Cause and Consequence of Tethering a SubTAD to Different Nuclear Compartments.

Detailed genomic contact maps have revealed that chromosomes are structurally organized in megabase-sized topologically associated domains (TADs) that encompass smaller subTADs. These domains segregate in the nuclear space to form active and inactive nuclear compartments, but cause and consequence of compartmentalization are largely unknown. Here, we combined lacO/lacR binding platforms with allele-specific 4C technologies to track their precise position in the three-dimensional genome upon recruitment of NANOG, SUV39H1, or EZH2. We observed locked genomic loci resistant to spatial repositioning and unlocked loci that could be repositioned to different nuclear subcompartments with distinct chromatin signatures. Focal protein recruitment caused the entire subTAD, but not surrounding regions, to engage in new genomic contacts. Compartment switching was found uncoupled from transcription changes, and the enzymatic modification of histones per se was insufficient for repositioning. Collectively, this suggests that trans-associated factors influence three-dimensional compartmentalization independent of their cis effect on local chromatin composition and activity.

Histone H1 eviction by the histone chaperone SET reduces cell survival following DNA damage.

Many chromatin remodeling and modifying proteins are involved in the DNA damage response, where they stimulate repair or induce DNA damage signaling. Interestingly, we identified that downregulation of the histone H1 (H1)-interacting protein SET results in increased resistance to a wide variety of DNA damaging agents. We found that this increased resistance does not result from alleviation of an inhibitory effect of SET on DNA repair but, rather, is the consequence of a suppressed apoptotic response to DNA damage. Furthermore, we provide evidence that the histone chaperone SET is responsible for the eviction of H1 from chromatin. Knockdown of H1 in SET-depleted cells resulted in re-sensitization of cells to DNA damage, suggesting that the increased DNA damage resistance in SET-depleted cells is the result of enhanced retention of H1 on chromatin. Finally, clonogenic survival assays showed that SET and p53 act epistatically in the attenuation of DNA damage-induced cell death. Taken together, our data indicate a role for SET in the DNA damage response as a regulator of cell survival following genotoxic stress.This article has an associated First Person interview with the first author of the paper.

Candidate methylation sites associated with endocrine therapy resistance in ER+/HER2- breast cancer.

BACKGROUND: Estrogen receptor (ER) positive breast cancer is often effectively treated with drugs that inhibit ER signaling, i.e., tamoxifen (TAM) and aromatase inhibitors (AIs). However, 30% of ER+ breast cancer patients develop resistance to therapy leading to tumour recurrence. Changes in the methylation profile have been implicated as one of the mechanisms through which therapy resistance develops. Therefore, we aimed to identify methylation loci associated with endocrine therapy resistance. METHODS: We used genome-wide DNA methylation profiles of primary ER+/HER2- tumours from The Cancer Genome Atlas in combination with curated data on survival and treatment to predict development of endocrine resistance. Association of individual DNA methylation markers with survival was assessed using Cox proportional hazards models in a cohort of ER+/HER2- tumours (N = 552) and two sub-cohorts corresponding to the endocrine treatment (AI or TAM) that patients received (N = 210 and N = 172, respectively). We also identified multivariable methylation signatures associated with survival using Cox proportional hazards models with elastic net regularization. Individual markers and multivariable signatures were compared with DNA methylation profiles generated in a time course experiment using the T47D ER+ breast cancer cell line treated with tamoxifen or deprived from estrogen. RESULTS: We identified 134, 5 and 1 CpGs for which DNA methylation is significantly associated with survival in the ER+/HER2-, TAM and AI cohorts respectively. Multi-locus signatures consisted of 203, 36 and 178 CpGs and showed a large overlap with the corresponding single-locus signatures. The methylation signatures were associated with survival independently of tumour stage, age, AI treatment, and luminal status. The single-locus signature for the TAM cohort was conserved among the loci that were differentially methylated in endocrine-resistant T47D cells. Similarly, multi-locus signatures for the ER+/HER2- and AI cohorts were conserved in endocrine-resistant T47D cells. Also at the gene set level, several sets related to endocrine therapy and resistance were enriched in both survival and T47D signatures. CONCLUSIONS: We identified individual and multivariable DNA methylation markers associated with therapy resistance independently of luminal status. Our results suggest that these markers identified from primary tumours prior to endocrine treatment are associated with development of endocrine resistance.

KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and is Resilient to TGFß1 Activation.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFß1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFß1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFß1-induced and uninduced PLOD2 expression.

The dynamics of early-state transcriptional changes and aggregate formation in a Huntington's disease cell model.

