
==== Front
Genetics
Genetics
genetics
Genetics
0016-6731
1943-2631
Oxford University Press US

38718207
10.1093/genetics/iyae074
iyae074
Investigation
Gene Expression
AcademicSubjects/SCI01180
AcademicSubjects/SCI01140
siRNA that participates in Drosophila dosage compensation is produced by many 1.688X and 359 bp repeats
Biswas Sudeshna Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Detroit, MI 48202, USA

Gurdziel Katherine Department of Pharmacology, Wayne State University, Integrative Bioscience Center (iBio), 6135 Woodward, Detroit, MI 48202, USA
Institute of Environmental Health Sciences, Wayne State University, Integrative Bioscience Center (iBio), 6135 Woodward, Detroit, MI 48202, USA

https://orcid.org/0000-0002-3727-983X
Meller Victoria H Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Detroit, MI 48202, USA

Bateman J Editor
Corresponding author: 5047 Gullen Mall, Detroit, MI 48202, USA. Email: victoria.meller@wayne.edu
Conflicts of interest The author(s) declare no conflict of interest.

7 2024
08 5 2024
08 5 2024
227 3 iyae07419 12 2023
29 4 2024
25 5 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.
2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Organisms with differentiated sex chromosomes must accommodate unequal gene dosage in males and females. Male fruit flies increase X-linked gene expression to compensate for hemizygosity of their single X chromosome. Full compensation requires localization of the Male-Specific Lethal (MSL) complex to active genes on the male X, where it modulates chromatin to elevate expression. The mechanisms that identify X chromatin are poorly understood. The euchromatic X is enriched for AT-rich, ∼359 bp satellites termed the 1.688X repeats. Autosomal insertions of 1.688X DNA enable MSL recruitment to nearby genes. Ectopic expression of dsRNA from one of these repeats produces siRNA and partially restores X-localization of MSLs in males with defective X recognition. Surprisingly, expression of double-stranded RNA from three other 1.688X repeats failed to rescue males. We reconstructed dsRNA-expressing transgenes with sequence from two of these repeats and identified phasing of repeat DNA, rather than sequence or orientation, as the factor that determines rescue of males with defective X recognition. Small RNA sequencing revealed that siRNA was produced in flies with a transgene that rescues, but not in those carrying a transgene with the same repeat but different phasing. We demonstrate that pericentromeric X heterochromatin promotes X recognition through a maternal effect, potentially mediated by small RNA from closely related heterochromatic repeats. This suggests that the sources of siRNAs promoting X recognition are highly redundant. We propose that enrichment of satellite repeats on Drosophilid X chromosomes facilitates the rapid evolution of differentiated sex chromosomes by marking the X for compensation.

dosage compensation
X recognition
Drosophila melanogaster; siRNA
small RNA
1.688X satellite repeats
roX
359 bp repeats
epigenetics
Wayne State University 10.13039/100006710 NIH 10.13039/501100012264
==== Body
pmcIntroduction

Many species have evolved highly differentiated X and Y chromosomes that create an imbalance in gene dosage between males and females (Disteche 2012). To ensure equivalent expression of X-linked genes, these species have evolved a process termed dosage compensation. Mammals achieve dosage compensation by inactivating one of the two X chromosomes in females, but Drosophila employ a different strategy (Charlesworth 1996; Lucchesi et al. 2005). Fruit fly males upregulate expression from their single X chromosome about two-fold (Lucchesi 1978). A ribonucleoprotein complex called the Male-Specific Lethal complex or Dosage Compensation Complex (MSL or DCC) is necessary for full dosage compensation in male flies (Gelbart and Kuroda 2009). The MSL complex is made up of five proteins, Male-Specific Lethal 1, -2 and -3 (MSL1; MSL2; and MSL3); Maleless (MLE) and Males absent On the First (MOF). MOF, a histone acetyltransferase, acetylates histone H4 at lysine 16 (H4K16Ac) (Hilfiker et al. 1997; Smith et al. 2000). Acetylation at this position is associated with active genes and decondensed chromatin (Grunstein 1997; Shogren-Knaak et al. 2006). H4K16 is highly enriched over the body of X-linked genes and this mark is thought to be responsible for transcriptional upregulation in males (Bone et al. 1994; Hilfiker et al. 1997; Akhtar and Becker 2000; Smith et al. 2000; Copur et al. 2018). In addition to these five proteins, one of two functionally redundant long noncoding RNAs, RNA on the X 1 and -2 (roX1 and roX2), also participate in MSL complex formation (Amrein and Axel 1997; Meller et al. 1997; Ilik et al. 2013). Simultaneous mutation of both roX genes disrupts X-localization of MSL proteins and leads to male lethality (Meller and Rattner 2002; Deng et al. 2005; Deng and Meller 2006).

Although the components of the dosage compensation complex and their functions are well studied, how the MSL complex selectively recognizes the X chromosome is not clear. One model suggests that the MSL complex forms on roX transcripts and is recruited to 200–300 X-linked Chromatin Entry Sites, also termed High Affinity Sites (CES, HAS) (Kelley et al. 1999; Park et al. 2002; Oh et al. 2003). CES are enriched for a 21 bp GA-rich MRE (MSL Recognition Element) motif (Alekseyenko et al. 2008). A zinc finger protein called Chromatin-Linked Adaptor for MSL Proteins (CLAMP), binds MRE sequences throughout the genome, but selectively recruits the MSL complex to X-linked CES (Soruco et al. 2013). The MSL complex then spreads to nearby genes through binding of MSL3 to the co-transcriptional H3K36me3 mark (Bell et al. 2007; Kind and Akhtar 2007; Larschan et al. 2007; Sural et al. 2008). Although autosomal MRE motifs bind CLAMP, they do not recruit the MSL complex. A CES variant termed PionX (Pioneering sites on the X) is reported to interact with CLAMP and a domain of MSL2 (Villa et al. 2016). These sites are among the first CES to be bound during the assembly of MS complex and are enriched on the X chromosome, suggesting a role in distinguishing the X chromosome and autosomes (Albig et al. 2019).

Both roX genes are X-linked and overlap CES. roX transgenes recruit compensation to nearby active genes when inserted on an autosome, suggesting a role in marking the X chromosome (Kelley et al. 1999; Henry et al. 2001; Joshi and Meller 2017). However, roX RNA produced from an autosomal transgene is incorporated into the MSL complex and travels to the X chromosome to rescue compensation in roX1 roX2 males (Meller and Rattner 2002). This indicates that roX genes are not sufficient to confer X-identity. A chromosome conformation study demonstrated that CES are spatially arranged close to each other in the male interphase nucleus (Ramírez et al. 2015). Long-range interactions near active X-linked genes are more pronounced in males compared to females, which suggests that chromosome organization contributes to X recognition and dosage compensation (Grimaud and Becker 2009; Pal et al. 2019).

