Distal and proximal cis-regulatory elements sense X chromosome dosage and developmental state at the Xist locus.

Developmental genes such as Xist, which initiates X chromosome inactivation, are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatiotemporal expression patterns. Xist integrates information on X chromosome dosage and developmental stage to trigger X inactivation in the epiblast specifically in female embryos. Through a pooled CRISPR screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist at the onset of random X inactivation. Chromatin profiling reveals that X-dosage controls the promoter-proximal region, while differentiation cues activate several distal enhancers. The strongest distal element lies in an enhancer cluster associated with a previously unannotated Xist-enhancing regulatory transcript, which we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. With this study, we start to disentangle how multiple, functionally distinct regulatory elements interact to generate complex expression patterns in mammals.

Modeling unveils sex differences of signaling networks in mouse embryonic stem cells.

For a short period during early development of mammalian embryos, both X chromosomes in females are active, before dosage compensation is ensured through X-chromosome inactivation. In female mouse embryonic stem cells (mESCs), which carry two active X chromosomes, increased X-dosage affects cell signaling and impairs differentiation. The underlying mechanisms, however, remain poorly understood. To dissect X-dosage effects on the signaling network in mESCs, we combine systematic perturbation experiments with mathematical modeling. We quantify the response to a variety of inhibitors and growth factors for cells with one (XO) or two X chromosomes (XX). We then build models of the signaling networks in XX and XO cells through a semi-quantitative modeling approach based on modular response analysis. We identify a novel negative feedback in the PI3K/AKT pathway through GSK3. Moreover, the presence of a single active X makes mESCs more sensitive to the differentiation-promoting Activin A signal and leads to a stronger RAF1-mediated negative feedback in the FGF-triggered MAPK pathway. The differential response to these differentiation-promoting pathways can explain the impaired differentiation propensity of female mESCs.

Kinetics of Xist-induced gene silencing can be predicted from combinations of epigenetic and genomic features.

To initiate X-Chromosome inactivation (XCI), the long noncoding RNA Xist mediates chromosome-wide gene silencing of one X Chromosome in female mammals to equalize gene dosage between the sexes. The efficiency of gene silencing is highly variable across genes, with some genes even escaping XCI in somatic cells. A gene's susceptibility to Xist-mediated silencing appears to be determined by a complex interplay of epigenetic and genomic features; however, the underlying rules remain poorly understood. We have quantified chromosome-wide gene silencing kinetics at the level of the nascent transcriptome using allele-specific Precision nuclear Run-On sequencing (PRO-seq). We have developed a Random Forest machine-learning model that can predict the measured silencing dynamics based on a large set of epigenetic and genomic features and tested its predictive power experimentally. The genomic distance to the Xist locus, followed by gene density and distance to LINE elements, are the prime determinants of the speed of gene silencing. Moreover, we find two distinct gene classes associated with different silencing pathways: a class that requires Xist-repeat A for silencing, which is known to activate the SPEN pathway, and a second class in which genes are premarked by Polycomb complexes and tend to rely on the B repeat in Xist for silencing, known to recruit Polycomb complexes during XCI. Moreover, a series of features associated with active transcriptional elongation and chromatin 3D structure are enriched at rapidly silenced genes. Our machine-learning approach can thus uncover the complex combinatorial rules underlying gene silencing during X inactivation.

Phosphorylation of the chromatin remodeling factor DPF3a induces cardiac hypertrophy through releasing HEY repressors from DNA.

DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure.

Rare and private variations in neural crest, apoptosis and sarcomere genes define the polygenic background of isolated Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Its genetic basis is demonstrated by an increased recurrence risk in siblings and familial cases. However, the majority of TOF are sporadic, isolated cases of undefined origin and it had been postulated that rare and private autosomal variations in concert define its genetic basis. To elucidate this hypothesis, we performed a multilevel study using targeted re-sequencing and whole-transcriptome profiling. We developed a novel concept based on a gene's mutation frequency to unravel the polygenic origin of TOF. We show that isolated TOF is caused by a combination of deleterious private and rare mutations in genes essential for apoptosis and cell growth, the assembly of the sarcomere as well as for the neural crest and secondary heart field, the cellular basis of the right ventricle and its outflow tract. Affected genes coincide in an interaction network with significant disturbances in expression shared by cases with a mutually affected TOF gene. The majority of genes show continuous expression during adulthood, which opens a new route to understand the diversity in the long-term clinical outcome of TOF cases. Our findings demonstrate that TOF has a polygenic origin and that understanding the genetic basis can lead to novel diagnostic and therapeutic routes. Moreover, the novel concept of the gene mutation frequency is a versatile measure and can be applied to other open genetic disorders.

