Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain.

Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA/ATTS family of transcription factors.

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check.

In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z-ring to drive septation. Spatial and temporal control of Z-ring formation is achieved by the Min and nucleoid occlusion (NO) systems. Unlike the well-studied Min system, less is known about the anti-DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)-mediated NO. SlmA contains a TetR-like DNA-binding fold, and chromatin immunoprecipitation analyses show that SlmA-binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA-FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti-parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA-DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z-ring formation at the correct place and time to effect NO.

CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

Wingless activity in the precursor cells specifies neuronal migratory behavior in the Drosophila nerve cord.

Neurons and their precursor cells are formed in different regions within the developing CNS, but they migrate and occupy very specific sites in the mature CNS. The ultimate position of neurons is crucial for establishing proper synaptic connectivity in the brain. In Drosophila, despite its extensive use as a model system to study neurogenesis, we know almost nothing about neuronal migration or its regulation. In this paper, I show that one of the most studied neuronal pairs in the Drosophila nerve cord, RP2/sib, has a complicated migratory route. Based on my studies on Wingless (Wg) signaling, I report that the neuronal migratory pattern is determined at the precursor cell stage level. The results show that Wg activity in the precursor neuroectodermal and neuroblast levels specify neuronal migratory pattern two divisions later, thus, well ahead of the actual migratory event. Moreover, at least two downstream genes, Cut and Zfh1, are involved in this process but their role is at the downstream neuronal level. The functional importance of normal neuronal migration and the requirement of Wg signaling for the process are indicated by the finding that mislocated RP2 neurons in embryos mutant for Wg-signaling fail to properly send out their axon projection.

STUDY OF REST AS A NEGATIVE REGULATOR OF P16INK4A

STUDY OF REST AS A NEGATIVE REGULATOR OF P16INK4A Monica Gireud, B.S. Thesis Advisor: Vidya Gopalakrishnan, Ph.D. The RE1 Silencing Transcription Factor (REST) is a negative regulator of neuronal differentiation. It is expressed ubiquitously in early embryos, but downregulated in neural progenitors concomitant with onset of neuronal differentiation in these cells. REST has been widely studied as a negative regulator of neuronal differentiation genes. Our recent work identified a novel role for REST in control of cell proliferation. However, the underlying molecular mechanism(s) are not known and is a focus of the current thesis project. Here, we provide evidence that REST signaling controls the expression of the cyclin-dependent kinase inhibitor, p16Ink4a, a negative regulator of the cell cycle and passage through G1. We determined that REST expression in the proliferating granule progenitors of the cerebellum and its lack of expression in the differentiated neurons is reciprocally correlated with that of p16Ink4a. Decline in REST levels in differentiating primary and neural stem cells immortalized with v-myc (NSC-M) granule progenitors in vitro was also associated with upregulation of p16Ink4a expression. Conversely, constitutive human REST transgene expression in NSC-M cells (NSC-MRs) blocked p16Ink4 upregulation, even under neuronal differentiation conditions. However, the lack of a consensus REST DNA binding RE1 element in the regulatory regions of p16Ink4a locus suggested an indirect regulation of p16Ink4a by REST. Based on work from other groups that showed repression of p16Ink4a transcription by the polycomb protein Bmi-1, and its negative regulation by microRNA-203 (miR-203) and our identification of a RE1 element in the downstream regulatory region of miR-203, we asked if the p16Ink4a expression was controlled by REST through a series of negative regulatory events involving miR-203 and Bmi-1. We observed that Bmi1 -expression mirrored that of REST and inversely correlated with that of miR-203 in the postnatal cerebellum and in vitro differentiated granule and NSC-M progenitors. In contrast, forced REST transgene expression in NSC-MR cells abrogated the decrease in Bmi-1 levels and elevation in miR-203 expression. Significant REST binding to the miR-203 RE1 element was also observed in NSC-M cells, indicating that REST had the potential to directly regulate miR-203 expression. In conclusion, our studies suggest a role for REST in control of cell cycle transit in neural progenitors through negative regulation of p16Ink4a. Further validation of these results in REST knockout mice is needed, and is ongoing.

