Exploring the Functional Relevance of Polymorphisms within the CD14 and IRF-1 Gene for Promoter Activity by Haplotype-Specific Chromatin Immunoprecipitation (HaploChIP)
by Jasmin Mertens
Date of Examination:2011-01-19
Date of issue:2011-02-16
Advisor:Prof. Dr. Sabine Mihm
Referee:Prof. Dr. Gerhard Hunsmann
Referee:PD Dr. Michael Hoppert
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Abstract
English
Genetic background may influence susceptibility and progression of various diseases. The IRF-1 promoter polymorphism rs2549009 (G/A) and the variant rs2569190 (C/T) within the CD14 gene have been described as being associated with various disease conditions. In vitro experiments (EMSA; reporter gene assays) showed that the T allele of CD14 rs2569190 favours transcription factor binding and that DNA elements containing the respective variant allele promote gene transcription. The IRF-1 rs2549009-carrying DNA element induces allele-specific constitutive expression levels of a luciferase reporter in various cell lines. To determine whether these single nucleotide polymorphisms (SNP) confer any functional effect on transcriptional activity in their natural genomic environment, analyses of allele-specific promoter activities were conducted by applying haplotype-specific chromatin immunoprecipitation (HaploChIP) on ex vivo-derived peripheral blood mononuclear cells (PBMC). Therefore, antibodies were used that are directed against serine 5 and serine 2 phosphorylated (ser5-P/ser2-P) isoforms of RNA polymerase II (RNAPII) that reflect binding and enzymatic activity at initiation and elongation stages of the transcription cycle, respectively. Ser5-P RNAPII was found to be preferentially recruited to the variant alleles of CD14 rs2569190 as well as of IRF-1 rs2549009, results that are in line with those from the above-mentioned in vitro experiments. The active (ser2-P) RNAPII, on the contrary, was found to not be preferentially bound to the wild-type or variant allele of CD14 rs2569190. Sequencing of the CD14 gene failed to reveal any so-far unknown SNP that could influence CD14 promoter activity. The genetic variant CD14 rs2569190 thus might be advantageous for transcription initiation. Endotoxin sensitivity associated with this polymorphism, however, does not appear to rely on allele-specific CD14 gene transcription at least in PBMC. Regarding IRF-1 rs2549009, a preferential binding of the ser2-P RNAPII to the G or A promoter variants or binding without any preference - depending on the donor - was observed in PBMC from healthy Caucasians. This finding actually accords with the varying results from different reporter gene assays and may explain the variance. Furthermore, the relative binding frequency of the active RNAPII to IRF-1 rs2549009 promoter variants was found to be closely related to the relative amount of allele-specific IRF-1 transcripts. These results provide evidence for a bidirectional IRF-1 gene expression that does not appear to be solely controlled by IRF-1 rs2549009 in cis, but may rely on a yet undetected polymorphism or haplotype or on environmental control in trans.
Keywords: genetic polymorphism; single nucleotide polymorphism (SNP); haplotype-specific chromatin immunoprecipitation (HaploChIP); promoter polymorphism
Schlagwörter: genetischer Polymorphismus; SNP; HaploChIP; Promotor-Polymorphismus