The cardiac transcription network modulated by Gata4, Mef2a, Nkx2.5, Srf, histone modifications, and microRNAs.

The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. We present a systems biology study integrating mRNA profiles with DNA-binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi-mediated knockdown. Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth. We provide insights into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, we present an Srf-centered transcription network providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, we show the combinatorial contribution of four DNA-binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders.

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of long-range enhancers.

X-chromosome inactivation (XCI) balances gene expression between the sexes in female mammals. Shortly after fertilization, upregulation of Xist RNA from one X chromosome initiates XCI, leading to chromosome-wide gene silencing. XCI is maintained in all cell types, except the germ line and the pluripotent state where XCI is reversed. The mechanisms triggering Xist upregulation have remained elusive. Here we identify GATA transcription factors as potent activators of Xist. Through a pooled CRISPR activation screen in murine embryonic stem cells, we demonstrate that GATA1, as well as other GATA transcription factors can drive ectopic Xist expression. Moreover, we describe GATA-responsive regulatory elements in the Xist locus bound by different GATA factors. Finally, we show that GATA factors are essential for XCI induction in mouse preimplantation embryos. Deletion of GATA1/4/6 or GATA-responsive Xist enhancers in mouse zygotes effectively prevents Xist upregulation. We propose that the activity or complete absence of various GATA family members controls initial Xist upregulation, XCI maintenance in extra-embryonic lineages and XCI reversal in the epiblast.

Epigenetic dynamics during capacitation of naïve human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some of the limitations of conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics of human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated hPSCs are transcriptionally closer to postimplantation epiblast than conventional hPSCs. This prompted us to comprehensively study epigenetic and related transcriptional changes during capacitation. Our results show that CpG islands, gene regulatory elements, and retrotransposons are hotspots of epigenetic dynamics during capacitation and indicate possible distinct roles of specific epigenetic modifications in gene expression control between naïve and primed hPSCs. Unexpectedly, PRC2 activity appeared to be dispensable for the capacitation. We find that capacitated hPSCs acquire an epigenetic state similar to conventional hPSCs. Significantly, however, the X chromosome erosion frequently observed in conventional female hPSCs is reversed by resetting and subsequent capacitation.

Evaluation of the LightCycler 1536 Instrument for high-throughput quantitative real-time PCR.

Quantitative real-time PCR (qPCR) is a frequently used, sensitive and accurate method to study gene expression profiles. However, its throughput was so far limited for routine laboratories to 384 reactions per run based on the limitations of the available instruments. Recently, the LightCycler 1536 Instrument was launched providing a high-throughput solution for qPCR with the analysis of 1536 reactions in approximately 45 min. We assessed the accuracy and sensitivity of this novel technology for the analysis of gene expression profiles in combination with the Innovadyne Nanodrop Express pipetting robot. We compared expression profiles obtained for 42 genes in 71 samples between the Universal ProbeLibrary and the LightCycler 1536 Instrument and SYBR Green I and the ABI PRISM 7900HT system. We found that the results were highly reproducible between both systems. Beside the higher throughput, the advantage of the LightCycler 1536 Instrument was the reduced consumption of reagents and sample material.

Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach.

BACKGROUND: X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signaling pathway, and delaying differentiation. RESULTS: To identify the genetic basis of these sex differences, we use a two-step CRISPR screening approach to comprehensively identify X-linked genes that cause the female pluripotency phenotype in murine embryonic stem cells. A primary chromosome-wide CRISPR knockout screen and three secondary screens assaying for different aspects of the female pluripotency phenotype allow us to uncover multiple genes that act in concert and to disentangle their relative roles. Among them, we identify Dusp9 and Klhl13 as two central players. While Dusp9 mainly affects MAPK pathway intermediates, Klhl13 promotes pluripotency factor expression and delays differentiation, with both factors jointly repressing MAPK target gene expression. CONCLUSIONS: Here, we elucidate the mechanisms that drive sex-induced differences in pluripotent cells and our approach serves as a blueprint to discover the genetic basis of the phenotypic consequences of other chromosomal effects.

Integrated analysis of Xist upregulation and X-chromosome inactivation with single-cell and single-allele resolution.

To ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is initiated during early development through upregulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and gene silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cell RNA-sequencing. Specifically, we assess the onset of random XCI in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify putative Xist regulators. Moreover, we show that transient Xist upregulation from both X chromosomes results in biallelic gene silencing right before transitioning to the monoallelic state, confirming a prediction of the stochastic model of XCI. Finally, we show that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known X-controlling element (Xce) effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

EPIGENE: genome-wide transcription unit annotation using a multivariate probabilistic model of histone modifications.

