Impairment of T cell development in deltaEF1 mutant mice.

Using the method of gene targeting in mouse embryonic stem cells, regulatory function of deltaEF1, a zinc finger and homeodomain-containing transcription factor, was investigated in vivo by generating the deltaEF1 mutant mice. The mutated allele of deltaEF1 produced a truncated form of the deltaEF1 protein lacking a zinc finger cluster proximal to COOH terminus. The homozygous deltaEF1 mutant mice had poorly developed thymi with no distinction of cortex and medulla. Analysis of the mutant thymocyte showed reduction of the total cell number by two orders of magnitude accompanying the impaired thymocyte development. The early stage intrathymic c-kit+ T precursor cells were largely depleted. The following thymocyte development also seemed to be affected as assessed by the distorted composition of CD4- or CD8-expressing cells. The mutant thymocyte showed elevated alpha4 integrin expression, which might be related to the T cell defect in the mutant mice. In the peripheral lymph node tissue of the mutant mice, the CD4-CD8+ single positive cells were significantly reduced relative to CD4+CD8-single positive cells. In contrast to T cells, other hematopoietic lineages appeared to be normal. The data indicated that deltaEF1 is involved in regulation of T cell development at multiple stages.

The delta-crystallin enhancer-binding protein delta EF1 is a repressor of E2-box-mediated gene activation.

The repressor delta EF1 was discovered by its action on the DC5 fragment of the lens-specific delta 1-crystallin enhancer. C-proximal zinc fingers of delta EF1 were found responsible for binding to the DC5 fragment and had specificity to CACCT as revealed by selection of high-affinity binding sequences from a random oligonucleotide pool. CACCT is present not only in DC5 but also in the E2 box (CACCTG) elements which are the binding sites of various basic helix-loop-helix activators and also the target of an unidentified repressor, raising the possibility that delta EF1 accounts for the E2 box repressor activity. delta EF1 competed with E47 for binding to an E2 box sequence in vitro. In lymphoid cells, endogenous delta EF1 activity as a repressor was detectable, and exogenous delta EF1 repressed immunoglobulin kappa enhancer by binding to the kappa E2 site. Moreover, delta EF1 repressed MyoD-dependent activation of the muscle creatine kinase enhancer and MyoD-induced myogenesis of 10T1/2 cells. Thus, delta EF1 counteracts basic helix-loop-helix activators through binding site competition and fulfills the conditions of the E2 box repressor. In embryonic tissues, the most prominent site of delta EF1 expression is the myotome. Myotomal expression as well as the above results argues for a significant contribution of delta EF1 in regulation of embryonic myogenesis through the modulation of the actions of MyoD family proteins.

Organization of the gene encoding transcriptional repressor deltaEF1 and cross-species conservation of its domains.

DeltaEF1 (delta-crystallin/E2-box factor 1) is a widely distributed repressor of transcription which binds at the E2-box sequence, CACCTG. It carries seven zinc fingers (Zf) in two clusters and a homeodomain in the middle as potential DNA-binding domains. We cloned the genomic gene encoding chicken deltaEF1 and analyzed its organization. The gene consisted of nine exons, the N-proximal Zf were encoded by exons 5 through 7, and the C-proximal Zf by exons 8 and 9. Exon 7 also coded for the large middle portion of the protein including the homeodomain. Promoter analysis and RNase-protection assay indicated that the gene is driven by a G+C-rich promoter without a TATA box, and the transcription start points (tsp) cluster around 20 bp from the start codon located in exon 1. cDNA and genomic sequences of the mouse delta EF1 were cloned and compared with the chicken sequence. The deduced amino acid (aa) sequence was highly conserved between the chicken and mouse deltaEF1, no only in DNA-binding motifs but also in other blocks (78% overall aa identity). More recently reported DNA-binding proteins, AREB6 (human) ZEB (human) and BZP (hamster), were attributed to homologues of deltaEF1, among which only AREB6 represented full-length sequence. It was also indicated that rodent deltaEF1 lacked exon 3.

Genetic control of testis development.

Sex determination refers to the decision of the bipotential early gonads to develop as either testes or ovaries during embryogenesis. In mammals, a single genetic trigger involved in this pivotal decision has been identified on the Y chromosome: the testis-determining gene SRY/Sry. During embryogenesis, SRY triggers the differentiation of Sertoli cells from the supporting cell precursor lineage which would otherwise give granulosa cells in ovaries. Several testis-specific events occur after SRY expression and the onset of Sertoli cell differentiation, notably Leydig cell differentiation, testis cord formation, and development of testis-specific vasculature. Although a number of genes involved in these events have been identified, how they relate to Sry action is poorly understood. Furthermore, even at the adult stage, some of these genes retain a key role in maintaining the testicular fate because conditional ablation of the genes leads to adult testis dysgenesis or transdifferentiation into an ovary. This sheds light on mammalian sex-reprogramming, despite the prevailing dogma that postnatal sex change does not occur in mammals. In this review, we summarize our current understanding of genetic pathways of testis determination and differentiation in mammals, particularly in the mouse and the human.

