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At least 181 records · Page 10Linked to original sources

PubMed2006

Evolution of cis elements in the differential expression of two Hoxa2 coparalogous genes in pufferfish (Takifugu rubripes).

Sequence divergence in cis-regulatory elements is an important mechanism contributing to functional diversity of genes during evolution. Gene duplication and divergence provide an opportunity for selectively preserving initial functions and evolving new activities. Many vertebrates have 39 Hox genes organized into four clusters (Hoxa-Hoxd); however, some ray-finned fishes have extra Hox clusters. There is a single Hoxa2 gene in most vertebrates, whereas fugu (Takifugu rubripes) and medaka (Oryzias latipes) have two coparalogous genes [Hoxa2(a) and Hoxa2(b)]. In the hindbrain, both genes are expressed in rhombomere (r) 2, but only Hoxa2(b) is expressed in r3, r4, and r5. Multiple regulatory modules directing segmental expression of chicken and mouse Hoxa2 genes have been identified, and each module is composed of a series of discrete elements. We used these modules to investigate the basis of differential expression of duplicated Hoxa2 genes, as a model for understanding the divergence of cis-regulatory elements. Therefore, we cloned putative regulatory regions of the fugu and medaka Hoxa2(a) and -(b) genes and assayed their activity. We found that these modules direct reporter expression in a chicken assay, in a manner corresponding to their endogenous expression pattern in fugu. Although sequence comparisons reveal many differences between the two coparalogous genes, specific subtle changes in seven cis elements of the Hoxa2(a) gene restore segmental regulatory activity. Therefore, drift in subsets of the elements in the regulatory modules is responsible for the differential expression of the two coparalogous genes, thus providing insight into the evolution of cis elements.

Animals↗

PubMed1997

Redox modulation of iron regulatory proteins by peroxynitrite.

Expression of several proteins of higher eukaryotes is post-transcriptionally regulated by interaction of iron-responsive elements (IREs) on their mRNAs and iron regulatory proteins (IRP1 and IRP2). IRP1 is a redox-sensitive iron-sulfur protein whose regulatory activity is modulated by iron depletion, synthesis of nitric oxide, or oxidative stress. IRP2 is closely related to IRP1, but it does not possess a [Fe-S] cluster. IRP2 is also regulated by intracellular iron level, but it is assumed that regulation is achieved by accelerated turn-over. In this report, the effect of peroxynitrite, a strong oxidant produced when nitric oxide and O-2 are biosynthesized simultaneously, on the RNA binding activity of IRP1 and IRP2 was investigated in vitro. Macrophage cytosolic extracts were exposed directly to a bolus addition of peroxynitrite or to SIN-1, which releases a continuous flux of peroxynitrite. Under these two experimental conditions, IRP1 lost its aconitase activity but did not gain increased capacity to bind IRE. However, addition of low amounts of the disulfide-reducing agent 2-ME during the binding assay revealed formation of a complex between IRP1 and IRE. Substrates of aconitase, which bind to the cluster of IRP1, prevented this effect, pointing to the [Fe-S] cluster as the target of peroxynitrite. Moreover, single mutation of the redox active Cys437 precluded oxidation of human recombinant IRP1 by SIN-1. Collectively, these results imply that peroxynitrite predisposes IRP1 to bind IREs under a suitable reducing environment. It is assumed that in addition to disrupting the cluster peroxynitrite also promotes disulfide bridge(s) between proximal cysteine residues in the vicinity of the IRE-binding domain, in particular Cys437. When exposed to peroxynitrite, IRP2 lost its spontaneous IRE binding activity, which was restored by further exposure to 2-mercaptoethanol, thus showing that peroxynitrite can also regulate IRP2 by a post-translational event.

Aconitate Hydratase↗

PubMed2003

Dynamic responses of protein homeostatic regulatory mechanisms to perturbations from steady state.

