Formaldehyde-assisted Isolation Of Regulatory Elements Research Articles

Anemia-Activated Cis Regulatory Requirements for Erythroid Regeneration

Acute anemia elicits broad transcriptional changes in erythroid progenitors and precursors. Whether anemia-activated transcription and underlying usage of cis-regulatory elements to control gene expression are unique to anemia or overlap with physiological erythropoiesis is an unresolved question regarding anemia response mechanisms. We previously discovered that a cis-regulatory transcriptional enhancer at the Samd14 locus (S14E), defined by a CANNTG-spacer-AGATAA (named E-box-GATA) composite motif and occupied by GATA1 and TAL1 transcription factors, is required for survival in severe anemia. Our working model proposes that the S14E is part of an ensemble of similar anemia-responsive cis-elements promoting erythroid precursor activities. To determine whether loci containing a cohort of S14E-like cis-elements are transcriptionally upregulated in anemia, we conducted single cell RNA-sequencing of Kit+ cells isolated from mouse spleen during the recovery phase of acute anemia. Single-cell regulatory network inference and clustering (SCENIC) analysis - used to predict transcription factors involved in regulatory mechanisms - revealed that anemia-activated transcripts co-expressed with Gata1 and Tal1 were localized in specific populations of erythroid cells. We identified 65 anemia-activated genes associated with GATA1/TAL1-occupied S14E-like cis-elements including Samd14, suggesting common transcriptional mechanisms are involved in restoring homeostasis. Using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), we found the chromatin accessibility of S14E-like cis-elements associated with synovial sarcoma X-2 interacting protein (Ssx2ip) and tubulin tyrosine ligase-like12 (Ttll12) was significantly increased in anemia. To test S14E-like function in transcriptional activation, we targeted these sequences using sgRNA-guided Cas9 and dCas9-KRAB in mouse and human erythroid cell lines (G1E, K562 and HUDEP-2). Both SSX2IP and TTLL12 S14E-like enhancers were required for maximal gene transcription. These data demonstrate that a cohort of S14E-like cis-elements promote anemia-activated gene expression. Ssx2ip is a poorly studied gene that is upregulated by anemia in erythroid precursors (19.2-fold, p<0.0001). To connect cis-regulatory activation in anemia with erythroid regeneration, we next tested Ssx2ip function in erythroid precursor cells isolated from a mouse model of acute anemia. shRNA mediated knockdown of Ssx2ip decreased the percentage of early stage (CD71lowTer119-) erythroid precursors 4.4-fold (p=0.001). Ssx2ip knockdown decreased burst-forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) colony forming potential of erythroid precursors by 1.3-fold (p=0.008) and 1.6-fold (p<0.0001), respectively. To investigate a possible role for Ssx2ip in cell proliferation, we conducted BrdU incorporation and cell proliferation assays. These experiments revealed that Ssx2ip is an important determinant of erythroid progenitor cell cycle progression, and proliferation in anemia. Together, we provide rigorous evidence that S14E-like cis-elements associate with anemia-activated genes, activate gene expression, and that the anemia-activated Ssx2ip gene plays important roles in erythroid precursor cells. Our results highlight a common mechanism of gene activation that regulates erythroid regeneration via S14E-like enhancers. Ongoing efforts will establish mechanisms of Ssx2ip activities in erythropoiesis and functional requirements of additional cohorts of S14E-like enhancers involved in gene regulation during anemia recovery. These findings provide a framework to understand anemia-specific transcriptional mechanisms, ineffective erythropoiesis, and anemia recovery. Given the role of Ssx2ip in erythroid regeneration, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia.

Genome-wide cancer-specific chromatin accessibility patterns derived from archival processed xenograft tumors.

Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.

TET3-mediated accumulation of DNA hydroxymethylation contributes to the activity-dependent gene expression of Rab3a in post-mitotic neurons.

