Competition analysis o f complex formation at the XR12 DNA fragment probe in PLB- 985 cells was performed using double-stranded, unlabelled DNA oligonucleotides
(Table 5.3). Complex formation was competed with homologous and non-homologous oligonucleotides in order to establish the specificity and exact location o f protein binding (Figure 5.4). Complex formation was stable in the presence o f 50-fold molar excess o f oligonucleotide containing a B cell-specific octamer binding site (OCT) (panel A, lane 11). Minimal competition o f complex formation occurred in the presence o f 50- fold molar excess o f homologous p47^^‘’^ oligonucleotide comprising -121 to -92bp o f
pj-omoter sequence, containing an ISRE m otif (I) (panel A, lane 3), but was presumed to be o f limited significance since increased competitor did not enhance competiton further (panel A, lane 12). Complex formation was not disrupted by 50 or 100-fold molar excess o f oligonucleotide I carrying a mutation o f the ISRE m otif (Imt) (panel A, lanes 4, 13). By contrast, complex formation was abolished by as little as 25- fold molar excess o f homologous competitor comprising p47^^‘^* promoter sequence from -52 to -29, containing the PU. 1 consensus binding site (?) (Figure 5.5). Complex formation was not disrupted by 50-fold molar excess o f oligonucleotide P carrying a mutation o f the PU .l core binding sequence that abrogates PU .l binding (120) (Pmt) (Fig. 5.4, panel A, lane 6; Fig. 5.5, lane 14). These results indicate that complex
1 2 3 4 5 6
Figure 5.3 Complex formation at the proximal 143bp p47^**’^ prom oter in PLB-985 myeloid and HeLa epithelial cells. EMSA was perform ed with a [^^P]-labelled double stranded DNA restriction
fragment probe containing 143bp o f the p47^^“ proximal prom oter (XR12) and 3pg nuclear extract from undifferentiated PLB-985 (lanes 4-6) or HeLa (lanes 1-3). Increasing am ounts o f probe (In g , lanes 3, 6; 2ng, lanes 2, 5; 3ng, lanes 1, 4) and poly dl-dC non-specific inhibitor (167pg/m l, lanes 3, 6; 333pg/ml,
lanes 2, 5; 500pg/m l, lanes 1, 4) were used. Specific com plex formation is indicated by C; free probe is indicated by F.
Oligonucleotide Position Nucleotide sequence prom oter P -5 2 to -2 9 AAAAGCGACTTCCTCTTTCCAGTG (3 6 1 ) Pmt -5 2 to -2 9 AAAAGCGACTGAATCTTTCCAGTG (1 2 0 ) 1 -121 to -9 2 GTCCCCAGAAACTGAAAGAAGAAGCAAAGC(3 6 1 ) Imt -121 to -9 2 GTCCCCAGAGGGCAGAAGAAGAAGCAAAGC (1 2 1 ) prom oter HAF -3 0 to -68 CTGCTGTTTTCATTTCCTCATTGGAAGAAGAAGCATAGT (3 3 5 ) 91 -2 6 2 t o -213 GTTATTTATCTCTTAGTTGTAGAAATTGGTTTCATTTTCCACTATGTTTA (1 2 1 ) 91m t -2 6 2 t o -213 GTTATTTATCTCTTAGTTGTAGAAATTGGTCCTGCCTTCCACTATGTTTA (1 2 1 )
FeyRI prom oter
GRR -1 6 9 t o -1 2 6 GCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAGGAACAT (3 6 2 )
PIE -1 0 4 to “84 GCAATTTCCTTCCTCTTTTCT (1 2 0 )
class I! HLA-DRa prom oter
OCT -6 2 to -3 5 AGAGTAATTGATTTGCATTTTAATGGTC (3 6 3 )
Table 5.3. Nucleotide sequence of double-stranded oligonucleotides used as [^^P|-labelled probes and unlabelled competitors for in vitro protein binding assays. Double stranded DNA was generated from overlapping, complementary primers (Chapter 2, section 2.2.2.6). Sense strand sequence in 5’ to 3’ direction is shown. Mutated sequence is shown in bold type.
