Transcription-Coupled Methylation of Histone H3 at Lysine 36 Regulates Dosage Compensation by Enhancing Recruitment of the MSL Complex in Drosophila melanogaster

ABSTRACT In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched toward the 3′ end of active genes, similar to the MSL proteins. Here, we have studied the link between histone H3 methylation and MSL complex targeting using RNA interference and chromatin immunoprecipitation. We show that trimethylation of histone H3 at lysine 36 (H3K36me3) relies on the histone methyltransferase Hypb and is localized promoter distal at dosage-compensated genes, similar to active genes on autosomes. However, H3K36me3 has an X-specific function, as reduction specifically decreases acetylation of histone H4 lysine 16 on the male X chromosome. This hypoacetylation is caused by compromised MSL binding and results in a failure to increase expression twofold. Thus, H3K36me3 marks the body of all active genes yet is utilized in a chromosome-specific manner to enhance histone acetylation at sites of dosage compensation.

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Absence of X-chromosome dosage compensation in the primordial germ cells of Drosophila embryos

Scientific Reports ◽

10.1038/s41598-021-84402-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ryoma Ota ◽

Makoto Hayashi ◽

Shumpei Morita ◽

Hiroki Miura ◽

Satoru Kobayashi

Keyword(s):

X Chromosome ◽

Germ Cells ◽

Dosage Compensation ◽

Sex Chromosome ◽

Primordial Germ Cells ◽

Histone H4 ◽

X Chromosomes ◽

Essential Components ◽

Msl Complex ◽

Male Specific

AbstractDosage compensation is a mechanism that equalizes sex chromosome gene expression between the sexes. In Drosophila, individuals with two X chromosomes (XX) become female, whereas males have one X chromosome (XY). In males, dosage compensation of the X chromosome in the soma is achieved by five proteins and two non-coding RNAs, which assemble into the male-specific lethal (MSL) complex to upregulate X-linked genes twofold. By contrast, it remains unclear whether dosage compensation occurs in the germline. To address this issue, we performed transcriptome analysis of male and female primordial germ cells (PGCs). We found that the expression levels of X-linked genes were approximately twofold higher in female PGCs than in male PGCs. Acetylation of lysine residue 16 on histone H4 (H4K16ac), which is catalyzed by the MSL complex, was undetectable in these cells. In male PGCs, hyperactivation of X-linked genes and H4K16ac were induced by overexpression of the essential components of the MSL complex, which were expressed at very low levels in PGCs. Together, these findings indicate that failure of MSL complex formation results in the absence of X-chromosome dosage compensation in male PGCs.

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Dosage Compensation of the X Chromosome: A Complex Epigenetic Assignment Involving Chromatin Regulators and Long Noncoding RNAs

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-062917-011816 ◽

2018 ◽

Vol 87 (1) ◽

pp. 323-350 ◽

Cited By ~ 35

Author(s):

Maria Samata ◽

Asifa Akhtar

Keyword(s):

X Chromosome ◽

Dosage Compensation ◽

Transcriptional Activation ◽

Gene Dosage ◽

Noncoding Rnas ◽

Long Noncoding Rnas ◽

Histone H4 ◽

Chromosome Conformation ◽

Msl Complex ◽

Male Specific

X chromosome regulation represents a prime example of an epigenetic phenomenon where coordinated regulation of a whole chromosome is required. In flies, this is achieved by transcriptional upregulation of X chromosomal genes in males to equalize the gene dosage differences in females. Chromatin-bound proteins and long noncoding RNAs (lncRNAs) constituting a ribonucleoprotein complex known as the male-specific lethal (MSL) complex or the dosage compensation complex mediate this process. MSL complex members decorate the male X chromosome, and their absence leads to male lethality. The male X chromosome is also enriched with histone H4 lysine 16 acetylation (H4K16ac), indicating that the chromatin compaction status of the X chromosome also plays an important role in transcriptional activation. How the X chromosome is specifically targeted and how dosage compensation is mechanistically achieved are central questions for the field. Here, we review recent advances, which reveal a complex interplay among lncRNAs, the chromatin landscape, transcription, and chromosome conformation that fine-tune X chromosome gene expression.