Two mechanisms in the action of repressor deltaEF1: binding site competition with an activator and active repression.

BACKGROUND: Counteraction between activators and repressors is crucial for the regulation of a number of cell-specific enhancers, where an activator and a repressor are mutually competitive in binding to the same site. DeltaEF1 is a repressor protein of delta1-crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator deltaEF3 from binding to the overlapped site. It has two zinc finger clusters N-fin and C-fin, close to N- and C-termini, respectively, and a homeodomain in the middle. deltaEF1 also binds to the E2-box sequence CACCTG, and represses E2-box-dependent enhancers. RESULTS: The mechanism of the repressor action of deltaEF1 was investigated by examining various deletion mutants of deltaEF1 for their activity to repress delta1-crystallin enhancer fragment HN which contained DC5 sequence and an additional activator site. Both zinc finger clusters were found to be essential for DNA binding and repression, but the homeodomain was not. In addition, the NR domain close to the N-terminus was required for full repression. The NR domain showed active repression when fused to the Gal4 DNA binding domain. Active repression by deltaEF1, dependent on the NR domain, was also demonstrated in a situation where the binding sites of deltaEF1 and deltaEF3 were separated. N-fin and C-fin in their isolated forms bind the 5'-(T/C)ACCTG-3' and 5'-(t/C)ACCT-3' sequences, respectively, while the homeodomain showed no DNA binding activity. An analysis of DNA binding of the delta(Int)F form, having both N-fin and C-fin, indicated that a single DNA binding domain is assembled from two zinc finger clusters. CONCLUSION: Two mechanisms are involved in the repressor action of deltaEF1. First, a binding site competition with an activator which depends on the integrity of both zinc finger clusters, and second, an active repression to silence an enhancer which is attributed to the NR domain.

Delta-crystallin enhancer binding protein delta EF1 is a zinc finger-homeodomain protein implicated in postgastrulation embryogenesis.

We investigated nuclear factors that bind to delta 1-crystallin enhancer core and regulate lens-specific transcription. A nuclear factor delta EF1, which binds to the essential element of the delta 1-crystallin enhancer core, was molecularly cloned from the chicken by a southwestern method. The protein organization of delta EF1 deduced from the cDNA sequence indicated that it has heterogeneous domains for DNA-binding, two widely separated zinc fingers and a homeodomain, analogous to Drosophila ZFH-1 protein. The C-terminal zinc fingers were found to be responsible for binding to the delta 1-crystallin enhancer core sequence. delta EF1 had proline-rich and acidic domains common to various transcriptional activators. During embryogenesis, delta EF1 expression was observed in the postgastrulation period in mesodermal tissues; initially, in the notochord, followed by somites, nephrotomes and other components. The expression level changed dynamically in a tissue, possibly reflecting the differentiation states of the constituent cells. Besides mesoderm, delta EF1 was expressed in the nervous system and the lens, but other ectodermal tissues and endoderm remained very low in delta EF1 expression. Cotransfection experiments indicated that this factor acts as a repressor of delta 1-crystallin enhancer. Possession of heterogeneous DNA-binding domains and its dynamic change of expression in embryogenesis strongly suggest that delta EF1 acts in multiple ways depending on the cell type and the gene under its regulation.

Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development.

Pax6 is a key transcription factor in eye development, particularly in lens development, but its molecular action has not been clarified. We demonstrate that Pax6 initiates lens development by forming a molecular complex with SOX2 on the lens-specific enhancer elements, e.g., the delta-crystallin minimal enhancer DC5. DC5 shows a limited similarity to the binding consensus sequence of Pax6 and is bound poorly by Pax6 alone. However, Pax6 binds cooperatively with SOX2 to the DC5 sequence, resulting in formation of a high-mobility form of ternary complex in vitro, which correlates with the enhancer activation in vivo. We observed Pax6 and SOX2-interdependent factor occupancy of DC5 in a chromatin environment in vivo, providing the molecular basis of synergistic activation by Pax6 and SOX2. Subtle alterations of the Pax6-binding-site sequence of DC5 or of the inter-binding-sites distance diminished the cooperative binding and caused formation of a non-functional low-mobility form complex, suggesting DNA sequence-guided and protein interaction-induced conformation change of the Pax6 protein. When ectopically expressed in embryo ectoderm, Pax6 and SOX2 in combination activate delta-crystallin gene and elicit lens placode development, indicating that the complex of Pax6 and SOX2 formed on specific DNA sequences is the genetic switch for initiation of lens differentiation.