Abstract:
Provided are systems, methods, and media that receive chromosome sequence data; select a first plurality of overlapping octamers from the chromosome sequence data; assign an enrichment score to each of the first plurality of overlapping octamers to produce a first set of enrichment scores; calculate a first average of the first set of enrichment scores; determine whether the first average is above a threshold; select a second plurality of overlapping octamers from the chromosome sequence data; assign an enrichment score to each of the second plurality of overlapping octamers to produce a second set of enrichment scores; calculate a second average of the second set of enrichment scores; determines whether the second average is above the threshold; and output data that indicates that a transcription factor binding site has been identified in connection with at least one of the first plurality of octamers and the second plurality of octamers.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims the benefit of U.S. Provisional Patent Application No. 61/349,131, filed May 27, 2010, which is hereby incorporated by reference herein in its entirety. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
       [0002]    This invention was made with government support under Grants U01 DK072504 and RO1 DK082590 awarded by the National Institute of Health. The government has certain rights in the invention. 
     
    
     TECHNICAL FIELD 
       [0003]    The disclosed subject matter relates to methods, systems, and media for identifying transcription factor binding sites. 
       BACKGROUND 
       [0004]    The dynamic process of gene regulation is essential for embryonic development and cellular function. Gene regulation is primarily mediated by the combinatorial effects of transcription factors interacting with cis-regulatory elements such as promoters and enhancers. Therefore, accurate identification of transcription factor binding sites within the genome is necessary to understand a wide range of cellular processes from cell differentiation to homeostasis to cancer. However, identifying these sites within the genome remains a complex biological and computational question. 
         [0005]    One of the challenges in predicting transcription factor binding sites is that identification of the strongest binding sequence, or consensus site, is not sufficient. Research analyzing genome wide transcription factor occupancy has shown that low affinity binding sites are also significantly occupied in both yeast and drosophila. Furthermore, transcription factors from the same family have been shown to bind identical high affinity sites, but distinct low affinity sites. Therefore, identification of both high and low affinity sites will aid in fully understanding transcription factor specificity within the genome. 
         [0006]    Nkx2.2 is a homeodomain transcription factor expressed in the ventral neural tube and the pancreas during development. A consensus sequence (T(t/c)AAGT(a/g)(c/g)TT) has been identified by SELEX and a corresponding position weight matrix (PWM) was generated and deposited in the TRANSFAC database. However, the predictive power of this PWM is low. More recently, a PWM for Nkx2.2 was generated using protein binding microarray technology. Protein Binding Microarrays use a mathematically constructed set of oligos to quantitatively measure protein-DNA binding for all possible octamers. 
         [0007]    The identification of transcription factor binding sites is an important biological question. To date, the majority of methods to detect these sites have focused on creating statistical models, such as position weight matrices, of transcription factor specificities. However, these models are limited due to the fact that they must make generalized assumptions about transcription factor binding properties that are not completely understood. Conversely, recent technologies have been developed such as ChIP-seq to look at genomic transcription factor occupancy. However, these technologies are technically difficult and limited by the lack of high quality antibodies for many transcription factors. 
         [0008]    Accordingly, new mechanisms for identifying transcription factor binding sites are needed. 
       SUMMARY 
       [0009]    Methods, systems, and media for identifying transcription factor binding sites in accordance with some embodiments are provided. In accordance with some embodiments, systems for identifying transcription factor binding sites are provided, the systems comprising at least one processor that: receives chromosome sequence data; selects a first plurality of overlapping octamers from the chromosome sequence data; assigns an enrichment score to each of the first plurality of overlapping octamers to produce a first set of enrichment scores; calculates a first average of the first set of enrichment scores; determines whether the first average is above a threshold; selects a second plurality of overlapping octamers from the chromosome sequence data; assigns an enrichment score to each of the second plurality of overlapping octamers to produce a second set of enrichment scores; calculates a second average of the second set of enrichment scores; determines whether the second average is above the threshold; and outputs data that indicates that a transcription factor binding site has been identified in connection with at least one of the first plurality of octamers and the second plurality of octamers. 
         [0010]    In accordance with some embodiments, methods for identifying transcription factor binding sites are provided, the methods comprising: receiving chromosome sequence data; selecting a first plurality of overlapping octamers from the chromosome sequence data; assigning an enrichment score to each of the first plurality of overlapping octamers to produce a first set of enrichment scores; calculating a first average of the first set of enrichment scores; determining whether the first average is above a threshold; selecting a second plurality of overlapping octamers from the chromosome sequence data; assigning an enrichment score to each of the second plurality of overlapping octamers to produce a second set of enrichment scores; calculating a second average of the second set of enrichment scores; determining whether the second average is above the threshold; and outputting data that indicates that a transcription factor binding site has been identified in connection with at least one of the first plurality of octamers and the second plurality of octamers. 
         [0011]    In accordance with some embodiments, computer readable media containing computer-executable instructions that, when executed by a processor, cause the processor to perform a method for identifying transcription factor binding sites are provided, the method comprising: receiving chromosome sequence data; selecting a first plurality of overlapping octamers from the chromosome sequence data; assigning an enrichment score to each of the first plurality of overlapping octamers to produce a first set of enrichment scores; calculating a first average of the first set of enrichment scores; determining whether the first average is above a threshold; selecting a second plurality of overlapping octamers from the chromosome sequence data; assigning an enrichment score to each of the second plurality of overlapping octamers to produce a second set of enrichment scores; calculating a second average of the second set of enrichment scores; determining whether the second average is above the threshold; and outputting data that indicates that a transcription factor binding site has been identified in connection with at least one of the first plurality of octamers and the second plurality of octamers. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]      FIG. 1A  shows an enrichment score (E-score) distribution table of Nkx2.2 in accordance with some embodiments. 
           [0013]      FIG. 1B  is a histogram showing the number of occurrences of each possible base in the first position for all possible E-scores in accordance with some embodiments. 
           [0014]      FIG. 1C  shows the results of an Electrophoretic Mobility Shift Assay (EMSA) experiment performed in accordance with some embodiments. 
           [0015]      FIG. 2A  is a flowchart showing a PBM-mapping process in accordance with some embodiments. 
           [0016]      FIG. 2B  shows the results of another EMSA experiment performed in accordance with some embodiments. 
           [0017]      FIG. 2C  shows the results of a Chromatin Immunoprecipitation (ChIP) experiment performed in accordance with some embodiments. 
           [0018]      FIGS. 3A-3C  show three graphs of the relative binding affinity versus prediction scores for PBM-mapping, TRANSFAC, and PBM-PWM in accordance with some embodiments. 
           [0019]      FIG. 4A  shows a schematic representation of the NeuroD promoter in accordance with some embodiments. 
           [0020]      FIG. 4B  shows the results of yet another EMSA experiment performed in accordance with some embodiments. 
           [0021]      FIGS. 5A-5F  are graphs showing relative binding affinity versus prediction score from PBM-mapping for groups of one, three, five, seven, and eight octamers in accordance with some embodiments. 
       
    
    
