Pioneer and PRDM transcription factors coordinate bivalent epigenetic states to safeguard cell fate.

Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative-lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF FOXA coordinates with PRDM1 TF to recruit nucleosome remodeling and deacetylation (NuRD) complexes and Polycomb repressive complexes (PRCs), which establish highly occupied, accessible nucleosome conformation with bivalent epigenetic states, thereby preventing precocious and alternative-lineage gene expression during human endoderm differentiation. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to form bivalent enhancers and repress cell differentiation programs in human pluripotent stem cells, suggesting that this may be a common and critical function of pioneer TFs. We propose that pioneer and PRDM TFs coordinate to safeguard cell fate through epigenetic repression mechanisms.

Protocol for establishing inducible CRISPR interference system for multiple-gene silencing in human pluripotent stem cells.

Inducible loss-of-function strategies are crucial for understanding gene function. However, creating inducible, multiple-gene knockout models is challenging and time-consuming. Here, we present a protocol for establishing a doxycycline-inducible CRISPR interference (CRISPRi) system to concurrently silence multiple genes in human induced pluripotent stem cells (hPSCs). We describe the steps for establishing host CRISPRi hPSCs, designing and cloning single-guide RNAs (sgRNAs) into a lentivirus plasmid, and establishing monoclonal CRISPRi hPSC lines transduced with sgRNAs. We also detail the procedures for selecting effective CRISPRi clones. For complete details on the use and execution of this protocol, please refer to Matsui et al.1.

Performance characteristics of a polyclonal enzyme immunoassay for the quantitation of Histoplasma antigen in human urine samples.

An antigen detection assay was prepared with rabbit anti- Histoplasma antibodies to detect and quantitate Histoplasma capsulatum antigen in urine samples. By using a 4-parameter curve fit, the assay calibration ranges from 2 to 1,000 enzyme immunoassay (EIA) units. We compared results for 99 urine samples with those of a reference laboratory, half of which tested positive or equivocal by that reference laboratory. Performance characteristics were further defined by studying the assay linearity, precision, percentage of positive agreement, and percentage of negative agreement. An acceptable correlation with the reference laboratory (R2=0.7174) was obtained with results ranging from less than 2 (negative samples) to 132 EIA units by the new method. Compared with the reference laboratory, the percentage of agreement for positive samples, excluding equivocal samples, was 92% and for negative samples was 98%. Crossreactivity occurred with culture filtrates of Paracoccidioides brasiliensis, Coccidioides immitis, and Blastomyces dermatiditis. No cross-reactivity was observed with Candida albicans or Aspergillus fumigatus culture filtrates. The current EIA for the detection and quantitation of H capsulatum antigen in urine specimens is a valid assay that agrees well with results from an established reference laboratory.