Modulation of PAX6 homeodomain function by the paired domain.

PAX6 is required for proper development of the eye, central nervous system, and nose. PAX6 has two DNA binding domains, a glycine-rich region that links the two DNA binding domains, and a transactivation domain. There is evidence that the different DNA binding domains of PAX6 have different target genes. However, it is not clear if the two DNA binding domains function independently. We have studied the effect of structural changes in the paired domain on the function of PAX6 mediated through its homeodomain. The R26G and I87R mutations have been reported in different human patients with clinically different phenotypes and are in the N- and the C-terminal halves of the paired domain, respectively. Surprisingly, we found that the I87R mutant protein not only lost the transactivation function but also failed to bind DNA by either of its DNA binding domains. In contrast, the R26G mutant protein lost DNA binding through its paired domain but had greater DNA binding and transactivation than wild-type PAX6 on homeodomain binding sites. Like R26G, the 5a isoform showed higher DNA binding than wild-type PAX6. This study demonstrates that the two subdomains of the paired domain influence the function of the homeodomain differentially and also provides an explanation for the difference in phenotypes associated with these mutations.

Tissue-specific and developmental regulation of the rat insulin II gene enhancer, RIPE3, in transgenic mice.

The rat insulin II gene enhancer, RIPE3 (-126 to -86), mediates beta-islet cell-specific activity in transfection assays. To investigate the in vivo activity of RIPE3, we generated mice carrying a transgene consisting of three copies of RIPE3 linked to a minimal chicken ovalbumin promoter in conjunction with sequences encoding the human growth hormone gene. 13 transgenic mice were obtained, 11 of which expressed the transgene, as determined by serum radioimmunoassay for human growth hormone. Expression of the transgene was assessed for cell specificity by immunocytochemistry. The pancreatic islet cells invariably stained for growth hormone, while the acinar and ductal cells did not. Staining of adjacent sections for insulin, glucagon, and somatostatin revealed that growth hormone was expressed in the beta-cell in all of the mice analyzed, but in some mice alpha-cells also contained growth hormone. RNase protection analysis revealed that the tissues that consistently express the transgene in these animals are the pancreas and brain. Developmental analysis revealed that the transgene was expressed in the pancreatic bud at embryonic day 9.5, corresponding to the temporal expression pattern of the insulin gene. These results signify that an element as small as 41 base pairs is capable of regulating pancreatic temporal and spatial gene expression in vivo.

BETA3, a novel helix-loop-helix protein, can act as a negative regulator of BETA2 and MyoD-responsive genes.

Using degenerate PCR cloning we have identified a novel basic helix-loop-helix (bHLH) transcription factor, BETA3, from a hamster insulin tumor (HIT) cell cDNA library. Sequence analysis revealed that this factor belongs to the class B bHLH family and has the highest degree of homology with another bHLH transcription factor recently isolated in our laboratory, BETA2 (neuroD) (J. E. Lee, S. M. Hollenberg, L. Snider, D. L. Turner, N. Lipnick, and H. Weintraub, Science 268:836-844, 1995; F. J. Naya, C. M. M. Stellrecht, and M.-J. Tsai, Genes Dev. 8:1009-1019, 1995). BETA2 is a brain- and pancreatic-islet-specific bHLH transcription factor and is largely responsible for the tissue-specific expression of the insulin gene. BETA3 was found to be tissue restricted, with the highest levels of expression in HIT, lung, kidney, and brain cells. Surprisingly, despite the homology between BETA2 and BETA3 and its intact basic region, BETA3 is unable to bind the insulin E box in bandshift analysis as a homodimer or as a heterodimer with the class A bHLH factors E12, E47, or BETA1. Instead, BETA3 inhibited both the E47 homodimer and the E47/BETA2 heterodimer binding to the insulin E box. In addition, BETA3 greatly repressed the BETA2/E47 induction of the insulin enhancer in HIT cells as well as the MyoD/E47 induction of a muscle-specific E box in the myoblast cell line C2C12. In contrast, expression of BETA3 had no significant effect on the GAL4-VP16 transcriptional activity. Immunoprecipitation analysis demonstrates that the mechanism of repression is via direct protein-protein interaction, presumably by heterodimerization between BETA3 and class A bHLH factors.

