Identification of upstream transcription factor binding sites in orthologous genes using mixed Student's t-test statistics

Background: Transcription factor (TF) regulates the transcription of DNA to messenger RNA by binding to upstream sequence motifs. Identifying the locations of known motifs in whole genomes is computationally intensive. Methodology/Principal Findings: This study presents a computational tool, named "Grit", for screening TF-binding sites (TFBS) by coordinating transcription factors to their promoter sequences in orthologous genes. This tool employs a newly developed mixed Student's t-test statistical method that detects high-scoring conserved and non-conserved binding sites among species. The program performs sequence scanning at a rate of 3.2 Mb/s on a quad-core Amazon server and has been benchmarked by the well-established ChIP-Seq datasets, putting Grit amongst the top-ranked TFBS predictors. It marginally outperforms the well-known transcription factor motif scanning tools, Pscan (4.8%) and FIMO (17.8%), in analyzing well-documented ChIP-Atlas human genome Chip-Seq datasets. Significance: Grit is a good alternative to current available motif scanning tools and is publicly available at http://www.thua45.cn/grit under an academic free license.

Ependymoma associated protein Zfta is expressed in immature ependymal cells but is not essential for ependymal development in mice

The fusion protein of uncharacterised zinc finger translocation associated (ZFTA) and effector transcription factor of tumorigenic NF-kB signalling, RELA (ZFTA-RELA), is expressed in more than two-thirds of supratentorial ependymoma (ST-EPN-RELA), but ZFTA’s expression profile and functional analysis in multiciliated ependymal (E1) cells have not been examined. Here, we showed the mRNA expression of mouse Zfta peaks on embryonic day (E) 17.5 in the wholemount of the lateral walls of the lateral ventricle. Zfta was expressed in the nuclei of FoxJ1-positive immature E1 (pre-E1) cells in E18.5 mouse embryonic brain. Interestingly, the transcription factors promoting ciliogenesis (ciliary TFs) (e.g., multicilin) and ZFTA-RELA upregulated luciferase activity using a 5’ upstream sequence of ZFTA in cultured cells. Zftatm1/tm1 knock-in mice did not show developmental defects or abnormal fertility. In the Zftatm1/tm1 E1 cells, morphology, gene expression, ciliary beating frequency and ependymal flow were unaffected. These results suggest that Zfta is expressed in pre-E1 cells, possibly under the control of ciliary TFs, but is not essential for ependymal development or flow. This study sheds light on the mechanism of the ZFTA-RELA expression in the pathogenesis of ST-EPN-RELA: Ciliary TFs initiate ZFTA-RELA expression in pre-E1 cells, and ZFTA-RELA enhances its own expression using positive feedback.

Tandem repeats ubiquitously flank and select translation initiation sites

Evolutionary divergence in cis-regulatory sequences impacts translation initiation sites (TISs). The implication of tandem repeats (TRs) in TIS selection remains elusive for the most part. Here we employed the TIS homology concept to study a possible link between all categories of TRs and TIS selection. Human and 83 other species were selected, and data was extracted on the entire protein-coding genes (n=1,611,368) and transcripts (n=2,730,515) annotated for those species from Ensembl 102. Two different weighing vectors were employed to assign TIS homology, and the results were assessed in 10-fold validation. On average, every TIS was flanked by 1.19 TRs of various categories within the 120 bp upstream sequence. We detected statistically significant excess of non-homologous TISs co-occurring with human-specific TRs, vice versa. We conclude that TRs are abundant cis elements in the upstream sequences of TISs across species, and there is a link between all categories of TRs and TIS selection.

