Carboxy-terminal DNA Binding Domain Research Articles

The Drosophila LIN54 homolog Mip120 controls two aspects of oogenesis.

ABSTRACTThe conserved multi-protein MuvB core associates with the Myb oncoproteins and with the RB-E2F-DP tumor suppressor proteins in complexes that regulate cell proliferation, differentiation, and apoptosis. Drosophila Mip120, a homolog of LIN54, is a sequence-specific DNA-binding protein within the MuvB core. A mutant of Drosophila mip120 was previously shown to cause female and male sterility. We now show that Mip120 regulates two different aspects of oogenesis. First, in the absence of the Mip120 protein, egg chambers arrest during the transition from stage 7 to 8 with a failure of the normal program of chromosomal dynamics in the ovarian nurse cells. Specifically, the decondensation, disassembly and dispersion of the endoreplicated polytene chromosomes fail to occur without Mip120. The conserved carboxy-terminal DNA-binding and protein-protein interaction domains of Mip120 are necessary but not sufficient for this process. Second, we show that a lack of Mip120 causes a dramatic increase in the expression of benign gonial cell neoplasm (bgcn), a gene that is normally expressed in only a small number of cells within the ovary including the germline stem cells.

Transcriptional Repression: The Dark Side of Myc

The (c-)Myc oncoprotein and its cousins, the N-Myc and L-Myc proteins, show all hallmarks of transcriptional activator proteins: Myc carries a carboxy-terminal DNA binding domain, which mediates sequence-specific binding to DNA. At its amino-terminus, Myc carries a transcriptional regulatory domain that strongly activates transcription when fused to an ectopic DNA binding domain; moreover, the strength of activation of different members of the Myc family correlates with their ability to transform rodent cells. Furthermore, activation of conditional alleles of Myc, either tetracycline or estrogen inducible, upregulates expression of a large number of genes, both in tissue culture and in transgenic animals. Indeed, many of these genes have essential roles in cell proliferation, cell growth, and metabolism; two of them, odc, encoding ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, and rpl24, encoding a constituent of the large ribosomal subunit, are haploinsufficient for Myc-induced lymphomagenesis but not for normal development, arguing very strongly that upregulation of both genes is critical for Myc-dependent tumor formation. Undoubtedly, therefore, Myc exerts part of its biological activities via transcriptional upregulation of a large number of target genes. One of the key issues in the field is whether there are additional biochemical activities of the Myc protein and, if so, whether and how they contribute to Myc biology. This review summarizes evidence demonstrating that Myc has the ability to repress transcription and that this may be an important function during oncogenic transformation.

Two distinct functions for the BTB domain of the CD4-differentiating factor Thpok (85.20)

Abstract Thpok is a zinc-finger (ZF) transcription factor essential for CD4 T cell development. Thpok promotes CD4 T helper lineage commitment, in part through the repression of genes necessary for CD8 differentiation. A member of the BTB-ZF subclass of ZF transcription factors, Thpok contains two conserved motifs: a carboxy-terminal ZF DNA binding domain, and an amino-terminal BTB motif. Although the BTB domain is required for Thpok to promote CD4 lineage differentiation, its contribution to Thpok function is not clear. In vitro analyses of the BTB domain of the prototype family member, Bcl6 (B cell lymphoma 6) revealed a role of the BTB domain in oligomerization and transcriptional repression. In the present study, we examined whether the Thpok BTB domain simply serves to promote dimerization of Thpok molecules, or whether it also serves to recruit other transcriptional regulators. We generated chimeric Thpok proteins by replacing the BTB domain of Thpok with that of other family members, or with a heterologous dimerization domain. All chimeric molecules containing the BTB domains tested blocked CD8 lineage specific gene expression; in contrast, the chimeric molecule carrying a heterologous dimerization domain was only partially functional. These results indicate that the BTB domain of Thpok not only promotes dimerization but it also serves to control transcription, presumably through interactions with transcriptional corepressors.

