Remotely acting SMCHD1 gene regulatory elements: in silico prediction and identification of potential regulatory variants in patients with FSHD

Background: Facioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of the D4Z4 macro-satellite repeat on chromosome 4q35 (FSHD1) or mutations in the SMCHD1 gene (FSHD2). Recent studies have shown that the clinical manifestation of FSHD1 can be modified by mutations in the SMCHD1 gene within a given family. The absence of either D4Z4 contraction or SMCHD1 mutations in a small cohort of patients suggests that the disease could also be due to disruption of gene regulation. In this study, we postulated that mutations responsible for exerting a modifier effect on FSHD might reside within remotely acting regulatory elements that have the potential to interact at a distance with their cognate gene promoter via chromatin looping. To explore this postulate, genome-wide Hi-C data were used to identify genomic fragments displaying the strongest interaction with the SMCHD1 gene. These fragments were then narrowed down to shorter regions using ENCODE and FANTOM data on transcription factor binding sites and epigenetic marks characteristic of promoters, enhancers and silencers

Probing photo-ionization: Experiments on positive streamers in pure gasses and mixtures

Positive streamers are thought to propagate by photo-ionization whose parameters depend on the nitrogen:oxygen ratio. Therefore we study streamers in nitrogen with 20%, 0.2% and 0.01% oxygen and in pure nitrogen, as well as in pure oxygen and argon. Our new experimental set-up guarantees contamination of the pure gases to be well below 1 ppm. Streamers in oxygen are difficult to measure as they emit considerably less light in the sensitivity range of our fast ICCD camera than the other gasses. Streamers in pure nitrogen and in all nitrogen/oxygen mixtures look generally similar, but become somewhat thinner and branch more with decreasing oxygen content. In pure nitrogen the streamers can branch so much that they resemble feathers. This feature is even more pronounced in pure argon, with approximately 10^2 hair tips/cm^3 in the feathers at 200 mbar; this density could be interpreted as the free electron density creating avalanches towards the streamer stem. It is remarkable that the streamer velocity is essentially the same for similar voltage and pressure in all nitrogen/oxygen mixtures as well as in pure nitrogen, while the oxygen concentration and therefore the photo-ionization lengths vary by more than five orders of magnitude. Streamers in argon have essentially the same velocity as well. The physical similarity of streamers at different pressures is confirmed in all gases; the minimal diameters are smaller than in earlier measurements.Comment: 28 pages, 14 figures. Major differences with v1: - appendix and spectra removed - subsection regarding effects of repetition frequency added - many more smaller change

Airway responsiveness after a single dose of salmeterol and during four months of treatment in children with asthma

Background: Inhalation of a single dose of the long-acting β2- adrenoceptor agonist salmeterol protects against methacholine-induced airway obstruction and other bronchoconstricting stimuli for at least 12 hours. Hypothetically, twice daily dosing of salmeterol may result in continuous protection. Objective: this study was designed to investigate the protective effect of a single dose of salmeterol and of continuous twice daily treatment on airway responsiveness to methacholine. Methods: In a double-blind, parallel study, salmeterol 50 μg twice daily was compared with salbutamol 200 μg twice daily. Thirty children with mild asthma, who had little or no bronchial obstruction and were hyperresponsive to methacholine (PD20 ≤ 150 μg) were allocated to receive either salmeterol or salbutamol. Airway responsiveness was measured before study entry, 12 hours after a single dose of drug was given, and monthly during 4 months of daily treatment. Measurements were always performed at the same time of the day, 12 hours after the last dose of medication was administered. Results: No significant differences in FEV1 were found between treatments at any time point. PD20 significantly increased after the first dose of salmeterol was given (geometric mean, 100 μg). Geometric mean PD20 values were significantly better during salmeterol treatment than during salbutamol treatment, 52 and 25 μg, respectively (p = 0.005). Conclusion: The protection provided by salmeterol during maintenance treatment was less than that provided after the first dose (p < 0.001). However, protection did not diminish during the 4- month treatment period and remained significant compared with baseline (p = 0.003)

Tomo-seq identifies SOX9 as a key regulator of cardiac fibrosis during ischemic injury

