5' Flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasite (by Lopeez-Rubio et al; 2007).
Introduction
The human malaria parasite Plasmodium.falciparum is responsible for morbidity and mortality of human (Dzikowski Ron 1, Frank Matthias 2 & 1 2006 March; Frank & Deitsch 2006), that because the protozoan of this parasite cause more than one million deaths a year (Frank & Deitsch 2006). However, the most prominent virulent protein or antigen expressed on the red blood cells surface called P.falciparum erythrocyte membrane protein 1 (PfEMP1) (Calderwood et al. 2003; Chookajorn et al. 2007; Dzikowski Ron 1, Frank Matthias & 1 2006 March; Frank & Deitsch 2006; Frank et al. 2006; Voss et al. 2006).
This protein is encoded by a wide spectrum of polymorphic gene family called var (Dzikowski Ron 1, Frank Matthias 2 & 1 2006 March; Lopez-Rubio et al. 2007), which are associated with cytoadherent and virulence phenotype of infected cell (Dzikowski Ron 1, Frank Matthias 2 & 1 2006 March). P.falciparum can maintain a single active var gene locus among many RBCs cycles and gained continuous switching to different loci to avoid the immune system responses (Comeaux & Duraisingh 2007). The multi-switching of PfEMP1 expression are the cause of changes in the transcription of each var genes (Chookajorn et al. 2007), also malaria parasites have ability to continuously switching var gene expression during the period of infection (Dzikowski Ron 1† et al. 31 August 2007). In addition, the other remaining var gene members are not transcribed and turn to be silenced (fig1) (Calderwood et al. 2003; Chookajorn et al. 2007). All var genes have two exons, the exon1 is long and encodes polymorphic extracellular part of protein, on the other hand exon2 is short and encodes a lots of conserved intracellular part (Chookajorn et al. 2007; Frank & Deitsch 2006).
Figure 1
This figure shows the Schematic of histone modifications associated with active transcription, bookmarking and silencing of var genes. Active var gene transcription (green) has been shown to be associated with an enrichment of H3K4me3 and H3K4me2 in the 5flanking region of the active var gene. Silenced var gene loci (red) have an enrichment of H3K9me3 in both their 5 flanking and coding regions. In later stages of the asexual life cycle, silenced var genes maintain an enrichment of H3K9me3 in their 5flanking and coding regions, while the previously active var locus appears to remain poised (yellow) for expression early in the next asexual life cycle by an enrichment of H3K4me2 in its 5flanking region. It is hypothesized that this mark confers transcriptional memory of the actively transcribed var gene locus in the preceding ring stages through many cell divisions. The figure and legend are taken from (Comeaux & Duraisingh 2007).
Furthermore, a majority of var genes have a promoter sequences type upsA, upsB and upsC, whereas two high conserved var genes uepE (var2csa) and upsD (var1csa) types (fig2) (Comeaux & Duraisingh 2007; Frank & Deitsch 2006; Lopez-Rubio et al. 2007), for instance, the differential expression of these promoter types is associated to disease, upsA and upsB var gene types are located subtelomerically (Voss et al. 2006). On the other hand, another study has suggested that upsA and upsE members are located subtelomerically (Comeaux & Duraisingh 2007), upsB and upsC genes are either chromosome central or subtelomeric and transcribed directed away from the tolemer (Comeaux & Duraisingh 2007; Voss et al. 2003) or display promoter activities and interact with DNA-binding proteins. Noteworthy, the complex process of antigenic variation is mediated by epigenetic factors in the absence of any controlled DNA rearrangements (Lopez-Rubio et al. 2007; W. 2005). Whereas, another study has shown that the expression of subtelomeric varCSA is programmed by unique DNA element of 1.8 kb (Vazquez-Macias et al. 2002). PfEMP1 mediate a binding to infected RBCs and to syncytiothrophoblast cell via chondroitin sulphat A (CSA) (Voss et al. 2003) and binding to cellular adhesion molecules on the surface of vascular endothelial cells such as CD36 (Vazquez-Macias et al. 2002). Furthermore, the variations of the var gene expression are depending on the alterations in the structure and subnuclear localization of chromatine as well as binding of the telomere combined protein Pfsir2 (W. 2005).