Nineteen hypothetical protein homeostatic regulatory mechanisms were constructed and analysed in terms of the rate at which they recovered from a perturbation in the steady-state concentration of any component. Systems were constructed to symbolize transcription/translation processes of the average protein from Escherichia coli (1000 copies of protein P along with 1 gene G per cell). In some model systems, G catalysed the synthesis of P directly, while in others G catalysed the synthesis of mRNA (called M), and M catalysed the synthesis of P in a subsequent step. Recovery rates for each regulatory mechanism were obtained by generating the corresponding system of differential equations, linearizing the system about the steady state, and determining eigenvalues of the associated coefficient matrix. The optimal rate of recovery for a given mechanism, R(D), was determined by combining random and gradient search approaches to find rate constants for which the system recovered fastest. Regulatory elements that improved dynamic regulation were identified. These consisted of negative feedback relationships that involved P binding to either G (to shut off the synthesis of P) or M (to stimulate its degradation). Regulation improved as increasing numbers of P's bound to either G or M; however, the binding to M was more effective. In other mechanisms PP dimers bound G. Dimer-binding mechanisms were roughly twice as effective in terms of regulation as those that bound P monomers. The effect of linking two regulatory "modules" was also investigated. Linking had no effect on R(D), but optimal rate constants for the linked system were similar to those of the unlinked modules, suggesting that it may be feasible to construct regulatory networks by linking individual modules of this type.

Animals↗

PubMed2006

Condition-specific coregulation with cis-regulatory motifs and modules in the mouse genome.

Deciphering genetic regulatory codes remains a challenge. Here, we present an effective approach to identifying in vivo condition-specific coregulation with cis-regulatory motifs and modules in the mouse genome. A resampling-based algorithm was adopted to cluster our microarray data of a stress response, which generated 35 tight clusters with unique expression patterns containing 811 genes of 5652 genes significantly altered. Database searches identified many known motifs within the 3-kb regulatory regions of 40 genes from 3 clusters and modules with six to nine motifs that were commonly shared by 60-100% of these genes. The upstream regulatory region contained the highest frequency of these common motifs. CisModule program predictions were comparable with the results from database searches and found four potentially novel motifs. This result indicates that these motifs and modules could be responsible for gene coregulation of the stress response in the lacrimal gland.

Algorithms↗

PubMed2006

Developmental mechanisms and cis-regulatory codes.

Complex networks of transcriptional interactions control the processes of animal development. These networks begin with broad positional information that patterns the cells of the early embryo, and end with precise expression profiles that provide the functions of fully differentiated cells. At the heart of these networks are cis-regulatory modules (CRMs), which contain binding sites for regulatory proteins and control the spatial and temporal expression of genes within the network. Recent studies in several model systems have begun to decipher the 'cis-regulatory codes' of CRMs involved in various developmental processes. These studies suggest that CRMs involved in regulating co-expressed genes share sequence characteristics that can be identified by in silico approaches. They also suggest that CRMs involved in specific types of developmental events have common binding site architectures, which can be linked to their specific functions.

Animals↗

PubMed2006

Marmoset CYP3A21, a model for human CYP3A4: protein expression and functional characterization of the promoter.

Due to its small size and the relative evolutionary proximity, the marmoset has been proposed as a model for studies of human drug interactions and metabolism. The current study investigated the expression and regulation of marmoset CYP3A using mass spectrometry and reporter gene techniques. Expression levels of hepatic marmoset CYP3A protein range from 51 to 123 pmol mg-1 total protein (mean 85.2 pmol mg-1, n = 10) and CYP3A21 is the dominant hepatic CYP3A protein in marmosets. The sequence similarity between human CYP3A4 and CYP3A21 across the first 7.5 kb of the cloned CYP3A21 promoter is 88% within the xenobiotic-responsive enhancer module (XREM) and the proximal promoter. Both regulatory modules confer transcriptional activation of CYP3A21-luciferase reporter gene constructs cotransfected with hPXR in intestinal LS174T cells. The pronounced response to rifampin and the moderate response to dexamethasone were similar to those observed with CYP3A4. Taken collectively, these data establish substantial similarities in expression and gene regulation between marmoset CYP3A21 and human CYP3A4. CYP3A21 may be an equivalent of CYP3A4 in New World monkeys, consistent with the phylogenetic relationship between these genes. The marmoset, therefore, appears to be a suitable in vivo model to study CYP3A4 function and regulation.

Amino Acid Sequence↗

PubMed2006

Computational identification of transcriptional regulatory elements in DNA sequence.