     DETAILED DESCRIPTION 
       [0022]    As is known in the art, the transcription factor Nkx2.2 binds a 10 base-pair sequence that was thought to contain an invariable “AAGT” core sequence. In accordance with some embodiments, a mechanism for identifying an alternative core sequence for a transcription factor (such as Nkx2.2) is provided. Using this mechanism, an alternative low-affinity core sequence with a wobble in the first position that contains “GAGT” has been identified. 
         [0023]    Berger M F, et al., “Variation in homeodomain DNA binding revealed by high-resolution analysis of sequence preferences,” Cell 133(7):1266-1276, 2008, which is hereby incorporated by reference herein in its entirety, published a protein binding microarray (PBM) analyzing the binding affinity of the Nkx2.2 homeodomain transcription factor. PBMs generate an enrichment score (E-score) with a range from −0.5 to 0.5 for every possible eight-base combination based on the relative intensity readouts from the microarray data. 
         [0024]      FIG. 1A  shows an E-score distribution table of octamers on Nkx2.2. In the rows of the table, octamers are divided into AAGT containing octamers, GAGT containing octamers, and all octamers as indicated in left column  102 . The number of octamers in each group with an E-score above 0.45 is shown in middle column  104 . The average of the E-scores from all octamers in each group is shown in right column  106 . 
         [0025]    In accordance with some embodiments, a mechanism for identifying an alternative core sequence for a transcription factor can operate as follows: First, all octamers with an E-score greater than 0.45 can be selected. As shown in the last row of column  104  of  FIG. 1A , 132 octamers were selected for Nkx2.2. In some embodiments, any other suitable threshold value (i.e., other than 0.45) can be used. Of the selected octamers, the octamers containing a known core sequence can be removed. For example, in embodiments in which the transcription factor is Nkx2.2, 96 (73%) octamers containing the canonical “AAGT” core sequence or its reverse compliment “ACTT” were removed. Any other suitable octamers can be removed or these octamers can be retained in some embodiments. An alternative core sequence can then be identified in the remaining octamers. For example, in embodiments in which the transcription factor is Nkx2.2, of the remaining 36 octamers, 33 (25% of the total) octamers had an alternative sequence “GAGT.” Two of the sequences originally classified as AAGT-containing octamers also had “GAGT” (AAGTGAGT and GAGTAAGT) while three octamers did not contain either core sequence. Finally, the average E-score for octamers containing AAGT, octamers containing GAGT, and all possible octamers can next be calculated to confirm that the average E-scores for the primary and alternative core sequences are significantly larger than the mean for all possible octamers. For example, in embodiments in which the transcription factor is Nkx2.2, AAGT and GAGT containing octamers had mean E-score values of 0.197 and 0.160, respectively, while all possible octamers had a mean E-score of only −0.029, as shown in column  106  of  FIG. 1A . 
         [0026]    As can be seen, the two identified core sequence motifs differ only in the first position. In order to determine whether significant enrichment can be seen with the other two possible first bases (e.g., TAGT and CAGT), a histogram 110 of the number of occurrences of each possible base in the first position (i.e., AAGT, GAGT, TAGT and CAGT) for all E-scores can be plotted as shown in  FIG. 1B . Each point in this histogram represents the percentage of total sites within a 0.10 bin that contains the given core sequence. As can be seen, there is a significant enrichment of only the AAGT and GAGT core sequences. 
         [0027]    In order to experimentally test the alternative GAGT binding site, Electrophoretic Mobility Shift Assay (EMSA) experiments were performed as shown in  FIG. 1C . 
         [0028]    The EMSA experiments were performed as follows: First, in vitro synthesized Nkx2.2 protein was made using the TNT Coupled Reticulolysate System (available from Promega Corporation). Probes were next prepared containing each of the predicted core sequences analyzed or a deleted core sequence. The sequences of each of the probes are listed in Table 1 of Appendix I. 
         [0029]    The probe containing the Nkx2.2 consensus sequence was prepared as described in Watada H, Mirmira R G, Kalamaras J, &amp; German M S, “Intramolecular control of transcriptional activity by the NK2-specific domain in NK-2 homeodomain proteins,” Proc Natl Acad Sci USA, 97(17):9443-9448, 2000, and Anderson K R, et al., “Cooperative transcriptional regulation of the essential pancreatic islet gene NeuroD1 (beta2) by Nkx2.2 and neurogenin 3,” J Biol Chem 284(45):31236-31248, 2009, which are hereby incorporated by reference herein in their entireties. 
         [0030]    Binding of each of the probes to the in vitro synthesized Nkx2.2 (Myc-Nkx2.2 TNT Protein) or alphaTC 1 nuclear extract with or without transfected Myc-Nkx2.2 was measured as follows. 
         [0031]    Probes were labeled by filling in 5′ overhangs with  32 P-dCTP. The binding buffer included 100 mM Tris HCl pH 7.5, 500 mM NaCl, 5 mM EDTA, 10 mM MgCl2, 40% glycerol, 5 mM DTT, 10×BSA, and 0.1 μg/μl of polydIdC. Binding reactions were incubated on ice for 45 minutes with 5 μl of in vitro synthesized protein and 25,000 CPMs, corresponding to approximately 1 fmol, of labeled probe. Samples were run on 5% non-denaturing polyacrylamide gels at 180 V for 1.5 hours in 1×TGE buffer (250 mM Tris base, 1.9 M glycine, and 10 mM EDTA). 
         [0032]    Bands were quantified using the integrated mean of a fixed window for each of the shifts using Photoshop Extended CS3 (available from Adobe Systems Inc.). Values were normalized to total probe (shifted probe+free probe). 
         [0033]    Binding of each probe was next compared to both the original consensus probe and a probe with a deleted core sequence. The GAGT containing probe showed significant binding with in vitro translated Nkx2.2 (TNT Nkx2.2) or nuclear extract from alphaTC1 cells with or without transfected Nkx2.2, although binding was weaker than the AAGT containing probe. 
         [0034]    Taken together, these experiments show that GAGT represents an alternative core sequence for Nkx2.2 binding sites, although its relative binding affinity is lower than the canonical AAGT core sequence. 
         [0035]    In accordance with some embodiments, protein binding microarray data can be mapped directly to the genome to identify putative binding sites, such as Nkx2.2 binding sites. 
         [0036]    The enrichment score (E-score) generated from the protein binding microarray can represent a semi-quantitative estimate of transcription factor binding affinity. In accordance with some embodiments, the E-score for each octamer can be mapped to the genome to predict Nkx2.2 binding sites. This mapping can be referred to a PBM-mapping. 
         [0037]    In accordance with some embodiments, single octamers with an E-score greater than 0.4 (or any other suitable threshold) can be mapped. 
         [0038]    In accordance with other embodiments, a moving average of seven (or any other suitable number) of octamers can be mapped to predict binding affinity with greater accuracy. Sequences with a moving average greater than a given threshold can then be deposited into a database and can be output to a display if desired. The threshold can be set to approximately 0.37 (or any other suitable value). 
         [0039]    A PBM-mapping process  200  that can be used in accordance with some embodiments is illustrated in  FIG. 2A . As shown, PBM data for a given transcription factor can be received at  210  and provided to a database of octamers and E-scores  212 . A genome sequence can also be received at  202 . Process  200  can then get a first (or the next) chromosome sequence of the genome at  204 . An array of seven overlapping octamers can next be formed at  206 . At  208 , E-scores can then be assigned to the octamers in the array based on the data in database  212 . Process  200  can then calculate an average E-score for the array of seven octamers at  214 . It can next be determined at  216  if the average E-score is above a given threshold (such as 0.37 or any other suitable value). If the average E-score is above the given threshold, a database  218  of binding sites can be updated with the array data, the average E-score, and/or any other suitable data. After database  218  is updated, or if it is determined at  216  that the average E-score is not above the given threshold, process  200  can then determine if the end of the chromosome has been reached at  220 . If it has not, then process  200  can, at  222 , delete the first octamer in the array, shift the contents of the array one position toward the former position of the first octamer, add the next octamer in the last position of the array, and loop back to  208 . Otherwise, if it is determined at  220  that the end of the chromosome has been reached, then process  200  can loop back to  204  to get the next chromosome sequence. 
         [0040]    Using this technique, complete analysis of the genome resulted in 3×10̂6 predicted sites, which falls within range of the expected number of transcription factor binding sites expected in the genome. In order to investigate sites that are most likely to be biologically relevant, a search for sites was limited to bound promoters (from 2.5 kb upstream to 1 kb downstream) of genes with expression levels significantly changed (e.g., more than two-fold) in Nkx2.2 null mice at e12.5 or e13.5 and one hundred and eleven novel Nkx2.2 binding site found. 
         [0041]    The results of sites within these promoters can be found in Table 2 of Appendix II. Binding sites were found in seven out of eight genes with increased expression and 24 out of 27 genes with decreased expression in the Nkx2.2 null pancreas. GAGT containing sites were highly represented in the predicted sites—confirming the ability of the technique to predict alternate sites. Twenty three sites, including six GAGT containing sites, were confirmed using EMSA analysis as shown in  FIG. 2B , and 24 sites were confirmed using Chromatin Immunoprecipitation (ChIP) as shown in  FIG. 2C . 
         [0042]    EMSA analysis of selected predicted sites was performed as described above except that probes spanning approximately 50-60 base pairs surrounding the predicted site were incubated with in vitro synthesized Nkx2.2, and the Nkx2.2 consensus probe and the consensus probe with the core sequence deleted were used as positive and negative controls, respectively. 
         [0043]    Confirmation of in vivo promoter occupancy at predicted sites by ChIP was performed using the Active Motif ChIP IT Express kit (available from Active Motif, Inc.). BetaTC6 cells were used for chromatin input and Nkx2.2 mouse monoclonal antibody was used for precipitations. BetaTC6 cells were grown in DMEM supplemented with 15% FBS. Approximately 1.5×10̂7 cells were crosslinked in 1% paraformaldehyde for five minutes at room temperature. Chromatin was then extracted and sheared by sonication using a Diagnode BioRuptor (8 min-30 sec ON/OFF) resulting in chromatin fragments from 200-800 base pairs long. The sheared chromatin was divided into six reactions and run independently. Pulldowns were done with 3 μg mouse anti-Nkx2.2 monoclonal antibody (available from Developmental Studies Hybridoma Bank). Enrichment is shown as fold change over IgG. Normal mouse IgG (available from Millipore Corporation) was used as a negative control. Occupancy of the predicted sites was tested by Sybr-Green qPCR (primers are listed in Table 3 of Appendix III). 