Regulation of Epstein-Barr Virus OriP Replication by Poly(ADP-Ribose) Polymerase 1

Poly(ADP-ribose) polymerase (PARP) is an abundant, chromatin-associated, NAD-dependent enzyme that functions in multiple chromosomal processes, including DNA replication and chromatin remodeling. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is a dynamic genetic element that confers stable episome maintenance, DNA replication initiation, and chromatin organization functions. OriP function depends on the EBV-encoded origin binding protein EBNA1. We have previously shown that EBNA1 is subject to negative regulation by poly(ADP-ribosyl)ation (PARylation). We now show that PARP1 physically associates with OriP in latently EBV-infected B cells. Short hairpin RNA depletion of PARP1 enhances OriP replication activity and increases EBNA1, origin recognition complex 2 (ORC2), and minichromosome maintenance complex (MCM) association with OriP. Pharmacological inhibitors of PARP1 enhance OriP plasmid maintenance and increase EBNA1, ORC2, and MCM3 occupancy at OriP. PARylation in vitro inhibits ORC2 recruitment and remodels telomere repeat factor (TRF) binding at the dyad symmetry (DS) element of OriP. Purified PARP1 can ribosylate EBNA1 at multiple sites throughout its amino terminus but not in the carboxy-terminal DNA binding domain. We also show that EBNA1 linking regions (LR1 and LR2) can bind directly to oligomers of PAR. We propose that PARP1-dependent PARylation of EBNA1 and adjacently bound TRF2 induces structural changes at the DS element that reduce EBNA1 DNA binding affinity and functional recruitment of ORC.

Multi-functional Dynamic Control of Bipolarity, Chromosome segregation and Spindle Elongation by a Novel Essential Chromatin Binding Klp in Fission Yeast

A subset of all known kinesin-like microtubule motor proteins (Klps) regulates spindle function for fidelity in chromosome segregation. While some families are ubiquitous, such as Kinesin-5 and Kinesin-14 Klps, others families seem restricted to multi-cellular eukaryotes, such as chromokinesins and MKlp1-like passenger proteins. We report a novel essential chromatin-binding Klp in fission yeast Schizosaccharomyces pombe Ckk1 (Cho1 and Kid-like kinesin). S. pombe Ckk1p has an N-terminal motor domain and carboxy-terminal tandem basic-Zip DNA binding domains. It localizes to chromosome arms in prometaphase and kinetochores in metaphase. In prometaphase to anaphase it also associates with overlapping anti-parallel microtubules of the spindle midzone. Passenger proteins show dynamic relocalization from chromosome arms to kinetochores to spindle midzone and regulate cytokinesis. Although Ckk1p has the conserved Cho-domain, it does not have actin binding domains and exhibits no cytokinesis defects. Ckk1p microtubule localization regulates microtubule sliding but not spindle midzone integrity, a role attributed to Ase1p in fission yeast. A single microtubule-binding site within the motor domain suggests that like MKlp1, protein association with the stalk is needed to mediate other microtubule interactions. Premature overexpression of Ckk1 can stabilize bipolar spindles in a compromised Kinesin-5 cut7-22ts strain, overriding the opposing effect of Kinesin-14 Pkl1. However in wild type cells overexpressed Ckk1p results in spindle collapse. Upon removal of pkl1, the ability of Ckk1p overexpression to cause spindle collapse is reduced, however pre-prophase spindle elongation occurs. The result is non-central, off-side anaphase A segregation of chromosomes. Ckk1p represents a novel chromatin-binding Klp in fission yeast, absent in budding yeast, that has multiple roles in stabilizing spindle assembly, equatorial chromosome alignment and spindle elongation and does not fall cleanly into either Kinesin-6 or Kinesin-10 families of Klps.

Amino acid residues in the GIY-YIG endonuclease II of phage T4 affecting sequence recognition and binding as well as catalysis.

Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.

Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

TAF1 Interacts with and Modulates Human Papillomavirus 16 E2-Dependent Transcriptional Regulation

High-risk human papillomavirus (HPV) infection is the main etiological factor in the development of cervical cancer and viral type 16 is the most frequently found in this neoplasia. The E2 protein plays a key role in viral DNA replication, transcription and genome maintenance. E2 is a sequence-specific DNA-binding protein that activates or represses the transcriptional activity of promoters depending on the distance from the E2-binding sites to the TATA box. The transactivation properties of E2 are modulated by the interaction with several cellular factors that regulate the recruitment of transcription factor IID. Here, we demonstrate by pull-down assays the in vitro interaction of HPV16 E2 and TAF1. The domain of TAF1 necessary for the binding maps into its amino region, while the carboxy-terminal DNA-binding domain and the transactivation domain of the E2 protein are involved in the interaction. By transient cotransfection assays on C-33 A cells, we demonstrated that TAF1 enhances the activation of an E2-dependent artificial promoter while overexpression of TAF1 alleviates the E2-dependent repression of a high-risk HPV long control region. The specific modification of the transcriptional activity of both promoters by TAF1 suggests that the interaction between these proteins could participate in the modulation of the transregulatory properties of E2, with important biological consequences.

The E8 repression domain can replace the E2 transactivation domain for growth inhibition of HeLa cells by papillomavirus E2 proteins

Continuous expression of the human papillomavirus (HPV) oncoproteins E6 and E7 is required for the growth of cervical cancer cell lines. So far, only the overexpression of the wild type papillomavirus E2 protein has been shown to induce growth arrest in HPV18-positive HeLa cells by repressing E6/E7 transcription. Growth arrest by E2 requires the aminoterminal transcription activation domain in addition to the carboxyterminal DNA-binding domain. Several papillomaviruses such as the carcinogenic HPV31 express in addition to E2 an E8(wedge)E2C fusion protein in which the E8 domain, which is required for repression of replication and transcription, replaces the E2 activation domain. In this report, we demonstrate that the HPV31 E8(wedge)E2C protein is able to inhibit the growth of HeLa cells but not of HPV-negative C33A cervical cancer cells. Growth repression by E8(wedge)E2C correlates with repression of the endogenous HPV18 E6/E7 promoter and the reappearance of E6- and E7-regulated p53, pRb and p21 proteins, suggesting that E8(wedge)E2C inhibits growth by reactivating dormant tumor suppressor pathways. Growth inhibition requires an intact E8 repression domain in addition to the carboxyterminal E2C DNA binding domain. Chromatin immunoprecipitation experiments suggest that the E8 repression domain enhances binding to the HPV18 promoter sequence in vivo. In summary, our results demonstrate that the small E8 repression domain can functionally replace the large E2 transactivation domain for growth inhibition of HeLa cervical cancer cells.

Pivotal Roles for the Receiver Domain in the Mechanism of Action of the Response Regulator RamR of Streptomyces coelicolor

The response regulator RamR activates expression of the ramCSAB operon, the source of the morphogenetic peptide SapB, and is therefore important for morphogenesis of the bacterium Streptomyces coelicolor. Like most response regulators, RamR consists of an amino-terminal receiver domain and a carboxy-terminal DNA binding domain. Four of five highly conserved active site residues known to be important in other response regulators are present in RamR: D12, D56 (the predicted site of phosphorylation), T84 and K105. Here, we show that in spite of this, RamR did not demonstrate an ability to autophosphorylate in vitro in the presence of small molecule phosphodonors. The unphosphorylated protein behaved as a dimer and bound cooperatively to three sites in the ramC promoter, one with very high affinity and two with lower affinity. On its own, the RamR DNA binding domain could not bind DNA but was able to interfere with the action of full length RamR in a manner suggesting direct protein-protein contact. Surprisingly, substitution of residues D12 or T84 had no effect on RamR function in vivo. In contrast, D56A and K105A substitutions caused defects in both dimer formation and DNA binding while the more conservative substitution, D56N permitted dimer formation but not DNA binding. L102 in RamR corresponds to a well-conserved tyrosine (or aromatic) residue that is important for function in the other response regulators. While a L102Y variant, which introduced the aromatic side-chain usually found at this position, functioned normally, L102A and L102W substitutions blocked RamR function in vivo. We show that these substitutions specifically impaired cooperative DNA binding by RamR at the lower affinity recognition sequences. The biochemical properties of RamR therefore differ markedly from those of other well-characterized response regulators.

