Molecular and cellular characterization of the Nmp4 nuclear matrix transcription factors

The regulation of collagen and matrix metalloproteinase expression in connective tissue is critical to normal bone physiology. We have discovered a family of proteins, Nmp4, that contribute to the mechanisms that underlie the osteoblast production of bone extracellular matrix (ECM). Splice variants of the Nmp4 gene include Cys₂His₂ zinc finger nuclear matrix transcription factors that regulate the type I collagen α1(I) polypeptide chain (COL1A1) promoter and several matrix metalloproteinase (MMP) genes. To better understand the functional role of Nmp4 in ECM gene regulation, we mapped the subcellular localization and the functional domains of these proteins. Immunocytochemistry indicates that Nmp4 isoforms reside in both the nucleus and the cytoplasm. In the cytoplasm, Nmp4 proteins localize to the mitochondria and golgi apparatus. In the nucleus, at least four Nmp4 isoforms are associated with the nuclear matrix. Western analysis using Nmp4 antibodies revealed a complex profile of protein bands in the nuclear, mitochondrial, and cytosolic fractions. Several of these bands were specific to particular intracellular domains. We further identified the zinc finger domain as a functional motif necessary for both nuclear localization and nuclear matrix targeting. To date, Nmp4 are the only Cys₂His ₂ zinc finger proteins known to bind homopolymeric poly(dA:dT) DNA. This unusual consensus sequence is found in numerous promoters of genes that encode extracellular matrix proteins and enzymes, including COL1A1 and several MMPs. Gel shift analysis showed that the Nmp4 zinc finger domain is necessary to mediate association with the minor groove of the COL1A1/ MMP DNA consensus element. Transactivation analysis indicates that allosteric effects, induced upon zinc finger association with the homopolymeric poly(dA:dT) minor groove, confer context-specific transactivation capacity to this unusual family of proteins. The significance of the present data is that it necessarily enlarges the known Nmp4 family from a group of nuclear matrix transcription factors that regulate ECM expression to a more far ranging class of intracellular proteins with a broader functional capacity in the osteoblast.