Genome-Wide Identification and Comparison of Cysteine Proteases in the Pollen Coat and Other Tissues in Maize

Cysteine proteases, belonging to the C1-papain family, play a major role in plant growth and development, senescence, and immunity. There is evidence to suggest that pollen cysteine protease (CP) (ZmCP03) is involved in regulating the anther development and pollen formation in maize. However, there is no report on the genome-wide identification and comparison of CPs in the pollen coat and other tissues in maize. In this study, a total of 38 homologous genes of ZmCP03 in maize were identified. Subsequently, protein motifs, conserved domains, gene structures, and duplication patterns of 39 CPs are analyzed to explore their evolutionary relationship and potential functions. The cis-elements were identified in the upstream sequence of 39 CPs, especially those that are related to regulating growth and development and responding to environmental stresses and hormones. The expression patterns of these genes displayed remarked difference at a tissue or organ level in maize based on the available transcriptome data in the public database. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that ZmCP03 was preferably expressed at a high level in maize pollen. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot, immunofluorescence and immunogold electron microscopy all validated the cellular localization of ZmCP03 in both the pollen coat and pollen cytoplasm. In addition, 142 CP genes from Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and cotton (Gossypium hirsutum), together with 39 maize CPs, were retrieved to analyze their evolution by comparing with orthologous genes. The results suggested that ZmCP03 was relatively conservative and stable during evolution. This study may provide a referential evidence on the function of ZmCP03 in pollen development and germination in maize.

Polymorphisms in coding and non-coding regions of rabbit (Oryctolagus cuniculus) myogenin (MyoG) gene

In animal breeding, selection based on growth is very often used, as this trait affects the profitability of animal production. Identification of polymorphisms within the genes affecting the growth process seems to be very important. Therefore, we decided to analyse rabbit myogenin (<em>MyoG</em> gene) for potential polymorphic sites and their association with growth and carcass traits in Termond White (TER), Belgian Giant Grey (BGG) and crossbred New Zealand White×Belgian Giant Grey (NZW×BGG) rabbits. We found three single nucleotide polymorphisms (SNPs) – in 5’ upstream sequence g.68679476 C&gt;T, in exon 1 – silent mutation g.68680096 T&gt;C and g.68680097 G&gt;A resulting in change of GTG triplet (valine) into ATG triplet (methionine). Association analysis showed that GG genotype weaning weight was statistically higher compared to GA in TER population (<em>P</em>=0.005), and that the hind parts for GG genotypes were heavier compared to those of GA (<em>P</em>=0.024), but association analysis of dissectible parts showed this was caused by higher bone weight (<em>P</em>=0.015). For g.68679476 C&gt;T in NZW×BGG population, the CC genotypes for fore (678±35) and hind part (615±29) weights were heavier compared to CT (588±16 and 549±13, respectively); moreover, association analysis of dissectible parts showed that weight of dissectible meat in hind part. Unfortunately, we did not find similar associations for other analysed breeds. For g.68679476 C&gt;T in NZWxBGG musculus longissimus lumborum pH leg after 24 h chilling (pH24L) were statistically lower for CC genotypes compared to CT (<em>P</em>=0.027). For g.68680097 G&gt;A in Termond White population L* value on the hind leg after 24 h chilling (L*24H) was higher for GA genotypes compared to GG (<em>P</em>=0.03), while for g.68679476 C&gt;T for musculus longissimus lumborum L* value after 24 h (L*24L) CC genotypes had higher value compared to CT (<em>P</em>=0.016) in BGG population. Moreover, in BGG population CT genotypes had higher weaning weight compared to CC (<em>P</em>=0.018). Our results show that SNPs within the <em>MyoG </em>gene may influence growth traits in some rabbit breeds, but the evolutionary conserved sequence may not be favourable for changes within coding sequences. For a better understanding thereof, additional analysis is required.

