Genome-wide analysis of gene regulation mechanisms during Drosophila spermatogenesis

Background During Drosophila spermatogenesis, testis-specific meiotic arrest complex (tMAC) and testis-specific TBP-associated factors (tTAF) contribute to activation of hundreds of genes required for meiosis and spermiogenesis. Intriguingly, tMAC is paralogous to the broadly expressed complex Myb-MuvB (MMB)/dREAM and Mip40 protein is shared by both complexes. tMAC acts as a gene activator in spermatocytes, while MMB/dREAM was shown to repress gene activity in many cell types. Results Our study addresses the intricate interplay between tMAC, tTAF, and MMB/dREAM during spermatogenesis. We used cell type-specific DamID to build the DNA-binding profiles of Cookie monster (tMAC), Cannonball (tTAF), and Mip40 (MMB/dREAM and tMAC) proteins in male germline cells. Incorporating the whole transcriptome analysis, we characterized the regulatory effects of these proteins and identified their gene targets. This analysis revealed that tTAFs complex is involved in activation of achi, vis, and topi meiosis arrest genes, implying that tTAFs may indirectly contribute to the regulation of Achi, Vis, and Topi targets. To understand the relationship between tMAC and MMB/dREAM, we performed Mip40 DamID in tTAF- and tMAC-deficient mutants demonstrating meiosis arrest phenotype. DamID profiles of Mip40 were highly dynamic across the stages of spermatogenesis and demonstrated a strong dependence on tMAC in spermatocytes. Integrative analysis of our data indicated that MMB/dREAM represses genes that are not expressed in spermatogenesis, whereas tMAC recruits Mip40 for subsequent gene activation in spermatocytes. Conclusions Discovered interdependencies allow to formulate a renewed model for tMAC and tTAFs action in Drosophila spermatogenesis demonstrating how tissue-specific genes are regulated

Selection of Diagnostically Significant Regions of the SLC26A4 Gene Involved in Hearing Loss

Screening pathogenic variants in the SLC26A4 gene is an important part of molecular genetic testing for hearing loss (HL) since they are one of the common causes of hereditary HL in many populations. However, a large size of the SLC26A4 gene (20 coding exons) predetermines the difficulties of its complete mutational analysis, especially in large samples of patients. In addition, the regional or ethno-specific prevalence of SLC26A4 pathogenic variants has not yet been fully elucidated, except variants c.919-2A>G and c.2168A>G (p.His723Arg), which have been proven to be most common in Asian populations. We explored the distribution of currently known pathogenic and likely pathogenic (PLP) variants across the SLC26A4 gene sequence presented in the Deafness Variation Database for the selection of potential diagnostically important parts of this gene. As a result of this bioinformatic analysis, we found that molecular testing ten SLC26A4 exons (4, 6, 10, 11, 13–17 and 19) with flanking intronic regions can provide a diagnostic rate of 61.9% for all PLP variants in the SLC26A4 gene. The primary sequencing of these SLC26A4 regions may be applied as an initial effective diagnostic testing in samples of patients of unknown ethnicity or as a subsequent step after the targeted testing of already-known ethno- or region-specific pathogenic SLC26A4 variants

Functional dissection of Drosophila melanogaster SUUR protein influence on H3K27me3 profile

Abstract Background In eukaryotes, heterochromatin replicates late in S phase of the cell cycle and contains specific covalent modifications of histones. SuUR mutation found in Drosophila makes heterochromatin replicate earlier than in wild type and reduces the level of repressive histone modifications. SUUR protein was shown to be associated with moving replication forks, apparently through the interaction with PCNA. The biological process underlying the effects of SUUR on replication and composition of heterochromatin remains unknown. Results Here we performed a functional dissection of SUUR protein effects on H3K27me3 level. Using hidden Markow model-based algorithm we revealed SuUR-sensitive chromosomal regions that demonstrated unusual characteristics: They do not contain Polycomb and require SUUR function to sustain H3K27me3 level. We tested the role of SUUR protein in the mechanisms that could affect H3K27me3 histone levels in these regions. We found that SUUR does not affect the initial H3K27me3 pattern formation in embryogenesis or Polycomb distribution in the chromosomes. We also ruled out the possible effect of SUUR on histone genes expression and its involvement in DSB repair. Conclusions Obtained results support the idea that SUUR protein contributes to the heterochromatin maintenance during the chromosome replication. A model that explains major SUUR-associated phenotypes is proposed

