Illuminating Epigenetics and Inheritance in the Immune System with Bioluminescence.

The remarkable process of light emission by living organisms has fascinated mankind for thousands of years. A recent expansion in the repertoire of catalytic luciferase enzymes, coupled with the discovery of the genes and pathways that encode different luciferin substrates, means that bioluminescence imaging (BLI) is set to revolutionize longitudinal and dynamic studies of gene control within biomedicine, including the regulation of immune responses. In this review article, we summarize recent advances in bioluminescence-based imaging approaches that promise to enlighten our understanding of in vivo gene and epigenetic control within the immune system.

Allele-specific control of replication timing and genome organization during development.

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus × castaneus mouse crosses and exploited the high single-nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (total nuclear RNA-seq), and chromatin accessibility (ATAC-seq). We also present HARP, a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of six different hybrid mESC clones with different genomes (C57BL/6, 129/sv, and CAST/Ei), parental configurations, and gender revealed significant RT asynchrony between alleles across ∼12% of the autosomal genome linked to subspecies genomes but not to parental origin, growth conditions, or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not as strongly with SNP density, gene expression, imprinting, or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types, including extraembryonic endoderm stem (XEN) cells, four male and female primary mouse embryonic fibroblasts (MEFs), and neural precursor cells (NPCs) differentiated in vitro from mESCs with opposite parental configurations. We found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs were largely lost in all differentiated cell types, accompanied by novel sites of allelic asynchrony at a considerably smaller proportion of the genome, suggesting that genome organization of homologs converges to similar folding patterns during cell fate commitment.

The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.

Bioluminescence imaging of Cyp1a1-luciferase reporter mice demonstrates prolonged activation of the aryl hydrocarbon receptor in the lung.

Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo.

Protein restriction during pregnancy alters Cdkn1c silencing, dopamine circuitry and offspring behaviour without changing expression of key neuronal marker genes.

We tracked the consequences of in utero protein restriction in mice throughout their development and life course using a luciferase-based allelic reporter of imprinted Cdkn1c. Exposure to gestational low-protein diet (LPD) results in the inappropriate expression of paternally inherited Cdkn1c in the brains of embryonic and juvenile mice. These animals were characterised by a developmental delay in motor skills, and by behavioural alterations indicative of reduced anxiety. Exposure to LPD in utero resulted in significantly more tyrosine hydroxylase positive (dopaminergic) neurons in the midbrain of adult offspring as compared to age-matched, control-diet equivalents. Positron emission tomography (PET) imaging revealed an increase in striatal dopamine synthesis capacity in LPD-exposed offspring, where elevated levels of dopamine correlated with an enhanced sensitivity to cocaine. These data highlight a profound sensitivity of the developing epigenome to gestational protein restriction. Our data also suggest that loss of Cdkn1c imprinting and p57KIP2 upregulation alters the cellular composition of the developing midbrain, compromises dopamine circuitry, and thereby provokes behavioural abnormalities in early postnatal life. Molecular analyses showed that despite this phenotype, exposure to LPD solely during pregnancy did not significantly change the expression of key neuronal- or dopamine-associated marker genes in adult offspring.

Loss of H3K9 trimethylation alters chromosome compaction and transcription factor retention during mitosis.

Recent studies have shown that repressive chromatin machinery, including DNA methyltransferases and polycomb repressor complexes, binds to chromosomes throughout mitosis and their depletion results in increased chromosome size. In the present study, we show that enzymes that catalyze H3K9 methylation, such as Suv39h1, Suv39h2, G9a and Glp, are also retained on mitotic chromosomes. Surprisingly, however, mutants lacking histone 3 lysine 9 trimethylation (H3K9me3) have unusually small and compact mitotic chromosomes associated with increased histone H3 phospho Ser10 (H3S10ph) and H3K27me3 levels. Chromosome size and centromere compaction in these mutants were rescued by providing exogenous first protein lysine methyltransferase Suv39h1 or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wild-type versus Suv39h1/Suv39h2 double-null mouse embryonic stem cells revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.

Endogenous bioluminescent reporters reveal a sustained increase in utrophin gene expression upon EZH2 and ERK1/2 inhibition.

