Methylation of histone H3 at lysine 36 (H3K36me3) marks active chromatin. The mark is interpreted by epigenetic readers that assist transcription and safeguard chromatin fiber integrity. In Drosophila, the chromodomain protein MSL3 binds H3K36me3 at X-chromosomal genes to implement dosage compensation. The PWWP-domain protein JASPer recruits the JIL1 kinase to active chromatin on all chromosomes. Because depletion of K36me3 had variable, locus-specific effects on the interactions of those readers, we systematically studied K36 methylation in a defined cellular model. Contrasting prevailing models, we found that K36me1, K36me2, and K36me3 each contribute to distinct chromatin states. Monitoring the changing K36 methylation landscape upon depletion of the three methyltransferases Set2, NSD, and Ash1 revealed local, context-specific methylation signatures. Each methyltransferase governs K36 methylation in dedicated genomic regions, with minor overlaps. Set2 catalyzes K36me3 predominantly at transcriptionally active euchromatin. NSD places K36me2/3 at defined loci within pericentric heterochromatin and on weakly transcribed euchromatic genes. Ash1 deposits K36me1 at putative enhancers. The mapping of MSL3 and JASPer suggested that they bind K36me2 in addition to K36me3, which was confirmed by direct affinity measurement. This dual specificity attracts the readers to a broader range of chromosomal locations and increases the robustness of their actions.

The acetyltransferase KAT5/Tip60 is an epigenetic regulator of transcription and the DNA damage response. In Drosophila, Tip60 acetylates histones as part of the DOM-A complex, but it is unclear whether it has other substrates. In this work, we comprehensively studied the functions of Tip60 in a Drosophila proliferative cell model. Depletion of Tip60 slows cell-cycle progression, but remaining viable cells resist mutagenic irradiation. The impaired proliferation is explained by reduced expression of critical cell-cycle genes. Tip60 binds their transcription start sites and Tip60-dependent acetylation of the histone variant H2A.V correlates with transcription activity. A potentially synergistic pathway for cell-cycle regulation involves the acetylation of proteins other than histones. The Tip60-dependent nuclear acetylome contains hundreds of proteins, many of which are involved in diverse aspects of cell growth and division, including replication, mitosis, gene expression, chromatin organization, and ribosome biogenesis. We hypothesize that Tip60 coordinates the proliferative state through histone and non-histone effectors. Reversible acetylation of diverse effector proteins bears potential for fine-tuning energy-intensive processes in response to stresses or nutritional shortcomings. Our study portrays the DOM-A/TIP60 complex as a general promoter of cell proliferation.

The phosphorylation of the histone variant H2AX to form γH2AX is an early and critical histone modification during the DNA damage response. This phosphorylation has proven to be a highly specific molecular marker for tracking the initiation and resolution of DNA damage. In this study, we investigate the roles of three phosphatases in removing the 'γ' phospho-epitope from H2AX in Drosophila Kc167 cells. We found that the bulk of the X-ray-induced γH2AX signal is erased by the PP2A-type phosphatase MTS (microtubule star).

The phosphorylation of the histone variant H2AX to form γH2AX is an early and critical histone modification during the DNA damage response. This phosphorylation has proven to be a highly specific molecular marker for tracking the initiation and resolution of DNA damage. In this study, we investigate the roles of three phosphatases in removing the 'γ' phospho-epitope from H2AX in Drosophila Kc167 cells. We found that the bulk of the X-ray-induced γH2AX signal is erased by the PP2A-type phosphatase MTS (microtubule star).

Acetylation of lysine 16 of histone H4 (H4K16ac) stands out among the histone modifications, because it decompacts the chromatin fiber. The metazoan acetyltransferase MOF (KAT8) regulates transcription through H4K16 acetylation. Antibody-based studies had yielded inconclusive results about the selectivity of MOF to acetylate the H4 N-terminus. We used targeted mass spectrometry to examine the activity of MOF in the male-specific lethal core (4-MSL) complex on nucleosome array substrates. This complex is part of the Dosage Compensation Complex (DCC) that activates X-chromosomal genes in male Drosophila. During short reaction times, MOF acetylated H4K16 efficiently and with excellent selectivity. Upon longer incubation, the enzyme progressively acetylated lysines 12, 8 and 5, leading to a mixture of oligo-acetylated H4. Mathematical modeling suggests that MOF recognizes and acetylates H4K16 with high selectivity, but remains substrate-bound and continues to acetylate more N-terminal H4 lysines in a processive manner. The 4-MSL complex lacks non-coding roX RNA, a critical component of the DCC. Remarkably, addition of RNA to the reaction non-specifically suppressed H4 oligo-acetylation in favor of specific H4K16 acetylation. Because RNA destabilizes the MSL-nucleosome interaction in vitro we speculate that RNA accelerates enzyme-substrate turn-over in vivo, thus limiting the processivity of MOF, thereby increasing specific H4K16 acetylation.

Extracts from Drosophila preblastoderm embryos (DREX) form the basis of a powerful in vitro chromatin reconstitution system that assembles entire genomes into complex chromatin with physiological nucleosome spacing and polymer condensation. As the zygotic genome has not yet been activated in preblastoderm embryos, the reconstitution extract lacks endogenous transcription factors (TFs) and the RNA polymerase machinery. At the same time, it contains high levels of ATP-dependent nucleosome sliding enzymes that render the reconstituted chromatin dynamic. The naïve chromatin can be used to determine the intrinsic DNA binding properties of exogenous, usually recombinant TFs (or DNA binding proteins in general) in a complex chromatin context. Recent applications of the system include the description of cooperation and competition of Drosophila pioneer TFs for composite binding sites, and the characterization of nucleosome interactions of mammalian pioneer TFs in the heterologous system.

