Guilt by association: non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila

Reviews

non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila

Guilt by association non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila Dosage compensation is a striking example of the interplay between gene-specific regulation and chromosomal architecture. This process has evolved to make X-linked gene expression equivalent in males with one X chromosome and females with two. Examining species at the molecular level has shown that dosage compensation is mediated by sex-specific factors that decorate the X chromosomes to regulate chromatin structure and gene expression. In Drosophila, dosage compensation is achieved, at least in part, through sitespecific histone H4 acetylation, which is modulated by a male- and X-specific protein complex. The discovery of non-coding RNAs that ‘paint’ dosage-compensated X chromosomes in mammals and in Drosophila suggests that RNAs play an intriguing, unexpected role in the regulation of chromatin structure and gene expression. n many organisms, the chromosomal basis for sex determination results in two sexes that carry different numbers of X chromosomes. In mammals, nematodes and Drosophila, embryos with two X chromosomes undergo female somatic development, whereas embryos with only one X and a Y (or XO in nematodes) differentiate as males. Early in development, compensatory mechanisms are employed to make X-chromosome expression similar in the two sexes, despite the difference in X-chromosome number. Dosage compensation is an essential process in one sex of each of these organisms, and it appears that the majority of X-chromosomal genes have acquired this additional level of regulation during the evolution of sex chromosomes1–5. In mammals, one X chromosome is inactivated in each female nucleus, whereas in nematodes, both X chromosomes are partially repressed in the hermaphrodite. By contrast, in Drosophila, most X-linked genes are upregulated in males. These unrelated solutions still provide a common theme for dosage compensation: In each case, the unique transcriptional state of the dosagecompensated X chromosome is the result of its unique chromatin composition. X-chromosome-specific molecules decorate the X chromosome and play a role in remodeling the chromatin structure of the dosagecompensated chromosome in one sex, but not in the other (Table 1, Fig. 1). Drosophila have an impressive list of such molecules, which includes male X-chromosome-specific proteins, non-coding RNAs and a specific modification of histone H4 (Ref. 4). The Drosophila proteins are required for the site-specific acetylation of histone H4, which is implicated in the upregulation of transcription in many systems6–8. The non-coding RNAs are thought to play a role in assembly of or targeting the protein complex to the X chromosome9,10. In nematodes, a distinct protein complex is

I

Carsten Stuckenholz [email protected] Yuji Kageyama [email protected] Mitzi I. Kuroda [email protected] Department of Molecular and Human Genetics, Department of Cell Biology, and Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. 454

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associated with the X chromosomes2 that downregulates transcription in XX individuals and is related to the 13S condensin (involved in chromosome condensation and segregation) that has been identified in frogs and yeast11,12. Finally, in mammals, no proteins have been identified to date that regulate dosage compensation3. By contrast, a 15 kb non-coding RNA, named Xist, is required for X inactivation in females, and spreads in cis to inactivate the chromosome13–16. There are also changes in the nucleosomes on the inactive X – they are enriched for a variant of histone H2A (Ref. 17) and their histones are underacetylated18. The most unconventional molecules implicated in chromosome-specific regulation are the non-coding RNAs. Xist is clearly required for X inactivation in mammals13,14 and recently the RNAs on X (roX) RNAs have been implicated in Drosophila dosage compensation9,10,19,20. Before focusing on these intriguing molecules, we will first review the known protein regulators of dosage compensation in Drosophila males.

The male-specific lethal complex binds to the Drosophila male X chromosome The X-chromosome-specific proteins that function in Drosophila dosage compensation are each essential for male viability and are collectively named the Male-specific lethal (MSL) proteins4. The five MSL proteins: Maleless (MLE), MSL1, MSL2, MSL3 and Males-absent on the first (MOF; Table 2), are present in all somatic nuclei during male development. Their importance in dosage compensation was first demonstrated through analysis of mle mutants, in which the male X fails to exhibit hypertranscription or the diffuse morphology typical of the wildtype X chromosome21–23. The MSL proteins are mosteasily visualized on the giant polytene chromosomes of the 0168-9525/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01855-7

