SU(VAR)3-7 Links Heterochromatin and Dosage Compensation in Drosophila

In Drosophila, dosage compensation augments X chromosome-linked transcription in males relative to females. This process is achieved by the Dosage Compensation Complex (DCC), which associates specifically with the male X chromosome. We previously found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. In this study, we examine the impact of change in levels of SU(VAR)3-7 on dosage compensation. We first demonstrate that the DCC makes the X chromosome a preferential target for heterochromatic markers. In addition, reduced or increased amounts of SU(VAR)3-7 result in redistribution of the DCC proteins MSL1 and MSL2, and of Histone 4 acetylation of lysine 16, indicating that a wild-type dose of SU(VAR)3-7 is required for X-restricted DCC targeting. SU(VAR)3-7 is also involved in the dosage compensated expression of the X-linked white gene. Finally, we show that absence of maternally provided SU(VAR)3-7 renders dosage compensation toxic in males, and that global amounts of heterochromatin affect viability of ectopic MSL2-expressing females. Taken together, these results bring to light a link between heterochromatin and dosage compensation

Global Analysis of the Relationship between JIL-1 Kinase and Transcription

The ubiquitous tandem kinase JIL-1 is essential for Drosophila development. Its role in defining decondensed domains of larval polytene chromosomes is well established, but its involvement in transcription regulation has remained controversial. For a first comprehensive molecular characterisation of JIL-1, we generated a high-resolution, chromosome-wide interaction profile of the kinase in Drosophila cells and determined its role in transcription. JIL-1 binds active genes along their entire length. The presence of the kinase is not proportional to average transcription levels or polymerase density. Comparison of JIL-1 association with elongating RNA polymerase and a variety of histone modifications suggests two distinct targeting principles. A basal level of JIL-1 binding can be defined that correlates best with the methylation of histone H3 at lysine 36, a mark that is placed co-transcriptionally. The additional acetylation of H4K16 defines a second state characterised by approximately twofold elevated JIL-1 levels, which is particularly prominent on the dosage-compensated male X chromosome. Phosphorylation of the histone H3 N-terminus by JIL-1 in vitro is compatible with other tail modifications. In vivo, phosphorylation of H3 at serine 10, together with acetylation at lysine 14, creates a composite histone mark that is enriched at JIL-1 binding regions. Its depletion by RNA interference leads to a modest, but significant, decrease of transcription from the male X chromosome. Collectively, the results suggest that JIL-1 participates in a complex histone modification network that characterises active, decondensed chromatin. We hypothesise that one specific role of JIL-1 may be to reinforce, rather than to establish, the status of active chromatin through the phosphorylation of histone H3 at serine 10

Schaltungen der Mikroelektronik fuer den Bereich der technischen Kommunikation Schlussbericht

With 50 figs., 22 refs.TIB: FR 1342 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Genetic Interaction between Mutations in c-Myb and the KIX Domains of CBP and p300 Affects Multiple Blood Cell Lineages and Influences Both Gene Activation and Repression

<div><p>Adult blood cell production or definitive hematopoiesis requires the transcription factor c-Myb. The closely related KAT3 histone acetyltransferases CBP (CREBBP) and p300 (EP300) bind c-Myb through their KIX domains and mice homozygous for a p300 KIX domain mutation exhibit multiple blood defects. Perplexingly, mice homozygous for the same KIX domain mutation in CBP have normal blood. Here we test the hypothesis that the CBP KIX domain contributes subordinately to hematopoiesis via a genetic interaction with c-Myb. We assessed hematopoiesis in mice bearing compound mutations of c-Myb and/or the KIX domains of CBP and p300, and measured the effect of KIX domain mutations on c-Myb-dependent gene expression. We found that in the context of a p300 KIX mutation, the CBP KIX domain mutation affects platelets, B cells, T cells, and red cells. Gene interaction (epistasis) analysis provides mechanistic evidence that blood defects in KIX mutant mice are consistent with reduced c-Myb and KIX interaction. Lastly, we demonstrated that the CBP and p300 KIX domains contribute to both c-Myb-dependent gene activation and repression. Together these results suggest that the KIX domains of CBP, and especially p300, are principal mediators of c-Myb-dependent gene activation and repression that is required for definitive hematopoiesis.</p> </div

Integrierte Bausteine fuer das dienstintegrierte, digitale Nachrichtennetz und Breitbandkommunikation

With 5 refs., 4 tabs., 45 figs.Standort: TIB Hannover: FR 585 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

The KIX domains of both CBP and p300 contribute to c-Myb transactivation function in a dose dependent manner.

