Widespread chromatin context-dependencies of DNA double-strand break repair proteins.

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. Examples of the former are BRCA2 and POLL, and of the latter the FANC complex and ATM. Moreover, in a diversity of human cancer types, loss of several of these proteins alters the distribution of pathway-specific mutations between heterochromatin and euchromatin. Together, these results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner.

Topoisomerase VI participates in an insulator-like function that prevents H3K9me2 spreading.

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically down-regulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some transposable element loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine synthase methionine adenosyl transferase 3 (MAT3), which is required for H3K9me2. A Topoisomerase VI defect affects MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a possible mechanistic explanation to the essential role of Topoisomerase VI in the delimitation of chromatin domains.

Nanodosimetric Calculations of Radiation-Induced DNA Damage in a New Nucleus Geometrical Model Based on the Isochore Theory.

Double-strand breaks (DSBs) in nuclear DNA represents radiation-induced damage that has been identified as particularly deleterious. Calculating this damage using Monte Carlo track structure modeling could be a suitable indicator to better assess and anticipate the side-effects of radiation therapy. However, as already demonstrated in previous work, the geometrical description of the nucleus and the DNA content used in the simulation significantly influence damage calculations. Therefore, in order to obtain accurate results, this geometry must be as realistic as possible. In this study, a new geometrical model of an endothelial cell nucleus and DNA distribution according to the isochore theory are presented and used in a Monte Carlo simulation chain based on the Geant4-DNA toolkit. In this theory, heterochromatin and euchromatin compaction are distributed along the genome according to five different families (L1, L2, H1, H2, and H3). Each of these families is associated with a different hetero/euchromatin rate related to its compaction level. In order to compare the results with those obtained using a previous nuclear geometry, simulations were performed for protons with linear energy transfers (LETs) of 4.29 keV/µm, 19.51 keV/µm, and 43.25 keV/µm. The organization of the chromatin fibers at different compaction levels linked to isochore families increased the DSB yield by 6-10%, and it allowed the most affected part of the genome to be identified. These new results indicate that the genome core is more radiosensitive than the genome desert, with a 3-8% increase in damage depending on the LET. This work highlights the importance of using realistic distributions of chromatin compaction levels to calculate radio-induced damage using Monte Carlo simulation methods.

Comparison of structural variants in the whole genome sequences of two Medicago truncatula ecotypes: Jemalong A17 and R108.

BACKGROUND: Structural variants (SVs) constitute a large proportion of the genomic variation that results in phenotypic variation in plants. However, they are still a largely unexplored feature in most plant genomes. Here, we present the whole-genome landscape of SVs between two model legume Medicago truncatula ecotypes-Jemalong A17 and R108- that have been extensively used in various legume biology studies. RESULTS: To catalogue SVs, we first resolved the previously published R108 genome assembly (R108 v1.0) to chromosome-scale using 124 × Hi-C data, resulting in a high-quality genome assembly. The inter-chromosomal reciprocal translocations between chromosomes 4 and 8 were confirmed by performing syntenic analysis between the two genomes. Combined with the Hi-C data, it appears that these translocation events had a significant effect on chromatin organization. Using both whole-genome and short-read alignments, we identified the genomic landscape of SVs between the two genomes, some of which may account for several phenotypic differences, including their differential responses to aluminum toxicity and iron deficiency, and the development of different anthocyanin leaf markings. We also found extensive SVs within the nodule-specific cysteine-rich gene family which encodes antimicrobial peptides essential for terminal bacteroid differentiation during nitrogen-fixing symbiosis. CONCLUSIONS: Our results provide a near-complete R108 genome assembly and the first genomic landscape of SVs obtained by comparing two M. truncatula ecotypes. This may provide valuable genomic resources for the functional and molecular research of legume biology in the future.

Chromatin Velocity reveals epigenetic dynamics by single-cell profiling of heterochromatin and euchromatin.

Recent efforts have succeeded in surveying open chromatin at the single-cell level, but high-throughput, single-cell assessment of heterochromatin and its underlying genomic determinants remains challenging. We engineered a hybrid transposase including the chromodomain (CD) of the heterochromatin protein-1α (HP-1α), which is involved in heterochromatin assembly and maintenance through its binding to trimethylation of the lysine 9 on histone 3 (H3K9me3), and developed a single-cell method, single-cell genome and epigenome by transposases sequencing (scGET-seq), that, unlike single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), comprehensively probes both open and closed chromatin and concomitantly records the underlying genomic sequences. We tested scGET-seq in cancer-derived organoids and human-derived xenograft (PDX) models and identified genetic events and plasticity-driven mechanisms contributing to cancer drug resistance. Next, building upon the differential enrichment of closed and open chromatin, we devised a method, Chromatin Velocity, that identifies the trajectories of epigenetic modifications at the single-cell level. Chromatin Velocity uncovered paths of epigenetic reorganization during stem cell reprogramming and identified key transcription factors driving these developmental processes. scGET-seq reveals the dynamics of genomic and epigenetic landscapes underlying any cellular processes.

Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy.

BACKGROUND: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. MATERIAL AND METHODS: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). RESULTS: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. CONCLUSION: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

Integrated analysis reveals the alterations that LMNA interacts with euchromatin in LMNA mutation-associated dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a serious cardiac heterogeneous pathological disease, which may be caused by mutations in the LMNA gene. Lamins interact with not only lamina-associated domains (LADs) but also euchromatin by alone or associates with the lamina-associated polypeptide 2 alpha (LAP2α). Numerous studies have documented that LMNA regulates gene expression by interacting with LADs in heterochromatin. However, the role of LMNA in regulating euchromatin in DCM is poorly understood. Here, we determine the differential binding genes on euchromatin in DCM induced by LMNA mutation by performing an integrated analysis of bioinformatics and explore the possible molecular pathogenesis mechanism. RESULTS: Six hundred twenty-three and 4484 differential binding genes were identified by ChIP-seq technology. The ChIP-seq analysis results and matched RNA-Seq transcriptome data were integrated to further validate the differential binding genes of ChIP-seq. Five and 60 candidate genes involved in a series of downstream analysis were identified. Finally, 4 key genes (CREBBP, PPP2R2B, BMP4, and BMP7) were harvested, and these genes may regulate LMNA mutation-induced DCM through WNT/ß-catenin or TGFß-BMP pathways. CONCLUSIONS: We identified four key genes that may serve as potential biomarkers and novel therapeutic targets. Our study also illuminates the possible molecular pathogenesis mechanism that the abnormal binding between LMNA or LAP2α-lamin A/C complexes and euchromatin DNA in LMNA mutations, which may cause DCM through the changes of CREBBP, PPP2R2B, BMP4, BMP7 expressions, and the dysregulation of WNT/ß-catenin or TGFß-BMP pathways, providing valuable insights to improve the occurrence and development of DCM.

Euchromatin histone methyltransferase II (EHMT2) regulates the expression of ras-related GTP binding C (RRAGC) protein.

Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been implicated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5- Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells. [BMB Reports 2020; 53(11): 576-581].

Free energy-based model of CTCF-mediated chromatin looping in the human genome.

In recent years, high-throughput techniques have revealed considerable structural organization of the human genome with diverse regions of the chromatin interacting with each other in the form of loops. Some of these loops are quite complex and may encompass regions comprised of many interacting chain segments around a central locus. Popular techniques for extracting this information are chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and high-throughput chromosome conformation capture (Hi-C). Here, we introduce a physics-based method to predict the three-dimensional structure of chromatin from population-averaged ChIA-PET data. The approach uses experimentally-validated data from human B-lymphoblastoid cells to generate 2D meta-structures of chromatin using a dynamic programming algorithm that explores the chromatin free energy landscape. By generating both optimal and suboptimal meta-structures we can calculate both the free energy and additionally the relative thermodynamic probability. A 3D structure prediction program with applied restraints then can be used to generate the tertiary structures. The main advantage of this approach for population-averaged experimental data is that it provides a way to distinguish between the principal and the spurious contacts. This study also finds that euchromatin appear to have rather precisely regulated 2D meta-structures compared to heterochromatin. The program source-code is available at https://github.com/plewczynski/looper.

A critical role of telomere chromatin compaction in ALT tumor cell growth.

ALT tumor cells often contain abundant DNA damage foci at telomeres and rely on the alternative lengthening of telomeres (ALT) mechanism to maintain their telomeres. How the telomere chromatin is regulated and maintained in these cells remains largely unknown. In this study, we present evidence that heterochromatin protein 1 binding protein 3 (HP1BP3) can localize to telomeres and is particularly enriched on telomeres in ALT cells. HP1BP3 inhibition led to preferential growth inhibition of ALT cells, which was accompanied by telomere chromatin decompaction, increased presence of C-circles, more pronounced ALT-associated phenotypes and elongated telomeres. Furthermore, HP1BP3 appeared to participate in regulating telomere histone H3K9me3 epigenetic marks. Taken together, our data suggest that HP1BP3 functions on telomeres to maintain telomere chromatin and represents a novel target for inhibiting ALT cancer cells.

Heterochromatin protein 1α interacts with parallel RNA and DNA G-quadruplexes.

