DNase I Hypersensitive Sites Research Articles

High-resolution mapping of open chromatin in the rice genome

Gene expression is controlled by the complex interaction of transcription factors binding to promoters and other regulatory DNA elements. One common characteristic of the genomic regions associated with regulatory proteins is a pronounced sensitivity to DNase I digestion. We generated genome-wide high-resolution maps of DNase I hypersensitive (DH) sites from both seedling and callus tissues of rice (Oryza sativa). Approximately 25% of the DH sites from both tissues were found in putative promoters, indicating that the vast majority of the gene regulatory elements in rice are not located in promoter regions. We found 58% more DH sites in the callus than in the seedling. For DH sites detected in both the seedling and callus, 31% displayed significantly different levels of DNase I sensitivity within the two tissues. Genes that are differentially expressed in the seedling and callus were frequently associated with DH sites in both tissues. The DNA sequences contained within the DH sites were hypomethylated, consistent with what is known about active gene regulatory elements. Interestingly, tissue-specific DH sites located in the promoters showed a higher level of DNA methylation than the average DNA methylation level of all the DH sites located in the promoters. A distinct elevation of H3K27me3 was associated with intergenic DH sites. These results suggest that epigenetic modifications play a role in the dynamic changes of the numbers and DNase I sensitivity of DH sites during development.

Quantitative analysis of genome-wide chromatin remodeling.

Recent high-throughput sequencing technologies have opened the door for genome-wide characterization of chromatin features at an unprecedented resolution. Chromatin accessibility is an important property that regulates protein binding and other nuclear processes. Here, we describe computational methods to analyze chromatin accessibility using DNaseI hypersensitivity by sequencing (DNaseI-seq). Although there are numerous bioinformatic tools to analyze ChIP-seq data, our statistical algorithm was developed specifically to identify significantly accessible genomic regions by handling features of DNaseI hypersensitivity. Without prior knowledge of relevant protein factors, one can discover genome-wide chromatin remodeling events associated with specific conditions or differentiation stages from quantitative analysis of DNaseI hypersensitivity. By performing appropriate subsequent computational analyses on a select subset of remodeled sites, it is also possible to extract information about putative factors that may bind to specific DNA elements within DNaseI hypersensitive sites. These approaches enabled by DNaseI-seq represent a powerful new methodology that reveals mechanisms of transcriptional regulation.

Integrating Structural and Functional Studies Leads to a New Model of β-Globin Activation That Suggests Distinct Initiation and Maintenance States

