Biology subjects

Becker, P. B.

Publications and source records attributed to Becker, P. B..

15 recordsLinked to original sources

bioRxiv2026

Dosage compensation defects due to roX RNA deletion are rescued by recalibration of X/autosome stoichiometry

Metazoa evolved regulatory networks to balance the expression of their sex chromosomes. In Drosophila, males have a single gene-rich X chromosome, whereas females have two. Balanced X/autosome expression is essential for viability, and in male flies is achieved by activation of genes on the X through the male-specific-lethal (MSL) dosage compensation complex (DCC). This ribonucleoprotein assembly contains long, non-coding roX RNAs. To dissect the functional requirements of roX in a cell-based system, we deleted the roX2 gene in male S2 cells and selected two independent lines lacking detectable roX RNA. In the absence of roX, the remaining MSL protein complex was unable to associate with known or newly identified binding sites and thus failed to activate transcription. Surprisingly, the X/autosome expression ratio appeared nevertheless compensated. Cytogenetic and genomic analyses revealed that both roX-deficient cell populations had acquired additional X chromosomes. Apparently, chromosome gains due to mis-segregation made up for the loss of DCC-mediated dosage compensation. Interestingly, ectopic expression of full-length roX2, but not of shortened derivatives, fully restored DCC binding and normalized the karyotype. These findings illustrate that X chromosome dosage compensation is critical for viability even in cultured cells and provide a striking example of rapid evolution under stringent selection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/707606v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1bbf907org.highwire.dtl.DTLVardef@1da43a9org.highwire.dtl.DTLVardef@903326org.highwire.dtl.DTLVardef@10e3865_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Reciprocal targeting of the unfolded protein response regulator Xbp1 and the Dom-A nucleosome remodeler in Drosophila

The DOM-A complex regulates cell growth and proliferation in Drosophila. Like the orthologous human P400 complex, DOM-A combines two epigenetic effectors: a SWR1-type histone exchange enzyme, Dom-A, and the Tip60 acetyltransferase. We found Xbp1, a conserved transcription regulator of the unfolded protein response (UPR), as tightly associated with immunopurified DOM-A and explored the functional implications of this interaction. We biochemically determined the Xbp1 DNA recognition motif in chromatin-reconstituted Drosophila genomes. Intersection of the chromatin binding profiles for Xbp1 and Dom-A in proliferating cells and reciprocal protein depletion studies revealed two distinct modes through which Xbp1 binds chromatin. Xbp1 recruits Dom-A to motif-bearing promoters of genes involved in the UPR, such as Xbp1, Hsc70-3 and Gp93, and activates their transcription. Xbp1 also localizes to hundreds of high-confidence Dom-A binding sites that lack Xbp1 recognition motifs. These interactions depend on Dom-A, pointing to a reverse targeting scenario. Upon depletion of Dom-A, Xbp1 protein levels, but not mRNA levels, are reduced. The Xbp1 may thus be stabilized upon binding to DOM-A. The complex interactions of Xbp1 and DOM-A in the genome bear potential to integrate signals from the UPR with the general, DOM-mediated regulation of cell growth and proliferation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/682518v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@4b60eborg.highwire.dtl.DTLVardef@c7f2d0org.highwire.dtl.DTLVardef@1164265org.highwire.dtl.DTLVardef@612ddd_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Intrinsic specificity of a 'core' Tip60 acetyltransferase complex in Drosophila

The lysine acetyltransferase Tip60 (KAT5) regulates gene expression through acetylation of histone N-terminal tail domains. We determined the intrinsic substrate selectivity of a recombinant, 4-subunit TIP60 core module from Drosophila melanogaster with synthetic nucleosome arrays. We compared matched arrays of nucleosomes containing either the replication-dependent histone H2A, or the variant H2A.V (H2A.Z in mammals), a prominent substrate of Tip60. Targeted mass spectrometry allowed to quantify acetylation of individual lysines in histones H2A, H2A.V and H4. Overall, H4 and H2A/H2A.V were equally well acetylated. The analysis comprehensively identified selected sites of acetylation, their relative acetylation levels, diacetylation patterns and revealed surprisingly different acetylation rates of individual lysines. We also applied this defined acetylation system to evaluate the effectiveness and selectivity of a TIP60 inhibitor, NU9056. Remarkably, the inhibitor shows variable effectiveness at different acetylation sites. Knowledge about the intrinsic substrate selectivity of Tip60 is a prerequisite for a mechanistic understanding of the enzymes mode of action.