A chromodomain protein mediates heterochromatin-directed piRNA expression

PIWI-interacting RNAs (piRNAs) play significant roles in suppressing transposons, maintaining genome integrity, and defending against viral infections. How piRNA source loci are efficiently transcribed is poorly understood. Here, we show that in Caenorhabditis elegans, transcription of piRNA clusters depends on the chromatin microenvironment and a chromodomain-containing protein, UAD-2. piRNA clusters form distinct focus in germline nuclei. We conducted a forward genetic screening and identified UAD-2 that is required for piRNA focus formation. In the absence of histone 3 lysine 27 methylation or proper chromatin-remodeling status, UAD-2 is depleted from the piRNA focus. UAD-2 recruits the upstream sequence transcription complex (USTC), which binds the Ruby motif to piRNA promoters and promotes piRNA generation. Vice versa, the USTC complex is required for UAD-2 to associate with the piRNA focus. Thus, transcription of heterochromatic small RNA source loci relies on coordinated recruitment of both the readers of histone marks and the core transcriptional machinery to DNA.

Borf: Improved ORF prediction in de-novo assembled transcriptome annotation

ABSTRACTORF prediction in de-novo assembled transcriptomes is a critical step for RNA-Seq analysis and transcriptome annotation. However, current approaches do not appropriately account for factors such as strand-specificity and incompletely assembled transcripts. Strand-specific RNA-Seq libraries should produce assembled transcripts in the correct orientation, and therefore ORFs should only be annotated on the sense strand. Additionally, start site selection is more complex than appreciated as sequences upstream of the first start codon need to be correctly annotated as 5’ UTR in completely assembled transcripts, or part of the main ORF in incomplete transcripts. Both of these factors influence the accurate annotation of ORFs and therefore the transcriptome as a whole. We generated four de-novo transcriptome assemblies of well annotated species as a gold-standard dataset to test the impact strand specificity and start site selection have on ORF prediction in real data. Our results show that prediction of ORFs on the antisense strand in data from stranded RNA libraries results in false-positive ORFs with no or very low similarity to known proteins. In addition, we found that up to 23% of assembled transcripts had no stop codon upstream and in-frame of the first start codon, instead comprising a sequence of upstream codons. We found the optimal length cutoff of these upstream sequences to accurately classify these transcripts as either complete (upstream sequence is 5’ UTR) or 5’ incomplete (transcript is incompletely assembled and upstream sequence is part of the ORF). Here, we present Borf, the better ORF finder, specifically designed to minimise false-positive ORF prediction in stranded RNA-Seq data and improve annotation of ORF start-site prediction accuracy. Borf is written in Python3 and freely available at https://github.com/betsig/borf.

Cis-regulatory elements of the cholinergic gene locus in the silkworm Bombyx mori

ABSTRACTGenes of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are encoded in the same gene locus, called the cholinergic gene locus. They are essential in cholinergic neurons to maintain their functional phenotype. The genomic structure of the cholinergic gene locus is conserved among invertebrates to mammals. However, it is still inconclusive how cholinergic genes express only in cholinergic neurons in insects. In this study, we analyzed the upstream sequence of cholinergic gene locus in the silkworm Bombyx mori to identify specific cis-regulatory regions. We found multiple enhancer regions that are localized within 1 kb upstream of the cholinergic gene locus. The combination of promoter assays using small deletions and bioinformatic analysis among insect species illuminates two conserved sequences in the cis-regulatory region: TGACGTA and CCAAT, which are known as the cAMP response element and CAAT box, respectively. We found that dibutyryl-cAMP, an analog of cAMP, influences the expression of ChAT. Tissue-specific expression analysis of transcriptional factors showed potential candidates that control the cholinergic gene locus expression. Our investigation provides new insight into the regulation mechanism of cholinergic neuron-specific gene machinery in this lepidopteran insect.

The formation of the bacterial RNA polymerase–promoter open complex involves a branched pathway

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)–σ70 holoenzyme to the promoter DNA. This initial RNAP–promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RPO) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RPO and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RPO formation. We find that the RPO pathway is branched, generating RPO complexes with different stabilities. The RNAP cleft loops, and especially the β’ rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond −35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RPO. The mechanistic heterogeneity of the RPO pathway may be a prerequisite for its regulation since such heterogeneity allows the amplification of small promoter sequence or transcription-factor-dependent changes in the free energy profile of the RPO pathway to large differences in transcription efficiency.