Reviews

non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila

Drosophila larva, where they colocalize in a banded pattern along the length of the male X chromosome (Fig. 1a). The proteins have been postulated to form a complex that is based on their colocalization and genetic interdependence – each MSL protein must be functional to detect complexes in a wild-type pattern on the X chromosome24–27. The association of MSL1, MSL2 and MSL3 has been examined in most detail28; the three proteins coimmunoprecipitate and cofractionate by gel-filtration and ion-exchange chromatography, and MSL1 interacts with MSL2 and MSL3 in the yeast two-hybrid system. MLE protein might not associate directly with the other MSL proteins, because biochemical studies suggest a looser or transient interaction. Association of the MSL proteins with the X chromosome correlates with its diffuse cytological appearance, which indicates a distinct chromatin structure29,30. Taken together, these observations provide substantial evidence that the X-chromosome-specific MSL proteins play an integral role in hypertranscription of the X chromosome4. An alternative model suggests that the relative levels of transcription from the X chromosome and the autosomes might be regulated indirectly by the MSL proteins, through sequestration of regulatory proteins from the autosomes31. The two very different models both predict that the MSL complex increases the relative ratio of X chromosome to autosomal gene expression in wild-type males, although by distinct biochemical mechanisms. Two key discoveries have provided a framework for understanding how the MSL proteins might catalyze a global change in X chromosomal architecture, leading to an increase in transcription. The first discovery was that the MSL proteins are required for the enrichment of a specific, post-translationally modified form of histone H4 on the male X, acetylated at Lysine 16 (H4Ac16)6,7. The second discovery was that the MOF protein is a putative histone acetyltransferase32. Recently, it has been established that histone acetylation is a commonly used mechanism by which transcription factors and co-activators assist RNA polymerase as it engages the generally repressive chromatin template8,33. The acetylation of histone H4, neutralizing a positive charge in its amino terminal tail, leads potentially to a diminished interaction between the core histone proteins and DNA, or between adjacent nucleosomes34. Thus, histone acetylation could allow easier access to the general factors required for expression of the diverse genes on the male X chromosome, resulting in hypertranscription. MLE, MSL1, MSL2 and MSL3 are thought to be critical for targeting MOF acetyltransferase activity to the X chromosome (Fig. 2). The MSL1 and MSL2 proteins are most central to the complex’s association with the X chromosome, because binding is completely abolished in msl1 and msl2 mutants. Moreover, neither MSL1 nor MSL2 will bind the X chromosome in the absence of the other, and MSL1 is dependent on MSL2 for stability26,35. The RING-finger domain of MSL2 binds the MSL1 protein, and residues around the first zinc-binding site of the MSL2 RING finger are critical for this interaction28. In mutants that lack either mle, msl3 or mof, the remaining MSL proteins can bind to approximately 30–40 sites scattered along the length of the X chromosome24–27. These sites have been proposed to act as chromatin entry sites, from which a fully functional and active MSL complex can normally spread in cis into flanking chromatin10. Spreading of

TABLE 1. Molecules that associate with dosage-compensated X chromosomes

Drosophila upregulated male X

Nematode downregulated hermaphrodite XX

Mammals inactive female X

Proteins

Nucleosome components

RNAs

MLE MSL1 MSL2 MSL3 MOF SDC-2 SDC-3 DPY-26 DPY-27 DPY-28 MIX-1 ?

8

H4Ac16

roX1 roX2

?

?

macroH2A hypoacetylated histones

Xist

Abbreviations: MLE, maleless; MSL, male specific-lethal; MOF, males-absent on the first; SDC, Sex and dosage compensation; DPY, dumpy; H4Ac16, Histone H4 acetylated at lysine 16; MIX, Mitosis and X-associated protein; roX, RNA on X; Xist, X inactive-specific transcript.

the complex, or its stable association with the normal complement of hundreds of sites on the X chromosome requires functional MSL3, MOF and MLE proteins.

FIGURE 1. X-chromosome association of Malespecific lethal 1 protein and roX1 RNA (a)

(b)

trends in Genetics

(a) The Male-specific lethal 1 (MSL1) protein is detected by immunofluorescence (red). (b) The RNA on X 1 (roX1) RNA is detected by in situ hybridization (red). In both (a) and (b), all chromosomes are counterstained with Hoechst 33258 (blue).

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non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila

TABLE 2. Sequence motifs of Drosophila dosagecompensation proteins MLE

Two double-stranded RNA-binding domains ATP-dependent RNA or DNA helicase (DExH family) Novel, acidic RING finger. Additional cysteine-rich region Two chromodomains Chromodomain C2HC-type zinc finger Histone acetyltransferase (MYST family)

MSL1 MSL2 MSL3 MOF

Abbreviations: MLE, maleless; MSL, male-specific-lethal; MOF, males absent on the first.

