Imperfect Tandem Repeats Research Articles

Functional Domains and Evolutionary History of the PMEL and GPNMB Family Proteins.

The ancient paralogs premelanosome protein (PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.

Identification of a large repetitive RTX immunogen in a highly virulent Rodentibacter heylii strain

Rodentibacter (R.) heylii is frequently detected in laboratory rodents. Repeats in toxin (RTX) toxins are considered important virulence factors of this major murine pathogen. We evaluated the virulence of a R.heylii strain negative for all known RTX toxin genes and Muribacter (M.) muris, a commensal in mice, in experimental infections of C57BL/6 and BALB/c mice. Experimental intranasal infection with 108 CFU of the pnxI-, pnxII- and pnxIII- R. heylii strain resulted in 75% and 100% mortality in C57BL/6 and BALB/c mice, respectively. In early losses, multiple internal organs were infected and purulent bronchopneumonia was the main pathology. Intranasal application of M. muris did not result in mortality or severe weight loss. Immunoproteomics led to the identification of a surface-associated and specific immunogen, which was designated as R. heylii immunogen A (RhiA) and which was exclusively recognised by sera obtained from mice infected with this R. heylii pathotype. RhiA is a 262.6 kDa large protein containing long imperfect tandem repeats and C-terminal RTX consensus sequences. Immunohistochemical analysis confirmed that this R.heylii pathotype expresses RhiA in the lower respiratory tract. In summary, this study describes a specific immunogen in a virulent R. heylii, strain which is an excellent antigen for pathotype-specific serological screenings and which might carry out RTX-related functions.

Novel In Silico Analyses of Repetitive Spider Silk Sequences to Understand the Evolution and Mechanical Properties of Fibrous Protein Materials

The mechanical properties of spider silks have diverged as spiders have diversely speciated. Because the main components of silks are proteins, it is valuable to investigate their sequences. However, silk sequences have been regarded as difficult information to analyze due to their imbalance and imperfect tandem repeats (ITR). Here, an in silico approach is applied to systemically analyze a group of silk sequences. It is found that every time new spider groups emerge, unique trimer motifs appear. These trimer motifs are used to find additional clues of evolution and to determine relationships with mechanical properties. For the first time, crucial evidence is provided that shows silk sequences coevolved with spider species and the mechanical properties of their fibers to adapt to new living environments. This novel approach can be used as a platform for analyzing other groups of ITR-harboring proteins and to obtain information for the design of tailor-made fibrous protein materials.

Isolation and genomic characterization of Culex theileri flaviviruses in field-collected mosquitoes from Turkey

Vector surveillance for the arthropod-borne infections has resulted in the isolation of a growing number of novel viruses, including several flavivirus strains that exclusively replicate in insects. This report describes the isolation and genomic characterization of four insect-specific flaviviruses from mosquitoes, previously collected from various locations in Turkey. C6/36 Aedes albopictus and Vero cell lines were inoculated with mosquito pools. On C6/36 cells, mild cytopathic effects, characterized as rounding and detachment, were observed in four pools that comprised female Culex theileri mosquitoes. Complete (3 isolates, 10,697 nucleotides) or near-complete (1 isolate, 10,452 nucleotides) genomic characterization was performed in these culture supernatants via next generation sequencing. All strains demonstrated high genetic similarities, with over 99% identity match on nucleotide and amino acid alignments, revealing them to be different isolates of the same virus. Sequence comparisons identified the closest relative to be the Culex theileri flavivirus (CTFV) strains, originally characterized in Portugal. Phylogenetic analyses demonstrated that the isolates remained distinct as a cluster but formed a monophyletic group with CTFV strains, and shared a common ancestor with Quang Binh or related Culex flaviviruses. The organization of the viral genome was consistent with the universal flavivirus structure and stem-loops; conserved motifs and imperfect tandem repeats were identified in the non-coding ends of the viral genomes. A potential ribosomal shifting site, resulting in the translation of an additional reading frame, was detected. The deduced viral polyprotein comprised 3357 amino acids and was highly-conserved. Amino acid variations, presumably associated with adaptive environmental pressures, were identified. These isolates comprise the first fully characterized insect-specific flaviviruses in Turkey. Their impact on West Nile virus circulation, which is also endemic in the study region, remains to be explored.

