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Metazoan transcription factors distinguish their response elements from a large excess of similar sequences. We explored underlying principles of DNA shape read-out and factor cooperativity in chromatin using a unique experimental system. We reconstituted chromatin on Drosophila genomes in extracts of preblastoderm embryos, mimicking the naïve state of the zygotic genome prior to developmental transcription activation. We then compared the intrinsic binding specificities of three recombinant transcription factors, alone and in combination, with GA-rich recognition sequences genome-wide. For MSL2, all functional elements reside on the X chromosome, allowing to distinguish physiological elements from non-functional ‘decoy’ sites. The physiological binding profile of MSL2 is approximated through interaction with other factors: cooperativity with CLAMP and competition with GAF, which sculpts the profile by occluding non-functional sites. An extended DNA shape signature is differentially read out in chromatin. Our results reveal novel aspects of target selection in a complex chromatin environment.

The dosage compensation complex (DCC) ofDrosophilaidentifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from theD. melanogastermodel: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. We found that inD. virilis, a species separated frommelanogasterby 40 million years of evolution, all principles are active but contribute differently to X specificity. Inmelanogaster, the DCC subunit MSL2 evolved intrinsic DNA-binding selectivity for rare PionX sites, which mark the X chromosome. Invirilis, PionX motifs are abundant and not X-enriched. Accordingly, MSL2 lacks specific recognition. Here, roX2 RNA plays a more instructive role, counteracting a nonproductive interaction of CLAMP and modulating DCC binding selectivity. Remarkably, roX2 triggers a stable chromatin binding mode characteristic of DCC. Evidently, X-specific regulation is achieved by divergent evolution of protein, DNA, and RNA components.

Histone variants are a universal means to alter the biochemical properties of nucleosomes, implementing local changes in chromatin structure. H2A.Z, one of the most conserved histone variants, is incorporated into chromatin by SWR1-type nucleosome remodelers. Here, we summarize recent advances toward understanding the transcription-regulatory roles of H2A.Z and of the remodeling enzymes that govern its dynamic chromatin incorporation. Tight transcriptional control guaranteed by H2A.Z nucleosomes depends on the context provided by other histone variants or chromatin modifications, such as histone acetylation. The functional cooperation of SWR1-type remodelers with NuA4 histone acetyltransferase complexes, a recurring theme during evolution, is structurally implemented by species-specific strategies.

The evolution of brain complexity correlates with an increased expression of long, noncoding (lnc) RNAs in neural tissues. Although prominent examples illustrate the potential of lncRNAs to scaffold and target epigenetic regulators to chromatin loci, only few cases have been described to function during brain development. We present a first functional characterization of the lncRNA

The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, we developed vitRIP, an unbiased, transcriptome-wide in vitro assay that reveals RNA binding specificity. We found that MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.

Understanding how the genome is structurally organized as chromatin is essential for understanding its function. Here, we review recent developments that allowed the readdressing of old questions regarding the primary level of chromatin structure, the arrangement of nucleosomes along the DNA and the folding of the nucleosome fiber in nuclear space. In contrast to earlier views of nucleosome arrays as uniformly regular and folded, recent findings reveal heterogeneous array organization and diverse modes of folding. Local structure variations reflect a continuum of functional states characterized by differences in post-translational histone modifications, associated chromatin-interacting proteins and nucleosome-remodeling enzymes. Technological advances have led to new insights into genome-wide arrangements of nucleosomes along the DNA and the folding of the chromosome fiber in nuclear space, revealing unexpected diversity.

Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to γH2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The γH2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range γH2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei.

Transcription regulators select their genomic binding sites from a large pool of similar, non-functional sequences. Although general principles that allow such discrimination are known, the complexity of DNA elements often precludes a prediction of functional sites. The process of dosage compensation in Drosophila allows exploring the rules underlying binding site selectivity. The male-specific-lethal (MSL) Dosage Compensation Complex (DCC) selectively binds to some 300 X chromosomal ‘High Affinity Sites’ (HAS) containing GA-rich ‘MSL recognition elements’ (MREs), but disregards thousands of other MRE sequences in the genome. The DNA-binding subunit MSL2 alone identifies a subset of MREs, but fails to recognize most MREs within HAS. The ‘Chromatin-linked adaptor for MSL proteins’ (CLAMP) also interacts with many MREs genome-wide and promotes DCC binding to HAS. Using genome-wide DNA-immunoprecipitation we describe extensive cooperativity between both factors, depending on the nature of the binding sites. These are explained by physical interaction between MSL2 and CLAMP. In vivo, both factors cooperate to compete with nucleosome formation at HAS. The male-specific MSL2 thus synergises with a ubiquitous GA-repeat binding protein for refined X/autosome discrimination.

Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In Drosophila, the single DOMINO chromatin regulator complex is thought to combine both activities via an unknown mechanism. Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. Drosophila generates the same diversity by alternative splicing of a single gene., eLife digest Cells contain a large number of proteins that control the activity of genes in response to various signals and changes in their environment. Often these proteins work together in groups called complexes. In the fruit fly Drosophila melanogaster, one of these complexes is called DOMINO. The DOMINO complex alters gene activity by interacting with other proteins called histones which influence how the genes are packaged and accessed within the cell. DOMINO works in two separate ways. First, it can replace certain histones with other variants that regulate genes differently. Second, it can modify histones by adding a chemical marker to them, which alters how they interact with genes. It was not clear how DOMINO can do both of these things and how that is controlled; but it was known that cells can make two different forms of the central component of the complex, called DOM-A and DOM-B, which are both encoded by the same gene. Scacchetti et al. have now studied fruit flies to understand the activities of these forms. This revealed that they do have different roles and that gene activity in cells changes if either one is lost. The two forms operate as part complexes with different compositions and only DOM-A includes the TIP60 enzyme that is needed to modify histones. As such, it seems that DOM-B primarily replaces histones with variant forms, while DOM-A modifies existing histones. This means that each form has a unique role associated with each of the two known behaviors of this complex. The presence of two different DOMINO complexes is common to flies and, probably, other insects. Yet, in other living things, such as mammals and yeast, their two roles are carried out by protein complexes originating from two distinct genes. This illustrates a concept called convergent evolution, where different organisms find different solutions for the same problem. As such, these findings provide an insight into the challenges encountered through evolution and the diverse solutions that have developed. They will also help us to understand the ways in which protein activities can adapt to different needs over evolutionary time.

In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, such as dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1’s targeting to chromatin depends on a PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). JASPer-JIL-1 (JJ)-complex is the major form of kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes, to modulate transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identify interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation but also coordinates chromatin-based regulation in the transcribed part of the genome., The chromosomal kinase JIL-1 is responsible for interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from heterochromatinisation. Here, the authors show that JIL-1 is stabilized and anchored to active genes and telomeric transposons by JASPer, which binds to H3K36me3 nucleosomes via its PWWP domain.

In Drosophila melanogaster males, X-chromosome monosomy is compensated by chromosome-wide transcription activation. We found that complete dosage compensation during embryogenesis takes surprisingly long and is incomplete even after 10 h of development. Although the activating dosage compensation complex (DCC) associates with the X-chromosome and MOF acetylates histone H4 early, many genes are not compensated. Acetylation levels on gene bodies continue to increase for several hours after gastrulation in parallel with progressive compensation. Constitutive genes are compensated earlier than developmental genes. Remarkably, later compensation correlates with longer distances to DCC binding sites. This time-space relationship suggests that DCC action on target genes requires maturation of the active chromosome compartment.

Regular successions of positioned nucleosomes, or phased nucleosome arrays (PNAs), are predominantly known from transcriptional start sites (TSSs). It is unclear whether PNAs occur elsewhere in the genome. To generate a comprehensive inventory of PNAs for Drosophila, we applied spectral analysis to nucleosome maps and identified thousands of PNAs throughout the genome. About half of them are not near TSSs and are strongly enriched for an uncharacterized sequence motif. Through genome-wide reconstitution of physiological chromatin in Drosophila embryo extracts, we uncovered the molecular basis of PNA formation. We identified Phaser, an unstudied zinc finger protein that positions nucleosomes flanking the motif. It also revealed how the global activity of the chromatin remodelers CHRAC/ACF, together with local barrier elements, generates islands of regular phasing throughout the genome. Our work demonstrates the potential of chromatin assembly by embryo extracts as a powerful tool to reconstitute chromatin features on a global scale in vitro.