Molecular characterization of the rat insulin enhancer-binding complex 3b2. Cloning of a binding factor with putative helicase motifs.

Cell-specific expression of the rat insulin II gene is in part mediated through an element located in the 5'-flanking region. The element, termed RIPE3b (-126 to -101), confers beta-cell-specific expression in conjunction with an adjacent element RIPE3a (-110 to -86). Here we report the characterization of one of the RIPE3b-binding complexes, 3b2. UV cross-linking analysis demonstrated that it is composed of at least three polypeptides: p58, p62, and p110. Furthermore, a cDNA was isolated via expression screening for binding to RIPE3b. Sequence analysis reveals that the encoded protein, designated Rip-1, possessed putative helicase motifs and a potential transcription activation domain. Overexpression of Rip-1 in cells greatly enhances the 3b2 binding complex, suggesting that Rip-1 is involved in the binding of 3b2.

Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor.

The insulin gene is one of the best paradigms of tissue-specific gene expression. It is developmentally regulated and is expressed exclusively in the pancreatic beta-cell. This restricted expression is directed by a tissue-specific enhancer, within the promoter, which contains an E-box sequence. The insulin E-box binds an islet-specific protein complex, termed 3a1. E-boxes bind proteins belonging to the basic helix-loop-helix (bHLH) family of transcription factors. The bHLH proteins function as potent transcriptional activators of tissue-specific genes by forming heterodimers between ubiquitous and cell-restricted family members. In addition, the cell-restricted bHLH members play an important role in specifying cell fate. To isolate the tissue-specific bHLH factor controlling insulin gene expression and study its role in islet cell differentiation, a modified yeast two-hybrid system was utilized to clone a novel bHLH factor, BETA2 (beta-cell E-box trans-activator 2), from a hamster insulin tumor (HIT) cell cDNA library. Northern analysis demonstrates that high-level expression of the BETA2 gene is restricted to pancreatic alpha- and beta-cell lines. As expected of tissue-specific bHLH members, BETA2 binds to the insulin E-box sequence with high affinity as a heterodimer with the ubiquitous bHLH factor E47. More importantly, antibody supershift experiments clearly show that BETA2 is a component of the native insulin E-box-binding complex. Transient transfection assays demonstrate that the BETA2/E47 heterodimer synergistically interacts with a neighboring beta-cell-specific complex to activate an insulin enhancer. In contrast, other bHLH factors such as MyoD and E47, which can bind to the insulin E-box with high affinity, fail to do so. Thus, a unique, cooperative interaction is the basis by which the insulin E-box enhancer discriminates between various bHLH factors to achieve tissue-specific activation of the insulin gene.

Transcriptional regulation of a hematopoietic proteoglycan core protein gene during hematopoiesis.

The expression of a hematopoietic proteoglycan core protein (HpPG) gene is up-regulated during the early stages of myeloblast differentiation at a time point coinciding with the beginning of granule genesis (Stellrecht, C. M., Mars, W. M., Miwa, H., Beran, M., and Saunders, G. F. (1991) Differentiation 48, 127-135). The mechanism of this up-regulatory event was investigated by analyzing the expression and regulation of the HpPG gene during the differentiation of the pluripotent hematopoietic cell line, K562. The level of HpPG gene expression in these cells was up-regulated approximately 10-fold upon 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation, as measured by Northern blot analysis. The HpPG gene's expression remained relatively unchanged during hemin-induced erythroid differentiation, further demonstrating the specificity of this regulatory event for granule-producing cell lineages. The effect of TPA induction on HpPG gene expression was also assessed during the differentiation of the myeloid leukemia cell line, HL-60. The expression of the gene was down-regulated approximately 20-fold upon TPA-induced differentiation into macrophage-like cells. In contrast, only a minimal decrease in HpPG gene expression was detected in gamma-interferon-induced monocyte differentiation. No detectable changes in expression levels were seen in HL-60 cells differentiated into granulocytes with retinoic acid or dimethyl sulfoxide. Nuclear runoff analysis demonstrated that the regulation of the HpPG gene is under transcriptional control in both TPA-induced differentiation systems.

