MSL2 variants lead to a neurodevelopmental syndrome with lack of coordination, epilepsy, specific dysmorphisms, and a distinct episignature.

Epigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing, here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay, intellectual disability, hypotonia, and motor issues such as coordination problems, feeding difficulties, and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions, including autism spectrum disorder, and to a lesser extent, seizures, connective tissue disease signs, sleep disturbance, vision problems, and other organ anomalies, were observed in affected individuals. As a molecular biomarker, a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably, while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac, global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless, MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features, a specific blood DNA episignature, and a distinct, MSL2-specific molecular etiology compared to other MSL complex-related disorders.

Loss of function of male-specific lethal 3 (Msl3) does not affect spermatogenesis in rodents.

BACKGROUND: Male-specific lethal 3 (Msl3) is a member of the chromatin-associated male-specific lethal MSL complex, which is responsible for the transcriptional upregulation of genes on the X chromosome in males of Drosophila. Although the dosage complex operates differently in mammals, the Msl3 gene is conserved from flies to humans. Msl3 is required for meiotic entry during Drosophila oogenesis. Recent reports indicate that also in primates, Msl3 is expressed in undifferentiated germline cells before meiotic entry. However, if Msl3 plays a role in the meiotic entry of mammals has yet to be explored. RESULTS: To understand, if Msl3a plays a role in the meiotic entry of mammals, we used mouse spermatogenesis as a study model. Analyses of single-cell RNA-seq data revealed that, in mice, Msl3 is mostly expressed in meiotic cells. To test the role of Msl3 in meiosis, we used a male germline-specific Stra8-iCre driver and a newly generated Msl3flox conditional knock-out mouse line. Msl3 conditional loss-of-function in spermatogonia did not cause spermatogenesis defects or changes in the expression of genes related to meiosis. CONCLUSIONS: Our data suggest that, in mice, Msl3 exhibits delayed expression compared to Drosophila and primates, and loss-of-function mutations disrupting the chromodomain of Msl3 alone do not impede meiotic entry in rodents.

Two distinct males absent on the first (MOF)-containing histone acetyltransferases are involved in the epithelial-mesenchymal transition in different ways in human cells.

Human males absent on the first (MOF), a histone acetyltransferase (HAT), forms male-specific lethal (MSL) and non-specific lethal (NSL), two multiprotein HATs, in cells. MSL was originally discovered in dosage compensation study in Drosophila that can specifically acetylate H4K16, while NSL can simultaneously catalyze the H4 at K5, K8, and K16 sites. However, comparative studies of the two HATs in regulating specific biological functions are rarely reported. Here, we present evidence to argue that MSL and NSL function in different ways in the epithelial-to-mesenchymal transition (EMT) process. At first, CRISPR/Cas9-mediated MSL1 (a key subunit of the MSL)-knockout (KO) and NSL3 (a key subunit of the NSL)-KO cells seem to prefer to grow in clusters. Interestingly, the former promotes cell survival and clonal formation, while the latter has the opposite effect on it. Cell staining revealed that MSL1-KO leads to multipolarized spindles, while NSL3-KO causes more lumen-like cells. Furthermore, in Transwell experiments, silencing of MSL1 promotes cell invasion in 293 T, MCF-7, and MDA-MB-231 cells. In contrast, the inhibitory effects on cell invasion are observed in the same NSL3-silenced cells. Consistent with this, mesenchymal biomarkers, like N-cadherin, vimentin, and snail, are negatively correlated with the expression level of MSL1; however, a positive relationship between these proteins and NSL3 in cells has been found. Further studies have clarified that MSL1, but not NSL3, can specifically bind to the E-box-containing Snail promoter region and thereby negatively regulate Snail transactivation. Also, silencing of MSL1 promotes the lung metastasis of B16F10 melanoma cells in mice. Finally, ChIP-Seq analysis indicated that the NSL may be mainly involved in phosphoinositide-mediated signaling pathways. Taken together, the MOF-containing MSL and NSL HATs may regulate the EMT process in different ways in order to respond to different stimuli.

Cloning and RNAi-mediated three lethal genes that can be potentially used for Chilo suppressalis (Lepidoptera: Crambidae) management.