The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains.^ To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed. ^

Analysis of a human placental lactogen gene promoter

Human placental lactogen (hPL) and human growth hormone (hGH) comprise a multigene family that share $>$90% nucleic acid sequence homology including 500 bp of 5$\sp\prime$ flanking sequence. Despite these similarities, hGH is produced in the anterior pituitary while hPL is expressed in the placenta. For most genes studied to date, regulation of expression occurs by alterations at the level of transcriptional initiation. Nuclear proteins bind specific DNA sequences in the promoter to regulate gene expression. In this study, the hPL$\sb3$ promoter was analyzed for DNA sequences that contribute to its expression. The interaction between the hPL$\sb3$ promoter and nuclear proteins was examined using nuclear extracts from placental and non-placental cells.^ To identify regulatory elements in the promoter of the hPL$\sb3$ gene, 5$\sp\prime$ deletion mutants were constructed by cleaving 1200 bp of upstream sequence with various restriction enzymes. These DNA fragments were ligated 5$\sp\prime$ to a promoterless bacterial gene chloramphenicol acetyltransferase (CAT) and transfected into JEG-3 cells, a human placental choriocarcinoma cell line. The level of CAT activity reflects the ability of the promoter mutants to activate transcription. Deletion of the sequence between $-$142 bp and $-$129 bp, relative to the start of transcription, resulted in an 8-fold decrease in CAT activity. Nuclear proteins from JEG-3, HeLa, and HepG2 (human liver cells), formed specific binding complexes with this region of the hPL$\sb3$ promoter, as shown by gel mobility shift assay. The $-$142 bp to $-$129 bp region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. Sp1-like proteins were identified by DNA binding assay, in the nuclear extracts of the three cell lines. A series of G nucleotides in the hPL$\sb3$ promoter regulatory region were identified by methylation interference assay to interact with the DNA-binding proteins and the pattern obtained is similar to that for other Sp1 binding sites that have been studied. This suggests that hPL$\sb3$ may be transcriptionally regulated by Sp1 or a Sp1-like transacting factor. ^

Transcriptional regulation of the invariant chain associated with MHC class II molecules

The invariant chain associated with the major histocompatibility complex (MHC) class II molecules is a non-polymorphic glycoprotein implicated in antigen processing and class II molecule intracellular transport. Class II molecules and invariant chain (In) are expressed primarily by B lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon gamma (IFN-$\gamma$) in a variety of cell types such as endothelial cells, fibroblasts, and astrocytes. In this study the cis-acting sequences involved in the constitutive, tissue-specific, and IFN-$\gamma$ induced expression of the human In gene were investigated and nuclear proteins which specifically bound these sequences were identified.^ To define promoter sequences involved in the regulation of the human In gene, 790 bp 5$\sp\prime$ to the initiation of transcription were subcloned upstream of the gene encoding chloramphenicol acetyl transferase (CAT). Transfection of this construct into In expressing and non-expressing cell lines demonstrated that this 790 bp In promoter sequence conferred tissue specificity to the CAT gene. Deletion mutants were created in the promoter to identify sequences important for transcription. Three regulatory regions were identified $-$396 to $-$241, $-$241 to $-$216, and $-$216 to $-$165 bp 5$\sp\prime$ to the cap site. Transfection into a human glioblastoma cell line, U-373 MG, and treatment with IFN-$\gamma$, demonstrated that this 5$\sp\prime$ region is responsive to IFN-$\gamma$. An IFN-$\gamma$ response element was sublocalized to the region $-$120 to $-$61 bp. This region contains homology to the interferon-stimulated response element (ISRE) identified in other IFN responsive genes. IFN-$\gamma$ induces a sequence-specific DNA binding factor which binds to an oligonucleotide corresponding to $-$107 to $-$79 bp of the In promoter. This factor also binds to an oligonucleotide corresponding to $-$91 to $-$62 of the interferon-$\beta$ gene promoter, suggesting this factor may be member of the IRF-1/ISGF2, IRF-2, ICSBP family of ISRE binding proteins. A transcriptional enhancer was identified in the first intron of the In gene. This element, located in a 2.6 kb BamHI/PstI fragment, enhances the IFN-$\gamma$ response of the promoter in U-373 MG. The majority of the In enhancer activity was sublocalized to a 550 bp region $\sim$1.6 kb downstream of the In transcriptional start site. ^

