Abstract
Using computer stress‐induced duplex destabilization (SIDD) analysis and binding experiments, we identified a S/MAR element (−599/−200 bp) (Hp‐S/MAR) adjacent to the cis‐element (−165/−56 bp) in the rat haptoglobin gene. We examined its functional interactions with the lamins and lamin‐associated proteins in the basal state and during acute‐phase (AP) response‐induced increased transcription. Colocalization, electrophoretic mobility shift assay (EMSA), and re‐electrophoresis of nucleoprotein complexes, South‐Western and Western blot analysis and coimunoprecipitation experiments revealed that the lamins, PARP‐1, C/EBPβ, and Hp‐S/MAR assembled higher order complexes through direct lamin‐Hp‐S/MAR and probably PARP‐1–Hp‐S/MAR interactions although C/EBPβ did not bind to the Hp‐S/MAR but established direct interaction with PARP‐1. The transition from constitutive to increased haptoglobin gene transcription during the AP response was associated with quantitative and qualitative changes in Hp‐S/MAR–protein interactions, respectively, observed as increased association of the lamin(s) with the Hp‐S/MAR and as the appearance of a 90 kDa Hp‐S/MAR‐binding protein. Also, during the AP response the contact between C/EBPβ and PARP‐1 established in the basal state was lost. DNA chromatography with the haptoglobin cis‐element and Western blot analysis suggests that PARP‐1 was a coactivator during constitutive and elevated transcription. The results show that the lamin components of the nuclear matrix form a network of functional, dynamic protein–protein and protein–Hp‐S/MAR associations with multiple partners, and underline the involvement of PARP‐1 in the regulation of haptoglobin gene transcription. We concluded that the interplay of these interactions fine tunes haptoglobin gene expression to meet the changing requirements of liver cells. J. Cell. Biochem. 107: 1205–1221, 2009. © 2009 Wiley‐Liss, Inc.