MPRAdecoder: Processing of the Raw MPRA Data With a priori Unknown Sequences of the Region of Interest and Associated Barcodes.

Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of numerous DNA regulatory elements and/or their mutant variants. The assays are based on the construction of reporter plasmid libraries containing two variable parts, a region of interest (ROI) and a barcode (BC), located outside and within the transcription unit, respectively. Importantly, each plasmid molecule in a such a highly diverse library is characterized by a unique BC-ROI association. The reporter constructs are delivered to target cells and expression of BCs at the transcript level is assayed by RT-PCR followed by next-generation sequencing (NGS). The obtained values are normalized to the abundance of BCs in the plasmid DNA sample. Altogether, this allows evaluating the regulatory potential of the associated ROI sequences. However, depending on the MPRA library construction design, the BC and ROI sequences as well as their associations can be a priori unknown. In such a case, the BC and ROI sequences, their possible mutant variants, and unambiguous BC-ROI associations have to be identified, whereas all uncertain cases have to be excluded from the analysis. Besides the preparation of additional "mapping" samples for NGS, this also requires specific bioinformatics tools. Here, we present a pipeline for processing raw MPRA data obtained by NGS for reporter construct libraries with a priori unknown sequences of BCs and ROIs. The pipeline robustly identifies unambiguous (so-called genuine) BCs and ROIs associated with them, calculates the normalized expression level for each BC and the averaged values for each ROI, and provides a graphical visualization of the processed data.

Fine gene expression regulation by minor sequence variations downstream of the polyadenylation signal.

The termination of transcription is a complex process that substantially contributes to gene regulation in eukaryotes. Previously, it was noted that a single cytosine deletion at the position + 32 bp relative to the single polyadenylation signal AAUAAA (hereafter the dC mutation) causes a 2-fold increase in the transcription level of the upstream eGFP reporter in mouse embryonic stem cells. Here, we analyzed the conservation of this phenomenon in immortalized mouse, human and drosophila cell lines and the influence of the dC mutation on the choice of the pre-mRNA cleavage sites. We have constructed dual-reporter plasmids to accurately measure the effect of the dC and other nearby located mutations on eGFP mRNA level by RT-qPCR. In this way, we found that the dC mutation leads to a 2-fold increase in the expression level of the upstream eGFP reporter gene in cultured mouse and human, but not in drosophila cells. In addition, 3' RACE analysis demonstrated that eGFP pre-mRNAs are cut at multiple positions between + 14 to + 31, and that the most proximal cleavage site becomes almost exclusively utilized in the presence of the dC mutation. We also identified new short sequence variations located within positions + 25.. + 40 and + 33.. + 48 that increase eGFP expression up to ~2-4-fold. Altogether, the positive effect of the dC mutation seems to be conserved in mouse embryonic stem cells, mouse embryonic 3T3 fibroblasts and human HEK293T cells. In the latter cells, the dC mutation appears to be involved in regulating pre-mRNA cleavage site selection. Finally, a multiplexed approach is proposed to identify motifs located downstream of cleavage site(s) that are essential for transcription termination.

Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries.

BACKGROUND: Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants. The assays are based on construction of highly diverse plasmid libraries containing two variable fragments, a region of interest (a sequence under study; ROI) and a barcode (BC) used to uniquely tag each ROI, which are separated by a constant spacer sequence. The sequences of BC-ROI combinations present in the libraries may be either known a priori or not. In the latter case, it is necessary to identify these combinations before performing functional experiments. Typically, this is done by PCR amplification of the BC-ROI regions with flanking primers, followed by next-generation sequencing (NGS) of the products. However, chimeric DNA molecules formed on templates with identical spacer fragment during the amplification process may substantially hamper the identification of genuine BC-ROI combinations, and as a result lower the performance of the assays. RESULTS: To identify settings that minimize formation of chimeric products we tested a number of PCR amplification parameters, such as conventional and emulsion types of PCR, one- or two-round amplification strategies, amount of DNA template, number of PCR cycles, and the duration of the extension step. Using specific MPRA libraries as templates, we found that the two-round amplification of the BC-ROI regions with a very low initial template amount, an elongated extension step, and a specific number of PCR cycles result in as low as 0.30 and 0.32% of chimeric products for emulsion and conventional PCR approaches, respectively. CONCLUSIONS: We have identified PCR parameters that ensure synthesis of specific (non-chimeric) products from highly diverse MPRA plasmid libraries. In addition, we found that there is a negligible difference in performance of emulsion and conventional PCR approaches performed with the identified settings.

