Linden Gearing

edgeR: a versatile tool for the analysis of shRNA-seq and CRISPR-Cas9 genetic screens

Pooled library sequencing screens that perturb gene function in a high-throughput manner are becoming increasingly popular in functional genomics research. Irrespective of the mechanism by which loss of function is achieved, via either RNA interference using short hairpin RNAs (shRNAs) or genetic mutation using single guide RNAs (sgRNAs) with the CRISPR-Cas9 system, there is a need to establish optimal analysis tools to handle such data. Our open-source processing pipeline in edgeR provides a complete analysis solution for screen data, that begins with the raw sequence reads and ends with a ranked list of candidate genes for downstream biological validation. We first summarize the raw data contained in a fastq file into a matrix of counts (samples in the columns, genes in the rows) with options for allowing mismatches and small shifts in sequence position. Diagnostic plots, normalization and differential representation analysis can then be performed using established methods to prioritize results in a statistically rigorous way, with the choice of either the classic exact testing methodology or generalized linear modeling that can handle complex experimental designs. A detailed users’ guide that demonstrates how to analyze screen data in edgeR along with a point-and-click implementation of this workflow in Galaxy are also provided. The edgeR package is freely available from http://www.bioconductor.org.

Transcriptional regulators Myb and BCL11A interplay with DNA methyltransferase 1 in developmental silencing of embryonic and fetal β-like globin genes

The clinical symptoms of hemoglobin disorders such as β‐thalassemia and sickle cell anemia are significantly ameliorated by the persistent expression of γ‐globin after birth. This knowledge has driven the discovery of important regulators that silence γ‐globin postnatally. Improved understanding of the γ‐ to β‐globin switching mechanism holds the key to devising targeted therapies for β‐hemoglobinopathies. To further investigate this mechanism, we used the murine erythroleukemic (MEL) cell line containing an intact 183‐kb human β‐globin locus, in which the Gγ‐ and β‐globin genes are replaced by DsRed and eGFP fluorescent reporters, respectively. Following RNA interference (RNAi)‐mediated knockdown of two key transcriptional regulators, Myb and BCL11A, we observed a derepression of γ‐globin, measured by DsRed fluorescence and qRT‐PCR (P < 0.001). Interestingly, double knockdown of Myb and DNA methyltransferase 1 (DNMT1) resulted in a robust induction of ε‐globin, (up to 20% of total β‐like globin species) compared to single knockdowns (P<0.001). Conversely, double knockdowns of BCL11A and DNMT1 enhanced γ‐globin expression (up to 90% of total β‐like globin species) compared to single knockdowns (P<0.001). Moreover, following RNAi treatment, expression of human β‐like globin genes mirrored the expression levels of their endogenous murine counterparts. These results demonstrate that Myb and BCL11A cooperate with DNMT1 to achieve developmental repression of embryonic and fetal β‐like globin genes in the adult erythroid environment.—Roosjen, M., McColl, B., Kao, B., Gearing, L. J., Blewitt, M. E., Vadolas, J. Transcriptional regulators Myb and BCL11A interplay with DNA methyltransferase 1 in developmental silencing of embryonic and fetal β‐like globin genes. FASEB J. 28, 28–1610 (1620). www.fasebj.org

Jarid2 regulates hematopoietic stem cell function by acting with polycomb repressive complex 2

Polycomb repressive complex 2 (PRC2) plays a key role in hematopoietic stem and progenitor cell (HSPC) function. Analyses of mouse mutants harboring deletions of core components have implicated PRC2 in fine-tuning multiple pathways that instruct HSPC behavior, yet how PRC2 is targeted to specific genomic loci within HSPCs remains unknown. Here we use short hairpin RNA-mediated knockdown to survey the function of PRC2 accessory factors that were defined in embryonic stem cells (ESCs) by testing the competitive reconstitution capacity of transduced murine HSPCs. We find that, similar to the phenotype observed upon depletion of core subunit Suz12, depleting Jarid2 enhances the competitive transplantation capacity of both fetal and adult mouse HSPCs. Furthermore, we demonstrate that depletion of JARID2 enhances the in vitro expansion and in vivo reconstitution capacity of human HSPCs. Gene expression profiling revealed common Suz12 and Jarid2 target genes that are enriched for the H3K27me3 mark established by PRC2. These data implicate Jarid2 as an important component of PRC2 that has a central role in coordinating HSPC function.

An in vivo model for analysis of developmental erythropoiesis and globin gene regulation

Expression of fetal γ‐globin in adulthood ameliorates symptoms of β‐hemoglobinopathies by compensating for the mutant β‐globin. Reactivation of the silenced γ‐globin gene is therefore of substantial clinical interest. To study the regulation of γ‐globin expression, we created the GG mice, which carry an intact 183‐kb human β‐globin locus modified to express enhanced green fluorescent protein (eGFP) from the Gγ‐globin promoter. GG embryos express eGFP first in the yolk sac blood islands and then in the aorta–gonad mesonephros and the fetal liver, the sites of normal embryonic hematopoiesis. eGFP expression in erythroid cells peaks at E9.5 and then is rapidly silenced (>95%) and maintained at low levels into adulthood, demonstrating appropriate developmental regulation of the human β‐globin locus. In vitro knockdown of the epigenetic regulator DNA methyltransferase‐1 in GG primary erythroid cells increases the proportion of eGFP+ cells in culture from 41.9 to 74.1%. Furthermore, eGFP fluorescence is induced > 3‐fold after treatment of erythroid precursors with epigenetic drugs known to induce γ‐globin expression, demonstrating the suitability of the Gγ‐globin eGFP reporter for evaluation of γ‐globin inducers. The GG mouse model is therefore a valuable model system for genetic and pharmacologic studies of the regulation of the β‐globin locus and for discovery of novel therapies for the P‐hemoglobinopathies.—McColl, B., Kao, B. R., Lourthai, P., Chan, K., Wardan, H., Roosjen, M., Delagneau, O., Gearing, L. J., Blewitt, M. E., Svasti, S., Fucharoen, S., Vadolas, J. An in vivo model for analysis of developmental erythropoiesis and globin gene regulation. FASEB J. 28, 2306–2317 (2014). www.fasebj.org