Rab3a, a subtype protein in the Rab3 family amongst the small G proteins, is closely associated with the learning and memory formation process. Various neuronal stimuli can induce the expression of Rab3a; however, how DNA modification is involved in regulating its expression is not fully understood. Ten-eleven translocation (TET) proteins can oxidate methylcytosine to hydroxymethylcytosine, which can further activate gene expression. Previous studies reported that TET-mediated regulation of 5hmC induced by learning is involved in neuronal activation. However, whether Tet protein regulates Rab3a is unknown. To understand the role of TET-mediated 5hmC on Rab3a in neuronal activation, we adopted a KCl-induced depolarization protocol in cultured primary cortical neurons to mimic neuronal activity in vitro. After KCl treatment, Rab3a and Tet3 mRNA expression were induced. Moreover, we observed a decrease in the methylation level and an increase of hydroxymethylation level surrounding the CpG island near the transcription start site of Rab3a. Furthermore, recently, Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) has proven powerful in identifying open chromatin in the genome of various eukaryotes. Using FAIRE-qPCR, we observed a euchromatin state and the increased occupancy of Tet3, H3K4me3, and H3K27ac at the promoter region of Rab3a after KCl treatment. Finally, by using shRNA to knockdown Tet3 prior KCl treatment, all changes mentioned above vanished. Thus, our findings elucidated that the neuronal activity-induced accumulation of hydroxymethylation, which Tet3 mediates, can introduce an active and permissive chromatin structure at Rab3a promoter and lead to the induction of Rab3a mRNA expression.

Epstein-Barr virus-positive gastric cancer involves enhancer activation through activating transcription factor 3.

Epstein‐Barr virus (EBV) is associated with particular forms of gastric cancer (GC). We previously showed that EBV infection into gastric epithelial cells induced aberrant DNA hypermethylation in promoter regions and silencing of tumor suppressor genes. We here undertook integrated analyses of transcriptome and epigenome alteration during EBV infection in gastric cells, to investigate activation of enhancer regions and related transcription factors (TFs) that could contribute to tumorigenesis. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) sequencing (‐seq) data revealed 19 992 open chromatin regions in putative H3K4me1+ H3K4me3− enhancers in EBV‐infected MKN7 cells (MKN7_EB), with 10 260 regions showing increase of H3K27ac. Motif analysis showed candidate TFs, eg activating transcription factor 3 (ATF3), to possibly bind to these activated enhancers. ATF3 was considerably upregulated in MKN7_EB due to EBV factors including EBV‐determined nuclear antigen 1 (EBNA1), EBV‐encoded RNA 1, and latent membrane protein 2A. Expression of mutant EBNA1 decreased copy number of the EBV genome, resulting in relative downregulation of ATF3 expression. Epstein‐Barr virus was also infected into normal gastric epithelial cells, GES1, confirming upregulation of ATF3. Chromatin immunoprecipitation‐seq analysis on ATF3 binding sites and RNA‐seq analysis on ATF3 knocked‐down MKN7_EB revealed 96 genes targeted by ATF3‐activating enhancers, which are related with cancer hallmarks, eg evading growth suppressors. These 96 ATF3 target genes were significantly upregulated in MKN7_EB compared with MKN7 and significantly downregulated when ATF3 was knocked down in EBV‐positive GC cells SNU719 and NCC24. Knockdown of ATF3 in EBV‐infected MKN7, SNU719, and NCC24 cells all led to significant decrease of cellular growth through an increase of apoptotic cells. These indicate that enhancer activation though ATF3 might contribute to tumorigenesis of EBV‐positive GC.

Formaldehyde-assisted isolation of regulatory DNA elements from Arabidopsis leaves.

