional firing , the intrinsic 
 dynamic behavior of the i - motif and its equilibrating conformational 
 forms will be even more accessible .
this may be important when larger 
 energetic barriers are present , such as in the disruption of the c  c base pairs .
the i - motif folding 
 and unfolding kinetics , the latter requiring disruption of base pairing , 
 is slow in comparison to rna elements like riboswitches .
however , in cellular promoter elements , where 
 i - motif - binding proteins are present together with dynamic forces 
 that result from negative superhelicity , the kinetics may be much 
 faster .
conformational transitions and biological consequences that occur 
 following mutually exclusive binding of imc-76 , imc-48 , and hnrnp 
 ll to the different equilibrating forms of the c - rich strand in the bcl2 promoter .
box a shows the two different major conformational 
 states of the c - rich strand in the bcl2 promoter 
 under different ph conditions .
acidic conditions drive formation of 
 the i - motif , and at ph 6.6 there is a conformational mixture of the 
 flexible hairpin and i - motif .
upon addition of imc-76 , the flexible 
 hairpin form is sequestered ( a to b ) , resulting in depletion of the 
 populations of the i - motif species .
conversely , imc-48 binds to the 
 central loop of the bcl2 i - motif to sequester this species , and then 
 the rrms 1 and 2 of the hnrnp ll , which are closely spaced apart , 
 are initially proposed to recognize and bind to both of the lateral 
 loops ( ii and v ) containing the cccg and cgcc sequences , which are 
 constrained in a single - stranded form ( a to c ) . following this recognition 
 event , there are hnrnp ll  driven changes in the interhelical 
 conformations such that the two lateral loops are forced apart so 
 that the further spaced apart rrms 2 and 3 or 4 are able to bind to 
 the 5 and 3 cccg and cgcc recognition sequences to 
 form a stable complex ( c to d ) .
last , hnrnp ll bound to the alternative 
 conformation of the c - rich strand causes transcriptional activation 
 of bcl2 ( d to e ) .
the consequence of competition between imc-76 and 
 hnrnp ll for the different conformational states of the c - rich strand 
 depletes the population undergoing the transition a to c to d to e 
 and repression of bcl2 gene expression .
alternatively , 
 binding of imc-48 to the bcl2 i - motif leads to an increased amount 
 of i - motif that is bound by hnrnp ll and transcriptional activation 
 ( a to c to d ) .
the recognition and subsequent 
 stable binding of hnrnp ll to the bcl2 i - motif was 
 more complex than we first anticipated . 

the hnrnp ll protein and its paralog hnrnp l share a 58% overall amino 
 acid identity and contain four classical rrms that are highly conserved . 

