Distinct mechanisms mediate X chromosome dosage compensation in Anopheles and Drosophila

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.

PRL Mutation Causing Alactogenesis: Insights into Prolactin Structure and Function Relationships

CONTEXT Isolated prolactin deficiency is a rare disorder manifesting as absence of puerperal lactation. We identified a two-generation family with 3 women experiencing alactogenesis. OBJECTIVE We hypothesized a heterozygous genetic mutation. DESIGN This was a family-based study. PATIENTS Two generations of women (proband, sister and niece) with puerperal alactogenesis and one control were studied. Prolactin levels in the three women ranged from 0.618 to 1.4 ng/mL (2.8-29.2 ng/mL). All the women had regular menstrual cycles during their reproductive years. The niece required fertility treatment to become pregnant and the proband and sister underwent menopause before age 45 years. INTERVENTION PRL exons 1-5 were sequenced. MAIN OUTCOME MEASURE We sought a heterozygous, deleterious gene variant with functional consequences. RESULTS We identified a heterozygous mutation (c.658C>T) changing CGA to TGA (p.Arg220Ter) in exon 5 of the prolactin gene. Transfection of PRL containing the stop gain mutation resulted in similar intracellular prolactin levels compared to PRL wild type, but little detectable immunoactive or bioactive prolactin in conditioned medium. Prolactin secretion was also impaired by a PRL stop gain mutation deleting both of the terminal cysteine amino acids (c.652A>T; p.Lys218Ter). CONCLUSIONS This is the first report of a PRL mutation causing familial prolactin deficiency and alactogenesis. The loss of the terminal cysteine resulted in failure of prolactin secretion. Secretion was not rescued by deleting the penultimate cysteine, with which it forms a disulfide bond. These data suggest that the PRL C terminal is critical for protein secretion.

A H3K9me2-Binding Protein AGDP3 Limits DNA Methylation and Transcriptional Gene Silencing in Arabidopsis

DNA methylation is critical for tuning gene expression to prevent potentially deleterious gene-silencing. The Arabidopsis DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required for the prevention of DNA hypermethylation at thousands of genomic loci. However, the mechanism recruiting ROS1 to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor required for ROS1-mediated DNA demethylation, and targets ROS1 to specific loci. We found that AGDP3 could bind to the H3K9me2 mark by its AGD12 cassette. The crystal structure of the AGDP3 AGD12 in complex with an H3K9me2 peptide reveals the molecular basis for the specific recognition, that the dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. Interestingly, a histidine residue located in the methylysine binding aromatic cage enables AGDP3 pH-dependent H3K9me2 binding capacity. Considering the intracellular pH correlates with the histone acetylation status, our results provide the molecular mechanism for the regulation of ROS1 DNA demethylase by the gene silencing H3K9me2 mark and the potential crosstalk with active histone acetylation mark.

Suboptimal Global Transcriptional Response Increases the Harmful Effects of Loss-of-Function Mutations

The fitness impact of loss-of-function mutations is generally assumed to reflect the loss of specific molecular functions associated with the perturbed gene. Here, we propose that rewiring of the transcriptome upon deleterious gene inactivation is frequently nonspecific and mimics stereotypic responses to external environmental change. Consequently, transcriptional response to gene deletion could be suboptimal and incur an extra fitness cost. Analysis of the transcriptomes of ∼1,500 single-gene deletion Saccharomyces cerevisiae strains supported this scenario. First, most transcriptomic changes are not specific to the deleted gene but are rather triggered by perturbations in functionally diverse genes. Second, gene deletions that alter the expression of dosage-sensitive genes are especially harmful. Third, by elevating the expression level of downregulated genes, we could experimentally mitigate the fitness defect of gene deletions. Our work shows that rewiring of genomic expression upon gene inactivation shapes the harmful effects of mutations.

