Characterization of the Unusual Bidirectional ves Promoters Driving VESA1 Expression and Associated with Antigenic Variation in Babesia bovis

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Rapid clonal antigenic variation in<jats:named-content content-type="genus-species">Babesia bovis</jats:named-content>involves the<jats:underline>v</jats:underline>ariant<jats:underline>e</jats:underline>rythrocyte<jats:underline>s</jats:underline>urface<jats:underline>a</jats:underline>ntigen-1 (VESA1) protein expressed on the infected-erythrocyte surface. Because of the significance of this heterodimeric protein for demonstrated mechanisms of parasite survival and virulence, there is a need to understand how expression of the<jats:italic>ves</jats:italic>multigene family encoding this protein is controlled. As an initial step toward this goal, we present here initial characterization of the<jats:italic>ves</jats:italic>promoter driving transcription of VESA1a and -1b subunits. A series of transfection constructs containing various sequence elements from the<jats:italic>in vivo</jats:italic>locus of active<jats:italic>ves</jats:italic>transcription (LAT) were used to drive expression of the firefly luciferase gene in a dual luciferase-normalized assay. The results of this approach reveal the presence of two bidirectional promoter activities within the 434-bp intergenic region (IGr), influenced by putative regulatory sequences embedded within the flanking<jats:italic>ves</jats:italic>1α and<jats:italic>ves</jats:italic>1β genes. Repressor-like effects on the apposing gene were observed for intron 1 of both<jats:italic>ves</jats:italic>1α and<jats:italic>ves</jats:italic>1β. This effect is apparently not dependent upon intronic promoter activity and acts only in<jats:italic>cis</jats:italic>. The expression of genes within the<jats:italic>ves</jats:italic>family is likely modulated by local elements embedded within<jats:italic>ves</jats:italic>coding sequences outside the intergenic promoter region in concert with chromatin modifications. These results provide a framework to help us begin to understand gene regulation during antigenic variation in<jats:named-content content-type="genus-species">B. bovis</jats:named-content>.</jats:p>

Unusual chromatin structure associated with monoparalogous transcription of the Babesia bovis ves multigene family

“…Previously, the LAT was shown to be the only transcriptionally active ves locus in the B. bovis C9.1 line genome (Allred et al, 2000; Al-Khedery and Allred, 2006; Zupanska et al, 2009; Xiao et al, 2010). Although there are numerous ves loci with similar divergent organization (Al-Khedery and Allred, 2006; Brayton et al, 2007), and IGrs from silent ves loci are capable of driving transcription in transient transfection assays (Wang et al, 2012), to date we have been unable to detect in situ transcriptional switching from the LAT to other ves loci, even when attempts were made to select for such switching (unpublished data). Regardless, to ensure continued expression of VESA1 derived from the LAT, parasites were periodically re-selected for immunoreactivity with the C9.1 line-specific monoclonal antibody, 4D9.1G1 (O’Connor et al, 1997, 1999b), to maintain population uniformity.…”

“…This possibility would raise a further logistical problem for ves genes, as most are clustered closely together, but the clusters are mostly small and scattered around the genome, without the usual bias to subtelomeric regions (Brayton et al, 2007). Moreover, the majority of ves genes within the clusters are organized functionally for bidirectional expression, having IGrs which are functional in vivo for expression of marker genes when present on episomal DNA (Wang et al, 2012). The monoparalogous nature of ves transcription (Zupanska et al, 2009), like that of most variant multigene families, raises the question how gene pairs within a cluster are individually and discriminately marked for transcription or silencing.…”

“…Within the divergently organized ves loci the distance is only 400–450 bp. Within this region are dual bidirectional promoter activities transcribing both ves 1α and ves 1β genes (Wang et al, 2012), with heavily overlapping 5′-UTRs (Allred et al, 2000; Wang et al, 2012). It remains to be determined critically whether the LAT IGr is able to transcribe simultaneously in both directions or must somehow alternate between transcribing ves 1α and ves 1β sequences.…”

“…The presence of paired inverted repeats and divergent organization lend the IGr of the LAT and similarly organized ves loci a quasi-palindromic character. Based upon mapping of the 5′-untranslated region (5′-UTR) of both ves 1α and ves 1β genes, transcription begins within this region, indicating that bidirectional promoter activity also resides there, an assertion supported by transfection studies (Wang et al, 2012). Palindromic sequences have been demonstrated in a number of organisms to provide promoter functions, sometimes even requiring extrusion as a cruciform or hairpin to facilitate transcription (Kim et al, 1998; Hughes et al, 2009).…”

“…The inverted repeats are highly conserved in sequence and are shared among IGrs from nearly all similarly organized ves genes (Al-Khedery and Allred, 2006). Due to the conservation of sequence and organization, IGrs from non-transcribed ves loci appear to be functionally equivalent to that derived from the locus of active ves gene transcription (LAT), and a preliminary characterization of ves promoters has confirmed this possibility (Wang et al, 2012). Despite such apparent functional equivalence, in vivo ves transcription appears to occur from a single locus among clonal parasites (Zupanska et al, 2009).…”