Synergistic Regulation of Competence Development inBacillus subtilisby Two Rap-Phr Systems

The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulator's ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Mol. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and RapF-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity.

Helicobacter pylori FlgR is an enhancer-independent activator of sigma54-RNA polymerase holoenzyme.

Helicobacter pylori FlgR activates transcription with sigma54-RNA polymerase holoenzyme (sigma54-holoenzyme) from at least five flagellar operons. Activators of sigma54-holoenzyme generally bind enhancer sequences located >70 bp upstream of the promoter and contact sigma54-holoenzyme bound at the promoter through DNA looping to activate transcription. H. pylori FlgR lacks the carboxy-terminal DNA-binding domain present in most sigma54-dependent activators. As little as 42 bp of DNA upstream of the flaB promoter and 26 bp of DNA sequence downstream of the transcriptional start site were sufficient for efficient FlgR-mediated expression from a flaB'-'xylE reporter gene in H. pylori, indicating that FlgR does not use an enhancer to activate transcription. Other examples of sigma54-dependent activators that lack a DNA-binding domain include Chlamydia trachomatis CtcC and activators from the other Chlamydia spp. whose genomes have been sequenced. FlgR from Helicobacter hepaticus and Campylobacter jejuni, which are closely related to H. pylori, appear to have carboxy-terminal DNA-binding domains, suggesting that the loss of the DNA-binding domain from H. pylori FlgR occurred after the divergence of these bacterial species. Removal of the amino-terminal regulatory domain of FlgR resulted in a constitutively active form of the protein that activated transcription from sigma54-dependent genes in Escherichia coli. The truncated FlgR protein also activated transcription with E. coli sigma54-holoenzyme in an in vitro transcription assay.

Ran modulates spindle assembly by regulating a subset of TPX2 and Kid activities including Aurora A activation.

Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin beta, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 we identified nuclear targeting information, microtubule-binding and Aurora A binding activities. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevented Aurora A activation and recruitment to microtubules. In addition we identified nuclear targeting information in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly.

Site-specific DNA cleavage of synthetic NarL sites by an engineered Escherichia coli NarL protein-1,10-phenanthroline cleaving agent.

The NarL response regulatory protein of Escherichia coli has been engineered by covalent modification with 1,10-phenanthroline (OP) to create a set of site-specific DNA-cleaving agents. This was accomplished by introducing single cysteine amino acid replacements at selected locations within the carboxy-terminal DNA-binding domain in or nearby the helix 8 to helix 9 region of the NarL protein using site-directed mutagenesis. Of 18 modified NarL-OP proteins made, 13 retained the ability to bind DNA as evidenced by gel mobility assays, whereas 10 of the 1,10-phenanthroline-modified proteins also exhibited specific cleavage activity for a synthetic NarL recognition sequence. These DNA-cleaving agents were divided into two groups based on the location of the cleavage sites. The first class set cleaved the DNA nearby the center of a synthetic 7-2-7 sequence composed of two NarL heptamer sites separated by a 2-bp spacer element. The second class cut the DNA at the periphery of the 7-2-7 sequence. The cleavage data are consistent with the ability of two NarL monomers to recognize and bind to the DNA in a head-to-head orientation. A second set of DNA-cleaving agents was constructed using the carboxy-terminal domain of NarL called NarL(C). Similar cleavage patterns were observed whether full-length NarL or NarL(C) was used. The availability of 1,10-phenanthroline-modified NarL and NarL(C) proteins opens up the possibility to explore the position, orientation, and number of NarL recognition sites at E. coli promoters predicted to contain multiple and complex arrangements of NarL-binding sites.