RNA interference (RNAi) has gained attention in recent years as a viable pest control strategy. Here, RNAi assays were performed to screen the potential functionality of genes in Chilo suppressalis, a serious pest of rice, and to determine their potential for developing a highly targeted molecular control approach. Potential homologs of NADH dehydrogenase (ND), glycerol 3-phosphate dehydrogenase (GPDH) and male specific lethal 3 (MSL3) were cloned from C. suppressalis, and their spatiotemporal gene expression evaluated. The expression of all three genes was higher in the pupal and adult stages than the larval stages and largely higher in the larval head compared to other tissues. Newly hatched larvae exhibited high mortalities and suppressed growth when fed bacteria producing double-stranded RNAs (dsRNAs) corresponding to the three target genes. This study provides insights into the function of ND, GPDH and MSL3 during C. suppressalis larval development and suggests that all may be candidate gene targets for C. suppressalis pest management.

Antiviral therapy may decrease HBx, affecting cccDNA and MSL2 in hepatocarcinogenesis.

Chronic hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC). Covalently closed circular DNA (cccDNA) is an intermediate in the life cycle of HBV. HBV-encoded X protein (HBx), a key viral oncoprotein, can be specifically ubiquitylated by male specific lethal 2 (MSL2), which causes upregulation of HBx activity and promotes transcription, cell proliferation and tumor growth. The present study compared the levels of cccDNA, MSL2 mRNA and HBx mRNA in tumor and peri-tumor tissues, and clarified the effect of antiviral therapy on these indicators. Levels of intrahepatic cccDNA, MSL2 mRNA and HBx mRNA were determined using quantitative PCR in patients with HBV-associated HCC who had undergone liver surgery. A total of 50 patients were included in the present study. Prior to surgery, 31 patients had undergone antiviral treatment. Intrahepatic cccDNA levels were significantly higher in the tumor tissues compared with the peri-tumor tissues (P=0.001), particularly in the hepatitis B e antigen-positive (P=0.008), tumor recurrence (P=0.002) and <3 cm tumor size (P=0.003) groups. Furthermore, in patients with preoperative cirrhosis, levels of cccDNA and MSL2 mRNA were significantly higher in tumor tissues compared with that in peri-tumor tissues (P<0.001 and P=0.023, respectively). The expression levels of HBx mRNA in antiviral-treated tumors and peri-tumor tissues were significantly lower compared with those in untreated tissues (P=0.026 and P=0.035). The levels of cccDNA and MSL2 mRNA in the HBx-positive group were significantly higher in tumor tissues compared with those in peri-tumor tissues (P=0.026 and P=0.013). In conclusion, cccDNA participated in the tumorigenesis of HBV-associated HCC, and antiviral therapy was found to modulate hepatocarcinogenesis by decreasing the levels of HBx to inhibit the tumorigenic effect of MSL2 and cccDNA.

no blokes Is Essential for Male Viability and X Chromosome Gene Expression in the Australian Sheep Blowfly.

It has been hypothesized that the Drosophila 4th chromosome is derived from an ancient X chromosome [1]. In the Australian sheep blowfly, Lucilia cuprina, the heterochromatic X chromosome contains few active genes and orthologs of Drosophila X-linked genes are autosomal. Of 8 X-linked genes identified previously in L. cuprina, 6 were orthologs of Drosophila 4th-chromosome genes [2]. The X-linked genes were expressed equally in males and females. Here we identify an additional 51 X-linked genes and show that most are dosage compensated. Orthologs of 49 of the 59 X-linked genes are on the 4th chromosome in D. melanogaster. Because painting of fourth (Pof) is important for expression of Drosophila 4th-chromosome genes [3], we used Cas9 to make a loss-of-function knockin mutation in an L. cuprina Pof ortholog we call no blokes (nbl). Homozygous nbl males derived from homozygous nbl mothers die at the late pupal stage. Homozygous nbl females are viable, fertile, and live longer than heterozygous nbl females. RNA expression of most X-linked genes was reduced in homozygous nbl male pupae and to a lesser extent in nbl females compared to heterozygous siblings. The results suggest that NBL could be important for X chromosome dosage compensation in L. cuprina. NBL may also facilitate gene expression in the heterochromatic environment of the X chromosome in both sexes. This study supports the hypothesis on the origin of the Drosophila 4th chromosome and that a POF-like protein was required for normal gene expression on the ancient X chromosome.