Eukaryotic gene transcription is associated with the eviction of nucleosomes and the formation of open chromatin, which enables the recruitment of transcriptional coactivators and other regulatory factors. Open chromatin is thus a hallmark of functional regulatory DNA elements in genomes. In recent years, formaldehyde-assisted isolation of regulatory elements (FAIRE) has proven powerful in identifying open chromatin in the genome of various eukaryotes, particularly yeast, human, and mouse. However, it has proven challenging to adapt the FAIRE protocol for use on plant material, and the few available protocols all have their drawbacks (e.g., applicability only to specific developmental stages). In this Protocol Extension, we describe a reliable FAIRE protocol for mature Arabidopsis (Arabidopsis thaliana) leaves that adapts the original protocol for use on plants. The main differences between this protocol extension and the earlier FAIRE protocol are an increased formaldehyde concentration in the chromatin crosslinking buffer, application of a repeated vacuum to increase crosslinking efficiency, and altered composition of the DNA extraction buffer. The protocol is applicable to leaf chromatin of unstressed and stressed plants and can be completed within 1 week. Here, we also describe downstream analysis using qPCR and next-generation sequencing. However, this Protocol Extension should also be compatible with downstream hybridization to a DNA microarray. In addition, it is likely that only minor adaptations will be necessary to apply this protocol to other Arabidopsis organs or plant species.

Atypical chromatin structure of immune-related genes expressed in chicken erythrocytes.

The major biological role of red blood cells is to carry oxygen to the tissues in the body. However, another role of the erythroid cell is to participate in the immune response. Mature erythrocytes from chickens express Toll-like receptors and several cytokines in response to stimulation of the immune system. We previously reported the application of a biochemical fractionation protocol to isolate highly enriched transcribed DNA from polychromatic erythrocytes from chickens. In conjunction with next-generation DNA, RNA sequencing, chromatin immunoprecipitation-DNA sequencing, and formaldehyde-assisted isolation of regulatory elements (FAIRE) sequencing, we identified the active chromosomal compartments and determined their structural signatures in relation to expression levels. Here, we present the detailed chromatin characteristics of erythroid genes participating in the innate immune response. Our studies revealed an atypical chromatin structure for several genes coding for Toll-like receptors, interleukins, and interferon regulatory factors. The body of these genes had nucleosome-free regions intermingled with nucleosomes modified with H3K4me3 and H3K27ac, suggesting a dynamic unstable chromatin structure. We further show that human genes involved in cell identity have gene bodies with the same chromatin-instability features as the chicken polychromatic erythrocyte genes participating in the innate immune response.

Synergism of open chromatin regions involved in regulating genes in Bombyx mori

The dynamic variability of transcription factors (TFs) and their binding sites makes it challenging to conduct genome-wide transcription regulation research. The silkworm Bombyx mori, which produces silk, is one of the most valuable model insects in the order Lepidoptera. The “opening” and “closing” of chromatin in different silk yield strains is associated with changes in silk production, making this insect a good model for studying the transcriptional regulation of genes. However, few studies have examined the open chromatin regions (OCRs) of silkworms, and studying OCR synergism and their function in silk production remains challenging. Here, we performed formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate OCRs from the silk glands of fifth-instar larvae of the DaZao and D872 strains. In total, 128,908 high confidence OCRs were identified and approximately 80% of OCRs were located in non-coding regions. OCRs upregulated adjacent genes and showed signal-dependent vulnerability to single-nucleotide polymorphisms. Mid- and low-signal OCRs were more likely to have single-nucleotide polymorphisms (SNP). Further, OCRs interacted with each other within a distance of 5 kb. We named the OCR interaction complex as the “cluster of related regions” (COREs). The functions of the CORE and its harbored OCRs showed some differences. Additionally, COREs enriched many silk protein synthesis-associated genes, some of which were upregulated. This study identified numerous high confidence regulation sites and synergistic regulatory modes of OCRs that affect adjacent genes. These results provide insight into silkworm transcriptional regulation and improve our understanding of cis-element cooperation.

Cooperation of PU.1 With IRF8 and NFATc1 Defines Chromatin Landscapes During RANKL-Induced Osteoclastogenesis.

