Matthias Cramer

The human transcriptional repressor protein NAB1: expression and biological activity.

The zinc finger protein early growth response 1 (Egr-1) is a transcriptional activator involved in the regulation of growth and differentiation. Egr-1 has a large activating domain and three zinc finger motifs that function as a DNA binding region. We show here that a third functional domain of the Egr-1 protein, localized between the extended activation domain and the zinc finger DNA binding region, acts as a transcriptional repressor domain when fused to a heterologous DNA binding domain (DBD). Through protein-protein interaction this inhibitory domain of Egr-1 brings the transcriptional corepressor NAB1 in close proximity to the transcription unit. NAB1 is expressed ubiquitously in human cell lines as shown by RNase protection mapping. Overexpression studies revealed that NAB1 is able to completely block transcription mediated by Egr-1. In addition, the transcriptional repression activity of a fusion protein containing the inhibitory domain of Egr-1 and the DBD of the yeast transcription factor GAL4 was increased by overexpression of NAB1. A fusion protein consisting of the DBD of GAL4 and the coding region of human NAB1 repressed transcription from model promoters with engineered upstream GAL4 binding sites. The GAL4-NAB1 fusion protein functioned from proximal and distal positions indicating that NAB1 displays transcriptional repressor activity at any position within the transcription unit. Thus, the biological function of the inhibitory domain of Egr-1 is solely to provide a docking site for NAB1 via protein-protein interaction.

Zebrafish hhex, nk2.1a, and pax2.1 regulate thyroid growth and differentiation downstream of Nodal-dependent transcription factors

During zebrafish development, the thyroid primordium initiates expression of molecular markers such as hhex and nk2.1a in the endoderm prior to pharynx formation. As expected for an endodermally derived organ, initiation of thyroid development depends on Nodal signalling. We find that it also depends on three downstream effectors of Nodal activity, casanova (cas), bonnie and clyde (bon), and faust (fau)/gata5. Despite their early Nodal-dependent expression in the endoderm, both hhex and nk2.1a are only required relatively late during thyroid development. In hhex and nk2.1a loss-of-function phenotypes, thyroid development is initiated and arrests only after the primordium has evaginated from the pharyngeal epithelium. Thus, like pax2.1, both hhex and nk2.1a have similarly late roles in differentiation or growth of thyroid follicular cells, and here, we show that all three genes act in parallel rather than in a single pathway. Our functional analysis suggests that these genes have similar roles as in mammalian thyroid development, albeit in a different temporal mode of organogenesis.

BIOLOGICAL ACTIVITY AND MODULAR STRUCTURE OF RE-1-SILENCING TRANSCRIPTION FACTOR (REST), A REPRESSOR OF NEURONAL GENES

The zinc finger protein RE-1-silencing transcription factor (REST)1 is a transcriptional repressor that represses neuronal genes in nonneuronal tissues. Transfection experiments of neuroblastoma cells using a REST expression vector revealed that synapsin I promoter activity is controlled by REST. The biological activity of REST was further investigated using a battery of model promoters containing strong promoters/enhancers and REST binding sites. REST functioned as a transcriptional repressor when REST binding motifs derived from the genes encoding synapsin I, SCG10, α1-glycine receptor, the β2-subunit of the neuronal nicotinic acetylcholine receptor, and the m4-subunit of the muscarinic acetylcholine receptor were present in the promoter region. No differences in the biological activity of these REST binding motifs tested were detected. Moreover, we found that REST functioned very effectively as a transcriptional repressor at a distance. Thus, REST represents a general transcriptional repressor that blocks transcription regardless of the location or orientation of its binding site relative to the enhancer and promoter. This biological activity could also be attributed to isolated domains of REST. Both repressor domains identified at the N and C termini of REST were transferable to a heterologous DNA binding domain and functioned from proximal and distal positions, similar to the REST protein.

Antigenic determinants shared between HLA-A, -B, -C antigens and H-2 class I molecules modified by bovine beta-2 microglobulin

The specificity of the mouse class I-specific antibody COB6-3 was examined in detail. It was found to react with the mouse class I molecules H-2Db, Kd, and Qa-2, and with human HLA-A, −B, −C antigens. The specificity pattern of COB6-3, despite its different origin, was similar to that of the monomorphic HLA class I-specific antibody W6/32. Cross-inhibition studies show that on human cells the antigenic determinants recognized by the two antibodies are situated close together and may be identical. On mouse cells, reactivity of both antibodies was generated upon replacement of mouse beta-2 microglobulin (B2m) with its bovine counterpart, but differences in specificity were observed using human B2m.

A structural somatic variant of the Kk antigen is generated by point mutation.

We have previously selected structural variants of the Kk antigen from a (C3 × D2)F1 T-cell lymphoma. Those mutants were identified by the loss of certain epitopes defined by monoclonal antibodies. The variant Kk molecule from HK13.S3 cells is no longer recognized by 40% of the trinitrophenyl-specific, Kk-restricted cytotoxic T lymphocytes. Here we report on the primary structure of the altered Kk molecules from the cell line HK13.S3. Comparison with the parental Kk reveals a single base pair exchange, GCG to GTG, that results in an alanine to valine exchange in position 40 of the protein. This observation emphasizes that minor structural alterations in class I molecules may have a strong effect on the H-2-restricted T-cell response.

A Monoclonal Antibody Induced by H-2 Syngeneic ConA Blasts: Its Reactivity Pattern with Mouse and Human MHC Class I Antigens

Class I antigens encoded by the major histocompatibility complex (MHC) are notable for their high level of interspecies homology. The monoclonal antibody COB6-3 highlights some aspects of this feature. The antibody’s production, characterization, and specificity are reviewed and summarized in this article.