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Characterization of a weakly expressed KIR2DL1 variant reveals a novel upstream promoter that controls KIR expression.

Wright PW, Li H, Huehn A, O'Connor GM, Cooley S, Miller JS, Anderson SK - Genes Immun. (2014)

Bottom Line: In order to identify novel genetic alterations associated with decreased KIR expression, a group of donors was characterized for KIR gene content, transcripts and protein expression.Surprisingly, there was no decrease in transcription from the KIR2DL1 proximal promoter.Altogether, these results indicate that distal transcripts are necessary for KIR2DL1 protein expression and are required for proper processing of sense transcripts from the bidirectional proximal promoter.

View Article: PubMed Central - PubMed

Affiliation: Basic Science Program, Leidos Biomedical Research Inc., Lab of Experimental Immunology, Frederick National Lab, Frederick, MD, USA.

ABSTRACT
Members of the human KIR (killer cell immunoglobulin-like receptor) class I major histocompatibility complex receptor gene family contain multiple promoters that determine the variegated expression of KIR on natural killer cells. In order to identify novel genetic alterations associated with decreased KIR expression, a group of donors was characterized for KIR gene content, transcripts and protein expression. An individual with a single copy of the KIR2DL1 gene but a very low level of gene expression was identified. The low expression phenotype was associated with a single-nucleotide polymorphism (SNP) that created a binding site for the inhibitory ZEB1 (Zinc finger E-box-binding homeobox 1) transcription factor adjacent to a c-Myc binding site previously implicated in distal promoter activity. Individuals possessing this SNP had a substantial decrease in distal KIR2DL1 transcripts initiating from a novel intermediate promoter located 230 bp upstream of the proximal promoter start site. Surprisingly, there was no decrease in transcription from the KIR2DL1 proximal promoter. Reduced intermediate promoter activity revealed the existence of alternatively spliced KIR2DL1 transcripts containing premature termination codons that initiated from the proximal KIR2DL1 promoter. Altogether, these results indicate that distal transcripts are necessary for KIR2DL1 protein expression and are required for proper processing of sense transcripts from the bidirectional proximal promoter.

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The KIR2DL1 SNP creates a ZEB-1 binding site. An oligonucleotide probe containing the wild-type (wt) or mutant (mut) distal promoter region was combined with nuclear extract from YT cells. The left panel shows the appearance of a novel band associated with the mutant SNP. The right panel shows the ability of several commercial anti-ZEB antibodies (ZEB1-1 to ZEB1-4, ZEB2), together with anti-C-Myc or MZF-1 controls to inhibit formation of the novel complex.
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Figure 3: The KIR2DL1 SNP creates a ZEB-1 binding site. An oligonucleotide probe containing the wild-type (wt) or mutant (mut) distal promoter region was combined with nuclear extract from YT cells. The left panel shows the appearance of a novel band associated with the mutant SNP. The right panel shows the ability of several commercial anti-ZEB antibodies (ZEB1-1 to ZEB1-4, ZEB2), together with anti-C-Myc or MZF-1 controls to inhibit formation of the novel complex.

Mentions: Examination of the distal SNPs associated with non-expression of KIR2DL1 revealed that the SNP at position -1206 created a binding site for the Zinc finger E-box-binding homeobox 1 (ZEB1) protein, a transcription factor that has been shown to inhibit IL2 gene expression20. ZEB1 requires two E-box motifs (CACCT) for efficient binding to DNA21, and the polymorphism generates a second E-box motif in close proximity to an E-box motif adjacent to the c-Myc/USF binding site (Figure 2) that has been previously implicated in the activation of KIR genes13. EMSA analysis indicated that ZEB1 binds to the E-box pair composed of the novel E-box generated by the polymorphism and the site located next to the c-Myc site (Figure 3). The generation of this novel ZEB1-binding site adjacent to the Myc site shown to enhance distal transcription suggested that the presence of the ZEB1 SNP might decrease activity of the KIR2DL1 distal promoter, reducing the probability of proximal promoter activation. Interestingly, all known KIR2DL5 alleles contain the SNP that generates the ZEB1-binding site, which may account for the low frequency of expression observed for KIR2DL5A*00122. Some KIR2DP1 alleles also contain this SNP, however the complete absence of KIR2DP1 transcripts precludes any observable effect of the ZEB1 SNP on the transcription of this gene.


