Specific quantification of inducible HIV-1 reservoir by RT-LAMP.

BACKGROUND: Strategies toward HIV-1 cure aim to clear, inactivate, reduce, or immunologically control the virus from a pool of latently infected cells such that combination antiretroviral therapy (cART) can be safely interrupted. In order to assess the impact of any putative curative interventions on the size and inducibility of the latent HIV-1 reservoir, robust and scalable assays are needed to precisely quantify the frequency of infected cells containing inducible HIV-1. METHODS: We developed Specific Quantification of Inducible HIV-1 by RT-LAMP (SQuHIVLa), leveraging the high sensitivity and specificity of RT-LAMP, performed in a single reaction, to detect and quantify cells expressing tat/rev HIV-1 multiply spliced RNA (msRNA) upon activation. The LAMP primer/probe used in SQuHIVLa was designed to exclusively detect HIV-1 tat/rev msRNA and adapted for different HIV-1 subtypes. RESULTS: Using SQuHIVLa, we successfully quantify the inducible viral reservoir in CD4+ T cells from people living with HIV-1 subtypes B and C on cART. The assay demonstrates high sensitivity, specificity, and reproducibility. CONCLUSIONS: SQuHIVLa offers a high throughput, scalable, and specific HIV-1 reservoir quantification tool that is amenable to resource-limited settings. This assay poses remarkable potential in facilitating the evaluation of potential interventional strategies toward achieving HIV-1 cure.

HIV infection remains challenging because the virus hides in certain cells, making it invisible to the immune system. This hidden virus forms what is called a latent HIV reservoir. If someone with HIV stops their antiviral therapy, the virus quickly re-emerges. Because of this, researchers are exploring various strategies to eliminate this reservoir and cure HIV. To evaluate these strategies, we need a method to measure the reservoir's size before and after trials. Our study introduces SQuHIVLa, a highly sensitive and specific method for quantifying the latent reservoir. SQuHIVLa could become a vital tool for monitoring HIV patients and assessing treatment effectiveness, bringing us closer to finding a cure.

PCID2 dysregulates transcription and viral RNA processing to promote HIV-1 latency.

HIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. Here, we characterize PCI domain-containing 2 (PCID2) protein, a subunit of the transcription and export complex 2 (TREX2) complex, to enforce transcriptional repression and post-transcriptional blocks to HIV-1 gene expression during latency. PCID2 bound the latent HIV-1 LTR (long terminal repeat) and repressed transcription initiation during latency. Depletion of PCID2 remodeled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and latency reversal. Immunoprecipitation coupled to mass spectrometry identified PCID2-interacting proteins to include negative viral RNA (vRNA) splicing regulators, and PCID2 depletion resulted in over-splicing of intron-containing vRNA in cell lines and primary cells obtained from PWH. MCM3AP and DSS1, two other RNA-binding TREX2 complex subunits, also inhibit transcription initiation and vRNA alternative splicing during latency. Thus, PCID2 is a novel HIV-1 latency-promoting factor, which in context of the TREX2 sub-complex PCID2-DSS1-MCM3AP blocks transcription and dysregulates vRNA processing.

The BAF complex inhibitor pyrimethamine reverses HIV-1 latency in people with HIV-1 on antiretroviral therapy.

Reactivation of the latent HIV-1 reservoir is a first step toward triggering reservoir decay. Here, we investigated the impact of the BAF complex inhibitor pyrimethamine on the reservoir of people living with HIV-1 (PLWH). Twenty-eight PLWH on suppressive antiretroviral therapy were randomized (1:1:1:1 ratio) to receive pyrimethamine, valproic acid, both, or no intervention for 14 days. The primary end point was change in cell-associated unspliced (CA US) HIV-1 RNA at days 0 and 14. We observed a rapid, modest, and significant increase in (CA US) HIV-1 RNA in response to pyrimethamine exposure, which persisted throughout treatment and follow-up. Valproic acid treatment alone did not increase (CA US) HIV-1 RNA or augment the effect of pyrimethamine. Pyrimethamine treatment did not result in a reduction in the size of the inducible reservoir. These data demonstrate that the licensed drug pyrimethamine can be repurposed as a BAF complex inhibitor to reverse HIV-1 latency in vivo in PLWH, substantiating its potential advancement in clinical studies.

