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1.5.1 - Introduction

The octamer motif (ATGCAAAT) or its inverse complement (A I I I G CAT) has been implicated as being the key regulatory element in a number of

gene promoters. For example it has been identified as being a key

functional element in the ubiquitously expressed U1 and U2 snRNA (Ares, et al. 1985) and histone H2B genes (Sive et al. 1986; Sive and Roeder, 1986). It has also been identified as a lymphoid specific element in both the upstream promoter and enhancer regions of the immunoglobulin genes (Wirth et al. 1987). In these cases the octamer motif acts in a positive manner determining the expression pattern of these genes. In other cells the octamer motif has been identified as being the functional element in the repression of gene expression. An example of such a case is the repression of herpes simplex virus (HSV) immediate early (IE) gene expression in neuronal cells (Kemp et al. 1990; Wheatley et al. 1991). The different effects of the octamer motif in different promoters and cell types are likely to be determined by the nature of the octamer binding proteins

present in each cell type (Lillycrop and Latchman, 1992). Early DNA

mobility shift studies identified two factors that bind indistinguishably to the octamer motif (Staudt et al. 1986). Both factors have subsequently been purified and cloned. The factors, Oct-1 (also known as NF-A1, NFIII, OBP100 and OTF1) and Oct-2 (also known as NF-A2 and OTF-2), are both members of the POU II subfamily. Oct-1 is ubiquitously expressed

whilst Oct-2 expression is highly restricted. The Oct-2 protein is

expressed as a number of alternatively spliced products each with a unique role in the regulation of genes carrying the octamer motif. The details of both Oct-1 and Oct-2 will be discussed below.

1.5.2 - Octamer binding POU II subfamily proteins

Oct-1, which is encoded by single gene found on mouse chromosome 1, is a 90-100 KDa ubiquitously expressed protein (Staudt et al. 1986; Sturm et al. 1988). It is responsible for widespread expression of housekeeping

genes where octamer binding activity is implicated. For example the

octamer binding ability of Oct-1 and its attenuation by phosphorylation has been implicated in the regulation of histone H2B protein levels during mitosis (Roberts et al. 1991 ; Segil et al. 1991 ; see 1.4.9).

Although originally defined as a B cell-specific octamer binding transcription factor (Singh et al. 1986; Staudt et al. 1986) mediating the activation of immunoglobulin genes (Lenardo et al. 1987) Oct-2 specific mRNAs have also been detected in neuronal cells, kidney, testis and

intestine (Hatzopoulos et al. 1990; He et al. 1989). Furthermore an

octamer binding protein with a mobility identical to lymphoid Oct-2 has been observed in DMA mobility shift assays on brain tissue (Scholer et al 1989). Transient transfection studies revealed that, in contrast to the lymphoid Oct-2, the neuronal forms of Oct-2 acted primarily as an inhibitor of transcription (Dent et al. 1991) and was responsible for the repression of the HSV IE gene expression in neuronal cells (Lillycrop et al. 1991). This repression of the HSV IE genes is dependent upon the octamer related TAATGARAT motif in the promoters of these genes (Wheatley et al. 1991). Although Oct-2 has been shown to be encoded by a single copy gene an alternative splicing mechanism has been identified in both lymphoid and neuronal cells which allows multiple isoforms of the protein to be generated (Wirth et al. 1991; Lillycrop and Latchman 1992). As a result of alternative splicing different isoforms are generated each with different effects on gene expression.

1.5.3 - Isoforms of POU II proteins

Initially six differentially expressed Oct-2 isoforms were identified in a B- cell library, all alternatively spliced from a single 14-exon gene (Wirth et al.