B
Molar excess Oligonucleotide competitor
50x" lOOx
I Imt P Pmt 91 91mt GRR PIE OCT I Imt GRR PIE
50x lOOx ^ H A F ^
c
ttmrn
C F F 1 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15F ig u re 5.4 C o m p etitio n an a ly sis o f com plex fo rm a tio n a t th e p ro x im a l 143bp p47^*'^ p ro m o te r. A, EMSA was perform ed with a [^^P]-labelled double stranded DNA restriction
fragment probe containing 143bp o f the p47^*°'' proxim al promoter (XR12) and 3fJ.g nuclear extract from undifferentiated PLB985 cells (lanes 2 to 15), or in the absence o f extract
(lane 1). Specific com plex form ation is indicated by C; free probe is indicated by F. Competition experiments were perform ed using 50-fold (lanes 3-11) or 100-fold (lanes 12-15) m olar excess o f double stranded unlabelled oligonucleotide as indicated. Oligonucleotide competitors were as follows: lanes 1 and 2, none; lanes 3 and 12, p47^^“ prom oter ISRE consensus m otif (I); lanes 4 and 13, oligonucleotide I with m utated ISRE m otif (Imt); lane 5, p47^^"'' promoter P U .l consensus binding site (P); lane 6, oligonucleotide P with m utated P U .l consensus binding site (Pmt); lane 7, gp9F ^'’'' prom oter M ID binding site (-262 to -213) containing ISRE (91); lane 8, oligonucleotide 91 with m utated ISRE ( 9 Im t);
lanes 9 and 14, FcyRI prom oter (-169 to -126) containing IFN-y response region (GRR) ; lanes 10 and 15, FcyRIb prom oter (-104 to -84) containing P U .l binding site (PIE); lane I I ,
human class II H L A -D R a prom oter (-65 to - 3 5 ) containing a B-cell specific octam er binding site (OCT). B, EMSA was perform ed with [^^P]-labelled double stranded DNA
restriction fragm ent probe X R12 and 3 |lg nuclear extract from undifferentiated PLB985 cells (lanes 1 to 3). Specific com plex formation is indicated by C; free probe is indicated by F. Com petition experim ents w ere perform ed using 50-fold (lane 2) or 100-fold (lane 3) molar excess o f HAF oligonucleotide (-30 to -68 gp91^*"^ prom oter sequence containing the HAF-1 binding site).
c—
F —
8
9
10
11
12
13 14
15
F ig u re 5.5 C o m p etitio n analysis o f com plex fo rm a tio n a t th e p roxim al 143bp p47'’''"^ p ro m o te r w ith hom ologous sequence. EMSA was perform ed with a [^^P]-labelled
double stranded DNA restriction fragm ent probe containing 143bp o f the p47^*’°’‘ proximal prom oter (XR12) and 3pg nuclear extract from undifferentiated PLB-985 cells (lanes 1 to 14), or in the absence o f extract (lane 15). Specific com plex formation is indicated by C; free probe is indicated by F. Oligonucleotide com petitors were as follows: lanes 2-7, p4 7P^«« prom oter P U .l consensus binding site (P); lanes 8-14, oligonucleotide P with mutated P U .l consensus binding site (Pmt); lane 15, none. O ligonucleotide com petitor was present in 1- to 50-fold m olar excess as follows: 1-fold, lanes 2, 9; 2-fold, lanes 3, 10; 5-fold, lanes 4, 11; 10-fold, lanes 5, 12; 25-fold, lanes 6, 13; 50-fold, lanes 7, 14, none,
lanes 1, 8. The direction o f increased com petitor is indicated by an open triangle.
formation is specific, and localised to the consensus PU .l binding site located between positions -38 and -43. In order to investigate the identity o f the specific complex formed at the XR12 fragment probe in PLB-985 cells, competition was performed with heterologous double-stranded oligonucleotides containing functional protein binding motifs (see Table 5.3). The ability o f functional PU .l binding sites to compete complex formation was investigated using oligonucleotides containing the PIE region o f the myeloid-specific FcRylb gene promoter (PIE) (120) and the HAF-1 binding site o f the gp91^^°^ promoter (HAF) (122). Complex formation was significantly disrupted by 50- fold molar excess o f both PIE (Fig. 5.4, panel A, lane 10) and HAF oligonucleotides (Fig. 5.4, panel B, lane 2). These results indicate that the specific complex formed on the XR12 fragment probe contains PU .l or a factor with a similar binding specificity.
The ability o f myeloid-specific interferon response motifs to compete complex formation at the XR12 fragment probe was investigated using oligonucleotides containing the IFN-y responsiveness-conferring m otif o f the FcRyl promoter (GRR), and the ISRE-containing MID m otif o f the gp9l^*^^ promoter (91) (see Fig. 4.19). Complex formation was not disrupted by 50- or 100-fold molar excess o f the GRR oligonucleotide (Fig. 5.4, panel A, lanes 9, 14) nor 50-fold molar excess o f the 91 oligonucleotide (Fig. 5.4, panel A, lane 7) or oligonucleotide comprising identical sequence to 91 except for mutation o f the ISRE m otif located at -234 to -222 (9 Imt) (Fig. 5.4, panel A, lane 8). These results suggest that the complex formed does not contain components o f the IFN-y-activating complex that interacts with the FcRyl GRR (GIRE-BP, see Table 5.2) (119) or lRF-2, TF-1^^^°^ or BID factors that interact with the gp91^^"^ MID m otif (121,342,343). In addition, these results suggest that an ISRE m otif overlapping the consensus PU .l binding site in opposite orientation does not contribute to complex formation.