No abstract

DNA vaccines have demonstrated antitumor efficacy in multiple preclinical models, but low immunogenicity has been observed in several human clinical trials. This has led to many approaches seeking to improve the immunogenicity of DNA vaccines. We previously reported that a DNA vaccine encoding the cancer-testis antigen SSX2, modified to encode altered epitopes with increased MHC class I affinity, elicited a greater frequency of cytolytic, multifunctional CD8+ T cells in non-tumor-bearing mice. In this report we sought to test if this optimized vaccine resulted in increased antitumor activity in mice bearing an HLA-A2-expressing tumor engineered to express SSX2. We found that immunization of tumor-bearing mice with the optimized vaccine elicited a surprisingly inferior antitumor effect relative to the native vaccine. Both native and optimized vaccines led to increased expression of PD-L1 on tumor cells, but antigen-specific CD8+ T cells from mice immunized with the optimized construct expressed higher PD-1. Splenocytes from immunized animals induced PD-L1 expression on tumor cells in vitro. Antitumor activity of the optimized vaccine could be increased when combined with antibodies blocking PD-1 or PD-L1, or by targeting a tumor line not expressing PD-L1. These findings suggest that vaccines aimed at eliciting effector CD8+ T cells, and DNA vaccines in particular, might best be combined with PD-1 pathway inhibitors in clinical trials. This may be particularly advantageous for vaccines targeting prostate cancer, a disease for which antitumor vaccines have demonstrated clinical benefit and yet PD-1 pathway inhibitors alone have shown little efficacy to date.

A gene's position in the genome can profoundly affect its expression because regional differences in chromatin modulate the activity of locally acting cis-regulatory sequences (CRSs). Here we study how CRSs and regional chromatin act in concert on a genome-wide scale. We present a massively parallel reporter gene assay that measures the activities of hundreds of different CRSs, each integrated at many specific genomic locations. Although genome location strongly affected CRS activity, the relative strengths of CRSs were maintained at all chromosomal locations. The intrinsic activities of CRSs also correlatedwith their activities in plasmid-based assays. We explain our data with a quantitative model in which expression levels are set by independent contributions from local CRSs and the regional chromatin environment, rather than by more complex sequenceor protein-specific interactions between these two factors. The methods we present will help investigators determine when regulatory information is integrated in a modular fashion and when regulatory sequences interact in more complex ways.Two types of cis-regulatory information control the production of mRNAs: local cisregulatory CRSs, such as promoters, enhancers and insulators 1-3 , and the regional chromatin surrounding a gene's location in the genome [4][5][6] . Although short CRSs are often sufficient to drive reporter genes in patterns that reflect their activity in the genome [7][8][9][10][11][12] , their activities are also heavily influenced by regional differences in chromatin state. Regional differences in chromatin underlie chromosome position effects, the phenomenon whereby the same gene is expressed at dramatically different levels depending on its location in the genome 6,13-25 .

, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). Many kindergarten through grade 12 (K-12) schools offering in-person learning have adopted strategies to limit the spread of SARS-CoV-2, the virus that causes COVID-19 (1). These measures include mandating use of face masks, physical distancing in classrooms, increasing ventilation with outdoor air, identification of close contacts,* and following CDC isolation and quarantine guidance † (2). A 2-week pilot investigation was conducted to investigate occurrences of SARS-CoV-2 secondary transmission in K-12 schools in the city of Springfield, Missouri, and in St. Louis County, Missouri, during December 7-18, 2020. Schools in both locations implemented COVID-19 mitigation strategies; however, Springfield implemented a modified quarantine policy permitting student close contacts aged ≤18 years who had school-associated contact with a person with COVID-19 and met masking requirements during their exposure to continue in-person learning. § Participating students, teachers, and staff members with COVID-19 (37) from 22 schools and their * A close contact was defined as any person who spent a cumulative total of ≥15 minutes in one 24-hour period within 6 ft of a person with COVID-19 while that person was potentially infectious, regardless of mask use. A person with COVID-19 was considered potentially infectious to others starting from 2 days before symptom onset (or if asymptomatic, 2 days before the collection of the first positive SARS-CoV-2 test specimen) until the person was isolated.

No abstract

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome.

DNA methylation undergoes dynamic changes during development and cell differentiation. Recent genome-wide studies discovered that tissue-specific differentially methylated regions (DMRs) often overlap tissue-specific distal cis-regulatory elements. However, developmental DNA methylation dynamics of the majority of the genomic CpGs outside gene promoters and CpG islands has not been extensively characterized. Here we generate and compare comprehensive DNA methylome maps of zebrafish developing embryos. From these maps we identify thousands of developmental stage-specific DMRs (dsDMR) across zebrafish developmental stages. The dsDMRs contain evolutionarily conserved sequences, are associated with developmental genes, and are marked with active enhancer histone post-translational modifications. Their methylation pattern correlates much stronger than promoter methylation with expression of putative target genes. When tested in vivo using a transgenic zebrafish assay, 20 out of 20 selected candidate dsDMRs exhibit functional enhancer activities. Our data suggest that developmental enhancers are a major target of DNA methylation changes during embryogenesis.

In embryonic stem cells (ESCs), a core transcription factor (TF) network establishes the gene expression program necessary for pluripotency. To address how interactions between four key TFs contribute to cis-regulation in mouse ESCs, we assayed two massively parallel reporter assay (MPRA) libraries composed of binding sites for SOX2, POU5F1 (OCT4), KLF4, and ESRRB. Comparisons between synthetic cis-regulatory elements and genomic sequences with comparable binding site configurations revealed some aspects of a regulatory grammar. The expression of synthetic elements is influenced by both the number and arrangement of binding sites. This grammar plays only a small role for genomic sequences, as the relative activities of genomic sequences are best explained by the predicted occupancy of binding sites, regardless of binding site identity and positioning. Our results suggest that the effects of transcription factor binding sites (TFBS) are influenced by the order and orientation of sites, but that in the genome the overall occupancy of TFs is the primary determinant of activity.