1991) (see Figure 1.6). These isoforms were named Oct-2.1 through to Oct-2.6. Oct-2.1 was found to be the predominant B-cell isoform (Wirth et al. 1991) whilst Oct-2.4 and Oct-2.5 were shown to be predominantly expressed in neuronal cells (Lillycrop and Latchman, 1992). As detailed in Figure 1.6, Oct-2.1 shares C-terminal homology with the Oct-2.2, Oct-2.3 and Oct-2.6 isoforms and N-terminal homology with Oct-2.4 and Oct-2.5. Thus Oct-2.2 is homologous to Oct-2.1 apart from a unique 48bp (16 amino acid) addition found at the N-terminus of exon 7. Likewise Oct-2.3 has a unique 66bp (22 amino acid) insert at the N-terminus of exon 4. Oct-2.6 also differs from Oct-2.1 at the N-terminal region where a frame deletion of 117bp (39 amino acids) removes exon 4 completely. No 0- terminal homology however is shared between Oct-2.1 and Oct-2.4. An alternative splicing event in exon 12 removes 136bp and introduces a stop codon 17 amino acids down stream of the deletion site. Likewise Oct-2.5 exhibits the splicing in of a 74bp exon 12 amino acids upstream of the stop codon utilised in the translation of isoforms Oct-2.1, Oct-2.2, Oct-2.3 and Oct-2.6. As a consequence of the resulting frame shift a further 132 new amino acids are added at the C-terminus. All six Oct-2 isoforms originally described in B-cells maintain an intact POU domain (Wirth et al. 1991).

Five further alternatively spliced isoforms of Oct-2 have subsequently been isolated. These are Oct-2c, Oct-2d (also known as mini-Oct-2), Oct-2 Oct-2A^^' and Oct-2AB. Oct-2c although essentially identical to Oct-2.1 lacks the last 12 C-terminal amino acids (Stoykova et al. 1992). Mini-Oct- 2 comprises just the DNA binding POU domain and like Oct-2c was originally found expressed in the brain (Stoykova et al. 1992). However later studies have identified much higher levels of mini-Oct-2 mRNA expressed in spleen cells (Liu et al. 1995).

Exons Oct-2.1 Oct-2.2 Oct-2.3 Oct-2.4 Oct-2.5 Oct-2.6 Oct-2A^" Oct-2A^'- Oct-2AB Oct-2C Mini-Oct-2

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Figure 1.6 - Alternative splicing of the Oct-2 primary transcript to produce mRNAs encoding the different Oct-2 isoforms

Schematic representation of the eleven Oct-2 isofoms (Wirth et al. 1991; Stoykova et al. 1992; Johansson et al. 1994). The top line shows the 14 exons utilised by the various isoforms. The position of the translation stop codons predicted for each isoforms is shown as a filled circle above the respective exon. The POU domian region (Exons 7 to 10) is shaded.

Since mini-Oct-2 is essentially just the POU DNA binding domain it is able to bind the octamer motif and may act to block the site, inhibiting or competing with other octamer binding factors. Levels of mini-Oct-2 mRNA have been shown to be down regulated in the presence of growth factors and up-regulated in response to differentiation-inducing stimuli (Liu et al. 1995). The final three Oct-2 isoforms were cloned following the treatment of B-lymphocytes with bacterial lipo-polyasaccaride (Johansson et al. 1994). Oct-2A"^^^ contains a highly basic region inserted between exons 4 and 5 whilst Oct-2A^^' entirely lacks exon 11. As a result a putative C- terminal leucine zipper region (Clerc et al. 1988) is disrupted and a frame shift is introduced which creates a novel truncated proline rich C-terminal

(Johansson et al. 1994). Finally Oct-2AB contains both the additional

region of exon 7 seen in Oct-2.2 and all of exon 13 uniquely seen in Oct- 2.5. Oct-2AB therefore encodes the largest Oct-2 protein and was found

to be preferentially expressed in B-cells stimulated with lipo-

polysaccharide and phorbol-di-butyrate. To date no unique roles have been attributed to these LPS induced forms of Oct-2 (Johansson et al. 1994).

