The immunoglobulin genes are expressed only in B-lineage cells, and even within this lineage, the genes are expressed at different rates during different developmental stages. As with other eukaryotic genes, three major classes of cis regulatory sequences in DNA regulate transcription of immunoglobu-lin genes:
■ Promoters: relatively short nucleotide sequences, extending about 200 bp upstream from the transcription initiation site, that promote initiation of RNA transcription in a specific direction
■ Enhancers: nucleotide sequences situated some distance upstream or downstream from a gene that activate transcription from the promoter sequence in an orientation-independent manner
■ Silencers: nucleotide sequences that down-regulate transcription, operating in both directions over a distance.
The locations of the three types of regulatory elements in germ-line immunoglobulin DNA are shown in Figure 5-19. All of these regulatory elements have clusters of sequence motifs that can bind specifically to one or more nuclear proteins.
Each VH and VL gene segment has a promoter located just upstream from the leader sequence. In addition, the Jk cluster and each of the DH genes of the heavy-chain locus are preceded by promoters. Like other promoters, the immunoglobulin promoters contain a highly conserved AT-rich sequence called the TATA box, which serves as a site for the binding of a number of proteins that are necessary for the initiation of RNA transcription. The actual process of transcription is performed by RNA polymerase II, which starts transcribing DNA from the initiation site, located about 25 bp downstream of the TATA box. Ig promoters also contain an essential and conserved octamer that confers B-cell specificity on the promoter. The octamer binds two transcription factors, oct-1, found in many cell types, and oct-2, found only in B cells.
While much remains to be learned about the function of enhancers, they have binding sites for a number of proteins, many of which are transcription factors. A particularly important role is played by two proteins encoded by the E2A gene which can undergo alternate splicing to generate two collaborating proteins, both of which bind to the ^ and k intronic enhancers. These proteins are essential for B-cell development and E2A knockout mice make normal numbers of T cells but show a total absence of B cells. Interestingly, trans-fection of these enhancer-binding proteins into a T cell line resulted in a dramatic increase in the transcription of ^ chain mRNA and even induced the T cell to undergo DH + JH ^ DHJH rearrangement. Silencers may inhibit the activity of Ig
P L VH P L VH DH JH E„ cm C8 cy3 cy1 CY2b CY2a Ce Ca 3'aE
Key = Promoter
Enhancer ^^ Silencer O
P L Vx2 JX2CX2 JX4 CX4 X2-4E
P L Vx1 JX3 CX3 JX1 CX1
Location of promoters (dark red), enhancers (green), and silencers (yellow) in mouse heavy-chain, k light-chain, and X light-chain germ-line DNA. Variable-region DNA rearrangement moves an enhancer close enough to a promoter that the en-
hancer can activate transcription from the promoter. The promoters that precede the DH cluster, a number of the C genes and the JX cluster are omitted from this diagram for the sake of clarity.
enhancers in non-B cells. If so, they could be important contributors to the high levels of Ig gene transcription that are characteristic of B cells but absent in other cell types.
One heavy-chain enhancer is located within the intron between the last (3') J gene segment and the first (5') C gene segment (C^), which encodes the ^ heavy chain. Because this heavy-chain enhancer (E^) is located 5' of the S^ switch site near C^, it can continue to function after class switching has occurred. Another heavy-chain enhancer (3'aE) has been detected 3' of the Ca gene segment. One k light-chain enhancer (Ek) is located between the Jk segment and the Ck segment, and another enhancer (3'kE) is located 3' of the Ck segment. The X light-chain enhancers are located 3' of CX4 and 3' of CX1. Silencers have been identified in heavy-chain and K-chain DNA, adjacent to enhancers, but not in X-chain DNA.
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