Figure 2
This figure shows the 5' flanking var2csa region and the initiation of transcription highlighted by the flag. The figure is taken from (Lopez-Rubio et al. 2007).
The var gene intron has bidirectional promoter activity and this promoter emanates to be essential for gene silencing (Frank et al. 2006; Gannoun-Zaki et al. 2005; Lopez-Rubio et al. 2007), some study has shown that the complete silencing of var gene is regulated by intron at 5' flanking region (Voss et al. 2003), which support the idea of the authors in this article. Each intron may silence a single promoter, intron-promoter pair is causing a silencing in each individual var gene, and this disruption of silencing one gene does not alter the transcriptional process of next var promoters (Frank et al. 2006). Therefore, this region is highly AT-rich and is adequate to silence an associated var promoter (Calderwood et al. 2003).
About the article
According to Molecular Microbiology (Lopez-Rubio et al; 2007) in this article , they have shown that tri-dimethylation of histone H3 lysine4 are bookmarking in the 5' upstream region of transcribed var gene during transient silence stage (POSISED) for restarting active transcription in the next cycle. This study has demonstrated the role histone marks modification according to the P.falciparum parasite proliferation during the ring stage and the mature stage. The data have shown that the activities of transcription a long 5' flanking var2csa gene region is very high during the ring stage CSA parasite, whereas there is no significant transcription during same stage with in CD36 parasite and mature stage (silenced). As well as it has highlighted the interaction of histone marks in the poised stat 24-48h which is the mature stage with in CSA parasite (fig3).
Figure 3
This figure shows P. falciparum transcriptional var gene states during the 48 h blood stage cycle. Transcription of a single var gene starts about 4 h after the erythrocyte invasion by free parasite forms called merozoites and lasts for approximately 12–14 h (ring stage). For the remaining time of the 48 h cycle (trophozoite and schizont stage), which is mainly devoted to multiple rounds of parasite DNA replication and differentiation to merozoites, var gene transcription ends until free merozoites establish a new round of asexual blood stage cycle. Activation of a particular var member was achieved by selecting infected erythrocytes expressing a var gene able to bind to CSA, called var2csa. The two different states of transcription of an active var loci during the blood stage cycle is called here ‘ON’ for being transcribed and ‘POISED’ for being transiently silent but ready to get reactivated in the next cycle. Stable silencing (‘OFF’ state) of the var2csa gene is achieved by selecting parasites that express another var gene able to bind to CD36. The legend and figure are taken from (Lopez-Rubio et al. 2007).
How the var2csa is recognized in the next life cycle to be transcribe in the absence of programmed DNA rearrangement while it was silence in the previous stage?
This study has shown that the 5' flanking var gene region nucleates epigenetic event strongly linked with sustenance of mono-allelic var gene expression pattern during the parasite proliferation, as well as they hypothesized that there is a cellular memory may be acquired from inheriting var locus enrich with H3K4me at 5' flanking region. Therefore, the initiation of active transcription is begin in the same marked var region, probably by elevating the levels of hitone trimethyl marks again to maintain the transcription. Another study has support this hypothesis by identifying the role epigenetic mark in silenced var genes and their contribution with transcriptional memory (Chookajorn et al. 2007). In contrast, another model respond that to the strict intron-promoter pairing is not only required for silencing, but as well as for demanding var gene recognition and mutually exclusive expression (Dzikowski Ron 1† et al. 31 August 2007).
Further question raise it self. Why flanking var region has chosen for study?
The answer according to the authors is, the var gene upstream regions are sufficient for controlling mutually exclusive transcription or mono-allelic expression in the absence of the coding region and sufficient for epigenetic silencing (Voss et al. 2006).
