Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single‐molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.

Highlights d Circadian depletion of GCs is required for reactivation of GR target genes d Ultradian and constant hormone treatments result in distinct RNA bursting patterns d The GR dwell time and bound fraction determine RNA burst duration and frequency d Stimulation with GCs induces co-bursting of proximal and distal transcription sites

This study presents performance specifications of an in-house developed human leukocyte antigen (HLA) typing assay using next-generation sequencing (NGS) on the Illumina MiSeq platform. A total of 253 samples, previously characterized for HLA-A, -B, -C, -DRB1 and -DQB1 were included in this study, which were typed at high-resolution using a combination of Sanger sequencing, sequence-specific primer (SSP) and sequence-specific oligonucleotide probe (SSOP) technologies and recorded at the two-field level. Samples were selected with alleles that cover a high percentage of HLA specificities in each of five different race/ethnic groups: European, African-American, Asian Pacific Islander, Hispanic and Native American. Sequencing data were analyzed by two software programs, Omixon's target and GenDx's NGSengine. A number of metrics including allele balance, sensitivity, specificity, precision, accuracy and remaining ambiguity were assessed. Data analyzed by the two software systems are shown independently. The majority of alleles were identical in the exonic sequences (third field) with both programs for HLA-A, -B, -C and -DQB1 in 97.7% of allele determinations. Among the remaining discrepant genotype calls at least one of the analysis programs agreed with the reference typing. Upon additional manual analysis 100% of the 2530 alleles were concordant with the reference HLA genotypes; the remaining ambiguities did not exceed 0.8%. The results demonstrate the feasibility and significant benefit of HLA typing by NGS as this technology is highly accurate, eliminates virtually all ambiguities, provides complete sequencing information for the length of the HLA gene and forms the basis for utilizing a single methodology for HLA typing in the immunogenetics labs.

No abstract

Very little is known about how lipid signaling regulates intima hyperplasia after vascular injury. Herein, we report that deletion and pharmacological inhibition of phospholipase D (PLD)2, which generates the signaling lipid phosphatidic acid (PA), reduced neointimal formation in the mouse carotid artery ligation model. PLD2 deficiency inhibits migration of vascular smooth muscle cells (VSMCs) into the intima in mice as well as migration and formation of membrane ruffles in primary VSMCs. PA specifically binds to the IQ motif–containing guanosine triphosphatase–activating protein 1 (IQGAP1) scaffold protein. The binding between PA and IQGAP is required for the plasma membrane recruitment of IQGAP1. Similar to PLD2 inhibition, knockdown of IQGAP1 blocks ruffle formation and migration in VSMCs, which are rescued by expression of the exogenous IQGAP1 but not the PA binding–deficient mutant. These data reveal that the PLD2‐PA‐IQGAP1 pathway plays an important role in VSMC migration and injury‐induced vascular remodeling, and implicate PLD2 as a candidate target for therapeutic interventions.—Wang, Z., Cai, M., Tay, L. W. R., Zhang, F., Wu, P., Huynh, A., Cao, X., Di Paolo, G., Peng, J., Milewicz, D. M., Du, G. Phosphatidic acid generated by PLD2 promotes the plasma membrane recruitment of IQGAP1 and neointima formation. FASEB J. 33, 6713–6725 (2019). http://www.fasebj.org