The NF-M transcription factor is related to C/EBP beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells.

Retroviral oncogenes encode nuclear regulators of gene expression or signal transduction molecules, such as protein kinases, which stimulate the activity of cellular transcription factors. Here we describe the cloning of NF-M, a myeloid-specific transcription factor related to C/EBP beta, which is a target of activated protein kinases. NF-M stimulates the expression of the gene encoding cMGF, a myeloid cell-specific growth factor, creating an autocrine growth loop crucial to oncogene transformation of myeloid cells. The NF-M protein bound directly to the cMGF gene promoter and activated its transcription, even in erythroid cells where the promoter is usually inactive. In addition, a truncated, dominant-negative form of NF-M inhibited cMGF expression in macrophages, indicating that NF-M is required for the normal activation of the gene. When multipotent hematopoietic progenitor cells were stimulated to differentiate, NF-M expression was induced at a very early stage, suggesting that the transcription factor plays a role in lineage commitment. The stimulation of transformed myelomonocytic cells or of normal peripheral blood macrophages with kinases or LPS or TPA respectively, led to the rapid redistribution of NF-M protein from the cell bodies to the nucleus, consistent with the notion that NF-M was directly affected by such treatments. Our data indicate that NF-M plays a key role in myelomonocytic differentiation, in signal transduction during macrophage activation and in the development of myelogenous leukemia.

Synergism in ternary complex formation between the dimeric glycoprotein p67SRF, polypeptide p62TCF and the c-fos serum response element.

Transcriptional regulation of the c-fos proto-oncogene requires the serum response element (SRE) which is complexed by a multi-protein assembly observed both in vitro and in vivo. Two protein factors, p67SRF and p62TCF (previously called p62), are required to interact with the SRE for efficient induction of c-fos by serum. By quantitative band shift electrophoresis we measure at least a 50-fold increase in SRE affinity for p67SRF/p62TCF over p67SRF alone. Stoichiometrically we determine that the ternary complex with p62TCF involves p67SRF in dimeric form. We demonstrate that p67SRF is a glycosylated nuclear transcription factor carrying terminal N-acetylglucosamine (GlcNAc) as a post-translational modification. A proteolytic limit digestion product, approximately 13 kd in size, was generated from the p67SRF-SRE complex. This p67SRF-core domain binds SRE, can dimerize with p67SRF and is still able to form a ternary complex with p62TCF. Therefore, three functional activities can be ascribed to this small p67SRF-core domain: specific DNA binding, dimerization and interaction with p62TCF. We demonstrate that these functions map within the p67SRF core fragment containing the region between amino acids 93 and 222.

Topoisomer gel retardation: detection of anti-Z-DNA antibodies bound to Z-DNA within supercoiled DNA minicircles.

Small DNA fragments of approximately 350 bp in length, either with or without d(CG)n tracts, are ligated into underwound DNA minicircles to generate topoisomeric rings with different topological linking numbers, Lk. These minicircles, differing by an Lk of one, can be separated by acrylamide gel electrophoresis. Furthermore, electrophoresis can be used to reveal DNA double helix conformational changes that are induced by supercoiling, such as left-handed Z-DNA. When anti-Z-DNA antibodies are added to such minicircles, their binding leads to a selective retardation of the electrophoretic migration of the Z-DNA containing circles. This effect is not seen with relaxed minicircles and those with insufficient torsional stress to induce a conformational transition. Thus the technique of 'topoisomer gel retardation' presents a very sensitive assay for the identification of proteins that selectively bind to DNA conformations stabilized by negative DNA supercoiling.

Disassembly and domain structure of the proteins in the signal-recognition particle.

The signal-recognition particle (SRP) is a ribonucleoprotein (RNP) complex consisting of six different polypeptide chains and a 7SL RNA. It participates in initiating the translocation of proteins across the membrane of the endoplasmic reticulum. SRP was disassembled in 2 M KCl into three components, one RNP composed of 7SL RNA and the 54-kDa and 19-kDa proteins, and two heterodimers consisting of the 72/68-kDa and the 14/9-kDa proteins respectively. The 54-kDa protein could be released from the RNP subparticle by chromatography on DEAE-Sepharose in Mg2+-depleted buffer, while the 19-kDa protein remained bound to the 7SL RNA. The domain structure of SRP proteins was probed by using mild elastase treatment and protein-specific antibodies. It was found that the 72, 68, 54 and 19-kDa SRP proteins were proteolytically processed in distinct steps. Most remarkably a protein fragment of 55-kDa, generated from the 72-kDa SRP protein, and a 35-kDa fragment from the 54-kDa SRP protein were both released from the RNP particle. Fragments generated from the 68-kDa protein and detectable with the anti-(68-kDa protein) antibody remained associated with the RNP particle. Cleavage of the SRP proteins by elastase at 2.5 micrograms/ml resulted in partial loss of activity, while 10 micrograms/ml caused complete inactivation of the particle. Neither the elongation arrest of IgG light chain nor its translocation across SRP-depleted microsomal membranes was promoted. The implications of these results on the possible interaction between the SRP subunits are discussed.