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by loss of function mutations in the dystrophin gene (Dmd), resulting in progressive muscle weakening. Here we modelled the longitudinal expression of endogenous Dmd, and its paralogue Utrn, in mice and in myoblasts by generating bespoke bioluminescent gene reporters. As utrophin can partially compensate for Dmd-deficiency, these reporters were used as tools to ask whether chromatin-modifying drugs can enhance Utrn expression in developing muscle. Myoblasts treated with different PRC2 inhibitors showed significant increases in Utrn transcripts and bioluminescent signals, and these responses were independently verified by conditional Ezh2 deletion. Inhibition of ERK1/2 signalling provoked an additional increase in Utrn expression that was also seen in Dmd-mutant cells, and maintained as myoblasts differentiate. These data reveal PRC2 and ERK1/2 to be negative regulators of Utrn expression and provide specialised molecular imaging tools to monitor utrophin expression as a therapeutic strategy for DMD.

Drug-induced loss of imprinting revealed using bioluminescent reporters of Cdkn1c.

Genomic imprinting is an epigenetically mediated mechanism that regulates allelic expression of genes based upon parent-of-origin and provides a paradigm for studying epigenetic silencing and release. Here, bioluminescent reporters for the maternally-expressed imprinted gene Cdkn1c are used to examine the capacity of chromatin-modifying drugs to reverse paternal Cdkn1c silencing. Exposure of reporter mouse embryonic stem cells (mESCs) to 5-Azacytidine, HDAC inhibitors, BET inhibitors or GSK-J4 (KDM6A/B inhibitor) relieved repression of paternal Cdkn1c, either selectively or by inducing biallelic effects. Treatment of reporter fibroblasts with HDAC inhibitors or GSK-J4 resulted in similar paternal Cdkn1c activation, whereas BET inhibitor-induced loss of imprinting was specific to mESCs. Changes in allelic expression were generally not sustained in dividing cultures upon drug removal, indicating that the underlying epigenetic memory of silencing was maintained. In contrast, Cdkn1c de-repression by GSK-J4 was retained in both mESCs and fibroblasts following inhibitor removal, although this impact may be linked to cellular stress and DNA damage. Taken together, these data introduce bioluminescent reporter cells as tools for studying epigenetic silencing and disruption, and demonstrate that Cdkn1c imprinting requires distinct and cell-type specific chromatin features and modifying enzymes to enact and propagate a memory of silencing.

Epigenetic changes induced by in utero dietary challenge result in phenotypic variability in successive generations of mice.

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.

Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction.

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.

Associations of perceived stress and state anger with symptom burden and functional status in patients with heart failure.

Background: Psychosocial stress and anger trigger cardiovascular events, but their relationship to heart failure (HF) exacerbations is unclear. We investigated perceived stress and anger associations with HF functional status and symptoms. Methods and Results: In a prospective cohort study (BETRHEART), 144 patients with HF (77% male; 57.5 ± 11.5 years) were evaluated for perceived stress (Perceived Stress Scale; PSS) and state anger (STAXI) at baseline and every 2 weeks for 3 months. Objective functional status (6-min walk test; 6MWT) and health status (Kansas City Cardiomyopathy Questionnaire; KCCQ) were also measured biweekly. Linear mixed model analyses indicated that average PSS and greater than usual increases in PSS were associated with worsened KCCQ scores. Greater than usual increases in PSS were associated with worsened 6MWT. Average anger levels were associated with worsened KCCQ, and increases in anger were associated with worsened 6MWT. Adjusting for PSS, anger associations were no longer statistically significant. Adjusting for anger, PSS associations with KCCQ and 6MWT remained significant. Conclusion: In patients with HF, both perceived stress and anger are associated with poorer functional and health status, but perceived stress is a stronger predictor. Negative effects of anger on HF functional status and health status may partly operate through psychological stress.

Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells.

BACKGROUND: Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice. RESULTS: Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging. CONCLUSIONS: Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors.

Visualizing Changes in Cdkn1c Expression Links Early-Life Adversity to Imprint Mis-regulation in Adults.

Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.

CHiCAGO: robust detection of DNA looping interactions in Capture Hi-C data.

Capture Hi-C (CHi-C) is a method for profiling chromosomal interactions involving targeted regions of interest, such as gene promoters, globally and at high resolution. Signal detection in CHi-C data involves a number of statistical challenges that are not observed when using other Hi-C-like techniques. We present a background model and algorithms for normalisation and multiple testing that are specifically adapted to CHi-C experiments. We implement these procedures in CHiCAGO ( http://regulatorygenomicsgroup.org/chicago ), an open-source package for robust interaction detection in CHi-C. We validate CHiCAGO by showing that promoter-interacting regions detected with this method are enriched for regulatory features and disease-associated SNPs.

The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex.

