In many ways, the alignment of ELA sequence with HLA demonstrated a great deal of organizational similarity and conserved synteny as expected (Gustafson et al., 2003). Sequence alignments also confirmed earlier mapping studies that found ELA class I sequences distributed in three clusters, including two class I loci within the class II region (Gustafson et al., 2003; Tallmadge et al., 2005). However, noteworthy differences were predicted. One of the striking results from the initial assembly of the ELA and extended regions from the equine genome project was that the size of the region was approximately 6.0 Mb in size, almost 1.3 Mb larger than the human HLA and extended regions. Furthermore, annotation of the ELA sequence identified 40 class I loci, many more than expected from serology, analysis of BAC clones, and gene expression studies (Ellis et al., 1995; Tallmadge et al., 2005, 2010).

Closer examination of the assembled sequence of ELA revealed that the increased size of ELA was largely due to two features apparently unique to the MHCs of horses when compared to other sequenced mammalian taxa. One feature is a gene desert of approximately 550 Kb at the boundary of the class II and class III regions from coordinates 20: 31,896,104..32,442,400 (Figure 5.1). This region contains a single annotated gene, C6orf10, also called testis-specific binding protein (TSBP), and two pseudogenes, one related to a tetraspanin 17-like sequence and the other to a Spi-c-like transcription factor. The chromosomal position and sequence homology of ELA C6orf10 to human C6orf10 indicates that these are orthologous loci. Alignment of the balance of the ELA desert sequence with whole genomic sequences of other species identified no comparable sequence at any location in any other species.

The second distinctive genomic feature in ELA is a large and strikingly conserved segmental duplication of at least 11 units, each about 45Kb in size, at the boundary of the ELA class I and III regions (Figure 5.2) (Brinkmeyer-Langford et al., 2010). Each unit in the segmental duplication contains one sequence related to a truncated form of the B-associated transcript 1 (Bat1) that aligns to the c-terminal domain of the helicase domain, and a second sequence with strong homology to class I sequences. The Bat-1 and class I sequences are regularly interspersed and arrayed largely in head to tail arrangement throughout the segmental duplication. The Bat-1 sequences are extraordinarily conserved within Equus caballus and among other Perissodactyl species (Brinkmeyer-Langford et al., 2010), arguing for some functional role(s) for these sequences. Twenty-four genes are predicted from the version 2.0 assembly to be contained within the segmental duplication. Three closely related sequences are chromosomally unassigned in the 2.0 assembly, indicating that the segmental duplication may include 14 repeating units and 30 annotatable genes.

Figure 5.2 Dotplot of repeat-masked sequence in the ELA segmental duplication region on ECA20 (NW_001867389.1; positions 30600000-31350000bp) aligned against itself using the bl2seq option of the NCBI BLAST website. At least 11 regions contain related sequences generally aligned head to tail. Three additional closely related sequences are found in the ChrUn database of the horse, indicating that 14 repeat units may reside within the segmental duplication.

The remarkable conservation of sequences within the segmental duplication feature among different Perissodactyl MHCs suggests that this region would contain functional genes. Evidence for gene expression has been sought using reverse transcription PCR and chromatin immunoprecipition sequencing (ChIP seq).

RT-PCR

We used locus-specific RT-PCR to seek transcripts from nine of the class I genes and four of the Bat-1 like genes in the segmental duplication (Table 5.1; Brinkmeyer-Langford et al., 2010). Sequence similarities precluded design of locus-specific primers for the remaining predicted genes. Transcripts for five of the nine class I sequences were successfully amplified from peripheral WBCs of at least some of several horses tested, while no transcripts were detected for any of the four tested BAT1-like sequences. Transcripts from the full-length BAT-1 sequence were identified as expected. These results indicate that at least some of the class I genes within the segmental duplication are transcriptionally active, including two genes previously identified (Ellis et al., 1995; Tallmadge et al., 2005). Consequently, it seems that most of the predicted genes within the segmental duplication are not expressed as mRNAs, although it is possible that some of these genes may demonstrate tissue-specific expression profiles not assessed by these studies.

Table 5.1 Summary of predicted genes and transcripts within the segmental duplication interrogated for gene expression by H3K4me3 ChIPseq and reverse transcription PCR.

Chromatin Modifications Associated with Transcription

Another approach to identifying regions of transcribed DNA is by sequencing the DNA bound to nucleosomes immunoprecipitated with antibodies specific for histone modifications that are predictive of open chromatin (ChIP). Using unpublished data graciously provided by S. Dindot and N. Cohen of Texas A&M University, we examined the gene desert and segmental duplication regions of ELA in whole genome ChIP seq data from anti-H3K4me3-immunoprecipitated chromatin of neutrophils obtained from a newborn foal. Histone 3 trimethylated at lysine 4 (H3K4me3) is a histone modification predictably associated with the 5′ transcribed regions of actively expressed genes in higher eukaryotes (Santos-Rosa et al., 2002; Schneider et al., 2003) and is useful as a surrogate for identifying expressed genes.

Representation of DNA sequences aligned to the gene desert coordinates ECA 20: 31,896,104..32,442,400 revealed low levels of H3K4me3-captured sequences over the entire span of the gene desert, consistent with the predictions of few or no expressed genes in this region of ELA. In contrast, the region of the segmental duplication (ECA 20:30,600,000..31,336,000) was highly enriched for H3K4me3 bound sequences, suggesting an abundance of actively transcribed DNA. Twenty-four predicted genes are located in the ELA segmental duplication and 17 of these sequences were located in regions that were moderately to highly represented in anti-H3K4me3 immunoprecipitates. These peaks corresponded to the locations of 13 predicted genes (Ensembl). Of the seven regions highly enriched in H3K4me3, four (57%) were also positive for RT-PCR transcripts. Of the six predicted genes in regions of low H3K4me3, none were detected as transcripts by RT-PCR. A summary of the evidence for gene expression in the segmental duplication is presented in Table 5.1.

Summary

As more detailed analyses of the vertebrate MHC become available, the picture emerging is of an organizationally constrained but structurally dynamic region evolving primarily by recombination and gene conversion. These processes act to sort and reshuffle combinations of genes that have been strongly selected for over evolutionary time frames. The early paradigm that mammalian MHCs were predictably arranged by gene content into three regions consisting of class I and class II genes flanking a class III region is proving to be overly simplistic. The disruption of the class II region of the ruminant MHC (Childers et al., 2006) and less dramatic rearrangements in the MHCs of cat (FLA) and dog (DLA) (Yuhki et al., 2007) promise to provide insights into the evolutionary processes at work in this important region of the genome. The unusual features now known to characterize the Perissodactyl MHCs add significantly to the list of diversifying structural changes present in the vertebrate MHC. Access to research material from a common domestic animal such as the horse, with deep pedigrees and well-developed formal biology, provides great potential to help unravel the puzzling structural and functional properties of the MHC.

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