Conserved DNA Elements Research Articles

Brain Evolution and Uniqueness in the Human Genome

Despite an ever-expanding database of sequenced mammalian genomes to be mined for clues, the emergence of the unique human brain remains an evolutionary enigma. In their new study, trawl the human genome and those of other mammals in search of short conserved DNA elements that show extremely rapid evolution only in humans. As they report in a recent issue of Nature, their scan yielded a gene for a novel noncoding RNA that adopts a human-specific structure and may regulate neurodevelopment.

Biotin Sensing in Saccharomyces cerevisiae Is Mediated by a Conserved DNA Element and Requires the Activity of Biotin-Protein Ligase

Biotin is a water-soluble vitamin that functions as a prosthetic group in carboxylation reactions. In addition to its role as a cofactor, biotin has multiple roles in gene regulation. We analyzed biotin effects on gene expression in the yeast Saccharomyces cerevisiae and demonstrated by microarray, Northern, and Western analyses that all yeast genes encoding proteins involved in biotin metabolism are up-regulated following biotin depletion. Many of these genes contain a palindromic promoter element that is necessary and sufficient for mediating the biotin response and functions as an upstream-activating sequence. Mutants lacking the plasma membrane biotin transporter Vht1p display constitutively high expression levels of biotin-responsive genes. However, they react normally to biotin precursors that do not require Vht1p for uptake. The biotin-like effect of precursors with regard to gene expression requires their intracellular conversion to biotin. This demonstrates that Vht1p does not act as a sensor for biotin and that intracellular biotin is crucial for gene expression. Mutants with defects in biotin-protein ligase, similar to vht1delta mutants, also display aberrantly high expression of biotin-responsive genes. Like vht1delta cells, they have reduced levels of protein biotinylation, but unlike vht1delta mutants, they possess normal levels of free intracellular biotin. This indicates that free intracellular biotin is irrelevant for gene regulation and identifies biotin-protein ligase as an important element of the biotin-sensing pathway in yeast.

Transcription factor GATA-1 potently represses the expression of the HIV-1 coreceptor CCR5 in human T cells and dendritic cells

AbstractCC chemokine receptor 5 (CCR5) is the major HIV-1 coreceptor and its expression levels are a critical determinant of HIV-1 infection. However, the molecular mechanisms of CCR5 regulation in primary targets of HIV-1 remain unknown. Despite binding to conserved DNA elements, we show that the transcription factors GATA binding protein 1 (GATA-1) and GATA-3 differentially suppress the expression of CCR5 in stem-cell–derived dendritic cells and primary human T-cell subsets. In addition, GATA-1 expression was also more potent than GATA-3 in suppressing T helper 1 (Th1)–associated genes, interferon-γ (IFNγ), and CXC chemokine receptor-3 (CXCR3). GATA-1, but not GATA-3, potently suppressed CCR5 transcription, thereby rendering human T cells resistant to CCR5-tropic HIV-1 infection. However, GATA-1 could also serve as a surrogate for GATA-3 in its canonic role of programming Th2 gene expression. These findings provide insight into GATA-3–mediated gene regulation during T-cell differentiation. Importantly, decoding the mechanisms of GATA-1–mediated repression of CCR5 may offer an opportunity to develop novel approaches to inhibit CCR5 expression in T cells.

A gene of the opsin family in the carotenoid gene cluster of Fusarium fujikuroi.

Opsins are membrane photoreceptors closely related to the heat-shock proteins of the HSP30 family. Their functions include light-driven ion pumping in archaea and light detection in algae and animals, using the apocarotenoid retinal as a light-absorbing prosthetic group. We describe a gene of Fusarium fujikuroi, carO, coding for a polypeptide resembling opsins and HSP30-like proteins and contiguous to the genes of the carotenoid pathway, carRA and carB. Transcription of carO is induced by light and is deregulated in carotenoid-overproducing mutants. The same regulation pattern is exhibited by carRA and carB; and common conserved DNA elements are found in the three promoters. Heat shock resulted in a modest induction of carO transcription, similar to the one exhibited by carB, confirming a common regulation. Targeted mutagenesis of carO produced no apparent phenotypic modification, including no change in the photoinduction of carotenoid biosynthesis.