         [0044]    All predicted sites were significantly increased over the IgG control. The housekeeping gene GapdH was used as a negative control and was not significantly enriched. Nkx6.2 −1441, nkx6.2 +669, Irs4 +1495 and Tm4sf4 +912 were not tested in ChIP for technical reasons. 
         [0045]    Tested sites were randomly selected from putative sites in bound promoter regions. In addition to the randomly selected sites, the following sites were also included: a site predicted by the PBM-mapping mechanism described herein that is located in the Region IV enhancer of the Pdx1 promoter, an additional Irs4 site downstream of the bound region (Irs4 +1495), and a previously published Nkx2.2 binding site in the insulin promoter that was the only published site not predicted the PBM-mapping mechanism described herein. 
         [0046]    Of the 28 sites tested by EMSA, only the insulin promoter site, the Nkx6.2 +669 site, and the glucagon −1080 site did not show detectable binding. Glucagon −1080 and Nkx6.2 +669 had an average E-score of 0.347 and 0.364, respectively, and represented the lowest scores of any predicted site tested. The Ins2 −144 site was below an original threshold with an average E-score of 0.233. 
         [0047]    In order to test whether the E-score is correlated with relative Nkx2.2 binding affinity, the relative binding affinity of Nkx2.2 binding in the EMSA experiments was quantified and graphed against the TRANSFAC PWM score, the PBM seed and wobble matrix score, and the E-score. The TRANSFAC PWM was developed from alignment of 23 sequences enriched using SELEX experiments. The PBM-PWM was based on microarray experiments, which provide data for all possible octamers. Numerous statistical corrections to the PWM model were not part of this study. 
         [0048]    As shown in  FIGS. 3A-3C , the highest score obtained from the EMSA probe was compared to relative binding affinity calculated from the EMSA shown in  FIG. 2B . Probes with more than one predicted site (Spk3 and Nkx2.2 −1503) were excluded. Scores from probes that were not bound in the EMSA (Gcg −1080, Nkx6.2 +669, and Ins2 −144) were plotted along the X-axis and not used for r-squared calculation.  FIG. 3A  uses the average E-score from seven overlapping octamers from PBM-mapping,  FIG. 3B  uses the average log-odds from TRANSFAC-PWM, and  FIG. 3C  uses the average Seed and Wobble matrix score from PBM-PWM. 
         [0049]    Single E-scores for the highest octamer and averages of three, five, six, seven, and eight octamer were tested as shown in  FIGS. 5A ,  5 B,  5 C,  5 D,  5 E, and  5 F, respectively. The average of seven overlapping scores showed the highest correlation with relative binding affinity (r-squared=0.666) and outperformed both the TRANSFAC PWM score (r-squared=0.305) and the PBM seed and wobble matrix score (r-squared=0.604) as can be seen from  FIGS. 3A-3C . Using a larger window of overlapping octamers resulted in a decrease in accuracy. Taken together, these experiments show that PBM-mapping represents a highly accurate prediction method to find genome wide binding sites. 
         [0050]    Although the above-described mechanism for determining transcription factor binding sites has been illustrated for Nkx2.2, this mechanism can additionally or alternatively be applied to other transcription factor binding sites to create composite transcription factor binding site maps across the entire genome. Generation of such a map can greatly aid work to identify cis-regulatory elements and understand gene regulation. PBM data is available for at least 391 non-redundant proteins from several species, as described in Newburger D E &amp; Bulyk M L, “UniPROBE: an online database of protein binding microarray data on protein-DNA interactions,” Nucleic Acids Res 37(Database issue):D77-82, 2009, which is hereby incorporated by reference herein in its entirety. However, adjustments to the mechanism may need to be made to account for different profiles of different classes of proteins. 
         [0051]    Although there is overlap between PWM based predictions and PBM mapping, two examples of promoters where the predictions are significantly different have been identified: NeuroD and Insulin. The functional control of the NeuroD promoter by Nkx2.2 is described in Anderson KR, et al., “Cooperative transcriptional regulation of the essential pancreatic islet gene NeuroD1 (beta2) by Nkx2.2 and neurogenin 3,” J Biol Chem 284(45):31236-31248, 2009, which is hereby incorporated by reference herein in its entirety. In the NeuroD promoter, the TRANSFAC-PWM for Nkx2.2 predicted two sites while PBM mapping predicted a novel site upstream of the two TRANSFAC predicted sites that were not bound in vitro or in vivo as illustrated in  FIG. 4A . However, EMSA analysis confirmed binding to the PBM mapping predicted site and not to the two TRANSFAC predicted sites as shown in  FIG. 4B . 
         [0052]    As shown in  FIG. 4B , EMSA analysis showed binding through both core sites, AAGT and GAGT. In this analysis, wildtype, AAGT mutant, GAGT mutant, and double mutant probes were incubated with in vitro translated Nkx2.2 or BetaTC6 nuclear extract. Supershifts were done using the monoclonal Nkx2.2 antibody. 
         [0053]    The PBM mapping site is unique because it is predicted to consist of two adjacent binding sites separated by four base pairs as illustrated in the schematic representation of the NeuroD promoter shown in  FIG. 4A . One binding site contains a canonical AAGT core sequence while the other has the GAGT core sequence identified as described above. However, EMSA experiments did not show dimerization of Nkx2.2 on the promoter. Mutation of each individual core sequence showed a reduction in binding and both sites must be mutated to completely ablate Nkx2.2 binding as shown in  FIG. 4B . Therefore, both sites contribute to Nkx2.2 binding, but dimer formation is prevented, possibly by steric hinderence. This may represent a unique mechanism to increase transcription factor occupancy on the promoter. 
         [0054]    An Nkx2.2 binding site in the insulin promoter (Ins2 −144) was previously published in Watada H, Mirmira R G, Kalamaras J, &amp; German M S, “Intramolecular control of transcriptional activity by the NK2-specific domain in NK-2 homeodomain proteins,” Proc Natl Acad Sci USA, 97(17):9443-9448, 2000, which is hereby incorporated by reference herein in its entirety. This site is the only published Nkx2.2 binding site not predicted by the process illustrated in  FIG. 2A  and described herein, but this site is predicted by the TRANSFAC PWM and the PBM seed and wobble matrix. Attempts to confirm Nkx2.2 binding to this site using EMSA as shown in  FIG. 2C  were unsuccessful. PBM mapping predicted a site 328 bases upstream of the previously published site (Ins2 −477) and was confirmed by EMSA as also shown in  FIG. 2C . ChIP analysis showed Nkx2.2 occupancy with primers for both the published and our predicted site, although occupancy was stronger on the PBM-mapping predicted site as shown in  FIG. 2D . However, the ChIP results are unable to completely distinguish between occupancy of both sites because of their close proximity. It is possible that Nkx2.2 could bind this site through cooperative binding with cofactors that would not have been seen in previous experiments. Therefore, an additional EMSA analysis using BetaTC6 nuclear extract was performed. In this subsequent analysis, Nkx2.2 containing complexes formed on both sites, but in vitro translated Nkx2.2 only bound to the upstream site. Therefore, it appears that Nkx2.2 may be stabilized on the Ins2 −144 site by interacting factors. 
         [0055]    Insulin expression is lost in the Nkx2.2 null mouse. However, mutation of the Ins2 −144 site resulted in a paradoxical increase in insulin expression. Therefore, luciferase assays were performed to assess Nkx2.2 function through the upstream Nkx2.2 binding site. Luciferase constructs were created to contain the 586 bases upstream of the Ins2 promoter. 
         [0056]    The insulin promoter from −585 to +2 was cloned into the pGL4.17 luciferase plasmid (available from Promega Corporation). Mutagenesis of the previously published and predicted Nkx2.2 binding sites was done using the Quickchange II mutagnesis kit (available from Agilent Technologies Inc., formerly Stratagene) with the following primers and their respective reverse compliment sequence: 
         [0000]    GGAGGAGGGACCATTGCCTTGCTGCCTGAATTC (Ins2 −144) and GACCTAGCACCAGGGGTTTGGAAACTGCAGC (Ins2 −477). A ratio of 10:1 (500 ng/50 ng) of pGL4:ins2 promoter/pRL-null plasmids were transfected using Fugene 6 transfection reagent (available from F. Hoffmann-La Roche Ltd.) into 5×10̂5 betaTC6 cells. After 48 hours, cells were harvested and assayed for luciferase activity using the dual luciferase assay kit (available from Promega Corporation). At least three independent experiments were performed in triplicate and the unpaired student t-test was used to measure significance of changes between sample conditions. 
         [0057]    Basal activity of the promoter was very high in BetaTC6 cells. Mutation of the upstream Nkx2.2 binding site resulted in a 50% reduction in activity, indicating that Nkx2.2 increases the rate of insulin production, but is not necessary for insulin expression. Mutation of the downstream site also resulted in a decrease in luciferase levels, contrary to what was previously published. These experiments show that Nkx2.2 activates the insulin promoter through both binding sites, but binds more strongly to the Ins2 −477 site. 
         [0058]    In accordance with some embodiments, the techniques described herein can be implemented at least in part in one or more computer systems. These computer systems can be any of a general purpose device such as a computer or a special purpose device such as a client, a server, etc. Any of these general or special purpose devices can include any suitable components such as a processor (which can be a microprocessor, digital signal processor, a controller, etc.), memory, communication interfaces, display controllers, input devices, etc. 
         [0059]    In some embodiments, any suitable computer readable media can be used for storing instructions for performing the processes described herein. For example, in some embodiments, computer readable media can be transitory or non-transitory. For example, non-transitory computer readable media can include media such as magnetic media (such as hard disks, floppy disks, etc.), optical media (such as compact discs, digital video discs, Blu-ray discs, etc.), semiconductor media (such as flash memory, electrically programmable read only memory (EPROM), electrically erasable programmable read only memory (EEPROM), etc.), any suitable media that is not fleeting or devoid of any semblance of permanence during transmission, and/or any suitable tangible media. As another example, transitory computer readable media can include signals on networks, in wires, conductors, optical fibers, circuits, any suitable media that is fleeting and devoid of any semblance of permanence during transmission, and/or any suitable intangible media. 
         [0060]    Although the invention has been described and illustrated in the foregoing illustrative embodiments, it is understood that the present disclosure has been made only by way of example, and that numerous changes in the details of implementation of the invention can be made without departing from the spirit and scope of the invention. For example, in some embodiments, rather than operating on octamers (which include 8 base pairs), a suitable portion of a DNA strand including any suitable number of base pairs (e.g., 10) can be used. Features of the disclosed embodiments can be combined and rearranged in various ways. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 APPENDIX I 
               
