The Huck Institutes of the Life Sciences

Thermodynamic and Structural Determinants of Differential Pdx1 Binding to Elements from the Insulin and IAPP Promoters

Bastidas M., Showalter S., (2013) Journal of Molecular Biology. Vol. 425, Issue 18, p.3360-3377

The Pdx1 homeodomain (Pdx1-HD) binds gene promoter elements containing the core 5’-TAAT-3’ DNA recognition sequence. We study Pdx1 binding to promoter sequences from two beta cell-specific genes, insulin and islet amyloid polypeptide (iapp). We show, by ITC, that Pdx1-HD binds preferentially to insulin promoter elements containing the sequence 5’-CTAAT-3’, over the IAPP elements containing the sequence 5’-TTAAT-3’.

 

In addition, we used the auto-ITC to measure the thermodynamic parameters of Pdx1-HD binding to various DNA sequences derived from a consensus sequence. Our calorimetric results show that Pdx1-HD binds preferentially to Gua or Thy in the first base pair 3’ the TAAT core, supporting the results obtained from other groups.

 

DNA Forward Sequence n Kd (nM) ΔH
  Group 1: Natural human promoter sequences      
Insulin A1 5´-AGGCCCTAATGGGCCA-3´ 0.9 ± 0.1 7 ± 2 -10.2 ± 0.1
Insulin A3 5´-AGACTCTAATGACCCG-3´ 0.9 ± 0.1 4 ± 1 -9.6 ± 0.1
Insulin E1 5´-AGCCATCTGCCGACCC-3´ 1.0 ± 0.1 1300 ± 300 -2.7 ± 0.2
IAPP A1 5´-GGAAATTAATGACAGA-3´ 1.1 ± 0.1 22 ± 5 -4.6 ± 0.1
IAPP A2 5´-ATGAGTTAATGTAATA-3´ 1.2 ± 0.1 27 ± 7 -5.7 ± 0.1
  Group 2: Consensus recognition sequences      
Consensus 5´-CCACTCTAATGAGTTC-3´ 0.9 ± 0.1 5 ± 1 -8.6 ± 0.1
Con. T1→A 5´- CCACTCAAATGAGTTC -3´ 1.0 ± 0.1 20 ± 4 -4.98 ± 0.04
Con. A2→T 5´- CCACTCTTATGAGTTC -3´ 1.0 ± 0.1 360 ± 50 -3.9 ± 0.1
Con. A3→T 5´- CCACTCTATTGAGTTC -3´ 1.0 ± 0.1 110 ± 20 -2.74 ± 0.05
Con. T4→A 5´- CCACTCTAAAGAGTTC -3´ 0.9 ± 0.1 64 ± 4 -6.27 ± 0.03
Con. 5´C→G 5´- CCACTGTAATGAGTTC -3´ 1.0 ± 0.1 10 ± 2 -7.45 ± 0.05
Con. 5´C→A 5´- CCACTATAATGAGTTC -3´ 1.0 ± 0.1 9 ± 1 -7.07 ± 0.04
Con. 5´C→T 5´- CCACTTTAATGAGTTC -3´ 0.9 ± 0.1 8 ± 1 -7.00 ± 0.04
Con. 3´G→A 5´- CCACTCTAATAAGTTC -3´ 1.1 ± 0.1 11 ± 3 -4.29 ± 0.04
Con. 3´G→T 5´- CCACTCTAATTAGTTC -3´ 1.1 ± 0.1 3.4 ± 0.9 -8.9 ± 0.1
Con. 3´G→C 5´- CCACTCTAATCAGTTC -3´ 1.1 ± 0.1 30 ± 3 -9.08 ± 0.05