A toolset to study functions of Cytosolic non-specific dipeptidase 2 (CNDP2) using Drosophila as a model organism.

BACKGROUND: Expression of the CNDP2 gene is frequently up- or down-regulated in different types of human cancers. However, how the product of this gene is involved in cell growth and proliferation is poorly understood. Moreover, our knowledge of the functions of the CNDP2 orthologs in well-established model organisms is scarce. In particular, the function of the D. melanogaster ortholog of CNDP2, encoded by the CG17337 gene (hereafter referred to as dCNDP2), is still unknown. RESULTS: This study was aimed at developing a set of genetic and molecular tools to study the roles of dCNDP2. We generated a dCNDP2 null mutation (hereafter ∆dCNDP2) using CRISPR/Cas9-mediated homologous recombination (HR) and found that the ∆dCNDP2 mutants are homozygous viable, morphologically normal and fertile. We also generated transgenic fly lines expressing eGFP-tagged and non-tagged dCNDP2 protein, all under the control of the UAS promoter, as well as polyclonal antibodies specific to dCNDP2. Using these tools, we demonstrate that only one of the two predicted dCNDP2 isoforms is expressed throughout the different tissues tested. dCNDP2 was detected in both the cytoplasm and the nucleus, and was found to be associated with multiple sites in the salivary gland polytene chromosomes. CONCLUSIONS: The dCNDP2 gene is not essential for fly viability under standard laboratory conditions. The subcellular localization pattern of dCNDP2 suggests that this protein might have roles in both the cytoplasm and the nucleus. The genetic and molecular tools developed in this study will allow further functional characterization of the conserved CNDP2 protein using D. melanogaster as a model system.

The large fraction of heterochromatin in Drosophila neurons is bound by both B-type lamin and HP1a.

BACKGROUND: In most mammalian cell lines, chromatin located at the nuclear periphery is represented by condensed heterochromatin, as evidenced by microscopy observations and DamID mapping of lamina-associated domains (LADs) enriched in dimethylated Lys9 of histone H3 (H3K9me2). However, in Kc167 cell culture, the only Drosophilla cell type where LADs have previously been mapped, they are neither H3K9me2-enriched nor overlapped with the domains of heterochromatin protein 1a (HP1a). RESULTS: Here, using cell type-specific DamID we mapped genome-wide LADs, HP1a and Polycomb (Pc) domains from the central brain, Repo-positive glia, Elav-positive neurons and the fat body of Drosophila third instar larvae. Strikingly, contrary to Kc167 cells of embryonic origin, in neurons and, to a lesser extent, in glia and the fat body, HP1a domains appear to overlap strongly with LADs in both the chromosome arms and pericentromeric regions. Accordingly, centromeres reside closer to the nuclear lamina in neurons than in Kc167 cells. As expected, active gene promoters are mostly not present in LADs, HP1a and Pc domains. These domains are occupied by silent or weakly expressed genes with genes residing in the HP1a-bound LADs expressed at the lowest level. CONCLUSIONS: In various differentiated Drosophila cell types, we discovered the existence of peripheral heterochromatin, similar to that observed in mammals. Our findings support the model that peripheral heterochromatin matures enhancing the repression of unwanted genes as cells terminally differentiate.

Piwi interacts with chromatin at nuclear pores and promiscuously binds nuclear transcripts in Drosophila ovarian somatic cells.

Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.

Drosophila SUUR protein associates with PCNA and binds chromatin in a cell cycle-dependent manner.

Drosophila SUUR (Suppressor of UnderReplication) protein was shown to regulate the DNA replication elongation process in endocycling cells. This protein is also known to be the component of silent chromatin in polyploid and diploid cells. To mark the different cell cycle stages, we used immunostaining patterns of PCNA, the main structural component of replication fork. We demonstrate that SUUR chromatin binding is dynamic throughout the endocyle in Drosophila salivary glands. We observed that SUUR chromosomal localization changed along with PCNA pattern and these proteins largely co-localized during the late S-phase in salivary glands. The hypothesized interaction between SUUR and PCNA was confirmed by co-immunoprecipitation from embryonic nuclear extracts. Our findings support the idea that the effect of SUUR on replication elongation depends on the cell cycle stage and can be mediated through its physical interaction with replication fork.