James Douglas Engel

Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation

Transcription factor Nrf2 (encoded by Nfe2l2) regulates a battery of detoxifying and antioxidant genes, and Keap1 represses Nrf2 function. When we ablated Keap1, Keap1-deficient mice died postnatally, probably from malnutrition resulting from hyperkeratosis in the esophagus and forestomach. Nrf2 activity affects the expression levels of several squamous epithelial genes. Biochemical data show that, without Keap1, Nrf2 constitutively accumulates in the nucleus to stimulate transcription of cytoprotective genes. Breeding to Nrf2-deficient mice reversed the phenotypic Keap1 deficiencies. These experiments show that Keap1 acts upstream of Nrf2 in the cellular response to oxidative and xenobiotic stress.

DNA-binding specificities of the GATA transcription factor family.

Members of the GATA family of transcription factors, which are related by a high degree of amino acid sequence identity within their zinc finger DNA-binding domains, each show distinct but overlapping patterns of tissue-restricted expression. Although GATA-1, -2, and -3 have been shown to recognize a consensus sequence derived from regulatory elements in erythroid cell-specific genes, WGATAR (in which W indicates A/T and R indicates A/G), the potential for more subtle differences in the binding preferences of each factor has not been previously addressed. By employing a binding selection and polymerase chain reaction amplification scheme with randomized oligonucleotides, we have determined the binding-site specificities of bacterially expressed chicken GATA-1, -2, and -3 transcription factors. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA-2 and GATA-3 but only poorly by GATA-1. These studies suggest that all three GATA factors are capable of mediating transcriptional effects via a common erythroid consensus DNA-binding motif. Furthermore, GATA-2 and GATA-3, because of their distinct expression patterns and broader DNA recognition properties, may be involved in additional regulatory processes beyond those of GATA-1. The definition of an alternative GATA-2-GATA-3 consensus sequence may facilitate the identification of new target genes in the further elucidation of the roles that these transcription factors play during development.

MafA Is a Key Regulator of Glucose-Stimulated Insulin Secretion

ABSTRACT MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic β cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.

Tissue-specific DNA cleavages in the globin chromatin domain introduced by DNAase I

Abstract Using recombinant chicken DNA clones as probes, we have investigated the DNAase I sensitivity of chromosomal DNA regions bordering the α- and β-globin structural genes. By both a blot hybridization assay and solution hybridization, we find that regions around these globin genes are preferentially sensitive (relative to the ovalbumin gene) to DNAase I after mild digestion of isolated red cell nuclei. These regions are resistant in cells that do not express globin. The preferential DNAase I sensitivity extends to at least 8 kb on the 3′ side of the β-globin gene cluster and to 6 or 7 kb on the 5′ side, where relatively resistant DNA fragments have been identified. Using low levels of DNAase I to titrate the sensitivities of coding and adjacent noncoding regions, it was observed that coding regions are organized into a very sensitive structure, while adjacent noncoding regions are organized into a moderately sensitive structure. The blot hybridization assay has also revealed that DNAase I introduces specific, double-stranded cuts into both the α- and β-globin gene clusters. Many of these cuts are tissue-specific. Several α gene-specific sites occur toward the 3′ side of the α-coding sequences. The β sites are different in embryonic and adult red cells. In embryonic cells, the cut occurs near the 5′ end of an embryonic β gene, while in adult cells there are two cuts, one at approximately 2 kb and the other at approximately 6 kb from the 5′ side of an adult gene. Based on the observation that the general region around the origin for replication and promotors for transcription in the SV40 minichromosome is also very sensitive to specific, double-stranded scissions by DNAase I, we speculate that the specific cuts in the globin domain may be structural modifications of the chromatin that are associated with origins for DNA replication or promotors for transcription.

Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system

Mouse embryos deficient in Gata3 die by 11 days post coitum (d.p.c.) from pathology of undetermined origin. We recently showed that Gata3-directed lacZ expression of a 625-kb Gata3 YAC transgene in mice mimics endogenous Gata3 expression, except in thymus and the sympathoadrenal system. As this transgene failed to overcome embryonic lethality (unpublished data and ref. 3) in Gata3−/− mice, we hypothesized that a neuroendocrine deficiency in the sympathetic nervous system (SNS) might cause embryonic lethality in these mutants. We find here that null mutation of Gata3 leads to reduced accumulation of Th (encoding tyrosine hydroxylase, Th) and Dbh (dopamine β-hydroxylase, Dbh) mRNA, whereas several other SNS genes are unaffected. We show that Th and Dbh deficiencies lead to reduced noradrenaline in the SNS, and that noradrenaline deficiency is a proximal cause of death in mutants by feeding catechol intermediates to pregnant dams, thereby partially averting Gata3 mutation-induced lethality. These older, pharmacologically rescued mutants revealed abnormalities that previously could not be detected in untreated mutants. These late embryonic defects include renal hypoplasia and developmental defects in structures derived from cephalic neural crest cells. Thus we have shown that Gata3 has a role in the differentiation of multiple cell lineages during embryogenesis.

Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer.

A family of transcriptional activators has recently been identified in chickens; these transcriptional activators recognize a common consensus motif (WGATAR) through a conserved C4 zinc finger DNA-binding domain. One of the members of this multigene family, cGATA-3, is most abundantly expressed in the T-lymphocyte cell lineage. Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms. The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer. We infer that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation.

Developmental regulation of β-globin gene switching

Abstract A chicken erythroid cell-specific enhancer is located in the intergenic region between the adult β- and embryonic e-globin genes. In this paper we show that the β-globin enhancer stimulates transcription of both genes. e-Globin is, however, inappropriately regulated since it is expressed in both embryonic and adult red blood cells. Appropriate stage-specific regulation is observed for both genes when they are present on one plasmid. By analysis of deletion and substitution mutants, we conclude that β-globin tissue- and developmental stage-specific regulation is mediated by interaction of the β-globin enhancer with a positive regulatory element within the adult β-globin promoter, the developmental stage selector element (SSE).

