Combinatorial Code Research Articles

A chromatin code for alternative splicing involving a putative association between CTCF and HP1α proteins.

BackgroundAlternative splicing is primarily controlled by the activity of splicing factors and by the elongation of the RNA polymerase II (RNAPII). Recent experiments have suggested a new complex network of splicing regulation involving chromatin, transcription and multiple protein factors. In particular, the CCCTC-binding factor (CTCF), the Argonaute protein AGO1, and members of the heterochromatin protein 1 (HP1) family have been implicated in the regulation of splicing associated with chromatin and the elongation of RNAPII. These results raise the question of whether these proteins may associate at the chromatin level to modulate alternative splicing.ResultsUsing chromatin immunoprecipitation sequencing (ChIP-Seq) data for CTCF, AGO1, HP1α, H3K27me3, H3K9me2, H3K36me3, RNAPII, total H3 and 5metC and alternative splicing arrays from two cell lines, we have analyzed the combinatorial code of their binding to chromatin in relation to the alternative splicing patterns between two cell lines, MCF7 and MCF10. Using Machine Learning techniques, we identified the changes in chromatin signals that are most significantly associated with splicing regulation between these two cell lines. Moreover, we have built a map of the chromatin signals on the pre-mRNA, that is, a chromatin-based RNA-map, which can explain 606 (68.55%) of the regulated events between MCF7 and MCF10. This chromatin code involves the presence of HP1α, CTCF, AGO1, RNAPII and histone marks around regulated exons and can differentiate patterns of skipping and inclusion. Additionally, we found a significant association of HP1α and CTCF activities around the regulated exons and a putative DNA binding site for HP1α.ConclusionsOur results show that a considerable number of alternative splicing events could have a chromatin-dependent regulation involving the association of HP1α and CTCF near regulated exons. Additionally, we find further evidence for the involvement of HP1α and AGO1 in chromatin-related splicing regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0141-5) contains supplementary material, which is available to authorized users.

Specification of Individual Adult Motor Neuron Morphologies by Combinatorial Transcription Factor Codes

How the highly stereotyped morphologies of individual neurons are genetically specified is not well understood. We identify six transcription factors (TFs) expressed in a combinatorial manner in seven post-mitotic adult leg motor neurons (MNs) that are derived from a single neuroblast in Drosophila. Unlike TFs expressed in mitotically active neuroblasts, these TFs do not regulate each other's expression. Removing the activity of a single TF resulted in specific morphological defects, including muscle targeting and dendritic arborization, and in a highly specific walking defect in adult flies. In contrast, when the expression of multiple TFs was modified, nearly complete transformations in MN morphologies were generated. These results show that the morphological characteristics of a single neuron are dictated by a combinatorial code of morphology TFs (mTFs). mTFs function at a previously unidentified regulatory tier downstream of factors acting in the NB but independently of factors that act in terminally differentiated neurons.

Identification of Biological Relevant Minor Histocompatibility Antigens within the B-lymphocyte-Derived HLA-Ligandome Using a Reverse Immunology Approach.

T-cell recognition of minor histocompatibility antigens (MiHA) not only plays an important role in the beneficial graft-versus-leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) but also mediates serious GVH complications associated with allo-SCT. Using a reverse immunology approach, we aim to develop a method enabling the identification of T-cell responses directed against predefined antigens, with the goal to select those MiHAs that can be used clinically in combination with allo-SCT. In this study, we used a recently developed MiHA selection algorithm to select candidate MiHAs within the HLA-presented ligandome of transformed B cells. From the HLA-presented ligandome that predominantly consisted of monomorphic peptides, 25 polymorphic peptides with a clinically relevant allele frequency were selected. By high-throughput screening, the availability of high-avidity T cells specific for these MiHA candidates in different healthy donors was analyzed. With the use of MHC multimer enrichment, analyses of expanded T cells by combinatorial coding MHC multimer flow cytometry, and subsequent single-cell cloning, positive T-cell clones directed to two new MiHA: LB-CLYBL-1Y and LB-TEP1-1S could be demonstrated, indicating the immunogenicity of these two MiHAs. The biologic relevance of MiHA LB-CLYBL-1Y was demonstrated by the detection of LB-CLYBL-1Y-specific T cells in a patient suffering from acute myeloid leukemia (AML) that experienced an anti-leukemic response after treatment with allo-SCT.