The nature of chromatin as regular succession of nucleosomes has gained iconic status. However, since most nucleosomes in metazoans are poorly positioned it is unknown to which extent the bulk genomic nucleosome repeat length (NRL) reflects the regularity and spacing of nucleosome arrays at individual loci. We describe a new approach to map nucleosome array regularity and spacing through sequencing oligonucleosome-derived DNA by Illumina sequencing as well as emergent nanopore-technology. This revealed modulation of array regularity and NRL depending on functional chromatin states independently of nucleosome phasing and even in unmappable regions. We also found that nucleosome arrays downstream of silent promoters are considerably more regular than those downstream of highly expressed ones, despite more extensive nucleosome phasing of the latter. Our approach is generally applicable and provides an important parameter of chromatin organisation that so far had been missing.

Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq). We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters. Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin. Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted.

Chromatin accessibility complex/ATP-utilizing chromatin assembly and remodeling factor help to establish basal transcriptional repression, conceivably through improving the regular spacing of nucleosomes in euchromatin., The chromatin remodeling complexes chromatin accessibility complex and ATP-utilizing chromatin assembly and remodeling factor (ACF) combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression remains unclear. Here, we explored the influence of Drosophila melanogaster chromatin accessibility complex/ACF on transcription by using complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of an Acf knock-out allele showed that only lowly expressed genes are derepressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together, our results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.

International audience; X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific lethal dosage compensation complex (DCC) identifies and binds to X-chromosomal high-affinity sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. Long-range, inter-domain interactions between DCC binding sites are stronger in males, suggesting that the complex refines chromatin organization. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.

SummaryRegular successions of positioned nucleosomes – phased nucleosome arrays (PNAs) – are predominantly known from transcriptional start sites (TSS). It is unclear whether PNAs occur elsewhere in the genome. To generate a comprehensive inventory of PNAs forDrosophila, we applied spectral analysis to nucleosome maps and identified thousands of PNAs throughout the genome. About half of them are not near TSS and strongly enriched for a novel sequence motif. Through genome-wide reconstitution of physiological chromatin inDrosophilaembryo extracts we uncovered the molecular basis of PNA formation. We identified Phaser, an unstudied zinc finger protein that positions nucleosomes flanking the new motif. It also revealed how the global activity of the chromatin remodeler CHRAC/ACF, together with local barrier elements, generates islands of regular phasing throughout the genome. Our work demonstrates the potential of chromatin assembly by embryo extracts as a powerful tool to reconstitute chromatin features on a global scalein vitro.

SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. We systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. We show that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange.

SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. InDrosophila melanogastera single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed, but not well documentedin vivo. It is also unclear, whether the two alternatively spliced isoforms,dom-Aanddom-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis causing female sterility. Therefore, we systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg chamber formation. We show that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, while incorporation in endoreplicating germline nurse cells is independent of DOM. In contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange.Summary StatementIsoforms of nucleosome remodeling factor Domino change chromatin structure by histone variant exchange to direct essential cellular processes in oocyte development.

The Chromatin Accessibility Complex (CHRAC) consists of the ATPase ISWI, the large ACF1 subunit and a pair of small histone-like proteins, CHRAC-14/16. CHRAC is a prototypical nucleosome sliding factor that mobilizes nucleosomes to improve the regularity and integrity of the chromatin fiber. This may facilitate the formation of repressive chromatin. Expression of the signature subunit ACF1 is restricted during embryonic development, but remains high in primordial germ cells. Therefore, we explored roles for ACF1 during Drosophila oogenesis. ACF1 is expressed in somatic and germline cells, with notable enrichment in germline stem cells and oocytes. The asymmetrical localization of ACF1 to these cells depends on the transport of the Acf1 mRNA by the Bicaudal-D/Egalitarian complex. Loss of ACF1 function in the novel Acf1(7) allele leads to defective egg chambers and their elimination through apoptosis. In addition, we find a variety of unusual 16-cell cyst packaging phenotypes in the previously known Acf1(1) allele, with a striking prevalence of egg chambers with two functional oocytes at opposite poles. Surprisingly, we found that the Acf1(1) deletion - despite disruption of the Acf1 reading frame - expresses low levels of a PHD-bromodomain module from the C-terminus of ACF1 that becomes enriched in oocytes. Expression of this module from the Acf1 genomic locus leads to packaging defects in the absence of functional ACF1, suggesting competitive interactions with unknown target molecules. Remarkably, a two-fold overexpression of CHRAC (ACF1 and CHRAC-16) leads to increased apoptosis and packaging defects. Evidently, finely tuned CHRAC levels are required for proper oogenesis.