The formation of the MSL complex is male-specific, although most of the components are synthesized in both sexes. MSL2 is the key male-limited component36–38 and ectopic expression of MSL2 in females results in the appearance of complete MSL complexes on both X chromosomes, and significant female lethality30. Expression of MSL2 leads to stabilization of MSL1 protein, which plays

FIGURE 2. Model for dosage compensation in Drosophila Male

Female

(X:Autosome Ratio = .5)

(X:Autosome Ratio = 1)

SXL

SXL

MSL1 RN

MSL2 MLE

MSL2

As

roX

MOF

Complex formation in presence of MSL2

MSL3

a central role in assembling the remaining MSL proteins30,35. Sex lethal (SXL), an RNA-binding protein, plays the pivotal role in prevention of MSL2 expression in female Drosophila. SXL is a female-specific protein that is regulated by the ratio of X chromosomes to autosomes in the early embryo and thus, plays a key regulatory role in sex determination and dosage compensation2. SXL represses translation of MSL2 through sequences in the 59- and 39untranslated regions of msl2 transcripts36,37,39. Regulation of MSL1 by SXL could also contribute to the sex-specificity of dosage compensation. MSL1 is highly dependent on MSL2 for its protein stability, but a subset of its transcripts also contain SXL-binding sites in their 39-untranslated regions40 and these are found to contribute to MSL1 repression35. Overcoming these control mechanisms by overexpressing MSL1 and MSL2 in females is sufficient to completely switch this regulatory pathway into the male mode35. Studies on MLE foreshadowed the discovery of noncoding RNAs that bind the X chromosome. The MLE protein has several predicted RNA-interaction domains, including two double-stranded RNA-binding domains, an ATP-dependent helicase domain and a glycine-rich region. In vitro, MLE has ATP-dependent RNA- and DNAhelicase activity, and an intact ATP-binding domain is critical for its function in vivo41. Although MLE colocalizes with the MSL complex and is required for association of the other MSL proteins along the length of the X chromosome, it is the only MSL for which strong biochemical interactions with the other complex members are so far lacking28. This might be explained by the observation that, uniquely, MLE is released from the X chromosome by RNase, suggesting that RNA bridges its interaction with the other MSL proteins42. Whereas the possibility that MLE interacts with nascent transcripts to direct the complex to active genes has not been excluded, the discovery of the roX RNAs lends credence to the idea that the MSL complex could contain one or more integral RNA components.

Non-coding RNAs: a role in chromatin structure? Binding to X chromatin Site-specific acetylation and hypertranscription

MSL3

RN A

X chromatin

s?

MLE

roX

MSL1

MSL2 MOF

trends in Genetics

The Male-specific lethal (MSL) complex only forms in males, because MSL2 is translationally repressed by the female-specific Sex lethal (SXL) protein. In males, the complex targets the X chromosome where Males-absent on the first (MOF) is believed to acetylate lysine 16 of histone H4 (indicated by solid black triangle). H4Ac16 is thought to cause a more open chromatin structure, resulting in a twofold increase in transcription. The inclusion of roX RNAs in the complex is hypothetical. Nucleosomes are represented schematically as discs with DNA wrapped around their circumference.

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The discovery of two non-coding RNAs that bind the Drosophila male X chromosome opens up new possibilities for how the MSL complex might target, assemble and function on the X chromosome (Fig. 1b). The two candidate RNA components of the MSL complex are roX1 and roX2. These male-specific RNAs are structurally similar to mRNAs because they are spliced and polyadenylated, but lack significant protein-coding potential. They are retained in the nucleus and colocalize with the MSL complex on the male X chromosome9,19,20. They only accumulate in animals that express a functional MSL complex19,20. Thus, in wild-type females or in msl mutant males, roX RNAs are not evident, whereas in females that ectopically express the MSL complex, roX RNAs accumulate on the X chromosomes. This regulation could occur at the level of MSL-dependent transcriptional control of roX RNAs or through stabilization of the RNAs by the MSL complex. Despite their similar regulation and localization, roX1 and roX2 RNAs show little primary sequence homology9,20 and are distinct in size (3.6 kb versus 1.1 kb), yet are both encoded by genes on the X chromosome20. One model under consideration is that the roX RNAs might be scaffolds for regulated assembly of