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

SUMMARYThe genomic regulatory programs that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer, and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic stem cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signaling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signaling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role of TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

Combinatorial code governing cellular responses to complex stimuli.

Cells adapt to their environment through the integration of complex signals. Multiple signals can induce synergistic or antagonistic interactions, currently considered as homogenous behaviours. Here, we use a systematic theoretical approach to enumerate the possible interaction profiles for outputs measured in the conditions 0 (control), signals X, Y, X+Y. Combinatorial analysis reveals 82 possible interaction profiles, which we biologically and mathematically grouped into five positive and five negative interaction modes. To experimentally validate their use in living cells, we apply an original computational workflow to transcriptomics data of innate immune cells integrating physiopathological signal combinations. Up to 9 of the 10 defined modes coexisted in context-dependent proportions. Each interaction mode was preferentially used in specific biological pathways, suggesting a functional role in the adaptation to multiple signals. Our work defines an exhaustive map of interaction modes for cells integrating pairs of physiopathological and pharmacological stimuli.

Transcriptional selectors, masters, and combinatorial codes: regulatory principles of neural subtype specification.

The broad range of tissue and cellular diversity of animals is generated to a large extent by the hierarchical deployment of sequence-specific transcription factors and co-factors (collectively referred to as TF's herein) during development. Our understanding of these developmental processes has been facilitated by the recognition that the activities of many TF's can be meaningfully described by a few functional categories that usefully convey a sense for how the TF's function, and also provides a sense for the regulatory organization of the developmental processes in which they participate. Here, we draw on examples from studies in Caenorhabditis elegans, Drosophila melanogaster, and vertebrates to discuss how the terms spatial selector, temporal selector, tissue/cell type selector, terminal selector and combinatorial code may be usefully applied to categorize the activities of TF's at critical steps of nervous system construction. While we believe that these functional categories are useful for understanding the organizational principles by which TF's direct nervous system construction, we however caution against the assumption that a TF's function can be solely or fully defined by any single functional category. Indeed, most TF's play diverse roles within different functional categories, and their roles can blur the lines we draw between these categories. Regardless, it is our belief that the concepts discussed here are helpful in clarifying the regulatory complexities of nervous system development, and hope they prove useful when interpreting mutant phenotypes, designing future experiments, and programming specific neuronal cell types for use in therapies. WIREs Dev Biol 2015, 4:505–528. doi: 10.1002/wdev.191For further resources related to this article, please visit the WIREs website.

Writing and Reading the CTD Code: In Vitro Reconstruction of Post‐Translational Modifications on RNA Polymerase II

Information flow within the cell is filtered through a vast array of codes. Vital biological data is compiled at the DNA, RNA, and protein levels with additional information relayed through an ever‐expanding repertoire of post‐transcriptional and post‐translational modifications (PTM). The PTM state of proteins can impact their recognition, structure, and function as well as relay important signals to influence cellular events, as found in histone code. A newly characterized coding system in eukaryotic transcription is “CTD code.” The C‐terminal Domain of RNA Polymerase II (CTD) is a unique structure composed of repeats of the consensus heptad YSPTSPS. These residues are subject to a variety of PTMs and specific factors are recruited depending on the PTM state of CTD. Particular PTM marks are linked to transcriptional events including initiation, elongation, and termination. The PTM state of CTD is generated and altered through the action of PTM enzymes such as kinases and phosphatases. Current technologies allow the determination of isolated and simplistic PTM marks along the CTD but lack the potential for the investigation of a combinatorial code or the identification of novel PTM marks. To overcome this we propose a bottom up approach to in vitro reconstruct the CTD code. We treat CTD substrate with PTM enzymes and characterize resultant modifications using a combination of molecular biology and mass spectrometry. Our novel mass spectrometry methodologies allow for heptad level resolution and characterization of individual and combinatorial PTM marks along CTD. Using this interdisciplinary cipher we can determine the lexicon for CTD code and better understand the impact of PTM on cellular communication.