Web-Based Bioinformatics Predictors: Recommendations to Assess Lysosomal Cholesterol Trafficking Diseases-Related Genes

Introduction The growing number of genetic variants of unknown significance (VUS) and availability of several in silico prediction tools make the evaluation of potentially deleterious gene variants challenging. Materials and Methods We evaluated several programs and software to determine the one that can predict the impact of genetic variants found in lysosomal storage disorders (LSDs) caused by defects in cholesterol trafficking best. We evaluated the sensitivity, specificity, accuracy, precision, and Matthew's correlation coefficient of the most common software. Results Our findings showed that for exonic variants, only MutPred1 reached 100% accuracy and generated the best predictions (sensitivity and accuracy = 1.00), whereas intronic variants, SROOGLE or Human Splicing Finder (HSF) generated the best predictions (sensitivity = 1.00, and accuracy = 1.00). Discussion Next-generation sequencing substantially increased the number of detected genetic variants, most of which were considered to be VUS, creating a need for accurate pathogenicity prediction. The focus of the present study is the importance of accurately predicting LSDs, with majority of previously unreported specific mutations. Conclusion We found that the best prediction tool for the NPC1, NPC2, and LIPA variants was MutPred1 for exonic regions and HSF and SROOGLE for intronic regions and splice sites.

Hybrid incompatibility caused by an epiallele

Hybrid incompatibility resulting from deleterious gene combinations is thought to be an important step toward reproductive isolation and speciation. Here, we demonstrate involvement of a silent epiallele in hybrid incompatibility. In Arabidopsis thaliana accession Cvi-0, one of the two copies of a duplicated histidine biosynthesis gene, HISN6A, is mutated, making HISN6B essential. In contrast, in accession Col-0, HISN6A is essential because HISN6B is not expressed. Owing to these differences, Cvi-0 × Col-0 hybrid progeny that are homozygous for both Cvi-0 HISN6A and Col-0 HISN6B do not survive. We show that HISN6B of Col-0 is not a defective pseudogene, but a stably silenced epiallele. Mutating HISTONE DEACETYLASE 6 (HDA6), or the cytosine methyltransferase genes MET1 or CMT3, erases HISN6B's silent locus identity, reanimating the gene to circumvent hisn6a lethality and hybrid incompatibility. These results show that HISN6-dependent hybrid lethality is a revertible epigenetic phenomenon and provide additional evidence that epigenetic variation has the potential to limit gene flow between diverging populations of a species.

Hybrid incompatibility caused by an epiallele

Hybrid incompatibility resulting from deleterious gene combinations is thought to be an important step towards reproductive isolation and speciation. Here we demonstrate involvement of a silent epiallele in hybrid incompatibility. In Arabidopsis thaliana strain Col-0, one of the two copies of a duplicated histidine biosynthesis gene, HISN6B is not expressed, for reasons that have been unclear, making its paralog, HISN6A essential. By contrast, in strain Cvi-0, HISN6B is essential because HISN6A is mutated. As a result of these differences, Cvi-0 × Col-0 hybrid progeny that are homozygous for both Col-0 HISN6B and Cvi-0 HISN6A do not survive. We show that HISN6B is not a defective pseudogene in the Col-0 strain, but a stably silenced epiallele. Mutating HISTONE DEACETYLASE 6 (HDA6) or the cytosine methyltransferase genes, MET1 or CMT3 erases HISN6B’s silent locus identity in Col-0, reanimating the gene such that hisn6a lethality and hybrid incompatibility are circumvented. These results show that HISN6-dependent hybrid lethality is a revertible epigenetic phenomenon and provide additional evidence that epigenetic variation has the potential to limit gene flow between diverging populations of a species.Significance statementDeleterious mutations in different copies of a duplicated gene pair have the potential to cause hybrid incompatibility between diverging subpopulations, contributing to reproductive isolation and speciation. This study demonstrates a case of epigenetic gene silencing, rather than pseudogene creation by mutation, contributing to a lethal gene combination upon hybridization of two strains of Arabidopsis thaliana. The findings provide direct evidence that naturally occurring epigenetic variation can contribute to incompatible hybrid genotypes, reducing gene flow between strains of the same species.

A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome

Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome for prophylaxis against aortic aneurysm progression, despite limited evidence for their efficacy and safety in the disorder. Unexpectedly, Marfan mice treated with CCBs show accelerated aneurysm expansion, rupture, and premature lethality. This effect is both extracellular signal-regulated kinase (ERK1/2) dependent and angiotensin-II type 1 receptor (AT1R) dependent. We have identified protein kinase C beta (PKCβ) as a critical mediator of this pathway and demonstrate that the PKCβ inhibitor enzastaurin, and the clinically available anti-hypertensive agent hydralazine, both normalize aortic growth in Marfan mice, in association with reduced PKCβ and ERK1/2 activation. Furthermore, patients with Marfan syndrome and other forms of inherited thoracic aortic aneurysm taking CCBs display increased risk of aortic dissection and need for aortic surgery, compared to patients on other antihypertensive agents.