Genomic Regulatory Elements Research Articles

Enhancer hijacking: Innovative ways of carcinogenesis

Enhancer elements are specific DNA sequences that play a crucial role in regulating gene expression. Located upstream or downstream in the genomic context, enhancers enhance the transcription of linked genes. This is achieved by providing binding sites for transcription factors and other proteins, acting as a nucleation point that promotes the assembly of the transcriptional machinery. What makes enhancers unique is that located even thousands of base pairs away, they can exert their function. They can act over long distances, looping to interact with the promoter region of their target genes. Enhancers are often involved in cell type-specific gene expression as different cells express different sets of genes by activating and repressing cell type-specific enhancers. Cancer cells seem to co-opt this mechanism and hijack it for their own survival. In this process, an aberrant enhancer element activates the transcription of oncogenes (genes that have the potential to cause cancer) due to some alterations in genomic structure or regulatory elements that happen in cancer, contributing to tumorigenesis and cancer progression. Enhancer hijacking often occurs through complex genomic rearrangements such as chromosomal translocations and other structural rearrangements such as deletions or amplifications.8 These alterations can bring a usually distant, far-located enhancer into proximity with an oncogene, leading to its inappropriate activation. This is often seen in various cancers, including leukemias and solid tumors. Enhancer hijacking and its effects on gene regulation in cancer are frequently studied using methods such as RNA sequencing, chromatin conformation capture, and CRISPR-Cas9. Finding an enhancer hijacking event may be used to inform treatment choices, especially in precision medicine settings, and may also function as a biomarker for particular cancer types.

Lentiviral vectors for precise expression to treat X-linked lymphoproliferative disease

X-linked lymphoproliferative disease (XLP1) results from SH2D1A gene mutations affecting the SLAM-associated protein (SAP). A regulated lentiviral vector (LV), XLP-SMART LV, designed to express SAP at therapeutic levels in T, NK, and NKT cells, is crucial for effective gene therapy. We experimentally identified 34 genomic regulatory elements of the SH2D1A gene and designed XLP-SMART LVs to emulate the lineage and stage-specific control of SAP. We screened them for their on-target enhancer activity in T, NK, and NKT cells and their off-target enhancer activity in B cell and myeloid populations. In combination, three enhancer elements increased SAP promoter expression up to 4-fold in on-target populations invitro. NSG-Tg(Hu-IL15) xenograft studies with XLP-SMART LVs demonstrated up to 7-fold greater expression in on-target cells over a control EFS-LV, with no off-target expression. The XLP-SMART LVs exhibited stage-specific T and NK cell expression in peripheral blood, bone marrow, spleen, and thymic tissues (mimicking expression patterns of SAP). Transduction of XLP1 patient CD8+ Tcells or BM CD34+ cells with XLP-SMART LVs restored restimulation-induced cell death and NK cytotoxicity to wild-type levels, respectively. These data demonstrate that it is feasible to create a lineage and stage-specific LV to restore the XLP1 phenotype by gene therapy.

Transitions in chromatin conformation shaped by fatty acids and the circadian clock underlie hepatic transcriptional reorganization in obese mice

The circadian clock system coordinates metabolic, physiological, and behavioral functions across a 24-h cycle, crucial for adapting to environmental changes. Disruptions in circadian rhythms contribute to major metabolic pathologies like obesity and Type 2 diabetes. Understanding the regulatory mechanisms governing circadian control is vital for identifying therapeutic targets. It is well characterized that chromatin remodeling and 3D structure at genome regulatory elements contributes to circadian transcriptional cycles; yet the impact of rhythmic chromatin topology in metabolic disease is largely unexplored. In this study, we explore how the spatial configuration of the genome adapts to diet, rewiring circadian transcription and contributing to dysfunctional metabolism. We describe daily fluctuations in chromatin contacts between distal regulatory elements of metabolic control genes in livers from lean and obese mice and identify specific lipid-responsive regions recruiting the clock molecular machinery. Interestingly, under high-fat feeding, a distinct interactome for the clock-controlled gene Dbp strategically promotes the expression of distal metabolic genes including Fgf21. Alongside, new chromatin loops between regulatory elements from genes involved in lipid metabolism control contribute to their transcriptional activation. These enhancers are responsive to lipids through CEBPβ, counteracting the circadian repressor REVERBa. Our findings highlight the intricate coupling of circadian gene expression to a dynamic nuclear environment under high-fat feeding, supporting a temporally regulated program of gene expression and transcriptional adaptation to diet.

