In silico analysis of regulatory and structural motifs of the ovine HSP90AA1 gene

Abstract

Gene promoters are essential regions of DNA where the transcriptional molecular machinery to produce RNA molecules is recruited. In this process, DNA epigenetic modifications can acquire a fundamental role in the regulation of gene expression. Recently, in a previous work of our group, functional features and DNA methylation involved in the ovine HSP90AA1 gene expression regulation have been observed. In this work, we report a combination of methylation analysis by bisulfite sequencing in several tissues and at different developmental stages together with in silico bioinformatic analysis of putative regulating factors in order to identify regulative mechanisms both at the promoter and gene body. Our results show a "hybrid structure" (TATA box + CpG island) of the ovine HSP90AA1 gene promoter both in somatic and non-differentiated germ tissues, revealing the ability of the HSP90AA1 gene to be regulated both in an inducible and constitutive fashion. In addition, in silico analysis showed that several putative alternative spliced regulatory motifs, exonic splicing enhancers (ESEs), and G-quadruplex secondary structures were somehow related to the DNA methylation pattern found. The results obtained here could help explain the differences in cell-type transcripts, tissue expression rate, and transcription silencing mechanisms found in this gene.

Keywords: HSP90AA1; In silico analysis; Methylation pattern; Regulatory motifs; Sheep; Structural features.

Fig. 1

Graphic representation of the ovine HSP90AA1 CpG island (based on the reference sequence from the NCBI database, DQ983231.1; 5917 bp). The X axis represents the base pairs, while the Y axis represents the percentage of GC (data obtained from the Sequence Manipulation Suite http://www.bioinformatics.org/sms/cpg_island.html). The putative CpG island spans through the core promoter including the heat shock element (HSE), TATA box, and initiator element, also the exon 1,intron 1, exon 2, and intron 2 (see top of the figure)

Fig. 2

Methylation pattern representation of the HSP90AA1 CGI across different tissues. Identifications (IDs) from tissues are as follows: M.C.A.B. blood samples from non-stressed adults of Manchega breed, M.H.S.B. blood samples from heat-stressed adults of Manchega breed, R.Y.H. heart samples from young animals of Rasa Aragonesa breed, M.A.B. brain sample from an adult animal of Manchega breed, M.A.L. liver sample from an adult of Manchega breed, M.Y.O. ovary samples from young animals of Manchega breed, R.Y.T. testicule samples from young animals of Rasa Aragonesa breed, M.Y.T. testicule sample from a young animal of Manchega breed, M.A.T. testicle sample from an adult animal of Manchega breed, and M.A.S. sperm samples from adult animals of Manchega breed. Numbers on the top of the figure indicate the position of the CpG cytosine relative to TSS. CpGs from −582 to +632 positions relative to TSS are un-methylated (not shown in the figure). The signs at the bottom of the figure indicate positions at the promoter region of the gene, exons (E), and introns (I). CpG at position −778 represents the 5′ boundary of the CGI in somatic and non-differentiated germ tissues. Differentiated germ tissues have no identified CGI border in 5′. CpG at position +1455 (3′ end) is methylated in all tissues. Patterns of epigenetic marks are specific of each tissue. The rs397514116 (g.660G > C) mutation creates an ASM in blood and an “allele-specific hemi-methylation” in somatic and non-differentiated germ tissues. Differentiated germ tissues have no ASM

Fig. 3

Graphic representation of ovine HSP90AA1 gene and their putative regulatory motifs related with transcription and splicing processes found in our in silico study. Every site is numbered relative to the TSS

Fig. 4

Tissue-specific transcripts from the ovine and human HSP90AA1 genes. Only tissues analyzed in the present work and their reverse strands are shown. The arrow indicates the direction of transcription. P1 (promoter), AP2, and AP3 (alternative promoters 2 and 3) indicate the type of promoter. Graphical representation by crosses of the each expression ratio is based on total alignments of RNAseq data (number of alignments + alignments omitted) with respect to the total alignments of liver (which is the tissue with the least number of alignments) (available at http://www.ensembl.org/Ovis_aries and http://www.ensembl.org/Homo_sapiens)