CTD Code: a Combinatorial Code for Eukaryotic Transcription

In eukaryotes, the C‐terminal domain of RNA polymerase II (CTD) orchestrates the temporal and spatial control of transcription and is involved in the epigenetic regulation of gene expression. Errors in CTD regulation can result in cell death, cancer and severe developmental defects. The CTD executes its function as transcription template through various post‐translational modifications on its heptad repeat sequences. Recently, novel modifications on new regulatory sites of CTD have been identified, setting the stage for the possibility of combinatorial mechanisms for transcription regulation. We focused on two well‐characterized modification of CTD, namely serine phosphorylation and prolyl isomerization, and discuss the interplay between the enzymes regulating these modification states. To address this question, we developed chemical compounds that can be used to mimic the cis and trans isomer states of proline residues in the CTD and therefore probe the selectivity of CTD binding proteins that are sensitive for this subtle variation. Our results established that the selectivity of prolyl isomerization state of CTD by phosphatases results in different regulatory effects and can lead to differentiated CTD phosphorylation states and eventually, transcription outcome. In particular, the phosphatases that specially recognize the minor species of cis proline will display enhanced activity upon prolyl isomerase regulation which is essential for accurate transcription termination. The application of such chemical probes can help us understand the molecular mechanism of the interplay between different post‐translational modifications with prolyl isomerization state and how that affects the conformational status of CTD in transcription.

Cadherins mediate sequential roles through a hierarchy of mechanisms in the developing mammillary body.

Expression of intricate combinations of cadherins (a family of adhesive membrane proteins) is common in the developing central nervous system. On this basis, a combinatorial cadherin code has long been proposed to underlie neuronal sorting and to be ultimately responsible for the layers, columns and nuclei of the brain. However, experimental proof of this particular function of cadherins has proven difficult to obtain and the question is still not clear. Alternatively, non-specific, non-combinatorial, purely quantitative adhesive differentials have been proposed to explain neuronal sorting in the brain. Do cadherin combinations underlie brain cytoarchitecture? We approached this question using as model a well-defined forebrain nucleus, the mammillary body (MBO), which shows strong, homogeneous expression of one single cadherin (Cdh11) and patterned, combinatorial expression of Cdh6, −8 and −10. We found that, besides the known combinatorial Cdh pattern, MBO cells are organized into a second, non-overlapping pattern grouping neurons with the same date of neurogenesis. We report that, in the Foxb1 mouse mutant, Cdh11 expression fails to be maintained during MBO development. This disrupted the combination-based as well as the birthdate-based sorting in the mutant MBO. In utero RNA interference (RNAi) experiments knocking down Cdh11 in MBO-fated migrating neurons at one specific age showed that Cdh11 expression is required for chronological entrance in the MBO. Our results suggest that neuronal sorting in the developing MBO is caused by adhesion-based, non-combinatorial mechanisms that keep neurons sorted according to birthdate information (possibly matching them to target neurons chronologically sorted in the same manner). Non-specific adhesion mechanisms would also prevent cadherin combinations from altering the birthdate-based sorting. Cadherin combinations would presumably act later to support specific synaptogenesis through specific axonal fasciculation and final target recognition.

Differential combinatorial coding of pheromones in two olfactory subsystems of the honey bee brain.

Neural coding of pheromones has been intensively studied in insects with a particular focus on sex pheromones. These studies favored the view that pheromone compounds are processed within specific antennal lobe glomeruli following a specialized labeled-line system. However, pheromones play crucial roles in an insect's life beyond sexual attraction, and some species use many different pheromones making such a labeled-line organization unrealistic. A combinatorial coding scheme, in which each component activates a set of broadly tuned units, appears more adapted in this case. However, this idea has not been tested thoroughly. We focused here on the honey bee Apis mellifera, a social insect that relies on a wide range of pheromones to ensure colony cohesion. Interestingly, the honey bee olfactory system harbors two central parallel pathways, whose functions remain largely unknown. Using optophysiological recordings of projection neurons, we compared the responses of these two pathways to 27 known honey bee pheromonal compounds emitted by the brood, the workers, and the queen. We show that while queen mandibular pheromone is processed by l-ALT (lateral antennal lobe tract) neurons and brood pheromone is mainly processed by m-ALT (median antennal lobe tract) neurons, worker pheromones induce redundant activity in both pathways. Moreover, all tested pheromonal compounds induce combinatorial activity from several AL glomeruli. These findings support the combinatorial coding scheme and suggest that higher-order brain centers reading out these combinatorial activity patterns may eventually classify olfactory signals according to their biological meaning.