the overall arrangement of the rrms in hnrnp l and hnrnp ll is similar , 
 such that in both cases they are separated by linkers of different 
 lengths so they can recognize either adjacent domains or ones spaced 
 further apart .
a combination of at least two rrms ( 1/2 or 2/3 ) is 
 required for the high - affinity binding of hnrnp l to rna .
the competition 
 experiments in figure 4a demonstrate that pre - organization 
 of the binding sequences in the i - motif lateral loops confers entropic 
 and kinetic advantages for hnrnp ll binding .
a comparison of the role 
 of hnrnp l in the rna switch that regulates vegf expression 
 with the role of hnrnp ll in the regulation of bcl2 expression provides two insightful analogies .
first , the hnrnp l binding site consists of 21 nucleotides 
 in mrna 3utr approximately equivalent to the 23 combined nucleotides 
 contained in the two lateral loops and the linker region recognized 
 by hnrnp ll .
second , the conformational change in the vegf 3utr is directed by two different signals , hnrnp l and an 
 inf--activated inhibitor of the translational complex , which 
 bind to two different rna conformers in a mutually exclusive manner , 
 just as hnrnp ll and imc-76 bind to the i - motif and flexible hairpin 
 in the bcl2 promoter . whether there is a transcriptional 
 factor equivalent to imc-76 remains to be determined .
the hnrnp 
 ll protein binds with high affinity to the bcl2 i - motif 
 ( 2070 pm ) , and sirna knockdown significantly decreased bcl2 expression ( figure 2a ) .
recognition 
 of the i - motif is through the 5 and 3 lateral loops , 
 but subsequent unfolding of the i - motif is presumably required before 
 a stable complex is formed ( figures 3 and 4 ) .
it is likely that both lateral loops are initially 
 recognized by adjacent rrms ( 1 and 2 ) before subsequent hnrnp ll  driven 
 changes in the interhelical conformation , so that the 5 and 
 3 lateral loops are driven apart to bind to the rrms spaced 
 further apart ( e.g. , 1 or 2 with 3 or 4 ) ( figure 8a  c and a  d ) . in cells the competition for the bcl2 i - motif species by imc-76 , which depletes this population , 
 reduces the amount of hnrnp ll bound to the bcl2 promoter , 
 as shown by chip analysis ( figure 7a ) , whereas 
 imc-48 produces the opposite effect ( figure 7b ) by constraining the i - motif structure . as further proof of our 
 overall proposal , imc-48 and imc-76
are antagonistic in their effects 
 at three different levels : in solution , as shown by nmr ; at the promoter 
 level ( figure 7c ) , as shown by chip analysis ; 
 and in cellular consequences , as shown by chemosensitization to cyclophosphamide .
the mechanism by which hnrnp ll acts as 
 a transcriptional factor 
 is as yet unknown . as noted
, hnrnp k activates myc transcription and binds to the ct elements in the promoter , probably 
 by mechanism similar to that for hnrnp ll .
at least two other factors 
 may be important in the mechanism for transcriptional activation by 
 hnrnp ll .
first , there is a ca element in the upstream region that 
 is a potential hnrnp l binding site ; the binding of the more ubiquitous 
 hnrnp l to this element may result in a looping structure with formation 
 of a heterodimer with hnrnp ll to activate transcription .
second , ddx21 , an rna helicase , also binds to 
 the i - motif ( table 1 ) or to an associated protein , 
 and this may be important in facilitating i - motif unfolding to activate 
 transcription .
our contribution shows 
 that the intrinsic dynamic state of the 
 i - motif , similar in many respects to the dynamic nature of rna , makes 
 the dynamic equilibrium of the noncanonical dna structure an attractive 
 target for small molecule control of gene expression .
it is important 
 to note that the biological effects we observe are correlations consistent 
 with the proposed mechanism rather than direct proof for i - motifs 
 in cells .
we propose that the correlated evidence between the solution 
 and the cellular effects of imc-48 and imc-76 in combination with 
 hnrnp ll are at this point as compelling as that provided for the 
 presence of g - quadruplexes in cells , with the exception of experiments 
 of the type recently published by the cambridge group using a fluorescent 
 antibody against g - quadruplexes . for 
 the bcl2 i - motif ,
the mutual exclusivity of imc-76 
 and hnrnp ll for targeting different conformational forms of the equilibrating 
 i - motif allows the repression of bcl2 gene expression 
 and chemosensitization of drug - resistant lymphoma and breast cancer 
 cells using a steroid molecule .
conversely , 
 enhanced expression of bcl2 mediated by compounds 
 related to imc-48 provides a means to protect against neurodegenerative 
 diseases , such as those found in cns disorders .
this brings the i - motif 
 into focus as an alternative structure to the g - quadruplex in promoter 
 elements as a therapeutic target .
it is anticipated that the tools 
 of the medicinal chemist can be harnessed to identify additional molecules 
 that function at this locus to control gene expression with important 
 therapeutic consequences .
all of the 
 following incubations , washes , and centrifugations ( 1 min at 500 g ) were performed at 4 c .
the biotinylated bcl2 i - motif wild - type and mutant oligomers ( 5-biotin - ttttcttttcccccacgccctctgctttgggaacccgggaggggcgcttacagccccgctcccgcccccttcctcccgcgcccgcccct-3 ) 
 and mutant oligomers ( 5-biotin - ttttcttttcccccacgccctctgctttgggaacccgggaggggcgcttacagttttgctcccgctttcttccttttgcgcccgcccct-3 ) 
 ( 4 g each ) were conjugated to washed streptavidin beads in 
 separate 1.5 ml eppendorf tubes in binding buffer b ( 1 mm dtt , 25 
 mm tris - hcl [ ph 7.6 ] , 50 mm nacl , 0.5 mm mgcl2 , 1 mm edta , 
 10% glycerol ) plus 1 protease inhibitor cocktail overnight , 
 rotating . following overnight incubation ,
the beads were washed in 
 buffer b. the mutant oligomer - conjugated beads were incubated with 
 500 g hela extract for 3 h , rotating .
the beads were centrifuged , 
 and supernatant was transferred to the wild - type oligomer - conjugated 
 beads and incubated for 3 h , rotating .
the mutant oligomer / bead / hela 
 extract complex was washed in buffer b , and supernatant from each 
 wash was transferred to the wild - type oligomer .
proteins were eluted 
 off the mutant oligomer - conjugated beads with successive washes of 
 an nacl gradient ( 0.12 m ) in buffer b , and each supernatant 
 was collected and combined .
the wild - type oligomer / bead / hela nuclear 
 extract complex was subjected to the same procedure of washing and 
 elution as that described for the mutant oligomer complex .
the eluted 
 proteins were processed by the bio5 proteomics core facility ( university 
 of arizona , bio5 institute , tucson , az ) .
the two protein samples were 
 subjected to sds page and visualized by coomassie and silver staining . 