The process of dosage compensation (DC) in Drosophila counterbalances the monosomy of the X chromosome in male flies by increasing the transcription from this unique chromosome in the two-fold range. Upon exclusive expression of male-specific lethal 2 (MSL2) in males, the dosage compensation machinery assembles on active X-chromosomal genes. Overexpression of MSL proteins leads to aberrant binding of complex components to autosomes. Accordingly, MSL levels have to be carefully regulated. Here we describe a new mechanism through which MSL2 can fulfill its role as the central regulator of the faithful biogenesis and functionality of the DC machinery. MSL2 is an E3 ligase that ubiquitylates itself and the other associated components when their stoichiometry is unbalanced, uncovering proteasome-dependent degradation as an additional layer of homeostatic control of MSL levels. Furthermore, systematic mapping of modification sites by mass spectrometry and chromatin interaction studies on the target protein MSL1 suggest that the role of MSL2-mediated ubiquitylation goes beyond proteolysis.

The MOF (males absent on the first)-containing NSL (non-specific lethal) complex binds to a subset of active promoters in Drosophila melanogaster and is thought to contribute to proper gene expression. The determinants that target NSL to specific promoters and the circumstances in which the complex engages in regulating transcription are currently unknown. Here, we show that the NSL complex primarily targets active promoters and in particular housekeeping genes, at which it colocalizes with the chromatin remodeler NURF (nucleosome remodeling factor) and the histone methyltransferase Trithorax. However, only a subset of housekeeping genes associated with NSL are actually activated by it. Our analyses reveal that these NSL-activated promoters are depleted of certain insulator binding proteins and are enriched for the core promoter motif ‘Ohler 5’. Based on these results, it is possible to predict whether the NSL complex is likely to regulate a particular promoter. We conclude that the regulatory capacity of the NSL complex is highly context-dependent. Activation by the NSL complex requires a particular promoter architecture defined by combinations of chromatin regulators and core promoter motifs.

Active chromatin remodelling is integral to the DNA damage response in eukaryotes, as damage sensors, signalling molecules and repair enzymes gain access to lesions. A variety of nucleosome remodelling complexes is known to promote different stages of DNA repair. The nucleosome sliding factors CHRAC/ACF of Drosophila are involved in chromatin organization during development. Involvement of corresponding hACF1-containing mammalian nucleosome sliding factors in replication, transcription and very recently also non-homologous end-joining of DNA breaks have been suggested. We now found that hACF1-containing factors are more generally involved in the DNA damage response. hACF1 depletion increases apoptosis, sensitivity to radiation and compromises the G2/M arrest that is activated in response to UV- and X-rays. In the absence of hACF1, γH2AX and CHK2ph signals are diminished. hACF1 and its ATPase partner SNF2H rapidly accumulate at sites of laser-induced DNA damage. hACF1 is also required for a tight checkpoint that is induced upon replication fork collapse. ACF1-depleted cells that are challenged with aphidicolin enter mitosis despite persistence of lesions and accumulate breaks in metaphase chromosomes. hACF1-containing remodellers emerge as global facilitators of the cellular response to a variety of different types of DNA damage.

The nucleosome remodelling complexes CHRAC and ACF of Drosophila are thought to play global roles in chromatin assembly and nucleosome dynamics. Disruption of the gene encoding the common ACF1 subunit compromises fly viability. Survivors show defects in chromatin assembly and chromatin-mediated gene repression at all developmental stages. We now show that ACF1 expression is under strict developmental control. The expression is strongly diminished during embryonic development and persists at high levels only in undifferentiated cells, including the germ cell precursors and larval neuroblasts. Constitutive expression of ACF1 is lethal. Cell-specific ectopic expression perturbs chromatin organisation and nuclear programmes. By monitoring heterochromatin formation during development, we have found that ACF1-containing factors are involved in the initial establishment of diversified chromatin structures, such as heterochromatin. Altering the levels of ACF1 leads to global and variegated deviations from normal chromatin organisation with pleiotropic defects.

