Richard L. Kelley

Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila.

Male-specific lethal-2 (msl-2) is a RING finger protein that is required for X chromosome dosage compensation in Drosophila males. Consistent with the formation of a dosage compensation protein complex, msl-2 colocalizes with the other MSL proteins on the male X chromosome and coimmunoprecipitates with msl-1 from male larval extracts. Ectopic expression of msl-2 in females results in the appearance of the other MSL dosage compensation regulators on the female X chromosomes and decreased female viability. We suggest that msl-2 RNA is the primary target of SxI regulation in the dosage compensation pathway and present a speculative model for the regulation of two distinct modes of dosage compensation by SxI.

Epigenetic Spreading of the Drosophila Dosage Compensation Complex from roX RNA Genes into Flanking Chromatin

The multisubunit MSL dosage compensation complex binds to hundreds of sites along the Drosophila single male X chromosome, mediating its hypertranscription. The male X chromosome is also coated with noncoding roX RNAs. When either msl3, mle, or mof is mutant, a partial MSL complex is bound at only approximately 35 unusual sites distributed along the X. We show that two of these sites are the roX1 and roX2 genes and postulate that one of their functions is to provide entry sites for the MSL complex to recognize the X chromosome. The roX1 gene provides a nucleation site for extensive spreading of the MSL complex into flanking chromatin even when moved to an autosome. The spreading can occur in cis or in trans between paired homologs. We present a model for how the dosage compensation complex recognizes X chromatin.

Ordered assembly of roX RNAs into MSL complexes on the dosage-compensated X chromosome in Drosophila

BACKGROUNDnIn the male Drosophila, the X chromosome is transcriptionally upregulated to achieve dosage compensation, in a process that depends on association of the MSL proteins with the X chromosome. A role for non-coding RNAs has been suggested in recent studies. The roX1 and roX2 RNAs are male-specific, non-coding RNAs that are produced by, and also found associated with, the dosage-compensated male X chromosome. Whether roX RNAs are physically part of the MSL complex has not been resolved.nnnRESULTSnWe found that roX RNAs colocalize with the MSL proteins and are highly unstable unless the MSL complex is coexpressed, suggesting a physical interaction. We were able to immunoprecipitate roX2 RNA from male tissue-culture cells with antibodies to the proteins Msl1 and Mle, consistent with an integral association with MSL complexes. Localization of roX1 and roX2 RNAs in mutants indicated an order of MSL-complex assembly in which roX2 RNA is incorporated early in a process requiring the Mle helicase. We also found that the roX2 gene, like roX1, is a nucleation site for MSL complex spreading into flanking chromatin in cis.nnnCONCLUSIONSnOur results support a model in which MSL proteins assemble at specific chromatin entry sites (including the roX1 and roX2 genes); the roX RNAs join the complex at their sites of synthesis; and complete complexes spread in cis to dosage compensate most genes on the X chromosome.

Noncoding RNA Genes in Dosage Compensation and Imprinting

Alleles silenced via imprinting are not thought to be packaged in densely compacted heterochromatin, but several imprinted genes have oppositely imprinted noncoding RNA partners nearby on the chromosome. Maternal expression of the Igf2r gene is reminiscent of the Xist/Tsix locus in that imprinting is tied to antisense transcription on the paternal chromosome (Wutz et al. 1997xWutz, A, Smrzka, O.W, Schweifer, N, Schellander, K, Wagner, E.F, and Barlow, D.P. Nature. 1997; 389: 745–749Crossref | PubMed | Scopus (421)See all ReferencesWutz et al. 1997) (Figure 1CFigure 1C). Perhaps the most intensively studied example of imprinting is the paternally expressed Ins2 Igf2 gene cluster that is linked to the maternally expressed H19 gene (Tilghman 1999xTilghman, S.M. Cell. 1999; 96: 185–193Abstract | Full Text | Full Text PDF | PubMed | Scopus (385)See all ReferencesTilghman 1999) (Figure 2AFigure 2A). Although no function has been found for the noncoding H19 RNA, it is conserved and abundantly expressed. Moreover, the Dlk1/Gtl2 locus was recently found to have a nearly identical arrangement of a paternally imprinted protein coding gene adjacent to a maternally imprinted noncoding RNA, suggesting some type of selection for these unusual RNAs (Schmidt et al. 2000xSchmidt, J.V, Matteson, P.G, Jones, B.K, Guan, X.-J, and Tilghman, S.M. Genes Dev. 2000; 14: 1997–2002PubMedSee all ReferencesSchmidt et al. 2000) (Figure 2BFigure 2B).Figure 2Reciprocal Imprinting of Adjacent Protein-Coding and Noncoding RNA Genes(A) Reciprocal imprinting is observed at the Igf2 locus where a single enhancer drives expression of either Ins2 and Igf2 from the paternal homolog, or noncoding H19 RNA from the maternal chromosome. The imprinting control region (ICR) contains CpG-rich sequences that serve as the binding site for the chromatin insulator CTCF. When CTCF occupies the ICR, the enhancer cannot act on distal genes, and H19 is transcribed. Paternal methylation of the ICR prevents CTCF binding allowing the enhancer to act on the Igf2 and Ins2 genes (5xHark, A.T, Schoenherr, C.J, Katz, D.J, Ingram, R.S, Levorse, J.M, and Tilghman, S.M. Nature. 2000; 405: 486–489Crossref | PubMed | Scopus (946)See all References, 3xBell, A.C and Felsenfeld, G. Nature. 2000; 405: 482–485Crossref | PubMed | Scopus (1003)See all References).(B) The paternally imprinted Dlk1 gene is upstream of a maternally imprinted Gtl2 gene, which makes a noncoding RNA. As with the Igf2/H19 locus, a differentially methylated CpG-rich region (filled circle) lies upstream on the noncoding RNA gene.View Large Image | View Hi-Res Image | Download PowerPoint SlideAs unconventional RNAs are being encountered in novel epigenetic regulatory mechanisms, one striking feature is that the site of synthesis is critical to function. In both mammals and flies, moving the Xist or roX genes from the X to autosomes redirects dosage compensation to the new sites of insertion. Antisense Tsix expression from one homolog cannot regulate the Xist allele from the other homolog, demonstrating exclusive cis activity. The same appears to be true within the Igf2r locus. Is it possible that RNA is a common epigenetic regulator? By analogy to the RNAs discovered so far, noncoding RNAs could function by repackaging a local segment of chromatin, or by capturing a complementary mRNA, or they could simply be by-products from some type of mutually exclusive action of linked promoters.‡E-mail: rkelley@bcm.tmc.edu (R. L. K.), mkuroda@bcm.tmc.edu (M. I. K.)

Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site

In Drosophila, dosage compensation is controlled by the male‐specific lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is specifically localized on the male X chromosome to increase its expression ∼2‐fold. We recently proposed a model for the targeted assembly of the MSL complex, in which initial binding occurs at ∼35 dispersed chromatin entry sites, followed by spreading in cis into flanking regions. Here, we analyze one of the chromatin entry sites, the roX1 gene, to determine which sequences are sufficient to recruit the MSL complex. We found association and spreading of the MSL complex from roX1 transgenes in the absence of detectable roX1 RNA synthesis from the transgene. We mapped the recruitment activity to a 217 bp roX1 fragment that shows male‐specific DNase hypersensitivity and can be preferentially cross‐linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is sufficient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kilobases into neighboring regions.

Equality for X Chromosomes

In many species, females possess two X chromosomes and males have one X chromosome. This difference is critical for the initial determination of sex. However, the X encodes many functions required equally in males and females; thus, X chromosome expression must be adjusted to compensate for the difference in dosage between the sexes. Distinct dosage compensation mechanisms have evolved in different species. A common theme in the Drosophila melanogaster and Caenorhabditis elegans systems is that a subtle alteration of chromatin structure may impose this modest, but vital adjustment of the X chromosome transcription level.

Transcription rate of noncoding roX1 RNA controls local spreading of the Drosophila MSL chromatin remodeling complex.

The dosage compensation complex in Drosophila is composed of at least five MSL proteins and two noncoding roX RNAs that bind hundreds of sites along the single male X chromosome. The roX RNAs are transcribed from X-linked genes and their RNA products paint the male X. The roX RNAs and bound MSL proteins can spread in cis from sites of roX transcription, but the mechanism controlling spreading is unknown. Here we find that cis spreading from autosomal roX1 transgenes is coupled to the level of roX transcription. Low to moderate transcription favors, and vigorous transcription abolishes local spreading. We constructed a roX1 minigene one third the size of wild type as a starting point for mutagenesis. This allowed us to test which evolutionarily conserved motifs were required for activity. One short repeat element shared between roX1 and roX2 was found to be particularly important. When all copies were deleted, the RNA was inactive and unstable, while extra copies seem to promote local spreading of the MSL complex from sites of roX1 synthesis. We propose that assembly of the MSL proteins onto the extreme 3 region of elongating roX1 transcripts determines whether the MSL complex spreads in cis.

The role of chromosomal RNAs in marking the X for dosage compensation

Both flies and mammals remodel the architecture of the X chromosome to achieve dosage compensation. A novel class of noncoding RNAs that paint entire chromosomes are centrally involved in this process. The genes encoding these unusual RNAs are themselves located on the X, and are key sites that target the X for dosage compensation.

X chromosome dosage compensation in Drosophila.

a colon length of 15,000 crypts. The mean width of the observed adenomas was seven crypts (although this figure is an overestimate of x, because it was observed after any collisions had occurred, which presumably had the effect of increasing x). We analyzed the model assuming different values of n and determining whether these values can account for two observations: (i) the final figure of 300 adenomas after any collisions have occurred, and (ii) the estimate of 76% polyclonality. In general, it is not possible to reconcile observations (i) and (ii). If n is sufficiently small to account for observation (i), then far too few collisions occur to account for observation (ii). Conversely, if n is large enough to account for observation (ii), then many more polyps than 300 result. For example, use an estimate for n, assuming that each polyclonal adenoma is formed of three original adenomas. In this case, n = 756, x = 7, and y

Autoregulation of the Drosophila Noncoding roX1 RNA Gene

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.