Optimal combinatorial batch code: Monotonicity, lower and upper bounds

A combinatorial batch code with parameters n , k , m is a system consisting of m subsets B 1 , B 2 ,..., B m of an n -element set such that any k elements can be retrieved by reading at most one (or in general, t ) elements from each subset. An optimization problem is to determine N ( n , k , m ), the minimum of | B 1 | + | B 2 | +··· + | B m |. This problem has both theoretical interests and strong practical motivation. In this article, we study the fuction N ( n , k , m ) and give a lower and upper bounds for N ( n , k , m ): for 2≤ k m ≤ n -3, if m + 1- k > [√ k + 1 ] then ( n - m ) k + m > N( n , k ; m ) ≥ 2 n - m + k -6 + [2√ k + 1 ; if m + 1- k k + 1 ] then ( n - m ) k + m ≥ N ( n , k , m )≥2 n -6 +[1+( k + 1)/ m - k + 1. We also determined that N ( m + 3,4, m )= m + 9 ( m ≥ 6), N (8, 4, 8)=15. Our results partly settle an open problem of Paterson et al.

Deciphering the Combinatorial DNA-binding Code of the CCAAT-binding Complex and the Iron-regulatory Basic Region Leucine Zipper (bZIP) Transcription Factor HapX

The heterotrimeric CCAAT-binding complex (CBC) is evolutionarily conserved in eukaryotic organisms, including fungi, plants, and mammals. The CBC consists of three subunits, which are named in the filamentous fungus Aspergillus nidulans HapB, HapC, and HapE. HapX, a fourth CBC subunit, was identified exclusively in fungi, except for Saccharomyces cerevisiae and the closely related Saccharomycotina species. The CBC-HapX complex acts as the master regulator of iron homeostasis. HapX belongs to the class of basic region leucine zipper transcription factors. We demonstrated that the CBC and HapX bind cooperatively to bipartite DNA motifs with a general HapX/CBC/DNA 2:1:1 stoichiometry in a class of genes that are repressed by HapX-CBC in A. nidulans during iron limitation. This combinatorial binding mode requires protein-protein interaction between the N-terminal domain of HapE and the N-terminal CBC binding domain of HapX as well as sequence-specific DNA binding of both the CBC and HapX. Initial binding of the CBC to CCAAT boxes is mandatory for DNA recognition of HapX. HapX specifically targets the minimal motif 5'-GAT-3', which is located at a distance of 11-12 bp downstream of the respective CCAAT box. Single nucleotide substitutions at the 5'- and 3'-end of the GAT motif as well as different spacing between the CBC and HapX DNA-binding sites revealed a remarkable promiscuous DNA-recognition mode of HapX. This flexible DNA-binding code may have evolved as a mechanism for fine-tuning the transcriptional activity of CBC-HapX at distinct target promoters.

Decoding the combinatorial aroma code of a commercial Cognac by application of the sensomics concept and first insights into differences from a German brandy.

