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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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Altered chromatin organization is associated with deletions in the 3′ intergenic region, independent of expression.(A)The relative nucleosome density across the PD1 gene was determined as described in Materials and Methods in L cells stably transfected with PD1cDNAint1-2/Bam, PD1cDNAint1-2/Sac and PD1cDNAint1-2/polyA and maintained in the absence of HAT medium. Although all three clones retain the respective transgenes, only the PD1cDNAint1-2/Bam line expresses PD1. The results shown were derived with clones B in Figure 5A. The location of primers used for determining nucleosomal occupancy are illustrated in the map below the graph. The abscissa corresponds to the sites amplified as indicated on the map. (B) Relative levels of histone H3 K9/14 acetylation, and (C) histone H3K4 dimethylation. Materials and Methods, primers and abscissa are the same as in (A). All results are representative of two to three ChIP of independent clones.
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pone-0006748-g007: Altered chromatin organization is associated with deletions in the 3′ intergenic region, independent of expression.(A)The relative nucleosome density across the PD1 gene was determined as described in Materials and Methods in L cells stably transfected with PD1cDNAint1-2/Bam, PD1cDNAint1-2/Sac and PD1cDNAint1-2/polyA and maintained in the absence of HAT medium. Although all three clones retain the respective transgenes, only the PD1cDNAint1-2/Bam line expresses PD1. The results shown were derived with clones B in Figure 5A. The location of primers used for determining nucleosomal occupancy are illustrated in the map below the graph. The abscissa corresponds to the sites amplified as indicated on the map. (B) Relative levels of histone H3 K9/14 acetylation, and (C) histone H3K4 dimethylation. Materials and Methods, primers and abscissa are the same as in (A). All results are representative of two to three ChIP of independent clones.

Mentions: Barrier elements are required to maintain nucleosome organization and histone modifications associated with an open chromatin structure [30]. Thus, the presence of barrier activity in the 3′ intergenic region downstream of the polyA site predicts that both the nucleosome organization and histone modifications of the class I gene will be affected by its absence. To determine whether chromatin changes accompany gene silencing, we assessed the effect of the barrier segment on the relative nucleosome density across the gene in stable L cell transfectant clones. In the selective medium, where expression is maintained for all constructs, all three showed similar pattern of uniformly low nucleosomal density (Figure S3). In the absence of selection, nucleosomal density remained uniformly low across the PD1 gene in PD1cDNAint1-2/Bam (which stably expressed PD1). In sharp contrast, PD1cDNAint1-2/polyA clones that lost expression acquired significantly increased nucleosomal density across the gene (Figure 7A). Thus, nucleosomal density across the class I gene correlates with expression status and with the presence of the full 730 bp intergenic segment containing 3′ barrier function.


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)

Altered chromatin organization is associated with deletions in the 3′ intergenic region, independent of expression.(A)The relative nucleosome density across the PD1 gene was determined as described in Materials and Methods in L cells stably transfected with PD1cDNAint1-2/Bam, PD1cDNAint1-2/Sac and PD1cDNAint1-2/polyA and maintained in the absence of HAT medium. Although all three clones retain the respective transgenes, only the PD1cDNAint1-2/Bam line expresses PD1. The results shown were derived with clones B in Figure 5A. The location of primers used for determining nucleosomal occupancy are illustrated in the map below the graph. The abscissa corresponds to the sites amplified as indicated on the map. (B) Relative levels of histone H3 K9/14 acetylation, and (C) histone H3K4 dimethylation. Materials and Methods, primers and abscissa are the same as in (A). All results are representative of two to three ChIP of independent clones.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2727697&req=5

pone-0006748-g007: Altered chromatin organization is associated with deletions in the 3′ intergenic region, independent of expression.(A)The relative nucleosome density across the PD1 gene was determined as described in Materials and Methods in L cells stably transfected with PD1cDNAint1-2/Bam, PD1cDNAint1-2/Sac and PD1cDNAint1-2/polyA and maintained in the absence of HAT medium. Although all three clones retain the respective transgenes, only the PD1cDNAint1-2/Bam line expresses PD1. The results shown were derived with clones B in Figure 5A. The location of primers used for determining nucleosomal occupancy are illustrated in the map below the graph. The abscissa corresponds to the sites amplified as indicated on the map. (B) Relative levels of histone H3 K9/14 acetylation, and (C) histone H3K4 dimethylation. Materials and Methods, primers and abscissa are the same as in (A). All results are representative of two to three ChIP of independent clones.
Mentions: Barrier elements are required to maintain nucleosome organization and histone modifications associated with an open chromatin structure [30]. Thus, the presence of barrier activity in the 3′ intergenic region downstream of the polyA site predicts that both the nucleosome organization and histone modifications of the class I gene will be affected by its absence. To determine whether chromatin changes accompany gene silencing, we assessed the effect of the barrier segment on the relative nucleosome density across the gene in stable L cell transfectant clones. In the selective medium, where expression is maintained for all constructs, all three showed similar pattern of uniformly low nucleosomal density (Figure S3). In the absence of selection, nucleosomal density remained uniformly low across the PD1 gene in PD1cDNAint1-2/Bam (which stably expressed PD1). In sharp contrast, PD1cDNAint1-2/polyA clones that lost expression acquired significantly increased nucleosomal density across the gene (Figure 7A). Thus, nucleosomal density across the class I gene correlates with expression status and with the presence of the full 730 bp intergenic segment containing 3′ barrier function.

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