Whole Exome Sequencing Reveals Homozygous Mutations in RAI1, OTOF, and SLC26A4 Genes Associated with Nonsyndromic Hearing Loss in Altaian Families (South Siberia)

Hearing loss (HL) is one of the most common sensorineural disorders and several dozen genes contribute to its pathogenesis. Establishing a genetic diagnosis of HL is of great importance for clinical evaluation of deaf patients and for estimating recurrence risks for their families. Efforts to identify genes responsible for HL have been challenged by high genetic heterogeneity and different ethnic-specific prevalence of inherited deafness. Here we present the utility of whole exome sequencing (WES) for identifying candidate causal variants for previously unexplained nonsyndromic HL of seven patients from four unrelated Altaian families (the Altai Republic, South Siberia). The WES analysis revealed homozygous missense mutations in three genes associated with HL. Mutation c.2168A>G (SLC26A4) was found in one family, a novel mutation c.1111G>C (OTOF) was revealed in another family, and mutation c.5254G>A (RAI1) was found in two families. Sanger sequencing was applied for screening of identified variants in an ethnically diverse cohort of other patients with HL (n = 116) and in Altaian controls (n = 120). Identified variants were found only in patients of Altaian ethnicity (n = 93). Several lines of evidences support the association of homozygosity for discovered variants c.5254G>A (RAI1), c.1111C>G (OTOF), and c.2168A>G (SLC26A4) with HL in Altaian patients. Local prevalence of identified variants implies possible founder effect in significant number of HL cases in indigenous population of the Altai region. Notably, this is the first reported instance of patients with RAI1 missense mutation whose HL is not accompanied by specific traits typical for Smith-Magenis syndrome. Presumed association of RAI1 gene variant c.5254G>A with isolated HL needs to be proved by further experimental studies.This work was supported by grant #2011Y1SA09 from the Chinese Academy of Sciences Fellowship for Young International Scientists and NSFC grant #31150110466 (to AYC), by the State Project #0324-2015-0004, and by grant #0324-2015-0031 from the Siberian Branch of the Russian Academy of Sciences (to OLP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

High Rates of Three Common GJB2 Mutations c.516G>C, c.-23+1G>A, c.235delC in Deaf Patients from Southern Siberia Are Due to the Founder Effect

The mutations in the GJB2 gene (13q12.11, MIM 121011) encoding transmembrane protein connexin 26 (Cx26) account for a significant portion of hereditary hearing loss worldwide. Earlier we found a high prevalence of recessive GJB2 mutations c.516G>C, c.-23+1G>A, c.235delC in indigenous Turkic-speaking Siberian peoples (Tuvinians and Altaians) from the Tyva Republic and Altai Republic (Southern Siberia, Russia) and proposed the founder effect as a cause for their high rates in these populations. To reconstruct the haplotypes associated with each of these mutations, the genotyping of polymorphic genetic markers both within and flanking the GJB2 gene was performed in 28 unrelated individuals homozygous for c.516G>C (n = 18), c.-23+1G>A (n = 6), or c.235delC (n = 4) as well as in the ethnically matched controls (62 Tuvinians and 55 Altaians) without these mutations. The common haplotypes specific for mutations c.516G>C, c.-23+1G>A, or c.235delC were revealed implying a single origin of each of these mutations. The age of mutations estimated by the DMLE+ v2.3 software and the single marker method is discussed in relation to ethnic history of Tuvinians and Altaians. The data obtained in this study support a crucial role of the founder effect in the high prevalence of GJB2 mutations c.516G>C, c.-23+1G>A, c.235delC in indigenous populations of Southern Siberia

Updated carrier rates for c.35delG (GJB2) associated with hearing loss in Russia and common c.35delG haplotypes in Siberia