The eukaryotic genome is functionally organized into domains of transcriptionally active euchromatin and domains of highly compact transcriptionally silent heterochromatin. Heterochromatin is constitutively assembled at repetitive elements that include the telomeres and centromeres. The histone code model proposes that HP1α forms and maintains these domains of heterochromatin through the interaction of its chromodomain with trimethylated lysine 9 of histone 3, although this interaction is not the sole determinant. We show here that the unstructured hinge domain, necessary for the targeting of HP1α to constitutive heterochromatin, recognizes parallel G-quadruplex (G4) assemblies formed by the TElomeric Repeat-containing RNA (TERRA) transcribed from the telomere. This provides a mechanism by which TERRA can lead to the enrichment of HP1α at telomeres to maintain heterochromatin. Furthermore, we show that HP1α binds with a faster association rate to DNA G4s of parallel topology compared to antiparallel G4s that bind slowly or not at all. Such G4-DNAs are found in the regulatory regions of several oncogenes. This implicates specific non-canonical nucleic acid structures as determinants of HP1α function and thus RNA and DNA G4s need to be considered as contributors to chromatin domain organization and the epigenome.

Activity-dependent neuroprotective protein recruits HP1 and CHD4 to control lineage-specifying genes.

De novo mutations in ADNP, which encodes activity-dependent neuroprotective protein (ADNP), have recently been found to underlie Helsmoortel-Van der Aa syndrome, a complex neurological developmental disorder that also affects several other organ functions 1 . ADNP is a putative transcription factor that is essential for embryonic development 2 . However, its precise roles in transcriptional regulation and development are not understood. Here we show that ADNP interacts with the chromatin remodeller CHD4 and the chromatin architectural protein HP1 to form a stable complex, which we refer to as ChAHP. Besides mediating complex assembly, ADNP recognizes DNA motifs that specify binding of ChAHP to euchromatin. Genetic ablation of ChAHP components in mouse embryonic stem cells results in spontaneous differentiation concomitant with premature activation of lineage-specific genes and in a failure to differentiate towards the neuronal lineage. Molecularly, ChAHP-mediated repression is fundamentally different from canonical HP1-mediated silencing: HP1 proteins, in conjunction with histone H3 lysine 9 trimethylation (H3K9me3), are thought to assemble broad heterochromatin domains that are refractory to transcription. ChAHP-mediated repression, however, acts in a locally restricted manner by establishing inaccessible chromatin around its DNA-binding sites and does not depend on H3K9me3-modified nucleosomes. Together, our results reveal that ADNP, via the recruitment of HP1 and CHD4, regulates the expression of genes that are crucial for maintaining distinct cellular states and assures accurate cell fate decisions upon external cues. Such a general role of ChAHP in governing cell fate plasticity may explain why ADNP mutations affect several organs and body functions and contribute to cancer progression1,3,4. Notably, we found that the integrity of the ChAHP complex is disrupted by nonsense mutations identified in patients with Helsmoortel-Van der Aa syndrome, and this could be rescued by aminoglycosides that suppress translation termination 5 . Therefore, patients might benefit from therapeutic agents that are being developed to promote ribosomal read-through of premature stop codons6,7.

Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals.

Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.

The Histone Acetyltransferase Mst2 Protects Active Chromatin from Epigenetic Silencing by Acetylating the Ubiquitin Ligase Brl1.

Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report a positive feedback loop that preserves the transcription-competent state of RNA polymerase II-transcribed genes. We found that Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3-marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription and prevents ectopic heterochromatin assembly. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

The histone deubiquitinase OTLD1 targets euchromatin to regulate plant growth.

Histone monoubiquitination is associated with active chromatin and plays an important role in epigenetic regulation of gene expression in plants. Deubiquitinating enzymes remove the ubiquitin group from histones and thereby contribute to gene repression. The Arabidopsis thaliana genome encodes 50 deubiquitinases, yet only 2 of them-UBP26 and OTLD1, members of the USP/UBP (ubiquitin-specific protease and ubiquitin-binding protein) and OTU (ovarian tumor protease) deubiquitinase families-are known to target histones. Furthermore, UBP26 is the only plant histone deubiquitinase for which the functional role has been characterized in detail. We used gain- and loss-of-function alleles of OTLD1 to examine its role in the plant life cycle and showed that OTLD1 stimulates plant growth, increases cell size, and induces transcriptional repression of five major regulators of plant organ growth and development: GA20OX, WUS, OSR2, ARL, and ABI5 OTLD1 associated with chromatin at each of these target genes and promoted the removal of euchromatic histone acetylation, ubiquitination, and methylation marks. Thus, these data indicate that OTLD1 promotes the concerted epigenetic regulation of a set of genes that collectively limit plant growth.

A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha.