Predicting regulatory mutations and their target genes by new computational integrative analysis: A study of follicular lymphoma

Mutations in DNA regulatory regions are increasingly being recognized as important drivers of cancer and other complex diseases. These mutations can regulate gene expression by affecting DNA-protein binding and epigenetic profiles, such as DNA methylation in genome regulatory elements. However, identifying mutation hotspots associated with expression regulation and disease progression in non-coding DNA remains a challenge. Unlike most existing approaches that assign a mutation score to individual single nucleotide polymorphisms (SNP), a mutation block (MB)-based approach was introduced in this study to assess the collective impact of a cluster of SNPs on transcription factor-DNA binding affinity, differential gene expression (DEG), and nearby DNA methylation. Moreover, the long-distance target genes of functional MBs were identified using a new permutation-based algorithm that assessed the significance of correlations between DNA methylation at regulatory regions and target gene expression. Two new Python packages were developed. The Differential Methylation Region (DMR-analysis) analysis tool was used to detect DMR and map them to regulatory elements. The second tool, an integrated DMR, DEG, and SNP analysis tool (DDS-analysis), was used to combine the omics data to identify functional MBs and long-distance target genes. Both tools were validated in follicular lymphoma (FL) cohorts, where not only known functional MBs and their target genes (BCL2 and BCL6) were recovered, but also novel genes were found, including CDCA4 and JAG2, which may be associated with FL development. These genes are linked to target gene expression and are significantly correlated with the methylation of nearby DNA sequences in FL. The proposed computational integrative analysis of multiomics data holds promise for identifying regulatory mutations in cancer and other complex diseases.

Genetic Implication of Prenatal GABAergic and Cholinergic Neuron Development in Susceptibility to Schizophrenia.

The ganglionic eminences (GE) are fetal-specific structures that give rise to gamma-aminobutyric acid (GABA)- and acetylcholine-releasing neurons of the forebrain. Given the evidence for GABAergic, cholinergic, and neurodevelopmental disturbances in schizophrenia, we tested the potential involvement of GE neuron development in mediating genetic risk for the condition. We combined data from a recent large-scale genome-wide association study of schizophrenia with single-cell RNA sequencing data from the human GE to test the enrichment of schizophrenia risk variation in genes with high expression specificity for developing GE cell populations. We additionally performed the single nuclei Assay for Transposase-Accessible Chromatin with Sequencing (snATAC-Seq) to map potential regulatory genomic regions operating in individual cell populations of the human GE, using these to test for enrichment of schizophrenia common genetic variant liability and to functionally annotate non-coding variants-associated with the disorder. Schizophrenia common variant liability was enriched in genes with high expression specificity for developing neuron populations that are predicted to form dopamine D1 and D2 receptor-expressing GABAergic medium spiny neurons of the striatum, cortical somatostatin-positive GABAergic interneurons, calretinin-positive GABAergic neurons, and cholinergic neurons. Consistent with these findings, schizophrenia genetic risk was concentrated in predicted regulatory genomic sequence mapped in developing neuronal populations of the GE. Our study implicates prenatal development of specific populations of GABAergic and cholinergic neurons in later susceptibility to schizophrenia, and provides a map of predicted regulatory genomic elements operating in cells of the GE.

Development of a New Model System to Study Long-Distance Interactions Supported by Architectural Proteins.

Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems. Drosophila became the first model organism in which architectural proteins that determine the activity of insulators were described. In Drosophila, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and attP-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in cis and trans only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer-promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the D. melanogaster model.