Expression pattern of a hematopoietic proteoglycan core protein gene during human hematopoiesis.

Expression of the hematopoietic proteoglycan core protein (HpPG) gene was examined in normal peripheral blood, normal bone marrow, and leukemic peripheral blood leukocytes samples to assess the expression pattern of the HpPG gene in these cells and to ascertain points of regulation of this gene during hematopoiesis. In situ hybridization to normal bone marrow and peripheral blood leukocytes demonstrated that the gene was expressed in the promyelocytes at a approximately two fold greater level than in the segmented neutrophils and the expression decreased as the granulocytes matured. The ratio of expression in the other leukocytes to expression in the segmented neutrophils were as follows: eosinophils/basophils approximately 7; monocytes approximately 2; lymphocytes less than 1. Expression of the HpPG gene during myeloblast differentiation was assessed by Northern blot analysis of acute myelogenous leukemia (AML) RNA samples. The expression of this gene, when compared to the levels in HL-60 cells, was approximately ten fold lower in the poorly differentiated blast cells obtained from three AML patients classified M"0". Conversely, the expression in the more differentiated blast cells obtained from 10 of 11 AML patients classified as M1 and M2 were at levels similar to the levels in HL-60 cells. The expression level found in eight lymphoid leukemias was approximately ten fold or more lower than in HL-60 cells. Gene copy number determination confirmed that the HpPG gene is present in one copy per haploid genome. Thus the HpPG gene's expression pattern denotes a single copy gene being differentially expressed during hematopoiesis with initial regulation occurring very early in this developmental process and an additional up-regulatory event occurring during granule genesis.

Localization of an abundant myeloid mRNA to individual leukocytes in mixed cell populations.

Differential screening of cDNA libraries with radiolabeled RNAs isolated from various sources provides a convenient way to identify cDNA clones representing RNAs that are more (or less) abundant in selected tissues. This strategy was previously used to isolate cDNA clones representing poly(A+)RNAs (mRNA) that are abundant in leukocytes from chronic myelogenous leukemia (CML) patients. One limitation with the initial experiments was that the RNAs were isolated from heterogenous cell populations and it was impossible to distinguish whether all of the cells were producing the abundant mRNAs or if a subset of the cell population was responsible for the majority of the RNA species. To resolve this important issue, we have directly hybridized radiolabeled cDNAs to the cellular RNAs of intact, morphologically distinguishable, primary hematopoietic cells. In the present study clone pC-A3, which represents an mRNA species that is abundant in the chronic phase of CML, was used to examine three samples from normal bone marrow, one sample from normal peripheral blood, and four samples from peripheral blood of leukemic patients (one Ph1 + AL, two CML in compensated phase, and one CML in accelerated phase). The results show that while C-A3 gene expression is detectable in cells from the granulocytic, monocytic, and lymphoid lineage, its relative abundance peaks at the level of the neutrophilic myelocytes and promyelocytes. Earlier myeloid precursors like myeloblasts or mature neutrophilic granulocytes show less labeling. Further, all maturation stages of eosinophils (Eos) and basophil (Baso) are highly labeled. This finding reinforces recent evidence that Eos and Baso share a common progenitor and suggests that these two cell types may have a stronger role than previously noted in the prominent myeloproliferative response that is characteristic of CML.

Localization of an abundant myeloid-related sequence.

Differential screening of a CML-derived cDNA library was used to isolate a cDNA clone representing an abundant mRNA in leukocytes of chronic phase, chronic myelogenous leukemia (CML) patients. This myeloid-related sequence (mrs) comprises approximately 2% of the chronic phase, CML mRNA. Hybridization of the radiolabeled cDNA to the cellular RNAs of intact, morphologically distinguishable, primary hematopoietic cells indicates that the primary cells producing mrs RNA are eosinophils (Eo), basophils (Baso), promyelocytes (Pro) and myelocytes (Mye). In the neutrophilic series, the abundance of mrs RNA decreases as the cells mature. The mrs RNA potentially encodes a 93 amino acid protein that has not been previously described. It has been localized to chromosome 8, region 8q21.1-23.