SUBJECT AREAS:
`MOLECULAR BIOLOGY
`BIOINFORMATICS
`STEM CELLS
`METHODS
`
`Received
`7 July 2011
`
`Accepted
`8 September 2011
`
`Published
`4 October 2011
`
`Correspondence and
`requests for materials
`should be addressed to
`P.A.L. (trish.labosky@
`vanderbilt.edu)
`
`* Current address:
`Department of Life and
`Physical Sciences
`305 Talley-Brady Hall,
`Fisk University,
`1000 Seventeenth
`Avenue North,
`Nashville, TN 37208
`
`A predicted hairpin cluster correlates
`with barriers to PCR, sequencing and
`possibly BAC recombineering
`
`Brian L. Nelms* & Patricia A. Labosky
`
`Vanderbilt University Medical Center, Department of Cell and Developmental Biology, Center for Stem Cell Biology, Program in
`Developmental Biology, Nashville, Tennessee, USA.
`
`Formation of higher-order structure of nucleic acids (hairpins or loops, for example) may impact not only
`gene regulation, but also molecular biology techniques and approaches critical for design and production of
`vectors needed for genetic engineering approaches. In the course of designing vectors aimed to modify the
`murine Foxd3locus through homologous recombination in embryonic stem cells, we discovered a 370
`nucleotide segment of DNA resistant to polymerase read-through. In addition to sequencing and PCR
`disruptions, we were unable to use BAC recombineering strategies to exchange sequences within the Foxd3
`locus. This segment corresponds to a putative DNA hairpin region just upstream of the 5’ untranslated
`region of Foxd3.This region is also highly conserved across vertebrate species, suggesting possible
`functional significance. Our findings provide a cautionary note for researchers experiencing technical
`challenges with BAC recombineering or other molecular biology methods requiring recombination or
`polymerase activity.
`
`R ecombination-mediated genetic engineering using bacterial artificial chromosomes (BAC recombineering)
`
`is a powerful approach for efficiently generating complex DNA constructs, transgenes and gene targeting
`vectors 1–4. Using this method, much larger fragments of DNA can be assembled, allowing for inclusion of
`distal upstream regulatory elements for analysis and the precise replacement of endogenous regions of the
`genome. The technique utilizes homologous recombination between short homology fragments in modified
`strains of E. coli expressing phage recombinase proteins. BAC recombineering also enables relatively rapid
`generation of complex constructs. We initially aimed to use BAC recombineering to generate DNA constructs
`with a goal of modifying the Foxd3 locus. In our attempts to produce these vectors, we encountered a conserved
`stretch of 370 nucleotides (nt) in the 5’ untranslated region (UTR) of the Foxd3 locus that was resistant to
`polymerase read-through during both PCR and sequencing reactions. In addition, we were also unable to achieve
`successful recombination over this region.
`High guanine-cytosine content (GC content) within DNA can lead to formation of secondary structure
`through structures such as DNA hairpins or loops. This secondary structure can be prohibitive to polymerase
`read-through under normal conditions, resulting in abrupt sequencing stops5–9. In addition to empirical evidence
`10, there is an abundance of anecdotal evidence (including online troubleshooting guides from multiple sequen-
`cing facilities) suggesting that abrupt stops in sequencing reads may also be the result of DNA hairpin structures.
`The 370 nt sequence we describe is predicted to assemble into secondary structure in the form of a cluster of DNA
`hairpins. We present a case here demonstrating that such a hairpin, while troublesome for sequencing, may also
`prohibit BAC recombineering.
