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Modeling of Epigenetic Modification-Induced Changes in CRX-dependent Genes cis-Regulatory Elements

Purpose DNA methylation is a well characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of synthesizing a strand of RNA from DNA, or transcription, is controlled by proteins that regulate RNA polymerase activity by binding to specific gene regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell specific gene expression. While much is known about the functions and DNA binding specificity of CRX, less is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements. Methods We used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO, PDE6B, PAX6, and LINE. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences. Results In this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to our DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate that changes to the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX dependent transcription. Conclusion Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

Conclusion: Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

INTRODUCTION
Epigenetic modification of genomic DNA and associated histone proteins are crucial regulatory signals allowing eukaryotic cells the ability to adapt to dynamic environmental conditions 1 . DNA methylation is the covalent addition of a methyl group to the C-5 position of cytosine bases in genomic DNA. This addition is catalyzed by structurally distinct DNA methyltransferases (Dnmt) enzyme family members 2,3 . In plant and vertebrate genomes, DNA methylation is required for normal development and function of organisms 4,5 . DNA methylation has been linked to many key processes in vertebrate genomes, such as X-chromosome inactivation 6 , regulation of tissue-specific gene expression 7 , and suppression of mobile element transposition 8 .
Dysregulation of DNA methylation-related epigenetic mechanisms is associated with human disease 9,10 . While DNA methylation has an increasingly appreciated role in complex genome regulation, the specific biochemical underpinnings of how this modification modulates the genome remain unclear.
Recent evidence demonstrates that DNA methylation regulates transcription within the retina.
Cone and rod photoreceptor-specific genes display cell-specific patterns of DNA methylation, which appear to play an important role in the establishment and maintenance of retinal cell typerestricted gene expression 11,12 . Furthermore, targeted retina-specific disruption of Dnmts in murine models result in abnormal development of retinal neurons and dysregulation of global retinal gene expression [13][14][15] . Collectively these findings hint at an important role for epigenetic modification of DNA during retinal differentiation and maturation. However, deciphering the mechanistic detail of this role is vital for gaining insight into retinal function, retinal regeneration, and developing novel therapeutic strategies for retinal degeneration.
The homeodomain transcription factor cone-rod homeobox (CRX) is required for proper maturation of both rod and cone photoreceptors 16,17 . Mutations in CRX result in the blinding retinal degenerative diseases cone-rod dystrophy, Leber congenital amaurosis, and retinitis pigmentosa 18 . CRX mediates complex photoreceptor-specific transcriptional networks through physical interaction with evolutionarily conserved DNA sequence motifs 19,20 . Despite the presence of hundreds of thousands of these cis-regulatory elements in the mouse genome, less than 6,000 functional CRX binding regions (CBRs) have been identified in the murine retina.
Furthermore, a subset of these CBRs display cell-type specific affinity in mouse rods and cones 19 . Beyond sequence context, genomic features responsible for regulating the spatial and temporal binding of CRX are poorly understood and represent a significant gap in our knowledge of photoreceptor development. One potential model for this differential binding is the dynamic epigenetic modification of the genome influencing local chromatin conformation and accessibility to transcriptional regulators such as CRX. Evidence in model systems demonstrates that several CRX-dependent genes have an inverse correlation between DNA methylation and gene expression 11,12 . Here, we expand on these findings by demonstrating a similar relationship for the first time in primary human retinal tissue. We also use molecular modeling to build evidence that reversible DNA methylation proximal to CRX binding motifs alters structural characteristics of the DNA double helix including minor and major groove width and DNA flexibility that may modulate CRX binding. Collectively, this study offers compelling evidence that DNA methylation plays a critical role in epigenetic regulation of human photoreceptor neurons and also provides insight into the biochemical interactions underlying this mode of regulation.

Tissue Collection
De-identified post-mortem human donor eyes procured from three individuals with no reported ocular disease (National Disease Research Interchange, Philadelphia, PA) were used to collect ocular tissues for gene-specific quantification of DNA methylation. A scalpel was used to pierce the limbus followed by collection of the cornea using scissors. Whole corneas were flash frozen and ground into a fine powder using a mortar and pestle then immediately transferred to nucleic acid extraction buffer and stored at -80˚C. Eyecups were further dissected with scissors by making four radial cuts exposing the retina. Cuts posterior to the ciliary margin were made liberating the retina from the anterior portion of the eye. Vitreous was removed and the retina was carefully peeled away from the eyecup using fine forceps. Whole retina from each donor was briefly washed in HBSS without calcium or magnesium to rinse away contaminating retinal pigment epithelial cells, placed in nucleic acid extraction buffer, vigorously vortexed, and stored at -80˚C.