Previous studies from our lab have established a role for small interfering RNA (siRNA) in X recognition (Menon and Meller 2012; Menon et al. 2014). There are hundreds 1.688X satellite repeats (CsCl density of 1.688 g/cm3) in short, tandem repeats in euchromatin of the Drosophila X chromosome (Hsieh and Brutlag 1979; Waring and Pollack 1987; DiBartolomeis et al. 1992; Kuhn et al. 2012). These are AT-rich, have a repeat unit of ∼359 bp and are dissimilar to CES. Many 1.688X repeats are transcribed and small RNA aligning to 1.688X repeats is present in early embryos and the maternal germ line, but rare or absent in later development (Menon et al. 2014). Ectopic expression of double-stranded RNA (dsRNA) from a repeat at cytological position 3F (1.6883F) dramatically rescued the survival of roX1 roX2 males and partially restored X-localization of the MSL proteins (Menon et al. 2014). Sequencing revealed substantial production of siRNAs upon 1.6883F dsRNA expression. In addition, autosomal transgenes containing 1–2 kb of DNA from 1.6883F, as well as repeats at 1A and 3C (1.6881A, 1.6883C) recruit MSL proteins in males and increase expression of genes up to 150 kb away (Joshi and Meller 2017; Deshpande and Meller 2018). This suggests a role for 1.688X DNA in marking the X, and for small RNA from these repeats in the process of X recognition.

Repeats at 1.6881A, 1.6883C, and 1.6884A were selected to create dsRNA-expressing transgenes as these repeats range from very similar to quite divergent from 1.6883F (89, 67 and 95% identity, respectively). Surprisingly, dsRNA-producing transgenes constructed with 1.6881A, 1.6883C, and 1.6884A sequences failed to rescue roX1 roX2 males (Menon et al. 2014, and unpublished). This was baffling because insertion of 1.6881A or 1.6883C DNA on an autosome attracts the compensation machinery to nearby genes, indicating that these repeats do contribute to marking the X for compensation (Joshi and Meller 2017). Intriguingly, 1.6883F is immediately distal to roX1. The Drosophila genome annotation depicts a roX1 transcript extending through the 1.6883F repeat, but we have been unable to detect this extended transcript by amplification of cDNA and believe that virtually all roX1 transcripts end at termination signals between roX1 and the 1.6883F repeats. Nevertheless, the proximity of these elements is provocative and suggested that the 1.6883F repeat might have a unique biological function. Notably, roX1 is expressed 1.5 h after egg laying (AEL) and supports initial X recognition (Meller 2003). In roX1 mutant males, X-localization of the MSL proteins is delayed until the onset of roX2 expression at 6 h AEL. These observations suggested that two RNA-producing elements at 3F might collaborate to drive initial X recognition.

It is possible that only siRNA from 1.6883F is capable of promoting X recognition and achieving rescue of roX1 roX2 males, and that sequence differences between the 1.688X repeats previously tested are critical. Alternatively, details of construction of the transgenes expressing dsRNA from each repeat could determine function. To address these questions, we designed studies to determine if repeat sequence determines biological activity, or if the details of transgene construction play a crucial role.

We show that dsRNA from the 1.6881A and 1.6883C repeats rescues roX1 roX2 males when the expressing transgenes are constructed with the same repeat phasing as used to generate the original 1.6883F dsRNA construct. Selection of these repeats enables direct comparisons between the original 1.6881A and 1.6883C dsRNA transgenes that fail to rescue roX1 roX2 males and reconstructed versions containing 1.6881A and 1.6883C sequences. Small RNA sequencing revealed that 1.6881A dsRNA is processed into small RNA when the phasing of the repeat matches that of the original 1.6883F dsRNA construct. In contrast, virtually no siRNA aligning to 1.6881A was detected in flies expressing dsRNA 1.6881A from the original transgene that does not rescue roX1 roX2 males. We conclude that the distinction between the repeats previously tested is due to details of transgene construction, not DNA sequence. This suggests that many of the 1.688X repeats on the X are capable of producing small RNA promoting X recognition. We provide evidence that the closely related pericentromeric 359 bp repeats, comprising 10 Mb of X heterochromatin, as well as shorter blocks on the second and third chromosomes, act maternally to promote X recognition, possibly through production of siRNAs that are transmitted to the zygote. These findings indicate high redundancy in the sources of satellite siRNAs that promote X recognition.

Materials and methods

Fly culture and genetics

Flies were maintained at room temperature on yeast and molasses media. Strains used in these studies are presented in Supplementary Table 1. Transgenes were injected by Rainbow Transgenics (Camarillo, CA). Three independent second chromosome insertions of each construct were selected for further studies. Integrations of roX1 and 1.6883F recruiting elements in the haf gene were previously described (Joshi and Meller 2017). Integrations were recombined with p[w+Sqh-Gal4]2 and homozygous adults mated to flies carrying reconstructed pWIZ transgenes. Groups of 60 larvae were collected for RNA isolation and RT-qPCR (primers in Supplementary Table 3). roX1ex33A roX2Δ Zhr1 recombinants were identified by single fly qPCR.

Generation of reconstructed transgenes

All dsRNA-expressing constructs were created using the pWIZ (White Intron Zipper) vector (Lee and Carthew 2003). Reconstructed repeats with the same size and repeat phasing as the original pWIZ-ds1.6883F transgene are designated by a superscript indicating the repeat followed by R (1.6881AR, 1.6883CR). Due to high AT content, the 1.6881AR repeat was synthesized as two gene blocks based on sequence downloaded from Flybase (FB2020; Öztürk-Çolak et al. 2024), synthesized by Integrated DNA Technologies and joined at a naturally occurring EcoRI site. Blocks were amplified, digested with EcoRI, ligated, digested with XbaI, gel-purified, ligated into pBlueScript, and confirmed by sequencing. Primers used for the generation and validation of transgenes are presented in Supplementary Table 2. Assembly of 1.6883CR was accomplished by PCR of overlapping fragments templated with an existing 901 bp 1.6883C amplicon (Menon et al. 2014). Gel-purified amplicons were mixed and subjected to five cycles of denaturation, annealing, and extension. This templated an amplification with flanking primers 3C_Fo and 3C_Ro. A product of the correct size was gel purified, digested with Xho1 and Not1, inserted into pBlueScript and confirmed by sequencing. 1.6881AR and 1.6883CR inserts were introduced sequentially into pWIZ and orientation was verified by sequencing after each step. Random insertions were mapped to chromosome and three independent insertions of teach transgene selected for further studies.

Rescue of roX1 roX2 males

Males homozygous for each reconstructed transgene were mated to roX1 roX2; p[w+Sqh-Gal4]2/+ virgins to produce offspring with and without the p[w+Sqh-Gal4]2 driver. Offspring were counted for 10 days following initial eclosion and eye color was used to distinguish flies with the p[w+Sqh-Gal4]2 driver. The driver had no effect on female survival. For this reason, the total number of daughters was used to calculate survival of each class of sons (Supplementary Tables 4 and 5).

RNA preparation

Groups of 60 third instar male larvae were homogenized in Trizol (Invitrogen). RNA was cleaned using the QIAGEN RNeasy kit and measured by nanodrop as previously described (Koya and Meller 2015). One μg of RNA was reverse transcribed using random hexamers and Invitrogen SuperScript IV reverse transcriptase.

