Mammalian transcriptional hotspots are enriched for tissue specific enhancers near cell type specific highly expressed genes and are predicted to act as transcriptional activator hubs

BACKGROUND: Transcriptional hotspots are defined as genomic regions bound by multiple factors. They have been identified recently as cell type specific enhancers regulating developmentally essential genes in many species such as worm, fly and humans. The in-depth analysis of hotspots across multiple cell types in same species still remains to be explored and can bring new biological insights. RESULTS: We therefore collected 108 transcription-related factor (TF) ChIP sequencing data sets in ten murine cell types and classified the peaks in each cell type in three groups according to binding occupancy as singletons (low-occupancy), combinatorials (mid-occupancy) and hotspots (high-occupancy). The peaks in the three groups clustered largely according to the occupancy, suggesting priming of genomic loci for mid occupancy irrespective of cell type. We then characterized hotspots for diverse structural functional properties. The genes neighbouring hotspots had a small overlap with hotspot genes in other cell types and were highly enriched for cell type specific function. Hotspots were enriched for sequence motifs of key TFs in that cell type and more than 90% of hotspots were occupied by pioneering factors. Though we did not find any sequence signature in the three groups, the H3K4me1 binding profile had bimodal peaks at hotspots, distinguishing hotspots from mono-modal H3K4me1 singletons. In ES cells, differentially expressed genes after perturbation of activators were enriched for hotspot genes suggesting hotspots primarily act as transcriptional activator hubs. Finally, we proposed that ES hotspots might be under control of SetDB1 and not DNMT for silencing. CONCLUSION: Transcriptional hotspots are enriched for tissue specific enhancers near cell type specific highly expressed genes. In ES cells, they are predicted to act as transcriptional activator hubs and might be under SetDB1 control for silencing. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12859-014-0412-0) contains supplementary material, which is available to authorized users

Cell type-selective disease-association of genes under high regulatory load

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3′ UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manne

Cell type-selective disease-association of genes under high regulatory load

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3'UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner

Investigating 3D genome organisation in Caenorhabditis elegans with Accessible Region Conformation Capture (ARC-C)

3C and its derivatives have been applied in various organisms to study chromatin architecture. However, these methods have limitations: most of them are limited to restriction-fragment resolution and all of them, with the exception of Hi-C, only survey a pre-defined subset of the genome. I developed a variant of Hi-C, named Accessible Region Conformation Capture (ARC-C), in C. elegans, which interrogates genome organisation at multiple scales genome-wide - from domains and compartments to high resolution interactions between regulatory elements. I applied ARC-C in wild-type Bristol N2, met-2 set-25 mutants that have no H3K9 methylation to study the effects on domain and compartment formation. In these mutants, compartmentalisation (i.e. inter-domain interactions) between H3K27me3-enriched regulated domains is reduced. I also used ARC-C in blmp-1 mutants to understand the role of BLMP-1 in chromatin looping. In blmp-1 mutants, interactions between putative BLMP-1 mediated loops for downregulated genes are significantly reduced. In wild-type worms, when surveying significant interactions at 500 bp resolution, I observe the presence of dense clusters of significant interactions anchored at high occupancy target (HOT) regions that I call “hubs.” Interestingly, the deletion of these hubs does not affect the transcription of linked or local genes. However, local interactions are altered and some extent of redundancy is observed. To improve our ARC-C protocol, I tested several variations with different enzymes and biotin-mediated streptavidin beads pulldown. In all, ARC-C revealed insights into genome organisation in C. elegans and I have made progress toward a next-generation version of the method.A*STAR National Science Scholarship (PhD

Identifying and characterizing transcriptional regulatory elements from chromosome conformation capture data

Which features on the chromatin are responsible for regulating gene transcription? Using promoter contacts obtained from chromosome conformation capture (3C) data as a readout for transcriptional regulation, I modeled how well histone modification marks and chromatin accessibility predict promoter contact frequency. I found that promoter contacts were often located in the same topologically associating domain and that the correlation between promoter contact frequency and each chromatin feature varied across promoter gene expression level, with poised promoters less constrained than active or silent promoters when forming contacts. I applied this knowledge to understand the molecular changes that occurred at several limb development enhancers in a mouse selective breeding experiment for longer tibia called “Longshanks.

Deciphering Regulatory Networks in the Mouse Genome

Regardless of all the major achievements in the field of genomics and in depth studies of the protein-coding genes, our knowledge about non-coding regions and their contribution in diseases remains incomplete. Large scale projects such as the ENCODE have produced a wealth of sequencing data which can be utilised to study epigenetic features associated with gene regulation. These studies have comprehensively identified regulatory elements such as enhancers in the human genome, but numerous questions still remain on their effect on gene function and disease causation. The aim of this thesis is to identify enhancer regulatory networks in the mouse genome and investigate their effect on mouse models of human diseases. In order to study enhancer regulation, I have taken two approaches. First, I have produced a catalogue of well-defined multiple enhancer types in a diverse range of mouse tissues and cell-types. By systematically comparing different enhancer types, I found that super- and typical-enhancers have different effect on gene expression, but both are preferentially associated with relevant tissue-type phenotypes. Also genes associated with super- and typical-enhancers exhibit no difference in phenotype effect size or pleiotropy. Second, by utilising publicly available regulatory annotations, my enhancer catalogue and omics data, I have investigated regulatory mechanisms associated with metabolic and circadian mouse models. Here I identified novel regulatory networks or enhancers or transcription factor binding sites pertaining to the mutant mice. In conclusion, my research has shown the usefulness of integrating enhancer annotations with an array of molecular data and has for the first time shown how different enhancer architectures influence gene function in the mouse genome. This study provides a valuable dataset to further characterise the mechanisms of gene regulation by enhancers in the mouse genome

