SPH Motif Research Articles

Quantitative Statistical Analysis of cis-Regulatory Sequences in ABA/VP1- and CBF/DREB1-Regulated Genes of Arabidopsis

We have developed a simple quantitative computational approach for objective analysis of cis-regulatory sequences in promoters of coregulated genes. The program, designated MotifFinder, identifies oligo sequences that are overrepresented in promoters of coregulated genes. We used this approach to analyze promoter sequences of Viviparous1 (VP1)/abscisic acid (ABA)-regulated genes and cold-regulated genes, respectively, of Arabidopsis (Arabidopsis thaliana). We detected significantly enriched sequences in up-regulated genes but not in down-regulated genes. This result suggests that gene activation but not repression is mediated by specific and common sequence elements in promoters. The enriched motifs include several known cis-regulatory sequences as well as previously unidentified motifs. With respect to known cis-elements, we dissected the flanking nucleotides of the core sequences of Sph element, ABA response elements (ABREs), and the C repeat/dehydration-responsive element. This analysis identified the motif variants that may correlate with qualitative and quantitative differences in gene expression. While both VP1 and cold responses are mediated in part by ABA signaling via ABREs, these responses correlate with unique ABRE variants distinguished by nucleotides flanking the ACGT core. ABRE and Sph motifs are tightly associated uniquely in the coregulated set of genes showing a strict dependence on VP1 and ABA signaling. Finally, analysis of distribution of the enriched sequences revealed a striking concentration of enriched motifs in a proximal 200-base region of VP1/ABA and cold-regulated promoters. Overall, each class of coregulated genes possesses a discrete set of the enriched motifs with unique distributions in their promoters that may account for the specificity of gene regulation.

Molecular cloning of a cDNA encoding human SPH-binding factor, a conserved protein that binds to the enhancer-like region of the U6 small nuclear RNA gene promoter.

Many vertebrate small nuclear RNA gene promoters contain an SPH motif in their distal control regions that can confer transcriptional stimulation by RNA polymerase II or RNA polymerase III. Using the human U6 gene SPH motif as a probe, we isolated a cDNA encoding human SPH-binding factor (hSBF) from a HeLa cell expression library. The coding region of hSBF is almost identical to ZNF143, a 626 amino acid, seven zinc finger protein of previously unknown function. Furthermore, the predicted amino acid sequence of hSBF is highly homologous to Xenopus laevis and mouse Staf proteins, that bind to SPH motifs and stimulate transcription of selenocysteine tRNA gene promoters. Recombinant hSBF expressed in vitro or from Escherichia coli bound specifically to the human U6 gene SPH motif as shown by DNase I footprinting and electrophoretic mobility shift assays using various mutant SPH sites as competitors. Antibodies prepared against recombinant hSBF inhibited assembly of native SBF-DNA complexes. Immunodepleted HeLa S100 transcription extract no longer supported elevated levels of transcription by RNA polymerase III from a U6 promoter containing an SPH motif, whereas addition of recombinant hSBF protein to the immunodepleted extract reconstituted stimulated transcription.

Determination of Occupancies of the SPH and GT-IIC Transcription Factor Binding Motifs in SV40: Evidence for Two Forms of Transcription Elongation Complex

Occupancies of the SPH and GT-IIC sequence motifs in the native SV40 late transcription elongation complex were determined by assessing blockage to restriction enzyme cleavage. Cleavages specific to the transcription elongation complex were quantified by radioactive extension labeling and polymerase run-off analysis. The SPH motif was assayed bySphI digestion and found to be unoccupied. In contrast, digestion withPvuII at the GT-IIC site was blocked in 36% of the complexes, indicating that approximately a third of the complexes are occupied by factor. This fractional occupancy indicates that there are at least two forms of SV40 late transcription elongation complexes, one form with the GT-IIC site occupied by a factor and another with the site vacant.

Cloning, structural analysis and mapping of the mouse selenocysteine tRNA([Ser]Sec) gene (Trsp).

The tRNA([Ser]Sec) molecule mediates the synthesis of selenoproteins by incorporating selenocysteine into specific UGA codons upon translation of mRNAs that encode selenocysteine-containing proteins. The mouse gene encoding tRNA([Ser]Sec) (Trsp) was isolated from a genomic library and sequenced. The mouse sequence is colinear with its tRNA product, and contains a C to T transition relative to the homologous genes in other vertebrates except rat. Transcriptional control motifs found 5' to the tRNA coding region included a TATA element, a PSE element and an SPH motif which is associated with an octamer motif. A Northern hybridization analysis showed highest expression in the testis, followed by thymus, spleen, kidney, ovary, brain, liver, heart and skeletal muscle. Surprisingly, the expression level was lowest in embryonic stem cells. These results suggest a tissue-specific transcriptional control. Using restriction fragment length variants (RFLVs) in interspecific backcross mice between Mus musculus (C3H strain) and Mus spretus, the Trsp gene was mapped to the proximal region of mouse Chr 7, cosegregating with octamer-binding transcription factor-2 (Otf2).