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Drosophila CLAMP is an essential protein with sex-specific roles in males and females

10.1101/042820 ◽

2016 ◽

Author(s):

Jennifer A. Urban ◽

Caroline A. Doherty ◽

William T. Jordan ◽

Jacob E. Bliss ◽

Jessica Feng ◽

...

Keyword(s):

X Chromosome ◽

Dosage Compensation ◽

Zinc Finger Protein ◽

Pericentric Heterochromatin ◽

Single Male ◽

Essential Protein ◽

Msl Complex ◽

Males And Females ◽

Male Specific

AbstractDosage compensation is a fundamental mechanism in many species that corrects for the inherent imbalance in X-chromosome copy number between XY males and XX females. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the Male Specific Lethal (MSL) complex to specific sequences along the length of the X-chromosome. The initial recruitment of MSL complex to the X-chromosome is dependent on a recently discovered zinc finger protein called Chromatin-Linked Adapter for MSL Proteins (CLAMP). However, further studies on the in vivo function of CLAMP remained difficult because the location of the gene in pericentric heterochromatin made it challenging to create null mutations or deficiencies. Using the CRISPR/Cas9 genome editing system, we generated the first null mutant in the clamp gene that eliminates expression of CLAMP protein. We show that CLAMP is necessary for both male and female viability. While females die at the third instar larval stage, males die earlier, likely due to the essential role of CLAMP in male dosage compensation. Moreover, we demonstrate that CLAMP promotes dosage compensation in males and represses key male-specific transcripts involved in sex-determination in females. Our results reveal that CLAMP is an essential protein with dual roles in males and females, which together assure that dosage compensation is a sex-specific process.

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Rapid Evolution of Autosomal Binding Sites of the Dosage Compensation Complex in Drosophila melanogaster and Its Association With Transcription Divergence

Frontiers in Genetics ◽

10.3389/fgene.2021.675027 ◽

2021 ◽

Vol 12 ◽

Author(s):

Aimei Dai ◽

Yushuai Wang ◽

Anthony Greenberg ◽

Zhongqi Liufu ◽

Tian Tang

Keyword(s):

Drosophila Melanogaster ◽

Dosage Compensation ◽

Binding Sites ◽

Protein Sequence ◽

Rapid Evolution ◽

Close Relative ◽

Sequence Evolution ◽

Male Specific Lethal ◽

Msl Complex ◽

Male Specific

How pleiotropy influences evolution of protein sequence remains unclear. The male-specific lethal (MSL) complex in Drosophila mediates dosage compensation by 2-fold upregulation of the X chromosome in males. Nevertheless, several MSL proteins also bind autosomes and likely perform functions not related to dosage compensation. Here, we study the evolution of MOF, MSL1, and MSL2 biding sites in Drosophila melanogaster and its close relative Drosophila simulans. We found pervasive expansion of the MSL binding sites in D. melanogaster, particularly on autosomes. The majority of these newly-bound regions are unlikely to function in dosage compensation and associated with an increase in expression divergence between D. melanogaster and D. simulans. While dosage-compensation related sites show clear signatures of adaptive evolution, these signatures are even more marked among autosomal regions. Our study points to an intriguing avenue of investigation of pleiotropy as a mechanism promoting rapid protein sequence evolution.

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Distinct mechanisms mediate X chromosome dosage compensation in Anopheles and Drosophila

Life Science Alliance ◽

10.26508/lsa.202000996 ◽

2021 ◽

Vol 4 (9) ◽

pp. e202000996

Author(s):

Claudia Isabelle Keller Valsecchi ◽

Eric Marois ◽

M Felicia Basilicata ◽

Plamen Georgiev ◽

Asifa Akhtar

Keyword(s):