`
`Results
`Discovery of a polymerase-resistant region of DNA upstream of the Foxd3 coding sequence. Sequencing of a
`plasmid containing the genomic Foxd3 coding region with flanking DNA (under both normal conditions and
`conditions for GC-rich templates), revealed that 3’ to 5’ sequencing reads came to an abrupt stop 442 nt upstream
`(position 2442) of the Foxd3 ATG, and 5’ to 3’ sequencing reads resulted in a sequencing stop precisely 811 nt
`upstream (position 2811) of the Foxd3 ATG (Figure 1). These two positions define a segment of 370 nt resistant
`to polymerase read-through (blue rectangle in Figure 1). Multiple primers flanking this segment were used
`(Primers 1–3 and 11–13 in Figure 1), but sequencing reads extending across this entire region were never
`obtained, with sequencing stops consistently occurring at the border of this 370 nt sequence. However, when
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`Figure 1 | A 370 nt region upstream of the Foxd3coding sequence is resistant to polymerase read-through during PCR or sequencing reactions. A
`schematic representing the murine Foxd3 locus shows the location of primers used for sequencing and PCR (arrows labeled 1–13). When primers outside the
`370 nt (numbers 1 – 3 and 11 – 13) were used to sequence this region, reads stopped abruptly at position 2811 for 5’ to 3’ sequencing or position 2442 for 3’
`to 5’ sequencing. Sequencing of DNA using primers located within the 370 nt region (numbers 4 – 10) continued successfully through the sequence. The scale
`bar shows increments of 500 nt, with position 1 indicating the A in the Foxd3 ATG. The 370 nt region is shown in blue. The table details locations of each
`primer and the positions where polymerase read-through ends abruptly, where applicable. Brackets indicate the positions of homology fragments A through
`D used for BAC recombineering. Abbreviations: ATG, start codon; nt, nucleotide; Seq, sequencing; TGA, stop codon; UTR, untranslated region.
`
`primers that anneal inside the 370 nt sequence were used to sequence
`out of the region, sequencing reads could easily extend through either
`border (Primers 4–10 in Figure 1). Using primers within this seg-
`ment, we were able to sequence across the region and verify that no
`abnormal intervening sequence was the cause of the sequencing
`stops. Similar results were obtained when sequencing from a BAC
`DNA template containing the Foxd3 locus.
`In parallel, we attempted PCR across this region with genomic
`DNA extracted from mouse tail biopsies and murine embryonic stem
`(ES) cells, in addition to DNA from BAC clones or Foxd3-containing
`plasmids. Without exception, these reactions were unsuccessful
`under multiple conditions; the PCR presumably failed due to the
`presence of the polymerase-resistant region. This stretch of sequence
`is GC-rich (see analysis in Figure 2); therefore we attempted PCR
`amplification of this region using multiple polymerases and kits with
`conditions tailored to GC-rich templates (more details in Methods
`Section). However, these attempts were universally unsuccessful. In
`contrast, similar to results with DNA sequencing reactions above,
`when we performed PCR with one primer that annealed within the
`370 nt sequence and one that annealed outside the 370 nt sequence
`(ex. Primers 9 or 10 with a primer external to the 370 nt region), we
`obtained amplicons that crossed the boundary of the 370 nt segment.
`
`Amplification across a boundary was only successful when one of the
`primers used for PCR was within the 370 nt sequence, suggesting that
`polymerases primed from external primers could not extend the
`amplicon through the putative boundary established by the 370 nt
`segment, but were able to travel along the DNA far enough to meet
`the strand amplified from the inside outward. We used this strategy
`to amplify the entire 370 nt sequence in two pieces.