Lamins are components of the peripheral nuclear lamina and interact with heterochromatic genomic regions, termed lamina-associated domains (LADs). In contrast to lamin B1 being primarily present at the nuclear periphery, lamin A/C also localizes throughout the nucleus, where it associates with the chromatin-binding protein lamina-associated polypeptide (LAP) 2 alpha. Here, we show that lamin A/C also interacts with euchromatin, as determined by chromatin immunoprecipitation of euchromatin- and heterochromatin-enriched samples. By way of contrast, lamin B1 was only found associated with heterochromatin. Euchromatic regions occupied by lamin A/C overlap with those bound by LAP2alpha, and lack of LAP2alpha in LAP2alpha-deficient cells shifts binding of lamin A/C toward more heterochromatic regions. These alterations in lamin A/C-chromatin interactions correlate with changes in epigenetic histone marks in euchromatin but do not significantly affect gene expression. Loss of lamin A/C in heterochromatic regions in LAP2alpha-deficient cells, however, correlated with increased gene expression. Our data show a novel role of nucleoplasmic lamin A/C and LAP2alpha in regulating euchromatin.

APOBEC-Induced Cancer Mutations Are Uniquely Enriched in Early-Replicating, Gene-Dense, and Active Chromatin Regions.

An antiviral component of the human innate immune system-the APOBEC cytidine deaminases-was recently identified as a prominent source of mutations in cancers. Here, we investigated the distribution of APOBEC-induced mutations across the genomes of 119 breast and 24 lung cancer samples. While the rate of most mutations is known to be elevated in late-replicating regions that are characterized by reduced chromatin accessibility and low gene density, we observed a marked enrichment of APOBEC mutations in early-replicating regions. This unusual mutagenesis profile may be associated with a higher propensity to form single-strand DNA substrates for APOBEC enzymes in early-replicating regions and should be accounted for in statistical analyses of cancer genome mutation catalogs aimed at understanding the mechanisms of carcinogenesis as well as highlighting genes that are significantly mutated in cancer.

Protection of the genome and central protein-coding sequences by non-coding DNA against DNA damage from radiation.

Non-coding DNA comprises a very large proportion of the total genomic content in higher organisms, but its function remains largely unclear. Non-coding DNA sequences constitute the majority of peripheral heterochromatin, which has been hypothesized to be the genome's 'bodyguard' against DNA damage from chemicals and radiation for almost four decades. The bodyguard protective function of peripheral heterochromatin in genome defense has been strengthened by the results from numerous recent studies, which are summarized in this review. These data have suggested that cells and/or organisms with a higher level of heterochromatin and more non-coding DNA sequences, including longer telomeric DNA and rDNAs, exhibit a lower frequency of DNA damage, higher radioresistance and longer lifespan after IR exposure. In addition, the majority of heterochromatin is peripherally located in the three-dimensional structure of genome organization. Therefore, the peripheral heterochromatin with non-coding DNA could play a protective role in genome defense against DNA damage from ionizing radiation by both absorbing the radicals from water radiolysis in the cytosol and reducing the energy of IR. However, the bodyguard protection by heterochromatin has been challenged by the observation that DNA damage is less frequently detected in peripheral heterochromatin than in euchromatin, which is inconsistent with the expectation and simulation results. Previous studies have also shown that the DNA damage in peripheral heterochromatin is rarely repaired and moves more quickly, broadly and outwardly to approach the nuclear pore complex (NPC). Additionally, it has been shown that extrachromosomal circular DNAs (eccDNAs) are formed in the nucleus, highly detectable in the cytoplasm (particularly under stress conditions) and shuttle between the nucleus and the cytoplasm. Based on these studies, this review speculates that the sites of DNA damage in peripheral heterochromatin could occur more frequently and may be removed by repetitive elements in non-coding DNA through the formation of eccDNAs and expelled out of the nucleus to the cytoplasm via the NPC. Therefore, this review proposes that the genome and central protein-coding sequences are doubly protected by non-coding DNA in peripheral heterochromatin against DNA damage from radiation, which may be a novel protective role of non-coding DNA in genome defense.

DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells.

Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.

Genetic Determinants of Epigenetic Patterns: Providing Insight into Disease.

The field of epigenetics and our understanding of the mechanisms that regulate the establishment, maintenance and heritability of epigenetic patterns continue to grow at a remarkable rate. This information is providing increased understanding of the role of epigenetic changes in disease, insight into the underlying causes of these epigenetic changes and revealing new avenues for therapeutic intervention. Epigenetic modifiers are increasingly being pursued as therapeutic targets in a range of diseases, with a number of agents targeting epigenetic modifications already proving effective in diseases such as cancer. Although it is well established that DNA mutations and aberrant expression of epigenetic modifiers play a key role in disease, attention is now turning to the interplay between genetic and epigenetic factors in complex disease etiology. The role of genetic variability in determining epigenetic profiles, which can then be modified by environmental and stochastic factors, is becoming more apparent. Understanding the interplay between genetic and epigenetic factors is likely to aid in identifying individuals most likely to benefit from epigenetic therapies. This goal is coming closer to realization because of continual advances in laboratory and statistical tools enabling improvements in the integration of genomic, epigenomic and phenotypic data.