Identification and annotation of all the functional elements in the genome, including genes and the regulatory sequences, is a fundamental challenge in genomics and computational biology. Since regulatory elements are frequently short and variable, their identification and discovery using computational algorithms is difficult. However, significant advances have been made in the computational methods for modeling and detection of DNA regulatory elements. The availability of complete genome sequence from multiple organisms, as well as mRNA profiling and high-throughput experimental methods for mapping protein-binding sites in DNA, have contributed to the development of methods that utilize these auxiliary data to inform the detection of transcriptional regulatory elements. Progress is also being made in the identification of cis-regulatory modules and higher order structures of the regulatory sequences, which is essential to the understanding of transcription regulation in the metazoan genomes. This article reviews the computational approaches for modeling and identification of genomic regulatory elements, with an emphasis on the recent developments, and current challenges.

Animals↗

PubMed2026

Noncoding transcription controls the developmental dynamics of long-range gene regulation.

The genomic regions regulating gene expression are often themselves transcribed into a variety of noncoding RNAs (ncRNAs). However, the regulatory roles of this noncoding transcription remain largely unknown. By using live imaging, we reveal that the sequential transcription of ncRNAs emanating from distinct regulatory elements underlies gene activation in Drosophila embryos. Single-allele co-visualization uncovers that optimal gene activation is achieved by only moderate levels of enhancer activity. Disrupting enhancer-associated ncRNAs causes precocious gene activation, providing evidence that ncRNAs control the timing of gene expression in development. We further show that enhancer transcription can regulate long-range interactions within complex regulatory landscapes. We propose that ncRNAs locally modulate regulatory element activity in cis to shape genome organization and orchestrate the temporal control of gene expression in development.

Journal Article↗

PubMed2026

Integrated analysis of ATAC-seq and RNA-seq reveals the TCP-ARF molecular module related to pathogenic process of phytoplasma infection in Paulownia fortunei.

BACKGROUND: Witches’ broom is an important disease of the Paulownia fortunei. Understanding the pathogenesis of witches’ broom is a prerequisite for its prevention and control. Phytoplasma is the pathogen of Paulownia witches’ broom. RESULTS: We investigated the changes in chromatin accessibility before and after phytoplasma infection in Paulownia fortunei by analyzing the DNA accessibility (ATAC-seq). In phytoplasma-infected P. fortunei (PFI) compared to healthy samples (PF), the closed regions of chromatin(1187 regions) were three times more than the open regions (352 regions). Fifty one percent of the accessible chromatin regions were overlapped with either H3K27ac or H3K9ac peaks. The closed regions were enriched in the conserved motif TGGGC[CT] that is recognized by the TCP transcription factor family. The closed regions in PFI are intersected with ARF family gene locus. The gene PfARF3 was verified to interact with the PfTCP23 transcription factor. The PfTCP23 was predicted to be interacted with the effector pawb44 in the pathogen of phytoplasma. CONCLUSIONS: The phytoplasma infection in P. fortunei is involved in the chromatin changes of the DNA accessibility and histone modification. The binding regions of TCP23 were found to be changed mostly in the accessibility between PFI and PF. The TCP-ARF module was found to be the possible regulatory module inducing the crinkled leaf trait.

RNA-Seq↗

PubMed2026

Light regulates capsaicinoid biosynthesis via the CaHY5-CaBBX2-CaACS8 module in pepper.

Capsaicinoids are a class of unique alkaloids that confer the pungent taste to pepper fruits. However, it remains largely unknown how light regulates the biosynthesis of capsaicinoids. We conducted a metabolic analysis on light- and dark-adapted pepper fruits. The results showed that dark-adapted pepper fruits had lower capsaicinoid contents and correspondingly downregulated transcription of capsaicinoid biosynthetic genes (CBGs), indicating that light plays a crucial role in capsaicinoid biosynthesis. Furthermore, silencing of CaHY5, a pivotal transcription factor gene in the light signaling pathway, decreased the content of capsaicinoid and suppressed the expression of CBGs, whereas transient overexpression of CaHY5 generated exactly opposite results. CaHY5 can bind to the G-box motif in the promoters of CaBBX2 and CaACS8, thereby enhancing their transcriptional levels. The activated CaBBX2 then binds to the T/G-box in the CaACS8 promoter to stimulate its expression. CaBBX2 or CaACS8 silencing led to decreased levels of capsaicinoids, while their transient overexpression produced increased capsaicinoid contents. Collectively, our results indicated that the light-activated CaHY5-CaBBX2-CaACS8 regulatory module plays a pivotal role in capsaicinoid biosynthesis. These findings provide new insights into the influence of light on capsaicinoid biosynthesis and potential targets for activation of this biosynthetic pathway in pepper.

Capsicum↗

PubMed2025

Foxi2 and Sox3 are master regulators controlling ectoderm germ layer specification.

