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

Abnormal Dosage Compensation of Reporter Genes Driven by the Drosophila Glass Multiple Reporter (GMR) Enhancer-Promoter

In Drosophila melanogaster the male specific lethal (MSL) complex is required for upregulation of expression of most X-linked genes in males, thereby achieving X chromosome dosage compensation. The MSL complex is highly enriched across most active X-linked genes with a bias towards the 3′ end. Previous studies have shown that gene transcription facilitates MSL complex binding but the type of promoter did not appear to be important. We have made the surprising observation that genes driven by the glass multiple reporter (GMR) enhancer-promoter are not dosage compensated at X-linked sites. The GMR promoter is active in all cells in, and posterior to, the morphogenetic furrow of the developing eye disc. Using phiC31 integrase-mediated targeted integration, we measured expression of lacZ reporter genes driven by either the GMR or armadillo (arm) promoters at each of three X-linked sites. At all sites, the arm-lacZ reporter gene was dosage compensated but GMR-lacZ was not. We have investigated why GMR-driven genes are not dosage compensated. Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation. Neither did proximity to a strong MSL binding site. However, replacement of the hsp70 minimal promoter with a minimal promoter from the X-linked 6-Phosphogluconate dehydrogenase gene did restore partial dosage compensation. Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females. GAGA and DREF have been implicated to play a role in dosage compensation. We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation. Further, it appears that the nature of the basal promoter and the presence of binding sites for specific factors influence the ability of a gene promoter to respond to the MSL complex

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

The (6;9) chromosome translocation, associated with a specific subtype of acute nonlymphocytic leukemia, leads to aberrant transcription of a target gene on 9q34.

The specific (6;9)(p23;q34) chromosomal translocation is associated with a defined subtype of acute nonlymphocytic leukemia (ANLL). The 9q34 breakpoint is located at the telomeric side of the c-abl gene. Through a combination of chromosome jumping, long-range mapping, and chromosome walking, the chromosome 9 breakpoints of several t(6;9) ANLL patients were localized within a defined region of 8 kilobases (kb), 360 kb telomeric of c-abl. Subsequent cDNA cloning revealed that this region represented an intron in the middle of a gene, called Cain (can), encoding a 7.5-kb transcript. Disruption of the can gene by the translocation resulted in the expression of a new 5.5-kb can mRNA from the 6p- chromosome. Isolation of chromosome 6 sequences showed that breakpoints on 6p23 also clustered within a limited stretch of DNA. These data strongly suggest a direct involvement of the translocation in the leukemic process of t(6;9) ANLL

Detection of a new submicroscopic Norrie disease deletion interval with a novel DNA probe isolated by differential Alu PCR fingerprint cloning.

Differential Alu PCR fingerprint cloning was used to isolate a DNA probe from the Xp11.4-->p11.21 region of the human X chromosome. This novel sequence, cpXr318 (DXS742), detects a new submicroscopic deletion interval at the Norrie disease locus (NDP). Combining our data with the consensus genetic map of the proximal short arm of the X chromosome, we propose the physical order Xcen-DXS14-DXS255-(DXS426, TIMP)-(DXS742-([MAOB-MAOA-DXS7], NDP)-DXS77-DXS228)-DXS209-DXS148-DXS196-++ +Xpter. The cpXr318 probe and a subclone from a cosmid corresponding to the DXS7 locus were converted into sequence-tagged sites. Finally, DXS742, DSX7, DXS77, and MAOA were integrated into a physical map spanning the Norrie disease locus

Mutation of the CH1 Domain in the Histone Acetyltransferase CREBBP Results in Autism-Relevant Behaviors in Mice

<div><p>Autism spectrum disorders (ASDs) are a group of neurodevelopmental afflictions characterized by repetitive behaviors, deficits in social interaction, and impaired communication skills. For most ASD patients, the underlying causes are unknown. Genetic mutations have been identified in about 25 percent of ASD cases, including mutations in epigenetic regulators, suggesting that dysregulated chromatin or DNA function is a critical component of ASD. Mutations in the histone acetyltransferase CREB binding protein (CBP, CREBBP) cause Rubinstein-Taybi Syndrome (RTS), a developmental disorder that includes ASD-like symptoms. Recently, genomic studies involving large numbers of ASD patient families have theoretically modeled CBP and its paralog p300 (EP300) as critical hubs in ASD-associated protein and gene interaction networks, and have identified <i>de novo</i> missense mutations in highly conserved residues of the CBP acetyltransferase and CH1 domains. Here we provide animal model evidence that supports this notion that CBP and its CH1 domain are relevant to autism. We show that mice with a deletion mutation in the CBP CH1 (TAZ1) domain (<i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>}) have an RTS-like phenotype that includes ASD-relevant repetitive behaviors, hyperactivity, social interaction deficits, motor dysfunction, impaired recognition memory, and abnormal synaptic plasticity. Our results therefore indicate that loss of CBP CH1 domain function contributes to RTS, and possibly ASD, and that this domain plays an essential role in normal motor function, cognition and social behavior. Although the key physiological functions affected by ASD-associated mutation of epigenetic regulators have been enigmatic, our findings are consistent with theoretical models involving CBP and p300 in ASD, and with a causative role for recently described ASD-associated CBP mutations.</p></div

<i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice show repetitive behaviors, hyperactivity, and less anxiety.

<p>(A,B) <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice display repetitive forelimb movements. White dots (A) indicate position of paws. Scores assigned in (B) represent the frequency of the repetitive movements. 0 = no forelimb repetitive movements (FRM); 1 = occasional FRM; 2 = continuous FRM. Mean ± SEM. N = 8 wild type (WT), 9 <i>CBP</i>^+/ΔCH1, 8 <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>}. (C) <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice show significantly increased self-grooming time. N = 14 WT, 21 <i>CBP</i>^+/ΔCH1, 13 <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>}. (D-F) <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice show increased travel distance (D), speed (E), and rearing (F) in a 30-min open field test. (G) <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice stay longer in the center of the open field arena. (H-I) <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice stay shorter in the closed arm, and enter less frequently the closed arm of an elevated plus maze. For (D-I), N = 19 WT, 24 <i>CBP</i>^+/ΔCH1, 16 <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>}. For (B-I) Asterisks indicate the p value for either Dunnett’s (in the repetitive movement assay) or Tukey (in the other tests) post hoc analysis after one-way ANOVA (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p< 0.0001). All the other pairings are not statistically different.</p

<i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice display motor dysfunction and abnormal recognition memory.

<p>(A) <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice fall faster in a wire hang assay. N = 15 wild type (WT), 21 <i>CBP</i>^+/ΔCH1, 14 <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>}. (B, C) <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice show significantly less grip strength for forelimbs only (B) or all four limbs (C). N = 15 WT, 21 <i>CBP</i>^+/ΔCH1, 14 <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>}. (D) <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice perform normally in a classic rotarod assay. N = 10 WT, 12 <i>CBP</i>^+/ΔCH1, 10 <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>}. (E,F) In a modified rotarod assay, in which the grips were eliminated from the rod surface, <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice perform normally when walking against the rod rotation (backward, E), but are impaired when walking with the rotation (forward, F). N = 42 WT, 27 <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>}. (G,H) In an object recognition test, <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>} mice have intact short-term recognition memory but impaired long-term memory. N = 17 WT, 12 <i>CBP</i>^{Δ<i>CH1/</i>Δ<i>CH1</i>}. Asterisks indicate the p value for the Student’s t-test (in the modified rotarod and the recognition memory test) or Tukey post hoc analysis after ANOVA in the other tests (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p< 0.0001). All the other pairings are not statistically different.</p