Combinatorial Interplay Research Articles

Alternative Splicing in Human Biology and Disease.

Alternative pre-mRNA splicing allows for the production of multiple mRNAs from an individual gene, which not only expands the protein-coding potential of the genome but also enables complex mechanisms for the post-transcriptional control of gene expression. Regulation of alternative splicing entails a combinatorial interplay between an abundance of trans-acting splicing factors, cis-acting regulatory sequence elements and their concerted effects on the core splicing machinery. Given the extent and biological significance of alternative splicing in humans, it is not surprising that aberrant splicing patterns can cause or contribute to a wide range of diseases. In this introductory chapter, we outline the mechanisms that govern alternative pre-mRNA splicing and its regulation and discuss how dysregulated splicing contributes to human diseases affecting the motor system and the brain.

In Silico Prediction of Transcription Factor Collaborations Underlying Phenotypic Sexual Dimorphism in Zebrafish (Danio rerio).

The transcriptional regulation of gene expression in higher organisms is essential for different cellular and biological processes. These processes are controlled by transcription factors and their combinatorial interplay, which are crucial for complex genetic programs and transcriptional machinery. The regulation of sex-biased gene expression plays a major role in phenotypic sexual dimorphism in many species, causing dimorphic gene expression patterns between two different sexes. The role of transcription factor (TF) in gene regulatory mechanisms so far has not been studied for sex determination and sex-associated colour patterning in zebrafish with respect to phenotypic sexual dimorphism. To address this open biological issue, we applied bioinformatics approaches for identifying the predicted TF pairs based on their binding sites for sex and colour genes in zebrafish. In this study, we identified 25 (e.g., STAT6-GATA4; JUN-GATA4; SOX9-JUN) and 14 (e.g., IRF-STAT6; SOX9-JUN; STAT6-GATA4) potentially cooperating TFs based on their binding patterns in promoter regions for sex determination and colour pattern genes in zebrafish, respectively. The comparison between identified TFs for sex and colour genes revealed several predicted TF pairs (e.g., STAT6-GATA4; JUN-SOX9) are common for both phenotypes, which may play a pivotal role in phenotypic sexual dimorphism in zebrafish.

Chromatin architecture reveals cell type-specific target genes for kidney disease risk variants

BackgroundCell type-specific transcriptional programming results from the combinatorial interplay between the repertoire of active regulatory elements. Disease-associated variants disrupt such programming, leading to altered expression of downstream regulated genes and the onset of pathological states. However, due to the non-linear regulatory properties of non-coding elements such as enhancers, which can activate transcription at long distances and in a non-directional way, the identification of causal variants and their target genes remains challenging. Here, we provide a multi-omics analysis to identify regulatory elements associated with functional kidney disease variants, and downstream regulated genes.ResultsIn order to understand the genetic risk of kidney diseases, we generated a comprehensive dataset of the chromatin landscape of human kidney tubule cells, including transcription-centered 3D chromatin organization, histone modifications distribution and transcriptome with HiChIP, ChIP-seq and RNA-seq. We identified genome-wide functional elements and thousands of interactions between the distal elements and target genes. The results revealed that risk variants for renal tumor and chronic kidney disease were enriched in kidney tubule cells. We further pinpointed the target genes for the variants and validated two target genes by CRISPR/Cas9 genome editing techniques in zebrafish, demonstrating that SLC34A1 and MTX1 were indispensable genes to maintain kidney function.ConclusionsOur results provide a valuable multi-omics resource on the chromatin landscape of human kidney tubule cells and establish a bioinformatic pipeline in dissecting functions of kidney disease-associated variants based on cell type-specific epigenome.

Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways.

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.

Computational identification of tissue-specific transcription factor cooperation in ten cattle tissues.