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies. Chromatin condensation does not impede nucleosome sliding by ISWI remodelers. Notably, ATP energy is used not only for remodeling but also for enzyme mobility and to prevent solidification of chromatin. A 'monkey-bar' model rationalizes the findings.

MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. We explore the nature of the cooperative binding to these GA-rich, composite sequence elements in reconstituted naïve embryonic chromatin. We found that the cooperativity requires physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. We find that the cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in our reconstitution, such as roX2 lncRNA.

SummaryUnwinding RNA secondary structures by RNA helicases is essential for RNA metabolism. How the basic unwinding reaction of DExH-type helicases is regulated by their accessory domains is unresolved. Here, we combine structural and functional analyses to address this challenge for the prototypic DExH RNA helicase maleless (MLE) fromDrosophila.We captured the helicase cycle of MLE with multiple structural snapshots. We discovered that initially, dsRBD2 flexibly samples substrate dsRNA and aligns it with the open helicase tunnel. Subsequently, dsRBD2 releases RNA and associates with the helicase core, leading to closure of the tunnel around ssRNA. Structure-based MLE mutations confirm the functional relevance of the structural model in cells. We propose a molecular model in which the dsRBD2 domain of MLE orchestrates large structural transitions that depend on substrate RNA but are independent of ATP.Our findings reveal the fundamental mechanics of dsRNA unwinding by DExH helicases with high general relevance for dosage compensation and specific implications for MLE’s human orthologue DHX9/RHA mechanisms in disease.

The basic helix–loop–helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members 1 . Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2 , 3 ). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry–exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A–H2B acidic patch 4 , the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes 5 – 7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

Long, non-coding (Inc) RNAs emerge as crucial regulators of gene expression. Their ability to interact with DNA and protein mediates the recruitment of regulators to defined chromosomal loci. LncRNAs are particularly abundant in brain tissues where they are involved in cell specification. We described a novel lncRNA, RUS (RNA upstream to Slitrk3), which interacts with epigenetic regulators and contributes to shaping the gene expression program of neural precursor cells towards neurogenesis.

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 LINC01322, which we term RUS for "RNA upstream of Slitrk3." The RUS gene is well conserved in mammals by sequence and synteny next to the neurodevelopmental gene Slitrk3. RUS is exclusively expressed in neural cells and its expression increases during neuronal differentiation of mouse embryonic cortical neural stem cells. Depletion of RUS locks neuronal precursors in an intermediate state towards neuronal differentiation resulting in arrested cell cycle and increased apoptosis. RUS associates with chromatin in the vicinity of genes involved in neurogenesis, most of which change their expression upon RUS depletion. The identification of a range of epigenetic regulators as specific RUS interactors suggests that the lncRNA may mediate gene activation and repression in a highly context-dependent manner.

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) of Drosophila identifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from the D. melanogaster model: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. We found that in D. virilis , a species separated from melanogaster by 40 million years of evolution, all principles are active but contribute differently to X specificity. In melanogaster , the DCC subunit MSL2 evolved intrinsic DNA-binding selectivity for rare PionX sites, which mark the X chromosome. In virilis , 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.

RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target selectivity and function of an individual protein. The presented in vitro RNA immunoprecipitation assay (vitRIP) uncovers intrinsic RNA binding specificities of isolated proteins using the total cellular RNA pool as a library. Total RNA extracted from cells or tissues is incubated with purified recombinant proteins, RNA-protein complexes are immunoprecipitated and bound transcripts are identified by deep sequencing or quantitative RT-PCR. Enriched RNA classes and the nucleotide frequency in these RNAs inform on the intrinsic specificity of the recombinant protein. The simple and versatile protocol can be adapted to other RNA binding proteins and total RNA libraries from any cell type or tissue. Graphic abstract: Figure 1. Schematic of the in vitro RNA immunoprecipitation (vitRIP) protocol.

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.

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.

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.

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.

Figure 1. : A. Effect of X-rays on larval hatching rate. Wild-type and AcfC embryos were irradiated with different X-ray doses (Gy). Each dot represents the % of embryos that hatched in a single experiment (N between 50 and 60 embryos). Horizontal bars represent the mean of the 4 biological replicates. B. Calculated p-values (null hypothesis: difference between means = 0) for the comparisons depicted in panel (A). C. Representative immunofluorescence images of irradiated (1 Gy) wild-type and AcfC embryos 0, 15 and 180 minutes after irradiation. Embryos were stained with DAPI and with antibodies against H2A.V and γH2A.V. Scale bar = 100 μm. D. Quantification of immunofluorescence images of wild-type and AcfC embryos from the time course after X-ray exposure as described in (C). Each dot represents the 7/30/2020 Open Access normalized γH2A.V signal (see methods) of one experiment (N between 8 and 15 embryos). Horizontal bars represent the mean of 3 biological replicates. E. Calculated p-values (null hypothesis: difference between means = 0) for the comparisons depicted in panel (D).