Gene expression governs cell fate, and is regulated via a complex interplay of transcription factors and molecules that change chromatin structure. Advances in sequencing-based assays have enabled investigation of these processes genome-wide, leading to large datasets that combine information on the dynamics of gene expression, transcription factor binding and chromatin structure as cells differentiate. While numerous studies focus on the effects of these features on broader gene regulation, less work has been done on the mechanisms of gene-specific transcriptional control. In this study, we have focussed on the latter by integrating gene expression data for the in vitro differentiation of murine ES cells to macrophages and cardiomyocytes, with dynamic data on chromatin structure, epigenetics and transcription factor binding. Combining a novel strategy to identify communities of related control elements with a penalized regression approach, we developed individual models to identify the potential control elements predictive of the expression of each gene. Our models were compared to an existing method and evaluated using the existing literature and new experimental data from embryonic stem cell differentiation reporter assays. Our method is able to identify transcriptional control elements in a gene specific manner that reflect known regulatory relationships and to generate useful hypotheses for further testing.

Cellular differentiation is a tightly controlled process that is essential for the development of a single celled embryo into a multicellular organism. This process is regulated primarily through changes in the dynamics of gene differentiation and is orchestrated by the interplay of various biological mechanisms such as chromatin accessibility, modifications and most importantly, binding of transcription factors. In this study we have generated, integrated and analysed a large multi-dimensional dataset for the in-vitro differentiation of mouse embryonic stem cells to macrophages. At the first step we successfully associate gene expression changes with important cell fate decisions that determine lineage specification. By interrogating DNaseI accessibility, active histone marks such as H3K27ac, H3K9ac and H3K4me3, repressive H3K27me3 histone mark and 16 transcription factors, in a unified manner, we show that we can map this four dimensional data to gene expression events, vis-a-vis cellular differentiation. Here, we also show how we systematically build up the complexity of the analysis and derive a simple yet highly informative core gene regulatory network for the transcriptions factors important in our developmental pathway. Follow-up experiments driven by the new hypotheses derived from our analyses shows that this dataset can be powerful in implicating new biological gene networks and pathways in definitive hematopoiesis. We have also provided these analyses as a user friendly website for the access of general scientific community.

Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.

Comprehensive study of transcriptional control processes will be required to enhance our understanding of both normal and malignant hematopoiesis. Modern sequencing technologies have revolutionized our ability to generate genome-scale expression and histone modification profiles, transcription factor (TF)-binding maps, and also comprehensive chromatin-looping information. Many of these technologies, however, require large numbers of cells, and therefore cannot be applied to rare hematopoietic stem/progenitor cell (HSPC) populations. The stem cell factor-dependent multipotent progenitor cell line HPC-7 represents a well-recognized cell line model for HSPCs. Here we report genome-wide maps for 17 TFs, 3 histone modifications, DNase I hypersensitive sites, and high-resolution promoter-enhancer interactomes in HPC-7 cells. Integrated analysis of these complementary data sets revealed TF occupancy patterns of genomic regions involved in promoter-anchored loops. Moreover, preferential associations between pairs of TFs bound at either ends of chromatin loops led to the identification of 4 previously unrecognized protein-protein interactions between key blood stem cell regulators. All HPC-7 data sets are freely available both through standard repositories and a user-friendly Web interface. Together with previously generated genome-wide data sets, this study integrates HPC-7 data into a genomic resource on par with ENCODE tier 1 cell lines and, importantly, is the only current model with comprehensive genome-scale data that is relevant to HSPC biology.

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis-regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.

Urochordates are the closest relatives of vertebrates and at the larval stage, possess a characteristic bilateral chordate body plan.In vertebrates, the genes that orchestrate embryonic patterning are in part regulated by highly conserved non-coding elements (CNEs), yet these elements have not been identified in urochordate genomes.Consequently the evolution of the cis-regulatory code for urochordate development remains largely uncharacterised.Here, we use genome-wide comparisons between C. intestinalis and C. savignyi to identify putative urochordate cis-regulatory sequences.Ciona conserved noncoding elements (ciCNEs) are associated with largely the same key regulatory genes as vertebrate CNEs.Furthermore, some of the tested ciCNEs are able to activate reporter gene expression in both zebrafish and Ciona embryos, in a pattern that at least partially overlaps that of the gene they associate with, despite the absence of sequence identity.We also show that the ability of a ciCNE to up-regulate gene expression in vertebrate embryos can in some cases be localised to short subsequences, suggesting that functional cross-talk may be defined by small regions of ancestral regulatory logic, although functional sub-sequences may also be dispersed across the whole element.We conclude that the structure and organisation of cis-regulatory modules is very different between vertebrates and urochordates, reflecting their separate evolutionary histories.However, functional cross-talk still exists because the same repertoire of transcription factors has likely guided their parallel evolution, exploiting similar sets of binding sites but in different combinations.

