Enhancers with tissue-specific activity are enriched in intronic regions

Tissue function and homeostasis reflect the gene expression signature by which the combination of ubiquitous and tissue-specific genes contribute to the tissue maintenance and stimuli-responsive function. Enhancers are central to control this tissue-specific gene expression pattern. Here, we explore the correlation between the genomic location of enhancers and their role in tissue-specific gene expression. We find that enhancers showing tissue-specific activity are highly enriched in intronic regions and regulate the expression of genes involved in tissue-specific functions, whereas housekeeping genes are more often controlled by intergenic enhancers, common to many tissues. Notably, an intergenic-to-intronic active enhancers continuum is observed in the transition from developmental to adult stages: the most differentiated tissues present higher rates of intronic enhancers, whereas the lowest rates are observed in embryonic stem cells. Altogether, our results suggest that the genomic location of active enhancers is key for the tissue-specific control of gene expression

Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages.

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays

Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing

An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control

Analysis of scRNA-Seq from drosophila developmental tissues

Organisms are formed by a huge variety of cell types. Conventional genomics approaches do not allow for the understanding of the diversity of cell populations forming tissues and organs. Recently, and with the aim of scrutinizing the cellular complexity of organisms, single-cell technologies have arisen, challenging the limitations of former methods. In this project, we have analyzed single-cell transcriptomics data generated in different laboratories and using a manifold of technologies. We first explored scRNA-Seq data produced by two independent groups on Drosophila melanogaster brain cells. Data from these reference datasets were generated using four different technologies, allowing for the assessment and correction of putative batch effects caused by the multiple platforms used. By implementing a number of pipelines in bash and R environments, we have processed these datasets from the mapping of raw fastq files to the identification of subpopulations of cells. Cells from the 8 identified clusters expressed several known marker genes involved in neuron and glia differentiation, permitting the full characterization of these populations. The pipeline implemented along this first part of the project was, afterwards, used to analyze scRNA-Seq data generated in our own lab on Drosophila wing imaginal discs. Within our dataset, we distinguished four clusters, although the low expression of known marker genes did not allow for a precise characterization of these populations. Still, we were able to identify several markers showing high variability between clusters, indicating that, indeed, these subpopulations represent different cell types within the wing.Los organismos están formados por una gran variedad de tipos celulares. Los enfoques genómicos convencionales no permiten comprender la diversidad de las poblaciones celulares que forman tejidos y órganos. Recientemente, y con el objetivo de examinar la complejidad celular de los organismos, han surgido tecnologías unicelulares, desafiando las limitaciones de los métodos anteriores. En este proyecto, hemos analizado los datos de transcriptómica unicelular generados en diferentes laboratorios y utilizando una variedad de tecnologías. Primero exploramos los datos de scRNA-Seq producidos por dos grupos independientes en células cerebrales Drosophila melanogaster. Los datos de estos conjuntos de datos de referencia se generaron utilizando cuatro tecnologías diferentes, lo que permite la evaluación y corrección de los supuestos efectos por lotes causados por las múltiples plataformas utilizadas. Al implementar una serie de tuberías en entornos bash y R, hemos procesado estos conjuntos de datos desde la asignación de archivos fastq sin procesar hasta la identificación de subpoblaciones de celdas. Las células de los 8 grupos identificados expresaron varios genes marcadores conocidos involucrados en la diferenciación de neuronas y glía, lo que permite la caracterización completa de estas poblaciones. La tubería implementada a lo largo de esta primera parte del proyecto fue, luego, utilizada para analizar datos scRNA-Seq generados en nuestro propio laboratorio en discos imaginales del ala Drosophila. Dentro de nuestro conjunto de datos, distinguimos cuatro grupos, aunque la baja expresión de genes marcadores conocidos no permitió una caracterización precisa de estas poblaciones. Aún así, pudimos identificar varios marcadores que muestran una alta variabilidad entre los grupos, lo que indica que, de hecho, estas subpoblaciones representan diferentes tipos de células.Els organismes estan formats per una gran varietat de tipus cel·lulars. Els enfocaments genòmics convencionals no permeten comprendre la diversitat de les poblacions cel·lulars que formen teixits i òrgans. Recentment, i amb l'objectiu d'examinar la complexitat cel·lular dels organismes, han sorgit tecnologies unicel·lulars, desafiant les limitacions dels mètodes anteriors. En aquest projecte, hem analitzat les dades de transcriptòmica unicel·lular generades en diferents laboratoris i utilitzant una varietat de tecnologies. Primer explorem les dades de scRNA-Seq produïts per dos grups independents en cèl·lules cerebrals Drosophila melanogaster. Les dades d'aquests conjunts de dades de referència es van generar utilitzant quatre tecnologies diferents, cosa que permet l'avaluació i correcció dels suposats efectes per lots causats per les múltiples plataformes utilitzades. En implementar una sèrie de canonades en entorns bash i R, hem processat aquests conjunts de dades des de l'assignació d'arxius fastq sense processar fins a la identificació de subpoblacions de cel·les. Les cèl·lules dels 8 grups identificats van expressar diversos gens marcadors coneguts involucrats en la diferenciació de neurones i glia, cosa que permet la caracterització completa d'aquestes poblacions. La canonada implementada al llarg d'aquesta primera part del projecte va ser, després, utilitzada per analitzar dades scRNA-Seq generats en el nostre propi laboratori en discos imaginals de l'ala Drosophila. Dins del nostre conjunt de dades, distingim quatre grups, tot i que la baixa expressió de gens marcadors coneguts no va permetre una caracterització precisa d'aquestes poblacions. Així i tot, vam poder identificar diversos marcadors que mostren una alta variabilitat entre els grups, el que indica que, de fet, aquestes subpoblacions representen diferents tipus de cèl·lules