`We discovered that this 370 nt sequence also interfered with
`attempts to recombine sequences overlapping with this region dur-
`ing BAC recombineering. Using traditional BAC recombineering 4 to
`generate constructs targeting the Foxd3 locus, we generated 4 homo-
`logy fragments of approximately 500 nt (Fragments A–D shown in
`Figure 1) to extract 5’ and 3’ homology arms of approximately 8.5 kb
`(from start of A [28549 to 28039] to end of B [2547 to 0] and 5 kb
`(from start of C [3665 to 4216] to end of D [7699 to 8239]), respec-
`tively. Fragment B contained the approximately 500 nt immediately
`upstream of the Foxd3 start codon (ATG), which included the entire
`326 nt 5’ UTR (labeled 5’ in Figure 1). The 5’ end of Fragment B
`partially overlapped with the 370 nt of proposed secondary structure
`we describe in this report. Although we were able to electroporate a
`BAC containing genomic Foxd3 sequence into EL350 cells, multiple
`attempts at BAC recombineering were unsuccessful. While some
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`Figure 2 | The GC-rich 370 nt region is conserved among multiple vertebrate species and correlates with enrichment of H3K4 methylation. Three
`stretches of 370 nt upstream of the Foxd3 coding region are analyzed: the center 370 nt polymerase-resistant region (segment#2 ) plus 370 nt flanking either side
`(segments #1 and #3 ). Analysis of GC-content shows that both the 370 nt polymerase-resistant region #2 and the 3’ 370 nt region #3 are highly GC-rich. A
`schematic representing the murine Foxd3 locus with the location of the regions below (#1 ,#2 and #3 ) is shown in green. Nucleotide BLAST analysis of these
`
`three regions is given in the table. Chlorocebus: Chlorocebus aethiops (monkey), Homo: Homo sapiens (human), Gallus: Gallus gallus (chicken), Danio: Danio rerio
`(zebrafish), Xenopus: Xenopus laevis (frog). Percentage (%) indicates percent of the query (370 nt) that was aligned, while ‘‘score’’ indicates the alignment score
`assigned by the blastn scoring matrix. The mouse Foxd3 locus is shown within the NCBI DCODE ECR browser identifying regions of homology across species
`
`shown by peaks. The 5’ UTR is not predicted in humans, but there is a conserved region of 5’ sequence. The 370 nt region#2 (large green arrow) is just upstream
`
`of the mouse 5’ UTR and is conserved in human, rat, opossum, dog, rhesus monkey, and chimpanzee. Blue represents conserved coding sequence, yellow
`represents conserved UTRs, green represents conserved simple repeats or transposons, and red represents conserved intergenic peaks. Tracks from the DCODE
`
`ECR Browser for the human FOXD3 locus were aligned with tracks from ENCODE data in the UCSC Genome Browser, demonstrating that region #2
`
`corresponds to peaks in H3K4me3 in two cell lines and a peak representing open chromatin, indicated by DNase hypersensitivity.
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`clones survived antibiotic selection, restriction enzyme digestion
`analyses of DNA prepared from recombinant clones with appropri-
`ate antibiotic resistance resulted in fragment patterns indicating
`smaller plasmid sizes than expected. Although fragments corres-
`ponding to the 3’ arm (between C and D) were found, fragments
`corresponding to pieces upstream of the 370 nt region were never
`found in the recombined BAC (between Fragments A and B). When
`these clones were sequenced, the DNA corresponding to Fragment B
`matched the expected sequence until position 2442, the same posi-
`tion of the precise stop observed in sequencing and PCR, where the
`subsequent sequence was unreadable.
`
`The 370 nt region is conserved among vertebrate species and
`highly conserved among mammals. When we examined homolog-
`ous regions upstream of the Foxd3 coding region in other vertebrates,
`we observed a significant degree of conservation over this 370 nt
`segment (Figure 2). Nucleotide BLAST analysis of the 370 nt
`sequence of the mouse genome revealed conservation with human
`(Homo), monkey (Chlorocebus), zebrafish (Danio), chicken (Gallus),
`and frog (Xenopus). In contrast, the same length of sequence imme-
`diately upstream (5’) of the 370 nt segment showed no significant
`conservation, and a 370 nt length of sequence immediately down-
`stream (3’) of the 370 nt region of interest showed only conservation
`with mammals (human and monkey) (Figure 2). Note: for simplicity,
`we will define these three fragments as #1 : the 370 upstream of
`the polymerase resistant region, #2 : the resistant region and #3 :
`the 370 nt immediately 3’ of the resistant region (diagrammed
`at the top of Figure 2). All three of these sequences are 5’ of the
`coding region although #3 is within the 5’ UTR. Using the NCBI
`DCODE software to identify ECRs (Evolutionarily Conserved
`Regions) upstream of human FOXD3 further demonstrated conser-
`vation of this region in mammalian species (Figure 2).