Repeat sequence analysis of Mycobacterium tuberculosis

Tuberculosis is a major cause of human death around the world and is caused by various strains of mycobacteria, usually Mycobacterium tuberculosis. The bacterium infects 1.8 billion people yearly which equals one-third of the world population. Pathogenic bacterial genomes contain many perfect, imperfect and approximate tandem repeats that can serve as marker for genotyping these pathogens. Tandem repeats are generated by duplications during successive generations which changes genome structures, thereby providing diversity and improving the fitness of the pathogen during infection. In the present study, we have retrieved reference genomes of 30 Mycobacterium tuberculosis strains and used Tandem Repeats Finder tool for the prediction of genome-wide repeats. The genomes of publicly available Mycobacterium tuberculosis strains varied in size from 4.38 MB to 4.42 MB. After trimming out low quality tandem repeats, we found the strain M. tuberculosis CCDC5079 to have the maximum tandem repeats density number (DN) while density length (DL) was found to be maximum in M. tuberculosis KIT87190.We found the lowest repeat density number and length in M. tuberculosis Erdman. This study would help in mapping and identification of pathogenic bacterial species and to understand the importance of tandem repeats in their evolution.

The O-glycomap of Lubricin, a Novel Mucin Responsible for Joint Lubrication, Identified by Site-specific Glycopeptide Analysis

The lubricative, heavily glycosylated mucin-like synovial glycoprotein lubricin has previously been observed to contain glycosylation changes related to rheumatoid and osteoarthritis. Thus, a site-specific investigation of the glycosylation of lubricin was undertaken, in order to further understand the pathological mechanisms involved in these diseases. Lubricin contains an serine/threonine/proline (STP)-rich domain composed of imperfect tandem repeats (EPAPTTPK), the target for O-glycosylation. In this study, using a liquid chromatography–tandem mass spectrometry approach, employing both collision-induced and electron-transfer dissociation fragmentation methods, we identified 185 O-glycopeptides within the STP-rich domain of human synovial lubricin. This showed that adjacent threonine residues within the central STP-rich region could be simultaneously and/or individually glycosylated. In addition to core 1 structures responsible for biolubrication, core 2 O-glycopeptides were also identified, indicating that lubricin glycosylation may have other roles. Investigation of the expression of polypeptide N-acetylgalactosaminyltransferase genes was carried out using cultured primary fibroblast-like synoviocytes, a cell type that expresses lubricin in vivo. This analysis showed high mRNA expression levels of the less understood polypeptide N-acetylgalactosaminyltransferase 15 and 5 in addition to the ubiquitously expressed polypeptide N-acetylgalactosaminyltransferase 1 and 2 genes. This suggests that there is a unique combination of transferase genes important for the O-glycosylation of lubricin. The site-specific glycopeptide analysis covered 82% of the protein sequence and showed that lubricin glycosylation displays both micro- and macroheterogeneity. The density of glycosylation was shown to be high: 168 sites of O-glycosylation, predominately sialylated, were identified. These glycosylation sites were focused in the central STP-rich region, giving the domain a negative charge. The more positively charged lysine and arginine residues in the N and C termini suggest that synovial lubricin exists as an amphoteric molecule. The identification of these unique properties of lubricin may provide insight into the important low-friction lubricating functions of lubricin during natural joint movement.

Mitochondrial genome of the tomato clownfish Amphiprion frenatus (Pomacentridae, Amphiprioninae)

The complete mitochondrial (mt) genome of the tomato clownfish Amphiprion frenatus was obtained in this study. The circular mtDNA molecule was 16,774 bp in size and the overall nucleotide composition of the H-strand was 29.72% A, 25.81% T, 15.38% G and 29.09% C, with an A + T bias. The complete mitogenome encoded 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region (D-loop), with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. The D-loop included termination associated sequence (TAS), central conserved domain (CCD) and conserved sequence block (CSB), and was composed of 6 complete continuity tandem repeat units and an imperfect tandem repeat unit.

