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

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