Aberrant transcriptional programs in combination with abnormal proliferative signaling drive leukemic transformation. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. Ets Related Gene (ERG) is a component of normal and leukemic stem cell signatures and high ERG expression is a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG promoters and +85 stem cell enhancer and a heptad of transcription factors that combinatorially regulate genes in HSCs. Gene expression signatures derived from ERG promoter-stem cell enhancer and heptad activity are associated with clinical outcome when ERG expression alone fails. We also show that the heptad signature is associated with AMLs that lack somatic mutations in NPM1 and confers an adverse prognosis when associated with FLT3 mutations. Taken together, these results suggest that transcriptional regulators cooperate to establish or maintain primitive stem cell-like signatures in leukemic cells and that the underlying pattern of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome.

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Summary Comprehensive analysis of cis-regulatory elements is key to understanding the dynamic gene regulatory networks that control embryonic development. While transgenic animals represent the gold standard assay, their generation is costly, entails significant animal usage, and in utero development complicates time-course studies. As an alternative, embryonic stem (ES) cells can readily be differentiated in a process that correlates well with developing embryos. Here, we describe a highly effective platform for enhancer assays using an Hsp68/Venus reporter cassette that targets to the Hprt locus in mouse ES cells. This platform combines the flexibility of Gateway® cloning, live cell trackability of a fluorescent reporter, low background and the advantages of single copy insertion into a defined genomic locus. We demonstrate the successful recapitulation of tissue-specific enhancer activity for two cardiac and two haematopoietic enhancers. In addition, we used this assay to dissect the functionality of the highly conserved Ets/Ets/Gata motif in the Scl+19 enhancer, which revealed that the Gata motif is not required for initiation of enhancer activity. We further confirmed that Gata2 is not required for endothelial activity of the Scl+19 enhancer using Gata2−/− Scl+19 transgenic embryos. We have therefore established a valuable toolbox to study gene regulatory networks with broad applicability.

The transcription factor Scl/Tal1 is a critical regulator of haematopoiesis and its precise spatiotemporal expression is necessary for proper developmental patterning in the mouse embryo. Several transcriptional cis-regulatory elements have been identified that determine the haematopoietic expression of Scl including the +19, -4 and +40 enhancers. Investigation of such regulatory elements allows determination of upstream regulators and assembly of TF cascades and networks.Assays currently used to assess putative haematopoietic enhancers include expression analysis of randomly integrated enhancer-linked reporter genes in transient F0 transgenic embryos and luciferase assays. However, transgenic embryos only provide qualitative data while luciferase assays are limited to population level analysis in non-developmental cell types. These assays therefore provide an incomplete picture of enhancer activity. The Scl+19 enhancer has been well characterised by these methods and others; it is sufficient to drive Scl expression during developmental haematopoiesis and contains a highly conserved Ets/Ets/Gata motif necessary for its activity. However, the cell type specificity and the quantitative effects of motif mutation in the Scl+19 during haematopoietic specification cannot be determined by current methods.We have developed a haematopoietic enhancer assay that utilises embryonic stem (ES) cell gene targeting for single copy integration of an enhancer-linked YFP reporter gene. Embryoid body differentiation of targeted ES cell lines provides an accurate model of haematopoietic specification and abrogates the need of transgenic mice. Flow cytometry of YFP and cell surface marker expression captures quantitative single cell data of enhancer activity and specificity. Gene targeting and analysis of mutated enhancers allows further dissection of their regulatory inputs.We will present use of this novel enhancer assay to define Scl+19 activity during haematopoietic specification and previously unidentified separable roles of the Ets and Gata motifs during this process. The transcription factor Scl/Tal1 is a critical regulator of haematopoiesis and its precise spatiotemporal expression is necessary for proper developmental patterning in the mouse embryo. Several transcriptional cis-regulatory elements have been identified that determine the haematopoietic expression of Scl including the +19, -4 and +40 enhancers. Investigation of such regulatory elements allows determination of upstream regulators and assembly of TF cascades and networks.Assays currently used to assess putative haematopoietic enhancers include expression analysis of randomly integrated enhancer-linked reporter genes in transient F0 transgenic embryos and luciferase assays. However, transgenic embryos only provide qualitative data while luciferase assays are limited to population level analysis in non-developmental cell types. These assays therefore provide an incomplete picture of enhancer activity. The Scl+19 enhancer has been well characterised by these methods and others; it is sufficient to drive Scl expression during developmental haematopoiesis and contains a highly conserved Ets/Ets/Gata motif necessary for its activity. However, the cell type specificity and the quantitative effects of motif mutation in the Scl+19 during haematopoietic specification cannot be determined by current methods.We have developed a haematopoietic enhancer assay that utilises embryonic stem (ES) cell gene targeting for single copy integration of an enhancer-linked YFP reporter gene. Embryoid body differentiation of targeted ES cell lines provides an accurate model of haematopoietic specification and abrogates the need of transgenic mice. Flow cytometry of YFP and cell surface marker expression captures quantitative single cell data of enhancer activity and specificity. Gene targeting and analysis of mutated enhancers allows further dissection of their regulatory inputs.We will present use of this novel enhancer assay to define Scl+19 activity during haematopoietic specification and previously unidentified separable roles of the Ets and Gata motifs during this process.