In the current issue of Mol Micro, Lopez and colleagues, they found that there was a competition between H3K9 methylation and H3K9 acetylation in the 5' flanking region, these histon marks associate epigenetically to suppression or enhancing var gene expression. The function of these two marks in this study is activating the expression pattern in CSA parasite (var2csa) during the ring stage (fig4), on the other hand the role of histone H3K9 acetylation is acting as antagonist to lysine4 methylation to achieve constantly silent var gene states along 5' flanking and coding regions, this reaction mainly happen in the mature stage with in CSA parasite (fig4).
Figure 4
This figure illustrates Schematic presentation of histone H3 marks linked to silent or active var genes. Histone modifications of the 5′ flanking region and exon 1 of an active (var2csaON or var2csaPOISED) and silent var gene loci (var2csaOFF) are shown. H3K9me3 is dynamically removed in the surrounding area of the transcription start site upon gene activation and is restored upon repression. Histone H3 marks at lysine 4 and 9 linked to active var genes peak in chromatin associated with 5′ flanking var region, supporting further the concept for a key role of this DNA element in the control of mono-allelic expression of var genes. Exon 2 is transcribed via a promoter-like sequence within the var intron sequence element in silent var genes and has been linked to var gene silencing. Chromatin associated with var intron and exon 2 has not been analysed due to the sequence homology of these regions with most members of the var gene family. .the figure and legend are taken from (Lopez-Rubio et al. 2007).
Furthermore, quantitative chromatin immunoperecipitations (qChIp) was used to illustrate the variation of histone methylation marks are enriched at the 5' flanking and coding regions of active and poised var gene. They have identified an increasing of histone H3 lysine4 dimethylation (fig5) and trimethylation (fig6) in the 5' flanking region of var locus active stage.
Figure 5
This figure shows the Dimethylated H3K4 levels at var2csa. A. Distribution of H3K4 dimethylation along the var2csa gene in CSA (white bars) and CD36 ring parasites (black bars). B. Distribution of H3K4 dimethylation along the var2csa gene in CSA (white bars) and CD36 mature parasites (black bars). .the figure and legend are taken from (Lopez-Rubio et al. 2007).
Figure 6
This figure demonstrates Trimethylated H3K4 levels at var2csa .A. Distribution of H3K4 trimethylation along the var2csa gene in CSA (white bars) and CD36 ring parasites (black bars). B. Distribution of H3K4 trimethylation along the var2csa gene in CSA (white bars) and CD36 mature parasites (black bars). .the figure and legend are taken from (Lopez-Rubio et al. 2007).
They also have noticed that H3K9me3 is enrich in the silent var gene coding region (fig4) as well as the enrichment of H3K9ac and H3K9me3 in the active and silence var2csa gene coding region and silent (fig7). The author and colleagues displayed data that H3K4me2 bookmarks of the active locus var gene during later mature stages for expression in the next life cycle (fig4).
Figure 7
This figure highlights Acetylated and methylated H3K9 levels at var2csa. A. Distribution of H3K9ac (white bars) and H3K9me3 (black bars) along the active var2csa gene in FCR3 CSA ring stage parasites. B. Distribution of H3K9ac (white bars) and H3K9me3 (black bars) along the silent var2csa gene in FCR3 CD36 ring stage parasites.the figure and legend are taken from (Lopez-Rubio et al. 2007).
Finally, they found strong reaction with antibodies targeted against H3K4me3 and H4K4me2 as well as strong antibodies reactivity aimed against H3K9ac. Whereas, aweak reactions were observed against H3K4 monomethyl by analysis of protein blot of acid-extracted histone preparation from proliferation cycle of the parasite.
Future impact
The understanding of P.falciparum epigenetic regulation is still vague. Comprehending the mechanisms of var gene expression may help to find better strategies to alert the adhesion properties of infected RBCs or exposure the parasite to the immune system of the host to elevate its clearance (Comeaux & Duraisingh 2007).
Authors have highlighted in this study that in future research will focus on the molecular machinery that able to read the histone marks at specific DNA region of the 5' flanking region and translate this finding into harmonized gene expression pattern of var gene.
Reviewed by:
EMAD AHMED SAIG
SN: 40858944
BIOC6006
SUPERVISOR: Dr/ PAUL EBERT
SEMESTER 1 2008-04-23
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