Characterization of a weakly expressed KIR2DL1 variant reveals a novel upstream promoter that controls KIR expression.

Wright PW, Li H, Huehn A, O'Connor GM, Cooley S, Miller JS, Anderson SK - Genes Immun. (2014)

The KIR2DL1 SNP creates a ZEB-1 binding site. An oligonucleotide probe containing the wild-type (wt) or mutant (mut) distal promoter region was combined with nuclear extract from YT cells. The left panel shows the appearance of a novel band associated with the mutant SNP. The right panel shows the ability of several commercial anti-ZEB antibodies (ZEB1-1 to ZEB1-4, ZEB2), together with anti-C-Myc or MZF-1 controls to inhibit formation of the novel complex.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4208966&req=5

Figure 3: The KIR2DL1 SNP creates a ZEB-1 binding site. An oligonucleotide probe containing the wild-type (wt) or mutant (mut) distal promoter region was combined with nuclear extract from YT cells. The left panel shows the appearance of a novel band associated with the mutant SNP. The right panel shows the ability of several commercial anti-ZEB antibodies (ZEB1-1 to ZEB1-4, ZEB2), together with anti-C-Myc or MZF-1 controls to inhibit formation of the novel complex.
Mentions: Examination of the distal SNPs associated with non-expression of KIR2DL1 revealed that the SNP at position -1206 created a binding site for the Zinc finger E-box-binding homeobox 1 (ZEB1) protein, a transcription factor that has been shown to inhibit IL2 gene expression20. ZEB1 requires two E-box motifs (CACCT) for efficient binding to DNA21, and the polymorphism generates a second E-box motif in close proximity to an E-box motif adjacent to the c-Myc/USF binding site (Figure 2) that has been previously implicated in the activation of KIR genes13. EMSA analysis indicated that ZEB1 binds to the E-box pair composed of the novel E-box generated by the polymorphism and the site located next to the c-Myc site (Figure 3). The generation of this novel ZEB1-binding site adjacent to the Myc site shown to enhance distal transcription suggested that the presence of the ZEB1 SNP might decrease activity of the KIR2DL1 distal promoter, reducing the probability of proximal promoter activation. Interestingly, all known KIR2DL5 alleles contain the SNP that generates the ZEB1-binding site, which may account for the low frequency of expression observed for KIR2DL5A*00122. Some KIR2DP1 alleles also contain this SNP, however the complete absence of KIR2DP1 transcripts precludes any observable effect of the ZEB1 SNP on the transcription of this gene.

Bottom Line: In order to identify novel genetic alterations associated with decreased KIR expression, a group of donors was characterized for KIR gene content, transcripts and protein expression.Surprisingly, there was no decrease in transcription from the KIR2DL1 proximal promoter.Altogether, these results indicate that distal transcripts are necessary for KIR2DL1 protein expression and are required for proper processing of sense transcripts from the bidirectional proximal promoter.

View Article: PubMed Central - PubMed

Affiliation: Basic Science Program, Leidos Biomedical Research Inc., Lab of Experimental Immunology, Frederick National Lab, Frederick, MD, USA.

ABSTRACT
Members of the human KIR (killer cell immunoglobulin-like receptor) class I major histocompatibility complex receptor gene family contain multiple promoters that determine the variegated expression of KIR on natural killer cells. In order to identify novel genetic alterations associated with decreased KIR expression, a group of donors was characterized for KIR gene content, transcripts and protein expression. An individual with a single copy of the KIR2DL1 gene but a very low level of gene expression was identified. The low expression phenotype was associated with a single-nucleotide polymorphism (SNP) that created a binding site for the inhibitory ZEB1 (Zinc finger E-box-binding homeobox 1) transcription factor adjacent to a c-Myc binding site previously implicated in distal promoter activity. Individuals possessing this SNP had a substantial decrease in distal KIR2DL1 transcripts initiating from a novel intermediate promoter located 230 bp upstream of the proximal promoter start site. Surprisingly, there was no decrease in transcription from the KIR2DL1 proximal promoter. Reduced intermediate promoter activity revealed the existence of alternatively spliced KIR2DL1 transcripts containing premature termination codons that initiated from the proximal KIR2DL1 promoter. Altogether, these results indicate that distal transcripts are necessary for KIR2DL1 protein expression and are required for proper processing of sense transcripts from the bidirectional proximal promoter.

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