BACKGROUND: Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG expansion in the Huntingtin (HTT) gene. Proteolytic cleavage of mutant huntingtin (Htt) protein with an expanded polyglutamine (polyQ) stretch results in production of Htt fragments that aggregate and induce impaired ubiquitin proteasome, mitochondrial functioning and transcriptional dysregulation. To understand the time-resolved relationship between aggregate formation and transcriptional changes at early disease stages, we performed temporal transcriptome profiling and quantification of aggregate formation in living cells in an inducible HD cell model. RESULTS: Rat pheochromocytoma (PC12) cells containing a stably integrated, doxycycline-inducible, eGFP-tagged N-terminal human Htt fragment with an expanded polyQ domain were used to analyse gene expression changes at different stages of mutant Htt aggregation. At earliest time points after doxycycline induction no detectable aggregates and few changes in gene expression were observed. Aggregates started to appear at intermediate time points. Aggregate formation and subsequent enlargement of aggregates coincided with a rapid increase in the number of differentially expressed (DE) genes. The increase in number of large aggregates coincided with a decrease in the number of smaller aggregates whereas the transcription profile reverted towards the profile observed before mutant Htt induction. Cluster-based analysis of the 2,176 differentially expressed genes revealed fourteen distinct clusters responding differently over time. Functional enrichment analysis of the two major gene clusters revealed that genes in the up-regulated cluster were mainly involved in metabolic (antioxidant activity and cellular ketone metabolic processes) and genes in the down-regulated cluster in developmental processes, respectively. Promoter-based analysis of the identified gene clusters resulted in identification of a transcription factor network of which several previously have been linked to HD. CONCLUSIONS: We demonstrate a time-resolved relationship between Htt aggregation and changes in the transcriptional profile. We identified two major gene clusters showing involvement of (i) mitochondrial dysfunction and (ii) developmental processes implying cellular homeostasis defects. We identified novel and known HD-linked transcription factors and show their interaction with known and predicted regulatory proteins. Our data provide a novel resource for hypothesis building on the role of transcriptional key regulators in early stages of HD and possibly other polyQ-dependent diseases.

Epigenetic imprinting during assisted reproductive technologies: The effect of temporal and cumulative fluctuations in methionine cycling on the DNA methylation state.

Assisted reproductive technology (ART) exposes gametes and embryos to an artificial environment that does not resemble the conditions of natural conception, and therefore might change epigenetic regulation of genes that are imprinted during development. In the present review, we discuss the relationship between susceptibility of specific genes to receive an altered epigenetic composition during ART processes, possibly via alterations in the biochemical folate and methionine cycle. We provide a comprehensive view of the current state of epigenetic patterning in ART-conceived healthy children and in Angelman syndrome (AS) and Beckwith-Wiedemann syndrome (BWS) patients. We illustrate that similar genes--that is, MEST, KCNQ1OT1, and IGF2--possess an altered DNA methylation profile in animal models, ART-conceived healthy children, and AS and BWS patients. The developmental stage at which these genes receive their epigenetic imprint appears to coincide with the specific moment that ART takes place. We highlight that ART procedures affect physiological levels of enzymes and substrates involved in the folate and methionine cycle thereby altering the DNA methylation state. Moreover, although the DNA methylation rate appears to be robust: (i) temporal imbalances coinciding with defined moments of epigenetic imprinting of specific genes affect the eventual DNA methylation state of those genes and (ii) cumulative ART effects on methionine and folate cycling can alter DNA methylation rates. These observations underscore the necessity to further investigate consequences of ART treatments on the epigenetic profile.

Concise review: the dynamics of induced pluripotency and its behavior captured in gene network motifs.

The flexibility of cellular identity is clearly demonstrated by the possibility to reprogram fully differentiated somatic cells to induced pluripotent stem (iPS) cells through forced expression of a set of transcription factors. The generation of iPS cells is of great interest as they provide a tremendous potential for regenerative medicine and an attractive platform to investigate pluripotency. Despite having gathered much attention, the molecular details and responsible gene regulatory networks during the reprogramming process are largely unresolved. In this review, we analyze the sequence and dynamics of reprogramming to construct a timeline of the molecular events taking place during induced pluripotency. We use this timeline as a road map to explore the distinct phases of the reprogramming process and to suggest gene network motifs that are able to describe its systems behavior. We conclude that the gene networks involved in reprogramming comprise several feedforward loops combined with autoregulatory behavior and one or more AND gate motifs that can explain the observed dynamics of induced pluripotency. Our proposed timeline and derived gene network motif behavior could serve as a tool to understand the systems behavior of reprogramming and identify key transitions and/or transcription factors that influence somatic cell reprogramming. Such a systems biology strategy could provide ways to define and explore the use of additional regulatory factors acting at defined gene network motifs to potentially overcome the current challenges of inefficient, slow, and partial somatic cell reprogramming and hence set ground of using iPS cells for clinical and therapeutic use.

Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes.

Despite significant advances made in epigenetic research in recent decades, many questions remain unresolved, especially concerning cause and consequence of epigenetic marks with respect to gene expression modulation (GEM). Technologies allowing the targeting of epigenetic enzymes to predetermined DNA sequences are uniquely suited to answer such questions and could provide potent (bio)medical tools. Toward the goal of gene-specific GEM by overwriting epigenetic marks (Epigenetic Editing, EGE), instructive epigenetic marks need to be identified and their writers/erasers should then be fused to gene-specific DNA binding domains. The appropriate epigenetic mark(s) to change in order to efficiently modulate gene expression might have to be validated for any given chromatin context and should be (mitotically) stable. Various insights in such issues have been obtained by sequence-specific targeting of epigenetic enzymes, as is presented in this review. Features of such studies provide critical aspects for further improving EGE. An example of this is the direct effect of the edited mark versus the indirect effect of recruited secondary proteins by targeting epigenetic enzymes (or their domains). Proof-of-concept of expression modulation of an endogenous target gene is emerging from the few EGE studies reported. Apart from its promise in correcting disease-associated epi-mutations, EGE represents a powerful tool to address fundamental epigenetic questions.

Single-Molecule Analysis of Transcription Dynamics to Understand the Relationship Between Epigenetic Alterations and Transcriptional Variability.

Heterogeneity in gene expression largely stems from the discontinuous nature of transcription, with transcripts being produced in bursts with defined frequencies. This cell-to-cell variability in transcription within isogenic cell populations is a known phenomenon across numerous genes. Multiple gene regulatory and epigenetic factors have been identified as key contributors to this pulsatile gene activity. Understanding the effects of epigenetic modulation on transcriptional cell-to-cell variability and kinetics of transcriptional activity is crucial for interpreting changes in treatment responsiveness. We present a detailed protocol that guides the assessment of fluctuations in gene expression induced by epigenetic modulation using single-molecule RNA in situ hybridization (smRNA FISH) combined with confocal microscopy imaging, data analysis, and quantification in breast cancer cells. Through smRNA FISH labeling, both mature and nascent transcripts are identified. Subsequently, the number of mature transcripts and the intensity and frequency of nascent transcripts are quantified, and these measurements are used to calculate the burst size and frequency for the labeled gene. By following this step-by-step methodology, insights are obtained into the intricate relationship between epigenetic alterations and the dynamic nature of gene expression in breast cancer cells.

Generation of Cell Lines Stably Expressing a dCas9-Fusion or sgRNA to Address Dynamics of Long-Term Effects of Epigenetic Editing.

Epigenetic modifications play a crucial role in regulating gene expression patterns. Through epigenetic editing approaches, the chromatin structure is modified and the activity of the targeted gene can be reprogrammed without altering the DNA sequence. By using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic repeats) platform with nuclease-deactivated dCas9 proteins to direct epigenetic effector domains (EDs) to genomic regulatory regions, the expression of the targeted gene can be modulated. However, the long-term stability of these effects, although demonstrated, remains unpredictable. The versatility and flexibility of (co-)targeting different genes with multiple epigenetic effectors has made the CRISPR/dCas9 platform the most widely used gene modulating technology currently available. Efficient delivery of large dCas9-ED fusion constructs into target cells, however, is challenging. An approach to overcome this limitation is to generate cells that stably express sgRNA(s) or dCas9-ED constructs. The sgRNA(s) or dCas9-ED stable cell lines can be used to study the mechanisms underlying sustained gene expression reprogramming by transiently expressing the other of the two constructs. Here, we describe a detailed protocol for the engineering of cells that stably express CRISPR/dCas9 or sgRNA. Creating a system where one component of the CRISPR/dCas9 is stably expressed while the other is transiently expressed offers a versatile platform for investigating the dynamics of epigenetic reprogramming.

Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing.

To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.

Compartmentalization of androgen receptor protein-protein interactions in living cells.