Complete mitochondrial genome of the yellowtail clownfish Amphiprion clarkii (Pisces: Perciformes, Pomacentridae)

In this study we determined the complete mitochondrial (mt) genome of the yellowtail clownfish Amphiprion clarkii using eight consensus primer pairs with a long PCR technique. The circular mtDNA molecule was 16,976 bp in size and the overall nucleotide composition of the H-strand was 29.15% A, 26.15% T, 15.67% G and 29.03% C, with an A + T bias. The complete mitogenome contained 13 protein-coding genes, 2 rRNAs, 22 tRNAs and 1 control region (D-loop), and the gene order was typical of vertebrate mitogenomes. We determined five complete continuity tandem repeat units and one imperfect tandem repeat, all located downstream in the control region.

Genome Reference and Sequence Variation in the Large Repetitive Central Exon of Human MUC5AC

Despite modern sequencing efforts, the difficulty in assembly of highly repetitive sequences has prevented resolution of human genome gaps, including some in the coding regions of genes with important biological functions. One such gene, MUC5AC, encodes a large, secreted mucin, which is one of the two major secreted mucins in human airways. The MUC5AC region contains a gap in the human genome reference (hg19) across the large, highly repetitive, and complex central exon. This exon is predicted to contain imperfect tandem repeat sequences and multiple conserved cysteine-rich (CysD) domains. To resolve the MUC5AC genomic gap, we used high-fidelity long PCR followed by single molecule real-time (SMRT) sequencing. This technology yielded long sequence reads and robust coverage that allowed for de novo sequence assembly spanning the entire repetitive region. Furthermore, we used SMRT sequencing of PCR amplicons covering the central exon to identify genetic variation in four individuals. The results demonstrated the presence of segmental duplications of CysD domains, insertions/deletions (indels) of tandem repeats, and single nucleotide variants. Additional studies demonstrated that one of the identified tandem repeat insertions is tagged by nonexonic single nucleotide polymorphisms. Taken together, these data illustrate the successful utility of SMRT sequencing long reads for de novo assembly of large repetitive sequences to fill the gaps in the human genome. Characterization of the MUC5AC gene and the sequence variation in the central exon will facilitate genetic and functional studies for this critical airway mucin.

Structural transitions in tau k18 on micelle binding suggest a hierarchy in the efficacy of individual microtubule‐binding repeats in filament nucleation

The protein tau is found in an aggregated filamentous state in the intraneuronal paired helical filament deposits characteristic of Alzheimer's disease and other related dementias and mutations in tau protein and mRNA cause frontotemproal dementia. Tau isoforms include a microtubule-binding domain containing either three or four imperfect tandem microtubule binding repeats that also form the core of tau filaments and contain hexapaptide motifs that are critical for tau aggregation. The tau microtubule-binding domain can also engage in direct interactions with detergents, fatty acids, or membranes, which can greatly facilitate tau aggregation and may also mediate some tau functions. Here, we show that the alternatively spliced second microtubule-binding repeat exhibits significantly different structural characteristics compared with the other three repeats in the context of the intact repeat domain. Most notably, the PHF6* hexapeptide motif located at the N-terminus of repeat 2 has a lower propensity to form strand-like structure than the corresponding PHF6 motif in repeat 3, and unlike PHF6 converts to partially helical structure in the micelle-bound state. Interestingly, the behavior of the Module-B motif, located at the beginning of repeat 4, resembles that of PHF6* rather than PHF6. Our observations, combined with previous results showing that PHF6* and Module-B are both less effective than PHF6 in nucleating tau aggregation, suggest a hierarchy in the efficacy of these motifs in nucleating tau aggregation that originates in differences in their intrinsic propensities for extended strand-like structure and the resistance of these propensities to changes in tau's environment.

Complete mitochondrial genome of the chocolate hind Cephalopholis boenak (Pisces: Perciformes)

The complete mitochondrial (mt) genome of the chocolate hind Cephalopholis boenak was obtained in this study, which is the first complete mitogenome in the genus Cephalopholis. The circular mtDNA molecule was 16 771 bp in size and the overall nucleotide composition of the H-strand was 29.58% A, 27.36% T, 15.84% G, and 27.22% C, with an A + T bias. The complete mitogenome encoded 13 protein-coding genes, 2 rRNAs, 22 tRNAs and 1 control region (D-loop), with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. The control region located between the tRNA Pro and tRNA Phe genes and the length was 1 064 bp, rich in A + T (70.11%); it was composed of 13 complete continuity tandem repeat units, 5′-TACATATCTATGTACTTAA-3′, and one imperfect tandem repeat.