More recently three isoforms of the Oct-1 protein have been identified from a murine pre-B cell cDNA library (Jaffe et al. 1995). These isoforms, Oct- 1a, Oct-1 b and Oct-1 c, show structural differences within the C-terminal of the protein. Thus Oct-1 a is very similar to human Oct-1, Whilst Oct-1 b contains an additional 72bp region within the serine/threonine rich C- terminal and lacks a 72bp region further downstream. The Oct-1 c isoform contains both 72bp regions. In all tissues but testis Oct-1 a was found to

be expressed in higher quantities that Oct-1 b. Although no specific

activities have been attributed to these isoforms it is clear that regulation of gene expression by Oct-1 may be more complex than originally thought (Jaffe et al. 1995).

1.5.4 - Differential activities of the Oct-2 isoforms

Studies of the Oct-2 isoforms have revealed differing functional activities depending upon the sequence of the octamer DNA binding site, the position of the motif within the promoter and cell type. Thus, for example, in fibroblast cells all of the six originally identified isoforms are able to activate heterologous gene promoters only carrying a consensus octamer motif (Wirth et al. 1991; Lillycrop and Latchman, 1992). The activation of such a promoter by Oct-2.5 in fibroblast cells has been shown to be dependant upon the position of the octamer motif since the positioning of the motif further upstream removes any activation (Lillycrop and Latchman, 1992). In contrast in neuronal ND7 cells all these isoforms down regulate expression of an octamer-containing promoter (Lillycrop and Latchman, 1992).

The study of the regulation of heterologous promoters carrying the octamer related TAATGARAT motif has led to interesting observations. Thus whilst Oct-2.1, Oct-2.2 and Oct-2.3 are able to activate this type of promoter in fibroblast cells, in neuronal cells they repress gene expression. This is in contrast to the Oct-2.4 and Oct-2.5 isoforms which repress TAATGARAT carrying promoters in both neuronal and fibroblast cell types (Lillycrop and Latchman, 1992). Structurally the Oct-2.4 and Oct-2.5 isoforms differ form the other isoforms at the C-terminal region. This is due to alternative splicing of the primary mRNA transcripts, which removes part of exon 12 and introduces a stop codon into the Oct2.4 mRNA producing a protein truncated at the C-terminal (see Figure 1.6). Similarly alternative splicing of the Oct-2.5 mRNA introduces exon 13 and a frameshift mutation which adds a further 132bp to the C-terminal disrupting the activation domain. The above examples demonstrate that regulation of gene expression by Oct-2 isoforms is dependent upon the cell type and the position within the promoter of the DNA binding site.

The sequence of DNA binding site is also important as has been seen for the differential activity of the isoforms with the octamer related

TAATGARAT motif. These effects are further demonstrated by the

tyrosine hydroxylase gene promoter, which contains a heptamer TAATGARAT-like motif and has been shown to be repressed by all Oct-2 isoforms in all cell types (Dawson et al. 1994).

The dichotomy that has been defined between the different Oct-2 isoforms, those which activate and those which repress octamer-like containing promoters may be explained by the presence or absence of different functional domains spliced into or out of the resulting active transcription factor. The different functional domains identified in Oct-2 are introduced and discussed below.

1.5.5 - The functional domains of Oct-2

Transient transfection studies using deletion constructs have identified a number of activator and repressor domains within the modular structure of Oct-2. Two distinct activation domains have been identified (Tanaka and Herr, 1990; Muller Immergluck et al. 1990; Gerster et al. 1990); i) an N- terminal glutamine rich domain and ii) a C-terminal domain rich in proline, serine and threonine.

These domains have been shown to synergise with each other to strongly activate transcription (Tanaka et al. 1994). The N-terminal glutamine rich activation domain, which is absent from mini-Oct-2, is also present in Oct- 1 whilst the C-terminal domain is not, differentiating Oct-1 from Oct-2 (Tanaka and Herr, 1990; Tanaka et al. 1994). The C-terminal activation domain is absent in Oct-2.4, Oct-2.5, Oct-2C, mini-Oct-2 and Oct-2A^^' due to alternative splicing of mRNA (see Figure 1.6). The lack of the C- terminal activation domain may in part explain the functional differences documented between the Oct-2.4 and Oct-2.5 isoforms. Thus Oct-2.1,

lack this intact domain may not direct activation of genes which specifically require the C-terminal activation domain and not the N-terminal activation

