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

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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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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Zinc Finger Protein Zn72D Promotes Productive Splicing of the maleless Transcript

Molecular and Cellular Biology ◽

10.1128/mcb.01415-07 ◽

2007 ◽

Vol 27 (24) ◽

pp. 8760-8769 ◽

Cited By ~ 9

Author(s):

Kathleen A. Worringer ◽

Barbara Panning

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Zinc Finger ◽

X Chromosome ◽

Dosage Compensation ◽

Fluorescent Protein ◽

Zinc Finger Protein ◽

General Factor ◽

Finger Protein ◽

Msl Complex

ABSTRACT In organisms with sex chromosomes, dosage compensation equalizes gene expression between the sexes. In Drosophila melanogaster males, the male-specific lethal (MSL) complex of proteins and two noncoding roX RNAs coat the X chromosome, resulting in a twofold transcriptional upregulation to equalize gene expression with that of females. How MSL complex enrichment on the X chromosome is regulated is not well understood. We performed an RNA interference screen to identify new factors required for dosage compensation. Using a Drosophila Schneider S2 cell line in which green fluorescent protein (GFP)-tagged MSL2 localizes to the X chromosome, we assayed ∼7,200 knockdowns for their effects on GFP-MSL2 distribution. One factor identified is the zinc finger protein Zn72D. In its absence, the MSL complex no longer coats the X chromosome. We demonstrate that Zn72D is required for productive splicing of the transcript for the MSL protein Maleless, explaining the dosage compensation defect. However, Zn72D is required for the viability of both sexes, indicating its functions are not sex specific. Consistent with this, Zn72D colocalizes with elongating RNA polymerase II, implicating it as a more general factor involved in RNA metabolism.

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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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Targeting of the dosage-compensated male X-chromosome during early Drosophila development

10.1101/671073 ◽

2019 ◽

Author(s):

LE Rieder ◽

WT Jordan ◽

EN Larschan

Keyword(s):

Early Development ◽

X Chromosome ◽

Dosage Compensation ◽

Binding Sites ◽

Zinc Finger Protein ◽

Multi Stage ◽

Compensation Factor ◽

Zygotic Transcription ◽

Male Specific ◽

Lethal Dosage

ABSTRACTThe essential process of dosage compensation, which corrects for the imbalance in X-linked gene expression between XX females and XY males, represents a key model for how genes are targeted for coordinated regulation. However, the mechanism by which dosage compensation complexes identify the X-chromosome during early development remained unknown because of the difficulty of sexing embryos prior to zygotic transcription. We used meiotic drive to sex Drosophila embryos prior to zygotic transcription and ChIP-seq to measure dynamics of dosage compensation factor targeting. The Drosophila Male-Specific Lethal dosage compensation complex (MSLc) requires the ubiquitous zinc-finger protein Chromatin-Linked Adaptor for MSL Proteins (CLAMP) to identify the X-chromosome. We observe a multi-stage process in which MSLc first identifies CLAMP binding sites throughout the genome followed by concentration at the strongest X-linked MSLc sites. We provide insight into the dynamic mechanism by which a large transcription complex identifies its binding sites during early development.

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The zinc finger protein CLAMP promotes long-range chromatin interactions that mediate dosage compensation of the Drosophila male X-chromosome

10.1101/2020.11.02.365122 ◽

2020 ◽

Author(s):

William Jordan ◽

Erica Larschan

Keyword(s):

Long Range ◽

Zinc Finger ◽

X Chromosome ◽

Dosage Compensation ◽

Zinc Finger Protein ◽

Dimensional Space ◽

Active Chromatin ◽

Single Male ◽

Finger Protein ◽

Genome Wide

SummaryDrosophila dosage compensation is an important model system for defining how active chromatin domains are formed. The Male-specific lethal dosage compensation complex (MSLc) increases transcript levels of genes along the length of the single male X-chromosome to equalize with that on the two female X-chromosomes. The strongest binding sites for MSLc cluster together in three-dimensional space independent of MSLc because clustering occurs in both sexes. CLAMP, a non-sex specific, ubiquitous zinc finger protein, binds synergistically with MSLc to enrich the occupancy of both factors on the male X-chromosome. Here, we demonstrate that CLAMP promotes the observed clustering of MSLc bindings sites. Genome-wide, CLAMP promotes interactions between active chromatin regions. Moreover, the X-enriched CLAMP protein more strongly promotes longer-range interactions on the X-chromosome than autosomes. Genome-wide, CLAMP promotes interactions between active chromatin regions together with other insulator proteins. Overall, we define how long-range interactions which are modulated by a locally enriched ubiquitous transcription factor promote hyper-activation of the X-chromosome to mediate dosage compensation.

