A new strategy for isolating genes controlling dosage compensation in Drosophila using a simple epigenetic mosaic eye phenotype

<p>Abstract</p> <p>Background</p> <p>The <it>Drosophila </it>Male Specific Lethal (MSL) complex contains chromatin modifying enzymes and non-coding <it>roX </it>RNA. It paints the male X at hundreds of bands where it acetylates histone H4 at lysine 16. This epigenetic mark increases expression from the single male X chromosome approximately twofold above what gene-specific factors produce from each female X chromosome. This equalises X-linked gene expression between the sexes. Previous screens for components of dosage compensation relied on a distinctive male-specific lethal phenotype.</p> <p>Results</p> <p>Here, we report a new strategy relying upon an unusual male-specific mosaic eye pigmentation phenotype produced when the MSL complex acts upon autosomal <it>roX1 </it>transgenes. Screening the second chromosome identified at least five loci, two of which are previously described components of the MSL complex. We focused our analysis on the modifier alleles of MSL1 and MLE (for 'maleless'). The MSL1 lesions are not simple nulls, but rather alter the PEHE domain that recruits the MSL3 chromodomain and MOF ('males absent on first') histone acetyltransferase subunits to the complex. These mutants are compromised in their ability to recruit MSL3 and MOF, dosage compensate the X, and support long distance spreading from <it>roX1 </it>transgenes. Yet, paradoxically, they were isolated because they somehow increase MSL complex activity immediately around <it>roX1 </it>transgenes in combination with wild-type MSL1 subunits.</p> <p>Conclusions</p> <p>We propose that these diverse phenotypes arise from perturbations in assembly of MSL subunits onto nascent <it>roX </it>transcripts. This strategy is a promising alternative route for identifying previously unknown components of the dosage compensation pathway and novel alleles of known MSL proteins.</p

Mutations in the Transcription Elongation Factor SPT5 Disrupt a Reporter for Dosage Compensation in Drosophila

<div><p>In <em>Drosophila</em>, the MSL (Male Specific Lethal) complex up regulates transcription of active genes on the single male X-chromosome to equalize gene expression between sexes. One model argues that the MSL complex acts upon the elongation step of transcription rather than initiation. In an unbiased forward genetic screen for new factors required for dosage compensation, we found that mutations in the universally conserved transcription elongation factor <em>Spt5</em> lower MSL complex dependent expression from the <em>miniwhite</em> reporter gene <em>in vivo</em>. We show that SPT5 interacts directly with MSL1 <em>in vitro</em> and is required downstream of MSL complex recruitment, providing the first mechanistic data corroborating the elongation model of dosage compensation.</p> </div

Genetic interactions between <i>Spt5</i> and dosage compensation.

<p>(A) Lowering SPT5 reduces male viability compared to sisters. All males were <i>roX1 roX2</i> double mutants and partially rescued by one copy of the <i>[GMroX1-75C]</i> transgene. Males wild type for all other loci are rescued 29%, but flies missing one copy of the indicated dosage compensation genes have reduced male viability. ** p<0.01 * p<0.05 Fisher exact test. Detailed results in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003073#pgen.1003073.s008" target="_blank">Table S2</a>. (B) Reducing <i>Spt5</i> rescues the sterility of <i>[H83M2]</i> females. <i>y w; Spt5^880C/CyO y+</i> females were mated to <i>w/Y; [w⁺ H83M2]/+</i> males. Only the adults eclosing during the first two days are indicated to measure delayed development. <i>[w⁺ H83M2]</i> escaper females produced few eggs and were sterile (black bar). [<i>w⁺ H83M2</i>] females heterozygous for <i>Spt5</i> regained fertility (gray bars). N = number of brothers recovered for each class. p calculated by Fisher exact test. (C) Males homozygous for the <i>[GMroX1]</i> transgene at 75C have a few pigmented sectors. (D) Singly, <i>Spt5</i> slightly reduces and (E) <i>msl1^P864L</i> dramatically increases local MSL activity. (F) When present together, <i>Spt5^S14F</i> blocks the increased activity of the <i>msl1^P864L</i> gain of function allele. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003073#pgen.1003073.s002" target="_blank">Figure S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003073#pgen.1003073.s003" target="_blank">Figure S3</a> for additional genetic analysis.</p

A model for dosage compensation.