Structure, dynamics and domain organization of the repeat protein Cin1 from the apple scab fungus

Venturia inaequalis is a hemi-biotrophic fungus that causes scab disease of apple. A recently-identified gene from this fungus, cin1 (cellophane-induced 1), is up-regulated over 1000-fold in planta and considerably on cellophane membranes, and encodes a cysteine-rich secreted protein of 523 residues with eight imperfect tandem repeats of ~60 amino acids. The Cin1 sequence has no homology to known proteins and appears to be genus-specific; however, Cin1 repeats and other repeat domains may be structurally similar. An NMR-derived structure of the first two repeat domains of Cin1 (Cin1-D1D2) and a low-resolution model of the full-length protein (Cin1-FL) using SAXS data were determined. The structure of Cin1-D1D2 reveals that each domain comprises a core helix–loop–helix (HLH) motif as part of a three-helix bundle, and is stabilized by two intra-domain disulfide bonds. Cin1-D1D2 adopts a unique protein fold as DALI and PDBeFOLD analysis identified no structural homology. A 15N backbone NMR dynamic analysis of Cin1-D1D2 showed that a short stretch of the inter-domain linker has large amplitude motions that give rise to reciprocal domain–domain mobility. This observation was supported by SAXS data modeling, where the scattering length density envelope remains thick at the domain–domain boundary, indicative of inter-domain dynamics. Cin1-FL SAXS data models a loosely-packed arrangement of domains, rather than the canonical parallel packing of adjacent HLH repeats observed in α-solenoid repeat proteins. Together, these data suggest that the repeat domains of Cin1 display a “beads-on-a-string” organization with inherent inter-domain flexibility that is likely to facilitate interactions with target ligands.

The mitogenome of Gammarus duebeni (Crustacea Amphipoda): A new gene order and non-neutral sequence evolution of tandem repeats in the control region

We determined the complete mitogenome sequence of Gammarus duebeni (Peracarida, Amphipoda). The mitogenome is circular and has a length of 15,651bp. The content corresponds to typical mitogenomes of metazoans. The gene order and transcriptional polarity of the protein-coding genes is identical to the pancrustacean ground pattern. Six tRNA genes are rearranged, making the gene order unique. Thus it will bring forward the understanding of mitogenome evolution within the Peracarida, for which much more derived gene orders are known. We postulate that the gene string trnA–trnS1 (AGN)–trnN–trnE–trnR constitutes an apomorphic character for the Amphipoda. In contrast to the relatively large genome size, we found two extremely truncated rRNA genes. The rrnL gene is the shortest (986bp) reported up until now for crustaceans. A six-time imperfect tandem repeat was observed within the control region. The inferred deterministic pattern of variation between the repeat units makes it likely, that functional constraints play an important role in the evolutionary dynamics and extant appearance of the repeat array.

Myb transcription factors in the oomycete Phytophthora with novel diversified DNA-binding domains and developmental stage-specific expression

Transcription factors containing two or three imperfect tandem repeats of the Myb DNA-binding domain (named R2R3 and R1R2R3, respectively) regulate important processes in growth and development. This study characterizes the structure, evolution, and expression of these proteins in the potato pathogen Phytophthora infestans and other oomycetes. P. infestans was found to encode five R2R3 and nine R1R2R3 transcription factor-like proteins, plus several with additional configurations of Myb domains. Sets of R2R3 and R1R2R3 orthologs are well-conserved in three Phytophthora species. Analyses of sites that bind DNA in canonical Myb transcription factors, such as mammalian c-Myb, revealed unusual diversification in the DNA recognition helices of the oomycete proteins. While oomycete R2R3 proteins contain c-Myb-like helices, R1R2R3 proteins exhibit either c-Myb-like or novel sequences. This suggests divergence in their DNA-binding specificities, which was confirmed by electrophoretic mobility shift assays. Eight of the P. infestans R2R3 and R1R2R3 genes are up-regulated during sporulation and three during zoospore release, which suggests their involvement in spore development. This is supported by the observation that an oomycete that does not form zoospores, Hyaloperonospora arabidopsidis, contains one-third fewer of these genes than Phytophthora.