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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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CLAMP directly interacts with MSL2 to facilitate Drosophila dosage compensation

10.1101/415455 ◽

2018 ◽

Author(s):

Evgeniya Tikhonova ◽

Anna Fedotova ◽

Artem Bonchuk ◽

Vladic Mogila ◽

Erica N. Larschan ◽

...

Keyword(s):

X Chromosome ◽

Dosage Compensation ◽

Protein Complexes ◽

Dna Binding Protein ◽

Zinc Finger Domain ◽

Dosage Compensation Complex ◽

Dna Interactions ◽

Male Specific Lethal ◽

Msl Complex ◽

Male Specific

AbstractThe binding of Drosophila male-specific lethal (MSL) dosage compensation complex exclusively to male X chromosome provides an excellent model system to understand mechanisms of selective recruitment of protein complexes to chromatin. Previous studies showed that the male-specific organizer of the complex, MSL2, and ubiquitous DNA-binding protein CLAMP are key players in the specificity of X chromosome binding. The CXC domain of MSL2 binds to genomic sites of MSL complex recruitment. Here we demonstrated that MSL2 directly interacts with the N-terminal zinc-finger domain of CLAMP. CLAMP-MSL2 and CXC-DNA interactions are cooperatively involved in recruitment of MSL complex to the X chromosome.

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Drosophila Dosage Compensation Loci Associate with a Boundary-Forming Insulator Complex

Molecular and Cellular Biology ◽

10.1128/mcb.00253-17 ◽

2017 ◽

Vol 37 (21) ◽

Cited By ~ 8

Author(s):

Emily G. Kaye ◽

Amina Kurbidaeva ◽

Daniel Wolle ◽

Tsutomu Aoki ◽

Paul Schedl ◽

...

Keyword(s):

X Chromosome ◽

Dosage Compensation ◽

Boundary Complex ◽

Bithorax Complex ◽

Male Specific Lethal ◽

Topologically Associated Domains ◽

Complex Boundary ◽

Recognition Elements ◽

Male Specific

ABSTRACT Chromatin entry sites (CES) are 100- to 1,500-bp elements that recruit male-specific lethal (MSL) complexes to the X chromosome to upregulate expression of X-linked genes in male flies. CES contain one or more ∼20-bp GA-rich sequences called MSL recognition elements (MREs) that are critical for dosage compensation. Recent studies indicate that CES also correspond to boundaries of X-chromosomal topologically associated domains (TADs). Here, we show that an ∼1,000-kDa complex called the late boundary complex (LBC), which is required for the functioning of the Bithorax complex boundary Fab-7, interacts specifically with a special class of CES that contain multiple MREs. Mutations in the MRE sequences of three of these CES that disrupt function in vivo abrogate interactions with the LBC. Moreover, reducing the levels of two LBC components compromises MSL recruitment. Finally, we show that several of the CES that are physically linked to each other in vivo are LBC interactors.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00006-08 ◽

2008 ◽

Vol 28 (10) ◽

pp. 3401-3409 ◽

Cited By ~ 38

Author(s):

Oliver Bell ◽

Thomas Conrad ◽

Jop Kind ◽

Christiane Wirbelauer ◽

Asifa Akhtar ◽

...

Keyword(s):

Drosophila Melanogaster ◽

X Chromosome ◽

Dosage Compensation ◽

Histone H3 ◽

The Body ◽

Histone H4 ◽

Histone H3 Methylation ◽

Msl Complex ◽

Male Specific ◽

Active Genes

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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