Quantitative RT-PCR

Amplification of cDNA was performed using Power SYBR Green PCR master mix on a Quantstudio3 qPCR system (ThermoFisher Scientific). Primers are presented in Supplementary Table 3. Values were normalized to dmn (DCTN2-p50). Fold change in expression was calculated relative to the yw lab reference strain using the efficiency corrected comparative quantification method (Pfaffl 2001). The high copy number of pericentric 359 bp repeats required serial dilution of qPCR template. To determine the influence of dsRNA expression on an autosomal gene with integrated recruiting elements, transgene insertions that achieved the strongest rescue of roX1 roX2 males were selected from the three independent insertions tested.

siRNA sequencing and alignment

Total RNA prepared as described above was shipped to Azenta Life Sciences for siRNA isolation and sequencing. Libraries were generated using the NEB Small RNA library Prep Kit (Ipswich, MA, USA) and Illumina 3′ and 5′ adapters. Index sequences were added by amplification and products of 145–160 bp were gel purified and sequenced on an Illumina NovaSeq (2 × 150 bp). Raw sequencing reads underwent quality analyses and adapter trimming using Trimmomatic (v0.30). Trimmed reads of 18 to 30 bp were aligned to the Drosophila genome (dm6) with bowtie2 with and default parameters (Hoskins et al. 2015). Reads were also aligned to the transcribed region of pWIZ-1.6881AR-inv using bowtie 1 (-l 10 -m 100) with zero mismatches.

Results

Details of transgene construction determine activity

The idea that the 1.6883F repeat, situated immediately distal to roX1, had special properties was intriguing. However, details of construction differed for the original pWIZ-ds1.6881A, pWIZ-ds1.6883C, and pWIZ-ds1.6883F transgenes (Menon et al. 2014). The amplified repeat segments were of different sizes (Fig. 1a and Supplementary Fig. 3). The ends of each amplicon also fell at different places in the 359 bp repeat unit, and thus each amplicon differed in repeat phasing. Many of the 1.688X repeats contain an EcoRI site that we arbitrarily designated as the boundary of individual repeats in tandem clusters. A two base pair change in 1.6883C converts EcoRI to SacI (see Supplementary Fig. 3). Finally, the orientation of repeat DNA introduced into pWIZ differed between pWIZ-ds1.6883F and the other two original constructs (Fig. 1a). We first performed PCR and sequencing on flies with the original pWIZ-ds1.6881A and pWIZ-ds1.6883C transgenes and confirmed that these were constructed as intended and had not been rearranged in transgenic flies. Reverse transcription and qPCR (RT-qPCR) of RNA from flies carrying pWIZ-ds1.6881A and the strong, near-ubiquitous p[w+Sqh-GAL4]2 driver confirmed expression, although at a lower level than the reference pWIZ-ds1.6883F insertion (Supplementary Fig. 1). We also validated substantial rescue of males with the severe roX1SMC17A roX2Δ mutations upon expression of pWIZ-ds1.6883F, but minimal rescue by pWIZ-ds1.6881A (Fig. 2b).

Fig. 1. Original and reconstructed 1.688X pWIZ transgenes. a) Left—1.6883F, 1.6881A, and 1.6883C amplicons used in original pWIZ-ds1.688X transgenes (Menon et al. 2014). Arrows represent ∼359 bp repeat units. EcoR1 sites present in many 1.688X repeats are arbitrarily designated as the endpoint of each repeat unit. The dotted line in 1.6883C represents a region with limited repeat homology. Center—orientation of inserts in original constructs. Right—dsRNAs produced by each construct. b) Inserts in reconstructed pWIZ-ds1.6881AR and pWIZ-ds1.6883CR are the same size, orientation, and phasing as the original pWIZ-ds1.6883F. The orientation of inserts in pWIZ-ds1.6881AR-inv and pWIZ-ds1.6883CR-inv is inverted.

Fig. 2. Expression of reconstructed ds1.688X transgenes rescues roX1SMC17A roX2Δ males. a) roX1SMC17A roX2Δ females heterozygous for the p[w+Sqh-GAL4]2 driver were mated to males carrying pWIZ transgenes. The recovery of male offspring was normalized by multiplying males of each category by two and dividing by the total number of daughters. b) The original pWIZ-ds1.6883F and pWIZ-ds1.6881A (hatched bars) and three independent insertions of each reconstructed transgenic (dark bars) were tested. The survival of males with each pWIZ transgene but without driver is shown by light gray bars. Error bars represent the SEM of four biological replicates. *P < 0.05; **P < 0.01, ***P < 0.001, as determined by Student’s t test.

This raises the question of whether the disparity in biological activity is due to differences in DNA sequence, expression levels, or to differences in the size, orientation, or phasing of repeat DNA in the pWIZ transgene. To address this, we constructed transgenes with 1.6881A and 1.6883C sequence but with the same size, orientation, and repeat phasing as pWIZ-ds1.6883F (see Materials and methods). This produced pWIZ-ds1.6881AR (89% sequence identity to 1.6883F) and pWIZ-ds1.6883CR (68% identity to 1.6883F) (see Supplementary Fig. 3 for sequence alignments). This strategy enables direct comparison to the original transgenes with the same repeat sequence in different phasing. To explore the role of orientation, we constructed pWIZ-ds1.6881AR-inv and pWIZ-ds1.6883CR-inv with the same inserts as pWIZ-ds1.6881AR and pWIZ-ds1.6883CR but inverted in orientation (Fig. 1b). Three independent second chromosome insertions of each new transgene were selected for further studies.

To confirm expression, total RNA was extracted from third instar male larvae carrying reconstructed transgenes and the p[w+Sqh-Gal4]2 driver (Supplementary Table 1). Primers to the w+ intron, present in the unprocessed transcript, were used to assess relative expression from each insertion (Supplementary Fig. 1). All transgenes were expressed but with variability due to insertion site. Nevertheless, the range of expression levels overlaps that of the original pWIZ-ds1.6881A and pWIZ-ds1.6883F transgenes.

To assess the biological activity of reconstructed transgenes, expression was driven in roX1SMC17A roX2Δ males (∼2–3% escapers) and roX1ex33A roX2Δ males (∼25% escapers). Survival of roX1SMC17A roX2Δ and roX1ex33 roX2Δ males expressing RNA from pWIZ-ds1.6881AR and pWIZ-ds1.6881AR-inv matched that achieved by the original pWIZ-ds1.6883F, reaching 30 and 65%, respectively (Fig. 2 and Supplementary Fig. 2; see Supplementary Tables 4 and 5 for raw data). Expression from pWIZ-ds1.6883CR and pWIZ-ds1.6883CR-inv also rescued males but to a somewhat lesser extent. A comparison of transgene expression and male survival reveals that the level of male rescue is largely independent of the level of RNA expression. For example, levels of ds1.6883CR vary more than 3-fold among strains used in this study, but rescue of roX1ex33 roX2Δ males is indistinguishable (Supplementary Figs. 1 and 2). This suggests that expression exceeds that necessary for the maximum achievable rescue of roX1 roX2 males by the dsRNAs produced. Taken together, these observations indicate that expression levels and differences in DNA sequence cannot account for the lack of male rescue by previously made pWIZ-ds1.6881A and pWIZ-ds1.6883C transgenes. Instead, the phasing of the repeat fragment introduced into pWIZ appears to determine the biological activity of transgenes that express ds1.688X RNA.