GENOME-WIDE DISCOVERY AND ANNOTATION OF HUMAN ENHANCERS RELEVANT TO DEVELOPMENT AND DISEASE

Ph.DDOCTOR OF PHILOSOPH

Genome-wide Transcriptional Characterization of the ETV6-RUNX1-positive Childhood Leukemia

Akuutti lymfoblastileukemia (ALL) on lasten yleisin syöpä. Useimmiten se saa alkunsa epäkypsästä B-solusta (preB), jossa tapahtuu tietty altistava geneettinen muutos. Yksi yleisimmistä muutoksista on translokaatio, joka johtaa ETV6-RUNX1 (E/R) fuusiogeenin syntymiseen. Leukemian puhkeamiseen vaaditaan lisäksi muita geneettisiä muutoksia, jotka usein osuvat B-solun identiteetille tärkeisiin geeneihin. DNA-vaurioiden lisäksi solun toiminta voi häiriintyä RNA-molekyylien ja proteiinien toiminnan muutoksista. E/R on epänormaali transkriptiotekijä ja sen suorat säätelykohteet ovat vielä jääneet epäselviksi. Tässä työssä tutkimme lasten prekursori B-ALL:ssa (preB-ALL) tapahtuvaa genominlaajuista geeniensäätelyä tarkastelemalla varhaista RNA-transkriptiota solulinjoissa ja potilasnäytteissä. E/R-fuusion kohdegeenien kartoittamista varten teimme solulinjamallin, jossa fuusion tuotantoa voidaan säädellä. Määritimme tehostaja-alueet tehostaja-RNA:iden (eRNA) ilmentymisen perusteella sekä niiden mahdolliset kohdegeenit perustuen signaalimuutosten samankaltaisuuteen. E/R- fuusion säätelemistä geeneistä kaksi kolmasosaa hiljeni suoran RUNX1-välitteisen DNA-sitoutumisen kautta. Lisäksi E/R vähensi B-solu-spesifisten tehostaja- alueiden luentaa. Osa geeneistä myös ilmentyi eri tavalla E/R-potilaiden leukemiasoluissa verrattuna muiden preB-ALL alityyppien potilaiden soluihin. RAG ja AID entsyymit on liitetty DNA-katkosten syntymiseen B-solu- leukemiassa ja niiden toimintaan tiedetään liittyvän avoimena oleva kromatiini. Tutkimme RNA-transkriptiota B-linjan soluissa keskittyen lasten leukemiassa usein nähtäviin DNA-katkoskohtiin. Huomasimme, että katkoskohtiin assosioituvat tietyt transkriptionaaliset ominaisuudet: RNA-polymeraasin pysähtyminen sekä yhtäaikainen geenienluenta päällekkäisiltä DNA-juosteilta. Nämä piirteet näyttävät altistavan DNA:n katkoksille erityisesti paljastamalla RAG-entsyymin rekombinaatiosignaalisekvenssejä. Huomasimme myös korkean RAG1-geenin luennan erityisesti E/R-potilailla sekä AID-entsyymiä koodaavan geenin epätavallisen luennan osalla korkean riskin preB-ALL potilaita. Tässä väitöskirjassa tunnistettiin E/R-fuusion genominlaajuisia säätelykohteita sekä toistuvien DNA-katkosten kohdille ominaisia transkriptionaalisia piirteitä lasten leukemiassa.Acute lymphoblastic leukemia (ALL) is the most common cancer affecting in childhood. It occurs typically in early B-lineage cells and is characterized by a few specific initiating genomic alterations. One of the most common alterations is the translocation resulting in the ETV6-RUNX1 (E/R) fusion gene. Progression to overt ALL requires additional genetic abnormalities that are recurrently found at essential B-cell lineage identity determining genes. Besides DNA, alterations in various RNA species and proteins could also have marked unwanted effects on cell behavior. E/R functions as an aberrant transcription factor but its direct target genes have thus far remained uncertain. We set out to study genome-wide gene regulation in childhood precursor B-ALL (preB-ALL) by studying nascent RNA transcription in cell lines and patient samples. For the examination of target sites, we generated a cell line model with an inducible E/R. We detected enhancer regions by the expression of eRNA transcripts and deciphered a possible target gene by correlating between expression level changes. Two thirds of the E/R-regulated genes were repressed by direct regulation via RUNX1 DNA binding. We further showed E/R-mediated downregulation of B-cell specific super-enhancers. Some of the regulated genes were observed to be differentially expressed among E/R patients when compared to other preB-ALL patients. RAG and AID are enzymes that have been linked to the genesis of secondary genetic alterations in B-cell leukemia. We explored the nascent RNA transcription across B-lymphoid cells at the genomic sites that are often deleted in childhood precursor B-ALL and noticed significant association with specific transcriptional features, namely RNA polymerase II stalling and convergent transcription. These features seem to expose the DNA to double strand breaks especially by revealing RAG recombination signal sequences. We noticed high RAG1 expression in the E/R subtype, and abnormal expression of AICDA among the non-classified precursor B-ALL cases. This thesis identifies genome-wide targets of the E/R fusion and specific transcriptional features that are associated with recurrent DNA breakpoint sites in childhood precursor B-ALL