Transgenerational Epigenetic Inheritance Mediated by a Core Promoter Element

Division Of Basic Sciences - Nci

Investigators

Abstract

Transcriptional control is achieved through interactions of transcription factors with core promoter elements, such as the CCAAT box, which is located about 60 bp upstream of transcription start sites in most genes. The CCAT box serves as a docking site for specific transcription factors like NF-Y and has been highly conserved from E.coli to humans. Although its regulatory role as a promoter element has been well established, structural roles for CCAAT boxes - either as boundary elements or in DNA looping - have not been described. We have shown previously that transgenic mice with a genomic DNA fragment of the major histocompability complex (MHC) class I gene, PD1, that encodes a cell surface glycoprotein which mediates immune surveillance, display normal tissue patterns of expression and cytokine responses. To assess the roles of core promoter elements in the regulation of expression of MHC class I genes, a series of transgenic mice with mutations either in the CCAAT box, the TATA box, Sp1 binding site or Inr were generated. Surprisingly, all of the mutant promoters were capable of supporting expression of the class I transgene (described in Barbash et al, submitted, 2013). Of 32 independently derived transgenic founder mice with CCAAT element mutations, only 6 failed to express the PD1 transgene; the remaining 26 founder mice all expressed the transgene at levels equal to or greater than the wild type. To investigate the basis for the expression of the CCAAT mutant promoter, we selected eight independently derived founders for breeding to establish lines of CCAAT (CCAATm) mutant transgenes; pups were monitored for expression of the PD1 protein by FACS analysis of their peripheral blood lymphocytes (PBL). Of the 8 founders, five of which expressed the transgene, all gave rise to off-spring that also expressed the transgene. Furthermore, all 8 lines also gave rise to off-spring that had the transgene but did not express PD1 on their PBL. In subsequent generations of inter se breeding, CCAATm mice that expressed the PD1 transgene gave rise to offspring that were genotype positive for the transgene but did not express it. Conversely, breeding of transgenic non-expressers mice inter se generated offspring that did express the transgene. This pattern of variegated expression continued in each line for up to 4 generations after which the phenotype was stable and remained stable even after 9 generations. Transgenerational variegation of transgene expression was not observed in any lines of mice with either the wild type promoter or any of the other core promoter mutants. The observed variegated expression among generations was not restricted to PBL: CCAATm transgenic mice that expressed the PD1 protein on their PBL also expressed RNA and protein in their tissues (i.e. spleen, kidney, brain). The CCAATm transgenic mice that did not express PD1 protein on their PBL, also did not express either PD1 protein or RNA in these tissues. However, the pattern of expression among expresser CCAATm transgenes differed from the wild type, with aberrantly high expression in kidney and brain, indicating that the CCAAT box also regulates tissue specific transcription. Expresser CCAATm transgenes, like the wild type promoter transgene and endogenous MHC class I genes, responded to gamma-interferon treatment with enhanced class I expression. In contrast, gamma-interferon treatment did not induce de novo expression of PD1 in the non-expresser CCAATm transgenic mice, although it did enhance endogenous H2-Kb expression. Thus, in the absence of a CCAAT box, global expression of the transgene is variegated and stochastic from generation to generation. The finding of variegated patterns of expression across generations of CCAATm transgenic lines suggested that the wild type CCAAT box normally functions to maintain epigenetic inheritance of expression. Therefore, we next asked whether the CCAAT element contributes to the regulation of chromatin organization or structure by comparing the profiles of CCAATm expresser and non-expresser mice. Whereas Pol II was associated with both wild type PD1 transgenic mice and CCAATm expressers at the promoter and across the gene body, Pol II was only detected upstream of the promoter in non-expressers. Accordingly, H3K4me3 and histone acetylation were observed across the transgene in the WT and CCAATm expresser, but not the non-expresser. Surprisingly, H3K9me3 was not detect across the CCAATm non-expresser transgene. Analysis of transcription factor binding showed decreased CTCF and increased C/EBP in the CCAATm non-expresser, relative to WT and CCAATm expresser; no significant changes in cohesin binding were observed. Interestingly, we observed marked changes in DNA looping amongst the WT, CCAATm expresser and non-expresser. Whereas all three lines have a predominant loop between -425 bp and exon 3, this loop is enhanced in the CCAATm expresser. Additionally, two new loops are formed in the CCAATm expresser that are not present in the other two lines, one of which has an anchor at exon 1, near the CCAAT box. These findings suggest that the CCAAT box regulates loop formation, presumably through the binding of cognate factors. The major CCAAT box binding factor is NF-Y, which regulates expression of both MHC class I and class II genes by nucleating the formation of an MHC enhanceosome. Indeed, whereas the WT transgene shows NF-Y binding to the CCAAT box, it is not found associated with either of the CCAAT mutants. Surprisingly, de novo association of NF-Y at exon 5 is observed in the CCAATm expresser mice. These findings raise the possibility that the aberrant binding of NF-Y at exon 5 in the CCAATm expresser nucleates an alternative enhanceosome to promote expression. Indeed, NF-Y has been shown to govern H3K4me3 levels and to play an important role in establishing open chromatin regions during development. To our knowledge, this is the first example of a single mutation resulting in the establishment of trans-generational epigenetic inheritance. These studies are being prepared for publication.