Transcription factors (TFs) are a special class of DNA-binding proteins that orchestrate gene transcription by recruiting other TFs, co-activators or co-repressors. Their combinatorial interplay in higher organisms maintains homeostasis and governs cell identity by finely controlling and regulating tissue-specific gene expression. Despite the rich literature on the importance of cooperative TFs for deciphering the mechanisms of individual regulatory programs that control tissue specificity in several organisms such as human, mouse, or Drosophila melanogaster, to date, there is still need for a comprehensive study to detect specific TF cooperations in regulatory processes of cattle tissues. To address the needs of knowledge about specific combinatorial gene regulation in cattle tissues, we made use of three publicly available RNA-seq datasets and obtained tissue-specific gene (TSG) sets for ten tissues (heart, lung, liver, kidney, duodenum, muscle tissue, adipose tissue, colon, spleen and testis). By analyzing these TSG-sets, tissue-specific TF cooperations of each tissue have been identified. The results reveal that similar to the combinatorial regulatory events of model organisms, TFs change their partners depending on their biological functions in different tissues. Particularly with regard to preferential partner choice of the transcription factors STAT3 and NR2C2, this phenomenon has been highlighted with their five different specific cooperation partners in multiple tissues. The information about cooperative TFs could be promising: i) to understand the molecular mechanisms of regulating processes; and ii) to extend the existing knowledge on the importance of single TFs in cattle tissues.

Combinatorial interplay of RNA-binding proteins tunes levels of mitochondrial mRNA in trypanosomes.

MRP1/2 is a heteromeric protein complex that functions in the trypanosomatid mitochondrion as part of the RNA editing machinery, which facilitates multiple targeted insertions and deletions of uridines. MRP1/2 was shown to interact with MRB8170, which initiates RNA editing by marking pre-edited mRNAs, while TbRGG2 is required for its efficient progression on pan-edited mRNAs. Both MRP1/2 and TbRGG2 are capable of modulating RNA–RNA interactions in vitro. As determined by using iCLIP and RIP-qPCR, RNAs bound to MRP1/2 are characterized and compared with those associated with MRB8170 and TbRGG2. We provide evidence that MRP1 and MRB8170 have correlated binding and similar RNA crosslinking peak profiles over minimally and never-edited mRNAs. Our results suggest that MRP1 assists MRB8170 in RNA editing on minimally edited mRNAs.

Intrathymic Notch3 and CXCR4 combinatorial interplay facilitates T-cell leukemia propagation

Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying “pre-leukemic” cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4+CD8+ thymocytes, possibly contributing to “pre-leukemic” cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4+CD8+ cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4+CD8+ cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4+CD8+ cells and such an expression is regulated by Notch3 through β-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4+CD8+ cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4+CD8+ cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of “pre-leukemic” CD4+CD8+ cells, by a “thymus-autonomous” mechanism.

Master Regulatory Transcription Factors in Plant Development: A Blooming Perspective.

Transcription factors that trigger major developmental decisions in plants and animals are termed "master regulators". Such master regulators are classically seen as acting on the top of a regulatory hierarchy that determines a complete developmental program, and they usually encode transcription factors. Here, we introduce master regulators of flowering time and flower development as examples to show how analysis of molecular interactions and gene-regulatory networks in plants has changed our view on the molecular mechanisms by which these factors control developmental processes. A picture has emerged that emphasizes a complex combinatorial interplay in determining cell-type transcriptional programs, and a high level of feedback control. The expression of master regulators themselves is usually regulated by multiple factors integrating environmental and endogenous spatiotemporal cues. Master regulatory transcription factors regulate gene expression by different mechanisms, including modifications in chromatin status in the bound regions. A poorly understood phenomenon is how developmental master regulators exert functions in different cell- and organ types. This is especially relevant for those factors that have important functions in several developmental processes.

Computational Identification of microRNAs binding to the Transcription factors related to Dopamine Neurons

The combinatorial interplay between transcription factors (TF's) and microRNAs (miRNAs) are involved in numerous biological processes including the pathogenesis of Parkinson's disease (PD). The dopamine neuron differentiation and survival are controlled by the levels of TFs, Nurr1, Foxa1, Lmx1a/b, En1/2, which are regulated by miRNAs. To predict the miRNAs regulating TFs expression concerning PD, three different algorithms; DIANA Micro-T CDS, TargetScan, and miRanda were used. The consensus prediction result is considered as satisfactory, when its statistically significant and it has a high probability to specific target. In three algorithms show 28, 9, 14, 16, 3 miRNAs for Nurr1, Foxa1, Lmx1a, En2, En1 respectively. While no identical miRNAs were observed for Lmx1b. using Fisher's exact test we juxtapose the results from three algorithms which take into account different aspects of binding microRNA to the target. In summary, the identified miRNAs strongly suggested to be involved in dopamine neuron differentiation and survival, which possibly regulate the expression of transcription factors and hence offer possibilities for further experimental validation, which may use diagnostic and prognostic biomarkers.

