Chip interacts with diverse homeodomain proteins and potentiates bicoid activity in vivo.

The Drosophila protein Chip potentiates activation by several enhancers and is required for embryonic segmentation. Chip and its mammalian homologs interact with and promote dimerization of nuclear LIM proteins. No known Drosophila LIM proteins, however, are required for segmentation, nor for expression of most genes known to be regulated by Chip. Here we show that Chip also interacts with diverse homeodomain proteins using residues distinct from those that interact with LIM proteins, and that Chip potentiates activity of one of these homeodomain proteins in Drosophila embryos and in yeast. These and other observations help explain the roles of Chip in segmentation and suggest a model to explain how Chip potentiates activation by diverse enhancers.

DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression.

High-level expression of the human growth hormone (hGH) gene is limited to somatotrope and lactosomatotrope cells of the anterior pituitary. We previously identified a locus control region (LCR) for the hGH gene composed of four tissue-specific DNase I-hypersensitive sites (HS) located between -14.6 kb and -32 kb 5' to the hGH transcription start site that is responsible for establishing a physiologically regulated chromatin domain for hGH transgene expression in mouse pituitary. In the present study we demonstrated that the LCR mediates somatotrope and lactosomatotrope restriction on an otherwise weakly and diffusely expressed hGH transgene. The subregion of the LCR containing the two pituitary-specific HS, HSI and HSII (-14.6 to -16.2 kb relative to the hGH promoter and denoted HSI,II), was found to be sufficient for mediating somatotrope and lactosomatotrope restriction, for appropriately timed induction of hGH transgene expression between embryonic days 15.5 and 16.5, and for selective extinction of hGH expression in mature lactotropes. When studied by cell transfection, the HSI,II fragment selectively enhanced transcription in a presomatotrope-derived cell line, although at levels (2- to 3-fold) well below that seen in vivo. The LCR activity of the HSI,II element was therefore localized by scoring transgene expression in fetal founder pituitaries at embryonic day 18.5. The data from these studies indicated that a 404-bp segment of the HSI,II region encodes a critical subset of LCR functions, including the establishment of a productive chromatin environment, cell-specific restriction and enhancement of expression, and appropriately timed induction of the hGH transgene during embryonic development.

Physical linkage of the human growth hormone gene cluster and the CD79b (Ig beta/B29) gene.

We have previously characterized a locus control region for the GH1 gene consisting of four DNase I hypersensitive sites (HS) located between 14.5 and 32 kb 5' to the GH1 gene transcription start site. Sequence analysis of the region between the GH1 gene and its most proximal HS (HSI) revealed a perfect match to the B-lymphocyte-specific CD79b gene. Restriction mapping and hybridization analysis of YAC and cosmid clones confirmed the close linkage of the CD79b gene to the hGH gene cluster and facilitated the assembly of a 100-kb physical map linking the hGH locus, the CD79b gene, and the more distant muscle-specific sodium channel alpha-subunit (SCN4A) gene.

Physical linkage of the human growth hormone gene cluster and the skeletal muscle sodium channel alpha-subunit gene (SCN4A) on chromosome 17.

The human growth hormone (GH) locus, a cluster of five genes, spans 47 kb on chromosome 17q22-q24. The skeletal muscle sodium channel alpha-subunit locus (SCN4A), a 32.5-kb gene, has previously been mapped to 17q23.1-q25.3. We demonstrate that both the GH gene cluster and the SCN4A gene colocalize to a single 525-kb yeast artificial chromosome (YAC) containing DNA derived from human chromosome 17. Restriction maps of two cosmids encompassing the 5' terminus of the GH locus and including up to 40 kb of 5'-flanking sequences demonstrate a perfect 20-kb overlap with previously published maps of the SCN4A gene. A 720-bp DNA segment, encompassing sequences 32.3 to 31.6 kb 5' to GH, was sequenced and found to be identical to exon 14 of SCN4A. These data demonstrate that the SCN4A gene and the entire GH gene cluster are contained within 100 kb on chromosome 17 and are separated by only 21.5 kb. Remarkably, this physical linkage between GH and SCN4A also reveals that multiple elements critical to tissue-specific transcriptional activation of the GH gene lie within the SCN4A gene.

In vitro evaluation of the differential cytotoxicity of keto-C-glycosides toward normal and malignant cells.

In a previous work, we have shown that some members of the family of keto-C-glycosides (KCGs) possess interesting biological properties as they exhibited cytotoxic effects at the nanomolar level on malignant cells. In this report, we selected six KCGs in order to investigate their selective cytotoxicity on several malignant epithelial and lymphoblastoid cells, as well as on their normal counterparts. For this purpose, we compared the activities of KCGs upon hepatoma cells and hepatocytes and upon lymphoma cells, normal lymphocytes and bone marrow cells. The tested drugs showed real discriminating cytotoxic effects since the cytotoxicity was several log greater on malignant than on non malignant cells. An in vitro comparative study of KCGs and some conventional chemotherapeutic agents showed that two of them were more potent than 5-fluorouracil, cis-platinum and etoposide. It is interesting to note that KCGs showed very low cytotoxic effects on either murine splenocytes, human peripheral blood lymphocytes or human bone marrow cells, indicating a weak immunosuppressive activity. The results presented here strongly suggest the selective cytotoxic activity of KCGs toward tumoral cells.