Does the three-dimensional (3D) conformation of interphase chromosomes merely reflect their function or does it actively contribute to gene regulation? The analysis of sex chromosomes that are subject to chromosome-wide dosage compensation processes promises new insight into this question. Chromosome conformations are dynamic and largely determined by association of distant chromosomal loci in the nuclear space or by their anchoring to the nuclear envelope, effectively generating chromatin loops. The type and extent of such interactions depend on chromatin-bound transcription regulators and therefore reflects function. Dosage compensation adjusts the overall transcription activity of X chromosomes to assure balanced expression in the two sexes. Initial analyses of mammalian and Drosophila X chromosomes have led to the hypothesis that their conformations may not only reflect their functional state but may in turn contribute to the coordination of chromosome-wide tuning of transcription.

The dosage compensation complex (DCC) in Drosophila globally increases transcription from the X chromosome in males to compensate for its monosomy. We discovered a male-specific conformation of the X chromosome that depends on the associations of high-affinity binding sites (HAS) of the DCC. The core DCC subunits MSL1–MSL2 are responsible for this male-specific organization. Contrary to emerging concepts, we found that neither DCC assembly nor the conformation of the male X chromosome are influenced by nuclear pore components. We propose that nuclear organization of HAS is central to the faithful distribution of the DCC along the X chromosome.

Chromatin serves to package, protect and organize the complex eukaryotic genomes to assure their stable inheritance over many cell generations. At the same time, chromatin must be dynamic to allow continued use of DNA during a cell's lifetime. One important principle that endows chromatin with flexibility involves ATP-dependent ‘remodeling’ factors, which alter DNA-histone interactions to form, disrupt or move nucleosomes. Remodeling is well documented at the nucleosomal level, but little is known about the action of remodeling factors in a more physiological chromatin environment. Recent findings suggest that some remodeling machines can reorganize even folded chromatin fibers containing the linker histone H1, extending the potential scope of remodeling reactions to the bulk of euchromatin.

Nucleosome-remodelling factors containing the ATPase ISWI, such as ACF, render DNA in chromatin accessible by promoting the sliding of histone octamers. Although the ATP-dependent repositioning of mononucleosomes is readily observable in vitro, it is unclear to which extent nucleosomes can be moved in physiological chromatin, where neighbouring nucleosomes, linker histones and the folding of the nucleosomal array restrict mobility. We assembled arrays consisting of 12 nucleosomes or 12 chromatosomes (nucleosomes plus linker histone) from defined components and subjected them to remodelling by ACF or the ATPase CHD1. Both factors increased the access to DNA in nucleosome arrays. ACF, but not CHD1, catalysed profound movements of nucleosomes throughout the array, suggesting different remodelling mechanisms. Linker histones inhibited remodelling by CHD1. Surprisingly, ACF catalysed significant repositioning of entire chromatosomes in chromatin containing saturating levels of linker histone H1. H1 inhibited the ATP-dependent generation of DNA accessibility by only about 50%. This first demonstration of catalysed chromatosome movements suggests that the bulk of interphase euchromatin may be rendered dynamic by dedicated nucleosome-remodelling factors.

The genomes of higher eukaryotes are carefully balanced systems of gene expression that compensate for the different numbers of sex chromosomes in the two sexes by adjusting gene expression levels. Different strategies for sex chromosome dosage compensation have evolved, which all involve modulating chromatin structure as a means to fine-tune transcription levels. As data accumulate, previous over-simplifications are being revised, and novel features of the compensation processes are gaining attention, many of which are of sufficient global validity to influence our view on gene expression beyond the realm of dosage compensation itself.

