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In vivo expression of MHC class I genes depends on the presence of a downstream barrier element.

Cohen H, Parekh P, Sercan Z, Kotekar A, Weissman JD, Singer DS - PLoS ONE (2009)

Bottom Line: Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene.Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications.Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Regulation of MHC class I gene expression is critical to achieve proper immune surveillance. In this work, we identify elements downstream of the MHC class I promoter that are necessary for appropriate in vivo regulation: a novel barrier element that protects the MHC class I gene from silencing and elements within the first two introns that contribute to tissue specific transcription. The barrier element is located in intergenic sequences 3' to the polyA addition site. It is necessary for stable expression in vivo, but has no effect in transient transfection assays. Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene. Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications. Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.

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Expression of a PD1 transgene is variegated in the absence of sequences 3′ to the SacI site.(A) PD1 expression is variegated on PBL among independent transgenic founder lines carrying a PD1cDNAint1-2 construct truncated at the SacI site. PBL from the different PD1cDNAint1-2/Sac transgenic lines were stained with either anti-PD1 antibody alone (left panels) or in combination with anti-B220 (B cells) (middle panels) or anti-Thy1 (T cells) (right panels). Shown are representative profiles of each of the groups. Numbers at the right show the number of founder lines having the same pattern of expression. The map of the construct is shown at the bottom. (B) RNA levels among the different tissues of non-expresser (left panels) and expresser (right panels) PD1cDNAint1-2/Sac transgenic lines were assessed by Northern using a probe that spans exons 2–3 and only minimally cross-reacts with endogenous MHC class I (top panels); level of expression was quantitated relative to 18S RNA (middle panels). RNA levels in various tissues of an independent set of mice were also measured in by qPCR and quantitated relative to 18S RNA (bottom panels). Results shown from individual mice are representative of 2–3 individuals tested for each line.
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pone-0006748-g004: Expression of a PD1 transgene is variegated in the absence of sequences 3′ to the SacI site.(A) PD1 expression is variegated on PBL among independent transgenic founder lines carrying a PD1cDNAint1-2 construct truncated at the SacI site. PBL from the different PD1cDNAint1-2/Sac transgenic lines were stained with either anti-PD1 antibody alone (left panels) or in combination with anti-B220 (B cells) (middle panels) or anti-Thy1 (T cells) (right panels). Shown are representative profiles of each of the groups. Numbers at the right show the number of founder lines having the same pattern of expression. The map of the construct is shown at the bottom. (B) RNA levels among the different tissues of non-expresser (left panels) and expresser (right panels) PD1cDNAint1-2/Sac transgenic lines were assessed by Northern using a probe that spans exons 2–3 and only minimally cross-reacts with endogenous MHC class I (top panels); level of expression was quantitated relative to 18S RNA (middle panels). RNA levels in various tissues of an independent set of mice were also measured in by qPCR and quantitated relative to 18S RNA (bottom panels). Results shown from individual mice are representative of 2–3 individuals tested for each line.

Mentions: Similar variegated expression was observed when the PD1 cDNAint1-2/PolyA, which does not retain barrier activity, was extended by 79 bp to a downstream Sac site (PD1 cDNAint1-2/Sac; Figure 3A, 4A). Of 10 founder lines generated from this construct, only two showed levels of cell surface expression on PBL comparable to the PD1cDNAint1-2/Bam mice, with a distribution between T and B cells that paralleled PD1cDNAint1-2/Bam transgenic lines (Figure 4A, compare with Figure 1B). The remaining lines either did not express PD1 (3/10) or had aberrant expression patterns (5/10) on whole PBL, T cells and B cells (Figure 4A).


In vivo expression of MHC class I genes depends on the presence of a downstream barrier element.

Cohen H, Parekh P, Sercan Z, Kotekar A, Weissman JD, Singer DS - PLoS ONE (2009)

Expression of a PD1 transgene is variegated in the absence of sequences 3′ to the SacI site.(A) PD1 expression is variegated on PBL among independent transgenic founder lines carrying a PD1cDNAint1-2 construct truncated at the SacI site. PBL from the different PD1cDNAint1-2/Sac transgenic lines were stained with either anti-PD1 antibody alone (left panels) or in combination with anti-B220 (B cells) (middle panels) or anti-Thy1 (T cells) (right panels). Shown are representative profiles of each of the groups. Numbers at the right show the number of founder lines having the same pattern of expression. The map of the construct is shown at the bottom. (B) RNA levels among the different tissues of non-expresser (left panels) and expresser (right panels) PD1cDNAint1-2/Sac transgenic lines were assessed by Northern using a probe that spans exons 2–3 and only minimally cross-reacts with endogenous MHC class I (top panels); level of expression was quantitated relative to 18S RNA (middle panels). RNA levels in various tissues of an independent set of mice were also measured in by qPCR and quantitated relative to 18S RNA (bottom panels). Results shown from individual mice are representative of 2–3 individuals tested for each line.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2727697&req=5

pone-0006748-g004: Expression of a PD1 transgene is variegated in the absence of sequences 3′ to the SacI site.(A) PD1 expression is variegated on PBL among independent transgenic founder lines carrying a PD1cDNAint1-2 construct truncated at the SacI site. PBL from the different PD1cDNAint1-2/Sac transgenic lines were stained with either anti-PD1 antibody alone (left panels) or in combination with anti-B220 (B cells) (middle panels) or anti-Thy1 (T cells) (right panels). Shown are representative profiles of each of the groups. Numbers at the right show the number of founder lines having the same pattern of expression. The map of the construct is shown at the bottom. (B) RNA levels among the different tissues of non-expresser (left panels) and expresser (right panels) PD1cDNAint1-2/Sac transgenic lines were assessed by Northern using a probe that spans exons 2–3 and only minimally cross-reacts with endogenous MHC class I (top panels); level of expression was quantitated relative to 18S RNA (middle panels). RNA levels in various tissues of an independent set of mice were also measured in by qPCR and quantitated relative to 18S RNA (bottom panels). Results shown from individual mice are representative of 2–3 individuals tested for each line.
Mentions: Similar variegated expression was observed when the PD1 cDNAint1-2/PolyA, which does not retain barrier activity, was extended by 79 bp to a downstream Sac site (PD1 cDNAint1-2/Sac; Figure 3A, 4A). Of 10 founder lines generated from this construct, only two showed levels of cell surface expression on PBL comparable to the PD1cDNAint1-2/Bam mice, with a distribution between T and B cells that paralleled PD1cDNAint1-2/Bam transgenic lines (Figure 4A, compare with Figure 1B). The remaining lines either did not express PD1 (3/10) or had aberrant expression patterns (5/10) on whole PBL, T cells and B cells (Figure 4A).

Bottom Line: Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene.Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications.Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Regulation of MHC class I gene expression is critical to achieve proper immune surveillance. In this work, we identify elements downstream of the MHC class I promoter that are necessary for appropriate in vivo regulation: a novel barrier element that protects the MHC class I gene from silencing and elements within the first two introns that contribute to tissue specific transcription. The barrier element is located in intergenic sequences 3' to the polyA addition site. It is necessary for stable expression in vivo, but has no effect in transient transfection assays. Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene. Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications. Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.

Show MeSH
Related in: MedlinePlus