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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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Sequences downstream of the MHC I promoter are required for proper surface expression on PBL in-vivo.(A) FACS profiles (right panels) of PD1 or CD2 surface expression on PBL of mice carrying the transgenes with the 1 Kb PD1 promoter ligated to the huCD2 reporter (PD1-CD2); full length PD1 (PD1/Bam) transgene or PD1 cDNA with the 3′ Bam HI fragment (PD1cDNA/Bam), cDNA containing introns 1 and 2 with a 3′ Bam HI fragment (PD1cDNAint1-2/Bam). (diagramed on left panels). PBL were stained with either anti-PD1 or anti-CD2 antibody, as described in Materials and Methods. Transgene copy numbers are indicated in parenthesis. Red curves represent staining of negative control C57/BL6 mice with the relevant antibody. Profiles are representative of all of the founders of each of the transgenic lines. (B) MHC class I expression patterns on B and T cells. Surface expression of MHC class I on B and T cells derived from mice transgenic for PD1/Bam and PD1cDNAint1-2/Bam was assessed by dual staining with anti-PD1 antibody and B220 (B cells) or Thy1.2 (T cells). The pattern of endogenous mouse MHC class I, Kb, expression on the PBL from PD1/Bam mice was determined by staining with an anti- Kb antibody. The Kb and PD1 antibodies do not cross-react (data not shown). The results are representative of multiple independent experiments. (C) PD1 RNA expression in PD1cDNAint1-2/Bam mice parallels that of PD1/Bam in different tissues. PD1 RNA levels in tissues of transgenic mice were determined by both Northern analysis using a probe that spans exons 2–3 and that only minimally cross-hybridizes with endogenous class I sequences. Relative levels of expression were quantitated by normalizing with an 18S RNA control probe (graphs below northern). This experiment is representative of three independent experiments. qPCR analyses are shown in Figure S1. (D) PD1 expression in PD1cDNA/Bam transgenic mice is aberrant. RNA from PBL (right panel) or various tissues was probed with a class I probe, by Northern as in (C) and normalized with GAPDH. The relative levels of expression in two individual PD1cDNA/Bam mice are shown in the left panel.
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pone-0006748-g001: Sequences downstream of the MHC I promoter are required for proper surface expression on PBL in-vivo.(A) FACS profiles (right panels) of PD1 or CD2 surface expression on PBL of mice carrying the transgenes with the 1 Kb PD1 promoter ligated to the huCD2 reporter (PD1-CD2); full length PD1 (PD1/Bam) transgene or PD1 cDNA with the 3′ Bam HI fragment (PD1cDNA/Bam), cDNA containing introns 1 and 2 with a 3′ Bam HI fragment (PD1cDNAint1-2/Bam). (diagramed on left panels). PBL were stained with either anti-PD1 or anti-CD2 antibody, as described in Materials and Methods. Transgene copy numbers are indicated in parenthesis. Red curves represent staining of negative control C57/BL6 mice with the relevant antibody. Profiles are representative of all of the founders of each of the transgenic lines. (B) MHC class I expression patterns on B and T cells. Surface expression of MHC class I on B and T cells derived from mice transgenic for PD1/Bam and PD1cDNAint1-2/Bam was assessed by dual staining with anti-PD1 antibody and B220 (B cells) or Thy1.2 (T cells). The pattern of endogenous mouse MHC class I, Kb, expression on the PBL from PD1/Bam mice was determined by staining with an anti- Kb antibody. The Kb and PD1 antibodies do not cross-react (data not shown). The results are representative of multiple independent experiments. (C) PD1 RNA expression in PD1cDNAint1-2/Bam mice parallels that of PD1/Bam in different tissues. PD1 RNA levels in tissues of transgenic mice were determined by both Northern analysis using a probe that spans exons 2–3 and that only minimally cross-hybridizes with endogenous class I sequences. Relative levels of expression were quantitated by normalizing with an 18S RNA control probe (graphs below northern). This experiment is representative of three independent experiments. qPCR analyses are shown in Figure S1. (D) PD1 expression in PD1cDNA/Bam transgenic mice is aberrant. RNA from PBL (right panel) or various tissues was probed with a class I probe, by Northern as in (C) and normalized with GAPDH. The relative levels of expression in two individual PD1cDNA/Bam mice are shown in the left panel.

Mentions: To determine whether the PD1 promoter contains all the regulatory elements necessary for normal in vivo expression, transgenic mice were generated from a construct containing the 1 Kb PD1 promoter segment ligated to a human CD2 reporter [29]. In contrast to the genomic PD1 transgenic lines, none of the nine independent PD1-CD2 lines expressed the CD2 reporter transgene (Figure 1A, top panel; Table 1). Similarly, when the class I promoter was ligated to a GFP reporter, none of 6 independent transgenic lines generated from this construct expressed GFP protein or RNA (J.Lovchick and D. Singer, unpublished observations). Thus, although the extended 1 Kb PD1 promoter is functional and regulated in transient transfection assays [3], [24], it is insufficient to direct stable class I transcription in transgenic mice, suggesting that sequences downstream of the transcription start site are necessary in vivo.