`
`The polymerase-resistant 370 nt region is GC-rich and predicted
`to form secondary structure. The presence of a segment of DNA
`resistant to polymerase read-through suggested secondary structures
`such as DNA hairpins 10. Secondary structure that inhibits sequen-
`cing or PCR reactions typically occurs in GC-rich regions. Therefore,
`we analyzed the GC-content of the 370 nt sequence compared to the
`two 370 nt segments immediately flanking it (regions #1 and #3 )
`(Figure 2). Although the 370 nt sequence is highly GC-rich (61% GC
`content) in contrast with region #1 (39% GC content), which is
`relatively GC-poor,
`its GC character is not unique within the
`Foxd3 locus, as region #3 is also highly GC-rich (71% GC content).
`To determine if the region of polymerase-resistance was due to the
`presence of secondary structure in the 370 nt, we analyzed this region
`with RNAfold secondary structure-predicting software 11 for DNA.
`We focused on the 1110 nt that includes the three regions discussed
`here (#1 ,#2 and #3 ). At 72 degrees Celsius (the extension temper-
`ature for polymerase), the 370 nt region of interest (#2 ) was pre-
`dicted by minimum free energy to form a tight cluster of hairpin
`structures (Figure 3A, boxed region). The nucleotides corresponding
`to the sequencing stop boundaries of this region (arrowheads in
`Figure 3B) were each located within a separate long predicted hairpin
`with the highest base-pairing probability, consistent with the pos-
`sibility that these long, stable hairpin arms defined the boundary for
`the 370 nt segment. While the GC-rich 370 nt region#3 segment was
`also predicted to form a series of hairpins, it lacked any strong, high
`base-pairing probability hairpins (Figure 3B). This analysis sug-
`gested the involvement of secondary structure as a cause for the
`precise stops during sequencing and interference during PCR and
`BAC recombineering of the Foxd3 locus. A second method using
`UNAfold prediction software, also predicted a hairpin that started
`at approximately the 2811 position (the same 5’ boundary corres-
`ponding to a barrier to sequencing and PCR).
`To determine if the barrier to DNA polymerase through this
`region was independent of the genomic or plasmid sequence context,
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`we took advantage of resident restriction enzyme sites (Figure 4A).
`Cutting the plasmid shown in Figure 4A with BspEI removed almost
`all (348 of 370 nts) of the predicted hairpin sequence (region #2
`shown in orange). When the BspEI fragment was removed from
`the vector (Figure 4B), we were then able to sequence across the
`remaining regions. The BspEI
`fragment was
`inserted into
`pBluescript (Figure 4C). The 5’ BspEI site is located 22 nucleotides
`into the predicted hairpin (region #2 in orange), therefore we
`would predict that the inserted BspEI fragment would have a dis-
`rupted 5’ hairpin boundary, but an intact 3’ hairpin boundary.
`Consistent with this, we were able to sequence through the
`hairpin with forward primers upstream of the predicted hairpin,
`but not with reverse primers downstream of the predicted hairpin.
`RNAfold analysis of secondary structure of this fragment further
`suggested that the 5’ hairpin boundary was disrupted (Figure 4D),
`resulting in a much weaker predicted hairpin than in intact Foxd3
`genomic DNA.
`An alternative secondary structure particularly common for GC-
`rich sequences is the G-quadruplex that can form via Hoogstein
`base-pairing among stretches of guanine nucleotides. These struc-
`tures occupy the proximal promoter of some genes and correlate with
`repression of gene expression 12. Although these structures would be
`an attractive alternative candidate for a conserved polymerase-
`resistant region, software prediction models of G-quadruplexing
`did not show any significant potential for G-quadruplexes in the
`370 nt region #2 sequence (data not shown).