             
             
               
                   
               
               
                 Table 1 
               
             
          
           
               
                 Probe 
                 Sequence 
               
               
                   
               
               
                 Chgb −1529 Forward 
                 GAACAAACAC AGGGTGACTC ATTGAAGTGT GATGCATGGC TAAAAGCAGA 
               
               
                   
               
               
                 Chgb −1529 Reverse 
                 AGTTCTGCTT TTAGCCATGC ATCACACTTC AATGAGTCAC CCTGTGTTTG 
               
               
                   
               
               
                 Chgb −217 Forward 
                 TGAGGTTAAA AGAGAGAGAG AATTTTGAAG TGTATCCTTT GGC 
               
               
                   
               
               
                 Chgb −217 Reverse 
                 AGGCCAAAGG ATACACTTCA AAATTCTCTC TCTCTTTTAA CC 
               
               
                   
               
               
                 Frzb −2290 Forward 
                 AGTCCAAATA TCTTAAGGAG ATAAACCACT TGAGAGGAGA CTTAATTC 
               
               
                   
               
               
                 Frzb −2290 Reverse 
                 TTGAGAATTA AGTCTCCTCT CAAGTGGTTT ATCTCCTTAA GATATTTGG 
               
               
                   
               
               
                 Gcg −1080 Forward 
                 AGACCATTGA AACAACTGGA GGAGTACTCT GACTGAACTT AATTCTTCAT 
               
               
                   
               
               
                 Gcg −1080 Reverse 
                 AGAATGAAGA ATTAAGTTCA GTCAGAGTAC TCCTCCAGTT GTTTCAATGG 
               
               
                   
               
               
                 Gcg −280 Forward 
                 ACGAAAAACT GCTAAAGTTC TCTCAAGTGA ATTTTGACGT CAAATGAGCC TAG 
               
               
                   
               
               
                 Gcg −280 Reverse 
                 AGACTAGGCT CATTTGACGT CAAAATTCAC TTGAGAGAAC TTTAGCAGTT TTT 
               
               
                   
               
               
                 Gcg −432 Forward 
                 AGTACACACA TATCAATAAC CCACTCATCC ACATTGTATG GAATAAATTT GTAT 
               
               
                   
               
               
                 Gcg −432 Reverse 
                 AGAATACAAA TTTATTCCAT ACAATGTGGA TGAGTGGGTT ATTGATATGT GTGT 
               
               
                   
               
               
                 Iapp −1184 Forward 
                 AGTGTAAAAA ATAAATTAAT TTTAAAAAAA ACACTTAAAC GTGAACACAT 
               
               
                   
               
               
                 Iapp −1184 Reverse 
                 TGTATGTGTT CACGTTTAAG TGTTTTTTTT AAAATTAATT TATTTTTTAC 
               
               
                   
               
               
                 Iapp −1355 Forward 
                 TGTCCTCAGG CCGCTACATA AAGGCACTCA AGAGACTGGA GGCCCCAGGG AGTTTGGAGG 
               
               
                   
               
               
                 Iapp −1355 reverse 
                 TGACCTCCAA ACTCCCTGGG GCCTCCAGTC TCTTGAGTGC CTTTATGTAG CGGCCTGAGG 
               
               
                   
               
               
                 Iapp −1955 Forward 
                 GTTAAGCTGG TATGGCTAGT TAAGTGGTTA TAGCTGACAT ATAATGTCT 
               
               
                   
               
               
                 Iapp −1955 Reverse 
                 TGAAGACATT ATATGTCAGC TATAACCACT TAACTAGCCA TACCAGCTT 
               
               
                   
               
               
                 Iapp +479 Forward 
                 TGTCCTCCTC ATCCTCTCTG TGGCACTGAA CCACTTGAGA GCTACACCTG 
               
               
                   
               
               
                 Iapp +479 Reverse 
                 TGACAGGTGT AGCTCTCAAG TGGTTCAGTG CCACAGAGAG GATGAGGAGG 
               
               
                   
               
               
                 Ins −144 Forward 
                 TGCTTTCTGC AGACCTAGCA CCAGGCAAGT GTTTGGAAAC TGCAGCT 
               
               
                   
               
               
                 Ins −144 reverse 
                 CTGAAGCTGC AGTTTCCAAA CACTTGCCTG GTGCTAGGTC TGCAGAA 
               
               
                   
               
               
                 Ins −471 forward 
                 AAGCAGAACT CAGGCAGCAA GGTACTTAAT GGTCCCTCCT TCTCCATC 
               
               
                   
               
               
                 Ins −471 Reverse 
                 AGAGATGGAG AAGGAGGGAC CATTAAGTAC CTTGCTGCCT GAGTTCT 
               
               
                   
               
               
                 Irs4 −111 Forward 
                 CCGCCTAGGC CCGCGTCCCC GCCCACTTCA CTGGGCTCAA GGCAGTGG 
               
               
                   
               
               
                 lrs4 −111 reverse 
                 TGCCCACTGC CTTGAGCCCA GTGAAGTGGG CGGGGACGCG GGCCTAGG 
               
               
                   
               
               
                 Irs4 +1495 Forward 
                 AGCCCTGGCT ACTGGAACCT TGGCCACTTG AGCCCCGTCC ACCTCCTGAG CCC 
               
               
                   
               
               
                 Irs4 +1495 reverse 
                 CCGGGGCTCA GGAGGTGGAC GGGGCTCAAG TGGCCAAGGT TCCAGTAGCC AGG 
               
               
                   
               
               
                 Mafa Forward 
                 TGTAACCAGG AGGCAGCCCC TCCAGCAAGC ACTTCAGTGT GCTCAGTGGG 
               
               
                   
               
               
                 Mafa reverse 
                 AACAGCCCCA CTGAGCACAC TGAAGTGCTT GCTGGAGGGG CTGCCTCCTG G 
               
               
                   
               
               