We then asked if differences in rescue of roX1 roX2 males reflect processing into small RNA. To assess this, we sequenced small RNA from male larvae expressing dsRNA from the original pWIZ-ds1.6881A, which does not rescue roX1 roX2 males, and pWIZ-ds1.6881AR-inv, which achieves strong rescue. Both transgenes contain sequence from 1.6881A introduced into pWIZ in the same orientation, but inserts differ in size and phasing. Small RNA from our lab reference strain (yw) was also sequenced. Consistent with previous studies, we detected minimal siRNA from the 1.6881A region in yw larvae (Menon et al. 2014). In contrast, abundant siRNAs from pWIZ-ds1.6881AR-inv aligned to 1.6881A as well as 1.6884A and other closely related 1.688X repeats on the X chromosome (Fig. 3a). No siRNA aligning to 1.6881A was detected in pWIZ-ds1.6881A males. However, endogenous small RNAs from other genomic regions, such as those near the hairpin RNA-producing esi-1 locus, were identified in all samples, demonstrating sample integrity (Fig. 3a). We then aligned all reads to the pWIZ-ds1.6881AR-inv transcript, permitting no mismatches. pWIZ-ds1.6881AR-inv produced abundant small RNAs aligning to both arms (Fig. 3b). However, few alignments to the insert are observed in control flies or those carrying pWIZ-ds1.6881A. In contrast, the SV40 terminator produces small RNAs in both transgene-carrying strains. We conclude that pWIZ-ds1.6881A produces transcript but the double-stranded regions created by the 1.6881A inserts are not effectively processed into siRNA. The reason for this striking disparity remains speculative.

Fig. 3. Biologically active and inactive dsRNAs differ in small RNA production. a) Alignment of small RNA from larvae carrying pWIZ-ds1.6881AR-inv (top; insertion 35) pWIZ-ds1.6881A (middle; insertion 2A) or no transgene (control, bottom). Two independent RNA preparations are shown for each genotype. The orientation of 1.6881A sequence within pWIZ is illustrated to the left. Tandem 1.6881A repeats proximal to tyn (left) are depicted by arrows. Related 1.6884A repeats are 4.3 kb distal to CG43689 (middle). All preparations contained abundant small RNA aligning to the autosomal, hairpin RNA-producing esi-1 locus (right). Alignments are depicted on a linear scale. b) Small RNAs were aligned to the transcript produced by pWIZ-ds1.6881AR-inv, schematically represented with 1.6881A sequence highlighted. The SV40 terminator is upstream of two polyadenylation sites marked with red arrow heads. Alignments to the pWIZ-ds1.6881AR-inv transcript are presented on a logarithmic scale. All alignments are viewed on igv genome browser (Robinson et al. 2011). Expression of all transgenes is driven by p[w+Sqh-Gal4]2.

Expression of reconstructed transgenes enhances compensation near an autosomal 1.6883F insertion

We postulate that the ability of reconstructed transgenes to rescue roX1 roX2 males involves small RNA-directed chromatin modification at hundreds of related sequences on the X chromosome. We previously demonstrated functional compensation of hemizygous autosomal genes near integrations of DNA from the 1.6881A, 1.6883C, and 1.6883F repeats (Joshi and Meller 2017). Less than 2 kb of 1.6883F DNA was capable of recruitment of MSL proteins detectable on polytene preparations and increased expression of active genes over 100 kb from an autosomal integration site (Deshpande and Meller 2018). The expression of nearby genes was enhanced upon production of ds1.6883F, revealing that small RNA from 1.6883F is able to promote recruitment at related genomic sequences (Deshpande and Meller 2018). We took advantage of autosomal integrations of 1.6883F and roX1 recruiting elements in the hattifattener (haf) gene (cytological position 22A3) to determine if expression of reconstructed dsRNA-expressing transgenes also enhances recruitment (Fig. 4a).

Fig. 4. Expression of reconstructed transgenes modulates expression near an autosomal 1.6883F integration. a) roX1 and 1.6883F recruiting elements are integrated in a 40 kb intron of the haf gene (arrow head). Primers to measure transcript accumulation amplify a haf exon and lie within a Rab3GP1 intron (verticle bar). b) Accumulation of transcripts in male larvae with integrated recruiting elements roX1, 1.6883F, or both is shown by gray bars (insertions [1.6883F]22A3, [roX1]22A3, and [roX1+1.6883F]22A3; Joshi and Meller 2017). Additional expression of ds1.6881AR or ds1.6881AR-inv (blue), and ds1.6883CR or ds1.6883CR-inv (green) increased haf expression only when 1.6883F was present in haf. Expression is normalized to dmn and that in lab reference yw males is set to 1 (line). The significance of expression with recruiting elements alone is with respect to yw males as indicated by asterisks. All other statistical comparisons are between males with recruiting elements alone and those with each recruiting element and dsRNA expression. Error bars represent SEM from three biological replicates. *P < 0.05; **P < 0.01, ***P < 0.001, as determined by Student’s t test.

We generated larvae that express reconstructed transgenes and have integrations of 1.6883F, roX1, or both elements in a haf intron ([1.6883F]22A3, [roX1]22A3, or [roX1&1.6883F]22A3, Supplementary Table 1). Accumulation of haf mRNA in male larvae was determined by qRT-PCR. Expression was normalized to dmn (DCTN2-p50) and expression in the reference yw strain set to one. Integration of 1.6883F, roX1, or both recruiting elements increased haf expression in males from 2- to 3-fold (gray bars, Fig. 4b). When only a roX1 recruiting element was present, no further increase was achieved upon expression of dsRNA from reconstructed transgenes. This agrees with the idea that recruitment of dosage compensation by the CES (present in roX1) and the 1.688X repeats occurs by different mechanisms (Joshi and Meller 2017). In contrast, haf expression increased 5-fold over that in the yw control when the 1.6883F recruiting element was integrated and dsRNAs were expressed. When both 1.6883F and roX1 were present, haf expression increased 6- to 10-fold over that of the control in response to dsRNA production (Fig. 4b and Supplementary Table 7). In this context, we see essentially no difference in response to transgenes expressing 1.6881A and 1.6883C dsRNA, even though 1.6883C shares only 68% identify with the 1.6883F recruiting element but 1.6881A shares 89%. We conclude that siRNAs from multiple 1.688X repeats are capable of promoting recruitment by related genomic sequences. This suggests that sources of siRNA that participate in X recognition are likely to be highly redundant. The levels of haf activation achieved are considerably higher than the 2-fold increase anticipated for full dosage compensation. This has been previously noted for autosomal roX1 insertions and is attributed to disruption of chromatin-based silencing upon recruitment of the MSL complex (Kelley and Kuroda 2003).