Cloning of an SNF2/SWI2-related Protein That Binds Specifically to the SPH Motifs of the SV40 Enhancer and to the HIV-1 Promoter

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

Characterization of a delayed early serum response region.

The proliferin (PLF) gene promoter provides a relatively simple model system for the study of growth-regulated gene expression in mouse cells. The promoter elements required for this serum-induced regulation have been identified and include an AP-1 site as well as an adjacent element comprised of three imperfect repeats that are similar in sequence to the simian virus 40 (SV40) Sph motif. Distinct protein complexes bound independently to the AP-1 and Sph elements, and both of these juxtaposed sites could be occupied simultaneously. Furthermore, serum stimulation of mouse fibroblasts resulted in similar increases in protein binding to the AP-1 and Sph elements. Consistent with this increase in AP-1 and Sph binding activity, the PLF AP-1 and Sph elements were independently able to confer serum responsiveness to a minimal promoter, and together these two elements acted synergistically in response to serum. Although several members of the AP-1 family were able to activate the PLF gene promoter in transient cotransfection experiments, the predominant AP-1 components interacting with the PLF gene promoter in serum-stimulated cells were Fra-1, JunB, and JunD. Analysis of the Sph element revealed that mutation of Sph repeats I or III abolished serum responsiveness of the PLF gene promoter, and mutation of Sph repeat III decreased protein binding to this element. Although the Sph element is similar in sequence to the SV40 element, the PLF Sph-binding factor is distinct from TEF-1, the factor that binds to the SV40 Sph motif.

Characterization of a delayed early serum response region.

The proliferin (PLF) gene promoter provides a relatively simple model system for the study of growth-regulated gene expression in mouse cells. The promoter elements required for this serum-induced regulation have been identified and include an AP-1 site as well as an adjacent element comprised of three imperfect repeats that are similar in sequence to the simian virus 40 (SV40) Sph motif. Distinct protein complexes bound independently to the AP-1 and Sph elements, and both of these juxtaposed sites could be occupied simultaneously. Furthermore, serum stimulation of mouse fibroblasts resulted in similar increases in protein binding to the AP-1 and Sph elements. Consistent with this increase in AP-1 and Sph binding activity, the PLF AP-1 and Sph elements were independently able to confer serum responsiveness to a minimal promoter, and together these two elements acted synergistically in response to serum. Although several members of the AP-1 family were able to activate the PLF gene promoter in transient cotransfection experiments, the predominant AP-1 components interacting with the PLF gene promoter in serum-stimulated cells were Fra-1, JunB, and JunD. Analysis of the Sph element revealed that mutation of Sph repeats I or III abolished serum responsiveness of the PLF gene promoter, and mutation of Sph repeat III decreased protein binding to this element. Although the Sph element is similar in sequence to the SV40 element, the PLF Sph-binding factor is distinct from TEF-1, the factor that binds to the SV40 Sph motif.

Promoter Strength and Structure Dictate Module Composition in RNA Polymerase III Transcriptional Activator Elements

RNA polymerase III transcription of genes with external promoters only (e.g. U6 snRNA) or containing in addition an internal B box (selenocysteine tRNA Sec) is stimulated by upstream elements; a distal sequence element (DSE) for U6 or an activator element in the tRNA Sec gene. In contrast to the composite structure of the DSE which requires an octamer motif, the Xenopus tRNA Sec activator element contains an SPH motif only. In vivo transcription is optimally stimulated by SPH in an absolute octamer-independent manner since adding octamer does not induce superstimulation. Experiments performed in the work presented here led to the following observations. Co-operation between SPH and octamer motifs can be detected in two distinct cases: first when these motifs are placed in front of B box-less tRNA Sec or U6 external promoters and second, if either element of the external promoter (proximal sequence element or TATA element), or the SPH motif itself, are altered. Altogether, our data provide evidence that an SPH motif can function alone in an optimized promoter only. In contrast, an octamer becomes indispensable when the basal promoter is weak or disabled. It follows that module composition of Pol III transcriptional activator elements is dependent on the structure and strength of the promoter. This reveals the existence of cross-talk between activator and promoter elements, mediated by the bound transcription factors, which are thus able to compensate for each other in order to allow successful assembly of the transcription complex.

Structural requirements for the functional activity of a U1 snRNA gene enhancer.