Quantitation of RNA using the polymerase chain reaction

Sequential use of reverse transcriptase and the polymerase chain reaction (RT-PCR) permits rapid and sensitive detection of specific RNAs. However, the greatest advantage of RT-PCR, its remarkable sensitivity, has also limited its usefulness in quantitative applications, since the effects of minor variations in reaction conditions from sample to sample are greatly magnified during the amplification process. Several recently developed techniques circumvent this problem, allowing accurate quantitation of RNA using RT-PCR.

Genetic Evidence that Small Maf Proteins Are Essential for the Activation of Antioxidant Response Element-Dependent Genes

ABSTRACT While small Maf proteins have been suggested to be essential for the Nrf2-mediated activation of antioxidant response element (ARE)-dependent genes, the extent of their requirement remains to be fully documented. To address this issue, we generated mafG::mafF double-mutant mice possessing MafK as the single available small Maf. Induction of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene was significantly impaired in double-mutant mice treated with butylated hydroxyanisole, while other ARE-dependent genes were less affected. Similarly, in a keap1-null background, where many of the ARE-dependent genes are constitutively activated in an Nrf2-dependent manner, only a subset of ARE-dependent genes, including NQO1, were sensitive to a simultaneous deficiency in MafG and MafF. Examination of single and double small maf mutant cells revealed that MafK also contributes to the induction of ARE-dependent genes. To obtain decisive evidence, we established mafG::mafK::mafF triple-mutant fibroblasts that completely lack small Mafs and turned out to be highly susceptible to oxidative stress. We found that induction in response to diethyl maleate was abolished in a wider range of ARE-dependent genes in the triple-mutant cells. These data explicitly demonstrate that small Mafs play critical roles in the inducible expression of a significant portion of ARE-dependent genes.

Looping, Linking, and Chromatin Activity: New Insights into β-globin Locus Regulation

Given our association with the looping and competition models (Choi and Engel 1988xChoi, O and Engel, J.D. Cell. 1988; 55: 17–26Abstract | Full Text PDF | PubMed | Scopus (166)See all ReferencesChoi and Engel 1988), we can readily incorporate these new data into predictive corollaries to the competition hypothesis; however, we note that these predictions can be applied to support linking as well. If the human locus is defined by intergenic elements that promote or restrict the ability of the LCR to activate transcription of genes within subdomains (Figure 2Figure 2), a primary layer of chromatin differentiation could serve to augment the positive or negative regulatory function of discrete transcriptional cis elements neighboring the genes. For example, we showed that deletion of the e-globin gene silencer from a YAC has very modest effects on e-globin gene expression in adult erythroid cells (Liu et al. 1997xLiu, Q, Bungert, J, and Engel, J.D. Proc. Natl. Acad. Sci. USA. 1997; 94: 169–174Crossref | PubMed | Scopus (43)See all ReferencesLiu et al. 1997), while the effect of its deletion in transgenic mice bearing only fragments of the locus is quite dramatic (Li et al. 1998xLi, Q, Blau, C.A, Clegg, C.H, Rohde, A, and Stamatoyannopoulos, G. J. Biol. Chem. 1998; 273: 17361–17367Crossref | PubMed | Scopus (19)See all ReferencesLi et al. 1998). We originally proposed that functional redundancy of cis elements could account for the lack of effects detected when the small e-globin gene silencer was deleted from a large transgenic YAC. The report of Gribnau et al. 2000xGribnau, J, Diderich, K, Pruzina, S, Calzolari, R, and Fraser, P. Mol. Cell. 2000; 5: 377–386Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesGribnau et al. 2000 support an additional alternative: that discrete cis element mutations are insufficient to confer full expressivity of local “silencer” or “enhancer” activities, and that these gene proximal elements can only act at the proper developmental stage when they do so within the context of regionally imposed (metastable) chromatin constraints. Thus, deletion of the e-globin silencer may not confer a significant positive transcriptional effect on the e-globin gene when examined in the context of the whole locus because the chromatin subdomain harboring e-globin is suppressed in adult erythroid cells (i.e., the primary effect of chromatin suppression dominates a secondary effect of silencer deletion).Figure 2Stage-Specific Activation within Subdomains of the β-globin Locus by an LCR HolocomplexChromatinized DNA status (either relatively open or closed, which is meant to reflect general sensitivity to DNase I) is depicted as spring shapes with the more condensed chromatin represented as tighter coiling. Individual LCR HS sites and genes are shown as spheres (shaded from yellow to red) and cylinders (blue), respectively. Purple lines show possible structural constraints generated by linkage of two putative boundary elements (purple spheres) and how this might contribute to the formation of chromosomal subdomains. The architectural constraints within the locus and/or the LCR holocomplex configuration may also influence which gene(s) can be activated by LCR at specific developmental stages (green arrows representing the domains that are rendered more accessible after differential orientation of the LCR in the primitive and definitive erythroid stages).View Large Image | View Hi-Res Image | Download PowerPoint SlideClearly there are a host of challenges remaining in this field, not the least of which concerns our ability to clearly define the caveats and possible limitations in interpreting each new study within the context of a huge body of existing literature. Lest we become complacent, its important to remember that each new year for the past three decades has been accompanied by often startling revelations about how globin diseases arise, how the locus is regulated, how LCRs function, and, most recently, how chromatin structure impinges on differential modulation of the genes. The formidable tasks of devising clear distinctions among the possible models, as well as executing defining experiments to distinguish among them, is still a challenge we face daily, and often optimistically.*E-mail: d-engel@nwu.edu.