Methylation Patterns in the 5’ Flanking Region of The Hoxb9 Gene in Sea Turtle Embryos with Congenital Malformations

Materials and Methods

Sample collection

A previous study was conducted at El Verde Camacho nesting beach in Mexico (23° 44’ 22” N, 106° 58’ 27” W) [20]. From this study, a catalog of congenital malformations was generated [20], tissue was sampled from the back of the neck and preserved in absolute ethanol at -20°C to perform the genetic analysis. Genomic DNA was extracted by the salting-out method [23] and stored at -20°C until use.

Locus-specific DNA methylation analysis

Isolation and characterization of the 5’ flanking region of the hoxb9 gene: The 5’ flanking region of the hoxb9 gene for the olive ridley sea turtle was isolated by PCR amplification. Primers were designed according to the coding region of the hoxb9 gene (HOXB9-201 ENSPSIT00000007772.1) reported for the Chinese soft-shelled turtle (Pelodiscus sinensis) identified on ENSEMBL database, and the first exon in the draft genome of the green sea turtle (Chelonia mydas) identified in the National Center for Biotechnology Information (NCBI) database; the sequences of both species were compared and primers were designed to amplify a fragment of about 948 bp (Table 1); the reverse primer was designed within the coding region of the hoxb9 gene, whereas the forward primer was designed in the putative promoter region. Amplified fragments were purified and sequenced in Macrogen Inc. (South Korea). Once the sequences were obtained, they were manually edited, and a BLAST analysis was performed against the genome of C. mydas to corroborate identity. Once the promoter sequence of the hoxb9 gene was obtained, CpG Islands (CGI) were identified with the Methprimer software [24], Transcription Factor Binding Sites (TFBS) were identified using the GPMiner software [25], which contains 4206 known binding sites that match upstream regions of human, mouse, rat, chimpanzee, and dog genes, and the Transcription Start Site (TSS) was identified with the TSSG software [26].

Table 1: Primers used to amplify the hoxb9 5’ flanking region.

Bisulfite conversion, amplification, cloning, and analysis of methylated CpG sites: For locus-specific DNA methylation analysis, four embryos were selected that presented multiple malformations, including Schistosomus Reflexus (SR): 1) SR-1811- IE39M, 2) SR-2029-E4M, 3) SR-2029-E1M, 4) SR-1257-IE9M and 3 normal embryos with no external malformations (Table 2). The DNA of normal and SR embryos was converted with sodium bisulfite using the EZ DNA Methylation Gold Kit™ (Zymo Research) following the manufacturer’s instructions. Since CGI-1 and CGI-2 were very close, primers were designed for a single fragment of about 334bp (Table 1). PCR products were gel-purified with the Wizard® SV Gel and PCR Clean-up System (Promega). Purified PCR products were ligated into a pGEM-T cloning vector (Promega). Transformation was performed by heat shock using Escherichia coli JM109 competent cells (Promega) and plasmid extraction was performed by alkaline lysis. Six to ten clones per embryo were sequenced using the T7 universal primer, and the difficult sequencing service provided by Macrogen Inc (Korea). The frequency of methylated CpG sites was examined using the QUMA (Quantification Tool for Methylation Analysis) software: (http://quma.cdb.riken.jp).

Table 2: Selected embryos to study DNA methylation in the 5’ flaking region of the hoxb9 gene.

Results

Isolation and characterization of the 5’-flanking regions of the hoxb9 gene

A fragment of 760 bp was obtained from genomic DNA of the olive ridley sea turtle. BLAST analysis indicated a >90% identity with the 5’-flanking regions (called putative promoter region) of the hoxb9 gene of the green sea turtle (C. mydas) and the chinese soft shelled turtle (P. sinensis). Nucleotide 363 (of the isolated fragment) was identified as a TSS (position +1) (Figure 1). The putative TATA box was located at nt +50 relative to the TSS. Fifteen putative transcription factor binding sites (TFBSs) were also identified: Pre-B-cell leukemia transcription factor 1 (PBX1_01), Nuclear transcription factor Y (NFY_Q6), Fetal Alz-50-Reactive Clone 1 (FAC1_01), MYB transcriptional activator (MYB_Q6), Neurofibromin 1 (NF1_Q6), Thyroid hormone receptor T3R (T3R_ Q6), Signal transducer and activator of transcription 3 (STAT3_02), Signal transducer and activator of transcription 5A (STAT5A_03), Signal transducer and activator of transcription 1 (STAT1_03), Signal transducer and activator of transcription 6 (STAT6_01), Signal transducer and activator of transcription 4 (STAT4_01), transcriptional repressor YY1 (YY1_01), Activator protein 1 (AP1_ Q4_01), Nuclear factor TCF11 (TCF11_01), Homeobox protein MEIS1 (MEIS1_01). Putative GC boxes were also found upstream the TSS (Figure 1).

Figure 1: Characterization of the putative promoter region of the hoxb9 gene.

