Solubility partitioning of C/EBPbeta on the rat hepatocyte nuclear matrix by hydrophobic interactions.
The greatest part of nuclear C/EBPbeta (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/EBPbeta to nuclear matrix proteins in isolated nuclei. The association of C/EBPbeta with the nuclear matrix resisted RNase and DNase treatment and extraction with protein sulfhydryl reducing agents combined with high ionic strength salt. C/EBPbeta 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/EBPbeta moieties were anchored to the nuclear matrix through hydrophobic protein-protein interactions with the lamins. Subsequent separation of nuclear matrix-associated C/EBPbeta into insoluble, reassembling, and soluble nuclear matrix protein (SNMP) fractions after a cycle of solubilization/reassembly pointed to the sub-partitioning of C/EBPbeta 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/EBPbeta on the nuclear matrix framework could represent a level of control of the general availability of regulatory proteins for establishing interactions with DNA.
Uskoković
Aleksandra
A
Molecular Biology Laboratory, Institute for Biological Research, 29. novembra 142, Belgrade, Serbia, 11060, Federal Republic of Yugoslavia.
Vidaković
Melita
M
Dinić
Svetlana
S
Ivanović-Maticć
Svetlana
S
Martinović
Vesna
V
Poznanović
Goran
G
D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D015530:Nuclear Matrix / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility
D022763:CCAAT-Enhancer-Binding Protein-alpha; D003432:Cross-Linking Reagents; D034521:Nuclear Matrix-Associated Proteins; D020033:Protein Isoforms; D005557:Formaldehyde