Nucleic Acid Purification
Genomic DNA was extracted from human ocular tissues using a Qiagen Allprep Kit (Hilden, Germany) per manufacturer's instructions. Briefly, lysates were homogenized using Qiashredder spin columns. Lysate flow through was then transferred to silica-based spin columns where genomic DNA and RNA were sequentially purified. Quality and quantity of DNAs were examined using UV spectrophotometry.

Bisulfite Pyrosequencing
Quantitative analysis of DNA methylation was measured using bisulfite pyrosequencing performed as previously described 14 was annealed to the purified single-stranded PCR product and pyrosequencing reactions were performed using the PyroMark Q24 Pyrosequencing System per manufacturer's instructions (Qiagen, Hilden, Germany). Percent DNA methylation at each CpG dinucleotide in the BS PCR amplicon was determined and averaged between biological triplicates and statistical significance between the two sample groups was determined using a one-tailed t-test with a significance threshold set at 0.01. All PCR and sequencing primers used in these experiments are shown Table 1.

Bioinformatics Analysis of CRX Binding Sites
Computationally predicted CRX binding regions (CBRs) in the human genome were generated from CBRs obtained from a previously published study of mouse photoreceptors 19 .
Experimentally validated mouse CBRs were aligned and mapped as a custom track to the human hg19 genome assembly using the LiftOver tool in the UCSC Genome Browser 22 . These data were overlaid with adult human retina RNA-sequencing transcriptome data as determined by

Homology Modeling of Human CRX Binding Site
The UniProt accession number O43186 was used for modeling and assembly of CRX 26 . The DNA binding domain of CRX, consisting of amino acids 39-98, was generated using Swiss Model 27 . Models of the CRX DNA binding domains were energy minimized in YASARA before docking experiments 28,29 .

Molecular Modeling of the Rhodopsin Enhancer Region and RHO Promoter Region
The RER enhancer region and RHO promoter sequences were obtained from UCSC Genome

CRX Docking
CRX was bound to RER and RHO sequences through HADDOCK to determine the interaction mechanism 31,32 . The previously modeled CRX DNA binding domain structure consisting of residues 39-98, was used in docking simulations with the energy minimized average structures from the promoter and RER simulations. Active residues for CRX include amino acids 40-46, 63, 69, 82, 84-85, and 88-93 26 . Active residues for the DNA molecules were bases 20-25 for the RER and 15-19 for the RHO promoter. CRX was then docked to the sequences using the Prediction interface on the HADDOCK server. We then used YASARA to determine the interactions occurring between the residues and the consensus sequence. Our epigenetic analysis of the human eye demonstrates tissue-specific patterns of DNA methylation in 5' regulatory sequences of photoreceptor-specific genes that are inversely correlated with mRNA expression. These and previous observations in other mammalian retinal model systems suggests a functional role for DNA methylation in repressing transcription at these loci. However, a mechanism for this repression remains uncharacterized. A commonality between RHO, PDE6B, and many other photoreceptor-specific genes, is that they are transcriptionally regulated by the homeodomain transcription factor CRX 19,34,35 . This observation led us to question whether DNA methylation plays a role in regulating temporal and cell-specific binding of CRX to cis-regulatory regions within photoreceptor genomes. To test this hypothesis, we used a computational approach to align previously determined genome-wide CRX binding regions (CBRs) in the rod rich wild type mouse retina to the human hg19 2009 genome assembly (Figure 2; Predicted CBRs). To further assess the functionality of these presumptive regulatory regions, predicted CBRs were searched for experimentally validated CRX binding sites (Figure 2A; Validated CRX Binding Sites) or sequences containing CRX binding motifs ( Figure 2B; Human CRX Binding Motifs). These analyses demonstrate that differentially methylated regions (DMRs) identified in this study are adjacent to experimentally validated CRX binding sites in the well-characterized RHO 5' promoter and Rhodopsin enhancer region (RER), as well as predicted CRX binding sites in the PDE6B 5' promoter. Given these results, we predict that differential methylation of these CpG sites may play a prominent role in modulating CRX binding to cis-regulatory elements upstream of RHO, PDE6B, and other CRXregulated genes.