MNESEDA: A prior-guided subgraph representation learning framework for predicting disease-related enhancers

Enhancers, as a class of functional genomic regulatory elements, activate transcription of their target genes and play significant roles in the pathogenesis of complex human diseases. Therefore, identifying associations between enhancers and diseases could help to illuminate the potential disease pathogenesis mechanism. However, current methods for identifying disease-related enhancers mainly focus on biological experiments, which are time-consuming and cost-intensive. It is crucial to develop an effective computational model for detecting novel enhancer-disease associations (EDAs). To this end, we propose a prior-guided maximum neighbor enclosing subgraph (MNES) representation learning framework named MNESEDA, for enhancer-disease association prediction, where maximum connected motif (MCM) discovery is a key ingredient that constrains the subsequent MNES extraction strategy. The proposed residual information fusion captures representation difference between motif subgraphs and alleviates the over-smoothing issue. Extensive experiments conducted on three datasets demonstrate the superior performance of MNESEDA over recent state-of-the-art methods and the satisfactory generalization and transfer ability. Further case studies exhibit the potential ability of MNESEDA to predict novel disease-related enhancers. Overall, MNESEDA could serve as an effective guide to elucidate pathogenesis and etiology of human diseases and decipher potential disease biomarkers.

Nuclease-free precise genome editing corrects MECP2 mutations associated with Rett syndrome.

Rett syndrome is an acquired progressive neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene which encodes a pleiotropic protein that functions as a global transcriptional regulator and a chromatin modifier. Rett syndrome predominantly affects heterozygous females while affected male hemizygotes rarely survive. Gene therapy of Rett syndrome has proven challenging due to a requirement for stringent regulation of expression with either over- or under-expression being toxic. Ectopic expression of MECP2 in conjunction with regulatory miRNA target sequences has achieved some success, but the durability of this approach remains unknown. Here we evaluated a nuclease-free homologous recombination (HR)-based genome editing strategy to correct mutations in the MECP2 gene. The stem cell-derived AAVHSCs have previously been shown to mediate seamless and precise HR-based genome editing. We tested the ability of HR-based genome editing to correct pathogenic mutations in Exons 3 and 4 of the MECP2 gene and restore the wild type sequence while preserving all native genomic regulatory elements associated with MECP2 expression, thus potentially addressing a significant issue in gene therapy for Rett syndrome. Moreover, since the mutations are edited directly at the level of the genome, the corrections are expected to be durable with progeny cells inheriting the edited gene. The AAVHSC MECP2 editing vector was designed to be fully homologous to the target MECP2 region and to insert a promoterless Venus reporter at the end of Exon 4. Evaluation of AAVHSC editing in a panel of Rett cell lines bearing mutations in Exons 3 and 4 demonstrated successful correction and rescue of expression of the edited MECP2 gene. Sequence analysis of edited Rett cells revealed successful and accurate correction of mutations in both Exons 3 and 4 and permitted mapping of HR crossover events. Successful correction was observed only when the mutations were flanked at both the 5' and 3' ends by crossover events, but not when both crossovers occurred either exclusively upstream or downstream of the mutation. Importantly, we concluded that pathogenic mutations were successfully corrected in every Rett line analyzed, demonstrating the therapeutic potential of HR-based genome editing.

HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies.

HIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during the "host phase" and the viral TF Tat during the "viral phase", which together facilitate the positive feedback loop required for exponential transcription, replication, and pathogenesis. The sequential action of these TFs poses a challenge to precisely delineate the contributions of the host and viral phases of the transcriptional program to guide future mechanistic and therapeutic studies. To address this limitation, we devised a genome engineering approach to mutate tat and create a genetically matched pair of Jurkat T cell clones harboring HIV-1 at the same integration site with and without Tat expression. By comparing the transcriptional profile of both clones, the transition point between the host and viral phases was defined, providing a system that enables the temporal mechanistic interrogation of HIV-1 transcription prior to and after Tat synthesis. Importantly, this CRISPR method is broadly applicable to knockout individual viral proteins or genomic regulatory elements to delineate their contributions to various aspects of the viral life cycle and ultimately may facilitate therapeutic approaches in our race towards achieving a functional cure.

PigBiobank: a valuable resource for understanding genetic and biological mechanisms of diverse complex traits in pigs.