Identification of CpG islands (CGIs) in the 5’-flanking region of the hoxb9 gene

Two CGIs were identified, CGI-1 of 109 bp (nt -120 to -12) and CGI-2 of 127 bp (nt +57 to +183) (Figure 2). A set of PCR primers (including CGI-1 and CGI-2) were designed to amplify bisulfitetreated DNA (Table 1). Primers amplified a fragment of 334 bp, which included 19 CpG sites, seven of which were within the CGI-1, and nine of them were in the CGI-2 (Figure 1 & 2). Characterization of the putative promoter region of the hoxb9 gene. CpG sites with methylation potential (yellow), possible transcription factor-binding sites (underlined in black), transcription start site (TSS, black arrow), TATA box (green rectangle) and GC box (red rectangles) are indicated. The blue box indicates the 334 bp fragment which includes the CpG island-1 (CGI-1; nt -120 to -12) and the CpG island-2 (CGI-2; nt +57 to +183). Regulatory features were identified by the GPMiner software [25]. Identification of CpG islands (light blue) in the putative promoter region of the hoxb9 gene of L. olivacea. A TSS was identified at nt 363 of the isolated fragment. CGI-1 was identified upstream of the TSS and CGI-2 in the 5 ‘UTR. CGIs were identified with the Methprimer software [24].

Figure 2: Identification of CpG islands (light blue) in the putative promoter region of the hoxb9 gene of L. olivacea.

Locus-specific DNA methylation in the putative promoter region of the hoxb9 gene

Bisulfite sequences from abnormal and normal embryos were compared. Conversion efficiency was 95-100% (Figure 3). Mosacism methylation was present in most cases (Table 3). A sequence of 319 bp was analyzed, which contained nineteen CpG sites Figure 3. Because most of the TFBS were identified upstream of both CGIs analyzed with bisulfite-PCR (Figure 1), it was not possible to identify methylated CpG sites in putative TFBS. All embryos analyzed (normal and abnormal) showed a particular methylation profile (Figure 3). Interestingly, there was a consistent pattern in which sites +128 and +136, located in the 5’ UTR region Figure 1 and 3 were methylated regardless of whether the embryos presented developmental defects, specifically site +128 was methylated in all embryos analyzed, which could suggest an important role of locus-specific methylation in the 5’ UTR of the hoxb9 gene related to embryonic development, but not related to any specific malformation. A different, individual pattern of methylated cytosines was also observed in the embryos, however, with such a small sample size and the variety of malformations observed is not possible to know if these methylation patterns are related to malformations or other developmental traits. Methylated cytosines were also identified in the non-CpG (CHG/CHH) context in both normal and abnormal embryos (Table 1). The role of non- CpG methylation in non-mammalian vertebrates is less understood and requires further research [27].

Figure 3: Methylation status in the putative promoter region of the hoxb9 gene from normal and malformed embryos (labeled as SR because all malformed embryos presented schistosomus reflexus). The black circles indicate the methylated cytosines in the CpG context.

Table 3: Frequency of clones with methylated cytosines in CpG and non-CpG (CHG/CHH) contexts of the putative promoter region of the hoxb9 gene in malformed (SR) and normal embryos.

Discussion

An apparent heterogeneous and individual methylation profile was observed in CGIs of the putative promoter region of the hoxb9 gene in both normal and malformed embryos. Bisulfite conversion efficiency of DNA was high (95 to 100%) but mosaicism was evident (Table 3). This may be due to the sampled region (the back of the neck) since it originates from ectoderm and mesoderm derivatives [28]. It is known that developmental genes regularly remain hypomethylated even if the locus is transcriptionally silent, although some methylation could be present [19], and this is consistent with our results in both normal and malformed embryos. Although heterogeneous, the methylation profile observed in the putative promoter region of hoxb9 shows a consistent pattern in two CpG sites (+128 and +136) that are repeated in normal and abnormal embryos (Figure 3) these methylated cytosines could play a role in the regulation of hoxb9 during development and may not be involved in developmental defects [29].

In this sense, it has been reported that the 5’ UTR region of several genes plays an important role in alternative splicing processes [30], it would be interesting to know if there is a relationship between the presence of these methylated cytosines and mRNA processing. In Hox genes, tissue-specific methylation of CGIs is essential for development [19]. The patterns of DNA methylation reflect signals established during development, and it is known that they can be modified in adult tissues with tumor progression [29,31]. In fact, it is well established that proteins of the Polycomb group modulate Hox gene expression by histone marks (e.g., methylation of histone 3 at lysine 27-H3K27me-) [32], so that DNA methylation could promote transcription by antagonizing genes from the Polycomb group [33], repressing gene expression. For instance, in mice with skeletal defects, hypermethylation of DNA at Hox loci was associated with the elimination of the SET domain of the Mll gene of the Tritorax group (antagonist of the Polycomb group) [34].

Currently, it is well established that environmental factors during embryonic development can cause different epigenetic modifications, such as changes in DNA methylation or posttranslational modification of histones; these factors may include nutrient deficiency, presence of pollutants, or even stress [35]. In sea turtles, temperature, humidity, and gas exchange during incubation may behave as teratogenic agents [36], affecting development through subtle epigenetic changes in Hox (and other developmental) genes with phenotypic implications [34,37,38]. No link was observed between methylation patterns in the putative hox promoter and congenital malformations in the embryos analyzed in this study; hox regulates the development of the spine in the thoracic-lumbar region, which could be implicated in SR, and it would be necessary to sample and analyze tissue from that region. It is also important to consider that the Hox genes act in a combinatorial fashion, and different Hox genes are required to shape each vertebra and the axial structure. In addition, paralogous Hox genes present functional compensation [39,40] therefore, the regulatory effects of Hox genes should be studied together, according to the Hox code and axial formula of each species.