Steroid receptors regulate gene expression in a ligand-dependent manner by binding specific DNA sequences. Ligand binding also changes the conformation of the ligand binding domain (LBD), allowing interaction with coregulators via LxxLL motifs. Androgen receptors (ARs) preferentially interact with coregulators containing LxxLL-related FxxLF motifs. The AR is regulated at an extra level by interaction of an FQNLF motif in the N-terminal domain with the C-terminal LBD (N/C interaction). Although it is generally recognized that AR coregulator and N/C interactions are essential for transcription regulation, their spatiotemporal organization is largely unknown. We performed simultaneous fluorescence resonance energy transfer and fluorescence redistribution after photobleaching measurements in living cells expressing ARs double tagged with yellow and cyan fluorescent proteins. We provide evidence that AR N/C interactions occur predominantly when ARs are mobile, possibly to prevent unfavorable or untimely cofactor interactions. N/C interactions are largely lost when AR transiently binds to DNA, predominantly in foci partly overlapping transcription sites. AR coregulator interactions occur preferentially when ARs are bound to DNA.

Spatially confined folding of chromatin in the interphase nucleus.

Genome function in higher eukaryotes involves major changes in the spatial organization of the chromatin fiber. Nevertheless, our understanding of chromatin folding is remarkably limited. Polymer models have been used to describe chromatin folding. However, none of the proposed models gives a satisfactory explanation of experimental data. In particularly, they ignore that each chromosome occupies a confined space, i.e., the chromosome territory. Here, we present a polymer model that is able to describe key properties of chromatin over length scales ranging from 0.5 to 75 Mb. This random loop (RL) model assumes a self-avoiding random walk folding of the polymer backbone and defines a probability P for 2 monomers to interact, creating loops of a broad size range. Model predictions are compared with systematic measurements of chromatin folding of the q-arms of chromosomes 1 and 11. The RL model can explain our observed data and suggests that on the tens-of-megabases length scale P is small, i.e., 10-30 loops per 100 Mb. This is sufficient to enforce folding inside the confined space of a chromosome territory. On the 0.5- to 3-Mb length scale chromatin compaction differs in different subchromosomal domains. This aspect of chromatin structure is incorporated in the RL model by introducing heterogeneity along the fiber contour length due to different local looping probabilities. The RL model creates a quantitative and predictive framework for the identification of nuclear components that are responsible for chromatin-chromatin interactions and determine the 3-dimensional organization of the chromatin fiber.

Functional Validation of the Putative Oncogenic Activity of PLAU.

Plasminogen activator, urokinase (PLAU) is involved in cell migration, proliferation and tissue remodeling. PLAU upregulation is associated with an increase in aggressiveness, metastasis, and invasion of several cancer types, including breast cancer. In patients, this translates into decreased sensitivity to hormonal treatment, and poor prognosis. These clinical findings have led to the examination of PLAU as a biomarker for predicting breast cancer prognosis and therapy responses. In this study, we investigated the functional ability of PLAU to act as an oncogene in breast cancers by modulating its expression using CRISPR-deactivated Cas9 (CRISPR-dCas9) tools. Different effector domains (e.g., transcription modulators (VP64, KRAB)) alone or in combination with epigenetic writers (DNMT3A/3L, MSssI) were fused to dCas9 and targeted to the PLAU promoter. In MDA-MB-231 cells characterized by high PLAU expression downregulation of PLAU expression by CRISPR-dCas9-DNMT3A/3L-KRAB, resulted in decreased cell proliferation. Conversely, CRISPR-dCas9-VP64 induced PLAU upregulation in low PLAU expressing MCF-7 cells and significantly increased aggressiveness and invasion. In conclusion, modulation of PLAU expression affected metastatic related properties of breast cancer cells, thus further validating its oncogenic activity in breast cancer cells.

Intranuclear sphingomyelin is associated with transcriptionally active chromatin and plays a role in nuclear integrity.

BACKGROUND INFORMATION: Sphingomyelin is one of the major phospholipids in the cell nucleus. However, its intranuclear distribution with regard to different functional nuclear domains as well as its possible involvement in the nuclear functional architecture remains to be elucidated. RESULTS: We carried out an ultrastructural cytochemical study of the intranuclear distribution of SM (sphingomyelin) using an in situ binding assay of neutral SMase (sphingomyelinase) conjugated to colloidal gold particles. The enzymatic labelling was carried out on ultrathin sections of different mammalian cells prepared by means of various fixation and resin-embedding protocols. Transmission electron microscopic analysis revealed preferential localization of SM within the PR (perichromatin region), a functionally important nucleoplasmic domain containing sites of pre-mRNA synthesis and processing. In the nucleolus, SM is mostly associated with the dense fibrillar component containing transcriptionally active ribosomal genes. Microinjection of enzymatically active SMase into living cells resulted in a rapid degradation of intranuclear structure. CONCLUSIONS: Our observations, supported by biochemical data, provide evidence for the involvement of SM in important nuclear functions. They bring additional information pointing out the PR as an essential functional nuclear domain. Furthermore, they suggest a role for SM in the internal nuclear architecture.