Abstract Background Mutations in GJB2 gene are a major causes of deafness and their spectrum and prevalence are specific for various populations. The well-known mutation c.35delG is more frequent in populations of Caucasian origin. Data on the c.35delG prevalence in Russia are mainly restricted to the European part of this country. We aimed to estimate the carrier frequency of c.35delG in Western Siberia and thereby update current data on the c.35delG prevalence in Russia. According to a generally accepted hypothesis, c.35delG originated from a common ancestor in the Middle East or the Mediterranean ~ 10,000–14,000 years ago and spread throughout Europe with Neolithic migrations. To test the c.35delG common origin hypothesis, we have reconstructed haplotypes bearing c.35delG and evaluated the approximate age of c.35delG in Siberia. Methods The carrier frequency of c.35delG was estimated in 122 unrelated hearing individuals living in Western Siberia. For reconstruction of haplotypes bearing c.35delG, polymorphic D13S141, D13S175, D13S1853 flanking the GJB2 gene, and intragenic rs3751385 were genotyped in deaf patients homozygous for c.35delG (n = 24) and in unrelated healthy individuals negative for c.35delG (n = 67) living in Siberia. Results We present updated carrier rates for c.35delG in Russia complemented by new data on c.35delG carrier frequency in Russians living in Western Siberia (4.1%). Two common D13S141-c.35delG-D13S175-D13S1853 haplotypes, 126-c.35delG-105-202 and 124-c.35delG-105-202, were reconstructed in the c.35delG homozygotes from Siberia. Moreover, identical allelic composition of the two most frequent c.35delG haplotypes restricted by D13S141 and D13S175 was established in geographically remote regions: Siberia and Volga-Ural region (Russia) and Belarus (Eastern Europe). Conclusions Distribution of the c.35delG carrier frequency in Russia is characterized by pronounced ethno-geographic specificity with a downward trend from west to east. Comparative analysis of the c.35delG haplotypes supports a common origin of c.35delG in some regions of Russia (Volga-Ural region and Siberia) and in Eastern Europe (Belarus). A rough estimation of the c.35delG age in Siberia (about 4800 to 8100 years ago) probably reflects the early formation stages of the modern European population (including the European part of the contemporary territory of Russia) since the settlement of Siberia by Russians started only at the end of sixteenth century

Different Rates of the SLC26A4-Related Hearing Loss in Two Indigenous Peoples of Southern Siberia (Russia)

Hereditary hearing loss (HL) is known to be highly locus/allelic heterogeneous, and the prevalence of different HL forms significantly varies among populations worldwide. Investigation of region-specific landscapes of hereditary HL is important for local healthcare and medical genetic services. Mutations in the SLC26A4 gene leading to nonsyndromic recessive deafness (DFNB4) and Pendred syndrome are common genetic causes of hereditary HL, at least in some Asian populations. We present for the first time the results of a thorough analysis of the SLC26A4 gene by Sanger sequencing in the large cohorts of patients with HL of unknown etiology belonging to two neighboring indigenous Turkic-speaking Siberian peoples (Tuvinians and Altaians). A definite genetic diagnosis based on the presence of biallelic SLC26A4 mutations was established for 28.2% (62/220) of all enrolled Tuvinian patients vs. 4.3% (4/93) of Altaian patients. The rate of the SLC26A4-related HL in Tuvinian patients appeared to be one of the highest among populations worldwide. The SLC26A4 mutational spectrum was characterized by the presence of Asian-specific mutations c.919-2A>G and c.2027T>A (p.Leu676Gln), predominantly found in Tuvinian patients, and c.2168A>G (p.His723Arg), which was only detected in Altaian patients. In addition, a novel pathogenic variant c.1545T>G (p.Phe515Leu) was found with high frequency in Tuvinian patients. Overall, based on the findings of this study and our previous research, we were able to uncover the genetic causes of HL in 50.5% of Tuvinian patients and 34.5% of Altaian patients

The results of the WES analysis and variants filtering for seven patients.

<p>The results of the WES analysis and variants filtering for seven patients.</p

Prevalence of mutations c.5254G>A (<i>RAI1</i>), c.1111C>G (<i>OTOF</i>), and c.2168A>G (<i>SLC26A4</i>) on the territory of the Altai Republic.

<p>(<b>A</b>) Territorial distribution of individuals homozygous or heterozygous for mutations denoted by fully or half-colored circle, correspondingly: c.5254G>A (<i>RAI1</i>)–by red; c.1111C>G (<i>OTOF</i>)–by blue; c.2168A>G (<i>SLC26A4</i>)–by green. (<b>B</b>) Distribution of studied Altaian patients (n = 93) and Altaian control sample (n = 120) on the territory of the Altai Republic.</p