                 Ngn3 −506 Forward 
                 CGCTCCTCCC AGCTGCCAGC CAAGAAGACA CTTGACTCCT TGATCGCTGG T 
               
               
                   
               
               
                 Ngn3 −506 Reverse 
                 TGAACCAGCG ATCAAGGAGT CAAGTGTCTT CTTGGCTGGC AGCTGGGAGG A 
               
               
                   
               
               
                 Nkx2.2 −1502 Forward 
                 GCTGCAAGTT TGCTACATAC CACTTGTTCG CCCCACTTAA CATCAGGAGT GGGCTT 
               
               
                   
               
               
                 Nkx2.2 −1502 Reverse 
                 GCTAAGCCCA CTCCTGATGT TAAGTGGGGC GAACAAGTGG TATGTAGCAA ACTTGC 
               
               
                   
               
               
                 Nkx2.2 −188 Forward 
                 CGCGTCGCTC TCGAGTCCAC ACACTTGAAA AGAGCCGTTT TAACAAATT 
               
               
                   
               
               
                 Nkx2.2 −188 Reverse 
                 ATGCAATTTG TTAAAACGGC TCTTTTCAAG TGTGTGGACT CGAGAGCGAC 
               
               
                   
               
               
                 Nkx2.2 −377 forward 
                 ACGTGTGGGC GGGTCTTGGG AGTCAAGTGG ATGAAGACAG TATTTG 
               
               
                   
               
               
                 Nkx2.2 −377 Reverse 
                 CTGCAAATAC TGTCTTCATC CACTTGACTC CCAAGACCCG CCCAC 
               
               
                   
               
               
                 Nkx2.2 −716 Forward 
                 GTCAATATTT TGGTTGAAGC TTAAGGATGA GTACTAGAAA TGACAAG 
               
               
                   
               
               
                 Nkx2.2 −716 Reverse 
                 TGACTTGTCA TTTCTAGTAC TCATCCTTAA GCTTCAACCA AAATATT 
               
               
                   
               
               
                 Nkx6.2 −1441 Forward 
                 AGCCACTTTA TGGCGGGAAC TGGAAATAAG TGCTGTGGTC CCGCTGACTT CT 
               
               
                   
               
               
                 Nkx6.2 −1441 Reverse 
                 TGCAGAAGTC AGCGGGACCA CAGCACTTAT TTCCAGTTCC CGCCATAAAG TG 
               
               
                   
               
               
                 Nkx6.2 +669 forward 
                 CCGAATCCCG CGCGGGCCAC TTACCGGAGC CGGCCAGTCG CGGGTCCCTC 
               
               
                   
               
               
                 Nkx6.2 +669 reverse 
                 CTGGAGGGAC CCGCGACTGG CCGGCTCCGG TAAGTGGCCC GCGCGGGATT 
               
               
                   
               
               
                 pdx1 −5877 site for 
                 TGCTCATGTG GGCAGAATTA AGTGGAATTA GCTAACAAAT TATATAAAAT 
               
               
                   
               
               
                 Pdx1 −5877 site rev 
                 TGAATTTTAT ATAATTTGTT AGCTAATTCC ACTTAATTCT GCCCACATGA 
               
               
                   
               
               
                 Spock3 −1041 Reverse 
                 GCAACAGGTG TGTCCCGTAT TCTGAGTACT TTGTTCTCAC TCGGGTCATA 
               
               
                   
               
               
                 Spock3 −1044 Forward 
                 AGTTATGACC CGAGTGAGAA CAAAGTACTC AGAATACGGG ACACACCTGT 
               
               
                   
               
               
                 Tm4sf4 −1723 forward 
                 GCCATTAGTG CCAATGACCC AGCACTCGAG GGTAGGGGGA GCACAGC 
               
               
                   
               
               
                 Tm4sf4 −1723 reverse 
                 ACTGGCTGTG CTCCCCCTAC CCTCGAGTGC TGGGTCATTG GCACTAATG 
               
               
                   
               
               
                 Tm4sf4 −5 Forward 
                 CTGAAGGCCT GCCGTAGTTG AGAAGTGAAG TGTCTCCAAG GTTCAAAGAA CT 
               
               
                   
               
               
                 Tm4sf4 −5 Reverse 
                 CAGAGTTCTT TGAACCTTGG AGACACTTCA CTTCTCAACT ACGGCAGGCC TT 
               
               
                   
               
               
                 Tm4sf4 +555 Forward 
                 AGCCCAGAGA ACCAAGCTAA TAGCCACTTG ATTATTTTAC TCTAGTCAAA TTGTG 
               
               
                   
               
               
                 Tm4sf4 +555 Reverse 
                 TGCCACAATT TGACTAGAGT AAAATAATCA AGTGGCTATT AGCTTGGTTC TCTGG 
               
               
                   
               
               
                 Tm4sf4 +912 Forward 
                 CGGCTGTTAG GTCTTGCCTG CCCCACTTAA GCCCCTGAGA CCTGAGGTCT 
               
               
                   
               
               
                 Tm4sf4 +912 Reverse 
                 TGAAGACCTC AGGTCTCAGG GGCTTAAGTG GGGCAGGCAA GACCTAACAG C 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                 APPENDIX II 
               
               
                   
               
               
                 Table 2 
               
               
                 Checking bound promoter regions from −2500 to +1000 bp. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Gcg (NM_008100) chr2: 62321710 (−) Fold change: e12.5: −19.95 
               
               
                 (FDR = 0.00) e13.5: −14.97 (FDR = 0.00) 
               
             
          
           
               
                   982 to 995 
                 ATGCCACTTCATAA 
                 PBM-score: 0.4068 
               
               
                   
               
               
                   787 to 800 
                 AAGGCACTTCAGAA 
                 PBM-score: 0.4205 
               
               
                   
               
               
                   271 to 284 
                 TCTCTAAGTAGTTT 
                 PBM-score: 0.3737 
               
               
                   
               
               
                   143 to 156 
                 ATAGTACTTAAACA 
                 PBM-score: 0.4108 
               
               
                   
               
               
                    23 to 36 
                 ACTTTGAGTGTGTC 
                 PBM-score: 0.3964 
               
               
                   
               
               
                  −293 to −280 
                 TCTCTCAAGTGAAT 
                 PBM-score: 0.3994 
               
               
                   
               
               
                  −445 to −432 
                 AACCCACTCATCCA 
                 PBM-score: 0.3715 
               
               
                   
               
               
                  −865 to −852 
                 ATCATAAGTATGTT 
                 PBM-score: 0.3764 
               
               
                   
               
             
          
           
               
                 Nkx2-2 (NM_001077632) chr2: 147012138 (−) Fold change: e12.5: −4.98 
               
               
                 (FDR = 0.00) e13.5: −13.25 (FDR = 0.00) 
               
             
          
           
               
                  −201 to −188 
                 GAGTCAAGTGGATG 
                 PBM-score: 0.4350 
               
               
                   
               
               
                  −390 to −377 
                 ACACACTTGAAAAG 
                 PBM-score: 0.4255 
               
               
                   
               
               
                  −729 to −716 
                 GGATGAGTACTAGA 
                 PBM-score: 0.4072 
               
               
                   
               
               
                 −1515 to −1502 
                 CATACCACTTGTTC 
                 PBM-score: 0.3808 
               
               
                   
               
               
                 −1529 to −1516 
                 GCCCCACTTAACAT 
                 PBM-score: 0.4148 
               
               
                   
               
             
          
           
               
                 Pyy (NM_145435) chr11: 101969090 (−) Fold change: e12.5: −7.64 
               
               
                 (FDR = 0.00) e13.5: −3.01 (FDR = 0.00) 
               
               
                   
               
               
                 Ghr1 (NM_021488) chr6: 113669874 (−) Fold change: e12.5: 6.48 
               
               
                 (FDR = 0.00) e13.5: 6.99 (FDR = 0.00) 
               
             
          
           
               
                   124 to 137 
                 TGACACTTATGAAT 
                 PBM-score: 0.3928 
               
               
                   
               
               
                  −129 to −116 
                 ACTAAGTACTCTTT 
                 PBM-score: 0.4308 
               
               
                   
               
             
          
           
               
                 Iapp (NM_010491) chr6: 142246944 (+) Fold change: e12.5: 5.21 
               
               
                 (FDR = 0.00) e13.5: 2.12 (FDR = 10.72) 
               
             
          
           
               
                 −1955 to −1942 
                 TAGTTAAGTGGTTA 
                 PBM-scorc: 0.4320 
               
               
                   
               
               
                 −1355 to −1342 
                 AAGGCACTCAAGAG 
                 PBM-score: 0.4294 
               