BACKGROUND: Understanding the transcriptome is critical for explaining the functional as well as regulatory roles of genomic regions. Current methods for the identification of transcription units (TUs) use RNA-seq that, however, require large quantities of mRNA rendering the identification of inherently unstable TUs, e.g. miRNA precursors, difficult. This problem can be alleviated by chromatin-based approaches due to a correlation between histone modifications and transcription. RESULTS: Here, we introduce EPIGENE, a novel chromatin segmentation method for the identification of active TUs using transcription-associated histone modifications. Unlike the existing chromatin segmentation approaches, EPIGENE uses a constrained, semi-supervised multivariate hidden Markov model (HMM) that models the observed combination of histone modifications using a product of independent Bernoulli random variables, to identify active TUs. Our results show that EPIGENE can identify genome-wide TUs in an unbiased manner. EPIGENE-predicted TUs show an enrichment of RNA Polymerase II at the transcription start site and in gene body indicating that they are indeed transcribed. Comprehensive validation using existing annotations revealed that 93% of EPIGENE TUs can be explained by existing gene annotations and 5% of EPIGENE TUs in HepG2 can be explained by microRNA annotations. EPIGENE outperformed the existing RNA-seq-based approaches in TU prediction precision across human cell lines. Finally, we identified 232 novel TUs in K562 and 43 novel cell-specific TUs all of which were supported by RNA Polymerase II ChIP-seq and Nascent RNA-seq data. CONCLUSION: We demonstrate the applicability of EPIGENE to identify genome-wide active TUs and to provide valuable information about unannotated TUs. EPIGENE is an open-source method and is freely available at: https://github.com/imbbLab/EPIGENE.

Altered microRNA and target gene expression related to Tetralogy of Fallot.

MicroRNAs (miRNAs) play an important role in guiding development and maintaining function of the human heart. Dysregulation of miRNAs has been linked to various congenital heart diseases including Tetralogy of Fallot (TOF), which represents the most common cyanotic heart malformation in humans. Several studies have identified dysregulated miRNAs in right ventricular (RV) tissues of TOF patients. In this study, we profiled genome-wide the whole transcriptome and analyzed the relationship of miRNAs and mRNAs of RV tissues of a homogeneous group of 22 non-syndromic TOF patients. Observed profiles were compared to profiles obtained from right and left ventricular tissue of normal hearts. To reduce the commonly observed large list of predicted target genes of dysregulated miRNAs, we applied a stringent target prediction pipeline integrating probabilities for miRNA-mRNA interaction. The final list of disease-related miRNA-mRNA pairs comprises novel as well as known miRNAs including miR-1 and miR-133, which are essential to cardiac development and function by regulating KCNJ2, FBN2, SLC38A3 and TNNI1. Overall, our study provides additional insights into post-transcriptional gene regulation of malformed hearts of TOF patients.

A symmetric toggle switch explains the onset of random X inactivation in different mammals.

Gene-regulatory networks control the establishment and maintenance of alternative gene-expression states during development. A particular challenge is the acquisition of opposing states by two copies of the same gene, as in the case of the long non-coding RNA Xist in mammals at the onset of random X-chromosome inactivation (XCI). The regulatory principles that lead to stable mono-allelic expression of Xist remain unknown. Here, we uncover the minimal regulatory network that can ensure female-specific and mono-alleleic upregulation of Xist, by combining mathematical modeling and experimental validation of central model predictions. We identify a symmetric toggle switch as the basis for random mono-allelic upregulation of Xist, which reproduces data from several mutant, aneuploid and polyploid mouse cell lines with various Xist expression patterns. Moreover, this toggle switch explains the diversity of strategies employed by different species at the onset of XCI. In addition to providing a unifying conceptual framework with which to explore XCI across mammals, our study sets the stage for identifying the molecular mechanisms needed to initiate random XCI.

Characterization of TBX20 in human hearts and its regulation by TFAP2.

The T-box family of transcription factors has been shown to have major impact on human development and disease. In animal studies Tbx20 is essential for the development of the atrioventricular channel, the outflow tract and valves, suggesting its potential causative role for the development of Tetralogy of Fallot (TOF) in humans. In the presented study, we analyzed TBX20 in cardiac biopsies derived from patients with TOF, ventricular septal defects (VSDs) and normal hearts. Mutation analysis did not reveal any disease causing sequence variation, however, TBX20 is significantly upregulated in tissue samples of patients with TOF, but not VSD. In depth analysis of TBX20 transcripts lead to the identification of two new exons 3' to the known TBX20 message resembling the mouse variant Tbx20a, as well as an extended 5'UTR. Functional analysis of the human TBX20 promoter revealed a 100 bp region that contains strong activating elements. Within this core promoter region we recognized functional binding sites for TFAP2 transcription factors and identified TFAP2 as repressors of the TBX20 gene in vitro and in vivo. Moreover, decreased TFAP2C levels in cardiac biopsies of TOF patients underline the biological significance of the pathway described. In summary, we provide first insights into the regulation of TBX20 and show its potential for human congenital heart diseases.