In the volatile fraction isolated from a commercial Cognac by means of extraction/SAFE distillation, 39 odor-active areas were detected, among which (E)-β-damascenone showed the highest flavor dilution (FD) factor of 2048 followed by 2- and 3-methylbutanol, (S)-2-methylbutanol, 1,1-diethoxyethane, ethyl methylpropanoate, and ethyl (S)-2-methylbutanoate, as well as 4-hydroxy-3-methoxybenzaldehyde (vanilla-like) and 2-phenylethanol. The quantitation of 37 odorants by stable isotope dilution assays and a calculation of odor activity values (OAV; ratio of concentration to odor threshold) resulted in 34 odorants with OAVs > 1. Among them (E)-β-damascenone, methylpropanal, ethyl (S)-2-methylbutanoate, ethyl methylpropanoate, and ethyl 3-methylbutanoate together with ethanol were established as key contributors to the Cognac aroma. Finally, the overall aroma of the Cognac could be mimicked by an aroma recombinate consisting of these 34 key odorants on the basis of their natural concentrations in the Cognac using an odorless matrix to simulate the influence of the nonvolatile constituents. A comparison of the FD factors of the key odorants identified in a German brandy to those in the Cognac suggested the pair (E)-β-damascenone and ethyl pentanoate as indicators to differentiate various Cognacs from German, French, and Spanish brandies. This was confirmed by calculating a ratio of the concentrations of (E)-β-damascenone to ethyl pentanoate for 12 Cognacs and 7 brandies from Germany and 2 from France and Spain, respectively.

Turán numbers and batch codes

Combinatorial batch codes provide a tool for distributed data storage, with possible application in reducing the computational overhead of private information retrieval protocol. Recently, Balachandran and Bhattacharya observed that the problem of constructing such uniform codes with some extremal properties can be formulated as a Turán-type question on hypergraphs. Here we establish general lower and upper bounds for this extremal problem, and also for its generalization where the forbidden family consists of those r-uniform hypergraphs H which satisfy the condition k≥|E(H)|>|V(H)|+q (for k>q+r and q>−r fixed). We also prove that, in the given range of parameters, the considered Turán function is asymptotically equal to the one restricted to |E(H)|=k, studied by Brown, Erdős and T. Sós. Both families contain some r-partite members–often called the ‘degenerate case’, characterized by the equality limn→∞ex(n,F)/nr=0–and therefore their exact order of growth is not known.

Scents and sensibility: how biology perceives chemistry

Everything we know about the world around us comes through our various sensory systems. Aristotle named five of these in human beings and for the last 2500 years that number has stuck. Even though it's not true. The classic five senses of Aristotle are vision, smell, taste, touch and hearing. We can add many others to these, either as subsets, or even as completely different senses. For example there is proprioception, the sense of your body's position in space, there is the vestibular system which tells you where your head is and in which way you pointed; touch should also include the thermal sensors for temperature and our multiple pain systems for acute, chronic, throbbing, aching, etc. types of pain. And the chemical senses, taste and smell, are in reality a group of multiple sensory systems capable of detecting and discriminating a wide variety of chemical structures and qualities.

On the nose: genetic and evolutionary aspects of smell.

On the nose: genetic and evolutionary aspects of smell.

The evolution of protein moonlighting: adaptive traps and promiscuity in the chaperonins.

Moonlighting proteins exhibit functions that are alternative to their main role in the cell. Heat-shock proteins, also known as molecular chaperones, are now recognized for their wide range of activities in and/or outside the cell, being prominent examples of moonlighting proteins. Chaperonins are highly conserved molecular chaperones that fold other proteins into their native conformation allowing them to carry out essential functions in the cell. Activities alternative to folding have been reported for the chaperonin (Cpn) 60 protein. Preservation of various alternative functions in one protein conflicts with the optimization of each of the functions. What evolutionary mechanisms have allowed the persistence of moonlighting proteins, and in particular the chaperonins, remains a mystery. In the present article, I argue that mechanisms that increase the resistance of phenotypes to genetic and environmental perturbations enable the persistence of a reservoir of genetic variants, each potentially codifying for a distinct function. Gene duplication is one such mechanism that has characterized the expansion and has been concomitant with the emergence of novel functions in these protein families. Indeed, Cpn60 performs a large list of folding-independent functions, including roles in the transmission of viruses from insects to plants and stimulation of the immune system, among others. In addition to the innovation promoted by gene duplication, I discuss that the Cpn60 protein comprises a hidden amino acid combinatorial code that may well be responsible for its ability to develop novel functions while maintaining an optimized folding ability. The present review points to a complex model of evolution of protein moonlighting.

Sox5 controls dorsal progenitor and interneuron specification in the spinal cord.

