In eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization.

The histone variant histone H2A.X comprises up to 25% of the H2A complement in mammalian cells. It is rapidly phosphorylated following exposure of cells to double-strand break (DSB) inducing agents such as ionising radiation. Within minutes of DSB generation, H2AX molecules are phosphorylated in large chromatin domains flanking DNA double-strand breaks (DSBs); these domains can be observed by immunofluorescence microscopy and are termed gammaH2AX foci. H2AX phosphorylation is believed to have a role mounting an efficient cellular response to DNA damage. Theoretical considerations suggest an essentially random chromosomal distribution of X-ray induced DSBs, and experimental evidence does not consistently indicate otherwise. However, we observed an apparently uneven distribution of gammaH2AX foci following X-irradiation with regions of the nucleus devoid of foci.

Here we show that the Ino80 chromatin remodeling complex (Ino80C) directly prevents euchromatin from invading transcriptionally silent chromatin within intergenic regions and at the border of euchromatin and heterochromatin. Deletion of Ino80C subunits leads to increased H3K79 methylation and noncoding RNA polymerase II (Pol II) transcription centered at the Ino80C-binding sites. The effect of Ino80C is direct, as it blocks H3K79 methylation by Dot1 in vitro. Heterochromatin stimulates the binding of Ino80C in vitro and in vivo. Our data reveal that Ino80C serves as a general silencing complex that restricts transcription to gene units in euchromatin.

Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA double-strand break (DSB) repair in Arabidopsis thaliana. Our experimental system includes targets for DSB induction in euchromatic and heterochromatic genomic regions of hybrid F1 plants, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and HR repair. We present a series of evidence showing that targeted DSBs can be repaired via HR using a homologous chromosome as the template in various chromatin contexts including in pericentric regions. Targeted crossover was rare, but gene conversion events were the most frequent outcome of HR and were found in both "hot and cold" regions. The length of the conversion tracts was variable, ranging from 5 to 7505 bp. In addition, a typical feature of these tracks was that they often were interrupted. Our findings pave the way for the use of targeted gene-conversion for precise breeding.

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.

Heterozygous mutations or deletions in the human Euchromatin histone methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a neurodevelopmental disorder that is characterized by autistic-like features and severe intellectual disability (ID). Neurodevelopmental disorders including ID and autism may be related to deficits in activity-dependent wiring of brain circuits during development. Although Kleefstra syndrome has been associated with dendritic and synaptic defects in mice and Drosophila, little is known about the role of EHMT1 in the development of cortical neuronal networks. Here we used micro-electrode arrays and whole-cell patch-clamp recordings to investigate the impact of EHMT1 deficiency at the network and single cell level. We show that EHMT1 deficiency impaired neural network activity during the transition from uncorrelated background action potential firing to synchronized network bursting. Spontaneous bursting and excitatory synaptic currents were transiently reduced, whereas miniature excitatory postsynaptic currents were not affected. Finally, we show that loss of function of EHMT1 ultimately resulted in less regular network bursting patterns later in development. These data suggest that the developmental impairments observed in EHMT1-deficient networks may result in a temporal misalignment between activity-dependent developmental processes thereby contributing to the pathophysiology of Kleefstra syndrome.

Direct analogs of chemically modified bases that carry important epigenetic information, such as 5-methylcytosine (m5C)/5-methyldeoxycytosine (5mC), 5-hydroxymethylcytosine (hm5C)/5-hydroxymethyldeoxycytosine (5hmC), and N6-methyladenosine (m6A)/N6-methyldeoxyadenosine (6mA), are detected in both RNA and DNA, respectively. The modified base N4-acetylcytosine (ac4C) is well studied in RNAs, but its presence and epigenetic roles in cellular DNA have not been explored.

The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. This implies the presence of insulator elements that separate adjacent domains, allowing them to maintain different chromatin structures. We show that the Fun30 chromatin remodeler, Fft3, is essential for maintaining a proper chromatin structure at centromeres and subtelomeres. Fft3 is localized to insulator elements and inhibits euchromatin assembly in silent chromatin domains. In its absence, euchromatic histone modifications and histone variants invade centromeres and subtelomeres, causing a mis-regulation of gene expression and severe chromosome segregation defects. Our data strongly suggest that Fft3 controls the identity of chromatin domains by protecting these regions from euchromatin assembly.