Abstract 349The human and mouse β-globin loci share a conserved structure in which the locus control region (LCR) and genes are flanked by three CTCF bound DNaseI hypersensitive sites (HSs); 3‘HS1 downstream, and 5‘HS5 and human and mouse orthologues HS-111 or HS-62 upstream. In mice HS-62 and 3‘HS1 delineate a DNase sensitive domain. During erythroid differentiation, high-level expression of the β-globin locus is associated with LCR-dependent re-localization of the locus from the nuclear periphery to the nucleoplasm, where it associates with foci of serine-phosphorylated PolII deemed transcription factories (TFs).To investigate the relationships among chromatin structure, nuclear localization and β-globin expression during human erythropoiesis, CD34 progenitor cells were differentiated and analyzed by ChIP-array, primary transcript FISH, immuno-FISH, and chromatin conformation capture, carbon copy (5C). Localization of the β-locus away from the nuclear periphery and to TFs, and detection of β-nascent transcripts are rare events at day 4 (proerythroblasts), whereas by day 15 (polychromatic erythroblasts), nearly all loci are centrally located, associated with TFs and actively expressing. Three megabase profiles of complementary active and repressive histone marks (H3 lysine 4 di-methylation (DiK4) and H3 lysine 27 tri-methylation) reveal that DiK4 is enriched in the LCR and adult genes in undifferentiated CD34 cells and nears maximal enrichment by day 4. Thus the chromatin landscape is set up prior to erythroid commitment and is increased at day 4, but shows little change with activation. These profiles also reveal a previously un-described 257kb domain spanning from HS-111 to +146 relative to the ε-gene cap, with CTCF bound at its boundaries.5C analysis reveals a high linkage frequency between the LCR and β-gene at day 4, prior to β-gene activation. Thus proximity may be necessary, but is not sufficient for high-level expression. In addition, the LCR and adult genes have frequent contact with surrounding regions, but interactions are sharply demarcated by HS-111 and +146, linking the above histone modification domain and 5C structure. The flanking regions HS-111, 3‘HS1 and +146 associate with the LCR and genes in an active chromatin hub (ACH)-like structure. By combining 5C with the Integrated Modeling Platform, a high-resolution three-dimensional (3D) model of chromatin structure was generated and revealed that the CTCF containing flanking regions, HS-111, 5’HS5, 3‘HS1 and +146 are in proximity and anchor loops of the intervening regions. The LCR and β-gene lie in close proximity (<100nm) within a tight chromatin globule. At day 15, 5C-interactions become more restricted. Throughout the 1Mb assayed there is a global decrease in linkages and, unlike the ACH model, associations of the flanking HSs with each other diminish. In contrast, the LCR and β-gene are more highly linked. While the distance between the LCR and β-gene remains under 100nm at day 15, most of the remaining 3D structure is less compact. One notable exception is with differentiation the LCR is in closer proximity to the β-gene 3‘enhancer. In addition, β-gene activation is associated with an increase in the contour length of the region, possibly correlating with DNaseI accessibility.In summary, our results reveal that acquisition of DiK4 precedes erythroid commitment. Enrichment of this active histone modification may occur in the nuclear periphery and is associated with a compact structure in which the flanking HSs, LCR and β-gene are in close proximity. Notably, close association of the LCR and β-gene precedes association with a TF and is not sufficient for expression. This suggests that if an ACH structure is important for function, its role may be limited to the initiation, but not the maintenance of gene expression. Alternatively, this compact structure may reflect the mafK-mediated recruitment of co-repressors to the LCR, as we demonstrated previously. The exchange of these repressors for activators could lead to the observed relocation from the periphery to TFs where high-level expression occurs and provides an explanation for the large change in expression that occurs despite a subtle change in the proximity of the LCR and β-gene. Preliminary studies on cell lines harboring intact, and LCR deficient chromosomes suggest the LCR may effect boundary element formation, domains of histone modifications and structure of the region. Disclosures:No relevant conflicts of interest to declare.

Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system

We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to -3861 bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning -3861 to -1569 bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately -1800 bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (-2112 to -1569 bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene.

Probabilistic modeling of Hi-C contact maps eliminates systematic biases to characterize global chromosomal architecture

Hi-C experiments measure the probability of physical proximity between pairs of chromosomal loci on a genomic scale. We report on several systematic biases that substantially affect the Hi-C experimental procedure, including the distance between restriction sites, the GC content of trimmed ligation junctions and sequence uniqueness. To address these biases, we introduce an integrated probabilistic background model and develop algorithms to estimate its parameters and renormalize Hi-C data. Analysis of corrected human lymphoblast contact maps provides genome-wide evidence for interchromosomal aggregation of active chromatin marks, including DNase-hypersensitive sites and transcriptionally active foci. We observe extensive long-range (up to 400 kb) cis interactions at active promoters and derive asymmetric contact profiles next to transcription start sites and CTCF binding sites. Clusters of interacting chromosomal domains suggest physical separation of centromere-proximal and centromere-distal regions. These results provide a computational basis for the inference of chromosomal architectures from Hi-C experiments.

Modulation of the Murine CD8 Gene Complex Following the Targeted Integration of Human CD2-Locus Control Region Sequences

The human CD2 (hCD2) locus control region (LCR) inserted in the mouse CD8 gene complex activates expression of the CD8 genes in T cell subsets in which the CD8 locus is normally silenced (e.g., CD4(+) single-positive T cells). In this article, we show that, in conditional mCD8/hCD2-LCR (CD8/LCR) knock-in mice, the continuous presence of the hCD2-LCR is required for this effect. Deletion of the inserted hCD2-LCR in a developmental stage and cell lineage-specific manner revealed that the temporary presence of the LCR during early development does not permanently alter the expression pattern of the CD8 genes. As a result, cells that have been affected by the insertion of the LCR can convert to their destined phenotype once the LCR is removed. DNaseI hypersensitive sites 1 and 2 of the hCD2-LCR influence the expression of the CD8 genes in a similar manner as does the full LCR, whereas insertion of hypersensitive site 3 alone of the LCR does not result in a changed expression pattern. This analysis revealed a dynamic interaction between the hCD2-LCR and the endogenous regulatory elements of the CD8 genes.

SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12

We have demonstrated that SlyA activates fimB expression and hence type 1 fimbriation, a virulence factor in Escherichia coli. SlyA is shown to bind to two operator sites (OSA1 and OSA2), situated between 194 and 167 base pairs upstream of the fimB transcriptional start site. fimB expression is derepressed in an hns mutant and diminished by a slyA mutation in the presence of H-NS only. H-NS binds to multiple sites in the promoter region, including two sites (H-NS2 and H-NS3) that overlap OSA1 and OSA2, respectively. Mutations that disrupt either OSA1 or OSA2 eliminate or reduce the activating effect of SlyA but have different effects on the level of expression. We interpret these results as reflecting the relative competition between SlyA and H-NS binding. Moreover we show that SlyA is capable of displacing H-NS from its binding sites in vitro. We suggest SlyA binding prevents H-NS binding to H-NS2 and H-NS3 and the subsequent oligomerization of H-NS necessary for full inhibition of fimB expression. In addition, we show that SlyA activates fimB expression independently of two other known regulators of fimB expression, NanR and NagC. It is demonstrated that the rarely used UUG initiation codon limits slyA expression and that low SlyA levels limit fimB expression. Furthermore, Western blot analysis shows that cells grown in rich-defined medium contain ∼1000 SlyA dimers per cell whereas those grown in minimal medium contain >20% more SlyA. This study extends our understanding of the role that SlyA plays in the host-bacterial relationship.

Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

Transcriptional and epigenetic effects of deleting large regions, alone or in combination, from their natural context in the chicken Ig-β gene

Previously, we used homologous recombination to delete six groups of cell-type-specific DNase I hypersensitive sites (DHSs), potential transcriptional and epigenetic regulators, scattered in and around the Ig-β gene from their natural context in B-lymphocyte-derived chicken DT40 cells. Simultaneous deletion of all six groups completely shut down transcription and epigenetic regulation of the Ig-β gene; therefore, the cooperation of the scattered regulatory regions was essential for transcription and epigenetic regulation. In this study, we regrouped the cell-type-specific DHSs of Ig-β, those in the original six deletions and three additional ones, into three larger regional groups—the long upstream region, the intron, and the long downstream region—and deleted these groups individually or in combination. Combinatorial deletion of all three regional groups decreased Ig-β mRNA levels to 0.4% of the control, which was significantly higher than < 0.1%, the level resulting from deletion of all six smaller groups. Histone H3 and H4 acetylation and H3K4 dimethylation levels at the Ig-β promoter were low in cells carrying deletions of all six smaller groups, but intermediate levels of acetylation and enhanced H3K4 dimethylation were observed in cells carrying deletions of all three larger groups. While CG methylation was definitely present at the Ig-β promoter in cells carrying all six smaller deletions, it was nearly absent from the Ig-β promoter in cells carrying all three larger deletions. Thus, combinatorial deletion of larger regulatory regions had less effect on transcription and epigenetic regulation at the chicken Ig-β gene than combinatorial deletion of shorter ones. Analysis of several combinatorial deletions, where combinations included two larger deletions and one smaller deletion, revealed the relative effects of each deletion on transcription of the Ig-β gene. Investigation of the CG methylation status at the Ig-β promoter in one combinatorial deletion demonstrated that USI was involved in the maintenance of CG methylation.

Nucleosome remodeling of regulatory region of rat tryptophan dioxygenase gene during transcription in vivo

Two DNase I-hypersensitive sites are identified in the regulatory region of the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically under the control of glucocorticoid hormones. One of these hypersensitive regions is identified at position −470 and the other is located in the vicinity of the first upstream gene. The micrococcal nuclease digestion DNA pattern of this region shows disturbances in the regular cleavage and accumulation of the DNA fragment with higher electrophoretic mobility than that of nucleosomal DNA. However, control experiments on the reconstruction of nucleosomal core particles in vitro have shown that the same DNA region could be involved in the formation of classic core particles. The obtained data indicate that, in vivo, in the regulatory tdo gene region, remodeling of the chromatin nucleosomal structure occurs in the state of native transcription and is accompanied by the destruction of canonic nucleosomes. These changes in the chromatin structure seem to reflect the state of the gene’s competence for transcription and the process of transcription.