Pericentromeric heterochromatin domains are maintained without accumulation of HP1.

The heterochromatin protein 1 (HP1) family is thought to be an important structural component of heterochromatin. HP1 proteins bind via their chromodomain to nucleosomes methylated at lysine 9 of histone H3 (H3K9me). To investigate the role of HP1 in maintaining heterochromatin structure, we used a dominant negative approach by expressing truncated HP1alpha or HP1beta proteins lacking a functional chromodomain. Expression of these truncated HP1 proteins individually or in combination resulted in a strong reduction of the accumulation of HP1alpha, HP1beta, and HP1gamma in pericentromeric heterochromatin domains in mouse 3T3 fibroblasts. The expression levels of HP1 did not change. The apparent displacement of HP1alpha, HP1beta, and HP1gamma from pericentromeric heterochromatin did not result in visible changes in the structure of pericentromeric heterochromatin domains, as visualized by DAPI staining and immunofluorescent labeling of H3K9me. Our results show that the accumulation of HP1alpha, HP1beta, and HP1gamma at pericentromeric heterochromatin domains is not required to maintain DAPI-stained pericentromeric heterochromatin domains and the methylated state of histone H3 at lysine 9 in such heterochromatin domains.

Time-Resolved Profiling Reveals ATF3 as a Novel Mediator of Endocrine Resistance in Breast Cancer.

Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express Estrogen Receptor α account for around 70% of cases and are mostly treated with targeted endocrine therapy. However, depending on the degree of severity of the disease at diagnosis, 10 to 40% of these tumors eventually relapse due to resistance development. Even though recent novel approaches as the combination with CDK4/6 inhibitors increased the overall survival of relapsing patients, this remains relatively short and there is a urgent need to find alternative targetable pathways. In this study we profiled the early phases of the resistance development process to uncover drivers of this phenomenon. Time-resolved analysis revealed that ATF3, a member of the ATF/CREB family of transcription factors, acts as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. ATF3 was found to be essential in controlling crucial processes such as proliferation, cell cycle, and apoptosis during the early response to treatment through the regulation of MAPK/AKT signaling pathways. Its essential role was confirmed in vivo in a mouse model, and elevated expression of ATF3 was verified in patient datasets, adding clinical relevance to our findings. This study proposes ATF3 as a novel mediator of endocrine resistance development in breast cancer and elucidates its role in the regulation of downstream pathways activities.

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation.

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.

Forensic DNA methylation profiling from minimal traces: How low can we go?

Analysis of human DNA methylation (DNAm) can provide additional investigative leads in crime cases, e.g. the type of tissue or body fluid, the chronological age of an individual, and differentiation between identical twins. In contrast to the genetic profile, the DNAm level is not the same in every cell. At the single cell level, DNAm represents a binary event at a defined CpG site (methylated versus non-methylated). The DNAm level from a DNA extract however represents the average level of methylation of the CpG of interest of all molecules in the forensic sample. The variance of DNAm levels between replicates is often attributed to technological issues, i.e. degradation of DNA due to bisulfite treatment, preferential amplification of DNA, and amplification failure. On the other hand, we show that stochastic variations can lead to gross fluctuation in the analysis of methylation levels in samples with low DNA levels. This stochasticity in DNAm results is relevant since low DNA amounts (1pg - 1ng) is rather the norm than the exception when analyzing forensic DNA samples. This study describes a conceptual analysis of DNAm profiling and its dependence on the amount of input DNA. We took a close look at the variation of DNAm analysis due to DNA input and its consequences for different DNAm-based forensic applications. As can be expected, the 95%-confidence interval of measured DNAm becomes narrower with increasing amounts of DNA. We compared this aspect for two different DNAm-based forensic applications: body fluid identification and chronological age determination. Our study shows that DNA amount should be well considered when using DNAm for forensic applications.

Cell-to-Cell Transcription Variability as Measured by Single-Molecule RNA FISH to Detect Epigenetic State Switching.

Single-molecule RNA fluorescent in situ hybridization (smRNA FISH) allows for the visualization, localization, and quantification of RNA transcripts within individual cells and tissues using custom-designed fluorescently labeled oligonucleotide probes. Here we describe a protocol for the preparation, imaging, and analysis of a smRNA FISH experiment that can be applied to any RNA of choice. We also provide insights as to how this powerful tool can be used to study epigenetic regulation, for example, following the epigenetic editing of genes.