Reviews

non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila

the MSL complex, and that the two RNAs have overlapping functions. In support of this model, males carrying a mutation in roX1, that are also deficient for a region encompassing roX2, fail to assemble MSL complexes on their X chromosomes early in development9. In addition to the idea that roX RNAs themselves play an integral role in dosage compensation, their sites of synthesis have been implicated in targeting of the MSL complex to the X chromosome10. The roX1 and roX2 genes map to 2 of the 30–40 chromatin entry sites along the length of the X chromosome that bind partial MSL complexes in the absence of msl3, mof or mle (Refs 10, 19, 20, 26). Intriguingly, MSL complexes are thought to spread from these sites into flanking X chromatin in cis. This new model is based on the observation that when roX1 transgenes are inserted on autosomes, they can attract MSL complexes, including roX RNAs, which then spread variably into autosomal sequences10. The roX RNAs were discovered as male-specific transcripts that were concentrated in the nuclei of adult neurons, which suggested a possible role in sexually dimorphic behavior19,20. However, male embryos and larvae accumulate roX1 RNA in the nuclei of most, if not all cells, which is more consistent with a function in a general process, such as dosage compensation9,19. The apparent concentration of roX RNAs in the adult nervous system has been questioned recently9. An additional intriguing observation is the location of genes predominantly expressed in females, adjacent to the male-specific roX genes. This arrangement suggests that, within each gene pair, regulation might occur by a mutually exclusive pattern, similar to the imprinted mammalian Igf2 (proteincoding) and H19 (non-coding RNA) genes20. However, transcripts from the opt1 gene, adjacent to roX1, accumulate to much-higher abundance in adult females than males because they are deposited maternally in large oocytes, a common expression pattern in Drosophila43. Thus, it is not clear what significance, if any, this gene organization has to the regulation of roX RNAs. The discovery of the association of roX RNAs with the X chromosome has prompted an examination of possible parallels between fly and mammalian dosage compensation (Table 3)44. The only known participant in X inactivation in mammals is Xist, a large non-coding RNA that originates from the region of the X chromosome that is required for its inactivation (the X inactivation centre; XIC). Although Xist RNA resembles a messenger RNA (it is large, alternatively spliced and polyadenylated), it does not encode a protein but, instead, acts in the nucleus to mediate X inactivation. The accumulation of Xist transcripts along the length of the X chromosome appears to be an essential step in chromosome inactivation3. An intriguing new non-coding RNA, antisense to Xist, was recently discovered at the XIC in mice and humans45. The Tsix transcript has no conserved open reading frame, and is detected solely in the nucleus at the XIC. Tsix is not observed to spread on either X chromosome. On the future inactive X chromosome, Tsix transcription disappears when Xist RNA begins to accumulate. By contrast, on the future active X chromosome, Tsix transcription persists until after Xist is no longer detectable, suggesting a role for Tsix in regulating the early steps of X inactivation45. roX genes and Xist are thought to work to achieve opposite goals: transcriptional upregulation versus tran-

TABLE 3. Comparison of roX and Xist RNAs roX RNA

Xist RNA

Similarities Spliced, polyadenylated non-coding RNAs Expressed from dosage compensated X ‘Paint’ dosage compensated X

Differences Paints X in cis and in trans At least 2 RNAs roX1 is dispensable Associated with transcriptional upregulation

Paints X in cis only Single RNA Xist is essential for X inactivation Causes transcriptional downregulation

scriptional inactivation. Nevertheless, their shared characteristics suggest a common function: they are non-coding RNAs that are thought to modulate chromatin architecture, and they are observed to spread in cis by unknown mechanisms from their sites of synthesis10,15. No systematic search for additional molecules of this class has been reported; these and most non-coding RNAs have been identified serendipitously. Finding more members of this class will be a significant challenge as current algorithms that search genome sequence data for open reading frames fail to identify non-coding RNAs. However, the extensive analysis of expressed sequences should identify more members of this class, which will be extremely helpful for studying the role, if any, that non-coding RNAs play in chromatin structure. In many ways, it is too early to propose specific models for how non-coding RNAs could regulate chromatin structure and gene expression. However, it would not be surprising to find that RNAs play an integral role in chromosome function. An attractive model for the origin of life is that it was once an ‘RNA world’, and support for this model comes from the many examples of the catalytic functions of RNAs that were originally thought to be the sole domain of proteins46. RNAs can form versatile three-dimensional structures and RNA species can be selected through in vitro experiments to bind with high affinities to a wide variety of novel substrates47. Whereas the overall prevalence of non-coding RNAs in any organism is not known, there has been extensive interest in the discoveries of non-coding RNAs in imprinted regions of the mammalian genome48. It is possible that one virtue of a regulatory RNA, as opposed to a protein, is that its site of synthesis on the chromosome could be a simple way to mark or target that specific region of the genome for regulation. We speculate that the roles of noncoding RNAs in the nucleus are just beginning to be realized.

