Transcriptional Regulation of Mammalian Genes in Vivo

Abstract

The past two decades have brought a major evolution in our understanding of promoter structure, transcription factors, and mechanisms by which transcriptional initiation is regulated in eukaryotes. Multiple approaches have been used to establish current models for transcriptional regulation, including the development of in vitro transcription systems using either naked DNA or reconstituted chromatin, genetic analysis of gene regulation in yeast and Drosophila, and in vivo analysis of either endogenous cellular genes or transiently transfected, exogenous gene promoters. It is now clear that chromatin structure, once considered to be transparent to the process of transcription, plays an important role in the regulation of gene expression (reviewed in Ref. 1). Since all cellular genes are packaged into ordered chromatin structures, an understanding of the mechanisms by which nucleoprotein structure influences transcription activation is necessary for a complete paradigm of gene regulation in higher eukaryotes.Studies on regulation of endogenous mammalian genes are challenging due to the lack of genetic techniques available in yeast and Drosophila systems, which allow targeted insertion of promoters and gene disruption. Structural analysis of integrated gene promoters in mammalian cells requires the time-consuming generation of multiple stable cell lines or pools, in which the integrated genes would be subject to position effects from surrounding chromatin. Therefore, the identification and characterization of factors involved in mammalian gene expression have been addressed primarily through the use of transient transfection assays. In this approach, exogenous plasmid DNA, usually promoter/reporter constructs and transcription factor expression vectors, is introduced into cultured cells and expressed transiently, without replication or integration into the cellular genome. A variety of transfection methods has been utilized, including calcium phosphate precipitation (2), DEAE-dextran (3), electroporation (4, 5), and liposomemediated transfer (6). These studies have resulted in an abundance of information about promoter structure (i.e. identification of cisacting elements), transcription factor structure and function, and the characterization of transcription factor interactions that are part of various cellular regulatory pathways. However, in light of the accumulating evidence indicating a role for chromatin structure in transcription, it is appropriate to question whether transiently transfected gene promoters are adequate models for transcriptional regulatory mechanisms active on endogenous genes in ordered, replicated chromatin. Structural Studies on Transiently Transfected DNAThe first issue to consider for transient templates is whether these molecules acquire physiologically spaced nucleosomes when introduced into cultured, mammalian cells. Since assembly of nucleosomes on endogenous genes is coupled to DNA replication, it is questionable whether transfected plasmids, which rarely carry mammalian replication...

Specific Binding of High-Mobility-Group I (HMGI) Protein and Histone H1 to the Upstream AT-Rich Region of the Murine Beta Interferon Promoter: HMGI Protein Acts as a Potential Antirepressor of the Promoter

“…4C and D). Chromatin is only partially reconstituted on transiently transfected plasmids, whereas it is fully reconstituted on DNA templates when they are integrated into the genome (34). The difference that we observed between transiently and stably transfected IFN-␤ promoters is therefore probably a consequence of chromatin which has been fully reconstituted only on stably transfected muIFN-␤ promoters.…”

“…Promoter derepression often requires chromatin remodelling (23). Chromatin is correctly reconstituted on stably integrated DNA templates, while it is incompletely organized on transiently transfected DNAs (21,34). We therefore analyzed the kinetics of virus-induced transcriptional activation of the wild-type and mutated muIFN-␤ promoters after stable integration into chromatin and compared them with the kinetics obtained with transiently transfected promoters.…”

p63α and ΔNp63α can induce cell cycle arrest and apoptosis and differentially regulate p53 target genes

“…This is not surprising and is consistent with our observation that p63a and DNp63a can induce some but not all endogenous p53 target genes (see Figure 6). In addition, it is well established that the activation of transfected promoter constructs does not always re¯ect the activation of endogenous genes where chromatin remodeling occurs (Smith and Hager, 1997). The chromatin structure of these endogenous p53 target genes may a ect p63-mediated transcription.…”

A critical assessment of the factors affecting reporter gene assays for promoter SNP function: a reassessment of −308 TNF polymorphism function using a novel integrated reporter system

“…In addition to the concentration effects outlined above, aberrant activity may also be caused by the lack of an appropriate chromatin conformation. 12 In many cases, long-range enhancer and silencer elements are not functional unless the reporter gene is integrated into the genome by either stable transfection or transgenic means. As the chromatin context in which a particular transcriptional element functions is likely to affect the interaction of the transcription factors with that element, the transient reporter gene assay, although a useful indicator of important regulatory regions, is not always an appropriate assay to assess the influence of small differences due to SNP variation, on the transcriptional activity of a particular gene.…”