<p>A highly simplified view separates transcription into phases of initiation controlled by gene-specific transcription factors (yellow), pausing of RNA polymerase II (blue) near the TSS by SPT4/5 (green), and release of pausing when P-TEFb phosphorylates both the CTD of RNAP and SPT5 leading to productive elongation. MSL complex (red) is attracted to the X chromosome by high affinity or chromatin entry sites (CES) scattered along the chromosome. The pioneer RNP may lay down new chromatin marks (hatch) characteristic of active genes. Some feature of active chromatin recruits MSL complex from local CES. During subsequent rounds of transcription MSL complex interacts with SPT5 to promote processivity.</p

MSL1 PEHE domain physically interacts with SPT5.

<p>(A) MSL1 PEHE domain (aa 751–1039) was expressed as GST (Glutathione S-transferase) fusion protein. SPT5 fragments N, M, and C were expressed as MBP (Maltose binding Protein) fusion proteins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003073#pgen.1003073-Andrulis1" target="_blank">[16]</a>. (B) Purified SPT5-MBP fragments immobilized on amylose beads were allowed to interact with either GST-MSL1PEHE or GST. After washing, the recovered proteins were analyzed anti-GST Westerns. The membrane was stripped and reprobed to visualize the MBP fusion proteins. The middle input panel corresponds to 10% of the input GST proteins visualized with anti-GST antibodies.</p

Isolation of <i>Spt5</i> mutations.

<p>(A) Male flies carrying the <i>[w⁺ GMroX1]</i> transgene inserted at the 2R telomere (60F) have sectored pigmentation due to dosage compensation at the transgene. (B) Males heterozygous for mutations in <i>Spt5</i> lose most red eye pigmentation due to reduced MSL complex activity. (C and D) <i>Spt5</i> mutants have no effect on the PEV line <i>In(1)w^m4</i>. (E) Genomic location of <i>Spt5</i> and flanking deficiencies. (F) New <i>Spt5</i> mutations failed to complement the previously reported <i>Spt5^MGE-3</i> allele <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003073#pgen.1003073-Mahoney1" target="_blank">[15]</a>. (G) SPT5 domain features. SR, Serine/Arginine; NGN, N-terminal NusG; KOW, Kyrpides, Ouzounis, Woese light green oval indicates only partial match to consensus; RGG, arginine glycine glycine repeats; CTR, C-Terminal Repeats similar to RNAPII. Screen design and results are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003073#pgen.1003073.s001" target="_blank">Figure S1</a>.</p

Ni(II) complex with bishydrazone ligand: synthesis, characterization, DNA binding studies and pro-apoptotic and pro-differentiation induction in human cancerous cell lines

A new Ni(II) complex, [Ni(L)(H2O)] (1), with diethyl 3,3'-(2,2'-(1,1'-(pyridine-2,6-diyl) bis(ethan-1-yl-1-ylidene)) bis(hydrazin-1-yl-2-ylidene)) bis(3-oxopropanoate) ligand (H2L) was synthesized as a potential chemotherapeutic agent. Polidentate ligand was coordinated to Ni(II) NNN-tridentately, in dianionic form, while monodentate water coordination completed square-planar geometry around metal. Structure in the solution was determined by NMR spectroscopy and the same coordination mode was observed in the solid state using IR spectroscopy and further verified by DFT calculations and electrochemical studies. Thermal stability of 1 was determined in both air and nitrogen atmosphere. Anticancer activity of 1 was investigated on acute monocytic leukemia (THP-1) and pancreatic adenocarcinoma (AsPC-1) cell lines. On THP-1 cells 1 induced powerful apoptotic response (ED50 = 10 +/- 3 mu M), which was revealed to be only partially caspase-dependent, with activation of caspase-8 as the dominant course. This suggested that experimentally validated covalent binding of 1 to DNA is not the only mechanism responsible for programmed cell death. This was supported with experiments on AsPC-1 cells. Although treatment of those cells with 1 resulted in poor apoptotic response, cell cycle changes showed concentration-dependent shifts indicating a dual mechanism of activity. This study also reviews the results of preliminary biological screening, which demonstrates that 1 displays a unique pattern of anticancer activity with at least two mechanisms involved