A key finding from early genomics research is the remarkable consistency in the number of protein-coding regions across diverse species. This has led many researchers to look to the cis-regulatory elements of genes as the fundamental influence behind evolving gene function and subsequent species diversification. Historically, since these elements are often located in vast intergenic and intronic regions of the genome, their identification has been recalcitrant. Now, with the deluge of whole-genome data from representatives of numerous eukaryotic lineages, various approaches have enabled us to begin to recognize features that characterize regulatory regions of the genome. Here we endeavour to collate these approaches in order to give an overview of the complexities involved in extrapolating regulatory signatures. The resource provided by the escalating richness of whole-genome datasets enables more sophisticated modelling of these regulatory signatures yet at the same time introduces increasing potential for noise. While we are only at the advent of making these discoveries, the next decade promises to be a very exciting and rewarding time for genome researchers.

Within the vertebrate lineage, a high proportion of duplicate genes have been retained after whole genome duplication (WGD) events. It has been proposed that many of these duplicate genes became indispensable because the ancestral gene function was divided between them. In addition, novel functions may have evolved, owing to changes in cis-regulatory elements. Functional analysis of the PAX2/5/8 gene subfamily appears to support at least the first part of this hypothesis. The collective role of these genes has been widely retained, but sub-functions have been differentially partitioned between the genes in different vertebrates. Conserved non-coding elements (CNEs) represent an interesting and readily identifiable class of putative cis-regulatory elements that have been conserved from fish to mammals, an evolutionary distance of 450 million years. Within the PAX2/5/8 gene subfamily, PAX2 is associated with the highest number of CNEs. An additional WGD experienced in the teleost lineage led to two copies of pax2, each of which retained a large proportion of these CNEs. Using a reporter gene assay in zebrafish embryos, we have exploited this rich collection of regulatory elements in order to determine whether duplicate CNEs have evolved different functions. Remarkably, we find that even highly conserved sequences exhibit more functional differences than similarities. We also discover that short flanking sequences can have a profound impact on CNE function. Therefore, if CNEs are to be used as candidate enhancers for transgenic studies or for multi-species comparative analyses, it is paramount that the CNEs are accurately delineated.

Whole genome duplication events are thought to have substantially contributed to organismal complexity, largely via divergent transcriptional regulation. Members of the vertebrate PAX2 , PAX5 and PAX8 gene subfamily derived from an ancient class of paired box genes and arose from such whole genome duplication events. These genes are critical in establishing the midbrain‐hindbrain boundary, specifying interneuron populations and for eye, ear and kidney development. Also PAX2 has adopted a unique role in pancreas development, whilst PAX5 is essential for early B‐cell differentiation. The contribution of PAX258 genes to their collective role has diverged across paralogues and the animal lineages, resulting in a complex wealth of literature. It is now timely to provide a comprehensive comparative overview of these genes and their ancient and divergent roles. We also discuss their fundamental place within gene regulatory networks and the likely influence of cis ‐regulatory elements over their differential roles during early animal development. Developmental Dynamics 238:2951–2974, 2009. © 2009 Wiley‐Liss, Inc.

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA.