Catchet-MS identifies IKZF1-targeting thalidomide analogues as novel HIV-1 latency reversal agents.

A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has been challenging. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to probe the differential protein composition of the latent and activated HIV-1 5'LTR. Catchet-MS identified known and novel latent 5'LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs. We demonstrate that, by targeting IKZF1 for degradation, these compounds reverse HIV-1 latency in CD4+ T-cells isolated from virally suppressed people living with HIV-1 and that they are able to synergize with other known LRAs.

A Two-Color Haploid Genetic Screen Identifies Novel Host Factors Involved in HIV-1 Latency.

To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using short hairpin RNA (shRNA)-mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV-1 latency. Chromatin immunoprecipitation assays indicated that CHD9, a chromodomain helicase DNA-binding protein, maintains HIV-1 latency via direct association with the HIV-1 5' long terminal repeat (LTR), and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-iodotubercidin, trametinib, and topiramate, targeting ADK, NF1, and GRIK5, respectively, were characterized for their latency reversal potential. While 5-iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4+ T cells, trametinib reversed latency in J-Lat cells but not in latent HIV-1 infected primary CD4+ T cells. Importantly, topiramate reversed latency in cell line models, in latently infected primary CD4+ T cells, and crucially in CD4+ T cells from three people living with HIV-1 (PLWH) under suppressive antiretroviral therapy, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency. IMPORTANCE A reservoir of latent HIV-1 infected cells persists in the presence of combination antiretroviral therapy (cART), representing a major obstacle for viral eradication. Reactivation of the latent HIV-1 provirus is part of curative strategies which aim to promote clearance of the infected cells. Using a two-color haploid screen, we identified 69 candidate genes as latency-maintaining host factors and functionally validated a subset of 10 of those in additional T-cell-based cell line models of HIV-1 latency. We further demonstrated that CHD9 is associated with HIV-1's promoter, the 5' LTR, while this association is lost upon reactivation. Additionally, we characterized the latency reversal potential of FDA compounds targeting ADK, NF1, and GRIK5 and identify the GRIK5 inhibitor topiramate as a viable latency reversal agent with clinical potential.

Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma.

The molecular events that drive hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV-infected organoids produced covalently closed circular DNA (cccDNA) and HBV early antigen (HBeAg), expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV-infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity and drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor sodium taurocholate co-transporting polypeptide (NTCP) after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the hepatocellular carcinoma (HCC) cohort on The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV-infected patients.

Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir.

An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). We used single-cell FISH-Flow technology to characterise the contribution of viral RNA to inducing cell death. The pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3 and upregulation of IFNß. DDX3 inhibition also resulted in the downregulation of BIRC5, critical to cell survival during HIV-1 infection, and selectively induced apoptosis in viral RNA-expressing CD4+ T cells but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir by quantitation of HIV-1 RNA, by FISH-Flow, RT-qPCR and TILDA. This study provides proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards the elimination of the inducible reservoir.

Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency.

A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

Novel genetic loci affecting facial shape variation in humans.

The human face represents a combined set of highly heritable phenotypes, but knowledge on its genetic architecture remains limited, despite the relevance for various fields. A series of genome-wide association studies on 78 facial shape phenotypes quantified from 3-dimensional facial images of 10,115 Europeans identified 24 genetic loci reaching study-wide suggestive association (p < 5 × 10-8), among which 17 were previously unreported. A follow-up multi-ethnic study in additional 7917 individuals confirmed 10 loci including six unreported ones (padjusted < 2.1 × 10-3). A global map of derived polygenic face scores assembled facial features in major continental groups consistent with anthropological knowledge. Analyses of epigenomic datasets from cranial neural crest cells revealed abundant cis-regulatory activities at the face-associated genetic loci. Luciferase reporter assays in neural crest progenitor cells highlighted enhancer activities of several face-associated DNA variants. These results substantially advance our understanding of the genetic basis underlying human facial variation and provide candidates for future in-vivo functional studies.

