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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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3′ intergenic sequences are required for stable in-vivo expression of the MHC I gene, PD1.(A) FACS profiles of CD2 surface expression on PBL of transgenic mice. Transgenic mice were generated with constructs containing the human CD2 reporter driven by the PD1 promoter (PD1/CD2) into which a segment 3′ to PD1 coding sequences was inserted in either the forward (PD1-CD2/Bam(+)) or reverse orientation (PD1-CD2/Bam(−)). The numbers in parentheses indicate the range of transgene copy numbers in different lines. (B) FACS profiles of huCD2 expression on B (B220) and T (Thy1.2) cells of PD1CD2/Bam(+) transgenic mice (pattern 1 from (A)). H-2Kb expression pattern in the same mice is shown as control. (C) M12 cells were transiently transfected with PD1/CD2 or PD1-CD2/Bam(+). Surface expression was determined by FACS analysis with anti-huCD2 antibody (left panels); relative surface expression (right panels) is the average level of expression and standard deviation among 3 independent transfections.
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pone-0006748-g002: 3′ intergenic sequences are required for stable in-vivo expression of the MHC I gene, PD1.(A) FACS profiles of CD2 surface expression on PBL of transgenic mice. Transgenic mice were generated with constructs containing the human CD2 reporter driven by the PD1 promoter (PD1/CD2) into which a segment 3′ to PD1 coding sequences was inserted in either the forward (PD1-CD2/Bam(+)) or reverse orientation (PD1-CD2/Bam(−)). The numbers in parentheses indicate the range of transgene copy numbers in different lines. (B) FACS profiles of huCD2 expression on B (B220) and T (Thy1.2) cells of PD1CD2/Bam(+) transgenic mice (pattern 1 from (A)). H-2Kb expression pattern in the same mice is shown as control. (C) M12 cells were transiently transfected with PD1/CD2 or PD1-CD2/Bam(+). Surface expression was determined by FACS analysis with anti-huCD2 antibody (left panels); relative surface expression (right panels) is the average level of expression and standard deviation among 3 independent transfections.

Mentions: The finding that in vivo expression of the PD1 gene does not depend on introns 1–6 suggested that the required downstream sequences are located within the 3′ intergenic region, which is present in all of the constructs that are expressed in vivo. To test this possibility, we next asked whether the 3′ terminus of the PD1/Bam clone could restore expression to the silent PD1-CD2 transgene (Figure 1A). To this end, a 1.3 Kb BamHI fragment encompassing the poly A addition site and 730 bp of 3′ intergenic sequences was inserted, in both orientations, downstream of the CD2 coding sequence in the PD1-CD2 construct (PD1-CD2/Bam(+) or PD1-CD2/Bam(−); Figure 2A, upper and lower panels, respectively). In contrast to the parental PD1-CD2 construct, all of the transgenic lines containing the PD1-CD2/Bam(+) construct (5/5) and a majority (7/8) of those with the PD1-CD2/Bam(−) expressed CD2, both on their PBL and in their tissues. (Figure 2A; Table 1). Those with the 3′ Bam segment in the forward orientation generally expressed cell surface CD2 protein at higher levels on PBL, despite their lower copy number (Figure 2A). Thus sequences downstream of the PD1 coding region contain regulatory elements necessary for expression.


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)

3′ intergenic sequences are required for stable in-vivo expression of the MHC I gene, PD1.(A) FACS profiles of CD2 surface expression on PBL of transgenic mice. Transgenic mice were generated with constructs containing the human CD2 reporter driven by the PD1 promoter (PD1/CD2) into which a segment 3′ to PD1 coding sequences was inserted in either the forward (PD1-CD2/Bam(+)) or reverse orientation (PD1-CD2/Bam(−)). The numbers in parentheses indicate the range of transgene copy numbers in different lines. (B) FACS profiles of huCD2 expression on B (B220) and T (Thy1.2) cells of PD1CD2/Bam(+) transgenic mice (pattern 1 from (A)). H-2Kb expression pattern in the same mice is shown as control. (C) M12 cells were transiently transfected with PD1/CD2 or PD1-CD2/Bam(+). Surface expression was determined by FACS analysis with anti-huCD2 antibody (left panels); relative surface expression (right panels) is the average level of expression and standard deviation among 3 independent transfections.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2727697&req=5

pone-0006748-g002: 3′ intergenic sequences are required for stable in-vivo expression of the MHC I gene, PD1.(A) FACS profiles of CD2 surface expression on PBL of transgenic mice. Transgenic mice were generated with constructs containing the human CD2 reporter driven by the PD1 promoter (PD1/CD2) into which a segment 3′ to PD1 coding sequences was inserted in either the forward (PD1-CD2/Bam(+)) or reverse orientation (PD1-CD2/Bam(−)). The numbers in parentheses indicate the range of transgene copy numbers in different lines. (B) FACS profiles of huCD2 expression on B (B220) and T (Thy1.2) cells of PD1CD2/Bam(+) transgenic mice (pattern 1 from (A)). H-2Kb expression pattern in the same mice is shown as control. (C) M12 cells were transiently transfected with PD1/CD2 or PD1-CD2/Bam(+). Surface expression was determined by FACS analysis with anti-huCD2 antibody (left panels); relative surface expression (right panels) is the average level of expression and standard deviation among 3 independent transfections.
Mentions: The finding that in vivo expression of the PD1 gene does not depend on introns 1–6 suggested that the required downstream sequences are located within the 3′ intergenic region, which is present in all of the constructs that are expressed in vivo. To test this possibility, we next asked whether the 3′ terminus of the PD1/Bam clone could restore expression to the silent PD1-CD2 transgene (Figure 1A). To this end, a 1.3 Kb BamHI fragment encompassing the poly A addition site and 730 bp of 3′ intergenic sequences was inserted, in both orientations, downstream of the CD2 coding sequence in the PD1-CD2 construct (PD1-CD2/Bam(+) or PD1-CD2/Bam(−); Figure 2A, upper and lower panels, respectively). In contrast to the parental PD1-CD2 construct, all of the transgenic lines containing the PD1-CD2/Bam(+) construct (5/5) and a majority (7/8) of those with the PD1-CD2/Bam(−) expressed CD2, both on their PBL and in their tissues. (Figure 2A; Table 1). Those with the 3′ Bam segment in the forward orientation generally expressed cell surface CD2 protein at higher levels on PBL, despite their lower copy number (Figure 2A). Thus sequences downstream of the PD1 coding region contain regulatory elements necessary for expression.

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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