An ensemble of regulatory elements controls Runx3 spatiotemporal expression in subsets of dorsal root ganglia proprioceptive neurons.

The Runx3 transcription factor is essential for development and diversification of the dorsal root ganglia (DRGs) TrkC sensory neurons. In Runx3-deficient mice, developing TrkC neurons fail to extend central and peripheral afferents, leading to cell death and disruption of the stretch reflex circuit, resulting in severe limb ataxia. Despite its central role, the mechanisms underlying the spatiotemporal expression specificities of Runx3 in TrkC neurons were largely unknown. Here we first defined the genomic transcription unit encompassing regulatory elements (REs) that mediate the tissue-specific expression of Runx3. Using transgenic mice expressing BAC reporters spanning the Runx3 locus, we discovered three REs-dubbed R1, R2, and R3-that cross-talk with promoter-2 (P2) to drive TrkC neuron-specific Runx3 transcription. Deletion of single or multiple elements either in the BAC transgenics or by CRISPR/Cas9-mediated endogenous ablation established the REs' ability to promote and/or repress Runx3 expression in developing sensory neurons. Our analysis reveals that an intricate combinatorial interplay among the three REs governs Runx3 expression in distinct subtypes of TrkC neurons while concomitantly extinguishing its expression in non-TrkC neurons. These findings provide insights into the mechanism regulating cell type-specific expression and subtype diversification of TrkC neurons in developing DRGs.

Understanding the combinatorial action of transcription factors and microRNA regulation from regions of open chromatin.

Transcriptional regulatory cascades are always triggered through the combinatorial interplay between transcription factors (TFs) and microRNAs (miRNAs) in eukaryotes. However, it is still a very substantial undertaking to dynamically profile their coordinated actions. In this work, we compared the differences in TFBS numbers between miRNA targets and non-targets, and found that miRNA targets tend to have more TFBS numbers. With the attempt to comprehensively understand the combinatorial action of TF and miRNA regulation from regions of open chromatin, we retrieved recently published DNase I hypersensitive sites (DHSs) across different human cell lines. The result showed that the differences are more statistically significant in DHS regions than non-DHS regions. Next, we trained classifiers for miRNA targets and non-targets. The result showed that TFBSs located in DHS regions achieved a competitive performance when discriminating miRNA targets and non-targets, whereas the performance of classifiers using TFBSs located in non-DHS regions is close to that of a random classifier. After the DHSs were divided into intergenic, transcription start sites (TSSs) and gene body DHS regions based on their genomic locations, only TFBSs located in TSS DHS regions provided a competitive performance. Our results provide us a clue that the coordinated activity of miRNAs and TFs describing the mechanism of gene expression control should be examined in a dynamic perspective.

Orchestration of ErbB3 signaling through heterointeractions and homointeractions.

Members of the ErbB family of receptor tyrosine kinases are capable of both homointeractions and heterointeractions. Because each receptor has a unique set of binding sites for downstream signaling partners and differential catalytic activity, subtle shifts in their combinatorial interplay may have a large effect on signaling outcomes. The overexpression and mutation of ErbB family members are common in numerous human cancers and shift the balance of activation within the signaling network. Here we report the development of a spatial stochastic model that addresses the dynamics of ErbB3 homodimerization and heterodimerization with ErbB2. The model is based on experimental measures for diffusion, dimer off-rates, kinase activity, and dephosphorylation. We also report computational analysis of ErbB3 mutations, generating the prediction that activating mutations in the intracellular and extracellular domains may be subdivided into classes with distinct underlying mechanisms. We show experimental evidence for an ErbB3 gain-of-function point mutation located in the C-lobe asymmetric dimerization interface, which shows enhanced phosphorylation at low ligand dose associated with increased kinase activity.

