Evaluation of genetic demultiplexing of single-cell sequencing data from model species.

Single-cell sequencing (sc-seq) provides a species agnostic tool to study cellular processes. However, these technologies are expensive and require sufficient cell quantities and biological replicates to avoid artifactual results. An option to address these problems is pooling cells from multiple individuals into one sc-seq library. In humans, genotype-based computational separation (i.e., demultiplexing) of pooled sc-seq samples is common. This approach would be instrumental for studying non-isogenic model organisms. We set out to determine whether genotype-based demultiplexing could be more broadly applied among species ranging from zebrafish to non-human primates. Using such non-isogenic species, we benchmark genotype-based demultiplexing of pooled sc-seq datasets against various ground truths. We demonstrate that genotype-based demultiplexing of pooled sc-seq samples can be used with confidence in several non-isogenic model organisms and uncover limitations of this method. Importantly, the only genomic resource required for this approach is sc-seq data and a de novo transcriptome. The incorporation of pooling into sc-seq study designs will decrease cost while simultaneously increasing the reproducibility and experimental options in non-isogenic model organisms.

Characterizing Primary transcriptional responses to short term heat shock in paired fraternal lymphoblastoid lines with and without Down syndrome.

Heat shock stress induces genome wide changes in transcription regulation, activating a coordinated cellular response to enable survival. Using publicly available transcriptomic and proteomic data sets comparing individuals with and without trisomy 21, we noticed many heat shock genes are up-regulated in blood samples from individuals with trisomy 21. Yet no major heat shock response regulating transcription factor is encoded on chromosome 21, leaving it unclear why trisomy 21 itself would cause a heat shock response, or how it would impact the ability of blood cells to subsequently respond when faced with heat shock stress. To explore these issues in a context independent of any trisomy 21 associated co-morbidities or developmental differences, we characterized the response to heat shock of two lymphoblastoid cell lines derived from brothers with and without trisomy 21. To carefully compare the chromatin state and the transcription status of these cell lines, we measured nascent transcription, chromatin accessibility, and single cell transcript levels in the lymphoblastoid cell lines before and after acute heat shock treatment. The trisomy 21 cells displayed a more robust heat shock response after just one hour at 42°C than the matched disomic cells. We suggest multiple potential mechanisms for this increased heat shock response in lymphoblastoid cells with trisomy 21 including the possibility that cells with trisomy 21 may exist in a hyper-reactive state due to chronic stresses. Whatever the mechanism, abnormal heat shock response in individuals with Down syndrome may hobble immune responses during fever and contribute to health problems in these individuals.

Lessons from eRNAs: understanding transcriptional regulation through the lens of nascent RNAs.

Nascent transcription assays, such as global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), have uncovered a myriad of unstable RNAs being actively produced from numerous sites genome-wide. These transcripts provide a more complete and immediate picture of the impact of regulatory events. Transcription factors recruit RNA polymerase II, effectively initiating the process of transcription; repressors inhibit polymerase recruitment. Efficiency of recruitment is dictated by sequence elements in and around the RNA polymerase loading zone. A combination of sequence elements and RNA binding proteins subsequently influence the ultimate stability of the resulting transcript. Some of these transcripts are capable of providing feedback on the process, influencing subsequent transcription. By monitoring RNA polymerase activity, nascent assays provide insights into every step of the regulated process of transcription.

Heat Shock Causes a Reversible Increase in RNA Polymerase II Occupancy Downstream of mRNA Genes, Consistent with a Global Loss in Transcriptional Termination.

Cellular transcriptional programs are tightly controlled but can profoundly change in response to environmental challenges or stress. Here we describe global changes in mammalian RNA polymerase II (Pol II) occupancy at mRNA genes in response to heat shock and after recovery from the stress. After a short heat shock, Pol II occupancy across thousands of genes decreased, consistent with widespread transcriptional repression, whereas Pol II occupancy increased at a small number of genes in a manner consistent with activation. Most striking, however, was loss of the Pol II peak near the 3' ends of mRNA genes, coupled to a gain in polymerase occupancy extending tens of kilobases downstream of 3' ends. Typical patterns of 3' end occupancy were largely restored 60 min after cells returned to normal growth temperatures. These changes in polymerase occupancy revealed a heat shock-induced loss of normal termination, which was potent, global, and reversible. The occupancy of the termination factor CPSF73 at the 3' ends of representative genes was reduced after heat shock, suggesting a mechanism for impaired termination. The data support a model in which heat shock induces widespread repression of transcriptional initiation and loss of transcription termination, which reverses as cells return to homeostasis.

Biochemical characterization of an isoprene synthase from Campylopus introflexus (heath star moss).

Each year, plants emit terragram quantities of the reactive hydrocarbon isoprene (2-methyl-1,3-butadiene) into the earth's atmosphere. In isoprene-emitting plants, the enzyme isoprene synthase (ISPS) catalyzes the production of isoprene from the isoprenoid intermediate dimethylallyl diphosphate (DMADP). While isoprene is emitted from all major classes of land plants, to date ISPSs from angiosperms only have been characterized. Here, we report the identification and initial biochemical characterization of a DMADP-dependent ISPS from the isoprene-emitting bryophyte Campylopus introflexus (heath star moss). The partially-purified C. introflexus ISPS (CiISPS) exhibited a Km for DMADP of 0.37 ± 0.28 mM, a pH optimum of 8.6 ± 0.5, and a temperature optimum of 40 ± 3 °C in vitro. Like ISPSs from angiosperms, the CiISPS required the presence of a divalent cation. However, unlike angiosperm ISPSs, the CiISPS utilized Mn(2+) preferentially over Mg(2+). Efforts are currently underway in our laboratory to further purify the CiISPS and clone the cDNA sequence encoding this novel enzyme. Our discovery of the first bryophyte ISPS paves the way for future studies concerning the evolutionary origins of isoprene emission in land plants and may help generate new bryophyte model systems for physiological and biochemical research on plant isoprene function.