prominent bands were excised from the gel and analyzed for protein 
 identification by lc / ms / ms .
the cdna of hnrnp 
 ll was purchased from open biosystems ( thermo scientific ) and subsequently 
 cloned into the pet28a protein expression vector ( novagen ) .
after 
 sequencing analysis to confirm the pet28a  hnrnp ll , this expression 
 construct was transformed into rosetta - gami b ( de3 ) plyss cells ( novagen ) . 

the expression of hnrnp ll was induced by 0.1 mm iptg ( isopropyl -d-1-thiogalactopyranoside ) overnight at room temperature . 

harvested cells were resuspended in a lysis buffer ( 50 mm nah2po4 [ ph 8.0 ] , 300 mm nacl , 1% triton x-100 , 1 mg / ml 
 lysozyme , and 1 protease inhibitor cocktail [ sigma , # 8465 ] ) 
 and underwent 10 cycles of the following : incubation on ice for 5 
 min , vortexing for 30 s , and sonication for 10 s. cell debris was 
 removed by centrifugation at 14  000 rpm for 30 min at 4 c , 
 and the supernatant was removed and incubated with hispur cobalt resin 
 ( thermo scientific ) while rotating for 30 min at 4 c to allow 
 for the selective binding of histidine - tagged hnrnp ll .
the resin 
 was washed by washing buffer a ( 50 mm nah2po4 with 0.4 protease inhibitor cocktail ) and b ( 50 mm nah2po4 [ ph 8.0 ] and 100 m nacl with 0.1 protease 
 inhibitor cocktail ) sequentially , and elution buffer ( 50 mm nah2po4 [ ph 8.0 ] , 300 mm nacl with 1 protease 
 inhibitor cocktail ) was used to separate hnrnp ll from resin .
purified 
 hnrnp ll was subjected to buffer exchange into a protein stock buffer 
 with 20 mm hepes - naoh ( ph 7.4 ) , 100 mm kcl , 10% glycerol , 2 mm dtt , 
 and 0.1% np-40 using a centricon ( millipore ) . purity of hnrnp ll was 
 confirmed by sypro ruby staining .
sequences 
 used for emsa experiments are shown 
 in figures 5 and 6 . 