`
`Discussion
`Here we describe a DNA sequence of 370 nt resistant to polymerase
`read-through, resulting in precise DNA sequencing stops and pre-
`venting PCR amplification across this region. This region is con-
`served and predicted to form secondary structure comprised of a
`cluster of hairpins. Regions of sequence that produce sequencing
`stops are usually attributed to GC-rich regions or secondary struc-
`ture, but consideration of such structures in the design of vectors has
`been generally under-studied and further analyses would be neces-
`sary to systematically determine if there is a defined effect of second-
`ary structure on homologous recombination at other loci or with
`other techniques. The presence of this type of region or secondary
`structure may be important to consider in other genes, especially if it
`has the potential
`to interfere with attempts to amplify these
`sequences by PCR or execute recombination across these regions,
`such as during BAC recombineering. We also acknowledge the pos-
`sibility that the predicted secondary structure could interfere with
`any of the other components needed for homologous recombination
`in addition to DNA polymerase13. In previously constructed Foxd3
`targeting vectors this area was cloned using restriction enzyme sites
`located outside the 370 nt sequence, avoiding the need for
`PCR amplification or homologous recombination in E. coli14–15.
`Although BAC recombineering and PCR at this specific area of the
`Foxd3 locus was unsuccessful, and the mechanism for hindrance of
`BAC recombineering is not precisely defined, we show here that
`another potential strategy to overcome the inability to PCR-amplify
`specific segments of polymerase-resistant DNA is to start from the
`middle and generate two amplicons that can later be pieced together.
`This may be an important alternative for researchers encountering
`difficulties with these commonly used polymerase-dependent
`approaches.
`Our observation and analysis of conservation of this region is
`consistent with the possibility that the predicted secondary structure
`may also have functional
`significance. This
`region may be
`important as part of the Foxd3 promoter or an enhancer element
`that regulates Foxd3 transcription in specific cell types. Interestingly,
`aligning the same sequence analyzed within the DCODE ECR
`Browser with tracks from the ENCODE (Encyclopedia of DNA
`Elements) Project using the UCSC Genome Browser showed the
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`Figure 3 | A predicted, stable DNA hairpin loop exists in the 370 nt polymerase-resistant region. (A) RNAfold analysis for DNA at 72 degrees Celsius
`performed on the 1110 bases of nucleotide sequence comprising regions#1 ,#2 , and#3 . (B) An enlargement of the region in A outlined with the dashed
`370 nt, region #1 (21181 to 2811) has almost no predicted secondary structure. The 370 nt polymerase-resistant region #2 (2811 to 2442)
`
`box. The structure shown is the predicted optimum structure for minimum free energy. Sequence is displayed from 5’ to 3’ counterclockwise. The first
`
`corresponds to a tight structure of clustered hairpins. The two long hairpin arms with highest base-pairing probability (orange to red) contain the
`boundaries of the polymerase-resistant region as marked with black arrowheads at 2442 and 2811 and bracketed at the bottom of the figure. The
`rainbow scale indicates a range of base-pairing probability from 0 to 1, violet to red.
`
`conserved 370 nt region #2 corresponded to peaks in trimethylation
`of Histone 3 Lysine 4 (H3K4me3) and DNase hypersensitivity,
`both indicators of open chromatin in two mesoderm-derived cell
`lines (GM12878 and K562) (Figure 2). This correlation could also
`explain the sequence conservation we observed. It is unknown
`whether this predicted structure occurs in vivo, where recruitment
`of specific DNA or histone binding factors could stabilize or elim-
`inate higher-order structures formed in this area. Although further
`functional analysis of this 370 nt segment is necessary to determine
`whether it impacts Foxd3 gene expression, we have identified
`an intriguing conserved element and offer the interesting idea
`that this potential DNA secondary structure may have functional
`significance.
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`Methods
`Sequencing. All DNA was sequenced by the Vanderbilt University Medical Center
`Genome Sciences Sanger DNA Sequencing laboratory using BigDye Terminator
`chemistry with resolution on an ABI 3730xl DNA Analyzer. In addition to standard
`sequencing protocols, we used protocols for GC-rich templates, including an increased
`denaturing temperature and additional enzyme. Primer sequences are listed in Figure 1.
`Templates used for sequencing included murine genomic Foxd3 fragments subcloned
`into a pBluescript backbone vector, and BAC DNA containing the Foxd3 locus. Two
`129S6 BAC clones containing Foxd3 (m355-P15 and m284-J23) were obtained from
`the RPCI-22 Mouse BAC library 16 (Research Genetics, Inc., now Invitrogen). A 129S7
`BAC clone containing Foxd3 (bMQ-388O11) was identified in the Ensembl genome
`browser and obtained from the Mouse bMQ BAC library (GeneService Ltd.).