Coupling Sequences Flanking Tn916 Do Not Determine the Affinity of Binding of Integrase to the Transposon Ends and Adjacent Bacterial DNA

Coupling sequences are the 6 bp flanking the conjugative transposon Tn916 and are thought to play a role in determining the frequency of conjugative transposition. The affinity of binding of a chimeric protein, which consisted of maltose binding protein fused to the carboxy-terminal DNA binding domain of Tn916 integrase (Int), to different double-stranded oligonucleotide substrates containing coupling sequences associated with high- and low-frequency conjugative transposition was measured using a competition binding assay. The relative affinity of the chimeric protein was unaffected by the nature of the coupling sequences tested. The same results were obtained when the coupling sequences were placed in a different surrounding sequence context. It therefore appears that the effects of different coupling sequences on the frequency of conjugative transposition are not due simply to differences in Int binding.

Functional interaction of the bovine papillomavirus E2 transactivation domain with TFIIB.

Induction of gene expression by the papillomavirus E2 protein requires its approximately 220-amino-acid amino-terminal transactivation domain (TAD) to interact with cellular factors that lead to formation of an activated RNA polymerase complex. These interaction partners have yet to be identified and characterized. The E2 protein localizes the transcription complex to the target promoter through its carboxy-terminal sequence-specific DNA binding domain. This domain has been reported to bind the basal transcription factors TATA-binding protein and TFIIB. We present evidence establishing a direct interaction between amino acids 74 to 134 of the E2 TAD and TFIIB. Within this region, the E2 point mutant N127Y was partially defective and W99C was completely defective for TFIIB binding in vitro, and these mutants displayed reduced or no transcriptional activity, respectively, upon transfection into C33A cells. Overexpression of TFIIB specifically restored transactivation by N127Y to close to wild-type levels, while W99C remained inactive. To further demonstrate the functional interaction of TFIIB with the wild-type E2 TAD, this region was fused to a bacterial DNA binding domain (LexA:E2:1-216). Upon transfection with increasing amounts of LexA:E2:1-216, there was reduction of its transcriptional activity, a phenomenon thought to result from titration of limiting factors, or squelching. Squelching of LexA:E2:1-216, or the wild-type E2 activator, was partially relieved by overexpression of TFIIB. We conclude that a specific region of the E2 TAD functionally interacts with TFIIB.

Mapping features of HIV-1 integrase near selected sites on viral and target DNA molecules in an active enzyme-DNA complex by photo-cross-linking.

The virally encoded integrase protein carries out retroviral integration, and to do so, it must make specific interactions with both viral and target DNA sequences. The retroviral integrase has three domains: an amino-terminal region of about 50 amino acids that contains a zinc finger-like motif, a tightly folded, phylogenetically conserved core domain that contains the active site, and a carboxy-terminal domain that can bind DNA in a nonspecific manner. The complete roles of the amino- and carboxyl-terminal domains have not yet been determined, but they appear to participate in multimerization and nonspecific or target DNA binding, respectively. The number and identity of integrase's DNA binding sites have been difficult to determine by conventional mutagenesis studies. In this report, we describe a photo-cross-linking approach to address these issues. Our findings suggest that HIV-1 integrase contacts with conserved features of the viral DNA end are likely to be mediated by residues in two peptides within the conserved core domain. Additional cross-links were seen between viral DNA and the carboxy-terminal DNA binding domain. Numerous sites in integrase, including peptides in each of the three domains, could be cross-linked to target DNA features. Integrase is known to function as a multimer, and it remains to be determined which specific contacts are in cis or trans with respect to the active site.

