Enrique Blanco

3D structures of individual mammalian genomes studied by single-cell Hi-C

The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.

Using geneid to Identify Genes

The unit describes the usage of geneid. geneid is a very efficient gene‐finding program. It allows for the analysis of large genomic sequences, including whole mammalian chromosomes. These sequences can be partially annotated, and geneid can be used to refine this initial annotation. Parameter configurations exist for a number of eukaryotic species. Geneid produces output in a variety of standard formats. The results, thus, can be processed by a variety of software tools, including visualization programs. Geneid software is in the public domain, and it is undergoing constant development. It is easy to install and use. Exhaustive benchmark evaluations show that geneid compares favorably with other existing gene finding tools.

Reconsidering the evolution of eukaryotic selenoproteins: a novel nonmammalian family with scattered phylogenetic distribution

While the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. The dual role of the UGA codon confounds the identification of novel selenoprotein genes. Here, we describe a comparative genomics approach that relies on the genome‐wide prediction of genes with in‐frame TGA codons, and the subsequent comparison of predictions from different genomes, wherein conservation in regions flanking the TGA codon suggests selenocysteine coding function. Application of this method to human and fugu genomes identified a novel selenoprotein family, named SelU, in the puffer fish. The selenocysteine‐containing form also occurred in other fish, chicken, sea urchin, green algae and diatoms. In contrast, mammals, worms and land plants contained cysteine homologues. We demonstrated selenium incorporation into chicken SelU and characterized the SelU expression pattern in zebrafish embryos. Our data indicate a scattered evolutionary distribution of selenoproteins in eukaryotes, and suggest that, contrary to the picture emerging from data available so far, other taxa‐specific selenoproteins probably exist.

Hnf1alpha (MODY3) controls tissue-specific transcriptional programs and exerts opposed effects on cell growth in pancreatic islets and liver.

ABSTRACT Heterozygous HNF1A mutations cause pancreatic-islet β-cell dysfunction and monogenic diabetes (MODY3). Hnf1α is known to regulate numerous hepatic genes, yet knowledge of its function in pancreatic islets is more limited. We now show that Hnf1a deficiency in mice leads to highly tissue-specific changes in the expression of genes involved in key functions of both islets and liver. To gain insights into the mechanisms of tissue-specific Hnf1α regulation, we integrated expression studies of Hnf1a-deficient mice with identification of direct Hnf1α targets. We demonstrate that Hnf1α can bind in a tissue-selective manner to genes that are expressed only in liver or islets. We also show that Hnf1α is essential only for the transcription of a minor fraction of its direct-target genes. Even among genes that were expressed in both liver and islets, the subset of targets showing functional dependence on Hnf1α was highly tissue specific. This was partly explained by the compensatory occupancy by the paralog Hnf1β at selected genes in Hnf1a-deficient liver. In keeping with these findings, the biological consequences of Hnf1a deficiency were markedly different in islets and liver. Notably, Hnf1a deficiency led to impaired large-T-antigen-induced growth and oncogenesis in β cells yet enhanced proliferation in hepatocytes. Collectively, these findings show that Hnf1α governs broad, highly tissue-specific genetic programs in pancreatic islets and liver and reveal key consequences of Hnf1a deficiency relevant to the pathophysiology of monogenic diabetes.

ABS: a database of Annotated regulatory Binding Sites from orthologous promoters.

Information about the genomic coordinates and the sequence of experimentally identified transcription factor binding sites is found scattered under a variety of diverse formats. The availability of standard collections of such high-quality data is important to design, evaluate and improve novel computational approaches to identify binding motifs on promoter sequences from related genes. ABS () is a public database of known binding sites identified in promoters of orthologous vertebrate genes that have been manually curated from bibliography. We have annotated 650 experimental binding sites from 68 transcription factors and 100 orthologous target genes in human, mouse, rat or chicken genome sequences. Computational predictions and promoter alignment information are also provided for each entry. A simple and easy-to-use web interface facilitates data retrieval allowing different views of the information. In addition, the release 1.0 of ABS includes a customizable generator of artificial datasets based on the known sites contained in the collection and an evaluation tool to aid during the training and the assessment of motif-finding programs.