To fully unlock the potential of pigs as both agricultural species for animal-based protein food and biomedical models for human biology and disease, a comprehensive understanding of molecular and cellular mechanisms underlying various complex phenotypes in pigs and how the findings can be translated to other species, especially humans, are urgently needed. Here, within the Farm animal Genotype-Tissue Expression (FarmGTEx) project, we build the PigBiobank (http://pigbiobank.farmgtex.org) to systematically investigate the relationships among genomic variants, regulatory elements, genes, molecular networks, tissues and complex traits in pigs. This first version of the PigBiobank curates 71885 pigs with both genotypes and phenotypes from over 100 pig breeds worldwide, covering 264 distinct complex traits. The PigBiobank has the following functions: (i) imputed sequence-based genotype-phenotype associations via a standardized and uniform pipeline, (ii) molecular and cellular mechanisms underlying trait-associations via integrating multi-omics data, (iii) cross-species gene mapping of complex traits via transcriptome-wide association studies, and (iv) high-quality results display and visualization. The PigBiobank will be updated timely with the development of the FarmGTEx-PigGTEx project, serving as an open-access and easy-to-use resource for genetically and biologically dissecting complex traits in pigs and translating the findings to other species.

Characterization of transcriptional enhancers in the chicken genome using CRISPR-mediated activation

DNA regulatory elements intricately control when, where, and how genes are activated. Therefore, understanding the function of these elements could unveil the complexity of the genetic regulation network. Genome-wide significant variants are predominantly found in non-coding regions of DNA, so comprehending the predicted functional regulatory elements is crucial for understanding the biological context of these genomic markers, which can be incorporated into breeding programs. The emergence of CRISPR technology has provided a powerful tool for studying non-coding regulatory elements in genomes. In this study, we leveraged epigenetic data from the Functional Annotation of Animal Genomes project to identify promoter and putative enhancer regions associated with three genes (HBBA, IRF7, and PPARG) in the chicken genome. To identify the enhancer regions, we designed guide RNAs targeting the promoter and candidate enhancer regions and utilized CRISPR activation (CRISPRa) with dCas9-p300 and dCas9-VPR as transcriptional activators in chicken DF-1 cells. By comparing the expression levels of target genes between the promoter activation and the co-activation of the promoter and putative enhancers, we were able to identify functional enhancers that exhibited augmented upregulation. In conclusion, our findings demonstrate the remarkable efficiency of CRISPRa in precisely manipulating the expression of endogenous genes by targeting regulatory elements in the chicken genome, highlighting its potential for functional validation of non-coding regions.

Wide-scale identification of novel/eliminated genes responsible for evolutionary transformations

BackgroundIt is generally accepted that most evolutionary transformations at the phenotype level are associated either with rearrangements of genomic regulatory elements, which control the activity of gene networks, or with changes in the amino acid contents of proteins. Recently, evidence has accumulated that significant evolutionary transformations could also be associated with the loss/emergence of whole genes. The targeted identification of such genes is a challenging problem for both bioinformatics and evo-devo research.ResultsTo solve this problem we propose the WINEGRET method, named after the first letters of the title. Its main idea is to search for genes that satisfy two requirements: first, the desired genes were lost/emerged at the same evolutionary stage at which the phenotypic trait of interest was lost/emerged, and second, the expression of these genes changes significantly during the development of the trait of interest in the model organism. To verify the first requirement, we do not use existing databases of orthologs, but rely purely on gene homology and local synteny by using some novel quickly computable conditions. Genes satisfying the second requirement are found by deep RNA sequencing. As a proof of principle, we used our method to find genes absent in extant amniotes (reptiles, birds, mammals) but present in anamniotes (fish and amphibians), in which these genes are involved in the regeneration of large body appendages. As a result, 57 genes were identified. For three of them, c-c motif chemokine 4, eotaxin-like, and a previously unknown gene called here sod4, essential roles for tail regeneration were demonstrated. Noteworthy, we established that the latter gene belongs to a novel family of Cu/Zn-superoxide dismutases lost by amniotes, SOD4.ConclusionsWe present a method for targeted identification of genes whose loss/emergence in evolution could be associated with the loss/emergence of a phenotypic trait of interest. In a proof-of-principle study, we identified genes absent in amniotes that participate in body appendage regeneration in anamniotes. Our method provides a wide range of opportunities for studying the relationship between the loss/emergence of phenotypic traits and the loss/emergence of specific genes in evolution.

Development of an Expression Vector Engineering Strategy Based on tDNA Insulator Element for the Stable Expression of Vascular Endothelial Growth Factor Receptor-Fc Fusion Protein.