DNA-dependent adenosine triphosphatase (helicaselike transcription factor) activates β-globin transcription in K562 cells

Correct developmental regulation of beta-like globin gene expression is achieved by preferential transcription of a gene at a given developmental stage, silencing of other beta-like gene promoters, and competition among these promoters for interaction with the locus control region (LCR). Several evolutionarily conserved DNA elements in the promoters of the beta-like genes and LCR have been studied in detail, and the role of their binding factors has been investigated. However, the beta-globin promoter includes additional evolutionarily conserved sequences of unknown function. The present study examined the properties of a 21-base pair (bp) promoter-conserved sequence (PCS) located at positions -115 to -136 bp relative to the transcription start site of the beta-globin gene. A helicaselike transcription factor (HLTF) belonging to the SWI2/SNF2 family of proteins binds to the PCS and a partly homologous sequence in the enhancer region of the LCR hypersensitive site 2 (HS2). Elevation of the level of HLTF in K562 erythroleukemic cells increases beta-promoter activity in transient transfection experiments, and mutations in the PCS that remove HLTF-binding regions abolish this effect, suggesting that HLTF is an activator of beta-globin transcription. Overexpression of HLTF in K562 cells does not affect the endogenous levels of gamma- and epsilon-globin message, but it markedly activates beta-globin transcription. In conclusion, this study reports a transcription factor belonging to the SWI2/SNF2 family, which preferentially activates chromosomal beta-globin gene transcription and which has not previously been implicated in globin gene regulation.

HSP-CBF Is an NF-Y-dependent Coactivator of the Heat Shock Promoters CCAAT Boxes

The cellular response to toxic stimuli is elicited through the expression of heat shock proteins, a transcriptional process that relies upon conserved DNA elements in the promoters: the Heat Shock Elements, activated by the heat shock factors, and the CCAAT boxes. The identity of the CCAAT activator(s) is unclear because two distinct entities, NF-Y and HSP-CBF, have been implicated in the HSP70 system. The former is a conserved ubiquitous trimer containing histone-like subunits, the latter a 110-kDa protein with an acidic N-terminal. We analyzed two CCAAT-containing promoters, HSP70 and HSP40, with recombinant NF-Y and HSP-CBF using electrophoretic mobility shift assay, protein-protein interactions, transfections and chromatin immunoprecipitation assays (ChIP) assays. Both recognize a common DNA-binding protein in nuclear extracts, identified in vitro and in vivo as NF-Y. Both CCAAT boxes show high affinity for recombinant NF-Y but not for HSP-CBF. However, HSP-CBF does activate HSP70 and HSP40 transcription under basal and heat shocked conditions; for doing so, it requires an intact NF-Y trimer as judged by cotransfections with a diagnostic NF-YA dominant negative vector. HSP-CBF interacts in solution and on DNA with the NF-Y trimer through an evolutionary conserved region. In yeast two-hybrid assays HSP-CBF interacts with NF-YB. These data implicate HSP-CBF as a non-DNA binding coactivator of heat shock genes that act on a DNA-bound NF-Y.

CSE4 genetically interacts with the Saccharomyces cerevisiae centromere DNA elements CDE I and CDE II but not CDE III. Implications for the path of the centromere dna around a cse4p variant nucleosome.

Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, we measured the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, we found that mutations throughout the Cse4p histone-fold domain caused surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, we propose a model that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome.

Identification of Conserved Potentially Regulatory Sequences of the SRY Gene from 10 Different Species of Mammals

We have sequenced the 5′ region of theSRYgene from human, chimpanzee, sheep, and mouse and from four additional mammalian species, not previously characterized (gorilla, gazelle, rat, and guinea pig). In order to identify conserved DNA elements potentially involved in the regulation of theSRYgene, the newly determined sequences were analyzed and compared to all mammalianSRYpromoter sequences available at present. Ten highly conserved potential regulatory elements have been identified in all 10 species (AP1, Barbie, GATA, Gfi1, cMyb, vMyb, NF1, Oct1, Sp1, and SRY). The known function of several of these regulatory elements fits well with the known expression of theSRYgene. However, except for the highly conserved coding HMG motif, only a short region close to the initiation of transcription in the humanSRYis conserved in the exact position along the gene in all the species analyzed. This lack of sequence identity at the orthologous positions is consistent with the suggested rapid evolution of theSRYgene. This relative lack of homology contrasts with a high sequence identity of the putative regulatory sequences found within each taxonomic group of species (primates, bovids, and rodents), which supports a common mechanism ofSRYexpression and possibly also a similar function.