Receptor activator of nuclear factor κB ligand (RANKL) induces osteoclast (OC) differentiation from bone marrow-derived macrophages (BMMs). The transcription factors nuclear factor of activated T cells 1 (NFATc1) and interferon regulatory factor (IRF) 8 play positive and negative roles, respectively, in this process. However, genomewide mapping of the active cis-regulatory elements regulating OC differentiation has not been performed, and little is known about the global landscape of OC-specific gene regulation. We used chromatin immunoprecipitation and formaldehyde-assisted isolation of regulatory elements followed by sequencing to show that PU.1 transcription factor binding motifs were overrepresented at active cis-regulatory regions in both murine BMMs and OCs, while IRF and NFAT binding motifs were selectively enriched at these regions in BMMs and OCs, respectively. We also found that RANKL induced the downregulation of Irf8 and upregulation of Nfatc1 expression, which was associated with dramatic alterations in histone modification. BMM-specific PU.1 binding sites were observed to overlap with IRF8 binding sites in BMMs, and this also occurred for OC-specific PU.1 binding sites and NFATc1 binding sites in OCs. The expression of genes with IRF8 peaks within BMM-specific PU.1 binding sites was significantly higher in BMMs than in OCs, while that of genes with NFATc1 peaks within OC-specific PU.1 binding sites was significantly higher in OCs than in BMMs. Our results suggest that PU.1 switches its transcription partner from IRF8 to NFATc1 and alters the binding regions during RANKL-induced osteoclastogenesis, which is associated with changes in epigenetic profiles and the control of cell type-specific gene expression. © 2019 American Society for Bone and Mineral Research.

High OGT activity is essential for MYC-driven proliferation of prostate cancer cells.

O-GlcNAc transferase (OGT) is overexpressed in aggressive prostate cancer. OGT modifies intra-cellular proteins via single sugar conjugation (O-GlcNAcylation) to alter their activity. We recently discovered the first fast-acting OGT inhibitor OSMI-2. Here, we probe the stability and function of the chromatin O-GlcNAc and identify transcription factors that coordinate with OGT to promote proliferation of prostate cancer cells.Methods: Chromatin immunoprecipitation (ChIP) coupled to sequencing (seq), formaldehyde-assisted isolation of regulatory elements, RNA-seq and reverse-phase protein arrays (RPPA) were used to study the importance of OGT for chromatin structure and transcription. Mass spectrometry, western blot, RT-qPCR, cell cycle analysis and viability assays were used to establish the role of OGT for MYC-related processes. Prostate cancer patient data profiled for both mRNA and protein levels were used to validate findings.Results: We show for the first time that OGT inhibition leads to a rapid loss of O-GlcNAc chromatin mark. O-GlcNAc ChIP-seq regions overlap with super-enhancers (SE) and MYC binding sites. OGT inhibition leads to down-regulation of SE-dependent genes. We establish the first O-GlcNAc chromatin consensus motif, which we use as a bait for mass spectrometry. By combining the proteomic data from oligonucleotide enrichment with O-GlcNAc and MYC ChIP-mass spectrometry, we identify host cell factor 1 (HCF-1) as an interaction partner of MYC. Inhibition of OGT disrupts this interaction and compromises MYC's ability to confer androgen-independent proliferation to prostate cancer cells. We show that OGT is required for MYC-mediated stabilization of mitotic proteins, including Cyclin B1, and/or the increased translation of their coding transcripts. This implies that increased expression of mRNA is not always required to achieve increased protein expression and confer aggressive phenotype. Indeed, high expression of Cyclin B1 protein has strong predictive value in prostate cancer patients (p=0.000014) while mRNA does not.Conclusions: OGT promotes SE-dependent gene expression. OGT activity is required for the interaction between MYC and HCF-1 and expression of MYC-regulated mitotic proteins. These features render OGT essential for the androgen-independent, MYC-driven proliferation of prostate cancer cells. Androgen-independency is the major mechanism of prostate cancer progression, and our study identifies OGT as an essential mediator in this process.

Using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) to Identify Functional Regulatory DNA in Insect Genomes.