The transcriptional enhancer of a chicken U1 small nuclear RNA (snRNA) gene contains a GC-box, an octamer motif, and an SPH motif that are recognized by the transcription factors Sp1, Oct-1, and SBF respectively. Previous work indicated that the octamer and the SPH motifs were both required for U1 gene enhancer activity in frog oocytes when the U1 gene was coinjected with a competing snRNA gene template. Here we show that neither two copies of the octamer motif, nor two copies of the SPH motif, can effectively substitute for the natural combination of octamer and SPH. Furthermore, neither the octamer nor the SPH motif (in the absence of the other) functioned efficiently in combination with a GC-box. Alteration of the spacing between the octamer and SPH motifs also reduced U1 template activity. Several potential cis-acting elements other than the SPH motif, with one possible exception among those tested, were unable to cooperate with the octamer motif to effectively enhance U1 gene expression. These results indicate that rather stringent structural requirements exist with respect to the essential cis-acting motifs present in the U1 enhancer, possibly reflecting the unique properties of the transcription complexes assembled on snRNA gene promoters.

Optimal tRNA((Ser)Sec) gene activity requires an upstream SPH motif.

The X. laevis tRNA((Ser)Sec) gene is different from the other tRNA genes in that its promoter contains two external elements, a PSE and a TATA box functionally equivalent to those of the U6 snRNA gene. Of the two internal promoters governing classical tRNA gene transcription, only subsists the internal B box. In this report, we show that the tRNA((Ser)Sec) contains in addition an activator element (AE) which we have mapped by extensive mutagenesis. Activation is only dependent on a 15 bp fragment residing between -209 and -195 and containing an SPH motif. In vitro, this element forms a complex with a nuclear protein which is different from the TEF-1 transcriptional activator that binds the SV40 Sph motifs. This AE is versatile since it shows capacity of activating a variety of genes in vivo, including U1 and U6 snRNAs and HSV thymidine kinase. Unexpectedly for an snRNA-related gene, the tRNA((Ser)Sec) is deprived of octamer or octamer-like motifs. The X.laevis tRNA((Ser)Sec) gene represents the first example of a Pol III snRNA-type gene whose activation of transcription is completely octamer-independent.

Octamer and SPH motifs in the U1 enhancer cooperate to activate U1 RNA gene expression.

The transcriptional enhancer of a chicken U1 small nuclear RNA gene has been shown to extend over approximately 50 base pairs of DNA sequence located 180 to 230 base pairs upstream of the U1 transcription initiation site. It is composed of multiple functional motifs, including a GC box, an octamer motif, and a novel SPH motif. The contributions of these three distinct sequence motifs to enhancer function were studied with an oocyte expression assay. Under noncompetitive conditions in oocytes, the SPH motif is capable of stimulating U1 RNA transcription in the absence of the other functional motifs, whereas the octamer motif by itself lacks this ability. However, to form a transcription complex that is stable to challenge by a second competing small nuclear RNA transcription unit, both the octamer and SPH motifs are required. The GC box, although required for full enhancer activity, is not essential for stable complex formation in oocytes. Site-directed mutagenesis was used to study the DNA sequence requirements of the SPH motif. Functional activity of the SPH motif is spread throughout a 24-base-pair region 3' of the octamer but is particularly dependent upon sequences near an SphI restriction site located at the center of the SPH motif. Using embryonic chicken tissue as a source material, we identified and partially purified a factor, termed SBF, that binds sequence specifically to the SPH motif of the U1 enhancer. The ability of this factor to recognize and bind to mutant enhancer DNA fragments in vitro correlates with the functional activity of the corresponding enhancer sequences in vivo.

Octamer and SPH Motifs in the U1 Enhancer Cooperate To Activate U1 RNA Gene Expression

The transcriptional enhancer of a chicken U1 small nuclear RNA gene has been shown to extend over approximately 50 base pairs of DNA sequence located 180 to 230 base pairs upstream of the U1 transcription initiation site. It is composed of multiple functional motifs, including a GC box, an octamer motif, and a novel SPH motif. The contributions of these three distinct sequence motifs to enhancer function were studied with an oocyte expression assay. Under noncompetitive conditions in oocytes, the SPH motif is capable of stimulating U1 RNA transcription in the absence of the other functional motifs, whereas the octamer motif by itself lacks this ability. However, to form a transcription complex that is stable to challenge by a second competing small nuclear RNA transcription unit, both the octamer and SPH motifs are required. The GC box, although required for full enhancer activity, is not essential for stable complex formation in oocytes. Site-directed mutagenesis was used to study the DNA sequence requirements of the SPH motif. Functional activity of the SPH motif is spread throughout a 24-base-pair region 3' of the octamer but is particularly dependent upon sequences near an SphI restriction site located at the center of the SPH motif. Using embryonic chicken tissue as a source material, we identified and partially purified a factor, termed SBF, that binds sequence specifically to the SPH motif of the U1 enhancer. The ability of this factor to recognize and bind to mutant enhancer DNA fragments in vitro correlates with the functional activity of the corresponding enhancer sequences in vivo.