Anàlisi de l'estructura de la cromatina del complex bitòrax de Drosophila melanogaster i contribució de les proteïnes dSAP18 i GAGA en la seva regulació

Consultable des del TDXTítol obtingut de la portada digitalitzadaEls gens homeòtics són els responsables del correcte desenvolupament dels organismes. A Drosophila, aquests gens es troben agrupats en dos clusters: el Complex Antenapèdia i el Complex Bitòrax (BX-C). El BX-C codifica per a tres gens homeòtics: l'Ultrabithorax, l'Abdominal A i l'Abdominal-B (Abd-B). L'expressió de l'Abd-B està conduïda per quatre dominis reguladors (iab-5 a iab-8) que controlen la seva expressió en els segments posteriors de l'organisme. En aquest treball, ens hem centrat en l'estudi dels elements reguladors que controlen l'expressió del l'Abd-B i en la contribució de les proteïnes dSAP18 i GAGA a la seva regulació. En primer lloc, assajos de digestió amb DNasaI realitzats a embrions de Drosophila ens han permès identificar diverses regions hipersensibles entre els dominis iab-5 i iab-6, suggerint l'existència de diversos elements reguladors en aquesta regió. Mitjançant un sistema en que el gen reporter white és control·lat pel seu propi promotor i enhancer, vam observar que la regió hipersensible més proximal promovia silenciament i Pairing-Sensitive Silencing (PSS) de l'expressió del gen reporter. Tant el silenciament com el PSS depenien de proteïnes del Grup Polycomb (Pc-G), indicant que aquesta regió actua com a un PRE (Polycomb Response Element). Hem anomenat aquesta regió iab6PRE. D'altra banda, hem observat que l'existència de regions lliures de nucleosomes als elements reguladors del BX-C és una característica molt conservada al llarg del desenvolupament de la mosca; també hi són presents a elements reguladors ectòpics i no depenen ni de l'estat transcripcional de la regió ni en el recrutament de proteïnes Pc i Trithorax (Trx) als PREs. No obstant, aquests llocs hipersensibles només estan presents a elements reguladors actius, ja que l'iniciador IAB6, que està implicat en la regulació del gen homèotic al principi del desenvolupament, mostra llocs lliures de nucleosomes a embrió, però no durant el desenvolupament larvari. Per tal d'analitzar l'organització nuclear del BX-C hem realitzat assajos de Chromosome Conformation Capture a cèl·lules S2 de Drosophila, que no expressen el gen Abd-B. Vam observar que l'element iab6PRE contacta amb el promotor del gen homeòtic així com amb d'altres elements de manteniment del complex, com l'MCP i el bxdPRE. L'element iniciador IAB6, en canvi, que es troba localitzat a només 5 Kb de l'iab6PRE, no interacciona amb cap d'aquests elements reguladors, suggerint que els elements de manteniment poden interaccionar a través de la formació de loops de cromatina. A més a més, hem vist que mosques mutants per a la proteïna dSAP18 mostren una disminució del silenciament conduït pels elements Fab-7 i iab7PRE en còpies ectòpiques; no obstant, aquesta disminució no és deguda ni a un canvi a les modificacions de les histones ni del patró de posicionament dels nucleosomes. Finalment, hem vist que una disminució de la quantitat de la proteïna GAGA mitjançant la inducció d'RNAs d'interferència a estadis tardans del desenvolupament larvari, promou la transformació homèotica del segment A6 a l'A5, suggerint una desregulació de l'expressió de l'Abd-B al segment A6. Aquesta desregulació indica que els patrons d'expressió establerts per les proteïnes Pc i Trx poden canviar al llarg del desenolupament de la mosca. No obstant, hem vist que la transformació tampoc és deguda a cap canvi en el patró d'hipersensibilitats a DNasaI ni a l'element Fab-7 ni a l'iab6PRE.The homeotic genes are responsible for the correct development of the organisms. In Drosophila, these genes are clustered in two complexes: the Antenapedia Complex and the Bithorax Complex (BX-C). The BX-C encodes for three homeotic genes: Ultrabithorax, Abdominal-A and Abdominal-B (Abd-B). The expression of the Abd-B is driven by 4 regulatory domains (iab-5 to iab-8), which control its expression at the most posterior segments of the organism. In this work, we have focused on the study of the regulatory elements that control Abd-B expression and the contribution of the dSAP18 and GAGA proteins to its regulation. First of all, DNaseI digestion assays performed in Drosophila embryos have allowed us to identify some hypersensitive regions among iab-5 and iab-6 domains, suggesting the existence of several regulatory elements within this region. By means of a reporter gene system in which the white gene was controlled by its own promoter and enhancer, we observed that the most proximal hypersensitive region promotes silencing and Pairing-Sensitive Silencing (PSS) of the reporter gene expression. Both silencing and PSS depend on Polycomb Group (Pc-G) proteins, indicating that this region acts as a PRE (Polycomb Response Element). We have named this region iab6PRE. On the other hand, we have observed that the existence of nucleosome-free regions on regulatory elements of the BX-C is a very conserved feature along the fly development; they are also present in ectopic regulatory elements and they do not depend either on the transcriptional state of the region or on the Pc and Trithorax (Trx) recruitment to PREs. However, these hypersensitive sites are present only at active regulatory elements, since the iniciator IAB6, which is implicated in the homeotic gene regulation at the beginning of the development, shows nucleosome free regions at embryo state, but not later during larval development. In order to analyse the nuclear organization of the BX-C, we have done Chromosome Conformation Capture assays in Drosophila S2 cells, which don't express the Abd-B gene. We observed that the iab6PRE element contacts with the homeotic gene promoter as well as with other maintenance elements of the complex, such as the MCP and the bxdPRE. The iniciator element IAB6, on the other hand, which is just located 5 Kb far from the iab6PRE, does not interact with any of these regulatory elements, suggesting that the maintenance elements may interact through the formation of chromatin loops. Furthermore, we have seen that dSAP18 mutant flies show a decrease in the silencing driven by Fab-7 and iab7PRE elements in ectopic copies; however, this decrease is not due to any change either in histone modification or in nucleosome position patterns. Finally, we have observed that a decrease in the amount of GAGA protein by inducing interference RNAs at later stages of larval development, promotes a homeotic transformation of the A6 segment to A5, suggesting a misregulation of the Abd-B expression in the A6 segment. This misregulation indicates that the expression patterns established by Pc and Trx proteins may change along the fly development. However, we have seen that the transformation is not due to any change in the DNaseI hypersensitive pattern neither of the Fab-7 nor of the iab6PRE elements