biochemistry↗

bioRxiv2025

The Tip60 acetylome is a hallmark of the proliferative state in Drosophila

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 arrests the cell cycle, 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.

molecular biology↗

bioRxiv2024

Genomic context-dependent histone H3K36 methylation by three Drosophila methyltransferases and implications for dedicated chromatin readers

Methylation of histone H3 at lysine 36 (H3K36me3) marks active chromatin. The mark is interpreted by epigenetic readers that assist transcription and safeguard the integrity of the chromatin fiber. The chromodomain protein MSL3 binds H3K36me3 to target X-chromosomal genes in male Drosophila for dosage compensation. The PWWP-domain protein JASPer recruits the JIL1 kinase to active chromatin on all chromosomes. Unexpectedly, depletion of K36me3 had variable, locus-specific effects on the interactions of those readers. This observation motivated a systematic and comprehensive study of K36 methylation in a defined cellular model. Contrasting prevailing models, we found that K36me1, K36me2 and K36me3 each represent independent chromatin states. A gene-centric view of the changing K36 methylation landscape upon depletion of the three methyltransferases Set2, NSD and Ash1 revealed local, context-specific methylation signatures. 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 regions with enhancer signatures. The genome-wide 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.

molecular biology↗

bioRxiv2023

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. 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 ISWIs mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWIs 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.

biochemistry↗

bioRxiv2022

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless

Unwinding 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) from Drosophila. 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 MLEs human orthologue DHX9/RHA mechanisms in disease.

molecular biology↗

bioRxiv2020

Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms

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. HighlightsO_LIIsoforms of DOMINO dictate the formation of distinct complexes C_LIO_LIDOM-B complex is the Drosophila SWR1 and incorporates H2A.V genome-wide C_LIO_LIDOM-A complex acetylates H4K12 and is the Drosophila NuA4 C_LI

molecular biology↗

bioRxiv2019

JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila

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, like dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1s targeting to chromatin depends on a new PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). The JASPer-JIL-1 (JJ)-complex is the major form of the kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes. Put in place, the complex modulates the 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 identified many new 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.

molecular biology↗

bioRxiv2018

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

biochemistry↗

bioRxiv2018

Factor cooperation for chromosome discrimination in Drosophila

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.\n\nThe process of dosage compensation in Drosophila allows exploring the rules underlying binding site selectivity. The male-specific-lethal (MSL) Dosage Compensation Complex 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.

molecular biology↗

bioRxiv2018

Genome-wide measurement of local nucleosome array regularity and spacing by nanopore sequencing

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.

genomics↗

bioRxiv2017

CHRAC/ACF Contribute to the Repressive Ground State of Chromatin

The chromatin remodeling complexes CHRAC and 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 is unclear. Here we explored the influence of Drosophila CHRAC/ACF on transcription by complementary gain- and loss-of-function approaches.\n\nTargeting 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 a Acf knock-out allele showed that only lowly expressed genes are de-repressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains.\n\nTaken together our results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.

molecular biology↗

bioRxiv2017

The Drosophila Dosage Compensation Complex activates target genes by chromosome looping within the active compartment

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific-lethal dosage compensation complex (DCC) identifies 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. 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.

genomics↗

bioRxiv2016

Genome-wide analysis of phased nucleosomal arrays reveals the functional characteristic of the nucleosome remodeler ACF

Regular 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 for Drosophila, 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 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 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 scale in vitro.

molecular biology↗