`
`PCR. Polymerase chain reaction across the 370 nt region of predicted secondary
`structure or from within this region was attempted with multiple protocols using
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`Figure 4 | Disruption or insertion of the polymerase-resistant region alters the barrier to sequencing read-through. (A–C) Removal of the majority of
`the sequence corresponding to the predicted hairpin from Foxd3-containing plasmids and insertion of a partially disrupted hairpin sequence into a
`pBluescript backbone. Successful sequencing read-through is indicated by a single black arrow, whereas unsuccessful read-through is indicated by a black
`arrow ending in a red X. (A) Schematic of a plasmid containing a 7642 bp EcoRI genomic Foxd3 fragment, showing the location of the EcoRI and BspEI sites.
`(B) Schematic of a vector generated by removal of the 938 bp and 123 bp BspEI fragments. (C) Schematic of 938 bp BspEI fragment inserted into the EcoRV
`site of pBluescript. (D) RNAfold secondary structure prediction for the 938 bp BspEI fragment alone indicates disruption of the 5’ hairpin boundary.
`
`different polymerases. This included GoTaq Flexi DNA polymerase (Promega), Pfu
`Ultra (Stratagene), LA Taq (Takara), and Expand Long Template Kit (Roche). For LA
`Taq, both GC Buffer I and GC Buffer II were used in an attempt to improve amp-
`lification through the polymerase-resistant region. For the Expand Long Template
`Kit, Systems 1, 2, and 3 were used. DNA templates used for PCR: genomic DNA
`isolated from tail biopsies from mice of either a predominantly C57B6 or 129S6
`genetic background, TL1 ES cells 17 (129S6), two distinct plasmids containing frag-
`ments of Foxd3 genomic sequence, and BAC DNA as described above.
`
`BAC recombineering. We electroporated a Foxd3-containing BAC into the E. coli
`strain EL350. We then electroporated an insertion vector containing two 500 bp
`homology fragments homologous to sequences 5’ of the Foxd3 start codon and 3’ of
`
`the Foxd3 stop codon (Figure 1) into the BAC-containing EL350 cells. Recombination
`events were identified by acquisition of antibiotic growth selection when compared to
`controls. Recombinant clones were selected and analyzed by PCR and restriction
`digests to determine whether the insertion sequence had been acquired. A retrieval
`vector was constructed by PCR-amplifying sequences corresponding to Fragments A
`and D (Figure 1) from mouse Foxd3 BAC DNA with primers that also added
`restriction enzyme sites (XbaI and SpeI for Fragment A, SacI and SacII for Fragment
`B) and cloning them into a modified pBluescript vector containing a diphtheria-toxin
`cassette (pBS.DT-A, a gift from the laboratory of Dr. Mark Magnuson). We then
`electroporated some of these clones with this retrieval vector containing homology
`fragments A and D, approximately 8.5 kb and 4.6 kb from the Foxd3 start and stop
`codons, respectively. Recombinant clones were selected by acquisition of antibiotic
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`resistance. However, because the first recombineering step did not generate clones
`with the full desired sequence, even successful recombination at this step could not
`result in clones with the complete target sequence.
`
`Annotation. The adenine nucleotide of the Foxd3 ATG start codon was designated
`position 1. Upstream (5’) nucleotides were assigned positions in negative numbers
`relating their distance from the Foxd3 ATG, whereas downstream (3’) nucleotides
`were designated by positive numbers indicating distance from the first A of the Foxd3
`ATG.