domain. An example of such a case is the activation of B cell

immunoglobulin genes from a distal enhancer site, activation of which Oct- 2.4 and Oct-2.5 are unable to mediate (Annweiler et al. 1994; Fried I and Matthias, 1995). Other genes have been identified which Oct-2.4 and Oct- 2.5 actually repress, rather than fail to activate. This repression has been mapped to. the N-terminal region of the Oct-2 protein (Annweiler et al. 1994; Lillycrop et al. 1994a; FriedI and Matthias, 1995, 1996). Three separate distinct inhibitory regions have been identified. Amino acids 161 to 181 have been mapped as critical for the repression of HSV IE gene promoters (Lillycrop et al. 1994a). A second domain has been identified between amino acids 42 and 64 (FriedI and Matthias, 1995, 1996), whilst a 22 amino acids domain, unique to the Oct-2.3 isoform, has been identified between animo acids 72 and 73 (numbering with respect to Oct-2.1) (Annweiler et al. 1994). Studies into the mechanisms of action of these three inhibitor regions are reported below (see Chapter 3).

The presence of the inhibitory regions within the essentially activating isoforms, Oct-2.1, Oct-2.2, Oct-2.3 and Oct-2.6 has led to the hypothesis that in the presence of the strong C-terminal activation domains the

repression mediated by these regions is overpowered. However in

isoforms Oct-2.4 and Oct-2.5 where no C-terminal activation domains are present the repression effect may be exhibited (Lillycrop and Latchman, 1992).

1.5.6 - Transcriptional co-activators of Oct-1 and Oct-2

Both Oct-1 and Oct-2 were originally identified as binding indistinguishably to the octamer motif. Activation domains have been identified in both transcription factors, however specific co-activators may be required for the activation of specific genes. Thus, for example, Oct-1 is alone able to activate the transcription of snRNA gene promoters by RNA polymerase II (Ares et al. 1987). However for Oct-1 to activate mRNA promoters, such

as the herpes simplex virus IE3 gene promoter, a co-activator is required. Thus although Oct-1 is able to bind to the TAATGARAT motif present in the HSV IE genes, only in the presence of the viral transactivator VP16 (also referred to as Vmw65 and aTIF) is Oct-1 able to frans-activate

mRNA transcription of such genes. VP16 contains a mRNA type

activation domain that after association with Oct-1 alters its activation properties. Furthermore the association of VP16 with the POU domain of Oct-1 stabilises the binding of Oct-1 to the low affinity TAATGARAT motif. In a similar manner the ubiquitously expressed Oct-1 is able to activate immunoglobulin gene expression, via the ATGCAAAT octamer motif, in a B-cell specific manner by functionally interacting with the B-cell specific co­

activator OCA-B (for Oct co-activator from B-cells). Although OCA-B

functions analogously to VP16 by providing a mRNA type activation domain and stabilising the binding of Oct-1 on the octamer, a recent study has revealed that OCA-B interacts with a separate surface of the Oct-1 POU domain. In fact both VP16 and OCA-B are able to associate with the Oct-1 POU domain simultaneously (Babb et al. 1997).

The Oct-2 transcription factor is also thought to be able to mediate activation in some cell types without a co-activator. However, in B-cells Oct-2 has been shown to require OCA-B to activate B-cell specific

immunoglobulin gene expression. Interestingly Oct-2 is unable to

associate with VP 16 and in fact is implicated in the maintenance of HSV latency by competing with the Oct-1 :VP 16 complex for TAATGARAT binding sites thus inhibiting expression of the IE genes. Within the POU- domain there are only seven amino acid differences between Oct-1 and

Oct-2 . Of these one at position 2 2 in the first a-helix of the POUh domain

has been shown to regulate the functional interaction of Oct-1 with VP16 (Lai et al 1992). Thus the presence of a glutamic acid at this position in Oct-1 allows Oct-1 and VP16 to associate. Oct-2, however, which has an

alanine residue at this position is unable to interact with VP16. The

Both Oct-1 and Oct-2 have also been shown to interact via the POU domain with the high mobility group (HMG) protein 2. This association results in a marked increase in the sequence specific DNA binding activity of both Oct-1 and Oct-2 proteins. These findings indicate that, like VP16 and OCA-B, one function of HMG2 is to support octamer transcription factors in their role as transcriptional activators (Zwilling et al. 1995).

