Transcriptional Regulation

The fact that the chromosomal location of recombinant genes within the host cell's genome can affect the expression of recombinant genes and lead to a phenomenon called the 'position effect', has been known for several years (Wilson et al. 1990). The 'position effect' or 'position effect variegation' occurs when a recombinant gene is inserted close to an endogenous promoter or enhancer, or alternatively into a region of inactive or silenced genomic DNA (i.e. heterochromatin region). Heterochromatin regions of DNA arise due to the formation of dense packaging of DNA and associated proteins, and the genes present within these highly condensed regions are usually transcriptionally inactive (Babu & Verma 1987; Wallrath 1998). Thus, the expression of any recombinant genes inserted into or close to such a region can be adversely affected (Henikoff 1990; Wilson et al. 1990; Wallrath 1998), and this phenomenon has had a significant influence on the design of vectors for stable expression of recombinant proteins.

Much research has been performed in relation to vector construct design, and the basic components (such as promoter elements, introns, polyadenylation sites, bacterial origins of replication) of a successful production vector for expression in mammalian cells are now well defined (see Chapter 5; Makrides 1999). However, in an attempt to increase production levels, and often as a direct result of the need to combat the problem of instability of production, various additional vector elements have been considered. Many of these elements are used to overcome the problem of the 'position effect' during the insertion of recombinant genes into a host cell's genome.

MOLECULAR MECHANISMS ASSOCIATED WITH INSTABILITY

MOLECULAR MECHANISMS ASSOCIATED WITH INSTABILITY

Figure 8.2 Potential sites for regulation of recombinant gene expression in mammalian cells. (Adapted from Barnes et al. (2003) with permission of John Wiley & Sons)

Insulator elements are naturally occurring DNA sequences that allow the region between them to act as an independent functional domain not affected by the surrounding genetic environment (Geyer 1997). These elements are often used to flank recombinant genes in order to suppress genomic position effects (Pikaart et al. 1998; Kwaks et al. 2003). However, to complicate the situation 'anti-insulator' elements have now been found that are thought to allow insulators to be selectively bypassed (Zhou & Levine 1999). Locus control regions (LCRs) are another type of DNA element that can buffer against the position effect (Needham et al. 1992; Li et al. 1999). LCRs are thought to aid in the opening of chromatin regions and are used to confer position-independent and copy number-dependent expression on recombinant genes (Li et al. 1999). In addition, research is also being performed to identify the chromatin elements that are associated with ubiquitously expressed 'housekeeping' genes. It is thought that the inclusion of these elements, termed UCOEs (ubiquitous/universal chromatin-opening elements), in expression vectors will permit integration-independent expression of recombinant proteins. It is hoped that such elements may alleviate the problem of chromatin-mediated silencing of recombinant genes (Benton et al. 2002; Antoniou et al. 2003).

Many studies have now been performed on the effect of chromosomal location on recombinant gene expression, and some of the most relevant studies from an industrial viewpoint have centred on CHO cells, particularly amplified CHO cells (Kim & Lee 1999). As mentioned before, the DHFR-CHO system affords the ability to amplify recombinant genes by inclusion of MTX in the culture medium. In many cases this can lead to a significant increase in product yield. However, this increase in yield is often counterbalanced by an increase in the occurrence of instability, particularly if the selective pressure of MTX is removed (Pallavicini et al. 1990; Kim et al. 1998a; Fann et al. 2000). It has been found that, without the continued selective pressure of MTX, amplified genes localized on extrachromosomal genetic structures called 'double minutes' are usually lost during mitosis (Wahl et al. 1982; Kaufman et al. 1983). However, incorporation into chromosomal DNA does not guarantee stability and such genes are often targets for genetic rearrangements, or may disrupt the integrity of endogenous genes upon insertion (Miele et al. 1989; Ruiz & Wahl 1990; Kim & Lee 1999). Studies in DHFR-CHO cells during long-term culture in the absence of MTX have indicated that incorporation of amplified recombinant genes close to (TTAGGG)n sequences (mammalian telomere repeat sequences), which are often found around the telomeric end of amplified arrays in CHO cells, may be associated with the stability of amplified genes (Yoshikawa et al. 2000b). A variety of gene amplification mechanisms has been proposed for mammalian cells (Windle & Wahl 1992; Stark 1993). However, there is not necessarily a linear relationship between levels of amplification and levels of expression, as excessive levels of the amplifying drug can be toxic to the cells and, as already discussed, the site of integration is important to expression levels and stability of expression. In amplified CHO cells, gradual stepwise increments in MTX exposure (rather than rapid exposure) have been reported to lead to amplified genes integrating into telomeric regions of chromosomes (Yoshikawa et al. 2000b).