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)

Sequences downstream of the MHC I promoter are required for proper surface expression on PBL in-vivo.(A) FACS profiles (right panels) of PD1 or CD2 surface expression on PBL of mice carrying the transgenes with the 1 Kb PD1 promoter ligated to the huCD2 reporter (PD1-CD2); full length PD1 (PD1/Bam) transgene or PD1 cDNA with the 3′ Bam HI fragment (PD1cDNA/Bam), cDNA containing introns 1 and 2 with a 3′ Bam HI fragment (PD1cDNAint1-2/Bam). (diagramed on left panels). PBL were stained with either anti-PD1 or anti-CD2 antibody, as described in Materials and Methods. Transgene copy numbers are indicated in parenthesis. Red curves represent staining of negative control C57/BL6 mice with the relevant antibody. Profiles are representative of all of the founders of each of the transgenic lines. (B) MHC class I expression patterns on B and T cells. Surface expression of MHC class I on B and T cells derived from mice transgenic for PD1/Bam and PD1cDNAint1-2/Bam was assessed by dual staining with anti-PD1 antibody and B220 (B cells) or Thy1.2 (T cells). The pattern of endogenous mouse MHC class I, Kb, expression on the PBL from PD1/Bam mice was determined by staining with an anti- Kb antibody. The Kb and PD1 antibodies do not cross-react (data not shown). The results are representative of multiple independent experiments. (C) PD1 RNA expression in PD1cDNAint1-2/Bam mice parallels that of PD1/Bam in different tissues. PD1 RNA levels in tissues of transgenic mice were determined by both Northern analysis using a probe that spans exons 2–3 and that only minimally cross-hybridizes with endogenous class I sequences. Relative levels of expression were quantitated by normalizing with an 18S RNA control probe (graphs below northern). This experiment is representative of three independent experiments. qPCR analyses are shown in Figure S1. (D) PD1 expression in PD1cDNA/Bam transgenic mice is aberrant. RNA from PBL (right panel) or various tissues was probed with a class I probe, by Northern as in (C) and normalized with GAPDH. The relative levels of expression in two individual PD1cDNA/Bam mice are shown in the left panel.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2727697&req=5

pone-0006748-g001: Sequences downstream of the MHC I promoter are required for proper surface expression on PBL in-vivo.(A) FACS profiles (right panels) of PD1 or CD2 surface expression on PBL of mice carrying the transgenes with the 1 Kb PD1 promoter ligated to the huCD2 reporter (PD1-CD2); full length PD1 (PD1/Bam) transgene or PD1 cDNA with the 3′ Bam HI fragment (PD1cDNA/Bam), cDNA containing introns 1 and 2 with a 3′ Bam HI fragment (PD1cDNAint1-2/Bam). (diagramed on left panels). PBL were stained with either anti-PD1 or anti-CD2 antibody, as described in Materials and Methods. Transgene copy numbers are indicated in parenthesis. Red curves represent staining of negative control C57/BL6 mice with the relevant antibody. Profiles are representative of all of the founders of each of the transgenic lines. (B) MHC class I expression patterns on B and T cells. Surface expression of MHC class I on B and T cells derived from mice transgenic for PD1/Bam and PD1cDNAint1-2/Bam was assessed by dual staining with anti-PD1 antibody and B220 (B cells) or Thy1.2 (T cells). The pattern of endogenous mouse MHC class I, Kb, expression on the PBL from PD1/Bam mice was determined by staining with an anti- Kb antibody. The Kb and PD1 antibodies do not cross-react (data not shown). The results are representative of multiple independent experiments. (C) PD1 RNA expression in PD1cDNAint1-2/Bam mice parallels that of PD1/Bam in different tissues. PD1 RNA levels in tissues of transgenic mice were determined by both Northern analysis using a probe that spans exons 2–3 and that only minimally cross-hybridizes with endogenous class I sequences. Relative levels of expression were quantitated by normalizing with an 18S RNA control probe (graphs below northern). This experiment is representative of three independent experiments. qPCR analyses are shown in Figure S1. (D) PD1 expression in PD1cDNA/Bam transgenic mice is aberrant. RNA from PBL (right panel) or various tissues was probed with a class I probe, by Northern as in (C) and normalized with GAPDH. The relative levels of expression in two individual PD1cDNA/Bam mice are shown in the left panel.
Mentions: To determine whether the PD1 promoter contains all the regulatory elements necessary for normal in vivo expression, transgenic mice were generated from a construct containing the 1 Kb PD1 promoter segment ligated to a human CD2 reporter [29]. In contrast to the genomic PD1 transgenic lines, none of the nine independent PD1-CD2 lines expressed the CD2 reporter transgene (Figure 1A, top panel; Table 1). Similarly, when the class I promoter was ligated to a GFP reporter, none of 6 independent transgenic lines generated from this construct expressed GFP protein or RNA (J.Lovchick and D. Singer, unpublished observations). Thus, although the extended 1 Kb PD1 promoter is functional and regulated in transient transfection assays [3], [24], it is insufficient to direct stable class I transcription in transgenic mice, suggesting that sequences downstream of the transcription start site are necessary in vivo.

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