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.

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.

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.

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 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. KeywordsATAC-seq/ CLAMP/ DNA-immunoprecipitation/ Dosage compensation/ MSL2

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.

31In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 32 phosphorylation and has been proposed to protect active chromatin from acquiring 33 heterochromatic marks, like dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show 34 that JIL-1's targeting to chromatin depends on a new PWWP domain-containing protein 35JASPer (JIL-1 Anchoring and Stabilizing Protein). The JASPer-JIL-1 (JJ)-complex is the 36 major form of the kinase in vivo and is targeted to active genes and telomeric transposons 37 via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes. Put in place, the 38 complex modulates the transcriptional output. JIL-1 and JJ-complex depletion in cycling cells 39 lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. 40Finally, we identified many new interactors of the endogenous JJ-complex and propose that 41 JIL-1 not only prevents heterochromatin formation, but also coordinates chromatin-based 42 regulation in the transcribed part of the genome. 43 Key Words: PWWP domain / histone phosphorylation / COMPASS / heterochromatin / BOD1 44 / Dpy-30L1 / Telomeres 45 46 Main text 47In mammals, several nuclear kinases contribute to phosphorylation of histone H3 at serine 48 10 (H3S10ph) in interphase, whereas in Drosophila melanogaster, the essential kinase JIL-1 49 is responsible for most of it 1 . The significance of interphase H3S10ph is often 50 underestimated because most H3S10 phosphorylation in asynchronous cell populations 51 stems from mitotic chromatin, where it is deployed by Aurora kinase B 2,3 . Originally, 52interphase H3S10ph has been associated, in combination with H3K9ac and H3K14ac, with 53 transcriptional activation of immediate early genes upon MAPK activation 4,5 . In Drosophila, 54interphase H3S10ph is enriched at the body of active genes 6 . In mammal, in the extreme 55 case of mouse embryonic stem cells (mESC), ~30% of the genome is enriched for H3S10ph 56 in interphase 7 . 57The current model is that JIL-1 protects euchromatin from heterochromatization 8 . According 58 to the phospho-methyl switch model for mitotic H3S10ph 9 , placing H3S10ph prevents H3K9 59 methylation and subsequent binding of heterochromatin components. JIL-1 phosphorylates 60 various H3 peptides with different methylation states, including H3K9me2/3, with a 61 comparable efficiency 6 , whereas histone methyltransferases of the Su(var)3-9 family are 62 inhibited by H3S10ph 10,11 . Several observations suggest that JIL-1 is important for the 63 balance between eu-and heterochromatin. The Su(var)3-1 alleles of JIL-1 gene, which lead 64 to the expression of JIL-1 truncated in its C-terminal domain (CTD), result in reduced 65 heterochromatin spreading at euchromatin-heterochromatin boundaries 12,13 . Conversely, in 66 the JIL-1 z2/z2 null mutant, heterochromatin components spread into euchromatin. The 67 spreading of H3K9me2 and HP1 is highest on the euchromatic part of the X chromosome in 68 both sexes 14 , the spreading of the 7-zinc-finger pr...

No abstract

No abstract