Biology subjects

Akhtar, A.

Publications and source records attributed to Akhtar, A..

4 recordsLinked to original sources

bioRxiv2018

Optimisation and validation of hydrogel-based brain tissue clearing shows uniform expansion across anatomical regions and spatial scales

Imaging of fixed tissue is routine in experimental neuroscience, but is limited by the depth of tissue that can be imaged using conventional methods. Optical clearing of brain tissue using hydrogel-based methods (e.g. CLARITY) allows imaging of large volumes of tissue and is rapidly becoming commonplace in the field. However, these methods suffer from a lack of standardised protocols and validation of the effect they have upon tissue morphology. We present a simple and reliable protocol for tissue clearing along with a quantitative assessment of the effect of tissue clearing upon morphology. Tissue clearing caused tissue swelling (compared to conventional methods), but this swelling was shown to be similar across spatial scales and the variation was within limits acceptable to the field. The results of many studies rely upon an assumption of uniformity in tissue swelling, and by demonstrating this quantitatively, research using these methods can be interpreted more reliably.

neuroscience↗

bioRxiv2018

The NSL complex mediated nucleosome landscape is required to maintain transcription fidelity and suppression of transcription noise

Nucleosomal organization at gene promoters is critical for transcription, with a nucleosome-depleted region (NDR) at transcription start sites (TSSs) being required for transcription initiation. How NDR and the precise positioning of the +1 nucleosome is maintained on active genes remains unclear. Here, we report that the Drosophila Non-Specific Lethal (NSL) complex is necessary to maintain this stereotypical nucleosomal organization at promoters. Upon NSL1 depletion, nucleosomes invade the NDRs at TSSs of NSL-bound genes. NSL complex member NSL3 binds to TATA-less promoters in a sequence-dependent manner. The NSL complex interacts with the NURF chromatin remodeling complex and is necessary and sufficient to recruit NURF to target promoters. The NSL complex is not only essential for transcription but is required for accurate TSS selection for genes with multiple TSSs. Further, loss of NSL complex leads to an increase in transcriptional noise. Thus, the NSL complex establishes a canonical nucleosomal organization that enables transcription and determines TSS fidelity.

genomics↗

bioRxiv2017

uvCLAP: a fast, non-radioactive method to identify in vivo targets of RNA-binding proteins

RNA-binding proteins (RBPs) play important and essential roles in eukaryotic gene expression regulating splicing, localization, translation and stability of mRNAs. Understanding the exact contribution of RBPs to gene regulation is crucial as many RBPs are frequently mis-regulated in several neurological diseases and certain cancers. While recently developed techniques provide binding sites of RBPs, they are labor-intensive and generally rely on radioactive labeling of RNA. With more than 1,000 RBPs in a human cell, it is imperative to develop easy, robust, reproducible and high-throughput methods to determine in vivo targets of RBPs. To address these issues we developed uvCLAP (UV crosslinking and affinity purification) as a robust, reproducible method to measure RNA-protein interactions in vivo. To test its performance and applicability we investigated binding of 15 RBPs from fly, mouse and human cells. We show that uvCLAP generates reliable and comparable data to other methods. Unexpectedly, our results show that despite their different subcellular localizations, STAR proteins (KHDRBS1-3, QKI) bind to a similar RNA motif in vivo. Consistently a point mutation (KHDRBS1Y440F) or a natural splice isoform (QKI-6) that changes the respective RBP subcellular localization, dramatically alters target selection without changing the targeted RNA motif. Combined with the knowledge that RBPs can compete and cooperate for binding sites, our data shows that compartmentalization of RBPs can be used as an elegant means to generate RNA target specificity.

molecular biology↗

bioRxiv2017

High-resolution TADs reveal DNA sequences underlying genome organization in flies

Eukaryotic chromatin is partitioned into domains called TADs that are broadly conserved between species and virtually identical among cell types within the same species. Previous studies in mammals have shown that the DNA binding protein CTCF and cohesin contribute to a fraction of TAD boundaries. Apart from this, the molecular mechanisms governing this partitioning remain poorly understood. Using our new software, HiCExplorer, we annotated high-resolution (570 bp) TAD boundaries in flies and identified eight DNA motifs enriched at boundaries. Known insulator proteins bind five of these motifs while the remaining three motifs are novel. We find that boundaries are either at core promoters of active genes or at non-promoter regions of inactive chromatin and that these two groups are characterized by different sets of DNA motifs. Most boundaries are present at divergent promoters of constitutively expressed genes and the gene expression tends to be coordinated within TADs. In contrast to mammals, the CTCF motif is only present on 2% of boundaries in flies. We demonstrate that boundaries can be accurately predicted using only the motif sequences, along with open chromatin, suggesting that DNA sequence encodes the 3D genome architecture in flies. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogeome.ie-freiburg.mpg.de.

genomics↗