Differential regulation of gene expression determines cell-type-specific function, making identification of the cis-regulatory elements that control gene expression a central goal of developmental biology. In addition, changes in the sequence of cis-regulatory elements are thought to drive changes in gene expression patterns between species, making comparisons of cis-regulatory element usage important for evolutionary biology as well. Due to the number of extant species and the incredible morphological diversity that they exhibit, insects are favorite model organisms for both developmental and evolutionary biologists alike. However, identifying cis-regulatory elements in insect genomes is challenging. Here, I describe a method termed FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements, followed by high-throughput sequencing) that can be used to identify functional DNA regulatory elements from developing insect tissues, genome-wide.

Identification of Aedes aegypti cis-regulatory elements that promote gene expression in olfactory receptor neurons of distantly related dipteran insects

BackgroundSophisticated tools for manipulation of gene expression in select neurons, including neurons that regulate sexually dimorphic behaviors, are increasingly available for analysis of genetic model organisms. However, we lack comparable genetic tools for analysis of non-model organisms, including Aedes aegypti, a vector mosquito which displays sexually dimorphic behaviors that contribute to pathogen transmission. Formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq) recently facilitated genome-wide discovery of putative A. aegypti cis-regulatory elements (CREs), many of which could be used to manipulate gene expression in mosquito neurons and other tissues. The goal of this investigation was to identify FAIRE DNA elements that promote gene expression in the olfactory system, a tissue of vector importance.ResultsEight A. aegypti CREs that promote gene expression in antennal olfactory receptor neurons (ORNs) were identified in a Drosophila melanogaster transgenic reporter screen. Four CREs identified in the screen were cloned upstream of GAL4 in a transgenic construct that is compatible with transformation of a variety of insect species. These constructs, which contained FAIRE DNA elements associated with the A. aegypti odorant coreceptor (orco), odorant receptor 1 (Or1), odorant receptor 8 (Or8) and fruitless (fru) genes, were used for transformation of A. aegypti. Six A. aegypti strains, including strains displaying transgene expression in all ORNs, subsets of these neurons, or in a sex-specific fashion, were isolated. The CREs drove transgene expression in A. aegypti that corresponded to endogenous gene expression patterns of the orco, Or1, Or8 and fru genes in the mosquito antenna. CRE activity in A. aegypti was found to be comparable to that observed in D. melanogaster reporter assays.ConclusionsThese results provide further evidence that FAIRE-seq, which can be paired with D. melanogaster reporter screening to test FAIRE DNA element activity in select tissues, is a useful method for identification of mosquito cis-regulatory elements. These findings expand the genetic toolkit available for the study of Aedes neurobiology. Moreover, given that the CREs drive comparable olfactory neural expression in both A. aegypti and D. melanogaster, it is likely that they may function similarly in multiple dipteran insects, including other disease vector mosquito species.

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

Arabidopsis RNA Polymerase V Mediates Enhanced Compaction and Silencing of Geminivirus and Transposon Chromatin during Host Recovery from Infection.

Plants employ RNA-directed DNA methylation (RdDM) and dimethylation of histone 3 lysine 9 (H3K9me2) to silence geminiviruses and transposable elements (TEs). We previously showed that canonical RdDM (Pol IV-RdDM) involving RNA polymerases IV and V (Pol IV and Pol V) is required for Arabidopsis thaliana to recover from infection with Beet curly top virus lacking a suppressor protein that inhibits methylation (BCTV L2-). Recovery, which is characterized by reduced viral DNA levels and symptom remission, allows normal floral development. Here, we used formaldehyde-assisted isolation of regulatory elements (FAIRE) to confirm that >90% of BCTV L2- chromatin is highly compacted during recovery, and a micrococcal nuclease-chromatin immunoprecipitation assay showed that this is largely due to increased nucleosome occupancy. Physical compaction correlated with augmented cytosine and H3K9 methylation and with reduced viral gene expression. We additionally demonstrated that these phenomena are dependent on Pol V and by extension the Pol IV-RdDM pathway. BCTV L2- was also used to evaluate the impact of viral infection on host loci, including repressed retrotransposons Ta3 and Athila6A Remarkably, an unexpected Pol V-dependent hypersuppression of these TEs was observed, resulting in transcript levels even lower than those detected in uninfected plants. Hypersuppression is likely to be especially important for natural recovery from wild-type geminiviruses, as viral L2 and AL2 proteins cause ectopic TE expression. Thus, Pol IV-RdDM targets both viral and TE chromatin during recovery, simultaneously silencing the majority of viral genomes and maintaining host genome integrity by enforcing tighter control of TEs in future reproductive tissues.IMPORTANCE In plants, RdDM pathways use small RNAs to target cytosine and H3K9 methylation, thereby silencing DNA virus genomes and transposable elements (TEs). Further, Pol IV-RdDM involving Pol IV and Pol V is a key aspect of host defense that can lead to recovery from geminivirus infection. Recovery is characterized by reduced viral DNA levels and symptom remission and thus allows normal floral development. Studies described here demonstrate that the Pol V-dependent enhanced viral DNA and histone methylation observed during recovery result in increased chromatin compaction and suppressed gene expression. In addition, we show that TE-associated chromatin is also targeted for hypersuppression during recovery, such that TE transcripts are reduced below the already low levels seen in uninfected plants. Thus, Pol IV-RdDM at once silences the majority of viral genomes and enforces a tight control over TEs which might otherwise jeopardize genome integrity in future reproductive tissue.