In vertebrates, germ layer specification represents a critical transition where pluripotent cells acquire lineage-specific identities. We identify the maternal transcription factors Foxi2 and Sox3 to be pivotal master regulators of ectodermal germ layer specification in Xenopus. Ectopic co-expression of Foxi2 and Sox3 in prospective endodermal tissue induces the expression of ectodermal markers while suppressing mesendodermal markers. Transcriptomics analyses reveal that Foxi2 and Sox3 jointly and independently regulate hundreds of ectodermal target genes. During early cleavage stages, Foxi2 and Sox3 pre-bind to key cis-regulatory modules (CRMs), marking sites that later recruit Ep300 and facilitate H3K27ac deposition, thereby shaping the epigenetic landscape of the ectodermal genome. These CRMs are highly enriched within ectoderm-specific super-enhancers (SEs). Our findings highlight the pivotal role of ectodermal SE-associated CRMs in precise and robust ectodermal gene activation, establishing Foxi2 and Sox3 as central architects of ectodermal lineage specification.

Ep300↗

PubMed2005

Logic functions of the genomic cis-regulatory code.

cis-Regulatory modules that control developmental gene expression process the regulatory inputs provided by the transcription factors for which they contain specific target sites. A prominent class of cis-regulatory processing functions can be modeled as logic operations. Many of these are combinatorial because they are mediated by multiple sites, although others are unitary. In this work, we illustrate the repertoire of cis-regulatory logic operations, as an approach toward a functional interpretation of the genomic regulatory code.

Animals↗

PubMed2003

The role of ERK2 docking and phosphorylation of PDE4 cAMP phosphodiesterase isoforms in mediating cross-talk between the cAMP and ERK signalling pathways.

PDE4 cAMP phosphodiesterases are widely expressed enzymes that serve as major regulators of cAMP signalling in cells. They provide targets for therapeutics having anti-inflammatory and cognitive-enhancing properties. ERK2 (extracellular-signal-regulated kinase 2) interacts with the PDE4 catalytic unit by binding to a KIM (kinase interaction motif) docking site located on an exposed beta-hairpin loop and an FQF (Phe-Gln-Phe) specificity site located on an exposed alpha-helix. These flank a site that allows phosphorylation by ERK, the functional outcome of which is orchestrated by the N-terminal UCR1/2 (upstream conserved region 1 and 2) modules. The three classes of PDE4 isoforms differ in these regulatory modules, allowing phosphorylation by ERK to lead to either inhibition or activation. ERK inhibition of long isoforms is regulated by a unique feedback control whereby elevated cAMP levels cause PKA (protein kinase A) to phosphorylate UCR1 and ablate the inhibitory action of ERK. PDE4 isoforms can also be found in complex with beta-arrestins where they provide a novel part of the cellular desensitization mechanism to receptor-mediated cAMP signalling. Stimulation of the beta(2)-adrenoceptor recruits beta-arrestins with bound PDE4, delivering an enzyme capable of degrading cAMP at its site of synthesis at the plasma membrane. Use of dominant negative PDE4 isoforms identifies that a major role of recruited PDE4 is to regulate plasma membrane PKA activity involved in phosphorylating the beta(2)-adrenoceptor. Recruited PDE4 thus desensitizes the ability of the beta(2)-adrenoceptor to activate ERK via G(i).

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

Brn1/2/4, the predicted midgut regulator of the endo16 gene of the sea urchin embryo.

A specific prediction of our detailed cis-regulatory analysis of the Strongylocentrotus purpuratus (Sp) endo16 gene was that the later expression of this gene would be driven by a midgut-specific transcriptional regulator. We have now identified this factor and determined some of its functions. The cDNA sequence reveals it to be a POU domain factor related closely to the mammalian factors Brain-1, -2, and -4. The factor was termed SpBrn1/2/4 (henceforth Brn1/2/4). Quantitative measurements of transcript prevalence show that the gene is first activated in the 20-h blastula, but there remain only about 100 molecules of brn1/2/4 mRNA per embryo (only a few per endoderm cell) until an abrupt 10-fold increase occurs as gastrulation begins. Measured in the same embryos, the late rise in prevalence of endo16 transcripts follows that of brn1/2/4 transcripts. As predicted by the endo16 model, brn1/2/4 expression is confined perfectly to the midgut, coincident with the domain of endo16 expression. The kinetics of accumulation of these transcripts indicates that the switch into the late phase of endo16 expression occurs when the brn1/2/4 transcript level nears its plateau (2000 molecules mRNA per embryo), after which each endo16 gene produces about 1 mRNA every 2 min (about 380 molecules mRNA per min in the whole embryo). Arrest of Brn1/2/4 translation by MASO treatment blocks the late phase of endo16 expression and specifically abolishes expression of cis-regulatory Module B of endo16, while not affecting Module A, also as predicted. The brn1/2/4 gene lies downstream of the regulatory genes executing post-gastrular specification of the midgut, as shown by further gene expression perturbation experiments which provide an initial glimpse of the underlying network architecture.