all oligomers for these experiments were purchased from eurofins mwg 
 operon and page - purified .
beer equation with molecular extinction 
 coefficients ( m cm ) as follows : 
 py39wt , 292  338 ; py39mutt , 319  216 ; pu39wt , 398  551 . 

the wild - type bcl-2 i - motif ( py39wt ) oligomer was end - labeled with 
 [ -p]-atp . the detailed procedure for labeling 

page - purified cold ( nonlabeled ) oligomers and 160 m hnrnp 
 ll as a final concentration were preincubated with 20 mm hepes ( ph 
 6.8 ) , 100 mm kcl , 2 mm mgcl2 , 1 mm edta , 1 mm dtt , 1 g/l 
 bsa , 0.1% tween 20 , 10% glycerol , and 0.01 g/l of poly(didc ) 
 for 20 min on ice , and end - labeled py39wt was added for 5 min . to determine the effect of imc-76 and imc-48 on binding of hnrnp 
 ll to the i - motif , end - labeled py39wt was incubated with several concentrations 
 of imc-76 in a buffer ( 20 mm mes [ ph 6.5 ] , 100 mm kcl , 4 mm mgcl2 , 1 mm dtt , 1 g/l bsa , 0.1% tween 20 , 10% glycerol , 
 and 0.01 g/l of poly[didc ] ) for 3 h at room temperature . 

160 m hnrnp ll was added and incubated for 10 min at 4 c . 
 the dna 
protein complex and free dna were visualized by 6% 
 native page ( 0.5 tbe and 1.25% glycerol in gel and running buffer ) 
 and phosphorimager scanning .
spr analyses were performed 
 on a biacore 
 t100 optical biosensor with cm5 sensor chips ( ge healthcare , piscataway 
 nj ) .
n - hydroxysuccinimide , 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide 
 hydrochloride , and ethanolamine , 1 m ( ph 8.5 ) were purchased from 
 ge healthcare .
biotinylated oligomers with wild - type and mutant sequences 
 ( py39wt : 5-biotinggcaccacagccccgctcccgcccccttcctcccgcgcccgcccctccgcgc3 
 and py39mutt : 5-biotinggcaccacagttttgcttttgctttcttccttttgcgtttgtttttccgcgc-3 ) 
 were purchased from eurofins mwg operon .
louis , mo ) ( sa ) was immobilized on a cm5 chip 
 using standard amine coupling .
briefly , carboxy groups on the chip 
 surfaces were activated with an injection of 0.05 m n - hydroxysuccinimide/0.2 m 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide 
 hydrochloride at a flow rate of 10 l / min for 7 min sa , diluted 
 in 10 mm naoac ( ph 5.5 ) , 20 g / ml , and pulsed over the surface 
 at a flow rate of 10 l / min until 1750 ru was achieved .
temperature 
 was 25 c , and running buffer was 10 mm hepes ( ph 7.4 ) , 150 mm 
 nacl , 0.05% tween 20 .
any remaining active esters were blocked by 
 injecting 1 m ethanolamine ( ph 8.5 ) for 7 min at 10 l / min . 

the sa surfaces were washed six times with 1 m nacl/50 mm naoh at 
 a flow rate of 20 l / min for 60 s. both active and reference 
 flow cells had sa .
the biotinylated oligomers were resuspended 
 in 10 mm tris ( ph 8.0 ) , 
 1 mm edta at 100 m , then diluted to 1 m in 20 mm hepes 
 ( ph 7.9 ) , 100 mm kcl , 2 mm mgcl2 , 1 mm edta , 1 mm dtt , 
 1 g/l bsa , 0.1% tween 20 , and 10% glycerol .
the diluted 
 oligomers were heated at 95 c for 5 min , cooled to rt , and centrifuged 
 at 16  000 g for 10 min at rt .
the supernatant 
 was diluted to 1 nm in the same buffer and injected over the active 
 surface at 10 l / min until 5 ru was captured .
hnrnp ll 
 was diluted into running buffer ( 20 mm hepes for ph 7.9 
 and ph 6.8 and 20 mm mes for ph 6.5 were used ) together with 100 mm 
 kcl , 2 mm mgcl2 , 1 mm edta , 1 mm dtt , 1 g/l 
 bsa , 0.1% tween 20 , and 10% glycerol .
analysis temperature was 18 c , and flow rate was 50 l / min . 