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

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.

Drosophila Sister-of-Sex-lethal is a repressor of translation.

The RNA-binding protein Sex-lethal (Sxl) is an important post-transcriptional regulator of sex determination and dosage compensation in female Drosophila To prevent the assembly of the MSL dosage compensation complex in female flies, Sxl acts as a repressor of male-specific lethal-2 (msl-2) mRNA translation. It uses two distinct and mutually reinforcing blocks to translation that operate on the 5' and 3' untranslated regions (UTRs) of msl-2 mRNA, respectively. While 5' UTR-mediated translational control involves an upstream open reading frame, 3' UTR-mediated regulation strictly requires the co-repressor protein Upstream of N-ras (Unr), which is recruited to the transcript by Sxl. We have identified the protein Sister-of-Sex-lethal (Ssx) as a novel repressor of translation with Sxl-like activity. Both proteins have a comparable RNA-binding specificity and can associate with uracil-rich RNA regulatory elements present in msl-2 mRNA. Moreover, both repress translation when bound to the 5' UTR of msl-2 However, Ssx is inactive in 3' UTR-mediated regulation, as it cannot engage the co-repressor protein Unr. The difference in activity maps to the first RNA-recognition motif (RRM) of Ssx. Conversion of three amino acids within this domain into their Sxl counterpart results in a gain of function and repression via the 3' UTR, allowing detailed insights into the evolutionary origin of the two proteins and into the molecular requirements of an important translation regulatory pathway.

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

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.

Dosage Compensation in Drosophila-a Model for the Coordinate Regulation of Transcription.

The sex chromosomes have special significance in the history of genetics. The chromosomal basis of inheritance was firmly established when Calvin Bridges demonstrated that exceptions to Mendel's laws of segregation were accompanied at the cytological level by exceptional sex chromosome segregation. The morphological differences between X and Y exploited in Bridges' experiments arose as a consequence of the evolution of the sex chromosomes. Originally a homologous chromosome pair, the degeneration of the Y chromosome has been accompanied by a requirement for increased expression of the single X chromosome in males. Drosophila has been a model for the study of this dosage compensation and has brought key strengths, including classical genetics, the exceptional cytology of polytene chromosomes, and more recently, comprehensive genomics. The impact of these studies goes beyond sex chromosome regulation, providing valuable insights into mechanisms for the establishment and maintenance of chromatin domains, and for the coordinate regulation of transcription.

Recent progress and open questions in Drosophila dosage compensation.

Sexual dimorphism is observed in many traits across diverse taxa, and often it is quite extreme. Within a species, individuals of opposing sex can appear strikingly different, reflecting differences at the molecular level that may be similarly striking. Among the most extreme cases of such molecular sexual dimorphism is the quantity of sex chromosomes that each sex possesses. Hemizygous sex chromosomes are common to many species, and various mechanisms have evolved to regulate transcriptional activity to ensure appropriate sex chromosome-to-autosome gene expression stoichiometry. Among the most thoroughly investigated of these mechanisms is Drosophila melanogaster's male-specific lethal (MSL) complex-mediated dosage compensation. In Drosophila, the male X chromosome transcription is upregulated approximately two-fold in somatic tissues to counterbalance the effects of sex chromosome hemizygosity on transcript abundance. Despite dramatic advances in our understanding of the Drosophila dosage compensation, many questions remain unanswered, and our understanding of its molecular underpinnings remains incomplete. In this review, we synthesize recent progress in the field as a means to highlight open questions, including how the MSL complex targets the X chromosome, how dosage compensation has shaped evolution of X-linked genes, and the degree to which MSL complex-mediated dosage compensation varies in activity across somatic tissues.

X-to-autosome expression and msl-2 transcript abundance correlate among Drosophila melanogaster somatic tissues.