Allele-specific long-distance regulation dictates IL-32 isoform switching and mediates susceptibility to HIV-1.

We integrated data obtained from HIV-1 genome-wide association studies with T cell-derived epigenome data and found that the noncoding intergenic variant rs4349147, which is statistically associated with HIV-1 acquisition, is located in a CD4+ T cell-specific deoxyribonuclease I hypersensitive region, suggesting regulatory potential for this variant. Deletion of the rs4349147 element in Jurkat cells strongly reduced expression of interleukin-32 (IL-32), approximately 10-kb upstream, and chromosome conformation capture assays identified a chromatin loop between rs4349147 and the IL-32 promoter validating its function as a long-distance enhancer. We generated single rs4349147-A or rs4349147-G allele clones and demonstrated that IL-32 enhancer activity and interaction with the IL-32 promoter are strongly allele dependent; rs4349147 -/A cells display reduced IL-32 expression and altered chromatin conformation as compared to rs4349147 G/- cells. Moreover, RNA sequencing demonstrated that rs4349147 G/- cells express a lower relative ratio of IL-32α to non-α isoforms than rs4349147 -/A cells and display increased expression of lymphocyte activation factors rendering them more prone to infection with HIV-1. In agreement, in primary CD4+ T cells, both treatment with recombinant IL-32γ (rIL-32γ) but not rIL-32α, and exogenous lentiviral overexpression of IL-32γ or IL-32ß but not IL-32α resulted in a proinflammatory T cell cytokine environment concomitant with increased susceptibility to HIV infection. Our data demonstrate that rs4349147-G promotes transcription of non-IL-32α isoforms, generating a proinflammatory environment more conducive to HIV infection. This study provides a mechanistic link between a HIV-associated noncoding DNA variant and the expression of different IL-32 isoforms that display discrete anti-HIV properties.

Allele-specific repression of Sox2 through the long non-coding RNA Sox2ot.

The transcription factor Sox2 controls the fate of pluripotent stem cells and neural stem cells. This gatekeeper function requires well-regulated Sox2 levels. We postulated that Sox2 regulation is partially controlled by the Sox2 overlapping long non-coding RNA (lncRNA) gene Sox2ot. Here we show that the RNA levels of Sox2ot and Sox2 are inversely correlated during neural differentiation of mouse embryonic stem cells (ESCs). Through allele-specific enhanced transcription of Sox2ot in mouse Sox2eGFP knockin ESCs we demonstrate that increased Sox2ot transcriptional activity reduces Sox2 RNA levels in an allele-specific manner. Enhanced Sox2ot transcription, yielding lower Sox2 RNA levels, correlates with a decreased chromatin interaction of the upstream regulatory sequence of Sox2 and the ESC-specific Sox2 super enhancer. Our study indicates that, in addition to previously reported in trans mechanisms, Sox2ot can regulate Sox2 by an allele-specific mechanism, in particular during development.

Transcription: Insights From the HIV-1 Promoter.

In this review, we cover transcription regulation of human immunodeficiency virus type 1 (HIV-1) gene expression, focusing on the invaluable contributions, made by HIV research over the years, toward the field of transcription. In this context, the HIV promoter can be considered to be a well-studied model promoter, which although a viral promoter, is subject to the same cellular regulatory mechanisms that modulate the transcriptional control of endogenous host cellular genes. The molecular control of HIV-1 transcription has been well studied and considerable knowledge toward development of alternative strategies for therapies aimed at eradicating both active but also latent HIV-1 has been obtained. Additionally, HIV-1 studies have provided insight into fundamental aspects of transcriptional regulation including transcriptional stochasticity, RNA polymerase II pausing, chromatin regulation of transcription, the role of the +1 nucleosome, the use of an RNA enhancer element, i.e., TAR, the discovery, and essential function of P-TEFb, and the super elongation complex in transcription elongation. These findings have been important not only in deciphering the mechanisms used by HIV-1 to regulate its gene expression and to establish and maintain HIV latency for therapeutic advancement, but were at the same time seminal in pushing the transcription field forward.

Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal.

Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal.

Nuclear positioning rather than contraction controls ordered rearrangements of immunoglobulin loci.

Progenitor-B cells recombine their immunoglobulin (Ig) loci to create unique antigen receptors. Despite a common recombination machinery, the Ig heavy and Ig light chain loci rearrange in a stepwise manner. We studied pre-pro-B cells and Rag(-/-) progenitor-B cells to determine whether Ig locus contraction or nuclear positioning is decisive for stepwise rearrangements. We found that both Ig loci were contracted in pro-B and pre-B cells. Igh relocated from the nuclear lamina to central domains only at the pro-B cell stage, whereas, Igκ remained sequestered at the lamina, and only at the pre-B cell stage located to central nuclear domains. Finally, in vitro induced re-positioning of Ig alleles away from the nuclear periphery increased germline transcription of Ig loci in pre-pro-B cells. Thus, Ig locus contraction juxtaposes genomically distant elements to mediate efficient recombination, however, sequential positioning of Ig loci away from the nuclear periphery determines stage-specific accessibility of Ig loci.

Genetics of skin color variation in Europeans: genome-wide association studies with functional follow-up.

In the International Visible Trait Genetics (VisiGen) Consortium, we investigated the genetics of human skin color by combining a series of genome-wide association studies (GWAS) in a total of 17,262 Europeans with functional follow-up of discovered loci. Our GWAS provide the first genome-wide significant evidence for chromosome 20q11.22 harboring the ASIP gene being explicitly associated with skin color in Europeans. In addition, genomic loci at 5p13.2 (SLC45A2), 6p25.3 (IRF4), 15q13.1 (HERC2/OCA2), and 16q24.3 (MC1R) were confirmed to be involved in skin coloration in Europeans. In follow-up gene expression and regulation studies of 22 genes in 20q11.22, we highlighted two novel genes EIF2S2 and GSS, serving as competing functional candidates in this region and providing future research lines. A genetically inferred skin color score obtained from the 9 top-associated SNPs from 9 genes in 940 worldwide samples (HGDP-CEPH) showed a clear gradual pattern in Western Eurasians similar to the distribution of physical skin color, suggesting the used 9 SNPs as suitable markers for DNA prediction of skin color in Europeans and neighboring populations, relevant in future forensic and anthropological investigations.

Allele-specific transcriptional regulation of IRF4 in melanocytes is mediated by chromatin looping of the intronic rs12203592 enhancer to the IRF4 promoter.

The majority of significant single-nucleotide polymorphisms (SNPs) identified with genome-wide association studies are located in non-coding regions of the genome; it is therefore possible that they are involved in transcriptional regulation of a nearby gene rather than affecting an encoded protein's function. Previously, it was demonstrated that the SNP rs12203592, located in intron 4 of the IRF4 gene, is strongly associated with human skin pigmentation and modulates an enhancer element that controls expression of IRF4. In our study, we investigated the allele-specific effect of rs12203592 on IRF4 expression in epidermal skin samples and in melanocytic cells from donors of different skin color. We focused on the characteristics and activity of the enhancer, and on long-range chromatin interactions in melanocytic cells homozygous and heterozygous for rs12203592. We found that, irrespective of the trans-activating environment, IRF4 transcription is strongly correlated with the allelic status of rs12203592, the activity of the rs12203592 enhancer and that the chromatin features depend on the rs12203592 genotype. Furthermore, we demonstrate that the rs12203592 enhancer physically interacts with the IRF4 promoter through an allele-dependent chromatin loop, and suggest that subsequent allele-specific activation of IRF4 transcription is stabilized by another allele-specific loop from the rs12203592 enhancer to an additional regulatory element in IRF4. We conclude that the non-coding SNP rs12203592 is located in a regulatory region and affects a wide range of enhancer characteristics, resulting into modulation of the enhancer's activity, its interaction with the IRF4 promoter and subsequent allele-specific transcription of IRF4. Our findings provide another example of a non-coding SNP affecting skin color by modulating enhancer-mediated transcriptional regulation.