DNA methylation is inversely
The CRX DNA binding domain interacts with DNA grooves but not known methylation sites DNA methylation is known to alter the three dimensional structure of double stranded DNA and interactions with DNA binding proteins such as transcription factors 36,37 . Computational modeling data suggest that the presence of a bulky methyl group in 5mC results in widening of the major groove and a concomitant narrowing of the minor groove 37 . Further, it has been experimentally validated that 5mC DNA bases biochemically mimic thymine bases due to similar hydrophobic interaction with major groove edge methyl groups 38 . More recent evidence has demonstrated a structural basis for affinity modulation of human transcription factors to methylated DNA via interactions of hydrophobic amino acid residues with methyl groups 36 .
Taken together these observations suggest a mechanism for DNA methylation to narrow minor groove width and increase available hydrophobic interactions of cis-regulatory elements as a reversible mode of modulating CRX binding. However, a lack of structural information on CRX and the interactions of CRX with DNA hinders development of a specific mechanism. To test the hypothesis that methylated DNA sequences are less favorable binding partners with CRX than their unmethylated counterparts, we generated a homology model of the CRX DNA binding domain and models of the RHO promoter and the RER. The CRX binding domain model shows the expected 3 helix bundle characteristic of a helix-turn-helix homeobox protein ( Figure 6). In both cases, DNA duplexes span one experimentally validated CRX binding site as well as one (promoter) or two (RER) CpG cytosine substrates for DNA methylation ( Figure 4+5). We then docked the model of CRX to the DNA models using HADDOCK 32 . The CRX binding site within the promoter is within the minor groove and the C-terminal helix of CRX fits into the groove making contacts with the DNA backbone. Glutamic acid 80, lysine 88, and arginine 90 are known disease causing mutations within CRX and in our model of CRX bound to DNA, we see that K88 and R90 make electrostatic interactions with DNA ( Figure 6) [39][40][41] . E80 makes contact with R69 and Q84, which contact the minor groove of DNA, suggesting a structural role for this amino acid ( Figure 6). Thus, we felt confident that our CRX model could provide insight into how CRX interacts with DNA and is regulated by methylation. We next repeated the docking to the CRX binding sequence in the RER. The best interaction score produced by HADDOCK placed CRX in the major groove side of the CRX binding sequence. In addition to the previous backbone interactions noted for the promoter, CRX makes interactions with the DNA bases via N89 and K93 ( Figure 6). Given that there currently is no structural information on CRX or these DNA sequences beyond the models we present here, we cannot exclude either binding mode.
What is clear is that CRX likely interacts with a single DNA groove and does not make direct contact with methylation sites at this locus. These data suggest a mechanism of inhibition based on direct interaction with the methyl CpGs is unlikely.

CpG Methylation enhances DNA flexibility
Our data showing that CpG methylation negatively regulates CRX binding raises the question of the molecular mechanism for binding inhibition. In addition to steric effects, CpG methylations have been proposed to change DNA dynamics and change the local structure of DNA, however which of these scenarios applies to CRX was not clear 36,37,42 . To address this question of the inhibition mechanism, we created a second set of DNA models that were methylated at the known sites and then equilibrated the models using molecular dynamics. We then analyzed the global and local effects of CpG methylation on the dynamics and structure of the DNA sequences and the CRX binding sites. Groove width statistics for these simulations are summarized in Table 2. While the mean and median values for the groove widths are similar, global analysis of the major and minor grooves shows that both structures are more dynamic (Table 2 and Figure 7). The increase in flexibility is indicated by the increased variance and standard deviations of the data measurements and visually by the wider distribution of values in the density plots ( Figure 7 and Table 2). Analysis of the ensemble volume ratio shows that the structures of the methylated promoter have a larger volume, characteristic of higher dynamics.
The effect of methylation is more apparent in the measurements that include just the CRX binding sequence, suggesting that the binding site is part of the dynamic region of DNA ( Figure   7). These observations indicate that methylation does not alter the structure of DNA or the CRX binding site within the RHO promoter but changes the dynamics of the structure.
We then performed these simulations and analyses using the RER sequence, which is methylated at sites on either side of the CRX binding motif. Methylation of the RER induced increased bending of the RER sequence in simulations relative to the unmodified sequence ( Figure 7C). Further analysis of the groove widths in the RER sequence shows that with methylation the major groove widens while the minor groove narrows relative to the unmodified form ( Figure 7 and Table 2). The standard deviation and variance for the major groove also increase, indicative of increased dynamics. The ensemble volume ratio increased by ~25% suggesting drastic changes in the dynamics of the RER upon methylation. The effect of methylation is especially apparent in the CRX binding site, which shows a significant population of major and minor groove widths in the methylated sequence simulations that is outside range values for the unmodified DNA sequence (Figure 7). Collectively, these data show that the presence of methylation alters the dynamics of DNA and the groove widths, possibly preventing efficient binding by CRX to the promoter and RER sequence.