Coordinate control of synaptic-layer specificity and rhodopsins in photoreceptor neurons

How neurons make specific synaptic connections is a central question in neurobiology. The targeting of the Drosophila R7 and R8 photoreceptor axons to different synaptic layers in the brain provides a model with which to explore the genetic programs regulating target specificity. In principle this can be accomplished by cell-type-specific molecules mediating the recognition between synaptic partners. Alternatively, specificity could also be achieved through cell-type-specific repression of particular targeting molecules. Here we show that a key step in the targeting of the R7 neuron is the active repression of the R8 targeting program. Repression is dependent on NF-YC, a subunit of the NF-Y (nuclear factor Y) transcription factor. In the absence of NF-YC, R7 axons terminate in the same layer as R8 axons. Genetic experiments indicate that this is due solely to the derepression of the R8-specific transcription factor Senseless (Sens) late in R7 differentiation. Sens is sufficient to control R8 targeting specificity and we demonstrate that Sens directly binds to an evolutionarily conserved DNA sequence upstream of the start of transcription of an R8-specific cell-surface protein, Capricious (Caps) that regulates R8 target specificity. We show that R7 targeting requires the R7-specific transcription factor Prospero (Pros) in parallel to repression of the R8 targeting pathway by NF-YC. Previous studies demonstrated that Sens and Pros directly regulate the expression of specific rhodopsins in R8 and R7. We propose that the use of the same transcription factors to promote the cell-type-specific expression of sensory receptors and cell-surface proteins regulating synaptic target specificity provides a simple and general mechanism for ensuring that transmission of sensory information is processed by the appropriate specialized neural circuits.

Transcription Factor Map Alignment of Promoter Regions

We address the problem of comparing and characterizing the promoter regions of genes with similar expression patterns. This remains a challenging problem in sequence analysis, because often the promoter regions of co-expressed genes do not show discernible sequence conservation. In our approach, thus, we have not directly compared the nucleotide sequence of promoters. Instead, we have obtained predictions of transcription factor binding sites, annotated the predicted sites with the labels of the corresponding binding factors, and aligned the resulting sequences of labels—to which we refer here as transcription factor maps (TF-maps). To obtain the global pairwise alignment of two TF-maps, we have adapted an algorithm initially developed to align restriction enzyme maps. We have optimized the parameters of the algorithm in a small, but well-curated, collection of human–mouse orthologous gene pairs. Results in this dataset, as well as in an independent much larger dataset from the CISRED database, indicate that TF-map alignments are able to uncover conserved regulatory elements, which cannot be detected by the typical sequence alignments.

Polycomb Regulates Mesoderm Cell Fate-Specification in Embryonic Stem Cells through Activation and Repression Mechanisms

Polycomb complexes (PRC1 and PRC2) are essential regulators of epigenetic gene silencing in embryonic and adult stem cells. Emerging evidence suggests that the core subunit composition regulates distinct biological processes, yet little is known about the mechanistic underpinnings of how differently composed Polycomb complexes instruct and maintain cell fate. Here we find that Mel18, also known as Pcgf2 and one of six Pcgf paralogs, uniquely regulates PRC1 to specify mesoderm cell fate in embryonic stem cells. Mechanistically, Mel18 functions as a classical Polycomb protein during early cardiac mesoderm differentiation by repressing pluripotency, lineage specification, late cardiac differentiation, and negative regulators of the BMP pathway. However, Mel18 also positively regulates expression of key mesoderm transcription factors, revealing an unexpected function of Mel18 in gene activation during cardiac differentiation. Taken together, our findings reveal that Mel18 is required to specify PRC1 function in both a context- and stage-specific manner.

Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis

The genome-wide localization and function of endogenous Dnmt3a and Dnmt3b in adult stem cells are unknown. Here, we show that in human epidermal stem cells, the two proteins bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce their associated enhancer RNAs. Both proteins prefer super-enhancers associated to genes that either define the ectodermal lineage or establish the stem cell and differentiated states. However, Dnmt3a and Dnmt3b differ in their mechanisms of enhancer regulation: Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function. Altogether, we reveal novel functions for Dnmt3a and Dnmt3b at enhancers that could contribute to their roles in disease and tumorigenesis.

Gene expression following induction of regeneration in Drosophila wing imaginal discs. Expression profile of regenerating wing discs

BackgroundRegeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen.ResultsUsing expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration.ConclusionsWe have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.