Regulatory Elements in Noncoding DNA in the Chromosome 9p21 Locus

1. Harismendy O, Notani D, Song X, Rahim NG, Tanasa B, Heintzman N, Ren B, Fu XD, Topol EJ, Rosenfeld MG, Frazer KA. 9p21 DNA variants associated with coronary artery disease impair interferon-g signalling response . Nature. 2011;470(7333):264–268. PMID: 21307941. ### Study Hypothesis A novel locus on chromosome 9p21 has been identified in numerous genome-wide association studies as the most highly associated with coronary artery disease (CAD) and myocardial infarction. Understanding the mechanism by which the locus influences disease has been a challenge because the locus lies entirely within noncoding DNA—in a so-called “gene desert” at a long distance from the closest genes. The authors hypothesized that common DNA variants in the locus alter transcriptional regulatory elements that engage in long-range interactions with and modulate expression of genes outside of the locus.1 ### How Was the Hypothesis Tested? The authors integrated multiple data sets to identify potential transcriptional regulatory elements in the 9p21 CAD locus. They searched in various types of human cells for chromatin modification “marks” of 3 types—promoters marked by enrichment of histone H3 trimethylated at lysine 4 (H3K4me3); insulators marked by CTCF-binding sites; and enhancers marked by enrichment of histone H3 monomethylated at lysine 4 (H3K4me1), or p300- and MED1-binding sites, or presence of DNase hypersensitivity sites. They …

Chromatin structure at active genes

For gene switching, chromatin must be manipulated, a process initiating at key cis-controlling elements, labelled operationally as DNaseI hypersensitive sites (DHS). This minireview series expands on how the remodelling of chromatin structure comes about and the consequences for gene activation and repression The conventional wisdom embodied (but not, in this age of rapid development, embalmed) in many textbooks has it that chromatin exists in cells either in a linker histone-containing condensed fibre of nucleosomes, diameter 30–40 nm, characteristic of heterochromatin, or as an open extended beads-on-a-string like structure, characteristic of all active genes. Even brief thought gives the lie to the second concept, since in very active tissues such as brain or stem cells in which, say, 40% of all genes are active, were they all fully extended throughout their transcribed length the nuclear volume could not possibly accommodate them. Since the chromatin of an active gene must be opened up at the moment of polymerase passage, it follows that continuous manipulation of chromatin structure must occur at active genes. Indeed, using the criterion of DNaseI accessibility we have recently shown that the overall state of certain active genes differs little from that of an immediately adjacent heterochromatin segment, experimentally demonstrated to be in the form of the 30 nm fibre. This time-averaged observation does not apply, however, to cis-controlling elements such as promoters and enhancers that, since the seminal observations of Hal Weintraub and Mark Groudine in 1976, have been shown to be locally hypersensitive to DNaseI digestion, i.e. in a more open conformation. Thus whereas the presence of bound histones, including the H1 linker, does not in general present a barrier to passage of the polymerase complex through an active gene, access to the DNA at cis elements requires a more open structure. It is worth emphasizing that DNaseI remains a valuable tool in studying local chromatin structure, not only for defining key control points as DNaseI hypersensitive sites, but also for locating the rare segments of open nucleosomal chromatin, i.e. genuine beads-on-a-string structures, sometimes found in the region of promoters or enhancers. This results from the fact that, whereas MNase digestion of nuclei always gives rise to conventional nucleosomal ladders, whether the underlying structure is the 30 nm fibre or open nucleosomal beads, DNaseI does not generate ladders from 30 nm chromatin fibres (the bulk of the chromatin) but it does from open beaded structures. Overall, the controlled manipulation of chromatin structure at such locations, in particular the displacement or even eviction of nucleosomes to permit the ordered recruitment of transcription factors, is a complex process requiring many interdependent components, in particular chromatin remodelling and histone modifying complexes. The following three minireviews address the problem of chromatin manipulation. The first, by Peter Cockerill, takes an overview of the chromatin structure at active genes and then illustrates this with the process of activating the human GM-CSF (granulocyte/macrophage colony stimulating factor) gene during T cell development, emphasizing how the binding of transcription factors is dependent on nucleosome displacement and is accompanied by the appearance of DNaseI hypersensitive sites. The paper by Gordon Hager and colleagues reviews how, following treatment of cells with steroid hormones, the chromatin structure of responsive genes changes in consequence of binding by the immediate hormone targets, the nuclear receptors. Finally, Stefan Dimitrov and his colleague Jan Bednar relate how the bare bone mechanics of chromatin manipulation could occur as nucleosomes and chromatin fibres are put under stress by pulling and tugging whilst held by optical and magnetic tweezers. In a field that can be confounded by the wide variation of experimental conditions, the authors very helpfully provide a summarizing table of published results.