The basic organization of somatosensory circuits in the spinal cord is already setup during the initial patterning of the dorsal neural tube. Extrinsic signals, such as Wnt and TGF-β pathways, activate combinatorial codes of transcription factors that are responsible for generating a pattern of discrete domains of dorsal progenitors (dp). These progenitors will give rise to distinct dorsal interneurons (dI). The Wnt/ βcatenin signaling pathway controls specification of dp/dI1-3 progenitors and interneurons. According to the current model in the field, Wnt/βcatenin activity seems to act in a graded fashion in the spinal cord, as different relative levels determine the identity of adjacent progenitors. However, it is not clear how this activity gradient is controlled and how the identities of dI1-3 are differentially regulated by Wnt signalling. We have determined that two SoxD transcription factors, Sox5 and Sox6, are expressed in restricted domains of dorsal progenitors in the neural tube. Using gain- and loss-of function approaches in chicken embryos, we have established that Sox5 controls cell fate specification of dp2 and dp3 progenitors and, as a result, controls the correct number of the corresponding dorsal interneurons (dI2 and dI3). Furthermore, Sox5 exerts its function by restricting dorsally Wnt signaling activity via direct transcriptional induction of the negative Wnt pathway regulator Axin2. By that way, Sox5 acts as a Wnt pathway modulator that contributes to sharpen the dorsal gradient of Wnt/βcatenin activity to control the distinction of two functionally distinct types of interneurons, dI2 and dI3 involved in the somatosensory relay.

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8 + T cell response

Anti-CTLA-4 treatment improves the survival of patients with advanced-stage melanoma. However, although the anti-CTLA-4 antibody ipilimumab is now an approved treatment for patients with metastatic disease, it remains unknown by which mechanism it boosts tumor-specific T cell activity. In particular, it is unclear whether treatment amplifies previously induced T cell responses or whether it induces new tumor-specific T cell reactivities. Using a combination ultraviolet (UV)-induced peptide exchange and peptide-major histocompatibility complex (pMHC) combinatorial coding, we monitored immune reactivity against a panel of 145 melanoma-associated epitopes in a cohort of patients receiving anti-CTLA-4 treatment. Comparison of pre- and posttreatment T cell reactivities in peripheral blood mononuclear cell samples of 40 melanoma patients demonstrated that anti-CTLA-4 treatment induces a significant increase in the number of detectable melanoma-specific CD8 T cell responses (P = 0.0009). In striking contrast, the magnitude of both virus-specific and melanoma-specific T cell responses that were already detected before start of therapy remained unaltered by treatment (P = 0.74). The observation that anti-CTLA-4 treatment induces a significant number of newly detected T cell responses-but only infrequently boosts preexisting immune responses-provides strong evidence for anti-CTLA-4 therapy-enhanced T cell priming as a component of the clinical mode of action.

Optimal combinatorial batch codes based on block designs

Batch codes, introduced by Ishai, Kushilevitz, Ostrovsky and Sahai, represent the distributed storage of an $$n$$ -element data set on $$m$$ servers in such a way that any batch of $$k$$ data items can be retrieved by reading at most one (or more generally, $$t$$ ) items from each server, while keeping the total storage over $$m$$ servers equal to $$N$$ . This paper considers a class of batch codes (for $$t=1$$ ), called combinatorial batch codes (CBCs), where each server stores a subset of a database. A CBC is called optimal if the total storage $$N$$ is minimal for given $$n,m$$ , and $$k$$ . A $$c$$ -uniform CBC is a combinatorial batch code where each item is stored in exactly $$c$$ servers. A $$c$$ -uniform CBC is called optimal if its parameter $$n$$ has maximum value for given $$m$$ and $$k$$ . Optimal $$c$$ -uniform CBCs have been known only for $$c\in \{2,k-1,k-2\}$$ . In this paper we present new constructions of optimal CBCs in both the uniform and general settings, for values of the parameters where tight bounds have not been established previously. In the uniform setting, we provide constructions of two new families of optimal uniform codes with $$c\sim \sqrt{k}$$ . Our constructions are based on affine planes and transversal designs.