Skittle: A 2-Dimensional Genome Visualization Tool

BackgroundIt is increasingly evident that there are multiple and overlapping patterns within the genome, and that these patterns contain different types of information - regarding both genome function and genome history. In order to discover additional genomic patterns which may have biological significance, novel strategies are required. To partially address this need, we introduce a new data visualization tool entitled Skittle.ResultsThis program first creates a 2-dimensional nucleotide display by assigning four colors to the four nucleotides, and then text-wraps to a user adjustable width. This nucleotide display is accompanied by a "repeat map" which comprehensively displays all local repeating units, based upon analysis of all possible local alignments. Skittle includes a smooth-zooming interface which allows the user to analyze genomic patterns at any scale.Skittle is especially useful in identifying and analyzing tandem repeats, including repeats not normally detectable by other methods. However, Skittle is also more generally useful for analysis of any genomic data, allowing users to correlate published annotations and observable visual patterns, and allowing for sequence and construct quality control.ConclusionsPreliminary observations using Skittle reveal intriguing genomic patterns not otherwise obvious, including structured variations inside tandem repeats. The striking visual patterns revealed by Skittle appear to be useful for hypothesis development, and have already led the authors to theorize that imperfect tandem repeats could act as information carriers, and may form tertiary structures within the interphase nucleus.

Conserved features of imprinted differentially methylated domains

Genomic imprinting is a conserved epigenetic phenomenon in eutherian mammals, with regards both to the genes that are imprinted and the mechanism underlying the expression of just one of the parental alleles. Epigenetic modifications of alleles of imprinted genes are established during oogenesis and spermatogenesis, and these modifications are then inherited. Differentially methylated domains (DMDs) of imprinted genes are the genomic sites of these inherited epigenetic imprints. We previously showed that CpG-rich imperfect tandem direct repeats within three different mouse DMDs ( Snurf/Snrpn, Kcnq1 and Igf2r), each with a unique sequence, play a central role in maintaining the differential methylation. This finding implicates repeat-related DNA structure, not sequence, in the imprinting mechanism. To better define the important features of this signal, we compared sequences of these three DMD tandem repeats among mammalian species. All DMD repeats contain short indirect repeats, many of which are organized into larger unit repeats. Even though the larger repeat units undergo deletion and addition during evolution (most likely through unequal crossovers during meiosis), the size of DMD tandem repeated regions has remained remarkably stable during mammalian evolution. Moreover, all three DMD tandem repeats have a high-CpG content, an ordered arrangement of CpG dinucleotides, and similar predicted secondary structures. These observations suggest that a structural feature or features of these DMD tandem repeats is the conserved DMD imprinting signal.

Fingerprinting fission yeast: polymorphic markers for molecular genetic analysis of Schizosaccharomyces pombe strains

The fission yeast Schizosaccharomyces pombe is widely used as a model eukaryote for cell and molecular studies but little is known of natural genetic variation in this species. In order to obtain informative molecular markers, imperfect tandem repeats, identified through bioinformatic methods, were tested for length polymorphism in six wild-type strains of Sch. pombe isolated from different substrates and geographical locations in Africa, America, Asia and Europe. Of 26 loci tested, 21 were multi-allelic, consistent with tandem repeat copy number variation. Eleven of these polymorphic tandem repeats are in regions encoding intracellular proteins. Most of the protein-coding repeats are not sited within structured domains but have non-regular predicted structure; one has a repeat unit length corresponding to integer turns of a predicted amphipathic alpha-helix secondary structure, suggesting that this repeat may be tolerated because copy number mutations change alpha-helix length but not orientation within the protein structure. In contrast to the differences observed between natural isolates of Sch. pombe, genetic strains were found to be essentially isogenic: only two polymorphic loci were detected out of 26 minisatellites and five microsatellites tested in 16 strains, including a hypervariable microsatellite in the med15 gene. The polymorphic tandem repeat markers identified in this study will prove useful for DNA fingerprinting and molecular analysis of natural genetic variation in Sch. pombe isolates.