Active transcription without histone modifications

Gene expression is regulated by proteins such as transcription factors, as well as by chromatin modifications on DNA and histones. Some histone modifications have been associated to transcriptional activation (i.e. H3K4me1, H3K4me3, H3K9ac, H3K27ac or H3K36me3) whereas others with gene silencing (H3K9me3 and H3K27me3). However, and challenging this premise, we have identified a set of genes in Drosophila melanogaster that are actively expressed in the absence of the canonical histone marks

Active transcription without histone modifications

Gene expression is regulated by proteins such as transcription factors, as well as by chromatin modifications on DNA and histones. Some histone modifications have been associated to transcriptional activation (i.e. H3K4me1, H3K4me3, H3K9ac, H3K27ac or H3K36me3) whereas others with gene silencing (H3K9me3 and H3K27me3). However, and challenging this premise, we have identified a set of genes in Drosophila melanogaster that are actively expressed in the absence of the canonical histone marks

Active transcription without histone modifications

Gene expression is regulated by proteins such as transcription factors, as well as by chromatin modifications on DNA and histones. Some histone modifications have been associated to transcriptional activation (i.e. H3K4me1, H3K4me3, H3K9ac, H3K27ac or H3K36me3) whereas others with gene silencing (H3K9me3 and H3K27me3). However, and challenging this premise, we have identified a set of genes in Drosophila melanogaster that are actively expressed in the absence of the canonical histone marks [1] [...]