Studies on the muscle-specific regulatory protein myogenin

Recently, a family of muscle-specific regulatory factors that includes myogenin, myoD, myf-5, and MRF-4 has been identified. They share a high degree of homology within a region that contains a basic and helix-loop-helix domain. Transfection of many non-muscle cell types with any one of these genes results in the activation of the entire myogenic program. To explore the mechanism through which myogenin regulates myogenesis, we have prepared antibodies against peptides specific to myogenin. Using these antibodies we show that myogenin is a 32 Kd phospho-protein which is localized to the nuclei of muscle cells. In vitro, myogenin oligomerizes with the ubiquitous enhancer binding factor E12, and acquires high affinity for an element of the core of the muscle creatine kinase (MCK) enhancer that is conserved among many muscle-specific genes. Myogenin synthesized in BC$\sb3$H1 and C2 muscle cell lines also binds to the same site in the enhancer. However, the MCK enhancer is not activated in 10T1/2 fibroblasts which have been transfected with a constitutive myogenin expression vector until growth factors have been removed from the media. This result indicates that mitogenic signals block the actions of myogenin.. Mutagenesis of the myogenin/E12 binding site in the MCK enhancer abolishes binding of the hetero-oligomer and prevents trans-activation of the enhancer by myogenin. By site directed mutagenesis of myogenin we have shown that the basic region consists of three clusters of basic residues, two of which are required for binding and activation of the myogenic program. Myogenic activation, but not DNA binding, is lost when the 10 residue region between the two required basic clusters is substituted with the corresponding region from E12, which also contains a similar basic and helix-loop-helix domain. Functional revertants of this substitution mutant have identified two amino acids which confer muscle specificity. The properties of myogenin suggest that it functions as a sequence-specific DNA binding factor that interacts directly with muscle-specific genes during myogenesis and contains within its basic domain a region which imparts myogenic activation and is separable from DNA binding. ^

Transcriptional regulation of the chicken MLC3f gene

The expression of the chicken fast skeletal myosin alkali light chain (MLC) 3f is subject to complex patterns of control by developmental and physiologic signals. Regulation over MLC3f gene expression is thought to be exerted primarily at the transcriptional level. The purpose of this dissertation was to identify cis-acting elements on the 5$\sp\prime$ flanking region of chicken MLC3f gene that are important for transcriptional regulation. The results show that the 5$\sp\prime$ flanking region of MLC3f gene contains multiple cis-acting elements. The nucleotide sequence of these elements demonstrates a high degree of conservation between different species and are also found in the 5$\sp\prime$ flanking regions of many muscle protein genes. The first regulatory region is located between $-$185 and $-$150 bp from the transcription start site and contains an AT-rich element. Linker scanner analyses have revealed that this element has a positive effect on transcription of the MLC3f promoter. Furthermore, when linked to a heterologous viral promoter, it can enhance reporter gene expression in a muscle-specific manner, independent of distance or orientation.^ The second regulatory region is located between $-$96 and $-$64 from the transcription start site. Sequences downstream of $-$96 have the capacity to drive muscle-specific reporter gene expression, although the region between $-$96 and $-$64 has no intrinsic enhancer-like activity. Linker scanner analyses have identified a GC-rich motif that required efficient transcription of the MLC3f promoter. Mutations to this region of DNA results in diminished capacity to drive reporter gene expression and is correlated with disruption of the ability to bind sequence-specific transcription factors. These sequence-specific DNA-binding proteins were detected in both muscle and non-muscle extracts. The results suggest that the mere presence or absence of transcription factors cannot be solely responsible for regulation of MLC3f expression and that tissue-specific expression may arise from complex interactions with muscle-specific, as well as more ubiquitous transcription factors with multiple regulatory elements on the gene. ^

H2B histone gene expression during {\it Xenopus laevis\/} development

Histone gene expression is replication-independent during oogenesis and early embryogenesis in amphibians; however, it becomes replication-dependent during later embryogenesis and remains replication-dependent through adulthood. In order to understand the mechanism for this switch in transcriptional regulation of histone gene expression during amphibian development, linker-scanning mutations were made in a Xenopus laevis H2B histone gene promoter by oligonucleotide site-directed mutagenesis and assayed by microinjection into oocytes and embryos. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CCAAT, and ATF motifs, required for maximal transcription in both oocytes and embryos. Factors binding to the CCAAT and ATF motifs are present in oocytes and embryos and increase slightly in abundance during early development. A sequence (CTTTACAT) in the frog H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is additionally required for maximal H2B transcription in frog embryos. Oocytes and embryos contain multiple octamer-binding proteins that are expressed in a sequential manner during early development. Sequences encoding three novel octamer-binding proteins were isolated from Xenopus cDNA libraries by virtue of their similarity with the DNA binding (POU) domain of the ubiquitously expressed transcription factor Oct-1. The protein encoded by one of these genes, termed Oct-60, was localized mainly in the cytoplasm of oocytes and was also present in early embryos until the gastrula stage of development. Proteins encoded by the other two genes, Oct-25 and Oct-91, were present in embryos after the mid-blastula stage of development and decreased by early neurula stage. The activity of the Xenopus H2B octamer motif in embryos is not specifically associated with increased binding by Oct-1 or the appearance of novel octamer-binding proteins but requires the presence of an intact CCAAT motif. We found that synergistic interactions among promoter elements are important for full H2B promoter activity. The results suggest that transcription of the Xenopus H2B gene is replication-dependent when it is activated at the mid-blastula stage of development and that replication-dependent H2B transcription is mediated by Oct-1. ^