               
                   
               
               
                 −1184 to −1171 
                 AAAACACTTAAACG 
                 PBM-score: 0.4021 
               
               
                   
               
               
                  −600 to −587 
                 AGGCTCTTGAGGGT 
                 PBM-score: 0.3832 
               
               
                   
               
               
                   479 to 492 
                 AACCACTTGAGAGC 
                 PBM-score: 0.4658 
               
               
                   
               
               
                   610 to 623 
                 AGAAGTACTTAAAG 
                 PBM-score: 0.4641 
               
               
                   
               
               
                   621 to 634 
                 AAGCTAAGTGGTTT 
                 PBM-score: 0.3938 
               
               
                   
               
             
          
           
               
                 Tm4sf4 (NM_145539) chr3: 57229380 (+) Fold change: e12.5: 4.52 
               
               
                 (FDR = 0.00) e13.5: 3.32 (FDR = 0.00) 
               
             
          
           
               
                 −1844 to −1831 
                 ATCTTCAAGAGTTG 
                 PBM-score: 0.3751 
               
               
                   
               
               
                 −1723 to −1710 
                 CAGCACTCGAGGGT 
                 PBM-scorc: 0.3895 
               
               
                   
               
               
                 −1261 to −1248 
                 TCTCTAAGTGTGTA 
                 PBM-scorc: 0.3722 
               
               
                   
               
               
                    −5 to 8 
                 AAGTGAAGTGTCTC 
                 PBM-score: 0.4144 
               
               
                   
               
               
                   483 to 496 
                 TTACTAAGTGGTTC 
                 PBM-score: 0.3914 
               
               
                   
               
               
                   555 to 568 
                 TAGCCACTTGATTA 
                 PBM-score: 0.4276 
               
               
                   
               
               
                   912 to 925 
                 GCCCCACTTAAGCC 
                 PBM-score: 0.3953 
               
               
                   
               
             
          
           
               
                 Tmem27 (NM_020626) chrX: 160528118 (+) Fold change: e12.5: −4.46 
               
               
                 (FDR = 0.00) e13.5: −2.80 (FDR = 0.00) 
               
             
          
           
               
                    24 to 37 
                 AGCTTTAAGTAGAG 
                 PBM-score: 0.3738 
               
               
                   
               
               
                   708 to 721 
                 TTCTTAAAGTACAC 
                 PBM-score: 0.3750 
               
               
                   
               
             
          
           
               
                 Chgb (NM_007694) chr2: 132607013 (+) Fold change: e12.5: −2.00 
               
               
                 (FDR = 0.35) e13.5: −4.09 (FDR = 0.00) 
               
             
          
           
               
                 −1529 to −1516 
                 TCATTGAAGTGTGA 
                 PBM-score: 0.3740 
               
               
                   
               
               
                  −988 to −975 
                 GGTAGAGTGCTTTC 
                 PBM-score: 0.3759 
               
               
                   
               
               
                  −217 to −204 
                 TTTTGAAGTGTATC 
                 PBM-score: 0.4064 
               
               
                   
               
               
                    61 to 74 
                 TACACACTTCAGAA 
                 PBM-score: 0.3789 
               
               
                   
               
             
          
           
               
                 Smarca4 (NM_011417) chr9: 21420612 (+) Fold change: e12.5: 3.58 
               
               
                 (FDR = 0.00) e13.5: 4.07 (FDR = 0.00) 
               
             
          
           
               
                 −1727 to −1714 
                 CAAGTGCTCTTAAC 
                 PBM-score: 0.4002 
               
               
                   
               
             
          
           
               
                 Ttr (NM_013697) chr18: 20823913 (+) Fold change: e12.5: −3.61 
               
               
                 (FDR = 0.00) e13.5: −2.44 (FDR = 0.00) 
               
             
          
           
               
                   174 to 187 
                 ACTAGAGTACTCAG 
                 PBM-score: 0.4257 
               
               
                   
               
               
                   913 to 926 
                 TCAACACTTATGTT 
                 PBM-score: 0.4159 
               
               
                   
               
             
          
           
               
                 Ins2 (NM_008387) chr7: 149865613 (−) Fold change: e12.5: −1.43 
               
               
                 (FDR = 1.54) e13.5: −3.36 (FDR = 0.00) 
               
             
          
           
               
                   340 to 353 
                 TCCTCCACTTCACG 
                 PBM-score: 0.3805 
               
               
                   
               
               
                    44 to 57 
                 GAGAAGAGTACCTT 
                 PBM-score: 0.3766 
               
               
                   
               
               
                  −477 to −464 
                 AAGGCACTTAATGG 
                 PBM-score: 0.4156 
               
               
                   
               
               
                  −702 to −689 
                 GCTTGGAGTGGTTG 
                 PBM-score: 0.3921 
               
               
                   
               
             
          
           
               
                 Ins1 (NM_008386) chr19: 52338812 (+) Fold change: e12.5: −1.53 
               
               
                 (FDR = 0.89) e13.5: −3.26 (FDR = 0.00) 
               
             
          
           
               
                 −1899 to −1886 
                 CAAGCACTTTAAAC 
                 PBM-score: 0.4042 
               
               
                   
               
               
                  −349 to −336 
                 CCATTAAGTACCTT 
                 PBM-score: 0.4194 
               
               
                   
               
               
                   −51 to −38 
                 CAATGAGTGCTTTC 
                 PBM-score: 0.3745 
               
               
                   
               
               
                   467 to 480 
                 CGTGAAGTGGAGGA 
                 PBM-score: 0.3805 
               
               
                   
               
               
                   837 to 850 
                 TAATTCAAGTATCT 
                 PBM-score: 0.4030 
               
               
                   
               
             
          
           
               
                 Slc38a5 (NM_172479) chrX: 7848517 (+) Fold change: e12.5: −3.23 
               
               
                 (FDR) = 0.00) e13.5: −3.22 (FDR = 0.00) 
               
             
          
           
               
                 −1643 to −1630 
                 AGAAGTACTCTTCA 
                 PBM-score: 0.4387 
               
               
                   
               
               
                 −1509 to −1496 
                 AGTGGCACTTCTAT 
                 PBM-score: 0.3921 
               
               
                   
               
               
                 −1330 to −1317 
                 ATTTTAAGTACCTA 
                 PBM-score: 0.4269 
               
               
                   
               
               
                    81 to 94 
                 TCCCACTTCAAATG 
                 PBM-score: 0.4017 
               
               
                   
               
             
          
           
               
                 Nepn (NM_025684) chr10: 52111413 (+) Fold change: e12.5: 3.12 
               
               
                 (FDR = 0.00) e13.5: 2.00 (FDR = 10.72) 
               
               
                   
               
               
                 Igfbp3 (NM_008343) chr11: 7113926 (−) Fold change: e12.5: −1.58 
               
               
                 (FDR = 0.00) e13.5: −3.07 (FDR = 0.00) 
               
             
          
           
               
                 −1092 to −1079 
                 TGGATGAGTGGTGG 
                 PBM-score: 0.3707 
               
               
                   
               
               
                 −1142 to −1129 
                 GATACTCTTGAGTT 
                 PBM-score: 0.3802 
               
               
                   
               
               
                 −1269 to −1256 
                 TGGTGAAGTGGACA 
                 PBM-score: 0.3737 
               
               
                   
               
             
          
           
               
                 Irf6 (NM_016851 chr1: 194979305 (+) Fold change: el2.5: −1.64 
               
               
                 (FDR = 0.00) e13.5: −2.93 (FDR = 0.00) 
               
             
          
           
               
                 −1335 to −1322 
                 ATTCAAGAGTGCAC 
                 PBM-score: 0.3950 
               
               
                   
               
               
                   334 to 347 
                 TCTTCAAGTAGTTT 
                 PBM-score: 0.4216 
               
               
                   
               
             
          
           
               
                 Vdac2 (NM_011695) chr14: 22650782 (+) Fold change: e12.5: −2.79 
               
               
                 (FDR = 0.00) e13.5: −1.72 (FDR = 12.29) 
               
             
          
           
               
                 −1520 to −1507 
                 CAGTACTTGAGTAG 
                 PBM-score: 0.4563 
               
               
                   
               
               
                 −1358 to −1345 
                 AGCTGAAGTGTCAG 
                 PBM-score: 0.3801 
               
               
                   
               
               
                   870 to 883 
                 GTTTAAAGTGCCAT 
                 PBM-score: 0.3774 
               
               
                   
               
             
          
           