Defining Regulatory Elements in the Human Genome Using Nucleosome Occupancy and Methylome Sequencing (NOMe-Seq).

NOMe-seq (nucleosome occupancy and methylome sequencing) identifies nucleosome-depleted regions that correspond to promoters, enhancers, and insulators. The NOMe-seq method is based on the treatment of chromatin with the M.CviPI methyltransferase, which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin (GpCm does not occur in the human genome and therefore there is no endogenous background of GpCm). Following bisulfite treatment of the M.CviPI-methylated chromatin (which converts unmethylated Cs to Ts and thus allows the distinction of GpC from GpCm) and subsequent genomic sequencing, nucleosome-depleted regions can be ascertained on a genome-wide scale. The bisulfite treatment also allows the distinction of CpG from CmpG (most endogenous methylation occurs at CpG dinucleotides) and thus the endogenous methylation status of the genome can also be obtained in the same sequencing reaction. Importantly, open chromatin is expected to have high levels of GpCm but low levels of CmpG; thus, each of the two separate methylation analyses serve as independent (but opposite) measures which provide matching chromatin designations for each regulatory element.NOMe-seq has advantages over ChIP-seq for identification of regulatory elements because it is not reliant upon knowing the exact modifications on the surrounding nucleosomes. Also, NOMe-seq has advantages over DHS (DNase hypersensitive site)-seq, FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)-seq, and ATAC (Assay for Transposase-Accessible Chromatin)-seq because it also gives positioning information for several nucleosomes on either side of each open regulatory element. Here, we provide a detailed protocol for NOMe-seq that begins with the isolation of chromatin, followed by methylation of GpCs with M.CviPI and treatment with bisulfite, and ending with the creation of next generation sequencing libraries. We also include sequencing QC analysis metrics and bioinformatics steps that can be used to identify nucleosome-depleted regions throughout the genome.

Enhancer identification and activity evaluation in the red flour beetle, Tribolium castaneum.

Evolution of cis-regulatory elements (such as enhancers) plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods for studying enhancers in species other than established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, Tribolium castaneum To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of Tribolium using FAIRE (formaldehyde-assisted isolation of regulatory elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and at each stage. In addition, comparison of the FAIRE data with sets of computationally predicted (i.e. supervised cis-regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using nubbin in the wing and hunchback in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in Tribolium, and in a cross-species context. Together, these advances will facilitate investigation of cis-evolution and morphological diversity in Tribolium and other insects.