Specific Differentially Methylated Domain Sequences Direct the Maintenance of Methylation at Imprinted Genes

Landmark features of imprinted genes are differentially methylated domains (DMDs), in which one parental allele is methylated on CpG dinucleotides and the opposite allele is unmethylated. Genetic experiments in the mouse have shown that DMDs are required for the parent-specific expression of linked clusters of imprinted genes. To understand the mechanism whereby the differential methylation is established and maintained, we analyzed a series of transgenes containing DMD sequences and showed that imperfect tandem repeats from DMDs associated with the Snurf/Snrpn, Kcnq1, and Igf2r gene clusters govern transgene imprinting. For the Igf2r DMD the minimal imprinting signal is two unit copies of the tandem repeat. This imprinted transgene behaves identically to endogenous imprinted genes in Dnmt1o and Dnmt3L mutant mouse backgrounds. The primary function of the imprinting signal within the transgene DMD is to maintain, during embryogenesis and a critical period of genomic reprogramming, parent-specific DNA methylation states established in the germ line. This work advances our understanding of the imprinting mechanism by defining a genomic signal that dependably perpetuates an epigenetic state during postzygotic development.

Rem, a New Transcriptional Activator of Motility and Chemotaxis in Sinorhizobium meliloti

The expression of 51 known genes clustered in the flagellar regulon of Sinorhizobium meliloti is organized as a three-class hierarchy: class IA comprises the master regulatory genes, visN and visR; class II, controlled by VisNR, comprises flagellar assembly and motility genes; and class III comprises flagellin and chemotaxis genes requiring class II for expression. The expression of visN-visR is constitutive throughout growth, whereas that of class II and class III genes is limited to exponential growth. A new OmpR-like, 25-kDa transcription factor, Rem, whose synthesis is confined to exponential growth, was shown to positively control swimming motility. No phosphorylation of the receiver domain of Rem was required for its activity. Gene expression in tester strains with known deficiencies placed the rem gene (class IB) below visN-visR (class IA) and above class II genes in the regulatory cascade. Footprinting analysis demonstrated that the Rem protein binds to class II gene promoters as well as to its own promoter, indicating that this protein is autoregulatory. An alignment of the Rem-protected DNA sequences revealed a conserved binding motif of imperfect tandem repeats overlapping a predicted -35 promoter box by 3 bp. This new promoter was confirmed by mapping the transcription start site of a typical class II gene, flgB, 5 nucleotides downstream of the -10 promoter box. The transcription of rem is under dual control of an upstream (Rem-activated) class II-type promoter and a downstream (VisNR-activated) sigma70-like promoter. The central role of Rem as the growth-dependent transcriptional activator intermediate between the master regulator, VisNR, and the flagellar and motility genes is a new distinguishing feature of the S. meliloti regulatory cascade.

Interaction between C/EBPβ and Tax down-regulates human T-cell leukemia virus type I transcription

The human T-cell leukemia virus type I (HTLV-I) Tax protein trans-activates viral transcription through three imperfect tandem repeats of a 21-bp sequence called Tax-responsive element (TxRE). Tax regulates transcription via direct interaction with some members of the activating transcription factor/CRE-binding protein (ATF/CREB) family including CREM, CREB, and CREB-2. By interacting with their ZIP domain, Tax stimulates the binding of these cellular factors to the CRE-like sequence present in the TxREs. Recent observations have shown that CCAAT/enhancer binding protein β (C/EBPβ) forms stable complexes on the CRE site in the presence of CREB-2. Given that C/EBPβ has also been found to interact with Tax, we analyzed the effects of C/EBPβ on viral Tax-dependent transcription. We show here that C/EBPβ represses viral transcription and that Tax is no more able to form a stable complex with CREB-2 on the TxRE site in the presence of C/EBPβ. We also analyzed the physical interactions between Tax and C/EBPβ and found that the central region of C/EBPβ, excluding its ZIP domain, is required for direct interaction with Tax. It is the first time that Tax is described to interact with a basic leucine-zipper (bZIP) factor without recognizing its ZIP domain. Although unexpected, this result explains why C/EBPβ would be unable to form a stable complex with Tax on the TxRE site and could then down-regulate viral transcription. Lastly, we found that C/EBPβ was able to inhibit Tax expression in vivo from an infectious HTLV-I molecular clone. In conclusion, we propose that during cell activation events, which stimulate the Tax synthesis, C/EBPβ may down-regulate the level of HTLV-I expression to escape the cytotoxic-T-lymphocyte response.