Discussion
Using cellular and in silico approaches, we addressed the regulation of CRX binding to genomic cis-regulatory elements and propose a model for how epigenetic DNA methylation may modulate this interaction. Bisulfite pyrosequencing of human ocular tissue showed that the RHO and PDE6B regulatory regions have lower levels of DNA methylation in the retina compared to corneal tissues (Figure 3). Given that CRX binds and regulates both genes, we postulated that CRX binding is inhibited by methylation of these regions upstream of the gene in non-expressing cells to block transcription. While this hypothesis will be best tested using biochemical methods, molecular modeling of the CRX DNA binding domain docked to CRX binding motifs within human RHO cis-regulatory elements were used in this study to demonstrate a basis for this model and to guide future biochemical experiments. To this end, our modeling data suggest that CRX interacts with the grooves of DNA and does not appear to make direct contact with methylation sites. Though CpG methyl sites do not occur within the CRX binding motifs analyzed in this study, molecular dynamics simulations demonstrated an increase in the overall structural dynamics and flexibility of CRX motifs adjacent to methyl CpG sites relative to data simulations of unmethylated sequences (Figure 7 and Table 2). Collectively, these data indicate that regional methylation of the RHO promoter and RER cis-regulatory elements may occlude CRX binding through alterations in the structure and dynamics of adjacent CRX binding motifs.
An additional possibility unexplored in this study is that DNA methylation is an indirect effector of CRX affinity to cis-regulatory elements. Changes in DNA methylation are known to induce other repressive epigenetic modifications such as histone deacetylation and histone methylation 43,44 . Future studies focused on in vitro binding assays using methylated and unmodified oligonucleotides will be useful in testing a direct role of DNA methylation in CRX binding affinity.
The role of DNA methylation on CRX affinity has not been previously explored, however the effect of methylation on DNA structure and interactions with DNA binding proteins has been extensively studied. DNA methylation is known to affect local and regional DNA/nucleosome structure. Recent work indicates that DNA methylation both reduces DNA flexibility and enhanced nucleosome stability 42,45,46 . Confoundingly, other finding suggest that DNA methylation increases DNA flexibility or acts as a physical block to transcription factor binding [47][48][49] . In a broad sense, our results are consistent with methylation increasing DNA flexibility with a few important clarifications. The first clarification regards positioning of CpG methyl sites. Methylation sites investigated in this study are outside of the CRX binding motif and therefore likely do not act as a direct physical barrier to protein binding as characterized in other studies 36 . Alternatively, our findings indicate that CpG methyl sites adjacent to CRX binding motifs change the dynamics of the major and minor grooves over the entire sequence region including the CRX binding motif (Figures 7). This mode of epigenetic regulation may be used by cells to dynamically modulate binding of many different transcription factors at a particular regulatory locus and represents a potentially new paradigm for exploring the role of DNA methylation in regulating gene expression.
The second clarification regards motif-specific epigenetic modulation of CRX-DNA interactions. Previous studies have indicated that the specific mechanism of interaction between proteins and methyl CpGs appears to be highly sequence and factor dependent. Global analysis of the effects of DNA methylation on several transcription factors shows that CpG methylation can have positive, neutral, and negative effects suggesting that the molecular mechanism may be a combination of effects on specific proteins binding to specific loci 36 . Our data also support a model for DNA methylation influencing motif-specific effects on local DNA-protein interactions. Our molecular dynamics data indicates a methylated promoter CpG site is less perturbed than the effect observed for methylated RER CpG sites (Figures 7). Given that the RHO promoter and RER have little sequence similarity outside of conserved transcription factor binding motifs, it is not surprising that we observe distinct effects on structure and dynamics. In fact, recent functional evidence has demonstrated that individual CRX binding motifs within the murine RHO locus encode differential transcriptional responses to CRX ranging from strong inhibition of transcription to strong transcriptional activation 50 . Our data indicate that the methyl CpG adjacent to the CRX binding motif in the RHO promoter increases the width of the minor groove (Table 2). Inversely, methyl CpGs adjacent to the RER CRX motif increases the width of the major groove in our simulations. This discrepancy may be due to a number of sequencerelated differences between the two cis-regulatory elements controlling human RHO transcription. Collectively, these data support the idea that the effect of DNA methylation on CRX binding is likely a combination of local and regional DNA sequence, distance of CpG methyl sites from CRX motifs, and the number of CpG methyl sites in or adjacent to CRX motifs. In this light, it is worth noting that though our data support a negative effect of DNA methylation on CRX binding at the RHO promoter and enhancer region, these finding do not preclude the possibility that DNA methylation may serve as a positive regulator of CRX binding at other loci in the genomes of retinal neurons. Future modeling studies of other CRX-regulated genes will be useful for further analyzing motif-specific epigenetic modulation of CRX affinity.
Modeling data reported here as well as in future studies will guide precision biochemical analysis of the interaction between CRX with methyl CpGs at diverse photoreceptor-specific cisregulatory elements.
Collaborative Research Grant awarded to RAE, and Burroughs Wellcome Fund Grant #1017506 awarded to RAE. Undergraduate students working on this project were also supported by endowed summer research scholarships provided by JMU alums Mr. and Mrs. Robert Crabtree and Jeffery E. Tickle to whom we thank for their generous support.       The sequence used in CRX/enhancer binding simulations is indicated as a black custom track.