association time was 300 
 s ( ph 7.9 and ph 6.8 ) or 420 s ( ph 6.5 ) .
dissociation time was 500 
 s. surfaces were regenerated with a 10 s pulse of 20 mm naoh at a 
 flow rate of 30 l / min , followed by a stabilization time of 
 350 s. raw data were reference subtracted , and buffer blanks 
 were subtracted 
 ( double referencing ) .
data were fit to a 1:1 binding model using a 
 global fit algorithm ( biacore t100 evaluation software ) to obtain 
 the kinetic parameters ka , kd , and kd .
py39wt and py39mutt were 
 diluted to 5 m with a buffer ( 50 mm mes [ ph 6.5 ] , 100 mm kcl , 
 2 mm mgcl2 , 1 mm edta , 1 mm dtt , 1 g/l bsa , 
 0.1% tween 20 , and 10% glycerol ) .
recombinant hnrnp ll was diluted 
 by protein stock buffer to desired concentrations to maintain consistent 
 buffer conditions in each sample .
cd spectra were baseline corrected 
 by subtracting a buffer alone or a buffer with protein .
for the br2 footprinting 
 of the bcl-2 i - motif and hnrnp ll complex , recombinant hnrnp ll was 
 incubated with end - labeled py39wt in a buffer ( 50 mm mes [ ph 6.5 ] , 
 4 mm mgcl2 , 100 mm kcl , 1 mm dtt , 1 g/l bsa , 
 0.1% tween 20 , 10% glycerol , and 0.02 g/l poly[didc ] ) 
 for 5 min at room temperature .
bromination was conducted by addition 
 of 0.1 mm bromine for 30 min at room temperature , and subsequently 
 a phenol / chloroform solution was added to interrupt the bromination 
 and remove the protein .
the pellet was washed with 80% etoh and treated with 
 10% piperidine at 93 c for 15 min to induce the bromination - specific 
 dna cleavage .
cleaved product was washed with water and visualized 
 by a 20% sequencing gel with 7 m urea .
sirnas ( i d : sasi_hs01_00171042 
 and sasi_hs01_00171043 ) targeting hnrnp ll ( sigma ) were diluted to 
 50 nm as a final concentration . as a negative control sirna , on - targetplus 
 nontargeting pool ( dharmacon ) was used . for the untreated control , 
 transfection reagent with media only was used .
mcf-7 cells ( 1.5  
 10 per well of a 12-well plate ) cultured in 10% fbs and 
 1% penicillin / streptomycin - supplemented rpmi were treated with hnrnp 
 ll sirna with fugene hd transfection reagent for 72 h. for determining 
 the knockdown effect of hnrnp ll along with imc-76 treatment , sirna 
 of hnrnp ll was transfected for 48 h followed by addition of imc-76 
 for 24 h. total rna was extracted using an rneasy purification kit 
 ( qiagen ) and quantitated by measuring absorbance at 260 nm .
the cdna 
 was synthesized by a reverse - transcription kit ( qiagen or takara with 
 gdna remover ) and used as templates for qpcr with taqman probes for 
 hnrnp ll ( hs00293181_m1 , fam - labeled ) , bcl-2 ( hs00608023_m1 , fam - labeled ) , 
 pdgfr- ( hs01019589_m1 , fam - labeled ) , kras ( hs00364282_m1 , fam - labeled ) , 
 and gapdh ( hs02758991_g1 , vic - labeled ) ( abi ) .
the ct values were obtained by rotor - gene q ( qiagen ) to analyze 
 the relative quantity of hnrnp ll and bcl-2 mrna compared to gapdh 
 as an internal control .
the pgl3-bcl-2 wild - type 
 construct was 
 prepared using the bcl-2 p1 promoter region from 35 to + 614 , 
 which includes the i - motif starting site .
the sequence was inserted 
 into the pgl3-basic vector at the kpni and nhei restriction 
 sites .
mcf-7 cells ( 1.5 
  10 ) were transfected with 500 ng of pgl3 construct , 
 10 ng of prl - tk , and 50 nm of negative control or hnrnp ll sirna by 
 fugene hd transfection reagent and incubated for 72 h. cells were 
 lysed by passive lysis buffer ( promega ) , and then supernatants were 
 subjected to dual - luciferase assays ( promega ) using an fb12 luminometer 
 ( berthold detection system ) .
data were normalized to the ratio of 
 firefly to renilla luciferase of sirna - treated sample and to sirna - untreated 
 control .
mcf-7 cells ( 5  10 ) and 
 bjab cells ( 1  10 ) were cultured overnight and then 
 treated for an additional 24 h with 0.5 of imc-76 or 2 m imc-48 . 