Gammaretroviral Integration into Nucleosomal Target DNA In Vivo

Some of the earliest studies of retroviral integration targeting reported that sites of gammaretroviral DNA integration were positively correlated with DNase I-hypersensitive sites in chromatin. This led to the suggestion that open chromatin was favorable for integration. More recent deep sequencing experiments confirmed that gammaretroviral integration sites and DNase I cleavage sites are associated in genome-wide surveys. Paradoxically, in vitro studies of integration show that nucleosomal DNA is actually favored over naked DNA, raising the question of whether integration target DNA in chromosomes is wrapped in nucleosomes or nucleosome free. In this study we examined gammaretroviral integration by infecting primary human CD4(+) T lymphocytes with a murine leukemia virus (MLV)-based retroviral vector or xenotropic murine leukemia virus-related virus (XMRV), and isolated 32,585 unique integration sites using ligation-mediated PCR and 454 pyrosequencing. CD4(+) T lymphocytes were chosen for study because of the particularly dense genome-wide mapping of chromatin features available for comparison. Analysis relative to predicted nucleosome positions showed that gammaretroviruses direct integration into outward-facing major grooves on nucleosome-wrapped DNA, similar to the integration pattern of HIV. Also, a suite of histone modifications correlated with gene activity are positively associated with integration by both MLV and XMRV. Thus, we conclude that favored integration near DNase I-hypersensitive sites does not imply that integration takes place exclusively in nucleosome-free regions.

