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

Methylation Patterns in the 5’ Flanking Region of The Hoxb9 Gene in Sea Turtle Embryos with Congenital Malformations. Abstract Sea turtles are wild animals whose embryonic development largely depends on environmental factors. The shell of sea turtles is a novelty among vertebrates and follows a Hox code and axial formula for the group. The hoxb9 gene is a transcription factor involved in the regulation of embryonic development, mainly during neural tube development and plays an important role in shaping the thoracic-lumbar transition of sea turtles. Although it is known that Hox genes are regulated by chromatin modulating proteins, DNA methylation in CpG islands may also contribute to gene regulation. Previous studies have shown a high prevalence of congenital malformations in the olive ridley sea turtle ( Lepidochelys olivacea ), including Schistosomus Reflexus (SR) syndrome. Here we studied the methylation profiles in the 5’ flanking region of hoxb9 comparing normal and abnormal sea turtle embryos. Our results indicate that methylation of the putative promoter region of hoxb9 in sea turtle embryos occurred, however methylated cytosines showed individual patterns, not directly related to the development of congenital malformations.


Introduction
Homeotic genes act as master regulators for the specification of body plans in development, regulating other genes and programming certain developmental pathways [1]. Hox genes encode transcription factors sharing a conserved DNA-binding domain, known as the homeodomain [2,3], regulating cell fate and vertebral identity during embryonic development [4]. The different Hox proteins are expressed in distinct, often overlapping, domains along the antero-posterior body axis of animal embryos [5]; therefore, these spatially constrained and overlapping expression domains represent a combinatorial code to generate regional diversity [6]. Interestingly, the reptile group presents a variety of shapes, and a complete inventory of Hox genes in crocodiles, turtles, snakes, and lizards have been reported [7]. Turtles have the most unusual body plan among amniotes, with a dorsal shell that fuses with the ribs [8]; for this group, as well as for other tetrapods, including mammals, birds, and amphibians, a total of 39 Hox genes have been reported, divided into four paralog groups (A, B, C and D) [7].
According to Böhmer & Werneburg [9], the Hox code in turtles is characterized by changes in spatial expression in the vertebral column compared to the closest phylogenetic group, the archosaurs (which includes crocodiles and birds), to give rise to the appearance of the unique shell of the turtle [8,9]. The Hoxb9 gene is involved in the regulation of embryonic development [10]. In mice, the homozygous elimination of the Hoxb1-Hoxb9 cluster causes abnormalities in the cervico-thoracic spine and defects in the sternum morphogenesis defects, including tracheo-esophageal abnormalities [12]. Also, in humans, de novo translocation has been reported with a breakpoint near the 5' end of the HOXB cluster in a patient with developmental delay and skeletal malformations [13]. Embryonic development is epigenetically regulated and Hox genes are no exception [14,15].
The transcriptional state of Hox genes is maintained by two groups of chromatins modulating proteins, acting in opposite directions: the Trithorax Group (TrxG) proteins, which activates transcription, and the Polycomb Group (PcG), which represses transcription [15][16][17].
The role of DNA methylation as a transcriptional regulator has been associated to the promoters of genes containing high density of CpG sites, called CpG islands (CGIs), which remain hypomethylated even if the locus is transcriptionally silent [18]. In Hox genes, CpG islands in promoters may contribute to gene regulation in cis [19]; in addition, CpG inter or intragenic islands are more susceptible to methylation, and present tissue-specific methylation in these same loci [19]. Bárcenas-Ibarra et al. [20,21] reported the presence of congenital malformations and Schistosomus Reflexus (SR) syndrome in the olive ridley sea turtle (Lepidochelys olivacea), a species considered vulnerable according to the IUCN Red List of Threatened Species [22]. This syndrome was identified in 31% of the embryos that showed congenital malformations, and its main characteristics are the lack of closure in the abdominal wall and alterations in the spinal cord [21]. Thus, the methylation profiles in the 5' flanking region of the hoxb9 gene were compared between embryos with multiple malformations, including SR, and their normal counterparts.

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.  (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].

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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].

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] [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 hoxb9 promoter and congenital malformations in the embryos analyzed in this study; hoxb9 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.