Human skin color is influenced by an intergenic DNA polymorphism regulating transcription of the nearby BNC2 pigmentation gene.

Single nucleotide polymorphisms (SNPs) found to be statistically significant when associated with human diseases, and other phenotypes are most often located in non-coding regions of the genome. One example is rs10765819 located in the first intron of the BNC2 gene previously associated with (saturation of) human skin color. Here, we demonstrate that a nearby intergenic SNP (rs12350739) in high linkage disequilibrium with rs10756819 is likely the causal DNA variant for the observed BNC2 skin color association. The highly conserved region surrounding rs12350739 functions as an enhancer element regulating BNC2 transcription in human melanocytes, while the activity of this enhancer element depends on the allelic status of rs12350739. When the rs12350739-AA allele is present, the chromatin at the region surrounding rs12350739 is inaccessible and the enhancer element is only slightly active, resulting in low expression of BNC2, corresponding with light skin pigmentation. When the rs12350739-GG allele is present however, the chromatin at the region surrounding rs12350739 is more accessible and the enhancer is active, resulting in a higher expression of BNC2, corresponding with dark skin pigmentation. Overall, we demonstrate the identification of the functional DNA variant that explains the BNC2 skin color association signal, providing another important step towards further understanding human pigmentation genetics beyond statistical association. We thus deliver a clear example of how an intergenic non-coding DNA variant modulates the regulatory potential of the enhancer element it is located within, which in turn results in allele-dependent differential gene expression affecting variation in common human traits.

Genetic variation in regulatory DNA elements: the case of OCA2 transcriptional regulation.

Mutations within the OCA2 gene or the complete absence of the OCA2 protein leads to oculocutaneous albinism type 2. The OCA2 protein plays a central role in melanosome biogenesis, and it is a strong determinant of the eumelanin content in melanocytes. Transcript levels of the OCA2 gene are strongly correlated with pigmentation intensities. Recent studies demonstrated that the transcriptional level of OCA2 is to a large extent determined by the noncoding SNP rs12913832 located 21.5 kb upstream of the OCA2 gene promoter. In this review, we discuss current hypotheses and the available data on the mechanism of OCA2 transcriptional regulation and how this is influenced by genetic variation. Finally, we will explore how future epigenetic studies can be used to advance our insight into the functional biology that connects genetic variation to human pigmentation.

Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions.

Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1-8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.

Transcription factor binding at enhancers: shaping a genomic regulatory landscape in flux.

The mammalian genome is packed tightly in the nucleus of the cell. This packing is primarily facilitated by histone proteins and results in an ordered organization of the genome in chromosome territories that can be roughly divided in heterochromatic and euchromatic domains. On top of this organization several distinct gene regulatory elements on the same chromosome or other chromosomes are thought to dynamically communicate via chromatin looping. Advances in genome-wide technologies have revealed the existence of a plethora of these regulatory elements in various eukaryotic genomes. These regulatory elements are defined by particular in vitro assays as promoters, enhancers, insulators, and boundary elements. However, recent studies indicate that the in vivo distinction between these elements is often less strict. Regulatory elements are bound by a mixture of common and lineage-specific transcription factors which mediate the long-range interactions between these elements. Inappropriate modulation of the binding of these transcription factors can alter the interactions between regulatory elements, which in turn leads to aberrant gene expression with disease as an ultimate consequence. Here we discuss the bi-modal behavior of regulatory elements that act in cis (with a focus on enhancers), how their activity is modulated by transcription factor binding and the effect this has on gene regulation.