Centromeres, sites of kinetochore assembly, are important for chromosome stability and integrity. Most eukaryotes have regional centromeres epigenetically specified by the presence of the histone H3 variant CENP-A. CENP-A chromatin is often surrounded by pericentromeric regions packaged into transcriptionally silent heterochromatin. Candida albicans, the most common human fungal pathogen, possesses small regional centromeres assembled into CENP-A chromatin. The chromatin state of C. albicans pericentromeric regions is unknown. Here, for the first time, we address this question. We find that C. albicans pericentromeres are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Pericentromeric chromatin is associated with nucleosomes that are highly acetylated, as found in euchromatic regions of the genome; and hypomethylated on H3K4, as found in heterochromatin. This intermediate chromatin state is inhibitory to transcription and partially represses expression of proximal genes and inserted marker genes. Our analysis identifies a new chromatin state associated with pericentromeric regions.

Transposable elements are found in the genomes of nearly all eukaryotes. The recent completion of the Release 3 euchromatic genomic sequence of Drosophila melanogaster by the Berkeley Drosophila Genome Project has provided precise sequence for the repetitive elements in the Drosophila euchromatin. We have used this genomic sequence to describe the euchromatic transposable elements in the sequenced strain of this species.

The activation of beta-cell genes, particularly of those encoding preproinsulin, requires an appropriate euchromatin (or "open") DNA template characterized by hypermethylation of Lys4 of histone H3. We hypothesized that this modification is maintained in islet beta-cells by the action of the histone methyltransferase Set7/9.

Major human alpha satellite DNA repeats are preferentially assembled within (peri)centromeric regions but are also dispersed within euchromatin in the form of clustered or short single repeat arrays. To study the evolutionary history of single euchromatic human alpha satellite repeats (ARs), we analyzed their orthologous loci across the primate genomes. The continuous insertion of euchromatic ARs throughout the evolutionary history of primates starting with the ancestors of Simiformes (45-60 Ma) and continuing up to the ancestors of Homo is revealed. Once inserted, the euchromatic ARs were stably transmitted to the descendant species, some exhibiting copy number variation, whereas their sequence divergence followed the species phylogeny. Many euchromatic ARs have sequence characteristics of (peri)centromeric alpha repeats suggesting heterochromatin as a source of dispersed euchromatic ARs. The majority of euchromatic ARs are inserted in the vicinity of other repetitive elements such as L1, Alu, and ERV or are embedded within them. Irrespective of the insertion context, each AR insertion seems to be unique and once inserted, ARs do not seem to be subsequently spread to new genomic locations. In spite of association with (retro)transposable elements, there is no indication that such elements play a role in ARs proliferation. The presence of short duplications at most of ARs insertion sites suggests site-directed recombination between homologous motifs in ARs and in the target genomic sequence, probably mediated by extrachromosomal circular DNA, as a mechanism of spreading within euchromatin.

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct "territories." Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.

Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.

Non-protein-coding DNA comprises the majority of animal genomes but its functions are largely unknown. We identified over 17,000 different tetranucleotide pairs in the Drosophila melanogaster genome that are over-represented at distances up to 100nt in conserved non-exonic sequences. Those exhibiting the highest information content in surrounding nucleotides were classified into five groups: tRNAs, motifs associated with histone genes, Suppressor-of-Hairy-wing binding sites, and two sets of previously unrecognized motifs (DLM3 and DLM4). There are hundreds to thousands of copies of DLM3 and DLM4, respectively, in the genome, located almost exclusively in non-coding regions. They have similar copy numbers among drosophilids, but are largely absent in other insects. DLM3 is likely a cis-regulatory element, whereas DLM4 sequences are capable of forming a short hairpin structure and are expressed as approximately 80nt RNAs. This work reports the existence of Drosophila genus-specific sequence motifs, and suggests that many more novel functional elements may be discovered in genomes using the general approach outlined herein.