Background: In vertebrates the “SONIC HEDGEHOG” signalling pathway has been implicated in cell-fate determination, proliferation and the patterning of many different cell types and organs. As the GLI family members (GLI1, GLI2 and GLI3) are key mediators of hedgehog morphogenetic signals, over the past couple of decades they have been extensively scrutinized by genetic, molecular and biochemical means. Thus, a great deal of information is currently available about the functional aspects of GLI proteins in various vertebrate species. To address the roles of GLI genes in diversifying the repertoire of the Hh signalling and deploying them for the vertebrate specifications, in this study we have examined the evolutionary patterns of vertebrate GLI sequences within and between species.Results: Phylogenetic tree analysis suggests that the vertebrate GLI1, GLI2 and GLI3 genes diverged after the separation of urochordates from vertebrates and before the tetrapods-bony fishes split. Lineage specific duplication events were also detected. Estimation of mode and strength of selection acting on GLI orthologs demonstrated that all members of the GLI gene family experienced more relaxed selection in teleost fish than in the mammalian lineage. Furthermore, the GLI1 gene appeared to have been exposed to different functional constraints in fish and tetrapod lineages, whilst a similar level of functional constraints on GLI2 and GLI3 was suggested by comparable average non-synonymous (Ka) substitutions across the lineages. A relative rate test suggested that the majority of the paralogous copies of the GLI family analyzed evolved with similar evolutionary rates except GLI1 which evolved at a significantly faster rate than its paralogous counterparts in tetrapods.Conclusions: Our analysis shows that sequence evolutionary patterns of GLI family members are largely correlated with the reported similarities and differences in the functionality of GLI proteins within and between the various vertebrate species. We propose that duplication and divergence of GLI genes has increased in the complexity of vertebrate body plan by recruiting the hedgehog signalling for the novel developmental tasks.

The zinc-finger transcription factor GLI3 is a key regulator of development, acting as a primary transducer of Sonic hedgehog (SHH) signaling in a combinatorial context dependent fashion controlling multiple patterning steps in different tissues/organs. A tight temporal and spatial control of gene expression is indispensable, however, cis-acting sequence elements regulating GLI3 expression have not yet been reported. We show that 11 ancient genomic DNA signatures, conserved from the pufferfish Takifugu (Fugu) rubripes to man, are distributed throughout the introns of human GLI3. They map within larger conserved non-coding elements (CNEs) that are found in the tetrapod lineage. Full length CNEs transiently transfected into human cell cultures acted as cell type specific enhancers of gene transcription. The regulatory potential of these elements is conserved and was exploited to direct tissue specific expression of a reporter gene in zebrafish embryos. Assays of deletion constructs revealed that the human-Fugu conserved sequences within the GLI3 intronic CNEs were essential but not sufficient for full-scale transcriptional activation. The enhancer activity of the CNEs is determined by a combinatorial effect of a core sequence conserved between human and teleosts (Fugu) and flanking tetrapod-specific sequences, suggesting that successive clustering of sequences with regulatory potential around an ancient, highly conserved nucleus might be a possible mechanism for the evolution of cis-acting regulatory elements.

The zinc-finger transcription factor GLI3 acts during vertebrate development in a combinatorial, context-dependent fashion as a primary transducer of sonic hedgehog (SHH) signaling. In humans, mutations affecting this key regulator of development are associated with GLI3-morphopathies, a group of congenital malformations in which forebrain and limb development are preferentially affected. We show that a non-coding element from intron two of GLI3, ultraconserved in mammals and highly conserved in the pufferfish Fugu, is a transcriptional enhancer. In transient transfection assays, it activates reporter gene transcription in human cell cultures expressing endogenous GLI3 but not in GLI3 negative cells. The identified enhancer element is predicted to contain conserved binding sites for transcription factors crucial for developmental steps in which GLI3 is involved. The regulatory potential of this element is conserved and was used to direct tissue-specific expression of a green fluorescent protein reporter gene in zebrafish embryos and of a beta-galactosidase reporter in transgenic mouse embryos. Time, location, and quantity of reporter gene expression are congruent with part of the pattern previously reported for endogenous GLI3 transcription.

Comparative genomics is currently one of the most popular approaches to study the regulatory architecture of vertebrate genomes. Fish-mammal genomic comparisons have proved powerful in identifying conserved non-coding elements likely to be distal cis-regulatory modules such as enhancers, silencers or insulators that control the expression of genes involved in the regulation of early development. The scientific community is showing increasing interest in characterizing the function, evolution and language of these sequences. Despite this, there remains little in the way of user-friendly access to a large dataset of such elements in conjunction with the analysis and the visualization tools needed to study them.