Acknowledgements We regret citing reviews in many instances rather than the primary literature owing to space limitations. We are grateful to R. Kelley and P. Gordadze for Fig. 1, V. Meller and P. Gordadze for unpublished data, and R. Kelley for critical reading of the manuscript. C.S. was supported by the Welch Foundation and the Graduate School of Baylor College of Medicine. Y.K. is an Associate and M.I.K. an Associate Investigator at the Howard Hughes Medical Institute. TIG November 1999, volume 15, No. 11

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Drosophila. EMBO J. 16, 2054–2060 33 Brownell, J.E. et al. (1996) T. thermophila histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell 84, 843–851 34 Luger, K. et al. (1997) Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389, 251–260 35 Chang, K.A. and Kuroda, M.I. (1998) Modulation of MSL1 abundance in female Drosophila contributes to the sex specificity of dosage compensation. Genetics 150, 699–709 36 Bashaw, G.J. and Baker, B.S. (1997) The regulation of the Drosophila msl-2 gene reveals a function for Sex lethal in translational control. Cell 89, 789–798 37 Kelley, R.L. et al. (1997) Sex lethal controls dosage compensation in Drosophila by a nonsplicing mechanism. Nature 387, 195–199 38 Zhou, S. et al. (1995) Male-specific-lethal 2, a dosage compensation gene of Drosophila that undergoes sex-specific regulation and encodes a protein with a RING finger and a metallothionein-like cluster. EMBO J. 14, 2884–2895 39 Kelley, R.L. and Kuroda, M.I. (1995) Equality for X chromosomes. Science 270, 1607–1610 40 Palmer, M.J. et al. (1993) The male specific lethal-one gene encodes a novel protein that associates with the male X chromosome in Drosophila. Genetics 134, 545–557 41 Lee, C-G. et al. (1997) The NTPase/helicase activities of Drosophila Maleless, an essential factor in dosage compensation. EMBO J. 16, 2671–2681 42 Richter, L. et al. (1996) RNA-dependent association of the Drosophila maleless protein with the male X chromosome. Genes Cells 1, 325–336 43 Roman, G. et al. (1998) The opt1 gene of Drosophila melanogaster encodes a proton-dependent dipeptide transporter. Am. J. Physiol. 275, C857–C869 44 Willard, H.F. and Salz, H.K. (1997) Remodelling chromatin with RNA. Nature 386, 228–229 45 Lee, J.T. et al. (1999) Tsix, a gene antisense to Xist at the X-inactivation centre. Nat. Genet. 21, 400–404 46 Nitta, I. et al. (1998) Reconstitution of peptide bond formation with Escherichia coli 23S ribosomal RNA domains. Science 281, 666–669 47 Tuerk, C. and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 48 Barlow, D.P. (1997) Competition – a common motif for the imprinting mechanism? EMBO J. 16, 6899–6905

Ci a complex transducer of the Hedgehog signal

Pedro Aza-Blanc [email protected] Thomas B. Kornberg tkornberg@ biochem.ucsf.edu Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA. 458

Recent progress has unveiled Cubitus interruptus (Ci) as a complex transcription factor whose diverse activities as an activator and repressor are regulated by its proteolysis, localization and concentration. The principal role of Ci is to elaborate the developmental program directed by the morphogen Hedgehog (Hh), and it uses its various activities to target the expression of key downstream genes to different spatial domains. Here, we highlight recent advances in the Ci story, and discuss remaining questions whose resolution promise to help explain how morphogens like Hh signal their distant targets. edgehog (Hh) is a morphogen that patterns the growth and development of vertebrates and invertebrates. Its distribution and concentration are critical to its function, and its synthesis and localization are both tightly regulated. Hh synthesis is limited to discrete groups of cells –

H

TIG November 1999, volume 15, No. 11

for instance, to the posterior (P) compartment [but not anterior (A) compartment] cells of the Drosophila embryo and imaginal discs1,2 (Box 1). Its distribution is strongly influenced by post-translational modifications3,4 that restrict its movement, or at least influence how signaling is 0168-9525/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01869-7