`
`Conservation analysis. NCBI Basic Local Alignment Search Tool (BLAST) analysis
`(http://blast.ncbi.nlm.nih.gov/Blast.cgi) 17 was performed independently for the
`370 nt region of polymerase resistance from the murine genome and the two flanking
`stretches of 370 nt. The blastn algorithm for somewhat similar sequences was used to
`query the nr/nt nucleotide collection. Maximum alignment scores were reported for
`the top vertebrate species. In the case where scores were not given, they are listed as
`‘‘below threshold’’. DCODE ECR browser (http://ecrbrowser.dcode.org) analysis 18
`was performed individually on the mouse Foxd3 (July 2007 mouse genome assembly
`– NCBI build 37/mm9) and human FOXD3 (March 2006 human genome assembly –
`NCBI build 36.1/hg18) loci. Within the ECR browser, pairwise alignments for con-
`servation analysis were selected for 11 available genomes: chimpanzee (Pan troglo-
`dytes), rhesus monkey (Macaca mulatta), mouse (Mus musculus), human (Homo
`sapiens), rat (Rattus norvegicus), dog (Canis familiaris), cow (Bos taurus), opossum
`(Monodelphis domestica), chicken (Gallus gallus), frog (Xenopus laevis), zebrafish
`(Danio rerio) and fugu pufferfish (Takifugu rubripes). For comparison with the
`mouse genome, the ECR browser window covered an interval of 13,869 nt, from
`positions 99313783 to 99327651 on mouse chromosome 4 (the Foxd3 transcribed
`region, including the 5’ and 3’ UTRs, is 99322990 to 99325362). For comparison with
`the human FOXD3 locus, the ECR browser window covered an interval of 13,786 nt,
`from positions 63552012 to 63565798 on human Chromosome 1 (FOXD3 tran-
`scribed region is 63561318-63563385). Parameters used for analysis and display
`included an ECR length of 100, ECR similarity of 90, layer height of 55 and a
`relative coordinate system. Tracks from human ENCODE data aligned to the same
`interval of FOXD3 (Chromosome 1: 63552012 to 63565798) from the March 2006
`assembly of the human genome were visualized within the UCSC genome browser
`(http://genome.ucsc.edu/cgi-bin/hgGateway). Tracks for H3K4 trimethylation and
`DNaseI hypersensitivity were selected from the Expression and Regulation track
`group. H3K4me3 data was obtained from ChIP-Seq experiments in GM12878 and
`K562 cell lines, while DNaseI hypersensitivity data displayed was from DNase-seq
`experiments in GM12878 cells; both indicate regions of open chromatin20-21.
`
`Prediction of secondary structure. RNAFOLD analysis was performed with the
`Vienna RNAfold Webserver (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) 11 on
`the 1110 nt upstream region of the mouse Foxd3 locus comprised of the 370 nt
`polymerase-resistant region and 370 nt flanking sequence on either side. Input
`parameters were entered for a linear DNA molecule (DNA parameters from 2004
`David Matthews model) at 72 degrees Celsius allowing for dangling energies on both
`sides of a helix in any case, using minimum free energy (MFE) and partition function
`fold algorithms 22. The output was visualized as an interactive secondary structure
`plot. Additional analysis of secondary structure was performed with the UNAFold
`program for two-state melting/folding located on the mfold/UNAFold webserver
`(http://mfold.rna.albany.edu/) 23. UNAFold parameters were set for DNA at
`72 degrees Celsius, 1M Na1, 0M Mg21, and for a linear molecule. Due to program
`limits, only 1000 nucleotides (rather than 1110) were entered; this sequence was the
`370 nt region of interest plus 315 nucleotides of flanking sequence on either side.
`
`Construction of plasmids. A 10.6 kb Foxd3 plasmid consisting of a backbone vector
`and an approximately 7.6 kb EcoRI genomic Foxd3 fragment (containing the entire
`Foxd3 coding sequence plus surrounding 5’ and 3’ genomic sequence) was digested
`with the enzyme BspEI, resulting in fragments of approximately 9.6, 0.9, and 0.1 kb
`that were separated using agarose gel electrophoresis. The 0.9 kb fragment spanned
`most (348 of 370 nt) of the predicted hairpin sequence and additional downstream
`sequence. The 9.6 kb band was gel purified, and this linear molecule with BspEI ends
`ligated to form a Foxd3 plasmid missing most of the predicted hairpin. The 0.9 kb
`fragment containing most of the hairpin was also purified and ligated into the EcoRV
`site of pBluescript forming a plasmid that contained most of the hairpin.
`
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