Complex experimental strategies involving in vitro reconstituted chromatin or simple chromatin interaction studies are much facilitated by immobilizing the nucleosomal arrays to paramagnetic beads. Chromatin-containing beads can be retrieved from a reaction mix solution on a magnet fast and quantitatively, effectively separating bound, loosely attached and unbound components efficiently. This chapter details a convenient strategy for immobilization of linear plasmid DNA on streptavidin-coated beads, the reconstitution of chromatin on such beads, and some fundamental handling procedures.

Chromatin remodeling complexes utilize the energy of ATP hydrolysis to change the packing state of chromatin, e.g. by catalysing the sliding of nucleosomes along DNA. Here we present simple models to describe experimental data of changes in DNA accessibility along a synthetic, repetitive array of nucleosomes during remodeling by the ACF enzyme or its isolated ATPase subunit, ISWI. We find substantial qualitative differences between the remodeling activities of ISWI and ACF. To understand better the observed behavior for the ACF remodeler, we study more microscopic models of nucleosomal arrays.

The term ‘nucleosome remodeling’ subsumes a large number of energy-dependent alterations of canonical nucleosome structure, catalyzed by dedicated ATPases in large multiprotein complexes. The importance of these factors for gene regulation and other processes with chromatin substrate have emerged from genetic studies. Mechanistic analyses of nucleosome remodeling by different enzymes provided a diverse, almost confusing phenomenology of ATP-dependent derangement of nucleosomes in vitro, suggesting that different remodeling machines follow different strategies to disrupt histone–DNA interactions. This review explores the alternative possibility that the rich phenomenology of nucleosome remodeling may be brought about by variations of one basic remodeling reaction.

The rRNA gene cluster consists of multiple transcription units. Half of these are active, while the other half are transcriptionally inactive. Previously, in vivo studies have demonstrated that silencing of ribosomal DNA (rDNA) is mediated by the chromatin remodeling NoRC (nucleolar remodeling complex). To explore the mechanisms underlying NoRC-directed silencing of rDNA transcription, we investigated the effect of recombinant NoRC on RNA polymerase I transcription on reconstituted chromatin templates. We show that NoRC interacts with the transcription terminator factor (TTF-I), and this interaction is required both for the binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome, thereby repressing RNA polymerase I transcription. This NoRC-directed rDNA repression requires the N terminus of histone H4. Repression is effective before preinitiation complex formation and as such is unable to exert an effect upon activated rDNA genes. Furthermore, the early steps of rDNA repression do not depend on DNA and histone modifications. These results reveal an important role for TTF-I in recruiting NoRC to rDNA and an active role for NoRC in the establishment of rDNA silencing.

Local differences in chromatin organisation may profoundly affect the activity of eukaryotic genomes. Regulation at the level of DNA packaging requires the targeting of structural proteins and histone-modifying enzymes to specific sites and their stable or dynamic interaction with the nucleosomal fiber. The "chromodomain", a domain shared by many regulators of chromatin structure, has long been suspected to serve as a module mediating chromatin interactions in a variety of different protein contexts. However, recent functional analyses of a number of different chromodomains revealed an unexpected diversity of interaction targets, including histones, DNA and even RNA. The chromodomains of today seem to have evolved from a common ancestral fold to fulfill various functions in different molecular contexts. Combining information gained from recent functional and structural studies of chromodomains with a bioinformatic classification of their structure could lead to the definition of sequence motifs with predictive quality for chromodomain function.

The organization of eukaryotic chromatin has a major impact on all nuclear processes involving DNA substrates. Gene expression is affected by the positioning of individual nucleosomes relative to regulatory sequence elements, by the folding of the nucleosomal fiber into higher-order structures and by the compartmentalization of functional domains within the nucleus. Because site-specific acetylation of nucleosomal histones influences all three aspects of chromatin organization, it is central to the switch between permissive and repressive chromatin structure. The targeting of enzymes that modulate the histone acetylation status of chromatin, in synergy with the effects mediated by other chromatin remodeling factors, is central to gene regulation.

dMi-2, the ATPase subunit of the Drosophila nucleosome remodelling and histone deacetylation (dNuRD) complex, was identified in a two-hybrid screen as an interacting partner of the transcriptional repressor, Tramtrack69 (Ttk69). A short region of Ttk69 is sufficient to mediate this interaction. Ttk69, but not the Ttk88 isoform, co-purifies with the dNuRD complex isolated from embryo extracts. dMi-2 and Ttk69 co-immunoprecipitate from embryonic extracts, indicating that they can associate in vivo. Both dMi-2 and Ttk69 co-localize at a number of discrete sites on polytene chromosomes, showing that they bind common target loci. We also demonstrate that dMi-2 and Ttk interact genetically, indicating a functional interaction in vivo. We propose that Ttk69 represses some target genes by remodelling chromatin structure through the recruitment of the dNuRD complex.