During the past decades, tremendous advances have occurred in manufacturing recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. Nevertheless, the production of stable high-producing cell lines has remained a major obstacle in the development process of the CHO cell line. It has been shown that genomic regulatory elements can promote cell line development efficiency by improving transgenes' productivity and stability. Such elements include insulators, ubiquitous chromatin opening elements, scaffold/matrix attachment regions, and antirepressors. In addition, tDNA elements are shown to act as insulators in mammalian cells. This study examines the effect of the tDNA insulator on stable expression of a vascular endothelial growth factor receptor-Fc fusion protein.

Chromosome conformation capture technologies as tools to detect structural variations and their repercussion in chromatin 3D configuration.

3D genome organization regulates gene expression in different physiological and pathological contexts. Characterization of chromatin structure at different scales has provided information about how the genome organizes in the nuclear space, from chromosome territories, compartments of euchromatin and heterochromatin, topologically associated domains to punctual chromatin loops between genomic regulatory elements and gene promoters. In recent years, chromosome conformation capture technologies have also been used to characterize structural variations (SVs) de novo in pathological conditions. The study of SVs in cancer, has brought information about transcriptional misregulation that relates directly to the incidence and prognosis of the disease. For example, gene fusions have been discovered arising from chromosomal translocations that upregulate oncogenes expression, and other types of SVs have been described that alter large genomic regions encompassing many genes. However, studying SVs in 2D cannot capture all their regulatory implications in the genome. Recently, several bioinformatic tools have been developed to identify and classify SVs from chromosome conformation capture data and clarify how they impact chromatin structure in 3D, resulting in transcriptional misregulation. Here, we review recent literature concerning bioinformatic tools to characterize SVs from chromosome conformation capture technologies and exemplify their vast potential to rebuild the 3D landscape of genomes in cancer. The study of SVs from the 3D perspective can produce essential information about drivers, molecular targets, and disease evolution.

Identification of genomic loci regulating platelet plasminogen activator inhibitor-1 in mice

BackgroundPlasminogen activator inhibitor-1 (PAI-1, Serpine1) is an important circulating fibrinolysis inhibitor. PAI-1 exists in 2 pools, packaged within platelet α-granules and freely circulating in plasma. Elevated plasma PAI-1 levels are associated with cardiovascular disease. However, little is known about the regulation of platelet PAI-1 (pPAI-1). ObjectivesWe investigated the genetic control of pPAI-1 levels in mice and humans. MethodsWe measured pPAI-1 antigen levels via enzyme-linked immunosorbent assay in platelets isolated from 10 inbred mouse strains, including LEWES/EiJ (LEWES) and C57BL/6J (B6). LEWES and B6 were crossed to produce the F1 generation, B6LEWESF1. B6LEWESF1 mice were intercrossed to produce B6LEWESF2 mice. These mice were subjected to genome-wide genetic marker genotyping followed by quantitative trait locus analysis to identify pPAI-1 regulatory loci. ResultsWe identified differences in pPAI-1 between several laboratory strains, with LEWES having pPAI-1 levels more than 10-fold higher than those in B6. Quantitative trait locus analysis of B6LEWESF2 offspring identified a major pPAI-1 regulatory locus on chromosome 5 from 136.1 to 137.6 Mb (logarithm of the odds score, 16.2). Significant pPAI-1 modifier loci on chromosomes 6 and 13 were also identified. ConclusionIdentification of pPAI-1 genomic regulatory elements provides insights into platelet/megakaryocyte-specific and cell type–specific gene expression. This information can be used to design more precise therapeutic targets for diseases where PAI-1 plays a role.

Automatic landmarking identifies new loci associated with face morphology and implicates Neanderthal introgression in human nasal shape

We report a genome-wide association study of facial features in >6000 Latin Americans based on automatic landmarking of 2D portraits and testing for association with inter-landmark distances. We detected significant associations (P-value <5 × 10−8) at 42 genome regions, nine of which have been previously reported. In follow-up analyses, 26 of the 33 novel regions replicate in East Asians, Europeans, or Africans, and one mouse homologous region influences craniofacial morphology in mice. The novel region in 1q32.3 shows introgression from Neanderthals and we find that the introgressed tract increases nasal height (consistent with the differentiation between Neanderthals and modern humans). Novel regions include candidate genes and genome regulatory elements previously implicated in craniofacial development, and show preferential transcription in cranial neural crest cells. The automated approach used here should simplify the collection of large study samples from across the world, facilitating a cosmopolitan characterization of the genetics of facial features.