               
                 Fbxw9 (NM_026791) chr8: 87584017 (+) Fold change: el2.5: −2.77 
               
               
                 (FDR = 0.00) e13.5: −1.85 (FDR = 2.56) 
               
             
          
           
               
                 −1884 to −1871 
                 CAGTTAAGTGTGCT 
                 PBM-score: 0.3959 
               
               
                   
               
               
                  −774 to −761 
                 GAGCACTTTAAGTG 
                 PBM-score: 0.4363 
               
               
                   
               
               
                   805 to 818 
                 CTTACAAGTGTTTG 
                 PBM-score: 0.3868 
               
               
                   
               
             
          
           
               
                 Neurog3 (NM_009719) chrl0: 61595837 (+) Fold change: e12.5: −2.66 
               
               
                 (FDR = 0.00) e13.5: −1.80 (FDR = 2.56) 
               
             
          
           
               
                 −1142 to −1129 
                 AACCTCTTAAGAGG 
                 PBM-score: 0.4253 
               
               
                   
               
               
                  −506 to −493 
                 AAGACACTTGACTC 
                 PBM-score: 0.4165 
               
               
                   
               
             
          
           
               
                 Pla2g1b (NM_011107) chr5: 115916274 (+) Fold change: e12.5: 2.66 
               
               
                 (FDR = 0.00) e13.5: 1.85 (FDR = 24.14) 
               
             
          
           
               
                  −429 to −416 
                 CAGAGCACTCATAC 
                 PBM-score: 0.3719 
               
               
                   
               
               
                   927 to 940 
                 CTCTGAAGTGTTAG 
                 PBM-score: 0.4065 
               
               
                   
               
             
          
           
               
                 Irx3 (NM_008393) chr8: 94325273 (−) Fold change: r12.5: −1.35 
               
               
                 (FDR = 7.71) e13.5: −2.56 (FDR = 0.00) 
               
               
                   
               
               
                 Gab1 (NM_021356) chr8: 83404378 (−) Fold change: e12.5: −2.52 
               
               
                 (FDR = 0.00) e13.5: −2.04 (FDR = 0.00) 
               
             
          
           
               
                 −1314 to −1301 
                 CCATAAAGTGCTTT 
                 PBM-score: 0.3757 
               
               
                   
               
               
                 −1565 to −1552 
                 ATTTAAAGTGTTGC 
                 PBM-score: 0.3920 
               
               
                   
               
             
          
           
               
                 Myt1 (NM_008665) chf2: 181501746 (+) Fold change: e12.5: −1.32 
               
               
                 (FDR = 0.89) e13.5: −2.39 (FDR = 0.00) 
               
             
          
           
               
                  −650 to −637 
                 TTTTAAAGTGTTTT 
                 PBM-score: 0.3969 
               
               
                   
               
             
          
           
               
                 Slc7a2 (NM_007514) chr8: 41947720 (+) Fold change: e12.5: −1.39 
               
               
                 (FDR = 4.32) e13.5: −2.06 (FDR = 0.00) 
               
             
          
           
               
                 −1979 to −1966 
                 TGGAGTACTACTCA 
                 PBM-score: 0.4042 
               
               
                   
               
               
                 −1854 to −1841 
                 CTGATAAGTGGATA 
                 PBM-score: 0.4337 
               
               
                   
               
               
                   754 to 767 
                 TAAGCACTTGAGTT 
                 PBM-score: 0.4478 
               
               
                   
               
               
                   807 to 820 
                 GCCTTGAGTACCTT 
                 PBM-score: 0.4056 
               
               
                   
               
             
          
           
               
                 S1c7a2 (NM_001044740) chr8: 41947746 (+) Fold change: e12.5: −1.39 
               
               
                 (FDR = 4.32) e13.5: −2.06 (FDR = 0.00) 
               
             
          
           
               
                 −1880 to −1867 
                 CTGATAAGTGGATA 
                 PBM-score: 0.4337 
               
               
                   
               
               
                   728 to 741 
                 TAAGCACTTGAGTT 
                 PBM-score: 0.4478 
               
               
                   
               
               
                   781 to 794 
                 GCCTTGAGTACCTT 
                 PBM-score: 0.4056 
               
               
                   
               
             
          
           
               
                 Cox6a1 (NM_007748) chr5: 115798964 (−) Fold change: e12.5: −1.30 
               
               
                 (FDR = 19.39) el3.5: −2.00 (FDR = 2.56) 
               
               
                   
               
               
                 Ela1 (NM_033612) chr15: 100518351 (−) Fold change: e12.5: 1.92 
               
               
                 (FDR = 4.32) e13.5: 1.97 (FDR = 11.77) 
               
             
          
           
               
                   491 to 504 
                 GTCTGAAGTGTCTG 
                 PBM-score: 0.4052 
               
               
                   
               
               
                    65 to 78 
                 TGATCCACTTACCA 
                 PBM-score: 0.3875 
               
               
                   
               
               
                  −195 to −182 
                 CATCCACTTAACCC 
                 PBM-score: 0.4058 
               
               
                   
               
               
                 −1249 to −1236 
                 AACTTGAGTGGCTC 
                 PBM-score: 0.4293 
               
               
                   
               
               
                 −1625 to −1612 
                 ATGCACTTGAAAAC 
                 PBM-score: 0.4248 
               
               
                   
               
             
          
           
               
                 Gast (NM_010257) chr11: 100195725 (+) Fold change: e12.5: −1.71 
               
               
                 (FDR = 0.00) e13.5: −1.94 (FDR = 0.00) 
               
             
          
           
               
                 −1993 to −1980 
                 GCAATTAAGTGGGG 
                 PBM-score: 0.4207 
               
               
                   
               
               
                 −1145 to −1132 
                 TATTAGAGTGGTTA 
                 PBM-score: 0.4030 
               
               
                   
               
               
                  −806 to −793 
                 TAACCACTTTAAGA 
                 PBM-score: 0.4277 
               
               
                   
               
               
                   495 to 508 
                 AGGAGTACTTATCA 
                 PBM-score: 0.4464 
               
               
                   
               
             
          
           
               
                 Dmwd (NM_010058) chr7: 19661548 (+) Fold change: e12.5: −1.87 
               
               
                 (FDR = 0.00) el3.5: −1.71 (FDR = 12.29) 
               
             
          
           
               
                  −858 to −845 
                 TCTCCACTCTTACA 
                 PBM-score: 0.3783 
               
               
                   
               
               
                  −627 to −614 
                 CTACACTTCACTCT 
                 PBM-score: 0.3885 
               
               
                   
               
             
          
           
               
                 Dsn1 (NM_025853) chr2: 156832811 (−) Fold change: e12.5: 1.87 
               
               
                 (FDR = 24.36) e13.5: −1.72 (FDR = 24.14) 
               
             
          
           
               
                  −380 to −367 
                 CCCTTAAGTACCTA 
                 PBM-score: 0.4500 
               
               
                   
               
             
          
           
               
                 Disp2 (NM_170593) chr2: 118605653 (+) Fold change: e12.5: −1.38 
               
               
                 (FDR = 0.89) e13.5: −1.76 (FDR = 2.56) 
               
             
          
           
               
                  −713 to −700 
                 TGCGCACTTAAAAG 
                 PBM-score: 0.3980 
               
               
                   
               
               
                   151 to 164 
                 TCGACACTTGATAA 
                 PBM-score: 0.4159 
               
               
                   
               
               
                   799 to 812 
                 ATGACACTTCATCT 
                 PBM-score: 0.3885 
               
               
                   
               
               
                   998 to 1011 
                 TTATTCAAGAGGGC 
                 PBM-score: 0.3705 
               
               
                   
               
             
          
           
               
                 Crp (NM_007768) chr1: 174628186 (+) Fold change: e12.5: −1.50 
               
               
                 (FDR = 0.00) e13.5: −1.68 (FDR = 15.36) 
               
             
          
           
               
                 −1809 to −1796 
                 TCTTCTTAAGTGAT 
                 PBM-score: 0.3840 
               
               
                   
               
               
                  −306 to −293 
                 ACACAAGTGCTCAT 
                 PBM-score: 0.3856 
               
               
                   
               
               
                   573 to 586 
                 TTTTGGAGTGGGTG 
                 PBM-score: 0.3882 
               
               
                   
               
             
          
           
               
                 Hmgn3 (NM_026122) chr9: 83040132 (−) Fold change: e12.5: −1.21 
               
               
                 (FDR = 14.88) e13.5: −1.65 (FDR = 12.29) 
               
             
          
           
               