Pericentromeric 359 bp satellites contribute to X recognition

Over 10 Mb of proximal X heterochromatin is composed of closely related 359 bp satellite repeats (Hsieh and Brutlag 1979; Lohe et al. 1993). The pericentromeric repeats are a potential source of siRNAs that participate in dosage compensation. To test this, we recombined the Zygotic hybrid Rescue (Zhr1) mutation, deleted for the pericentromeric 359 bp repeats, with roX1ex33 roX2Δ (Sawamura and Yamamoto 1993). Recombinants were validated by qPCR of the 359 repeats. roX1ex33 roX2Δ Zhr1 and roX1ex33 roX2Δ Zhr+ virgins were mated to males from our yw reference strain and the eclosion of males from each mating was calculated based on female survival. Control roX1ex33 roX2Δ Zhr+ sons eclosed at 27% (Fig. 5a and Supplementary Table 6). However, roX1ex33 roX2Δ Zhr1 sons were recovered at 10–15%, suggesting that the pericentric 359 bp repeats also contribute to compensation.

Fig. 5. Pericentromeric 359 bp satellites influence dosage compensation. a) Eclosion of roX1ex33 roX2Δ males that are wild-type for Zhr (Zhr+) and two independent roX1ex33 roX2Δ Zhr1 recombinants. Error bars represent SEM of four biological replicates. b) roX1ex33 roX2Δ Zhr1/+ mothers are mated to reference yw males (+++/Y) and male offspring pooled for DNA extraction and qPCR of 359 bp repeats. c) Abundance of 359 bp repeats in roX1ex33 roX2Δ Zhr+ (left) is set to 1. Zhr1 (center) lacks the 359 bp repeat array (see Supplementary Table 2 for primers). Pooled sons from roX1ex33 roX2Δ Zhr1/+ mothers (right) reveal approximately equal numbers of Zhr1 and Zhr+ sons. Error bars represent SEM from four biological replicates. **P < 0.01, ***P < 0.001, as determined by Students t test.

Zhr+ produces a maternally transmitted factor, presumably small RNA, that is necessary for assembly of the 359 bp repeats into heterochromatin in the zygote (Yuan and O’Farrell 2016; Ferree and Barbash 2009). This raised the possibility that maternal deposition of small RNA from the 359 bp repeats might also influence dosage compensation. To determine if this effect was maternal or zygotic, we mated roX1ex33 roX2Δ females heterozygous for Zhr1 (roX1ex33 roX2Δ Zhr1/+) to yw males. We reasoned that if the benefit of wild-type Zhr+ was zygotic, most eclosing male offspring would carry this allele. However, if Zhr+ confers a maternal effect, equal numbers of Zhr1 and Zhr+ sons are expected. To estimate the fraction of sons with each allele, male offspring were pooled for DNA extraction and the abundance of 359 bp repeats determined by qPCR (Fig. 5b). Amplification reveals approximately equal recovery of each class of sons (Fig. 5c). Lack of enrichment for Zhr+ in male offspring suggests that the 359 bp repeats act maternally to support the survival of roX1 roX2 sons.

Discussion

Recognition of X-linked genes is essential for dosage compensation, but how this is accomplished is not fully understood in any system. At least two classes of recruiting elements, the CES and 1.688X satellites, act to attract the compensation machinery the Drosophila X chromosome but do so by distinct mechanisms. For example, CLAMP binds to and is necessary for recruitment by the CES, but loss of CLAMP does not reduce recruitment by 1.688X repeats (Makki and Meller 2024). Conversely, genes in the siRNA pathway support recruitment by 1.688X repeats but are dispensable for the CES. We postulate that cooperation between multiple classes of recruiting elements achieves faithful recognition of the X chromosome by the MSL complex. Both CES and 1.688X repeats engage in long-range interactions, suggesting that organization of the X chromosome may contribute to selective identification (Ramírez et al. 2015; Sproul et al. 2020).

The involvement of the siRNA pathway in X recognition, abundance of 1.688X repeats on the X chromosome, and rescue of roX1 roX2 males by expression from the pWIZ-ds1.6883F transgene, suggested the involvement of an siRNA-directed chromatin modification system (Menon and Meller 2012; Menon et al. 2014). Mutation of a single copy of genes involved in production of siRNA and chromatin modification, including Dicer2, Ago2, and Su(var)3-9, enhance the male lethality of roX1 roX2 mutations supporting this idea (Menon and Meller 2012; Deshpande and Meller 2018). In accord with this, histone 3 di-methylated on lysine 9 (H3K9me2) is enriched on 1.688X repeats, and this mark is enhanced around an autosomal 1.6883F insertion upon expression of ds1.6883F RNA (Deshpande and Meller 2018). Recruitment of the MSL complex and upregulation of autosomal genes near insertions of 1.6881A or 1.6883C DNA is also enhanced by ds1.6883F RNA, suggesting that 1.688X repeats function redundantly to mark the X (Joshi and Meller 2017). Although these observations implicated 1.688X siRNA in X recognition, the fact that expression from the original pWIZ-ds1.6881A and pWIZ-ds1.6883C transgenes failed to rescue roX1 roX2 males raised questions regarding the origin of the siRNAs (Menon et al. 2014). The proximity of 1.6883F and roX1 is intriguing and suggests that 1.6883F might have a unique function. However, we have been unable to find evidence to support this. roX1 roX2 mutants carrying roX1VM18A, a deletion of the essential 3′ end of roX1, the entire 1.6883F repeat and part of echinus, do not exhibit more complete male lethality than other severe roX1 mutants that retain the 1.6883F repeats (Menon et al. 2014). Transcripts spanning roX1 and 1.6883F are depicted in the Drosophila genome annotation, but we have been unable to detect evidence of these longer transcripts in embryos or adults and suspect that annotation is based on rare read through transcripts. Nor have we discovered that the proximity of the 1.6883F repeats and roX1 is important to their function as each is capable of acting independently to recruit compensation to nearby genes (Joshi and Meller 2017). Demonstration of roX1 roX2 male rescue by reconstructed transgenes that express double-stranded 1.6881A and 1.6883C RNAs, and evidence that the pericentromeric 359 bp repeats also contribute to dosage compensation, suggests high redundancy in the sources of small RNAs that support X recognition.

The striking difference in biological activity between the original ds1.6883F, ds1.6881A, and ds1.6883C transgenes now appears due to an inability of cells to process dsRNA from the original transgenes. As phasing of the repeats in the dsRNA was identified as most likely responsible for this, it is possible that dsRNAs with some phasings adopt a conformation that impedes Dicer processing. Dicer preferentially processes substrates based on kinetic and thermodynamic characteristics and sequence composition may also influence Dicer cleavage (Vermeulen et al. 2005; Lee et al. 2023). Modeling of dsRNA secondary structures failed to reveal large differences between the most energetically favorable conformations of ds1.6881A and ds1.6881AR-inv. Ineffective RNAi has also been linked to degradation of dsRNA by endogenous nucleases (Singh et al. 2017). Understanding why siRNA production from the original pWIZ-1.6881A and pWIZ-1.6883C transgenes fails will require further studies to explore these ideas. We also acknowledge that the phasing in the original pWIZ-1.6883F transgene may not be optimal for small RNA production, leaving open the possibility that even higher activity might be obtained by trial and error. But the current studies do reveal that the 1.6883F satellite is not uniquely able to produce siRNA that supports X recognition as this property is shared by other 1.688X repeats and likely also the pericentric 359 bp satellites.