treatment with dmso served as the control . to determine the antagonistic 
 effect of two compounds , mcf-7 cells ( 34  10 )
were treated with dmso or 2 m of imc-76 for 24 h. 
 the next day , dmso - treated cells were administered with dmso or imc-76 , 
 and imc-76-treated cells were administered with 2 or 4 m of 
 imc-48 with fresh media for 24 h. the composition of the buffers used 
 for this chip assay is the same as those of the ez chip kit ( millipore ) . 

cells were treated with formaldehyde ( 1% ) to cross - link proteins to 
 dna for 13 min at rt .
mcf-7 and bjab cells were lysed with 1% sds 
 buffer and sonicated to fragment chromosomal dna into 500 
 base pairs for 15 and 45 cycles , respectively . sheared chromosomal 
 dna was diluted with chip dilution buffer and precleaned with protein 
 g - coupled dynabeads ( invitrogen ) for 2 h at 4 c .
overnight ip 
 with 4 g of igg ( cell signaling , # 2729s ) , acetyl - histone h3 
 ( millipore , # 06 - 599 ) , sp1 ( cell signaling , # 5931s ) , or hnrnp ll ( cell 
 signaling , # 4783s ) antibodies at 4 c was followed by addition 
 of protein g - coupled dynabeads for 90 min 4 c .
immunoprecipitants 
 were washed with low salt , high salt , and licl immune complex wash 
 buffer .
elution with vortexing for 30 min at rt and reverse cross - linking 
 with 200 mm nacl at 65 c overnight were performed sequentially . 

the dna was purified using a pcr purification kit ( qiagen ) , and sybr 
 green i qpcr analysis was performed with rotor - gene q ( qiagen ) to 
 determine relative quantity of dna using primers to specifically amplify 
 the 3 to 103 base pairs from the bcl-2 i - motif  forming 
 region within the promoter ( bcl-2 p1 promoter region , 5-aggagggctctttctttcttctt-3 
 [ forward ] and 5-gtgcctgtcctcttacttcattct-3 [ reverse ] ) . 