The fact that the site of incorporation of a recombinant gene within the host cell's genome is important to its level of expression is exploited by targeted integration. Targeted integration using site-specific recombination is employed by, for example, the Cre/loxP and Flp/FRT systems. A vector containing a reporter gene flanked by a target sequence (e.g. loxP or FRT) is inserted randomly into the host cell. Cells that show high levels of reporter gene expression are selected and targeted with a second vector containing the desired gene of interest flanked by the same target sequences. A recombinase (e.g. Cre, or Flp) then ensures that the reporter gene is removed from the genome and the gene of interest is inserted in its place (Bode et al. 2000; Koch et al. 2000).

Chromatin and proteins important for the control of transcription can undergo a variety of modifications. In particular two of these modifications are thought to be important for stability of recombinant expression; methylation and acetylation. Various studies of the relationship between these epigenetic mechanisms and stability of production have been made. Methylation typically occurs at cytosine residues that are immediately 5' to a guanosine residue at, or near to, promoters. It is now widely accepted that methylation of transfected DNA can play a significant role in the regulation of expression. In particular, methylation is known to cause repression of gene expression and it is thought to be a major cause of the maintenance of the silenced heterochro-matin regions of DNA. Conversely, hypomethylation of the DNA surrounding gene promoters is often associated with increased transcriptional activity (Razin & Cedar 1991; Paulin et al. 1998; Mielke et al. 2000). There are examples of methylation leading to decreased expression of

MOLECULAR MECHANISMS ASSOCIATED WITH INSTABILITY

transfected genes in mammalian cells, for example instability in BHK cells has been associated with transcriptional shut-off through methylation of regulatory elements of the transfected gene (Mielke et al. 2000). Acetylation, the addition of acetyl groups to lysine residues of histones (DNA binding proteins that, together with DNA, form the basic subunit of chromatin, the nu-cleosome), reduces their affinity for DNA and hence results in unfolding of the chromatin structure. This in turn leads to enhanced accessibility of DNA to proteins involved in transcription (Garcia-Ramirez et al. 1995; Grunstein 1997; Hansen et al. 1998). The precise mechanism by which acetylation regulates transcription is still elusive (Kouzarides 2000). However, it is now well established that transcriptionally active genes are usually associated with histone acetylation whereas this is not the case for transcriptionally inactive genes and silenced regions of DNA such as heterochromatin (Turner et al. 1992; Braunstein et al. 1996). In addition, silencers and the transcription factors that bind to them (i.e. repressor proteins) can have a significant role in the regulation of transcription. These proteins can affect, amongst other things, the general assembly of the transcription complex, chromatin structure, intron splicing, cytoplasmic retention of transcription factors and the activity of positive-acting transcription factors (Clark & Docherty 1993; Ogbourne & Antalis 1998).

As well as modifications to the DNA and associated proteins, it is also important to remember that the actual structure of the chromatin can play a role in the regulation of transcription (Wolffe & Guschin 2000). In eukaryotes, DNA is first condensed into units called nucleosomes and then into higher order chromatin structures (Woodcock & Dimitrov 2001). This degree of condensation can hinder the association between the DNA and the transcription complex. Hence, before transcription can be activated chromatin remodelling has to occur (Kingston et al. 1996; Felsenfeld 1996; Urnov & Wolffe 2001).

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