The genome is packed into the cell nucleus in the form of chromatin. Biochemical approaches have revealed that chromatin is packed within domains, which group into larger domains, and so forth. Such hierarchical packing is equally visible in super-resolution microscopy images of large-scale chromatin organization. While previous work has suggested that chromatin is partitioned into distinct domains via microphase separation, it is unclear how these domains organize into this hierarchical packing. A particular challenge is to find an image analysis approach that fully incorporates such hierarchical packing, so that hypothetical governing mechanisms of euchromatin packing can be compared against the results of such an analysis. Here, we obtain 3D STED super-resolution images from pluripotent zebrafish embryos labeled with improved DNA fluorescence stains, and demonstrate how the hierarchical packing of euchromatin in these images can be described as multiplicative cascades. Multiplicative cascades are an established theoretical concept to describe the placement of ever-smaller structures within bigger structures. Importantly, these cascades can generate artificial image data by applying a single rule again and again, and can be fully specified using only four parameters. Here, we show how the typical patterns of euchromatin organization are reflected in the values of these four parameters. Specifically, we can pinpoint the values required to mimic a microphase-separated state of euchromatin. We suggest that the concept of multiplicative cascades can also be applied to images of other types of chromatin. Here, cascade parameters could serve as test quantities to assess whether microphase separation or other theoretical models accurately reproduce the hierarchical packing of chromatin.

In most eukaryotes, histone methylation patterns regulate chromatin architecture and function: methylation of histone H3 lysine-9 (H3K9) demarcates heterochromatin, whereas H3K4 methylation demarcates euchromatin. We show here that the S. pombe JmjC-domain protein Lid2 is a trimethyl H3K4 demethylase responsible for H3K4 hypomethylation in heterochromatin. Lid2 interacts with the histone lysine-9 methyltransferase, Clr4, through the Dos1/Clr8-Rik1 complex, which also functions in the RNA interference pathway. Disruption of the JmjC domain alone results in severe heterochromatin defects and depletion of siRNA, whereas overexpressing Lid2 enhances heterochromatin silencing. The physical and functional link between H3K4 demethylation and H3K9 methylation suggests that the two reactions act in a coordinated manner. Surprisingly, crossregulation of H3K4 and H3K9 methylation in euchromatin also requires Lid2. We suggest that Lid2 enzymatic activity in euchromatin is regulated through a dynamic interplay with other histone-modification enzymes. Our findings provide mechanistic insight into the coordination of H3K4 and H3K9 methylation.

C3G (RapGEF1) is a ubiquitously expressed guanine nucleotide exchange factor that functions in signaling pathways regulating cell proliferation, apoptosis, and actin reorganization. It is essential for differentiation and early embryonic development in mice. Overexpressed C3G shows predominant cytoplasmic localization, but endogenous C3G is a component of nuclear fractions in a variety of cell types. Coexpression of importin-α and inhibition of nuclear export by leptomycin B resulted in predominant nuclear localization of C3G. Functional NLSs, NES, and GSK3-β-dependent phosphorylation regulate its dynamic nuclear localization. C3G translocates to the nucleus in response to myogenic differentiation and sublethal dose of cisplatin. C3G is associated with chromatin and nuclear matrix fractions. Cells with C3G localized in the nucleus showed peripheralization of heterochromatin and reduced histone modifications associated with euchromatin. Short hairpin RNA-mediated depletion of C3G in epithelial cells resulted in reduced expression of CDK inhibitors and the histone demethylase KDM5A. Myoblast clones with CRISPR/Cas9-mediated knockout of C3G failed to show repression of histone marks and did not show up-regulation of myosin heavy chain and myotube formation when grown in differentiation medium. Our results document regulated nucleocytoplasmic exchange of C3G in response to physiological stimuli and provide insights into nuclear functions for C3G.

A detailed understanding of the principles of the structural organization of genetic material is of great importance for elucidating the mechanisms of differential regulation of genes in development. Modern ideas about the spatial organization of the genome are based on a microscopic analysis of chromatin structure and molecular data on DNA-DNA contact analysis using Chromatin conformation capture (3C) technology, ranging from the "polymer melt" model to a hierarchical folding concept. Heterogeneity of chromatin structure depending on its functional state and cell cycle progression brings another layer of complexity to the interpretation of structural data and requires selective labeling of various transcriptional states under nondestructive conditions. Here, we use a modified approach for replication timing-based metabolic labeling of transcriptionally active chromatin for ultrastructural analysis. The method allows pre-embedding labeling of optimally structurally preserved chromatin, thus making it compatible with various 3D-TEM techniques including electron tomography. By using variable pulse duration, we demonstrate that euchromatic genomic regions adopt a fiber-like higher-order structure of about 200 nm in diameter (chromonema), thus providing support for a hierarchical folding model of chromatin organization as well as the idea of transcription and replication occurring on a highly structured chromatin template.

The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.