BMI-1 repression of the CD8 +T cell effector program is essential for memory formation

Abstract NaïveCD8 +T cell activation results in a program of proliferation and differentiation that results in large scale changes in phenotype and function that is critical for pathogen control and establishment of immunological memory. While these changes are underpinned by structural and biochemical changes in genome regulatory elements, the mechanisms that mediate these changes are not fully elucidated. Polycomb Repressive complexes (PRC) are chromatin remodeling complexes implicated in silencing genes encoding crucial developmental regulators. We demonstrated that upregulation of PRC1 components occurs rapidly upon naïve CD8 +T cell activation and was associated with maintenance of ubiquitination at key effector transcriptional regulators. Importantly, in vitro activation of PRC1-deficient CD8 +T cells resulted in greater proliferation and more rapid acquisition of effector function compared to wildtype cells. Use of two different viral infection models demonstrated that T cell specific PRC1 deficiency resulted in a greater effector expansion and effector differentiation with establishment of dysfunctional memory T cell populations. Strikingly, even after acute infection, the resulting memory T cells exhibited phenotypic and molecular markers of T cell exhaustion and were unable to be recalled. Our data suggest that PRC1 regulation early after CD8 +T cell activation is essential for restraining early effector T cell commitment and is a key step in establishing immunological CD8 +T cell memory. Supported by grants from National Health and Medical Research Council of Australia (Ideas Grant 2020562).

Massively Parallel Reporter Assays for High-Throughput In Vivo Analysis of Cis-Regulatory Elements.

The rapid improvement of descriptive genomic technologies has fueled a dramatic increase in hypothesized connections between cardiovascular gene expression and phenotypes. However, in vivo testing of these hypotheses has predominantly been relegated to slow, expensive, and linear generation of genetically modified mice. In the study of genomic cis-regulatory elements, generation of mice featuring transgenic reporters or cis-regulatory element knockout remains the standard approach. While the data obtained is of high quality, the approach is insufficient to keep pace with candidate identification and therefore results in biases introduced during the selection of candidates for validation. However, recent advances across a range of disciplines are converging to enable functional genomic assays that can be conducted in a high-throughput manner. Here, we review one such method, massively parallel reporter assays (MPRAs), in which the activities of thousands of candidate genomic regulatory elements are simultaneously assessed via the next-generation sequencing of a barcoded reporter transcript. We discuss best practices for MPRA design and use, with a focus on practical considerations, and review how this emerging technology has been successfully deployed in vivo. Finally, we discuss how MPRAs are likely to evolve and be used in future cardiovascular research.

Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus

Sox2 expression in mouse embryonic stem cells (mESCs) depends on a distal cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of interdependence remain a mystery. We analyzed the endogenous Sox2 locus using Big-IN to scarlessly integrate large DNA payloads incorporating deletions, rearrangements, and inversions affecting single or multiple DHSs, as well as surgical alterations to transcription factor (TF) recognition sequences. Multiple mESC clones were derived for each payload, sequence-verified, and analyzed for Sox2 expression. We found that two DHSs comprising a handful of key TF recognition sequences were each sufficient for long-range activation of Sox2 expression. By contrast, three nearby DHSs were entirely context dependent, showing no activity alone but dramatically augmenting the activity of the autonomous DHSs. Our results highlight the role of context in modulating genomic regulatory element function, and our synthetic regulatory genomics approach provides a roadmap for the dissection of other genomic loci.

A Kaleidoscope of Keratin Gene Expression and the Mosaic of Its Regulatory Mechanisms

Keratins are a family of intermediate filament-forming proteins highly specific to epithelial cells. A combination of expressed keratin genes is a defining property of the epithelium belonging to a certain type, organ/tissue, cell differentiation potential, and at normal or pathological conditions. In a variety of processes such as differentiation and maturation, as well as during acute or chronic injury and malignant transformation, keratin expression undergoes switching: an initial keratin profile changes accordingly to changed cell functions and location within a tissue as well as other parameters of cellular phenotype and physiology. Tight control of keratin expression implies the presence of complex regulatory landscapes within the keratin gene loci. Here, we highlight patterns of keratin expression in different biological conditions and summarize disparate data on mechanisms controlling keratin expression at the level of genomic regulatory elements, transcription factors (TFs), and chromatin spatial structure.