Transcriptomic analysis of the Non-Obstructive Azoospermia (NOA) to address gene expression regulation in human testis

Govindkumar, Balagannavar; Basavaraju, Kavyashree; Bajpai, Akhilesh Kumar; Davuluri, Sravanthi; Shruthi, Kannan; Srini, Vasan S; Chandrashekar, Darshan S.; Chitturi, Neelima; Acharya, Kshitish K.

doi:10.1080/19396368.2023.2176268

Cited by 3 publications

(2 citation statements)

References 94 publications

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“…Single-cell RNA-seq generally provides little information on transcript variant usage. We therefore decided to analyze bulk RNA-seq data of testicular samples isolated from either control subjects or patients with non-obstructive azoospermia (NOA), where germ cell counts are sometimes very low [ 26 ]. Exon quantification was performed to compare relative AGO1 and AGO1-V2 mRNA levels in control testes (set at 1) versus NOA testes that lack germ cells, as indicated by the reduced expression of MAGEA1 and MAGEA4 germ cell markers ( Figure 1 D).…”

Section: Resultsmentioning

confidence: 99%

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DNA Hypomethylation Underlies Epigenetic Swapping between AGO1 and AGO1-V2 Isoforms in Tumors

Fain,

Wangermez,

Loriot

et al. 2024

Epigenomes

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Human tumors progress in part by accumulating epigenetic alterations, which include gains and losses of DNA methylation in different parts of the cancer cell genome. Recent work has revealed a link between these two opposite alterations by showing that DNA hypomethylation in tumors can induce the expression of transcripts that overlap downstream gene promoters and thereby induce their hypermethylation. Preliminary in silico evidence prompted us to investigate if this mechanism applies to the locus harboring AGO1, a gene that plays a central role in miRNA biogenesis and RNA interference. Inspection of public RNA-Seq datasets and RT-qPCR experiments show that an alternative transcript starting 13.4 kb upstream of AGO1 (AGO1-V2) is expressed specifically in testicular germ cells, and becomes aberrantly activated in different types of tumors, particularly in tumors of the esophagus, stomach, and lung. This expression pattern classifies AGO1-V2 into the group of “Cancer-Germline” (CG) genes. Analysis of transcriptomic and methylomic datasets provided evidence that transcriptional activation of AGO1-V2 depends on DNA demethylation of its promoter region. Western blot experiments revealed that AGO1-V2 encodes a shortened isoform of AGO1, corresponding to a truncation of 75 aa in the N-terminal domain, and which we therefore referred to as “∆NAGO1”. Interestingly, significant correlations between hypomethylation/activation of AGO1-V2 and hypermethylation/repression of AGO1 were observed upon examination of tumor cell lines and tissue datasets. Overall, our study reveals the existence of a process of interdependent epigenetic alterations in the AGO1 locus, which promotes swapping between two AGO1 protein-coding mRNA isoforms in tumors.

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Section: Resultsmentioning

confidence: 99%

“…Fastq files of the control testis samples (normal or varicocele, n = 4) and the NOA testis samples (n = 8) were obtained from SRA [ 26 ] and processed as described above. All accession numbers are listed in Supplementary Table S2 .…”

Section: Methodsmentioning

confidence: 99%

DNA Hypomethylation Underlies Epigenetic Swapping between AGO1 and AGO1-V2 Isoforms in Tumors

Fain,

Wangermez,

Loriot

et al. 2024

Epigenomes

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Dysregulation of MTFR2, ATP5IF1 and BAK1 in Sertoli cells relates to idiopathic non-obstructive azoospermia via inhibiting mitochondrial fission and inducing mitochondrial dysfunction