an upstream region (   3456 base pairs ) from 
 this i - motif - forming region was also 
 amplified to serve as a negative control for normalization using the 
 primer pair 5-aggttggggccatggtttact-3 ( forward ) and 
 5-cagcctgggtgacagactgatac-3 ( reverse ) .
melting analysis 
 of pcr product showed only one detectable tm ( data now shown ) , and double normalizations were performed to obtain 
 data ( 2 ) .
ct values were calculated by 
 subtracting ct values of negative region 
 ( ct  ct  neg ) and then ct values were 
 obtained by normalizing to ct of 
 input ( ct  ct  input ) . to determine if imc-76 and imc-48 
 affect the transcription level of sp1 and hnrnp ll , qpcr was conducted 
 using rotor - gene q ( qiagen ) .
mcf-7 cells ( 1.5  10 ) and bjab cells ( 3  10 ) were treated with 2 m 
 imc-76 and imc-48 for 24 h. total rna extraction , cdna synthesis , 
 and qpcr were performed using the gene - specific taqman probes . the 
 specificity and ip quality of sp1 and hnrnp ll antibodies
are demonstrated 
 by the manufacturer and further verified by ip ( supplemental figure 4 ) . for further verification of 
 the specificity of these antibodies with mcf-7 cells ,
nuclei isolated by kit ( sigma , nuc101 ) 
 were incubated with ripa buffer ( cell signaling , # 9806 ) for 15 min 
 on ice .
after homogenization 
 using qiashredder ( qiagen ) , the extract was centrifuged at 14  00 
 rpm at 4 c for 15 min .
the supernatant was precleaned with 50 
 l of protein g - coupled magnetic beads ( invitrogen , 10003d ) 
 at 4 c for 1 h. protein concentration was determined by bradford 
 assay , and then the supernatant was diluted to 1 g/l 
 for sp1 and 12.5 g/l for hnrnp ll by lysis buffer . for 
 ip
, antibodies for sp1 and hnrnp ll were added to reach a 1:100 and 
 1:10 dilution , respectively . as a negative control ,
0.51 
 g of igg , optimized to adjust the heavy chain signal in ip 
 samples of igg and sp1 by western blot , was used .
binding of antibodies 
 was conducted at 4 c overnight for sp1 and 2 h at rt for hnrnp 
 ll .
protein g - coupled magnetic beads with 1% bsa were added and incubated 
 for 1 h to precipitate the immunocomplex .
the beads carrying the immunocomplex 
 were washed by lysis buffer three times . to dissociate the immunocomplex , 
 25 l of laemmli buffer
was added , heated at 95 c for 
 5 min , and subjected to sds - page ( 6% or 8% ) . for western blot analysis , 

proteins were transferred to pvdf membrane in tbs buffer with 20% 
 meoh . after blocking the membrane with 2% bsa/2% nonfat milk in tbs - t 
 ( 0.1% tween 20 ) for 1 h , sp1 antibody with 1:1000 dilution and hnrnp 
 ll antibody with 1:300 in 1%
bsa / tbs - t were treated overnight at 4 
 c . as a secondary antibody , goat antirabbit igg ( h+l ) dylight 
 680
was diluted into 1:10  000 in 1% bsa / tbs - t and incubated 
 for 1 h at rt .
 in a companion paper ( doi : 10.021/ja410934b ) we demonstrate that the c - rich 
 strand of the cis - regulatory element in the bcl2 promoter element 
 is highly dynamic in nature and can form either an i - motif or a flexible 
 hairpin . under physiological conditions these two secondary dna structures 
 are found in an equilibrium mixture , which can be shifted by the addition 
 of small molecules that trap out either the i - motif ( imc-48 ) or the 
 flexible hairpin ( imc-76 ) . in cellular experiments we demonstrate 
 that the addition of these molecules has opposite effects on bcl2 gene expression and furthermore that these effects 
 are antagonistic . in this contribution 
 we have identified a transcriptional 
 factor that recognizes and binds to the bcl2 i - motif 
 to activate transcription . the molecular basis for the recognition 
 of the i - motif by hnrnp ll is determined , and we demonstrate that 
 the protein unfolds the i - motif structure to form a stable single - stranded 
 complex . in subsequent experiments 
 we show that imc-48 and imc-76 
 have opposite , antagonistic effects on the formation of the hnrnp 
 ll  i - motif complex as well as on the transcription factor occupancy 
 at the bcl2 promoter . for the first time we propose 
 that the i - motif acts as a molecular switch that controls gene expression 
 and that small molecules that target the dynamic equilibrium of the 
 i - motif and the flexible hairpin can differentially modulate gene 
 expression . 
Introduction
Results and Discussion
Conclusion
Methods