Question

Abstract: A marked difference exists in the inducibility of the gene inducible NO synthase (iNOS) between humans and rodents.(Inducibility refers to the degree to which the expression of a gene can be changed.) The rodent iNOS gene can be induced to be expressed at high levels by several types of cytokines (a type of protein generated by immune cells), but these same cytokines have little to no effect on the expression of the human iNOS gene. A compelling molecular explanation for why human iNOS is resistant to induction has not been reported. In this study we present evidence that the hyporesponsiveness of the human iNOS promoter is based in part on epigenetic silencing.

Based on the information provided in the abstract, would you expect to find a higher percentage of CpG methylation in the promoter for the human iNOS gene or the mouse iNOS gene? Explain your reasoning.

Answer 1

The human iNOS promoter is one of the largest and most complex promoters known today indicative of a tightly controlled iNOS gene expression.
A TATA box is located 30 bp upstream of the transcription start site and exon 2 contains the ATG initiation codon which lies in a Kozak consensus sequence, but about 6% of the cytokine-inducible iNOS transcripts in human macrophages and epithelial cells start at multiple transcription initiation sites ,some extending several hundred base pairs upstream from the main TATA-directed initiation site . Further diversity in the iNOS mRNAs is gained by alternative splicing. Five distinct alternative splicing regions have been found ,one of which leads to the distinct deletion of exon 5 with a translational frame shift leading to a stop codon in exon 6 yielding a premature iNOS product of 134 amino acids. This deletion is abundant in cerebellum, suggesting a specific tissue-related function. Losses of exon 8 and 9, exon 9–11, or exon 15 and 16 by alternative splicing are in frame deletions. Exon 15–16 deletion leads to iNOS proteins missing the FMN binding site. Alternative splicing of exon 1 together with the different transcription initiation sites leads to variable lengths of the 5′ untranslated region (UTR) in a minor fraction of the iNOS mRNA . Human iNOS mRNA has a long and complex 5′UTR containing eight partially overlapping open reading frames prior to the start codon AUG. For other genes open reading frames in the 5′UTR of a specific RNA have been shown to inhibit its translation in a tissue-specific manner . Moreover, the 3′UTRs in exon 27 of both human and murine iNOS mRNA also bear regulatory functions . Both contain a UUAUUUAU motif that is common to a variety of cytokine and oncogene mRNAs . This motif has been shown to confer RNA instability, resulting in rapid degradation (thereby lowering basal promoter activity in transfection studies ). Comparison of the 3′ ends of the iNOS cDNAs from murine and human cells revealed poor sequence conservation within the 3′UTR except for these AU segments. The 3′UTR of murine iNOS mRNA contains two of these copies, while the human mRNA contains two additional elements. At least six nucleotides of these copies match the consensus motif. Rapid degradation due to the conserved AU-rich octanucleotide sequences results in transient expression of iNOS mRNA with a half life of about 6 h in murine cells. In the RAW 264 macrophage cell line two different 3′ ends have been found , indicating that mRNAs with different stabilities may be produced via alternative splicing. Exclusively in the human iNOS gene, the poly(A) signal, a GT-rich region, is located 10 bp downstream from the poly(A) site in the 3′ flanking region, while the usual poly(A) signal (AATAAA) is missing . Differences in gene expression and mRNA stability due to these two distinct signals are still not yet known.