Female hybrids generated by mating D. melanogaster males to D. simulans females, a species lacking the large block of 359 bp repeats on the X chromosome, fail to assemble heterochromatin over the 359 bp repeats and experience mitotic catastrophe when the D. melanogaster X chromosome fails to segregate (Ferree and Barbash 2009; Yuan and O'Farrell 2016). The maternal factor present in D. melanogaster eggs is suspected to be small RNA from the 359 bp repeats. Zhr1 lacks 359 bp repeats, thus rescuing mitosis in the daughters of D. melanogaster Zhr1 males mated to D. simulans females (Sawamura and Yamamoto 1993). Our studies suggest that the pericentric 359 bp repeats also play a maternal role in identification of the X chromosome for dosage compensation. Interestingly, a strong modifier of roX1 roX2 male lethality was previously mapped to the proximal X (Stuckenholz et al. 2003). Our findings suggest that this modifier could be an allelic variant of Zhr. The observation that small RNAs aligning to 1.688X satellites are abundant in early embryos but absent from wild-type larvae raises the question of when these small RNAs are produced (Menon et al. 2014). Many euchromatic 1.688X repeats are transcribed in male larvae but small RNAs aligning to these repeats are rare in this stage (Menon et al. 2014). Heterochromatic satellites are transcribed in the female germ line and processed into small RNA (Wei et al. 2021). It is possible that the primary source of the small RNAs active in dosage compensation are maternal. This is consistent with a role for satellite repeats and siRNA in identification of X chromatin during the establishment of dosage compensation in the zygote.

The X chromosomes of many Drosophilids are enriched with chromosome-specific repetitive sequences. These are up to 50 times more abundant than autosome-specific repeats in some species (Gallach 2014). Strikingly, the neo-X chromosome in D. pseudoobscura (arm XR) shares this enrichment, suggesting that acquisition of satellite repeats is an early step in the evolution of differentiated sex chromosomes. Repetitive DNA, marked by turnover in sequence and copy number, is implicated in the evolution of sex chromosomes (Smith 1976; Charlesworth et al. 1994; Bayes and Malik 2009; Gallach 2014). Theoretical models of sex chromosome evolution usually focus on accumulation of mutations on the Y chromosome, erosion of coding potential and the subsequent need to increase expression of hemizygous X-linked genes in males (Charlesworth 1996). Both the CES and X chromosome-specific satellite repeats rapidly populate neo-X chromosomes created by sex chromosome and autosomal fusions (Alekseyenko et al. 2013; Ellison and Bachtrog 2013; Gallach 2014). Given the power of CES and satellite repeats to attract compensation to nearby genes, it is possible that the acquisition of compensation precedes the loss of some Y-linked homologs. This could produce over expression that would exert a selective pressure promoting loss of the Y-linked homolog, accelerating sex chromosome differentiation.

Supplementary Material

iyae074_Supplementary_Data

Acknowledgments

We thank Maggie Lauria Sneideman, Reem Makki, and Marissa Ann Steiner for unwavering support. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study.

Data Availability

Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Sequence data can be downloaded from http://www.ncbi.nlm.nih.gov/bioproject/1050379 with accession PRJNA1050379.

Supplemental material available at GENETICS online.

Funding

This research was supported by a Wayne State University summer dissertation award to S.B. and National Institute of General Medical Sciences award GM093110 to V.H.M.
==== Refs
Literature cited