Molecular and biochemical analysis of the {\it Drosophila\/} segmentation gene {\it runt\/}

Genetic evidence has indicated that the segmentation gene runt plays a key role in regulating gene expression of the pair-rule genes hairy, even-skipped, and fushi tarazu. In contrast to other pair-rule genes, sequence data of the runt open reading frame did not reveal homologies to DNA-binding motifs of known transcriptional regulatory proteins. This thesis project examined several properties of the runt gene based on the sequence of the transcription unit, including the subcellular localization of the protein in vivo, its ability to bind DNA, and the functionality of a putative nucleotide binding domain.^ A runt-specific antibody was generated and used to demonstrate that runt is localized in the nucleus. Since the precise overlap of the pair-rule stripes is thought to be critical for the determination of cellular identity along the anterior-posterior axis, phasing of early runt expression in the blastoderm was examined with regard to the segmentation genes hairy, even-skipped, and fushi tarazu. runt was also expressed at later stages of embryogenesis, including expression in neuroblasts, and ganglion mother cells of the developing nervous system. Expression at this stage was required for the subsequent formation of specific neurons and runt was extensively expressed in the central and peripheral nervous systems.^ Several experiments were done to address the biochemical function of the runt protein. A direct interaction of runt with DNA was first examined. Although bacterial expressed runt was found to bind dsDNA-cellulose, subsequent experiments failed to detect sequence-specific interactions with DNA. Inter-species conservation of the putative nucleotide binding domain suggested that this region was functionally important, and runt protein bound a labeled ATP analog with high affinity in vitro. Finally, the effect of substitution of a critical residue of the nucleotide binding domain on runt activity was examined in vivo. Ectopic expression of the mutant protein indicated that this conserved substitution altered, but did not eliminate, runt activity as evaluated by segmentation phenotype and viability. ^

Regulation of Spec genes in aboral ectoderm cells of sea urchin {\it Strongylocentrotus purpuratus}

The Spec genes of the sea urchin Stronylocentrotus purpuratus serves as an excellent model for studying cell type-specific gene expression during early embryogenesis. The Spec1/Spec2 genes encode cytosolic calcium-binding proteins related to the calmodulin/troponin C/myosin light chain superfamily. Members of the Spec gene family are activated shortly after the sixth cleavage as the lineage-specific founder cells giving rise to aboral ectoderm are established, and the accumulation of the Spec mRNAs is limited exclusively to aboral ectoderm cell lineages. In this dissertation, the transcriptional regulation of the Spec genes was studied. Sequence comparisons of the Spec gene 5$\sp\prime$ flanking regions showed that a DNA block of approximately 800 bp from the 3$\sp\prime$ end of the first exon to the 5$\sp\prime$ end of a repetitive DNA element, termed RSR, was highly conserved. In Spec2a, the conserved region was a continuous stretch of DNA, but in Spec1 and Spec2c, DNA insertions interrupt the conserved sequence block and alter the relative placement of the RSR element and other 5$\sp\prime$ flanking DNA. Thus, drastic rearrangements have occurred within the putative control regions of the Spec genes. In vivo expression experiments using the sea urchin embryo gene-transfer system showed that while the 5$\sp\prime$ flanking regions of all three Spec genes conferred proper temporal activation to the reporter CAT gene, only the Spec2a 5$\sp\prime$ flanking region could restrict lacZ gene expression to aboral ectoderm cells. However, the Spec2a conserved region alone was not sufficient to confer proper spatial expression, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. A major positive regulatory region, defined as the RSR enhancer, was identified between base pairs $-$631 and $-$443 on Spec2a. The RSR enhancer was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. DNaseI footprinting and band-shift analysis of the RSR enhancer revealed multiple DNA-elements. One of the elements, an A/T-rich sequence called the A/T palindrome was studied in detail. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose DNA-binding specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutated A/T palindromes are incapable of binding the 45-kDa protein and lower promoter activity by 8-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells. ^