biostudies-arrayexpressAntonio LentiniGallus gallushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14390Sex-chromosome dosage represents a challenge for heterogametic species to maintain correct proportion of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZW/ZZ system) have been found to lack efficient compensation at RNA level, challenging the necessity of resolving major gene-dosage discrepancies in avian cells. Through allele-resolved multiome analyses, we comprehensively examined dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Remarkably, this revealed that females exhibit upregulation of their single Z through increased transcriptional burst frequency similar to mammalian X-upregulation, and that Z-protein levels are further balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrate remarkable conservation of the genomic encoding of burst kinetics between mammals and birds. Our study uncovers new mechanisms for achieving sex-chromosome dosage compensation and highlights the importance of gene-dosage balance across diverse species.biostudies-arrayexpressSequencing - The libraries were pooled in equimolar amounts and paired-end sequencing was performed on an Illumina Nextseq 550 instrument to obtain ~ 20 million paired-end reads per sample.Library Construction - Library amplification was performed by adding 30μl of PCR master mix to the purified DNA (25μl 2x NEBNext High Fidelity PCR Master Mix, 2.5μl Ad1_noMX (common i5 Nextera adapter primer ), 2.5μl Ad2 (unique i7 Nextera adapter primer). The libraries were amplified for 11 cycles using the following cycling conditions: 72°C for 5 min, 98°C for 30s, 11x (98°C for 10s, 63°C for 30s, 72°C for 1 min), 4°C on hold. The final libraries were fragment size-selected by double-sided 0.5x/1.3x bead purification using homemade 22% PEG magnetic beads. Briefly, 25μl of room temperature Ampure XP beads were added to each sample (beads-to-sample ratio = 0.5) and incubated for 10 min after thorough resuspension. The samples were placed on a magnetic rack and the supernatant was removed and transferred to a new microcentrifuge tube containing 65μl room temperature Ampure XP beads (beads-to-sample (original volume) ratio = 1.3). After thorough mixing, the samples were incubated at room temperature for 10 min and placed on a magnetic rack for 5 min. The supernatant was discarded and the beads were washed twice with 200μl of freshly-prepared 80% ethanol. After ethanol removal, the samples were air-dried for 5 min and the libraries eluted in 20μl of nuclease-free water.Nucleic Acid Extraction - The supernatant was removed and the samples were resuspended in 50μl transposition mixture (25μl 2x TD buffer, 1.5μl Tn5 (27uM) 16.5 μl 1x PBS, 0.5 μl 1% digitonin, 0.5μl 10% Tween-20, 7μl water). Tagmentation was performed in a thermoshaker at 37°C for 30 min at 1000rpm. The transposed DNA was purified using the Zymo DNA Clean and Concentrator-5 kit following the manufacturer’s instructions, and eluted in 21μl of elution buffer.Growth Protocol - CEFs were grown on 0.1% gelatin-coated plates in complete growth media (10% Fetal Bovine Serum [Gibco], 100U/ml Penicillin - 100ug/ml Streptomycin, 1mM non-essential amino acids [Gibco], 1mM Sodium pyruvate [Gibco]) to 80% confluency before collection.Sample Collection - For each replicate, 100000 cells were pelleted at 500g for 5 minutes at 4°C. The supernatant was carefully removed and the pellet was resuspended in 50μl ATAC-RSB lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) and incubated on ice for 10 min. The lysis buffer was washed out by adding 1ml of ATAC-RSB wash buffer containing 0.1% Tween but no NP-40 or digitonin and the samples inverted 3 times to mix and the nuclei pelleted at 500 RCF at 4°C for 10 min.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Aligned reads were then converted to BAM format, mate pair information was fixed, duplicate read pairs were marked, and the BAM output was sorted according to read coordinates using biobambam2 (v.2.0.87, bamsort fixmates=1, markduplicates=1). Replicate merging was performed using sambamba merge (v. 0.7.0). SNPs were called from the ATAC-seq data using bcftools (v. 1.10.2, mpileup --ignore-RG -Ou -a AD,DP --max-depth 8000 | call --threads 64 -mv -Oz --ploidy-file –samples-file | filter -g 5 -i 'TYPE=\\"snp\\" & QUAL>10 & INFO/DP>5 & GT=\\"het\\" & MAF>0.1'). Next, the GRCg6a genome was N-masked for SNP positions using bcftools (consensus --mask) and data was realigned to the N-masked reference using bowtie2 as described above. Peak calling on all replicates was performed using Genrich in ATAC-seq mode (v.0.6, settings: -j, -r, -d 150, -e W,MT, -E lowmap), PCR duplicates were removed, the cut sites were expanded to 150 bp and reads from the W chromosome and the mitochondria were excluded, and it was repeated both with and without low mappability regions. Genrich requires input sorted by query name which was done by biobambam2 bamsort. All peaks were combined and replicated peaks within 100bp were merged and counted using bedtools (v.2.30.0, merge -c 1 -o count -d 100) and peaks present with >1 counts were kept. Distance to nearest TSS for each peak was annotated using bedtools (v.2.30.0, closest). Peak quantification of proper read pairs, both excluding and including low-mapping regions, and removing PCR duplicates was calculated by deepTools (v.3.5.4.post1, multiBamSummary --samFlagInclude 2, --ignoreDuplicates, -bl lowmap). Genome-wide signal pileups was normalised using deepTools (bamCoverage --normalizeUsing RPGC, --effectiveGenomeSize 1058535536, --ignoreDuplicates, --samFlagInclude 2, -ignore MT, --minFragmentLength 38, --maxFragmentLength 2000). These only included proper read pairs, mitochondrial entries were removed as well as PCR duplicates, and only fragments between 38 and 2000 bp lengths were kept. The pileups were also made by containing, and excluding, low mapping regions, for further use and visualisations. Effective genome size of GRCg6a used for normalisation was obtained from Genrich. Enrichment 5kb around TSS was calculated using deeptools (computeMatrix -a 5000, -b 5000 -R Gallus_gallus.GRCg6a.100.gtf).Sequence Alignment - Raw ATAC-seq data was converted to FASTQ format using bcl2fastq (v.2.20.0.422). Sequencing adapter removal was performed using fastp (v.0.20.0). The resulting trimmed reads were aligned to the GRCg6a genome using Bowtie 2 (v.2.5.1, settings: -N 1, -X 2000, --very-sensitive).UnknownTranscriptomicsGenomicsProteomicsNextSeq 550ATAC-seqGallus gallusAntonio LentiniBjörn ReiniusNatali PapanicolaoufalseMulti-layer dosage compensation of the avian Z chromosome (ATAC-seq)Sex-chromosome dosage represents a challenge for heterogametic species to maintain correct proportion of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZW/ZZ system) have been found to lack efficient compensation at RNA level, challenging the necessity of resolving major gene-dosage discrepancies in avian cells. Through allele-resolved multiome analyses, we comprehensively examined dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Remarkably, this revealed that females exhibit upregulation of their single Z through increased transcriptional burst frequency similar to mammalian X-upregulation, and that Z-protein levels are further balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrate remarkable conservation of the genomic encoding of burst kinetics between mammals and birds. Our study uncovers new mechanisms for achieving sex-chromosome dosage compensation and highlights the importance of gene-dosage balance across diverse species.2025-06-30T00:00:00Z2024-08-27T16:50:49.62Z2024-08-27T16:50:49.62ZE-MTAB-14390ERP163594E-MTAB-14393E-MTAB-14392E-MTAB-14391EFO_0002944EFO_0007045EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184