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Genome-wide open chromatin regions and their effects on the regulation of silk protein genes in Bombyx mori

Nucleosome-depleted open chromatin regions (OCRs) often harbor transcription factor (TF) binding sites that are associated with active DNA regulatory elements. To investigate the regulation of silk-protein genes, DNA molecules isolated from the silk glands of third-day fifth-instar silkworm larvae and embryo-derived (BmE) cells were subjected to formal dehyde-assisted isolation of regulatory elements (FAIRE) and high-throughput sequencing. In total, 68,000 OCRs were identified, and a number of TF-binding motifs were predicted. In particular, OCRs located near silk-protein genes contained potential binding sites for functional TFs. Moreover, many TFs were found to bind to clusters of OCRs upstream of silk-protein genes, and to regulate the expression of these genes. The expression of silk protein genes may be related not only to regulating TFs (such as fkh, Bmdimm, and Bmsage), but also to developmental and hormone-induced TFs (such as zen, eve, Br, and eip74ef). Elucidation of genome-wide OCRs and their regulatory motifs in silk protein genes will provide valuable data and clues for characterizing the mechanisms of transcriptional control of silk protein genes.

Analysis of chromatin accessibility in human epidermis identifies putative barrier dysfunction-sensing enhancers.

To identify putative gene regulatory regions that respond to epidermal injury, we mapped chromatin dynamics in a stratified human epidermis during barrier maturation and disruption. Engineered skin substitutes (ESS) cultured at the air-liquid interface were used as a model of developing human epidermis with incomplete barrier formation. The epidermal barrier stabilized following engraftment onto immunocompromised mice, and was compromised again upon injury. Modified formaldehyde-assisted isolation of regulatory elements (FAIRE) was used to identify accessible genomic regions characteristic of monolayer keratinocytes, ESS in vitro, grafted ESS, and tape-stripped ESS graft. We mapped differentiation- and maturation-associated changes in transcription factor binding sites enriched at each stage and observed overrepresentation of AP-1 gene family motifs in barrier-deficient samples. Transcription of TSLP, an important effector of immunological memory in response to allergen exposure, was dramatically elevated in our barrier-deficient samples. We identified dynamic DNA elements that correlated with TSLP induction and may contain enhancers that regulate TSLP. Two dynamic regions were located near the TSLP promoter and overlapped with allergy-associated SNPs rs17551370 and rs2289877, strongly implicating these loci in the regulation of TSLP expression in allergic disease. Additional dynamic chromatin regions ~250kb upstream of the TSLP promoter were found to be in high linkage disequilibrium with allergic disease SNPs. Taken together, these results define dynamic chromatin accessibility changes during epidermal development and dysfunction.

Dosage compensation and sex-specific epigenetic landscape of the X chromosome in the pea aphid

Background Heterogametic species display a differential number of sex chromosomes resulting in imbalanced transcription levels for these chromosomes between males and females. To correct this disequilibrium, dosage compensation mechanisms involving gene expression and chromatin accessibility regulations have emerged throughout evolution. In insects, these mechanisms have been extensively characterized only in Drosophila but not in insects of agronomical importance. Aphids are indeed major pests of a wide range of crops. Their remarkable ability to switch from asexual to sexual reproduction during their life cycle largely explains the economic losses they can cause. As heterogametic insects, male aphids are X0, while females (asexual and sexual) are XX.ResultsHere, we analyzed transcriptomic and open chromatin data obtained from whole male and female individuals to evaluate the putative existence of a dosage compensation mechanism involving differential chromatin accessibility of the pea aphid’s X chromosome. Transcriptomic analyses first showed X/AA and XX/AA expression ratios for expressed genes close to 1 in males and females, respectively, suggesting dosage compensation in the pea aphid. Analyses of open chromatin data obtained by Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE-seq) revealed a X chromosome chromatin accessibility globally and significantly higher in males than in females, while autosomes’ chromatin accessibility is similar between sexes. Moreover, chromatin environment of X-linked genes displaying similar expression levels in males and females—and thus likely to be compensated—is significantly more accessible in males.ConclusionsOur results suggest the existence of an underlying epigenetic mechanism enhancing the X chromosome chromatin accessibility in males to allow X-linked gene dose correction between sexes in the pea aphid, similar to Drosophila. Our study gives new evidence into the comprehension of dosage compensation in link with chromatin biology in insects and newly in a major crop pest, taking benefits from both transcriptomic and open chromatin data.