X Chromosome ◽

Dosage Compensation ◽

Gene Dosage ◽

Histone H4 ◽

Ecological Constraints ◽

Evolutionary Trajectory ◽

X Chromosomes ◽

Deleterious Gene ◽

Msl Complex ◽

High Degree

Sex chromosomes induce potentially deleterious gene expression imbalances that are frequently corrected by dosage compensation (DC). Three distinct molecular strategies to achieve DC have been previously described in nematodes, fruit flies, and mammals. Is this a consequence of distinct genomes, functional or ecological constraints, or random initial commitment to an evolutionary trajectory? Here, we study DC in the malaria mosquito Anopheles gambiae. The Anopheles and Drosophila X chromosomes evolved independently but share a high degree of homology. We find that Anopheles achieves DC by a mechanism distinct from the Drosophila MSL complex–histone H4 lysine 16 acetylation pathway. CRISPR knockout of Anopheles msl-2 leads to embryonic lethality in both sexes. Transcriptome analyses indicate that this phenotype is not a consequence of defective X chromosome DC. By immunofluorescence and ChIP, H4K16ac does not preferentially enrich on the male X. Instead, the mosquito MSL pathway regulates conserved developmental genes. We conclude that a novel mechanism confers X chromosome up-regulation in Anopheles. Our findings highlight the pluralism of gene-dosage buffering mechanisms even under similar genomic and functional constraints.

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Painting of Fourth and the X-Linked 1.688 Satellite in D. melanogaster Is Involved in Chromosome-Wide Gene Regulation

Cells ◽

10.3390/cells9020323 ◽

2020 ◽

Vol 9 (2) ◽

pp. 323

Author(s):

Samaneh Ekhteraei-Tousi ◽

Jacob Lewerentz ◽

Jan Larsson

Keyword(s):

X Chromosome ◽

Dosage Compensation ◽

Rapid Evolution ◽

Regulatory Mechanisms ◽

Expression Of Genes ◽

Male Specific Lethal ◽

Msl Complex ◽

Specific Regulation ◽

Male Specific ◽

Genomic Regions

Chromosome-specific regulatory mechanisms provide a model to understand the coordinated regulation of genes on entire chromosomes or on larger genomic regions. In fruit flies, two chromosome-wide systems have been characterized: The male-specific lethal (MSL) complex, which mediates dosage compensation and primarily acts on the male X-chromosome, and Painting of fourth (POF), which governs chromosome-specific regulation of genes located on the 4th chromosome. How targeting of one specific chromosome evolves is still not understood; but repeated sequences, in forms of satellites and transposable elements, are thought to facilitate the evolution of chromosome-specific targeting. The highly repetitive 1.688 satellite has been functionally connected to both these systems. Considering the rapid evolution and the necessarily constant adaptation of regulatory mechanisms, such as dosage compensation, we hypothesised that POF and/or 1.688 may still show traces of dosage-compensation functions. Here, we test this hypothesis by transcriptome analysis. We show that loss of Pof decreases not only chromosome 4 expression but also reduces the X-chromosome expression in males. The 1.688 repeat deletion, Zhr1 (Zygotic hybrid rescue), does not affect male dosage compensation detectably; however, Zhr1 in females causes a stimulatory effect on X-linked genes with a strong binding affinity to the MSL complex (genes close to high-affinity sites). Lack of pericentromeric 1.688 also affected 1.688 expression in trans and was linked to the differential expression of genes involved in eggshell formation. We discuss our results with reference to the connections between POF, the 1.688 satellite and dosage compensation, and the role of the 1.688 satellite in hybrid lethality.

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Evidence that MSL-mediated dosage compensation in Drosophila begins at blastoderm

Development ◽

10.1242/dev.122.9.2751 ◽

1996 ◽

Vol 122 (9) ◽

pp. 2751-2760 ◽

Cited By ~ 1

Author(s):

A. Franke ◽

A. Dernburg ◽

G.J. Bashaw ◽

B.S. Baker

Keyword(s):

X Chromosome ◽

Dosage Compensation ◽

Dependent Manner ◽

Gene Products ◽

Histone H4 ◽

Single Male ◽

Somatic Tissues ◽

Male Specific Lethal ◽

Male Specific ◽

Blastoderm Stage

In Drosophila equalization of the amounts of gene products produced by X-linked genes in the two sexes is achieved by hypertranscription of the single male X chromosome. This process, dosage compensation, is controlled by a set of male-specific lethal (msl) genes, that appear to act at the level of chromatin structure. The properties of the MSL proteins have been extensively studied in the polytene salivary gland chromosomes where they bind to the same set of sites along the male X chromosome in a co-dependent manner. Here we report experiments that show that the MSL proteins first associate with the male X chromosome as early as blastoderm stage, slightly earlier than the histone H4 isoform acetylated at lysine 16 is detected on the X chromosome. MSL binding to the male X chromosome is observed in all somatic tissues of embryos and larvae. Binding of the MSLs to the X chromosome is also interdependent in male embryos and prevented in female embryos by the expression of Sex-lethal (Sxl). A delayed onset of binding of the MSLs in male progeny of homozygous mutant msl-1 or mle mothers coupled with the previous finding that such males have an earlier lethal phase supports the idea that msl-mediated dosage compensation begins early in embryogenesis. Other results show that the maleless (MLE) protein on embryo and larval chromosomes differs in its reactivity with antibodies; the functional significance of this finding remains to be explored.