BACKGROUND: Chromatin-modifying complexes have key roles in regulating various aspects of neural stem cell biology, including self-renewal and neurogenesis. The methyl binding domain 3/nucleosome remodelling and deacetylation (MBD3/NuRD) co-repressor complex facilitates lineage commitment of pluripotent cells in early mouse embryos and is important for stem cell homeostasis in blood and skin, but its function in neurogenesis had not been described. Here, we show for the first time that MBD3/NuRD function is essential for normal neurogenesis in mice. RESULTS: Deletion of MBD3, a structural component of the NuRD complex, in the developing mouse central nervous system resulted in reduced cortical thickness, defects in the proper specification of cortical projection neuron subtypes and neonatal lethality. These phenotypes are due to alterations in PAX6+ apical progenitor cell outputs, as well as aberrant terminal neuronal differentiation programmes of cortical plate neurons. Normal numbers of PAX6+ apical neural progenitor cells were generated in the MBD3/NuRD-mutant cortex; however, the PAX6+ apical progenitor cells generate EOMES+ basal progenitor cells in reduced numbers. Cortical progenitor cells lacking MBD3/NuRD activity generate neurons that express both deep- and upper-layer markers. Using laser capture microdissection, gene expression profiling and chromatin immunoprecipitation, we provide evidence that MBD3/NuRD functions to control gene expression patterns during neural development. CONCLUSIONS: Our data suggest that although MBD3/NuRD is not required for neural stem cell lineage commitment, it is required to repress inappropriate transcription in both progenitor cells and neurons to facilitate appropriate cell lineage choice and differentiation programmes.

Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome.

The Polycomb repressive complexes PRC1 and PRC2 maintain embryonic stem cell (ESC) pluripotency by silencing lineage-specifying developmental regulator genes. Emerging evidence suggests that Polycomb complexes act through controlling spatial genome organization. We show that PRC1 functions as a master regulator of mouse ESC genome architecture by organizing genes in three-dimensional interaction networks. The strongest spatial network is composed of the four Hox gene clusters and early developmental transcription factor genes, the majority of which contact poised enhancers. Removal of Polycomb repression leads to disruption of promoter-promoter contacts in the Hox gene network. In contrast, promoter-enhancer contacts are maintained in the absence of Polycomb repression, with accompanying widespread acquisition of active chromatin signatures at network enhancers and pronounced transcriptional upregulation of network genes. Thus, PRC1 physically constrains developmental transcription factor genes and their enhancers in a silenced but poised spatial network. We propose that the selective release of genes from this spatial network underlies cell fate specification during early embryonic development.

A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element.

Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation by mechanisms that are poorly understood. Here we show that a switch in forward and reverse noncoding transcription from the Drosophila melanogaster vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both noncoding RNAs inhibit PRC2 histone methyltransferase activity, but, in vivo, only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. We propose that the interaction of RNAs with PRC2 is differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of noncoding RNAs occurs at several hundred Polycomb-binding sites in fly and vertebrate genomes. This work identifies a previously unreported and potentially widespread class of PRE/TREs that switch function by switching the direction of noncoding RNA transcription.

Impaired in vitro erythropoiesis following deletion of the Scl (Tal1) +40 enhancer is largely compensated for in vivo despite a significant reduction in expression.

The Scl (Tal1) gene encodes a helix-loop-helix transcription factor essential for hematopoietic stem cell and erythroid development. The Scl +40 enhancer is situated downstream of Map17, the 3' flanking gene of Scl, and is active in transgenic mice during primitive and definitive erythropoiesis. To analyze the in vivo function of the Scl +40 enhancer within the Scl/Map17 transcriptional domain, we deleted this element in the germ line. Scl(Δ40/Δ40) mice were viable with reduced numbers of erythroid CFU in both bone marrow and spleen yet displayed a normal response to stress hematopoiesis. Analysis of Scl(Δ40/Δ40) embryonic stem (ES) cells revealed impaired erythroid differentiation, which was accompanied by a failure to upregulate Scl when erythropoiesis was initiated. Map17 expression was also reduced in hematopoietic tissues and differentiating ES cells, and the Scl +40 element was able to enhance activity of the Map17 promoter. However, only Scl but not Map17 could rescue the Scl(Δ40/Δ40) ES phenotype. Together, these data demonstrate that the Scl +40 enhancer is an erythroid cell-specific enhancer that regulates the expression of both Scl and Map17. Moreover, deletion of the +40 enhancer causes a novel erythroid phenotype, which can be rescued by ectopic expression of Scl but not Map17.