An alternatively spliced polycistronic mRNA encoding cyclic adenosine 3',5'-monophosphate (cAMP)-responsive transcription factor CREB (cAMP response element-binding protein) in human testis extinguishes expression of an internally translated inhibitor CREB isoform.

Cyclic AMP response element-binding protein (CREB) regulates the expression of cAMP-responsive genes. In the rat testis, several isoforms of CREB arise from alternative exon splicing that occurs cyclically during the 12-day cell association cycles of spermatogenesis. Insertion of alternatively spliced exon W into CREB mRNA during spermatogenesis results in a polycistronic RNA that encodes two novel internally translated CREB repressor isoforms called I-CREBs, consisting of the carboxy-terminal DNA-binding domain devoid of the transactivation domains. Here we report the alternative splicing of an additional novel exon Z in CREB mRNA expressed in human but not in mouse or rat testis. Insertion of exon Z abolishes the synthesis of one of the two inhibitor CREBs due to the introduction of an inframe stop codon within exon Z. We show that exon Z is not spliced into mRNAs in mouse and rat testes due to the evolution of mutations in the splice signals flanking exon Z. These findings suggest that the splicing in of exon Z may be part of a human-specific mechanism to regulate cAMP-dependent regulatory pathways in spermatogenesis by extinguishing the expression of a CREB repressor.

Monoclonal Antibodies Directed against the Amino-Terminal Domain of Human TBP Cross-React with TBP from Other Species

The TATA box-binding protein (TBP) is a key transcription factor required for transcription by all three eukaryotic RNA polymerases. It consists of a conserved carboxy-terminal DNA binding domain and a highly divergent amino terminal domain. TBP and different sets of TBP-associated factors (TAFs) constitute at least four multisubunit complexes referred to as SL1, TFIID, TFIIIB, and SNAPC. SL1, TFIID, and TFIIIB are required for transcription by RNA polymerases I, II, and III, respectively, while the SNAP complex is involved in transcription of the small nuclear RNA (snRNA) genes by RNA polymerases II and III. TBP also associates with a number of basal transcription factors such as TFIIA and TFIIB, and with several regulatory factors such as VP16, E1A, and p53. Here we describe the characterization of a panel of monoclonal antibodies (MAbs) directed against the amino-terminal domain of human TBP. These MAbs recognize different TBP epitopes, some of which have been precisely defined. Different MAbs recognize different TBP-containing complexes and several of them crossreact with TBP from other species. These antibodies can be used to purify TBP-containing complexes in a functional form and should be useful to identify new protein-protein interactions involving TBP.

A New Cys2/His2 Zinc Finger Gene, rKr2, Is Expressed in Differentiated Rat Oligodendrocytes and Encodes a Protein with a Functional Repressor Domain

The function of the vertebrate nervous system is dependent on the proper myelination of its fiber tracts. Myelin of the CNS is produced by oligodendrocytes. To identify gene regulatory proteins expressed in this particular glial cell type, we isolated cDNAs coding for Cys2/His2 zinc finger proteins from a rat oligodendrocyte cDNA library. One clone, named rKr2 (rKr for rat Krüppel-type protein), encodes a protein with 19 carboxy-terminal zinc finger domains and an amino-terminal Krüppel-associated box domain. This amino-terminal domain of the rKr2 protein behaved as a strong transcriptional repressor module when fused to the DNA binding domain of yeast GAL4 and tested on an appropriate reporter construct. High levels of rKr2 mRNA in adult rat tissues were found only in the CNS and testis; in the CNS, the message was predominantly expressed in differentiated oligodendrocytes. The modular structure of the rKr2 protein (carboxy-terminal DNA binding domain, amino-terminal repressor module) and its expression pattern suggest that it acts as a sequence-specific transcriptional repressor in the myelin-producing glial cells of the CNS.