Solubility partitioning of C/EBPβ on the rat hepatocyte nuclear matrix by hydrophobic interactions

Abstract

The greatest part of nuclear C/EBP (a major 35 kD protein, 30 and 38 kD isoforms) was observed to partition with the nuclear matrix. Cross-linking experiments with formaldehyde suggested that the association reflected the in situ juxtapositioning of C/EBP to nuclear matrix proteins in isolated nuclei. The association of C/EBP with the nuclear matrix resisted RNase and DNase treatment and extraction with protein sulfhydryl reducing agents combined with high ionic strength salt. C/EBP displayed a proclivity to extensively reassemble with the filament-forming nuclear matrix proteins after a cycle of solubilization with urea, followed by its removal by dialysis. These findings suggest that the C/EBP moieties were anchored to the nuclear matrix through hydrophobic protein-protein interactions with the lamins. Subsequent separation of nuclear matrix-associated C/EBP into insoluble, reassembling, and soluble nuclear matrix protein (SNMP) fractions after a cycle of solubilization/reassembly pointed to the sub-partitioning of C/EBP on the nuclear matrix. DNA affinity chromatography using the rat haptoglobin gene cis-element and SNMP revealed the binding of p35 during basal transcription, and p35 and p30 during elevated haptoglobin gene transcription in the course of the acute-phase (AP) response. It was concluded that the appearance of cis-element-binding p30 in the SNMP fraction resulted from its increased solubility (decreased hydrophobicity) and inability to reassociate with the lamins during urea removal. The observed solubility partitioning of C/EBP on the nuclear matrix framework could represent a level of control of the general availability of regulatory proteins for establishing interactions with DNA.