Akhtar  A, Becker  PB. 2000. Activation of transcription through histone H4 acetylation by MOF, an acetyltransferase essential for dosage compensation in Drosophila. Mol Cell. 5 (2 ):367–375. doi:10.1016/s1097-2765(00)80431-1.10882077
Albig  C, Tikhonova  E, Krause  S, Maksimenko  O, Regnard  C, Becker  PB. 2019. Factor cooperation for chromosome discrimination in Drosophila. Nucleic Acids Res.  47 (4 ):1706–1724. doi:10.1093/nar/gky1238.30541149
Alekseyenko  AA, Ellison  CE, Gorchakov  AA, Zhou  Q, Kaiser  VB, Toda  N, Walton  Z, Peng  S, Park  PJ, Bachtrog  D, et al  2013. Conservation and de novo acquisition of dosage compensation on newly evolved sex chromosomes in Drosophila. Genes Dev. 27 (8 ):853–858. doi:10.1101/gad.215426.113.23630075
Alekseyenko  AA, Peng  S, Larschan  E, Gorchakov  AA, Lee  OK, Kharchenko  P, McGrath  SD, Wang  CI, Mardis  ER, Park  PJ, et al  2008. A sequence motif within chromatin entry sites directs MSL establishment on the Drosophila X chromosome. Cell. 134 (4 ):599–609. doi:10.1016/j.cell.2008.06.033.18724933
Amrein  H, Axel  R. 1997. Genes expressed in neurons of adult male Drosophila. Cell. 88 (4 ):459–469. doi:10.1016/s0092-8674(00)81886-3.9038337
Bayes  JJ, Malik  HS. 2009. Altered heterochromatin binding by a hybrid sterility protein in Drosophila sibling species. Science. 326 (5959 ):1538–1541. doi:10.1126/science.1181756.19933102
Bell  O, Wirbelauer  C, Hild  M, Scharf  AN, Schwaiger  M, MacAlpine  DM, Zilbermann  F, van Leeuwen  F, Bell  SP, Imhof  A, et al  2007. Localized H3K36 methylation states define histone H4K16 acetylation during transcriptional elongation in Drosophila. EMBO J.  26 (24 ):4974–4984. doi:10.1038/sj.emboj.7601926.18007591
Bone  JR, Lavender  J, Richman  R, Palmer  MJ, Turner  BM, Kuroda  MI. 1994. Acetylated histone H4 on the male X chromosome is associated with dosage compensation in Drosophila. Genes Dev. 8 (1 ):96–104. doi:10.1101/gad.8.1.96.8288132
Charlesworth  B . 1996. The evolution of chromosomal sex determination and dosage compensation. Curr Biol. 6 (2 ):149–162. doi:10.1016/s0960-9822(02)00448-7.8673462
Charlesworth  B, Sniegowski  P, Stephan  W. 1994. The evolutionary dynamics of repetitive DNA in eukaryotes. Nature. 371 (6494 ):215–220. doi:10.1038/371215a0.8078581
Copur  Ö, Gorchakov  A, Finkl  K, Kuroda  MI, Müller  J. 2018. Sex-specific phenotypes of histone H4 point mutants establish dosage compensation as the critical function of H4K16 acetylation in Drosophila. Proc Natl Acad Sci U S A. 115 (52 ):13336–13341. doi:10.1073/pnas.1817274115.30530664
Deng  X, Meller  VH. 2006. Rox RNAs are required for increased expression of X-linked genes in Drosophila melanogaster males. Genetics. 174 (4 ):1859–1866. doi:10.1534/genetics.106.064568.17028315
Deng  X, Rattner  BP, Souter  S, Meller  VH. 2005. The severity of roX1 mutations is predicted by MSL localization on the X chromosome. Mech Dev. 122 (10 ):1094–1105. doi:10.1016/j.mod.2005.06.004.16125915
Deshpande  N, Meller  VH. 2018. Chromatin that guides dosage compensation is modulated by the siRNA pathway in Drosophila melanogaster. Genetics. 209 (4 ):1085–1097. doi:10.1534/genetics.118.301173.29921620
DiBartolomeis  SM, Tartof  KD, Jackson  FR. 1992. A superfamily of Drosophila satellite related (SR) DNA repeats restricted to the X chromosome euchromatin. Nucleic Acids Res. 20 (5 ):1113–1116. doi:10.1093/nar/20.5.1113.1549474
Disteche  CM . 2012. Dosage compensation of the sex chromosomes. Annu Rev Genet. 46 (1 ):537–560. doi:10.1146/annurev-genet-110711-155454.22974302
Ellison  CE, Bachtrog  D. 2013. Dosage compensation via transposable element mediated rewiring of a regulatory network. Science. 342 (6160 ):846–850. doi:10.1126/science.1239552.24233721
Ferree  PM, Barbash  DA. 2009. Species-specific heterochromatin prevents mitotic chromosome segregation to cause hybrid lethality in Drosophila. PLoS Biol. 7 (10 ):e1000234. doi:10.1371/journal.pbio.1000234.19859525
Gallach  M . 2014. Recurrent turnover of chromosome-specific satellites in Drosophila. Genome Biol Evol. 6 (6 ):1279–1286. doi:10.1093/gbe/evu104.24846631
Gelbart  ME, Kuroda  MI. 2009. Drosophila dosage compensation: a complex voyage to the X chromosome. Development. 136 (9 ):1399–1410. doi:10.1242/dev.029645.19363150
Grimaud  C, Becker  PB. 2009. The dosage compensation complex shapes the conformation of the X chromosome in Drosophila. Genes Dev. 23 (21 ):2490–2495. doi:10.1101/gad.539509.19884256
Grunstein  M . 1997. Histone acetylation in chromatin structure and transcription. Nature. 389 (6649 ):349–352. doi:10.1038/38664.9311776
Henry  RA, Tews  B, Li  X, Scott  MJ. 2001. Recruitment of the male-specific lethal (MSL) dosage compensation complex to an autosomally integrated roX chromatin entry site correlates with an increased expression of an adjacent reporter gene in male Drosophila. J Biol Chem. 276 (34 ):31953–31958. doi:10.1074/jbc.M103008200.11402038
Hilfiker  A, Hilfiker-Kleiner  D, Pannuti  A, Lucchesi  JC. 1997. Mof, a putative acetyl transferase gene related to the Tip60 and MOZ human genes and to the SAS genes of yeast, is required for dosage compensation in Drosophila. EMBO J. 16 (8 ):2054–2060. doi:10.1093/emboj/16.8.2054.9155031
Hoskins  RA, Carlson  JW, Wan  KH, Park  S, Mendez  I, Galle  SE, Booth  BW, Pfeiffer  BD, George  RA, Svirskas  R. 2015. The Release 6 reference sequence of the Drosophila melanogaster genome. Genome Res. 25 (3 ):445–458. doi:10.1101/gr.185579.114.25589440
Hsieh  T, Brutlag  D. 1979. Sequence and sequence variation within the 1.688g/cm3 satellite DNA of Drosophila melanogaster. J Mol Biol. 135 (2 ):465–481. doi:10.1016/0022-2836(79)90447-9.231676
Ilik  IA, Quinn  JJ, Georgiev  P, Tavares-Cadete  F, Maticzka  D, Toscano  S, Wan  Y, Spitale  RC, Luscombe  N, Backofen  R, et al  2013. Tandem stem-loops in roX RNAs act together to mediate X chromosome dosage compensation in Drosophila. Mol Cell. 51 (2 ):156–173. doi:10.1016/j.molcel.2013.07.001.23870142
Joshi  SS, Meller  VH. 2017. Satellite repeats identify X chromatin for dosage compensation in Drosophila melanogaster males. Curr Biol. 27 (10 ):1393–1402.e2. doi:10.1016/j.cub.2017.03.078.28457869
Kelley  RL, Kuroda  MI. 2003. The Drosophila roX1 gene can overcome silent chromatin by recruiting the male-specific lethal dosage compensation complex. Genetics. 164 (2 ):565–574. doi:10.1093/genetics/164.2.565.12807777
Kelley  RL, Meller  VH, Gordadze  PR, Roman  G, Davis  RL, Kuroda  MI. 1999. Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell. 98 (4 ):513–522. doi:10.1016/s0092-8674(00)81979-0.10481915
Kind  J, Akhtar  A. 2007. Cotranscriptional recruitment of the dosage compensation complex to X-linked target genes. Genes Dev. 21 (16 ):2030–2040. doi:10.1101/gad.430807.17699750
Koya  SK, Meller  VH. 2015. Modulation of heterochromatin by male specific lethal proteins and roX RNA in Drosophila melanogaster males. PLoS One. 10 (10 ):e0140259. doi:10.1371/journal.pone.0140259.26468879
Kuhn  GC, Küttler  H, Moreira-Filho  O, Heslop-Harrison  JS. 2012. The 1.688 repetitive DNA of Drosophila: concerted evolution at different genomic scales and association with genes. Mol Biol Evol. 29 (1 ):7–11. doi:10.1093/molbev/msr173.21712468