U4B snRNA gene enhancer activity requires functional octamer and SPH motifs

Expression of the chicken U4B small nuclear RNA (snRNA) gene is stimulated by a transcriptional enhancer located approximately 190-227 base pairs upstream of the transcription start site. This enhancer is composed of at least two functional motifs: an octamer (binding site for Oct-1) and an SPH motif. We now report that these two motifs functionally cooperate to stimulate U4B snRNA gene expression, and both are required for the formation of a stable transcription complex. Expression in frog oocytes of 24 different point mutant constructions indicates that the functional SPH motif is at least 15 base pairs in length. It is a recognition site for a sequence specific DNA-binding protein, termed SBF, purified from chicken embryonic nuclear extracts. The ability of the mutant SPH motif constructions to be recognized by SBF in vitro correlates with their transcriptional activities, suggesting that SBF mediates the stimulatory effect of the U4B SPH motif. These results are similar to our recent findings on the chicken U1 gene enhancer, which also contains adjacent binding sites for Oct-1 and SBF. These studies, together with evolutionary considerations and sequence comparisons among snRNA gene enhancers, suggest that cooperativity between octamer and SPH motifs could be a widely-employed mechanism for generating vertebrate snRNA gene enhancer activity.

The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence

We have purified a protein (TEF-1) that specifically binds to two sequence unrelated motifs (GT-IIC and Sph) of the simian virus 40 (SV40) enhancer. TEF-1 binds cooperatively to templates containing tandem but not inverted or spacéd repeats of its cognate motifs. This cooperative binding correlates with the ability of the tandem repeats to generate enhancer activity in vivo. In contrast, TEF-1 and a second SV40 enhancer binding protein, TEF-2, bind independently to templates containing the cognate motifs of both proteins (GT-1 and either GT-IIC or Sph motifs) even though these motifs cooperate in enhancer activity in vivo. These results allow us to distinguish different classes of enhancer factors.

Transcriptional signals of a U4 small nuclear RNA gene

The signals controlling the expression of a chicken U4 small nuclear RNA (snRNA) gene have been studied by microinjection into Xenopus oocytes. At least two distinct regions in the 5'-flanking DNA contribute to U4B RNA gene expression. The proximal regulatory element, which is inactivated by a 5'-flanking DNA deletion to position -38, provides a basal level of U4B RNA synthesis. The distal regulatory region, centered near position -200, acts as a transcriptional enhancer. It provides a 4-5 fold stimulation of U4B RNA gene expression above the basal level, and, like mRNA enhancers, is composed of multiple functional motifs. One of these, the octamer sequence ATGCAAAG, has previously been recognized as an important element of U1 and U2 snRNA gene enhancers, as well as being involved in the expression of a number of mRNA genes. However, the octamer sequence is not sufficient for U4B enhancer activity. An additional element, an "Sph motif," is located 12 base pairs downstream of the octamer and is an essential component of the U4B enhancer. Transcriptional competition studies indicate that the U4B and U1 snRNA genes utilize a common set of transcription factors.

Cell-type specific protein binding to the enhancer of simian virus 40 in nuclear extracts.

Enhancers are cis-acting activators of transcription from homologous or heterologous promoter elements of viral and cellular genes (see refs 1-6 for reviews). The activity of the simian virus 40 (SV40) (refs 7-9) and immunoglobulin heavy-chain gene (IgH) (refs 10, 11) enhancers has been reproduced to some extent in vitro and appears to be mediated by trans-acting factors both in vitro and in vivo. The SV40 enhancer consists of multiple sequence motifs in two domains, A and B (Fig. 1, see ref. 14): domain B contains GT-I and -II and two TC motifs, of which only TC-II is important for enhancer activity in HeLa cells; and domain A contains the P and the two Sph motifs, the repetition of which generates the sequence 5'-ATGCAAAG-3', similar to the 'octameric' sequence of the IgH enhancer, (Fig. 4i; refs 14, 16), where it is important for enhancing activity. Each SV40 enhancer motif is a binding site for a protein or proteins present in HeLa cell nuclear extracts. Unlike the SV40 enhancer, which is active in HeLa and lymphoid B cells, the IgH enhancer is preferentially active in B cells, suggesting that not all the trans-acting factors necessary for its activity are present in HeLa cells. However, the IgH enhancer can compete with the SV40 enhancer in vitro in HeLa or lymphoid cell extracts and in vivo in B cells. Here we show that both human HeLa and BJA-B lymphoid B-cell nuclear extracts contain proteins that bind to specific, sometimes overlapping, motifs of the SV40 enhancer. Some binding is cell-specific, suggesting that it is not the same set of sequence motifs and proteins that is responsible for the enhancer activity in the two cell types. This is confirmed by our results obtained in vivo with mutated SV40 enhancers.