<p>(A) Schematic showing approximate locations of CBP and p300 domains [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082684#B91" target="_blank">91</a>]. Nuclear receptor interaction domain (NRID), the CREB-binding domain (KIX), Cys/His-rich region-1 (CH1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082684#B57" target="_blank">57</a>] or transcriptional-adaptor zinc-finger-1, TAZ1), bromodomain (Br), plant homeodomain (PHD), HAT enzymatic region, zinc-binding domain (ZZ), TAZ2 (aka CH3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082684#B92" target="_blank">92</a>]), and the nuclear receptor binding domain (NCBD). (B) Alignment of the KIX domains of mouse and human p300 and CBP. Amino acids mutated in the KIX domain mutants are indicated with arrows. (C) Pulldown assay with the c-Myb transactivation domain (aa186-325) fused to GST using extracts from wild type (WT) and <i>CBP</i>^KIX/KIX mouse embryonic fibroblasts. Buffer C is extraction buffer without protein. (D) Transient transfection assay showing Gal-c-Myb 186-325 transactivation function is reduced in mouse embryonic fibroblasts having the indicated number of <i>CBP</i> and p300 mutant KIX alleles (one (<i>p300^+/KIX</i> or <i>CBP^+/KIX</i>), two (<i>CBP^KIX/KIX, p300^KIX/KIX</i> or <i>p300^+/KIX</i>;<i>CBP^+/KIX</i>) or three (<i>p300^+/KIX</i>;<i>CBP^KIX/KIX</i> or <i>p300^KIX/KIX</i>;<i>CBP</i>^+/KIX) KIX); wild type MEFs indicated; one MEF isolate of each genotype except wild type (N=2); (mean ± SEM). </p

Triple-KIX p300^<i>+/KIX</i>; <i>CBP</i>^KIX/KIX adult mice have defective hematopoiesis affecting multiple lineages.

<p>Peripheral blood counts from 1-4 month old C57BL/6J x 129Sv F1 wild type (WT) and KIX mutant mice (N=10-22). Counts from automated Hemavet complete blood count. For B cells and T cells, total lymphocyte counts from complete blood count were parsed using flow cytometry to determine the percentage of lymphocytes that were positive for B220 (B cells), CD3 (T cells), CD4 and CD8 (T cell subsets). Asterisks indicate significant p value by pairwise Tukey post test following one way ANOVA (* p<0.05, ** p<0.01, *** p<0.001). </p

Both c-Myb activated and repressed endogenous genes are sensitive to mutation of the KIX domains of CBP and p300 in thymocytes.

<p>(A,B) Affymetrix gene array of wild type (WT) and triple-KIX p300^<i>+/KIX</i><i>; CBP</i>^KIX/KIX CD4⁺CD8⁺ double positive (DP) thymocytes. Average signal; N=4 mice for each genotype. Probe sets marked in black were identified from data presented in Yuan <i>et </i><i>al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082684#B70" target="_blank">70</a>]. and indicate probe sets in which <i>c-Myb^flox/flox</i>;Cd4-Cre;<i>Tcrα</i>^<i>-/-</i> DP thymocytes were at least twofold higher (A) or at least 50% lower (B) than control cells. For our data sets (black and blue icons), no probes are shown with a signal below 3.4 (twice background). For the Myb-dependent gene set from Yuan et al. used here, probes selected had P<0.05 between Myb null and control DP thymocytes, and none were used where all signals were below 6. Axes scales are log₂. </p