Background: Cardiac ischemic injury induces a pathological remodeling response, which can ultimately lead to heart failure. Detailed mechanistic insights into molecular signaling pathways relevant for different aspects of cardiac remodeling will support the identification of novel therapeutic targets. Methods: While genome-wide transcriptome analysis on diseased tissues has greatly advanced our understanding of the regulatory networks that drive pathological changes in the heart, this approach has been disadvantaged by the fact that the signals are derived from tissue homogenates. Here we used tomo-seq to obtain a genome-wide gene expression signature with high spatial resolution spanning from the infarcted area to the remote to identify new regulators of cardiac remodeling. Cardiac tissue samples from patients suffering from ischemic heart disease were used to validate our findings. Results: Tracing transcriptional differences with a high spatial resolution across the infarcted heart enabled us to identify gene clusters that share a comparable expression profile. The spatial distribution patterns indicated a separation of expressional changes for genes involved in specific aspects of cardiac remodeling, like fibrosis, cardiomyocyte hypertrophy, and calcium-handling (Col1a2, Nppa, and Serca2). Subsequent correlation analysis allowed for the identification of novel factors that share a comparable transcriptional regulation pattern across the infarcted tissue. The strong correlation between the expression levels of these known marker genes and the expression of the co-regulated genes could be confirmed in human ischemic cardiac tissue samples. Follow-up analysis identified SOX9 as common transcriptional regulator of a large portion of the fibrosis-related genes that become activated under conditions of ischemic injury. Lineage-tracing experiments indicated the majority of COL1-positive fibroblasts to stem from a pool of SOX9-expressing cells and in vivo loss of Sox9 blunted the cardiac fibrotic response upon ischemic injury. The co-localization between SOX9 and COL1 could also be confirmed in patients suffering from ischemic heart disease. Conclusions: Based on the exact local expression cues, tomo-seq can serve to reveal novel genes and key transcription factors involved in specific aspects of cardiac remodeling. Using tomo-seq we were able to unveil the unknown relevance of SOX9 as key regulator of cardiac fibrosis, pointing to SOX9 as potential therapeutic target for cardiac fibrosis

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors

Hominin-specific regulatory elements selectively emerged in oligodendrocytes and are disrupted in autism patients

Speciation is associated with substantial rewiring of the regulatory circuitry underlying the expression of genes. Determining which changes are relevant and underlie the emergence of the human brain or its unique susceptibility to neural disease has been challenging. Here we annotate changes to gene regulatory elements (GREs) at cell type resolution in the brains of multiple primate species spanning most of primate evolution. We identify a unique set of regulatory elements that emerged in hominins prior to the separation of humans and chimpanzees. We demonstrate that these hominin gains perferentially affect oligodendrocyte function postnatally and are preferentially affected in the brains of autism patients. This preference is also observed for human-specific GREs suggesting this system is under continued selective pressure. Our data provide a roadmap of regulatory rewiring across primate evolution providing insight into the genomic changes that underlie the emergence of the brain and its susceptibility to neural disease

Transcriptomic and Epigenomic Profiling of Histone Deacetylase Inhibitor Treatment Reveals Distinct Gene Regulation Profiles Leading to Impaired Neutrophil Development

The clinical use of histone deacetylase inhibitors (HDACi) for the treatment of bone marrow failure and hematopoietic malignancies has increased dramatically over the last decades. Nonetheless, their effects on normal myelopoiesis remain poorly evaluated. Here, we treated cord blood derived CD34+ progenitor cells with two chemically distinct HDACi inhibitors MS-275 or SAHA and analyzed their effects on the transcriptome (RNA-seq), epigenome (H3K27ac ChIP-seq) and functional and morphological characteristics during neutrophil development. MS-275 (entinostat) selectively inhibits class I HDACs, with a preference for HDAC1, while SAHA (vorinostat) is a non-selective class I/II HDACi. Treatment with individual HDACi resulted in both overlapping and distinct effects on both transcriptome and epigenome, whereas functional effects were relatively similar. Both HDACi resulted in reduced expansion and increased apoptosis in neutrophil progenitor cells. Morphologically, HDACi disrupted normal neutrophil differentiation what was illustrated by decreased percentages of mature neutrophils. In addition, while SAHA treatment clearly showed a block at the promyelocytic stage, MS-275 treatment was characterized by dysplastic features and skewing towards the monocytic lineage. These effects could be mimicked using shRNA-mediated knockdown of HDAC1. Taken together, our data provide novel insights into the effects of HDAC inhibition on normal hematopoietic cells during neutrophil differentiation. These findings should be taken into account when considering the clinical use of MS-275 and SAHA, and can be potentially utilized to tailor more specific, hematopoietic-directed HDACi in the future

Recently Evolved Enhancers Emerge with High Interindividual Variability and Less Frequently Associate with Disease

Mutations in non-coding regulatory DNA such as enhancers underlie a wide variety of diseases including developmental disorders and cancer. As enhancers rapidly evolve, understanding their function and configuration in non-human disease models can have important clinical applications. Here, we analyze enhancer configurations in tissues isolated from the common marmoset, a widely used primate model for human disease. Integrating these data with human and mouse data, we find that enhancers containing trait-associated variants are preferentially conserved. In contrast, most human-specific enhancers are highly variable between individuals, with a subset failing to contact promoters. These are located further away from genes and more often reside in inactive B-compartments. Our data show that enhancers typically emerge as instable elements with minimal biological impact prior to their integration in a transcriptional program. Furthermore, our data provide insight into which trait variations in enhancers can be faithfully modeled using the common marmoset

High-throughput mapping of regulatory DNA

Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9–based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ~40 kb of cis-regulatory genomic space and uses knock-in green fluorescent protein (GFP) reporters to read out gene activity. Using this approach, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. We identify proximal and distal regulatory elements necessary for expression of four embryonic stem cell–specific genes. We show a consistent contribution of neighboring gene promoters to gene expression and identify unmarked regulatory elements (UREs) that control gene expression but do not have typical enhancer epigenetic or chromatin features. We compare thousands of functional and nonfunctional genotypes at a genomic location and identify the base pair–resolution functional motifs of regulatory elements.National Institutes of Health (U.S.) (1U01HG007037