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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 polyA site.(A) The positions of the polyA, SacI and BamHI sites in the PD1cDNAint1-2/Bam construct are diagrammed and constructs generated from truncation at these sites indicated. Numbers indicate the position relative to Hind III site at +1. (B)PD1 expression is variegated on PBL among transgenic lines carrying a PD1cDNAint1-2 construct truncated at the polyA site. PBL from the different PD1cDNAint1-2/polyA 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 PD1 profiles of each of the groups. The map of the construct is shown at the bottom. (C) RNA levels in different tissues of non-expresser and expresser PD1cDNAint1-2/polyA transgenic lines were assessed by qPCR. Results shown from individual mice are representative of 2–3 individuals tested for each line. (The small amounts of RNA detected in the thymus are not reproducibly observed.)
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pone-0006748-g003: Expression of a PD1 transgene is variegated in the absence of sequences 3′ to the polyA site.(A) The positions of the polyA, SacI and BamHI sites in the PD1cDNAint1-2/Bam construct are diagrammed and constructs generated from truncation at these sites indicated. Numbers indicate the position relative to Hind III site at +1. (B)PD1 expression is variegated on PBL among transgenic lines carrying a PD1cDNAint1-2 construct truncated at the polyA site. PBL from the different PD1cDNAint1-2/polyA 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 PD1 profiles of each of the groups. The map of the construct is shown at the bottom. (C) RNA levels in different tissues of non-expresser and expresser PD1cDNAint1-2/polyA transgenic lines were assessed by qPCR. Results shown from individual mice are representative of 2–3 individuals tested for each line. (The small amounts of RNA detected in the thymus are not reproducibly observed.)

Mentions: The minimal 3′ sequences necessary for barrier activity were determined by 3′ truncation of PD1 cDNAint1-2/Bam intergenic sequence to the poly A addition site (PD1 cDNAint1-2/PolyA) (Figure 3A). Most of the transgenic founders generated with this construct (14/21) either failed to express in any tissue or displayed a variegated pattern of expression on PBL (Figure 3B, C, left panels). Only 7 of the founders displayed low level PD1 expression on the surface of PBL and a normal pattern of RNA expression in B and T cells and in the tissues (Figure 3B right panels, Figure 3C, Table 2).


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 polyA site.(A) The positions of the polyA, SacI and BamHI sites in the PD1cDNAint1-2/Bam construct are diagrammed and constructs generated from truncation at these sites indicated. Numbers indicate the position relative to Hind III site at +1. (B)PD1 expression is variegated on PBL among transgenic lines carrying a PD1cDNAint1-2 construct truncated at the polyA site. PBL from the different PD1cDNAint1-2/polyA 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 PD1 profiles of each of the groups. The map of the construct is shown at the bottom. (C) RNA levels in different tissues of non-expresser and expresser PD1cDNAint1-2/polyA transgenic lines were assessed by qPCR. Results shown from individual mice are representative of 2–3 individuals tested for each line. (The small amounts of RNA detected in the thymus are not reproducibly observed.)
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006748-g003: Expression of a PD1 transgene is variegated in the absence of sequences 3′ to the polyA site.(A) The positions of the polyA, SacI and BamHI sites in the PD1cDNAint1-2/Bam construct are diagrammed and constructs generated from truncation at these sites indicated. Numbers indicate the position relative to Hind III site at +1. (B)PD1 expression is variegated on PBL among transgenic lines carrying a PD1cDNAint1-2 construct truncated at the polyA site. PBL from the different PD1cDNAint1-2/polyA 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 PD1 profiles of each of the groups. The map of the construct is shown at the bottom. (C) RNA levels in different tissues of non-expresser and expresser PD1cDNAint1-2/polyA transgenic lines were assessed by qPCR. Results shown from individual mice are representative of 2–3 individuals tested for each line. (The small amounts of RNA detected in the thymus are not reproducibly observed.)
Mentions: The minimal 3′ sequences necessary for barrier activity were determined by 3′ truncation of PD1 cDNAint1-2/Bam intergenic sequence to the poly A addition site (PD1 cDNAint1-2/PolyA) (Figure 3A). Most of the transgenic founders generated with this construct (14/21) either failed to express in any tissue or displayed a variegated pattern of expression on PBL (Figure 3B, C, left panels). Only 7 of the founders displayed low level PD1 expression on the surface of PBL and a normal pattern of RNA expression in B and T cells and in the tissues (Figure 3B right panels, Figure 3C, Table 2).

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