The Combinatorics of Automorphisms and Opposition in Generalised Polygons

We investigate the combinatorial interplay between automorphisms and opposition in (primarily finite) generalised polygons. We provide restrictions on the fixed element structures of automorphisms of a generalised polygon mapping no chamber to an opposite chamber. Furthermore, we give a complete classification of automorphisms of finite generalised polygons which map at least one point and at least one line to an opposite, but map no chamber to an opposite chamber. Finally, we show that no automorphism of a finite thick generalised polygon maps all chambers to opposite chambers, except possibly in the case of generalised quadrangles with coprime parameters.

Improved bottom-up strategy to efficiently separate hypermodified histone peptides through ultra-HPLC separation on a bench top Orbitrap instrument.

Histone post-translational modifications (hPTMs) play a crucial role in modulating chromatin structure and enforcing specific functional states on the underlying genome. Through the design of ad hoc analytical methods, MS has contributed significantly in the dissection of hPTMs, exhibiting specific strengths in identifying novel marks and assessing their combinatorial interplay. However, the comprehensive analysis of all individual isoforms of some hypermodified histone regions remains highly challenging with conventional proteomics platforms. Since complex hPTM patterns have unique functional outcomes on the genes, the implementation of new MS-proteomics solutions can boost epigenetic research. Here, we assessed the effectiveness of a new analytical platform-which combines ultra high-performance LC (UHPLC) with high-resolution MS/MS analysis-in dissecting hypermodified regions from macrophage core histones. We compared the resolving power of this configuration with a standard setup based on HPLC-MS/MS and focused on two case-study peptides, H3 (27-40) and H4 (4-17). We observed that the novel platform resolves a much larger set of distinct peptide isoforms; among them some were resolved for the first time. A comprehensive analysis of hPTMs from macrophages was then carried out at basal state and upon lipopolysaccharide induction, to profile their temporal change in bulk chromatin during the inflammatory response.

Biochemical Characterization of Diverse Stat5b-Binding Enhancers That Mediate Growth Hormone-Activated Insulin-Like Growth Factor-I Gene Transcription

Many of the biological effects of growth hormone (GH) are mediated by insulin-like growth factor I (IGF-I), a 70-amino acid secreted peptide whose gene expression is rapidly induced by GH via the Stat5b transcription factor. We previously identified multiple evolutionarily conserved GH-activated chromosomal binding domains for Stat5b within the rat Igf1 locus, and proposed that they could regulate IGF-I gene activity. Here we investigate the biochemical and functional characteristics of these putative long-range transcriptional enhancers. Each element contained 2 or 3 individual Stat5b recognition sequences that could bind Stat5b in vitro, but with affinities that varied over a >100-fold range. Full transcriptional responsiveness to GH required that all Stat5b sites be intact within an individual enhancer. Replacement of a single lower-affinity Stat5b sequence with a higher-affinity one increased in vitro binding of Stat5b, and boosted transcriptional potency of the entire element to GH. As enhanced transcriptional activity involved changes in only one or two nucleotides within an enhancer DNA segment, there appears to be remarkable specificity and sensitivity in the ability of Stat5b to transform DNA binding activity into transcriptional function. Stat5b was able to stimulate the transcriptional activity of two enhancers in the absence of GH, indicating that individual Stat5b-regulated elements possess distinct functional features. We conclude that combinatorial interplay among multiple Stat5b-binding response elements with distinguishable biochemical properties is responsible for highly regulated control of IGF-I gene activity by GH.

Thecis-regulatory code of Hox function inDrosophila

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, our understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, we have identified binding regions for the Hox TF Deformed (Dfd) in the Drosophila genome. Our analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. We also find that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, our results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo.