                   136 to 149 
                 AACACACTCGAGGG 
                 PBM-score: 0.3803 
               
               
                   
               
               
                  −217 to −204 
                 TTTCCACTTCACTG 
                 PBM-score: 0.3928 
               
               
                   
               
               
                 −1941 to −1928 
                 ATGGTACTTGAGGT 
                 PBM-score: 0.4237 
               
               
                   
               
             
          
           
               
                 Hmgn3 (NM_175074) chr9: 83040212 (−) Fold change: e12.5: −1.21 
               
               
                 (FDR = 14.88) e13.5: −1.65 (FDR = 12.29) 
               
             
          
           
               
                   216 to 229 
                 AACACACTCGAGGG 
                 PBM-score: 0.3803 
               
               
                   
               
               
                  −137 to −124 
                 TTTCCACTTCACTG 
                 PBM-score: 0.3928 
               
               
                   
               
               
                 −1861 to −1848 
                 ATGGTACTTGAGGT 
                 PBM-score: 0.4237 
               
               
                   
               
             
          
           
               
                 Rdh16 (NM_009040) chr10: 127238208 (+) Fold change: e12.5: −1.51 
               
               
                 (FDR = 0.35) e13.5: −1.59 (FDR = 19.07) 
               
             
          
           
               
                 −1376 to −1363 
                 AACAAGAGTGTCCA 
                 PBM-score: 0.3777 
               
               
                   
               
               
                  −571 to −558 
                 GGCCACTTGAGATC 
                 PBM-score: 0.4434 
               
               
                   
               
             
          
           
               
                 Spock3 (NM_023689) chr8: 65430243 (+) Fold change: e12.5: NA 
               
               
                 (FDR = NA) e13.5: 2.3 (FDR = 1.0) 
               
             
          
           
               
                 −1516 to −1503 
                 TTTTTGAAGTAGAG 
                 PBM-score: 0.3767 
               
               
                   
               
               
                 −1057 to −1044 
                 CAAAGTACTCAGAA 
                 PBM-score: 0.3905 
               
               
                   
               
             
          
           
               
                 Nkx6-2 (NM_183248) chr7: 146768692 (−) Fold change: e12.5: NA 
               
               
                 FDR = NA) el3.5: 8.3 (FDR = 0.0) 
               
             
          
           
               
                 −1431 to −1418 
                 AAGCCACTTTATGG 
                 PBM-score: 0.3850 
               
               
                   
               
               
                 −1454 to −1441 
                 GAAATAAGTGCTGT 
                 PBM-score: 0.3912 
               
               
                   
               
             
          
           
               
                 Irs4 (NM_010572) chrX: 138159760 (−) Fold change: e12.5: NA 
               
               
                 (FDR = NA) e13.5: 4.9 (FDR = 0.0) 
               
             
          
           
               
                  −124 to −111 
                 CGCCCACTTCACTG 
                 PBM-score: 0.3953 
               
               
                   
               
             
          
           
               
                 Frzb (NM_011356) chr2: 80287553 (−) Fold change: e12.5: NA 
               
               
                 (FDR = NA) e13.5: 3.2 (FDR = 19.3) 
               
             
          
           
               
                   922 to 935 
                 CGGTACTTGATGAG 
                 PBM-score: 0.4107 
               
               
                   
               
               
                  −693 to −680 
                 AGCCCACTTTAAAG 
                 PBM-score: 0.3983 
               
               
                   
               
               
                 −1625 to −1612 
                 GAACTCAAGAGGTT 
                 PBM-score: 0.3961 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 APPENDIX III 
               
             
             
               
                   
               
               
                 Table 3: 
               
             
          
           
               
                 Primer 
                 Sequence 
               
               
                   
               
               
                 Chgb −217 For 
                 CACCAATTATGTGTGCTCCAA 
               
               
                   
               
               
                 Chgb −217 Rev 
                 GGAATCTCCTACCCGACGTA 
               
               
                   
               
               
                 Chgb −1529 For 
                 GGGAACAAACACAGGGTGAC 
               
               
                   
               
               
                 Chgb −1529 Rev 
                 TCACTACCCTATTCCCATTTTCA 
               
               
                   
               
               
                 Frzb −2290 For 
                 TCCGAATTTTGGGTTTGTTG 
               
               
                   
               
               
                 Frzb −2290 Rev 
                 AAAACTGGCTGGTGGAAATG 
               
               
                   
               
               
                 Gcg −280/−432 For 
                 TCTCCCCACAAAGAGAATACAAA 
               
               
                   
               
               
                 Gcg −280/−432 Rev 
                 CCCTTGATTTGGTATTTGGC 
               
               
                   
               
               
                 Gcg −1080 For 
                 GTAGCTCCACACCCACCAGT 
               
               
                   
               
               
                 Gcg −1080 Rev 
                 TGACAAGACCACAGCGTTTC 
               
               
                   
               
               
                 Iapp −1955 For 
                 CCAGTGGTTAAGCTGGTATGG 
               
               
                   
               
               
                 Iapp −1955 Rev 
                 TATTGCAAATGCCACTCCTG 
               
               
                   
               
               
                 Iapp −1184/−1355 For 
                 GAGAAGCTGAAAATCGACGC 
               
               
                   
               
               
                 Iapp −1184/−1355 Rev 
                 GGCCTCCAGTCTCTTGAGTG 
               
               
                   
               
               
                 Iapp +479 For 
                 CAGCTGTCCTCCTCATCCTC 
               
               
                   
               
               
                 Iapp +479 Rev 
                 TCTCATAGCCAGGATTTGCTT 
               
               
                   
               
               
                 Irs4 −111 For 
                 GACGGTCACGTGTTGTTTTG 
               
               
                   
               
               
                 Irs4 −111 Rev 
                 GATGCACCGTGGTTTTAAGG 
               
               
                   
               
               
                 Ngn3 −506 For 
                 GGTTGCACACACATTTCCTG 
               
               
                   
               
               
                 Ngn3 −506 Rev 
                 TCTTTTGGCTCAGAGAGGGA 
               
               
                   
               
               
                 Nkx2-2 −188/−377 For 
                 CGGCTCTTTTCAAGTGTGTG 
               
               
                   
               
               
                 Nkx2-2 −188/−377 Rev 
                 GTGAAATTGTGGGTTTTGGG 
               
               
                   
               
               
                 Nkx2-2 −716 For 
                 CTGGCATGTCCAAGCCTATT 
               
               
                   
               
               
                 Nkx2-2 −716 Rev 
                 GCTGGTGGTTCCCTAAACAA 
               
               
                   
               
               
                 Nkx2-2 −1502/−1516 For 
                 GGACTAAGGCAACCCAAACA 
               
               
                   
               
               
                 Nkx2-2 −1502/−1516 Rev 
                 GAGGTACGAGGCTGCAAGTT 
               
               
                   
               
               
                 Pdx1 −5877 For 
                 CAAGCACACAGTAGGTGTTCTC 
               
               
                   
               
               
                 Pdx1 −5877 Rev 
                 TGCCTCTGACTGTGTCCCACT 
               
               
                   
               
               
                 Spock3 −1044 For 
                 ATCATCTAAAAGTTATGACCCGAG 
               
               
                   
               
               
                 Spock3 −1044 Rev 
                 TGAATTACATATGTCAGGCAAGC 
               
               
                   
               
               
                 Tm4sf4 −1723 For 
                 GGGAGATGATGCAGTGGGTACG 
               
               
                   
               
               
                 Tm4sf4 −1723 Rev 
                 TTCAGGGGCAGTCACACTTAGAC 
               
               
                   
               
               
                 Tm4sf4 −5 For 
                 GGCCTGCCGTACTTGAGAAG 
               
               
                   
               
               
                 Tm4sf4 −5 Rev 
                 CACAGGAAAGCACAGAGATCAAAGG 
               
               
                   
               
               
                 Tm4sf4 +483/+555 For 
                 CCCTTTCTATTCGCGGCTGG 
               
               
                   
               
               
                 Tm4sf4 +483/+555 Rev 
                 CTTACAGCTTCTGTGTCCCTTCAT 
               
               
                   
               
               
                 Mafa For 
                 CACCCCAGCGAGGGCTGATTTAATT 
               
               
                   
               
               
                 Mafa Rev 
                 AGCAAGCACTTCAGTGTGCTCAGTG 
               
               
                   
               
               
                 GapdH For 
                 CGCATCTTCTTGTGCAGTGCCAG 
               
               
                   
               
               
                 GapdH Rev 
                 TACGGGACGAGGCTGCAGGAG