Determinants of Disease Penetrance in PRPF31-Associated Retinopathy

Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms.

Elevated Alu retroelement copy number among workers exposed to diesel engine exhaust

BackgroundMillions of workers worldwide are exposed to diesel engine exhaust (DEE), a known genotoxic carcinogen. Alu retroelements are repetitive DNA sequences that can multiply and compromise genomic stability. There is some evidence linking altered Alu repeats to cancer and elevated mortality risks. However, whether Alu repeats are influenced by environmental pollutants is unexplored. In an occupational setting with high DEE exposure levels, we investigated associations with Alu repeat copy number.MethodsA cross-sectional study of 54 male DEE-exposed workers from an engine testing facility and a comparison group of 55 male unexposed controls was conducted in China. Personal air samples were assessed for elemental carbon, a DEE surrogate, using NIOSH Method 5040. Quantitative PCR (qPCR) was used to measure Alu repeat copy number relative to albumin (Alb) single-gene copy number in leucocyte DNA. The unitless Alu/Alb ratio reflects the average quantity of Alu repeats per cell. Linear regression models adjusted for age and smoking status were used to estimate relations between DEE-exposed workers versus unexposed controls, DEE tertiles (6.1–39.0, 39.1–54.5 and 54.6–107.7 µg/m3) and Alu/Alb ratio.ResultsDEE-exposed workers had a higher average Alu/Alb ratio than the unexposed controls (p=0.03). Further, we found a positive exposure–response relationship (p=0.02). The Alu/Alb ratio was highest among workers exposed to the top tertile of DEE versus the unexposed controls (1.12±0.08 SD vs 1.06±0.07 SD, p=0.01).ConclusionOur findings suggest that DEE exposure may contribute to genomic instability. Further investigations of environmental pollutants, Alu copy number and carcinogenesis are warranted.

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.

The Epithelial adhesin 1 tandem repeat region mediates protein display through multiple mechanisms

ABSTRACT The pathogenic yeast Candida glabrata is reliant on a suite of cell surface adhesins that play a variety of roles necessary for transmission, establishment and proliferation during infection. One particular adhesin, Epithelial Adhesin 1 [Epa1p], is responsible for binding to host tissue, a process which is essential for fungal propagation. Epa1p structure consists of three domains: an N-terminal intercellular binding domain responsible for epithelial cell binding, a C-terminal GPI anchor for cell wall linkage and a serine/threonine-rich linker domain connecting these terminal domains. The linker domain contains a 40-amino acid tandem repeat region, which we have found to be variable in repeat copy number between isolates from clinical sources. We hypothesized that natural variation in Epa1p repeat copy may modulate protein function. To test this, we recombinantly expressed Epa1p with various repeat copy numbers in S. cerevisiae to determine how differences in repeat copy number affect Epa1p expression, surface display and binding to human epithelial cells. Our data suggest that repeat copy number variation has pleiotropic effects, influencing gene expression, protein surface display and shedding from the cell surface of the Epa1p adhesin. This study serves to demonstrate repeat copy number variation can modulate protein function through a number of mechanisms in order to contribute to pathogenicity of C. glabrata.

Variation in repeat copy number of the Epithelial adhesin 1 tandem repeat region leads to variable protein display through multiple mechanisms

The pathogenic yeast Candida glabrata is reliant on a suite of cell surface adhesins that play a variety of roles necessary for transmission, establishment, and proliferation during infection. One particular adhesin, Epithelial Adhesin 1 [Epa1p], is responsible for binding to host tissue, a process which is essential for fungal propagation. Epa1p structure consists of three domains: an N-terminal intercellular binding domain responsible for epithelial cell binding, a C-terminal GPI anchor for cell wall linkage, and a serine / threonine-rich linker domain connecting these terminal domains. The linker domain contains a 40-amino acid tandem repeat region, which we have found to be variable in repeat copy number between isolates from clinical sources. We hypothesized that natural variation in Epa1p repeat copy may modulate protein function. To test this, we recombinantly expressed Epa1p with various repeat copy numbers in S. cerevisiae to determine how differences in repeat copy number affect Epa1p expression, surface display, and binding to human epithelial cells. Our data suggest that repeat copy number variation has pleiotropic effects, influencing gene expression, protein surface display, shedding from the cell surface, and host tissue adhesion of the Epa1p adhesin. Understanding these links between repeat copy number variants and mechanisms of infection provide new understanding of the variety of roles of repetitive proteins contribute to pathogenicity of C. glabrata.

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

The adjustment of transcription and translation rates to variable needs is of utmost importance for the fitness and survival of living cells. We have previously shown that the global transcription rate for RNA polymerase II is regulated differently in cells presenting symmetrical or asymmetrical cell division. The budding yeast Saccharomyces cerevisiae adopts a particular strategy to avoid that the smaller daughter cells increase their total mRNA concentration with every generation. The global mRNA synthesis rate lowers with a growing cell volume, but global mRNA stability increases. In this paper, we address what the solution is to the same theoretical problem for the RNA polymerase I synthesis rate. We find that the RNA polymerase I synthesis rate strictly depends on the copy number of its 35S rRNA gene. For cells with larger cell sizes, such as a mutant cln3 strain, the rDNA repeat copy number is increased by a mechanism based on a feed-back mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of the rRNA genes at the rDNA locus in a volume-dependent manner.

