A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes

The study of eukaryotic gene transcription depends on methods to discover distal cis-acting control sequences. Comparative bioinformatics is one powerful strategy to reveal these domains, but still requires conventional wet-bench techniques to elucidate their specificity and function. The DNase I hypersensitivity assay (DHA) is also a method to identify regulatory domains, but can also suggest their function. Technically however, the classical DHA is constrained to mapping gene loci in small increments of ∼20 kb. This limitation hinders efficient and comprehensive analysis of distal gene regions. Here, we report an improved method termed mega-DHA that extends the range of existing DHAs to facilitate assaying intervals that approach 100 kb. We demonstrate its feasibility for efficient analysis of single-copy genes within a large and complex genome by assaying 230 kb of the human ADAMTS14-perforin-paladin gene cluster in four experiments. The results identify distinct networks of regulatory domains specific to expression of perforin and its two neighboring genes

Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs

Activation of naive CD8+ T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) γ expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-γ, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs

In Vivo RNA Interference Screens Identify Regulators of Antiviral CD4+ and CD8+ T Cell Differentiation

SummaryClassical genetic approaches to examine the requirements of genes for T cell differentiation during infection are time consuming. Here we developed a pooled approach to screen 30–100+ genes individually in separate antigen-specific T cells during infection using short hairpin RNAs in a microRNA context (shRNAmir). Independent screens using T cell receptor (TCR)-transgenic CD4+ and CD8+ T cells responding to lymphocytic choriomeningitis virus (LCMV) identified multiple genes that regulated development of follicular helper (Tfh) and T helper 1 (Th1) cells, and short-lived effector and memory precursor cytotoxic T lymphocytes (CTLs). Both screens revealed roles for the positive transcription elongation factor (P-TEFb) component Cyclin T1 (Ccnt1). Inhibiting expression of Cyclin T1, or its catalytic partner Cdk9, impaired development of Th1 cells and protective short-lived effector CTL and enhanced Tfh cell and memory precursor CTL formation in vivo. This pooled shRNA screening approach should have utility in numerous immunological studies

Unraveling the perforin gene in cytotoxic lymphocytes: The transcriptional territory of perforin and elements required for its locus control region

Natural killer (NK) cells and cytotoxic T-lymphocytes (CTL) directly recognize and eliminate virally infected and malignant host cells. The principal machinery they use in this capacity is the focused exocytosis of cytotoxic granules containing the lytic molecule perforin. The regulation of perforin expression during development and activation of NK cells and CTL is determined by transcription of the perforin gene. Nevertheless, 45kb of the human perforin locus does not contain the regulatory sequences sufficient to drive this process physiologically. Consequently, previous studies suggested that even more distal regulatory regions were required for normal perforin gene expression. To identify these potentially distal elements, we developed a new approach to conventional Deoxyribonuclease I hypersensitivity (DHS) assays that facilitates mapping DHS sites across 100kb regions in a single experiment. This method was combined with chromosome transfer to determine the long-range changes that occur to the chromatin structure of perforin and its neighboring genes during its de novo epigenetic and transcriptional activation. To that end, human chromosome 10 was transferred from fibroblasts, where perforin is epigenetically silent, to CTL where mouse perforin is expressed. The transchromosomal perforin gene was activated de novo and expressed at physiological levels in the CTL; this process was not co-regulated with its neighboring genes. Chromatin remodeling occurred across 150kb during epigenetic reprogramming and established a locus-wide domain over the perforin gene that was comprised of 15 DHS sites. We show with transgenes in the CTL model, and in lymphoid cells differentiated from transgenic mouse embryonic stem (ES) cells that this 150kb domain includes a locus control region (LCR), suggesting perforin was controlled physiologically. An engineered deletion in the transgenes and a spontaneous deletion in one transchromosomal perforin allele demonstrates that four distal regulatory sites are required for LCR function. These studies establish the complete network of cis-acting sequences responsible for transcriptional control of the cytolytic gene perforin. They are fundamental for continued dissection of the genetic and epigenetic mechanisms governing development and activation of the cytotoxic program in NK cells and CTL

Evaluating the success of an inpatient PA and NP program through trends in ED consults.

OBJECTIVE: We investigated the effect of an inpatient physician associate/assistant (PA) and NP program on consult volume, length of stay (LOS), and ED returns. METHODS: A retrospective observational study of 4,118 orthopedic ED consults was conducted from January 2017 to March 2022. Univariate statistics were used to evaluate outcomes between cohorts and multivariate regression to evaluate the odds of an LOS of less than 24 hours. RESULTS: After implementation of the PA and NP program, surgeon consults steadily declined and orthopedic consults increased markedly. Statistically significant differences were found in LOS of less than 24 hours and ED arrival-to-discharge time. Adjusting for case mix, patients were 47% more likely to be discharged within 24 hours. Survey results noted that more than 80% of surgeons felt on-call workload, disruptions to clinic and surgical schedules decreased, and quality of care increased. CONCLUSIONS: Implementation of an inpatient PA and NP program reduced orthopedic surgeon consults and hospital LOS while improving surgeon satisfaction with on-call workload, schedule disruptions, and quality of care