Modification of histones can have a dramatic impact on chromatin structure and function. Acetylation of lysines within the N-terminal tail of the histone octamer marks transcriptionally active regions of the genome whereas deacetylation seems to play a role in transcriptional silencing. Recently, the methylation of the histone tails has also been shown to be important for transcriptional regulation and chromosome structure. Here we show by immunoaffinity purification that two activities important for chromatin-mediated gene silencing, the histone methyltransferase SU(VAR)3-9 and the histone deacetylase HDAC1, associate in vivo. The two activities cooperate to methylate pre-acetylated histones. Both enzymes are modifiers of position effect variegation and interact genetically in flies. We suggest a model in which the concerted histone deacetylation and methylation by a SU(VAR)3-9/HDAC1-containing complex leads to a permanent silencing of transcription in particular areas of the genome.

A multicellular organism is made up of a variety of different cell types and tissues. This organization is accomplished by a well-concerted action of different regulatory molecules, which--in a very hierarchical manner--influence the expression of certain cell-specific genes. Many of those regulators are transcription factors, which directly influence the expression of the controlled gene by binding to a specific DNA sequence within its promoter or enhancer region. This binding then leads to an enhancement or a decrease in the rate of transcription of that particular gene and eventually regulates the production of the corresponding polypeptide. One major obstacle to the binding of these transcription factors is the fact that DNA is not readily accessible in the eukaryotic nucleus. It is associated with a class of very basic proteins called histones. This complex of histones and DNA is called chromatin.

Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control.

Contains fulltext : 129327.pdf (Publisher’s version ) (Open Access) ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates. We also perform assays with arrays reconstituted with hyperacetylated or trypsinized histones and isolated histone (H3/H4)(2) tetramers. The results define three groups of the ATP-dependent family of remodeling enzymes. In addition we investigate the ability of an acidic activator to recruit remodeling complexes to nucleosomal arrays. We propose that ATP-dependent chromatin remodeling enzymes share a common reaction mechanism and that a key distinction between complexes is in their mode of regulation or recruitment.

The proteins of the Polycomb group (PcG) are required for maintaining regulator genes, such as the homeotic selectors, stably and heritably repressed in appropriate developmental domains. It has been suggested that PcG proteins silence genes by creating higher-order chromatin structures at their chromosomal targets, thus preventing the interaction of components of the transcriptional machinery with their cis-regulatory elements. An unresolved issue is how higher order-structures are anchored at the chromatin base, the nucleosomal fiber. Here we show a direct biochemical interaction of a PcG protein—the Polycomb (PC) protein—with nucleosomal core particles in vitro. The main nucleosome-binding domain coincides with a region in the C-terminal part of PC previously identified as the repression domain. Our results suggest that PC, by binding to the core particle, recruits other PcG proteins to chromatin. This interaction could provide a key step in the establishment or regulation of higher-order chromatin structures.

In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chromatin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation.

The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin. We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions. The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes. Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes. The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs.

The Drosophila GAGA factor binds specifically to the sequence GAGAG, and synergises with nucleosome remodelling factor to remodel chromatin in vitro. It consists of an N-terminal domain (POZ/BTB) which mediates protein-protein interactions, a central region which contains the DNA-binding domain, and a C-terminal glutamine-rich region. It is shown that the glutamine-rich region is responsible for the formation of fibres in vitro which, on the basis of their tinctorial properties and CD spectra, may be classified as amyloid fibres. A large structural change, probably resulting in β-sheet structure, is observed upon fibre formation. Mutants containing the central region, either alone or together with the glutamine-rich region, are largely lacking in secondary structure but they bind specifically to the cognate DNA and are able to remodel chromatin in vitro. Consequently, neither the N-terminal domain nor the C-terminal glutamine-rich regions of the GAGA factor are necessary for chromatin remodelling in vitro.