Comparative DNA methylation and gene expression analysis identifies novel genes for structural congenital heart diseases.

AIMS: For the majority of congenital heart diseases (CHDs), the full complexity of the causative molecular network, which is driven by genetic, epigenetic, and environmental factors, is yet to be elucidated. Epigenetic alterations are suggested to play a pivotal role in modulating the phenotypic expression of CHDs and their clinical course during life. Candidate approaches implied that DNA methylation might have a developmental role in CHD and contributes to the long-term progress of non-structural cardiac diseases. The aim of the present study is to define the postnatal epigenome of two common cardiac malformations, representing epigenetic memory, and adaption to hemodynamic alterations, which are jointly relevant for the disease course. METHODS AND RESULTS: We present the first analysis of genome-wide DNA methylation data obtained from myocardial biopsies of Tetralogy of Fallot (TOF) and ventricular septal defect patients. We defined stringent sets of differentially methylated regions between patients and controls, which are significantly enriched for genomic features like promoters, exons, and cardiac enhancers. For TOF, we linked DNA methylation with genome-wide expression data and found a significant overlap for hypermethylated promoters and down-regulated genes, and vice versa. We validated and replicated the methylation of selected CpGs and performed functional assays. We identified a hypermethylated novel developmental CpG island in the promoter of SCO2 and demonstrate its functional impact. Moreover, we discovered methylation changes co-localized with novel, differential splicing events among sarcomeric genes as well as transcription factor binding sites. Finally, we demonstrated the interaction of differentially methylated and expressed genes in TOF with mutated CHD genes in a molecular network. CONCLUSION: By interrogating DNA methylation and gene expression data, we identify two novel mechanism contributing to the phenotypic expression of CHDs: aberrant methylation of promoter CpG islands and methylation alterations leading to differential splicing.

reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4(+) memory T cells.

The combinatorial action of co-localizing chromatin modifications and regulators determines chromatin structure and function. However, identifying co-localizing chromatin features in a high-throughput manner remains a technical challenge. Here we describe a novel reChIP-seq approach and tailored bioinformatic analysis tool, normR that allows for the sequential enrichment and detection of co-localizing DNA-associated proteins in an unbiased and genome-wide manner. We illustrate the utility of the reChIP-seq method and normR by identifying H3K4me3 or H3K27me3 bivalently modified nucleosomes in primary human CD4(+) memory T cells. We unravel widespread bivalency at hypomethylated CpG-islands coinciding with inactive promoters of developmental regulators. reChIP-seq additionally uncovered heterogeneous bivalency in the population, which was undetectable by intersecting H3K4me3 and H3K27me3 ChIP-seq tracks. Finally, we provide evidence that bivalency is established and stabilized by an interplay between the genome and epigenome. Our reChIP-seq approach augments conventional ChIP-seq and is broadly applicable to unravel combinatorial modes of action.

PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3.

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.

Identification and functional analysis of CITED2 mutations in patients with congenital heart defects.

Recent reports have demonstrated that mice lacking the transcription factor Cited2 die in utero showing various cardiac malformations. We present for the first time functionally relevant mutations of CITED2 in patients with congenital heart defects (CHDs). CITED2 encodes a CREBBP/EP300 interacting transcriptional modulator of HIF1A and TFAP2. To study the potential impact of sequence variations in CITED2 for CHDs in humans, we screened a cohort of 392 well-characterized patients and 192 control individuals using DHPLC, sequencing, and Amplifluor genotyping techniques. We identified 15 CITED2 nucleotide alterations. Seven of these alterations were found only in CHD patients and were not detected in controls, including three mutations leading to alterations of the amino acid sequence (p.Ser170_Gly178del, p.Gly178_Ser179ins9, and p.Ser198_Gly199del). All three of these amino acid changing mutations cluster in the serine-glycine-rich junction of the protein, to which no functionality had heretofore been assigned. Here we show that these mutations significantly reduce the capacity of CITED2 to transrepress HIF1A, and that the p.Ser170_Gly178del mutation significantly diminishes TFAP2C coactivation. This reveals a modifying role for the serine-glycine-rich region in CITED2 function. In summary, the observation of these mutations in patients with septal defects indicates that CITED2 has a causative impact in the development of CHD in humans.