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Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum.

Voss TS, Tonkin CJ, Marty AJ, Thompson JK, Healer J, Crabb BS, Cowman AF - Mol. Microbiol. (2007)

Bottom Line: However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation.Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression.Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Australia.

ABSTRACT
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, undergoes antigenic variation and plays an important role in chronic infection and severe malaria. Only a single var gene is transcribed per parasite, and epigenetic control mechanisms are fundamental in this strategy of mutually exclusive transcription. We show that subtelomeric upsB var gene promoters carried on episomes are silenced by default, and that promoter activation is sufficient to silence all other family members. However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation. Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression. Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.

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Low-resolution chromatin analysis of episomal upsB promoters. Native chromatin was digested with MNase followed by indirect end-labelling. Southern blots were hybridized with a 257 bp hdhfr probe to detect MNase-sensitive sites in the silenced and activated upsB promoters on pHBupsBR. A map of the 4172 bp XbaI/SphI fragment of pHBupsBR containing the upsB promoter is shown on the right. MNase-sensitive sites are highlighted with respect to the hdhfr start codon. The positions of the cis-acting elements SPE1 and SPE2 and the rep20 repeat region are indicated. The arrow depicts the MNase-resistant region (−1760 to −2320) containing five direct SPE2 repeats (−2093 to −2231) (Voss et al., 2003). Asterisks identify sites that are also preferentially cut in naked plasmid DNA (Fig. S3). Circles represent a proposed nucleosomal organization.
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fig05: Low-resolution chromatin analysis of episomal upsB promoters. Native chromatin was digested with MNase followed by indirect end-labelling. Southern blots were hybridized with a 257 bp hdhfr probe to detect MNase-sensitive sites in the silenced and activated upsB promoters on pHBupsBR. A map of the 4172 bp XbaI/SphI fragment of pHBupsBR containing the upsB promoter is shown on the right. MNase-sensitive sites are highlighted with respect to the hdhfr start codon. The positions of the cis-acting elements SPE1 and SPE2 and the rep20 repeat region are indicated. The arrow depicts the MNase-resistant region (−1760 to −2320) containing five direct SPE2 repeats (−2093 to −2231) (Voss et al., 2003). Asterisks identify sites that are also preferentially cut in naked plasmid DNA (Fig. S3). Circles represent a proposed nucleosomal organization.

Mentions: We recently reported differences in local chromatin structure between the silenced and active states of a subtelomeric transgene in P. falciparum (Duraisingh et al., 2005). Here we have investigated whether such alterations are also important in the regulation of var gene promoters. Indirect end-labelling of native chromatin in 3D7/upsBR parasites revealed a pattern of MNase-sensitive sites, which might be indicative for a number of positioned nucleosomes (Fig. 5). A number of MNase-sensitive sites were also preferentially cut in naked plasmid DNA (Fig. S3), and due to the low-resolution mapping strategy employed here, it remains unknown whether these sites are truly positioned between adjacent nucleosomes. Two highly accessible sites are located within the region containing the transcriptional start site (−515 to −804) (Voss et al., 2003), followed by a protected area within the 5′ untranslated region. A 200 bp region in the promoter (−940 to −1140) contains three consecutive MNase-sensitive sites, positioned directly downstream of the SPE1 regulatory element (−1127 to −1171) (Voss et al., 2003). A 500 bp region upstream (−1760 to −2320) was highly resistant to digestion even at high enzyme concentrations. This region contains the regulatory SPE2-repeat array (−2093 to −2231) (Voss et al., 2003), suggesting that the SPE2 binding activity may play a key role in chromatin organization and epigenetic regulation. However, we neither observed significant differences between the silenced and active states of upsB (Fig. 5) or upsC promoters (data not shown), nor did we detect any effects conferred by rep20 repeats or the var intron on the local chromatin structure of var gene promoters (data not shown).


Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum.

Voss TS, Tonkin CJ, Marty AJ, Thompson JK, Healer J, Crabb BS, Cowman AF - Mol. Microbiol. (2007)

Low-resolution chromatin analysis of episomal upsB promoters. Native chromatin was digested with MNase followed by indirect end-labelling. Southern blots were hybridized with a 257 bp hdhfr probe to detect MNase-sensitive sites in the silenced and activated upsB promoters on pHBupsBR. A map of the 4172 bp XbaI/SphI fragment of pHBupsBR containing the upsB promoter is shown on the right. MNase-sensitive sites are highlighted with respect to the hdhfr start codon. The positions of the cis-acting elements SPE1 and SPE2 and the rep20 repeat region are indicated. The arrow depicts the MNase-resistant region (−1760 to −2320) containing five direct SPE2 repeats (−2093 to −2231) (Voss et al., 2003). Asterisks identify sites that are also preferentially cut in naked plasmid DNA (Fig. S3). Circles represent a proposed nucleosomal organization.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169929&req=5

fig05: Low-resolution chromatin analysis of episomal upsB promoters. Native chromatin was digested with MNase followed by indirect end-labelling. Southern blots were hybridized with a 257 bp hdhfr probe to detect MNase-sensitive sites in the silenced and activated upsB promoters on pHBupsBR. A map of the 4172 bp XbaI/SphI fragment of pHBupsBR containing the upsB promoter is shown on the right. MNase-sensitive sites are highlighted with respect to the hdhfr start codon. The positions of the cis-acting elements SPE1 and SPE2 and the rep20 repeat region are indicated. The arrow depicts the MNase-resistant region (−1760 to −2320) containing five direct SPE2 repeats (−2093 to −2231) (Voss et al., 2003). Asterisks identify sites that are also preferentially cut in naked plasmid DNA (Fig. S3). Circles represent a proposed nucleosomal organization.
Mentions: We recently reported differences in local chromatin structure between the silenced and active states of a subtelomeric transgene in P. falciparum (Duraisingh et al., 2005). Here we have investigated whether such alterations are also important in the regulation of var gene promoters. Indirect end-labelling of native chromatin in 3D7/upsBR parasites revealed a pattern of MNase-sensitive sites, which might be indicative for a number of positioned nucleosomes (Fig. 5). A number of MNase-sensitive sites were also preferentially cut in naked plasmid DNA (Fig. S3), and due to the low-resolution mapping strategy employed here, it remains unknown whether these sites are truly positioned between adjacent nucleosomes. Two highly accessible sites are located within the region containing the transcriptional start site (−515 to −804) (Voss et al., 2003), followed by a protected area within the 5′ untranslated region. A 200 bp region in the promoter (−940 to −1140) contains three consecutive MNase-sensitive sites, positioned directly downstream of the SPE1 regulatory element (−1127 to −1171) (Voss et al., 2003). A 500 bp region upstream (−1760 to −2320) was highly resistant to digestion even at high enzyme concentrations. This region contains the regulatory SPE2-repeat array (−2093 to −2231) (Voss et al., 2003), suggesting that the SPE2 binding activity may play a key role in chromatin organization and epigenetic regulation. However, we neither observed significant differences between the silenced and active states of upsB (Fig. 5) or upsC promoters (data not shown), nor did we detect any effects conferred by rep20 repeats or the var intron on the local chromatin structure of var gene promoters (data not shown).

Bottom Line: However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation.Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression.Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Australia.

ABSTRACT
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, undergoes antigenic variation and plays an important role in chronic infection and severe malaria. Only a single var gene is transcribed per parasite, and epigenetic control mechanisms are fundamental in this strategy of mutually exclusive transcription. We show that subtelomeric upsB var gene promoters carried on episomes are silenced by default, and that promoter activation is sufficient to silence all other family members. However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation. Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression. Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.

Show MeSH
Related in: MedlinePlus