Susanne Baumhueter

Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.

During development cell types arise through the activation or repression of classes of specific genes. One hypothesis is that this phenomenon is realized by tissue-specific factors playing a role at the transcription level. Recently we have described a liver-specific nuclear protein, hepatocyte nuclear factor 1, that appears to be involved in the transcription of the fibrinogen and alpha 1-antitrypsin genes. In this report we describe the purification of hepatocyte nuclear factor 1 and demonstrate that it interacts with essential promoter regions of many liver-specific genes, including albumin, alpha-fetoprotein, and transthyretin. This finding suggests that hepatocyte nuclear factor 1 could be one factor necessary for establishing the liver phenotype. We also show that this protein binds to the promoter of the surface-antigen gene of the hepatitis B virus, a virus characterized by a high degree of hepatotropism.

The coordinate expression of genes during development and differentiation is thought to be accomplished by common transcription factors operating on the promoters of families of coexpressed genes. HNF-1 is a transcriptional factor involved in the expression of genes in the liver and was originally defined as playing a major role in coordinating the expression of the linked fibrinogen genes. We have isolated cDNA clones for HNF-1 using oligonucleotides prepared to the sequence of the purified protein. The sequence of HNF-1 shares homeo domain, as well as short acidic and basic sequences with the POU family of transcriptional activators. Peptides from the protein interacting with the albumin proximal element, or B box (APF), and the factor interacting with the alpha 1-antitrypsin promoter (LF-B1) are found in the predicted sequence of HNF-1. HNF-1 mRNA is not present in the dedifferentiated hepatoma variant, C2, but reappears upon selection for gluconeogenesis coincident with the re-expression of liver-specific genes. Finally, the mRNA is not present in somatic cell hybrids in which liver-specific gene expression is extinguished. In contrast to earlier published results, we find that in addition to being present in the liver, HNF is expressed in the kidney, intestine, and spleen, but not in other tissues. This pattern of expression mirrors the complex pattern of expression of many genes, such as alpha-fetoprotein, alpha 1-antitrypsin, and fibrinogen, whose promoters contain HNF-1 sites. These data indicate that HNF-1 is a more broadly acting transcription factor than has been indicated by previous work.

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34-null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34-null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.