Chromatin has shifted into the focus of attention as a key to understanding the regulation of nuclear processes such as transcription. Protein machines have been described that use the energy of ATP to render chromatin dynamic and hence active, but which may also be involved in chromatin assembly. The discovery of three different Drosophila nucleosome remodeling complexes that contain imitation switch (ISWI), an ATPase with a high degree of sequence conservation from yeast to human, points to a central function of this ATPase in chromatin dynamics.

A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for those acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell-free system to reconstitute chromatin with increased histone acetylation levels, we directly tested for a causal role of histone acetylation in transcription by RNA pol II. Chromatin, containing either control or acetylated histones, was reconstituted to comparable nucleosome densities and characterized by electron microscopy after psoralen cross-linking as well as by in vitro transcription. While H1-containing control chromatin severely repressed transcription of our model hsp26 gene, highly acetylated chromatin was significantly less repressive. Acetylation of histones, and particularly of histone H4, affected transcription at the level of initiation. Monitoring the ability of the transcription machinery to associate with the promoter in chromatin, we found that heat shock factor, a crucial regulator of heat shock gene transcription, profited most from histone acetylation. These experiments demonstrate that histone acetylation can modulate activator access to their target sites in chromatin, and provide a causal link between histone acetylation and enhanced transcription initiation of RNA pol II in chromatin.

Increased acetylation at specific N-terminal lysines of core histones is a hallmark of active chromatin in vivo , yet the structural consequences of acetylation leading to increased gene activity are only poorly defined. We employed a new approach to characterize the effects of histone acetylation: A Drosophila embryo-derived cell-free system for chromatin reconstitution under physiological conditions was programmed with exogenous histones to assemble hyperacetylated or matching control chromatin of high complexity. Hyperacetylated chromatin resembled unmodified chromatin at similar nucleosome density with respect to its sensitivity toward microccal nuclease, its nucleosomal repeat length, and the incorporation of the linker histone H1. In contrast, DNA in acetylated chromatin showed an increased sensitivity toward DNase I and a surprisingly high degree of conformational flexibility upon temperature shift pointing to profound alterations of DNA/histone interactions. This successful reconstitution of accessible and flexible chromatin outside of a nucleus paves the way for a thorough analysis of the causal relationship between histone acetylation and gene function.

Transcription by RNA polymerase II is highly regulated at the level of initiation and elongation. Well-documented transcription activation mechanisms, such as the recruitment of TFIID and TFIIB, control the early phases of preinitiation complex formation. The heat shock genes provide an example for transcriptional regulation at a later step: in nuclei TFIID can be detected at the TATA box prior to heat induction. Using cell-free systems for chromatin reconstitution and transcription, we have analyzed the mechanisms by which heat shock factor (HSF) increases transcription of heat shock genes in chromatin. HSF affected transcription of naked DNA templates in multiple ways: (i) by speeding up the rate of preinitiation complex formation, (ii) by increasing the number of productive templates, and (iii) by increasing the reinitiation rate. Under the more physiological conditions of potentiated chromatin templates, HSF affected only the reinitiation rate. Activator-dependent reinitiation of transcription, obviating the slow assembly of the TFIID-TFIIA complex on a promoter, may be especially crucial for genes requiring a fast response to inducers.

Eukaryotic ribosomal gene promoters are preceded by a terminator element which is recognized by the transcription termination factor TTF-I. We have studied the function of this promoter-proximal terminator and show that binding of TTF-I is the key event which leads to ATP-dependent nucleosome remodeling and transcriptional activation of mouse rDNA pre-assembled into chromatin. We have analyzed TTF-I mutants for their ability to bind to free or nucleosomal DNA, and show that the DNA binding domain of TTF-I on its own is not sufficient for interaction with chromatin, indicating that specific protein features exist that endow a transcription factor with chromatin binding and remodeling properties. This first analysis of RNA polymerase I transcription in chromatin provides a clue for the function of the upstream terminator and establishes a dual role for TTF-I both as a termination factor and a chromatin-specific transcription activator.