The Mitochondrial DNA of Allomyces macrogynus: The Complete Genomic Sequence from an Ancestral Fungus

We have determined the complete nucleotide sequence of the circular mitochondrial DNA (mtDNA) of the chytridiomycete fungus, Allomyces macrogynus(57,473 bp; A + T content 60.5%). The identified genes that are typical for most fungal mitochondria include those for the large ( rnl) and small subunit ( rns) ribosomal RNAs, a complete set of 25 tRNAs, three ATPase subunits ( atp6, atp8and atp9), apocytochrome b(cob), three subunits of the cytochrome oxidase complex ( cox1, cox2and cox3), and seven subunits of the NADH dehydrogenase complex ( nad1, nad2, nad3, nad4, nad4L, nad5and nad6). A total of 28 introns of both groups are found, some of which contain open reading frames (ORFs) coding for potential endonucleases (group I) or reverse-transcriptases (group II). All mitochondrial genes are transcribed from the same DNA strand, as is the case in many other eufungi. Particular features of the A. macrogynus mtDNA include: (1) the first documented case of a fungal mitochondrial ribosomal protein gene ( rps3) that is clearly identified by similarity with bacterial homologues; (2) four unique ORFs; (3) the presence of an insert in the atp6gene that may have been acquired by interspecific transfer; (4) more than 67 short, highly structured and conserved DNA elements inserted in intergenic spacers, introns, and variable regions of the rnland rnsgenes: these elements are unusually G + C rich; (5) rRNA structures that resemble more closely those of eubacteria than their counterparts in other fungal mitochondria. The high degree of conservation of the A. macrogynusmitochondrial rRNA secondary structures, the existence of a mitochondrial rps3gene (common to protist but unique in fungal mtDNAs), and phylogenetic relationships inferred from highly conserved protein genes, demonstrate consistently the ancestral character of this fungal mitochondrial genome.

Identification of conserved potential regulatory sequences of the protamine-encoding P1 genes from ten different mammals

In order to detect regulatory conserved DNA elements within the protamine 1-encoding gene ( P1) promoter, we have sequenced this region from the rat, guinea pig, gorilla, orangutan, anubis baboon and red monkey P1 genes and compared it to the homologous human, bull, boar and mouse nucleotide (nt) sequences. We demonstrate the presence of a consensus sequence, HSMCYTCAYAAT ( Prot1C: protamine 1 consensus), from nt position — 64 to position — 53 in all P1 genes whose promoter sequences are now known. We also show that sequences similar to Prot1C are found in the promoter region of other testis-specific genes, such as the transition protein 1-encoding gene promoter which is thought to have derived from the P1 genes. The relevance of this conserved element in the expression of P1 genes is strongly supported by the recent demonstration of a mouse testis trans-acting factor [Tet-1; Tamura et al., J. Biol. Chem. 267 (1992) 4327–4332] which binds and matches in the mouse the first 11 bp of the corresponding consensus Prot1C sequence reported here. Another highly conserved element (TGTGAGG) has been identified 20 ± 3 nt upstream from ProtlC. This sequence forms a perfect palindrome with the central 7 nt of Prot1C and is absent in the homologous region of other genes. Further upstream, at positions −113 to −132, a third highly conserved region is present (MATGCCCATATWTGGRCAYG) which is similar to the c- fos SRE (serum-response element) and contains the central core common to all SREs. This element has not been found in the homologous region of other sperm-specific genes. (H, A or C or T; M, A or C; R, A or G; S, C or G; W, A or T; Y, C or T.)

In vivo genomic footprint of a yeast centromere.

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.

In Vivo Genomic Footprint of a Yeast Centromere

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.