Adipogenic hotspots: where the action is

EMBO J 30 8, 1459–1472 (2011); published online March222011 [PMC free article] [PubMed] Transcription factors (TFs) orchestrate cell-fate decisions by programming the expression of many genes, including those of other TFs that further drive lineage specification. C/EBP proteins and glucocorticoid receptor (GR) activate transcription of the master regulator of adipocyte differentiation, PPARγ. A study in this issue of The EMBO Journal sheds light on how C/EBPs, GR, STAT5, and RXR cooperate during early adipogenesis to globally remodel the epigenome. Adipocytes, the major lipid-containing component of adipose tissue, benefit metabolism by coordinating lipid and glucose homoeostasis. They are post-mitotic, terminally differentiated cells derived from mesenchymal precursors. In cell culture models, temporary exposure of preadipocytes to a mix of insulin, glucocorticoid, and an inducer of cAMP signaling triggers adipogenesis, changing the expression of hundreds of structural genes, and a variety of TFs (Farmer, 2006). Upon their induction, C/EBPs and PPARγ occupy sites near most of the genes that are upregulated during adipogenesis, including their own and each other's genes, forming a transcription network that helps to explain how they establish and maintain the adipocyte phenotype (Lefterova et al, 2008; Nielsen et al, 2008). Comparative analysis of chromatin state maps at different stages of adipogenesis has recently provided further insight into an adipogenic transcription network (Mikkelsen et al, 2010; Steger et al, 2010). Thousands of putative preadipoctye- and adipocyte-specific cis-regulatory regions were identified through examination of several histone modifications associated with transcriptionally active or repressed chromatin. Bioinformatics identified enriched TF motifs, and PLZF, SRF (Mikkelsen et al, 2010) and GR (Steger et al, 2010) were validated as sequence-specific regulators of adipogenesis. Mandrup and colleagues now present a genome-wide analysis of chromatin remodeling events occurring during adipocyte differentiation (Siersbaek et al, 2011). By monitoring differential sensitivity to DNase I, they detected dynamic changes to chromatin, identified enriched TF motifs at DNase I hypersensitive sites (DHSs), and validated binding of C/EBP proteins, GR, RXR, and STAT5 to the regions remodeled at early time points. Remarkably, these TFs co-localize at over 900 regions of open chromatin, which they termed ‘hotspots'. Most of the hotspots are transiently open only during the early stages of adipogenesis, and many of these correspond to enhancer regions marked by transient histone acetylation, TF binding, and coactivator recruitment that define an epigenomic transition state during adipogenesis (Figure 1A) (Steger et al, 2010). Figure 1 Chromatin opening during adipogenesis. Temporary exposure of preadipocytes to adipogenic stimuli induces DNase I hypersensitive ‘hotspots' with characteristics of enhancers including binding of multiple TFs, coactivator recruitment (e.g. p300), ... Interestingly, chromatin sites that open early and remain open through adipogenesis are frequently occupied by PPARγ in adipocytes (Figure 1B), suggesting that early adipogenic TFs may facilitate its binding. It is important to note, however, that most PPARγ-binding sites lie outside of early DHSs, leading to the possibility that PPARγ does not require pioneer factors to open up chromatin before gaining access to these targets (Figure 1C). Indeed, forced expression of PPARγ in preadipocytes caused binding to regions normally occupied only in adipocytes, which was accompanied by increased DNA accessibility and histone acetylation (Lefterova et al, 2010). It is intriguing to compare adipogenic hotspots with another recently identified DNA element termed highly occupied target (HOT) regions (Gerstein et al, 2010; modENCODE Consortium et al, 2010). HOT regions bind many different TFs (at least 15 in Caenorhabditis elegans) but are not enriched in the known motifs to which they bind, suggesting that the interactions are indirect. Adipogenic hotspots appear to be functionally distinct from HOT regions, as they display enrichment for motifs and tissue-specific expression of targets. Yet, the existence of hotspots and HOT regions indicates that genomic binding of many TFs may not be distributed evenly across sites containing cognate binding motifs, but rather, concentrated at a smaller number of regions of open chromatin. This may in part explain the specificity of TF binding that goes far beyond simple recognition of high-affinity motifs in the genome (Carroll et al, 2005). Adipogenic hotspots likely regulate gene expression, but it is unknown whether they function solely in this regard. Could TF occupancy accompanied by increased chromatin accessibility and enriched histone modification define all active enhancers? An answer awaits development of high-throughput methods that determine the function of putative cis-regulatory elements. Indeed, although many DHSs co-localize with active histone modifications, thousands are unmarked, and it is possible that various combinations of chromatin features may signal distinct three-dimensional structures that positively or negatively regulate mRNA and/or non-coding RNA synthesis as well as DNA replication and repair. Future studies will need to determine all the action going on at the hotspots.

The AP-1 binding sites located in the pol gene intragenic regulatory region of HIV-1 are important for viral replication.

Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.

DT40 knock-out and knock-in studies determine the regions necessary and sufficient for transcription and epigenetic conversion of the chicken Ig-β gene

The chicken Ig-β locus is organized by three cell-type-specific genes and two ubiquitously expressed genes. B-cell-specific DNase I hypersensitive sites (DHS) in that locus, including three present inside the flanking gene, were grouped into six regions and deleted. The deletions decreased Ig-β mRNA content to <0.1% of that of normal DT40 cells and converted epigenetic parameters such as histone modifications, CG methylation and DNase I hypersensitivity into inactive states. Knocked-in DHS regions into knock-out cells reactivated both transcription of the Ig-β gene and epigenetic parameters. Thus, the collaboration of the scattered regulatory regions was essential and sufficient not only for B-cell-specific transcription of the Ig-β gene, but also for the conversion of epigenetic parameters. On the basis of the knock-in studies, we determined the regions involved in the conversion and maintenance of the epigenetic parameters. These scattered regulatory regions were limited in vicinity such as in an intron of the gene, in the intergenic regions and in the introns of a flanking gene.