Larschan  E, Alekseyenko  AA, Gortchakov  AA, Peng  S, Li  B, Yang  P, Workman  JL, Park  PJ, Kuroda  MI. 2007. MSL complex is attracted to genes marked by H3K36 trimethylation using a sequence-independent mechanism. Mol Cell. 28 (1 ):121–133. doi:10.1016/j.molcel.2007.08.011.17936709
Lee  YS, Carthew  RW. 2003. Making a better RNAi vector for Drosophila: use of intron spacers. Methods (San Diego, Calif.). 30 (4 ):322–329. doi:10.1016/s1046-2023(03)00051-3.12828946
Lee  YY, Kim  H, Kim  VN. 2023. Sequence determinant of small RNA production by DICER. Nature. 615 (7951 ):323–330. doi:10.1038/s41586-023-05722-4.36813957
Lohe  AR, Hilliker  AJ, Roberts  PA. 1993. Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics. 134 (4 ):1149–1174. doi:10.1093/genetics/134.4.1149.8375654
Lucchesi  JC . 1978. Gene dosage compensation and the evolution of sex chromosomes. Science. 202 (4369 ):711–716. doi:10.1126/science.715437.715437
Lucchesi  JC, Kelly  WG, Panning  B. 2005. Chromatin remodeling in dosage compensation. Annu Rev Genet.  39 (1 ):615–651. doi:10.1146/annurev.genet.39.073003.094210.16285873
Makki  R, Meller  VH. 2024. Identification of X chromatin is modulated by complementary pathways in Drosophila melanogaster. G3 (Bethesda). [Online ahead of print] doi:10.1093/g3journal/jkae057.
Meller  VH . 2003. Initiation of dosage compensation in Drosophila embryos depends on expression of the roX RNAs. Mech Dev.  120 (7 ):759–767. doi:10.1016/s0925-4773(03)00157-6.12915227
Meller  VH, Rattner  BP. 2002. The roX genes encode redundant male-specific lethal transcripts required for targeting of the MSL complex. EMBO J. 21 (5 ):1084–1091. doi:10.1093/emboj/21.5.1084.11867536
Meller  VH, Wu  KH, Roman  G, Kuroda  MI, Davis  RL. 1997. Rox1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system. Cell. 88 (4 ):445–457. doi:10.1016/s0092-8674(00)81885-1.9038336
Menon  DU, Coarfa  C, Xiao  W, Gunaratne  PH, Meller  VH. 2014. siRNAs from an X-linked satellite repeat promote X-chromosome recognition in Drosophila melanogaster. Proc Natl Acad Sci U S A. 111 (46 ):16460–16465. doi:10.1073/pnas.1410534111.25368194
Menon  DU, Meller  VH. 2012. A role for siRNA in X-chromosome dosage compensation in Drosophila melanogaster. Genetics. 191 (3 ):1023–1028. doi:10.1534/genetics.112.140236.22554892
Oh  H, Park  Y, Kuroda  MI. 2003. Local spreading of MSL complexes from roX genes on the Drosophila X chromosome. Genes Dev. 17 (11 ):1334–1339. doi:10.1101/gad.1082003.12782651
Öztürk-Çolak  A, Marygold  SJ, Antonazzo  G, Attrill  H, Goutte-Gattat  D, Jenkins  VK, Matthews  BB, Millburn  G, dos Santos  G, Tabone  CJ, et al  2024. The FlyBase consortium, FlyBase: updates to the Drosophila genes and genomes database. Genetics. 227 (1 ):1–8. doi:10.1093/genetics/iyad211.
Pal  K, Forcato  M, Jost  D, Sexton  T, Vaillant  C, Salviato  E, Mazza  EMC, Lugli  E, Cavalli  G, Ferrari  F. 2019. Global chromatin conformation differences in the Drosophila dosage compensated chromosome X. Nat Commun. 10 (1 ):5355. doi:10.1038/s41467-019-13350-8.31767860
Park  Y, Kelley  RL, Oh  H, Kuroda  MI, Meller  VH. 2002. Extent of chromatin spreading determined by roX RNA recruitment of MSL proteins. Science. 298 (5598 ):1620–1623. doi:10.1126/science.1076686.12446910
Pfaffl  MW . 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29 (9 ):e45. doi:10.1093/nar/29.9.e45.11328886
Ramírez  F, Lingg  T, Toscano  S, Lam  KC, Georgiev  P, Chung  H-R, Lajoie  BR, de Wit  E, Zhan  Y, de Laat  W, et al  2015. High-affinity sites form an interaction network to facilitate spreading of the MSL complex across the X chromosome in Drosophila. Mol Cell. 60 (1 ):146–162. doi:10.1016/j.molcel.2015.08.024.26431028
Robinson  JT, Thorvaldsdóttir  H, Winckler  W, Guttman  M, Lander  ES, Getz  G, Mesirov  JP. 2011. Integrative genomics viewer. Nat Biotechnol.  29 (1 ):24–26. doi:10.1038/nbt.1754.21221095
Sawamura  K, Yamamoto  MT. 1993. Cytogenetical localization of Zygotic hybrid rescue (Zhr), a Drosophila melanogaster gene that rescues interspecific hybrids from embryonic lethality. Mol Gen Genet. 239 (3 ):441–449. doi:10.1007/BF00276943.8316215
Shogren-Knaak  M, Ishii  H, Sun  JM, Pazin  MJ, Davie  JR, Peterson  CL. 2006. Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science. 311 (5762 ):844–847. doi:10.1126/science.1124000.16469925
Singh  IK, Singh  S, Mogilicherla  K, Shukla  JN, Palli  SR. 2017. Comparative analysis of double-stranded RNA degradation and processing in insects. Sci Rep. 7 (1 ):17059. doi:10.1038/s41598-017-17134-2.29213068
Smith  GP . 1976. Evolution of repeated DNA sequences by unequal crossover. Science. 191 (4227 ):528–535. doi:10.1126/science.1251186.1251186
Smith  ER, Pannuti  A, Gu  W, Steurnagel  A, Cook  RG, Allis  CD, Lucchesi  JC. 2000. The Drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation. Mol Cell Biol. 20 (1 ):312–318. doi:10.1128/mcb.20.1.312-318.2000.10594033
Soruco  MM, Chery  J, Bishop  EP, Siggers  T, Tolstorukov  MY, Leydon  AR, Sugden  AU, Goebel  K, Feng  J, Xia  P, et al  2013. The CLAMP protein links the MSL complex to the X chromosome during Drosophila dosage compensation. Genes Dev. 27 (14 ):1551–1556. doi:10.1101/gad.214585.113.23873939
Sproul  JS, Khost  DE, Eickbush  DG, Negm  S, Wei  X, Wong  I, Larracuente  AM. 2020. Dynamic evolution of euchromatic satellites on the X chromosome in Drosophila melanogaster and the simulans clade. Mol Biol Evol. 37 (8 ):2241–2256. doi:10.1093/molbev/msaa078.32191304
Stuckenholz  C, Meller  VH, Kuroda  MI. 2003. Functional redundancy within roX1, a noncoding RNA involved in dosage compensation in Drosophila melanogaster. Genetics. 164 (3 ):1003–1014. doi:10.1093/genetics/164.3.1003.12871910
Sural  TH, Peng  S, Li  B, Workman  JL, Park  PJ, Kuroda  MI. 2008. The MSL3 chromodomain directs a key targeting step for dosage com- pensation of the Drosophila melanogaster X chromosome. Nat Struct Mol Biol.  15 (12 ):1318–1325. doi:10.1038/nsmb.1520.19029895
Vermeulen  A, Behlen  L, Reynolds  A, Wolfson  A, Marshall  WS, Karpilow  J, Khvorova  A. 2005. The contributions of dsRNA structure to Dicer specificity and efficiency. RNA. 11 (5 ):674–682. doi:10.1261/rna.7272305.15811921
Villa  R, Schauer  T, Smialowski  P, Straub  T, Becker  PB. 2016. Pionx sites mark the X chromosome for dosage compensation. Nature. 537 (7619 ):244–248. doi:10.1038/nature19338.27580037
Waring  GL, Pollack  JC. 1987. Cloning and characterization of a dispersed, multicopy, X chromosome sequence in Drosophila melanogaster. Proc Natl Acad Sci U S A. 84 (9 ):2843–2847. doi:10.1073/pnas.84.9.2843.3106978
Wei  X, Eickbush  DG, Speece  I, Larracuente  AM. 2021. Heterochromatin-dependent transcription of satellite DNAs in the Drosophila melanogaster female germline. eLife. 10 :e62375. doi:doi:10.7554/eLife.62375.34259629
Yuan  K, O’Farrell  PH. 2016. TALE-light imaging reveals maternally guided, H3K9me2/3-independent emergence of functional heterochromatin in Drosophila embryos. Genes Dev. 30 (5 ):579–593. doi:10.1101/gad.272237.115.26915820