Tafsir Gender Jawa: Telaah Tafsir Al-Iklil Fi Ma’ani Al-Tanzil Karya Misbah Mustafa

Penelitian ini dilatarbelakangi oleh adanya sebuah pemikiran tafsir dalam tradisi Jawa yang menempatkan perempuan dalam posisi yang lebih banyak bergerak di wilayah domestik. Karena persepsi mufassir yang melihat kedudukan laki-laki lebih berpotensi daripada perempuan, maka perempuan kurang diberi ruang dalam sektor publik. Di dalam salah satu karya tafsir dari tradisi Jawa yakni al-Iklil fi Ma’ani al-Tanzil karya Misbah Mustafa, ditemukan pemahaman bahwa peran laki-laki lebih utama daripada perempuan melalui serangkaian tafsir terhadap ayat-ayat gender. Melalui analisis struktur sosial terhadap beberapa tema gender seperti asal-usul penciptaan manusia, poligami, dan kepemimpinan laki-laki ataupun perempuan, pemikiran Misbah Mustafa terpola dengan jelas. Dengan menggunakan metode deskriptif-analitis, hasil penelitian menunjukkan bahwa pemikiran Misbah Mustafa dalam tafsir al-Iklil cenderung mengulang-ulang, menukil dan melegitimasi pendapat para ulama tradisional normatif yang kebanyakan mensubordinasikan kedudukan perempuan. Tulisan ini merefleksikan ke-arah bagaimana konstruksi sosial dan budaya mempengaruhi pola penafsiran Misbah Mustafa dalam karyanya tafsir al-Iklil.[This research is motivated by an interpretive thought in the Javanese tradition that places women in a position that is more engaged in domestic sphere. Because the perception of mufassir who see the position of men has more potential than women, women are less given space in the public sector. In one of the interpretations of Javanese tradition, al-Iklil fi Ma’ani al-Tanzil by Misbah Mustafa, it is found that the role of men is more important than women through a series of interpretations of gender verses. Through the analysis of social structure on several gender themes such as the origin of human creation, polygamy, and male or female leadership, Misbah Mustafa’s thoughts were clearly patterned. By using a descriptive-analytical method, the results of the study show that Misbah Mustafa’s thinking in the interpreting the Quran tends to repeat, copy and legitimize the opinions of traditional normative scholars who mostly subordinate the position of women. This paper also shows how social and cultural construction influence Misbah Mustafa’s interpretation patterns in his work, al-Iklil.]

Isw2 and Ino80 chromatin remodeling factors regulate chromatin, replication, and copy number at the yeast ribosomal DNA locus

In the budding yeast Saccharomyces cerevisiae, ribosomal RNA genes are encoded in a highly repetitive tandem array referred to as the ribosomal DNA (rDNA) locus. The yeast rDNA is the site of a diverse set of DNA-dependent processes, including transcription of ribosomal RNAs by RNA Polymerases I and III, transcription of non-coding RNAs by RNA Polymerase II, DNA replication initiation, replication fork blocking, and recombination-mediated regulation of rDNA repeat copy number. All of this takes place in the context of chromatin, but relatively little is known about the roles played by ATP-dependent chromatin remodeling factors at the yeast rDNA. In this work, we report that the Isw2 and Ino80 chromatin remodeling factors are targeted to this highly repetitive locus. We characterize for the first time their function in modifying local chromatin structure, finding that loss of these factors affects the occupancy of nucleosomes in the 35S ribosomal RNA gene and the positioning of nucleosomes flanking the ribosomal origin of replication. In addition, we report that Isw2 and Ino80 promote efficient firing of the ribosomal origin of replication and facilitate the regulated increase of rDNA repeat copy number. This work significantly expands our understanding of the importance of ATP-dependent chromatin remodeling for rDNA biology.Author SummaryTo satisfy high cellular demand for ribosomes, genomes contain many copies of the genes encoding the RNA components of ribosomes. In the budding yeast Saccharomyces cerevisiae, these ribosomal RNA genes are located in the “ribosomal DNA locus”, a highly repetitive array that contains approximately 150 copies of the same unit, in contrast to the single copies that suffice for most genes. This repetitive quality creates unique regulatory needs. Chromatin structure, the packaging and organization of DNA, is a critical determinant of DNA-dependent processes throughout the genome. ATP-dependent chromatin remodeling factors are important regulators of chromatin structure, and yet relatively little is known about how members of this class of protein affect DNA organization or behavior at the rDNA. In this work, we show that the Isw2 and Ino80 chromatin remodeling factors regulate two features of chromatin structure at the rDNA, the occupancy and the positioning of nucleosomes. In addition, we find that these factors regulate two critical processes that function uniquely at this locus: DNA replication originating from within the rDNA array, and the regulated increase of rDNA repeat copy number.