Parachuting in the epigenome: the biology of gene vector insertion profiles in the context of clinical trials

Retroviral pre-integration complexes (PICs) provide a most efficient mechanism to integrate foreign DNA into cellular chromatin. This has made retrovirus-based vectors a preferred tool for gene delivery in therapeutic settings where the chromosomal integration of a recombinant expression cassette that encodes a protein of interest can lead to a long-lasting correction of monogenetic diseases (so-called gene addition strategy). However, the efficiency of retroviral gene addition comes at the expense of a lack of precision in the choice of the integration site. Insertional mutagenesis with potential activation of proto-oncogenes as a first hit in a multistep scenario of cancer development thus represents one of the major hurdles to a more widespread exploration of gene-based treatments (Kustikova et al, 2010). To date, four clinical trials were reported to be associated with severe adverse reactions induced by insertional mutagenesis in haematopoietic stem and progenitor cells (HSC/P); two targeting the X-linked form of severe combined immunodeficiency (SCID-X1), one targeting chronic granulomatous disease, and most recently another trial exploring gene therapy for the Wiskott–Aldrich-Syndrome. In contrast, in the SCID caused by mutations in the gene encoding the metabolic enzyme adenosine deaminase (ADA), retroviral gene addition so far has been free of such complications. Furthermore, numerous trials using similar gene vectors to transfer genes into mature T cells have not been complicated by clonal outgrowth. This explains the great interest in a deeper understanding of retroviral vector–host interactions in the therapeutic setting of SCID-ADA.

A BEAF dependent chromatin domain boundary separates myoglianin and eyeless genes of Drosophila melanogaster

Precise transcriptional control is dependent on specific interactions of a number of regulatory elements such as promoters, enhancers and silencers. Several studies indicate that the genome in higher eukaryotes is divided into chromatin domains with functional autonomy. Chromatin domain boundaries are a class of regulatory elements that restrict enhancers to interact with appropriate promoters and prevent misregulation of genes. While several boundary elements have been identified, a rational approach to search for such elements is lacking. With a view to identifying new chromatin domain boundary elements we analyzed genomic regions between closely spaced but differentially expressed genes of Drosophila melanogaster. We have identified a new boundary element between myoglianin and eyeless, ME boundary, that separates these two differentially expressed genes. ME boundary maps to a DNaseI hypersensitive site and acts as an enhancer blocker both in embryonic and adult stages in transgenic context. We also report that BEAF and GAF are the two major proteins responsible for the ME boundary function. Our studies demonstrate a rational approach to search for potential boundaries in genomic regions that are well annotated.

Integration profile of retroviral vector in gene therapy treated patients is cell‐specific according to gene expression and chromatin conformation of target cell

The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor ‘vector-on-host’ effects in gene therapy (GT) trials but also provides crucial information about ‘host-on-vector’ influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT.

Dynamic reprogramming of chromatin accessibility during Drosophilaembryo development

BackgroundThe development of complex organisms is believed to involve progressive restrictions in cellular fate. Understanding the scope and features of chromatin dynamics during embryogenesis, and identifying regulatory elements important for directing developmental processes remain key goals of developmental biology.ResultsWe used in vivo DNaseI sensitivity to map the locations of regulatory elements, and explore the changing chromatin landscape during the first 11 hours of Drosophila embryonic development. We identified thousands of conserved, developmentally dynamic, distal DNaseI hypersensitive sites associated with spatial and temporal expression patterning of linked genes and with large regions of chromatin plasticity. We observed a nearly uniform balance between developmentally up- and down-regulated DNaseI hypersensitive sites. Analysis of promoter chromatin architecture revealed a novel role for classical core promoter sequence elements in directing temporally regulated chromatin remodeling. Another unexpected feature of the chromatin landscape was the presence of localized accessibility over many protein-coding regions, subsets of which were developmentally regulated or associated with the transcription of genes with prominent maternal RNA contributions in the blastoderm.ConclusionsOur results provide a global view of the rich and dynamic chromatin landscape of early animal development, as well as novel insights into the organization of developmentally regulated chromatin features.