Amino Acid Sequence↗

PubMed1999

Functional promoter modules can be detected by formal models independent of overall nucleotide sequence similarity.

MOTIVATION: Gene regulation often depends on functional modules which feature a detectable internal organization. Overall sequence similarity of these modules is often insufficient for detection by general search methods like FASTA or even Gapped BLAST. However, it is of interest to evaluate whether modules, often known from experimental analysis of single sequences, are present in other regulatory sequences. RESULTS: We developed a new method (FastM) which combines a search algorithm for individual transcription factor binding sites (MatInspector) with a distance correlation function. FastM allows fast definition of a model of correlated binding sites derived from as little as a single promoter or enhancer. ModelInspector results are suitable for evaluation of the significance of the model. We used FastM to define a model for the experimentally verified NFkappaB/IRF1 regulatory module from the major histocompatibility complex (MHC) class I HLA-B gene promoter. Analysis of a test set of sequences as well as database searches with this model showed excellent correlation of the model with the biological function of the module. These results could not be obtained by searches using FASTA or Gapped BLAST, which are based on sequence similarity. We were also able to demonstrate association of a hypothetical GRE-GRE module with viral sequences based on analysis of several GenBank sections with this module. AVAILABILITY: The WWW version of FastM is accessible at: http://www.gsf.de/cgi-bin/fastm. pl and http://genomatix.gsf.de/cgi-bin/fastm2/fastm.pl

Algorithms↗

PubMed2006

G protein-coupled receptor kinase 2-mediated phosphorylation of downstream regulatory element antagonist modulator regulates membrane trafficking of Kv4.2 potassium channel.

Downstream regulatory element antagonist modulator (DREAM)/potassium channel interacting protein (KChIP3) is a multifunctional protein of the neuronal calcium sensor subfamily of Ca2+-binding proteins with specific roles in different cell compartments. In the nucleus, DREAM acts as a Ca2+-dependent transcriptional repressor, and outside the nucleus DREAM interacts with Kv4 potassium channels, regulating their trafficking to the cell membrane and their gating properties. In this study we characterized the interaction of DREAM with GRK6 and GRK2, members of the G protein-coupled receptor kinase family of proteins, and their phosphorylation of DREAM. Ser-95 was identified as the site phosphorylated by GRK2. This phosphorylation did not modify the repressor activity of DREAM. Mutation of Ser-95 to aspartic acid, however, blocked DREAM-mediated membrane expression of the Kv4.2 potassium channel without affecting channel tetramerization. Treatment with the calcineurin inhibitors FK506 and cyclosporin A also blocked DREAM-mediated Kv4.2 channel trafficking and calcineurin de-phosphorylated GRK2-phosphorylated DREAM in vitro. Our results indicate that these two Ca2+-dependent posttranslational events regulate the activity of DREAM on Kv4.2 channel function.

Calcineurin↗

PubMed1997

Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity.

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase. 32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitro transcription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [gamma-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.

Amino Acid Sequence↗

PubMed1995

Unraveling the modular design of glutamate-gated ion channels.

Glutamate receptors that function as ligand-gated ion channels are essential components of cell-cell communication in the nervous system. Despite a wealth of information concerning these receptors, details of their structure are just beginning to emerge. We propose that glutamate receptors comprise four modules: two modules that are related to bacterial periplasmic-binding proteins, one module that is related to the pore-forming region of K+ channels, and one regulatory module of unknown origin. A K(+)-channel-like domain inserted into a crucial region of a periplasmic-binding protein-like domain suggests a mechanism for transduction of binding energy to channel opening. This modular design also suggests an evolutionary link between a ligand-gated ion-channel family and voltage-gated ion channels.

Amino Acid Sequence↗