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

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

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

The <i>Drosophila Over Compensating Males</i> Gene Genetically Inhibits Dosage Compensation in Males

<div><p>Male <i>Drosophila</i> are monosomic for the X chromosome, but survive due to dosage compensation. They use the Male Specific Lethal (MSL) complex composed of noncoding <i>roX</i> RNA and histone modifying enzymes to hypertranscribe most genes along the X ∼1.6–1.8 fold relative to each female allele. It is not known how the MSL complex achieves this precise adjustment to a large and diverse set of target genes. We carried out a genetic screen searching for novel factors that regulate dosage compensation in flies. This strategy generated thirty alleles in a previously uncharacterized gene, <i>over compensating males</i> (<i>ocm</i>) that antagonizes some aspect of MSL activity. The mutations were initially recovered because they derepressed an MSL-dependent eye color reporter. Null <i>ocm</i> mutations are lethal to both sexes early in development revealing an essential function. Combinations of hypomorphic <i>ocm</i> alleles display a male specific lethality similar to mutations in the classic <i>msl</i> genes, but <i>ocm</i> males die due to excessive, rather than lack of dosage compensation. Males that die due to very low MSL activity can be partially rescued by <i>ocm</i> mutations. Likewise, males that would die from <i>ocm</i> mutations can be rescued by reducing the dose of various <i>msl</i> and <i>roX</i> genes. <i>ocm</i> encodes a large nuclear protein that shares a novel cysteine rich motif with known transcription factors.</p> </div

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

Identification of <i>CG3363</i> as <i>ocm</i>.

<p>A. Genomic region near 60C showing predicted genes and five BACs used to rescue <i>ocm</i>. The BACs shown in black failed to rescue <i>ocm</i> mutations, but 117N13 (yellow) rescued viability and fertility of multiple <i>ocm</i> alleles. B. Conceptual translation of <i>ocm</i> reveals a protein with distinct motifs conserved between other Drosophila species (colored boxes) separated by diverged regions (thin line). The hatched box indicates the segment of OCM used to raise antibodies. C. Similar coding regions are found in the genomes of some other insects. The Bombyx alignment is taken from genomic DNA lacking cDNA support, so the exact alignment is uncertain. D. The Cys431 region (red) is also found in chordate Mga. E. Several Daphnia proteins contain Cys431 motifs. A second cys-rich region (orange) is also present in two of the Daphnia proteins.</p

<i>roX1</i> RNA is needed to sustain endogenous <i>roX1</i> transcription in males.

<p>X chromosomes from neighboring cells display a mosaic pattern in which the MSL complex either succeeded (arrowhead) or failed (arrow) to paint the X from <i>roX2</i>; <i>msl2</i>; <i>H83M2</i> female (A) and male (B) salivary glands. (C) Endogenous <i>roX1</i> and <i>H83roX1Δ39</i> transcripts (Orange) and antisense riboprobe recognizing only full length <i>roX1</i> (green). Whole mount <i>roX1</i> FISH using the internal probe on salivary glands from (D) wild type male, (E) wild type female, (F) <i>roX1⁺ roX2⁻</i>/Y; <i>msl2</i>; <i>H83M2</i> mosaic male, (G) <i>roX1⁺roX2⁻</i>/Y; <i>msl2; H83M2 H83-roX1Δ39</i>/+ male, (H) <i>roX1⁻ roX2 ⁻</i>/Y; <i>msl2; H83M2 H83-roX1Δ39</i>/+ male. The X chromosomes in G are fully painted in all cells with MSL complex relying upon <i>roX1-Δ39</i> RNA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002564#pgen.1002564.s001" target="_blank">Figure S1D</a>), but the truncated <i>roX1</i> RNA is not recognized by the internal probe.</p

Null alleles of <i>ocm</i> results in decreased cell numbers in the fly eyes.

<p>Different <i>ocm</i> alleles were assayed for cell viability in <i>Drosophila</i> eye. (A-B) <i>ocm^S1590F</i>, a weak hypomorphic allele of <i>ocm</i> does not affect cell growth. Other hypomorphic alleles, <i>ocm^G1646E</i> and <i>ocm^V1286D</i> were also tested but they are indistinguishable to <i>ocm^S1590F</i> (data not shown). Increasingly severe alleles (C–D) <i>ocm^V1334D</i>, (E–F) <i>ocm¹⁶⁶</i>^{Δ<i>11 bp</i>} (null allele) result in fly eyes dramatically reduced in size.</p

Mutations in <i>ocm</i> increase MSL activity around mosaic <i>roX1</i> transgenes.

<p>A and B. The <i>GMroX1-75C</i> reporter shows mosaic eye pigmentation in males, but females have pure white eyes. C and D. Reducing <i>ocm</i> activity increases eye pigmentation in males (more MSL activity) but has no effect on females who lack dosage compensation. E and F. The increased eye pigmentation seen in <i>ocm</i> males requires full MSL1 activity. G-J. A different <i>roX1</i> mosiac reporter displays the same male-specific <i>ocm</i> phenotype. K-N. Position effect variegation as measured by <i>In(1) w</i>^m4 is not affected by <i>ocm</i> mutations. The exact phenotypes of the flies are in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060450#s4" target="_blank">methods</a> section. O. Several hypomorphic <i>ocm</i> allelic combinations produce abundant females, but few or no males. X axis is viability. Allelic designations indicate codons affected. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060450#pone.0060450.s006" target="_blank">Table S2</a> for details.</p