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Transposon insertions causing constitutive Sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing.

Genetics ◽

10.1093/genetics/139.2.631 ◽

1995 ◽

Vol 139 (2) ◽

pp. 631-648

Author(s):

M Bernstein ◽

R A Lersch ◽

L Subrahmanyan ◽

T W Cline

Keyword(s):

Drosophila Melanogaster ◽

X Chromosome ◽

Dosage Compensation ◽

Rna Splicing ◽

Wild Type ◽

Gain Of Function ◽

Positive Regulators ◽

Sex Lethal ◽

Male Specific ◽

Transposon Insertions

Abstract Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle "on" vs. "off" reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. "Male-lethal" (SxlM) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, SxlM haplo-X animals (chromosomal males) die because of improper dosage compensation, and SxlM chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Five independent spontaneous SxlM alleles were shown previously to be transposon insertions into what was subsequently found to be the region of regulated sex-specific Sxl RNA splicing. We show that these five alleles represent three different mutant types: SxlM1, SxlM3, and SxlM4. SxlM1 is an insertion of a roo element 674 bp downstream of the translation-terminating male-specific exon. SxlM3 is an insertion of a hobo transposon (not 297 as previously reported) into the 3' splice site of the male exon, and SxlM4 is an insertion of a novel transposon into the male-specific exon itself. We show that these three gain-of-function mutants differ considerably in their ability to bypass the sex determination signal, with SxlM4 being the strongest and SxlM1 the weakest. This difference is also reflected in effects of these mutations on sex-specific RNA splicing and on the rate of appearance of SXL protein in male embryos. Transcript analysis of double-mutant male-viable SxlM derivatives in which the SxlM insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these SxlM alleles is a consequence of an alteration of the structure of the pre-mRNA that allows some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of SxlM alleles appears to depend on the mutant alleles' responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce.

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Regulation of Histone H4 Lys16 Acetylation by Predicted Alternative Secondary Structures in roX Noncoding RNAs

Molecular and Cellular Biology ◽

10.1128/mcb.00219-08 ◽

2008 ◽

Vol 28 (16) ◽

pp. 4952-4962 ◽

Cited By ~ 31

Author(s):

Seung-Won Park ◽

Mitzi I. Kuroda ◽

Yongkyu Park

Keyword(s):

Drosophila Melanogaster ◽

X Chromosome ◽

Noncoding Rnas ◽

Loop Structure ◽

Histone H4 ◽

Stem Loop ◽

Functional Conservation ◽

Stem Loop Structure ◽

Msl Complex ◽

H4k16 Acetylation

ABSTRACT Despite differences in size and sequence, the two noncoding roX1 and roX2 RNAs are functionally redundant for dosage compensation of the Drosophila melanogaster male X chromosome. Consistent with functional conservation, we found that roX RNAs of distant Drosophila species could complement D. melanogaster roX mutants despite low homology. Deletion of a conserved predicted stem-loop structure in roX2, containing a short GUb (GUUNUACG box) in its 3′ stem, resulted in a defect in histone H4K16 acetylation on the X chromosome in spite of apparently normal localization of the MSL complex. Two copies of the GUb sequence, newly termed the “roX box,” were functionally redundant in roX2, as mutants in a single roX box had no phenotype, but double mutants showed reduced H4K16 acetylation. Interestingly, mutation of two of three roX boxes in the 3′ end of roX1 RNA also reduced H4K16 acetylation. Finally, fusion of roX1 sequences containing a roX box restored function to a roX2 deletion RNA lacking its cognate roX box. These results support a model in which the functional redundancy between roX1 and roX2 RNAs is based, at least in part, on short GUUNUACG sequences that regulate the activity of the MSL complex.

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