1.5.7 - Gene regulation by Oct-1 and Oct-2

As previously discussed Oct-1 regulates the expression of snRNA genes (Ares et al. 1987) and the histone H2B gene (Fletcher et al. 1987; LaBella et al. 1988). Oct-1 has also been demonstrated to play a role in the regulation of a number of human lymphokine genes including interleukin 3 (IL-3), interleukin 5 (IL-5) and granulocyte macrophage-CSF (GM-CSF) (Kaushansky et al. 1994).

Although Oct-2 alone was initially believed to stimulate B-cell specific immunoglobulin gene expression (Muller et al. 1988) further studies have revealed that Oct-1 is also able to mediate cell specific immunoglobulin

expression. Both transcription factors mediate activation by protein-

protein interaction with the B cell specific co-activator OCA-B (Luo and Roeder, 1995; Strubin et al. 1995). Oct-1 and Oct-2 both also regulate interleukin 2 (IL-2) and interleukin 4 (IL-4) gene expression (Pfeuffer et al. 1994). Herpes simplex virus immediate early genes are activated by Oct-1 and repressed by Oct-2 (O'Hare et al. 1988; Lillycrop et al. 1993) whilst the reverse is the case for the human papilloma virus 16 and 18 promoters whose transcription Oct-1 represses whilst Oct-2 activates (Hopper-Seyler et al. 1991; Morris et al. 1993a, 1993b).

Oct-1 has been shown to mediate the activation of the murine mammary tumour virus (MMTV) promoter (Bruggemeier et al. 1991). Within the nervous system Oct-1 is essential for the activity of the gonadotrophin- releasing hormone (GnRH) neuron specific enhancer (Clark and Mellon, 1995). Although targeted deletion of the Oct-1 locus has not been

performed the disruption of a neighbouring locus, encoding the murine CD3 eta gene, which resulted in Oct-1 dysregulation, has been performed (Koyasu et al. 1994). The only reported consequence of these knockout experiments is a reduced birth rate in CD3 eta null mutant mice which is proposed to be the result of alterations in Oct-1 gene expression (Koyasu et al. 1994).

Studies of Oct-2 knockout mice have revealed that Oct-2, although not required for the early stages of B cell development, is essential for later B cell maturation and postnatal survival. Oct-2 null mice die within a few hours of birth for unclear reasons (Corcoran et al. 1993). Interestingly only those genes which Oct-2 regulates without interaction with a co-activator

are not expressed in Oct-2 knockout mice. The genes which Oct-2

regulates by interaction with a co-activator appear to be regulated by Oct- 1 when Oct-2 is absent. In B-lymphocytes Oct-2 has been shown to critically regulate the cell surface glycoprotein gene CD36 and as a result may influence cell differentiation (Konig et al. 1995). The cysteine-rich secreted protein 3 gene (Crisp 3) is also regulated by Oct-2 in pre-B cells

(Pfisterer et al. 1996). A mouse homologue of the human

monocyte/neutrophil elastase inhibitor (mEI) and two uncharacterised cDNAs, named Novi and Nov2, have also recently been shown to be regulated, in part, by Oct-2 (Pfisterer et al. 1997). Within neuronal cells Oct-2 represses tyrosine hydroxylase (TH) gene expression via a heptamer/TAATGARAT-like motif in the gene promoter (Dawson et al.

1994; Deans et al. 1995). Tyrosine hydroxylase is the rate limiting

enzyme in the synthesis of catecholamine neurotransmitters, such as adrenaline, in both the peripheral and central nervous systems (Nagatsu et al. 1964). Dysregulation of TH has been implicated in the onset of neurological disorders including Parkinson's disease, schizophrenia and

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