Wei,

Feng,

Dou

et al. 2023

Biology of Reproduction

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Non-obstructive azoospermia (NOA) affects more than 10% of infertile men with over 70% patients are idiopathic with uncharacterized molecular mechanisms which is referred as iNOA. In this study, we checked the morphology of Sertoli cell (SC) mitochondria in testis biopsies from patients with iNOA and patients with obstructive azoospermia (OA) who have normal spermiogenesis. The expression of 104 genes controlling mitochondria fission and fusion were analyzed in three gene expression datasets including a total of 60 patients with NOA. The levels of 7 candidate genes were detected in testis biopsies from 38 patients with iNOA and 24 patients with OA who have normal spermatogenesis by RT-qPCR. Cell viability, apoptosis, mitochondria membrane potential, ATP production, oxygen consumption and mitochondria morphology were examined in primary human SCs. Mouse spermatogonial stem cells (SSCs) were used to detect the cell supporting capacity of SCs. We observed that patients with iNOA had elongated mitochondria. MTFR2 and ATP5IF1 were downregulated, whereas BAK1 was upregulated in iNOA testis and SCs. SCs from patients with iNOA had reduced viability, mitochondria membrane potential, ATP production, oxygen consumption rate (OCR), glycolysis and increased apoptosis. Knockdown MTFR2 in SCs increased the mitochondria size. Knockdown ATP5IF1 did not change mitochondrial morphology but increased ATP hydrolysis. Overexpression of BAK1 reduced membrane potential and upregulated cell apoptosis. The dysregulation of all these three genes contributed to the dysfunction of SCs which provides a clue for iNOA treatment.

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Comparative Analysis of DNA Structural Parameters and the Corresponding Computational Tools to Differentiate Regulatory DNA Motifs and Promoters

Manivelan,

Kavyashree,

Sadanandan

et al. 2024

Preprint

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Analyzing and distinguishing functionally distinct DNA regions is essential for various applications, including predicting DNA motifs and promoters, and unraveling the mechanisms of gene expression regulation in disease conditions. However, existing methods for such analysis require refinement. Despite the value of DNA Structural Parameters (DSPs), users often struggle to objectively select parameters and tools, especially given the limited options available. This study addresses this challenge by thoroughly investigating DSP-tool combinations - particularly the local structural parameters that can be analyzed via web-interfaces, with a goal to discern hu-man DNA motifs and promoters. What sets this study apart is its examination of regulatory sequences that vary based on associated gene expression patterns or the presence or absence of specific motifs. Through the execution of over half a million queries, the study identified DSP-tool combinations that consistently outperformed others in differentiating DNA sequences across various types of analyses. Notably, the 'propeller twist' emerged as a standout DSP, while DNAshape, complemented by DNAshapeR scripts, demonstrated exceptional performance among the tools across four distinct types of analyses: testing motifs, sequences post motif insertion, comparing promoters with control sequences, and analyzing promoters of genes either up- or down-regulated under disease conditions. Significant alterations were observed in the values of multiple DSPs for 100-nucleotide-long promoter and control sequences following the insertion of single motifs such as triplex target sites (TTS), quadruplex-forming structural motifs (QSFM), and transcription factor binding sites (TFBS). However, no single combination proved universally successful. For instance, when analyzing promoters associated with differential gene expression, different combinations proved effective across various human diseases. These findings provide valuable insights for making informed selections among available options for sequence-based queries. Moreover, the report includes minor utilities aimed at enhancing the efficient utilization of DSP-tool combinations.

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Transcriptomic analysis of the Non-Obstructive Azoospermia (NOA) to address gene expression regulation in human testis

Cited by 3 publications

References 94 publications

DNA Hypomethylation Underlies Epigenetic Swapping between AGO1 and AGO1-V2 Isoforms in Tumors

DNA Hypomethylation Underlies Epigenetic Swapping between AGO1 and AGO1-V2 Isoforms in Tumors

Dysregulation of MTFR2, ATP5IF1 and BAK1 in Sertoli cells relates to idiopathic non-obstructive azoospermia via inhibiting mitochondrial fission and inducing mitochondrial dysfunction

Comparative Analysis of DNA Structural Parameters and the Corresponding Computational Tools to Differentiate Regulatory DNA Motifs and Promoters

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