Regulation of alternative splicing within the supraspliceosome

Alternative splicing is a fundamental feature in regulating the eukaryotic transcriptome, as ~95% of multi-exon human Pol II transcripts are subject to this process. Regulated splicing operates through the combinatorial interplay of positive and negative regulatory signals present in the pre-mRNA, which are recognized by trans-acting factors. All these RNA and protein components are assembled in a gigantic, 21 MDa, ribonucleoprotein splicing machine - the supraspliceosome. Because most alternatively spliced mRNA isoforms vary between different cell and tissue types, the ability to perform alternative splicing is expected to be an integral part of the supraspliceosome, which constitutes the splicing machine in vivo. Here we show that both the constitutively and alternatively spliced mRNAs of the endogenous human pol II transcripts: hnRNP A/B, survival of motor neuron (SMN) and ADAR2 are predominantly found in supraspliceosomes. This finding is consistent with our observations that the splicing regulators hnRNP G as well as all phosphorylated SR proteins are predominantly associated with supraspliceosomes. We further show that changes in alternative splicing of hnRNP A/B, affected by up regulation of SRSF5 (SRp40) or by treatment with C6-ceramide, occur within supraspliceosomes. These observations support the proposed role of the supraspliceosome in splicing regulation and alternative splicing.

Nuclei of Tsuga canadensis: Role of Flavanols in Chromatin Organization

Needle primordia of Tsuga canadensis (hemlock) arising from flank meristems of a shoot apex, form cell lineages consisting of four or eight cells. Within a recently established lineage there is striking uniformity in the pattern of nuclear flavanols. This fact points to an identical transcriptional expression of these flavanols during cell cycling. However two lineages, even if located close together within the same meristem, can be very different in the expression of both cell shape and nuclear flavanol pattern, indicating that epigenetic positional signals are operating in a collective specification of cell lineage development. There is a wide range of nuclear flavanol patterning from a mosaic-like distribution in an activated cell type to a homogenous appearance in silenced cell types. Single cells deriving from lineages are desynchronized because they underlie a signaling network at a higher tissue level which results in stronger epigenetic modifications of their nuclear flavanols. As an extreme case of epigenetic modulation, transient drought conditions caused a drastic reduction of nuclear flavanols. Upon treatment with sucrose or cytokinin, these nuclear flavanols could be fully restored. Analytical determination of the flavanols revealed 3.4 mg/g DW for newly sprouting needles and 19.6 mg/g DW for anthers during meiosis. The roughly 6-fold difference in flavanols is apparently a reflection of the highly diverging organogenetic processes. Collectively, the studies provide strong evidence for combinatorial interplay between cell fate and nuclear flavanols.

Advances in analysis of transcriptional regulatory networks

A transcriptional regulatory network represents a molecular framework in which developmental or environmental cues are transformed into differential expression of genes. Transcriptional regulation is mediated by the combinatorial interplay between cis-regulatory DNA elements and trans-acting transcription factors, and is perhaps the most important mechanism for controlling gene expression. Recent innovations, most notably the method for detecting protein-DNA interactions genome-wide, can help provide a comprehensive catalog of cis-regulatory elements and their interaction with given trans-acting factors in a given condition. A transcriptional regulatory network that integrates such information can lead to a systems-level understanding of regulatory mechanisms. In this review, we will highlight the key aspects of current knowledge on eukaryotic transcriptional regulation, especially on known transcription factors and their interacting regulatory elements. Then we will review some recent technical advances for genome-wide mapping of DNA-protein interactions based on high-throughput sequencing. Finally, we will discuss the types of biological insights that can be obtained from a network-level understanding of transcription regulation as well as future challenges in the field.

Gastrointestinal peptides and regulation of gastric acid secretion

Update on the role of gastrointestinal peptides in regulating gastric acid secretion. A novel transgenic mouse that expresses the entire human gastrin gene locus in G-cells of gastrin-null mice will facilitate investigation of gastrin gene regulatory elements. Isolation of a highly homogeneous population of G-cells permits the elucidation of stimulatory and inhibitory ligands without the confounding presence of other neuroendocrine cells. The use of somatostatin receptor knockout mice demonstrated the plasticity of gastric acid regulatory mechanisms and compensation by upregulation of the galanin pathway which inhibits secretion by enterochromaffin-like cells. The importance of adenosine in regulating somatostatin release was shown using adenosine receptor knockout mice. The importance of gastrointestinal peptides for regulating gastric acid is evident. Ongoing investigations will characterize the mechanisms underlying actions of these agents on gastric acid secretion, particularly with regard to their combinatorial effects and interplay with other acid-regulating pathways.