Hypothesis: The removal of V H gene downstream sequences is required for its expression in mature B cells

In immunoglobulin producing cells only the rearranged V H gene is expressed, while all the other germline V H genes are silent. On the other hand, it was shown that unrearranged V H gene segments are expressed at a high level in the early stages of B lymphocyte differentiation. This expression is independent of the Ig heavy chain enhancer element. It seems, therefore, that unrearranged V H gene expression is depressed in mature B cells by a mechanism that will not operate on the rearranged functional heavy chain gene. We have shown that a highly conserved DNA segment is present upstream to several mouse and human V H genes. DNA sequencing downstream to germline genes revealed a DNA element related to the one described at the 5' of the V H genes. A model is proposed in which the 5' and 3' conserved segments are functional in the regulation of V H gene expression. The model suggests the existence of a protein in mature B cells which recognizes the 5' and 3' conserved DNA elements and prevents germline V H gene expression by binding to these segments. V H-D-J H joining results in the deletion of the 3' flanking sequences of the rearranged V H gene but not of other upstream V H genes. This would result in the release of promoter suppression in the case of the rearranged gene, while all other V H genes would remain transcriptionally silent.

Evolution of the autosomal chorion cluster inDrosophila. II. Chorion gene expression and sequence comparisons of thes16 ands19 genes in evolutionarily distant species

We present a total of 13 kb of DNA sequences, encompassing autosomal chorion genes and their flanking DNA in four species of the genus Drosophila. Against a background of extensive divergence in introns and even in parts of the coding regions, islands of strong conservation are evident in the proximal 5' flanking and 5' untranslated sequences. An extragenic region of strong conservation is seen downstream of the last chorion gene in the autosomal cluster. The conserved DNA elements may be related to the conserved regulatory features of this cluster, including gene amplification and tissue- and temporally regulated transcription.

A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae.

Saccharomyces cerevisiae centromeres contain a conserved region ranging from 111 to 119 base pairs (bp) in length, which is characterized by the three conserved DNA elements CDEI, CDEII, and CDEIII. We isolated a 125-bp CEN6 DNA fragment (named ML CEN6) containing only these conserved elements and assayed it completely separated from its chromosomal context on circular minichromosomes and on a large linear chromosome fragment. The results show that this 125-bp CEN6 DNA fragment is by itself sufficient for complete mitotic and meiotic centromere functions.

A 125-Base-Pair CEN6 DNA Fragment Is Sufficient for Complete Meiotic and Mitotic Centromere Functions in Saccharomyces cerevisiae

Saccharomyces cerevisiae centromeres contain a conserved region ranging from 111 to 119 base pairs (bp) in length, which is characterized by the three conserved DNA elements CDEI, CDEII, and CDEIII. We isolated a 125-bp CEN6 DNA fragment (named ML CEN6) containing only these conserved elements and assayed it completely separated from its chromosomal context on circular minichromosomes and on a large linear chromosome fragment. The results show that this 125-bp CEN6 DNA fragment is by itself sufficient for complete mitotic and meiotic centromere functions.

Distamycins inhibit the binding of OTF-1 and NFE-1 transfactors to their conserved DNA elements.

We investigated the effects of the antiviral agent distamycin A and of a distamycin derivative (FCE 24517) which possesses antineoplastic activity on the binding of some regulatory proteins to DNA. Both compounds inhibited the binding to DNA of the ubiquitous octamer binding factor OTF 1 and of the erythroid specific GATAAG protein (NFE 1). This was shown using the electrophoretic mobility shift assay on a DNA fragment of human gamma-globin gene promoter (-156 to -201), on the same fragment with a point mutation (T to C mutation) known to have an increased affinity of binding for NFE 1, on a DNA fragment of human histone H2B promoter and on a DNA fragment of mouse alpha 1 globin promoter. The ability of distamycin or of FCE 24517 to inhibit the binding was specific for AT-rich sequences since neither drug inhibited the binding of nuclear protein factors to the sequence CCACACCC of the human beta globin gene. Binding to DNA was investigated by evaluating the drugs' ability to protect selected sequences from DNase I digestion (DNase footprinting). Distamycins binding was highly preferential for DNA sequences containing stretches of AT. These studies indicate that chemicals which have a high degree of DNA sequence-specific binding can selectively inhibit the binding of regulatory proteins to DNA. These effects might be responsible for modification of the transcription of specific genes and might to some extent account for these drugs' antiviral and antineoplastic activities. This approach offers potential for the investigation of new such drugs.