In Drosophila melanogaster, the male-specific lethal (MSL) complex has been studied extensively for its role in upregulating male X-linked genes. Recent advances in high-throughput technologies have improved our understanding of how the MSL complex mediates dosage compensation through chromosome-wide chromatin modifications. Most studies, however, have focused on cell line models that cannot reflect any potential heterogeneity of in vivo dosage compensation. Comparisons between cell line and organismal gene-level dosage compensation upregulation suggest the possibility of variation in MSL complex activity among somatic tissues. We hypothesize the degree, up to but not exceeding 2-fold, to which the MSL complex upregulates male X-linked genes varies quantitatively by tissue type. In this model, MSL complex abundance acts as a rheostat to control the extent of upregulation. Using publicly available expression data, we provide evidence for our model in Drosophila somatic tissues. Specifically, we find X-to-autosome expression correlates with the tissue-specific expression of msl-2 which encodes an essential male-specific component of the MSL complex. This result suggests MSL complex mediated dosage compensation varies quantitatively by tissue type. Furthermore, this result has consequences for models explaining the organismal-scale molecular and evolutionary consequences of MSL-mediated dosage compensation.

Are we there yet? Initial targeting of the Male-Specific Lethal and Polycomb group chromatin complexes in Drosophila.

Chromatin-binding proteins must navigate the complex nuclear milieu to find their sites of action, and a constellation of protein factors and other properties are likely to influence targeting specificity. Despite considerable progress, the precise rules by which binding specificity is achieved have remained elusive. Here, we consider early targeting events for two groups of chromatin-binding complexes in Drosophila: the Male-Specific Lethal (MSL) and the Polycomb group (PcG) complexes. These two serve as models for understanding targeting, because they have been extensively studied and play vital roles in Drosophila, and their targets have been documented at high resolution. Furthermore, the proteins and biochemical properties of both complexes are largely conserved in multicellular organisms, including humans. While the MSL complex increases gene expression and PcG members repress genes, the two groups share many similarities such as the ability to modify their chromatin environment to create active or repressive domains, respectively. With legacies of in-depth genetic, biochemical and now genomic approaches, the MSL and PcG complexes will continue to provide tractable systems for understanding the recruitment of multiprotein chromatin complexes to their target loci.

A genome-wide screen identifies genes that affect somatic homolog pairing in Drosophila.

In Drosophila and other Dipterans, homologous chromosomes are in close contact in virtually all nuclei, a phenomenon known as somatic homolog pairing. Although homolog pairing has been recognized for over a century, relatively little is known about its regulation. We performed a genome-wide RNAi-based screen that monitored the X-specific localization of the male-specific lethal (MSL) complex, and we identified 59 candidate genes whose knockdown via RNAi causes a change in the pattern of MSL staining that is consistent with a disruption of X-chromosomal homolog pairing. Using DNA fluorescent in situ hybridization (FISH), we confirmed that knockdown of 17 of these genes has a dramatic effect on pairing of the 359 bp repeat at the base of the X. Furthermore, dsRNAs targeting Pr-set7, which encodes an H4K20 methyltransferase, cause a modest disruption in somatic homolog pairing. Consistent with our results in cultured cells, a classical mutation in one of the strongest candidate genes, pebble (pbl), causes a decrease in somatic homolog pairing in developing embryos. Interestingly, many of the genes identified by our screen have known roles in diverse cell-cycle events, suggesting an important link between somatic homolog pairing and the choreography of chromosomes during the cell cycle.

Further analysis on the male-specific lethal mutations that affect dosage compensation in Drosophila melanogaster.

The male-specific lethal genes (msl) of D. melanogaster represent a set of genes whose functions are required for the specific X chromosome hypertranscription in males (dosage compensation). We have carried out the clonal analysis of one of those msl mutations: msl-3 b. Clones homozygous for msl-3 b are deleterious; this mutation presents cell autonomy and in the cases where msl clones appeared in sexually dimorphic regions (5th and 6th tergites) they do not show sexual transformation. Moreover, the lethal phase and the growth dynamics (measured by the protein content during larval growth) are the same for male larvae homozygous for one msl mutation (msl-1) or three msl mutations (msl-2 msl-1 mle), i.e. the msl mutations do not show additive effects. This paper considers the possible interactions between the msl genes that bring about dosage compensation.