Autoregulation of the Drosophila Noncoding roX1 RNA Gene Chiat Koo Lim1, Richard L. Kelley1,2* 1 Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, United States of America, 2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America Abstract Most genes along the male single X chromosome in Drosophila are hypertranscribed about two-fold relative to each of the two female X chromosomes. This is accomplished by the MSL (male-specific lethal) complex that acetylates histone H4 at lysine 16. The MSL complex contains two large noncoding RNAs, roX1 (RNA on X) and roX2, that help target chromatin modifying enzymes to the X. The roX RNAs are functionally redundant but differ in size, sequence, and transcriptional control. We wanted to find out how roX1 production is regulated. Ectopic DC can be induced in wild-type (roX1+ roX2+) females if we provide a heterologous source of MSL2. However, in the absence of roX2, we found that roX1 expression failed to come on reliably. Using an in situ hybridization probe that is specific only to endogenous roX1, we found that expression was restored if we introduced either roX2 or a truncated but functional version of roX1. This shows that pre-existing roX RNA is required to positively autoregulate roX1 expression. We also observed massive cis spreading of the MSL complex from the site of roX1 transcription at its endogenous location on the X chromosome. We propose that retention of newly assembled MSL complex around the roX gene is needed to drive sustained transcription and that spreading into flanking chromatin contributes to the X chromosome targeting specificity. Finally, we found that the gene encoding the key male-limited protein subunit, msl2, is transcribed predominantly during DNA replication. This suggests that new MSL complex is made as the chromatin template doubles. We offer a model describing how the production of roX1 and msl2, two key components of the MSL complex, are coordinated to meet the dosage compensation demands of the male cell. Citation: Lim CK, Kelley RL (2012) Autoregulation of the Drosophila Noncoding roX1 RNA Gene. PLoS Genet 8(3): e1002564. doi:10.1371/journal.pgen.1002564 Editor: Jeannie T. Lee, Massachusetts General Hospital, Howard Hughes Medical Institute, United States of America Received September 22, 2011; Accepted January 15, 2012; Published March 15, 2012 Copyright: 2012 Lim, Kelley. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The initial phase of this work was supported by NIH grant 5RO1GM071538 to RLK and completed with funds from Baylor College of Medicine. Funding for the BCM Cytometry and Cell Sorting Core came from the NIH (NCRR grant S10RR024574, NIAID AI036211, and NCI P30CA125123). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rkelley@bcm.tmc.edu Introduction Some long noncoding RNAs have the ability to recruit chromatin modifying enzymes to specific genes thereby controlling their expression [1]. Other noncoding RNAs behave as transcrip- tional enhancers to flanking protein coding genes [2]. The two roX RNAs that participate in dosage compensation of the single male X chromosome in Drosophila are some of the best characterized examples of noncoding RNAs that target chromatin remodeling enzymes to large domains [3]. The roX RNAs assemble into a complex containing at least five MSL protein subunits that bind actively transcribed genes along the male X chromosome, but not autosomes or the two X chromosomes in females [4]. This has been termed the dosage compensation complex or the MSL complex. One function of the complex is acetylation of histone H4 at lysine 16, carried out by the MOF (males absent on first) histone acetyltransferase resulting in an essential ,two-fold increase in transcription [5–8]. Another modification is ubiquitylation of histone H2B at K34 by the MSL2 RING finger protein [9]. Flies carry two roX genes that differ greatly in size and sequence [10]. The roX1 gene is located on the X chromosome at polytene band 3F and produces a 3.7 kb RNA. The roX2 gene is located at 10C on the X and makes a ,600 nt RNA. Both RNAs ‘paint’ the length of the male X in a banded pattern [11]. The only obvious sequence similarity between them is limited to short repeated elements near the 39 end of each gene [12]. These repeats are essential for function and predicted to fold into conserved secondary structures [13,14]. Neither roX RNA is maternally deposited in eggs. Zygotic transcription of roX1 RNA occurs in both male and female embryos beginning at blastoderm [15]. Females lose roX1 RNA midway through embryogenesis, but males maintain expression through adulthood. By contrast, roX2 RNA first appears a few hours after roX1 but only in male embryos [16]. Despite the vast differences in size, sequence, and regulation, the two roX RNAs are functionally redundant [17]. Little is known about how production of the roX RNAs and MSL protein subunits are coordinated. Unusual cis spreading behavior of MSL complex from sites of autosomal roX transgene has been attributed to cotranscriptional assembly of free MSL subunits onto growing nascent roX transcripts [13,18,19], although direct biochemical evidence is lacking. Most MSL protein subunits are made in both males and females, except for MSL2 which is translationally repressed in females by the action of SXL [20,21]. MSL2 is a RING finger protein that binds DNA in a sequence independent manner through a second cysteine-rich motif [22]. The H83M2 transgene removes the 59 and 39 UTRs containing SXL binding sites from msl2 mRNA and has been used extensively to drive ectopic MSL2 expression in females [5,11,15,17,23–29]. Forcing females to make MSL2 using the H83M2 transgene induces production of roX RNAs, resulting in ectopic dosage PLoS Genetics | www.plosgenetics.org 1 March 2012 | Volume 8 | Issue 3 | e1002564