Biology subjects

Ehmsen, K. T.

Publications and source records attributed to Ehmsen, K. T..

3 recordsLinked to original sources

bioRxiv2021

Glucocorticoid receptor collaborates with pioneer factors and AP-1 to execute genome-wide regulation

AO_SCPLOWBSTRACTC_SCPLOWThe glucocorticoid receptor (GR) regulates transcription through binding to specific DNA motifs, particularly at enhancers. While the motif to which it binds is constant across cell types, GR has cell type-specific binding at genomic loci, resulting in regulation of different genes. The presence of other bound transcription factors (TFs) is hypothesized to strongly influence where GR binds. Here, we addressed the roles of other TFs in the glucocorticoid response by comparing changes in GR binding and nascent transcription at promoters and distal candidate cis-regulatory elements (CCREs) in two distinct human cancer cell types. We found that after glucocorticoid treatment, GR binds to thousands of genomic loci that are primarily outside of promoter regions and are potentially enhancers. The majority of these GR binding sites are cell-type specific, and they are associated with pioneer factor binding. A small fraction of GR occupied regions (GORs) displayed increased bidirectional nascent transcription, which is a characteristic of many active enhancers, after glucocorticoid treatment. Non-promoter GORs with increased transcription were specifically enriched for AP-1 binding prior to glucocorticoid treatment. These results support a model of transcriptional regulation in which multiple classes of TFs are required. The pioneer factors increase chromatin accessibility, facilitating the binding of GR and additional factors. AP-1 binding poises a fraction of accessible sites to be rapidly transcribed upon glucocorticoid-induced GR binding. The coordinated activity of multiple TFs then results in cell type-specific changes in gene expression. We anticipate that many models of inducible gene expression also require multiple distinct TFs that act at multiple steps of transcriptional regulation.

genomics↗

bioRxiv2019

Definition of alleles and altered regulatory motifs across Cas9-edited cell populations

BackgroundGenetic alteration of candidate response elements at their native chromosomal loci is the only valid determinant of their potential transcriptional regulatory activities. Targeted DNA cleavage by Cas9 coupled with cellular repair processes can produce arrays of alleles that can be defined by massively parallel sequencing by synthesis (SBS), presenting an opportunity to generate and survey edited cell populations that include informative alterations. Such editing efforts commonly rely on subclonal enrichment to isolate cells with preferred genotypic properties at target loci; short nucleotide adducts (indices/barcodes) allow PCR-amplified molecules from diverse sample sources to be pooled, sequenced, and demultiplexed to resolve source-specific content. Not widely available, however, are capabilities for barcoding thousands of clones, or for automated analysis of individual candidate regulatory loci PCR-amplified and sequenced from a genetically heterogeneous population--specifically, imputation of discrete genotype(s) by allele definition and abundance, and identification of altered regulatory factor binding motifs.\n\nResultsWe describe a panel of 192 8-nucleotide barcode primers compatible with Illumina(R) sequencing platforms, and the application of these barcodes to genotypic analysis of Cas9-edited clones. Permutations of the ninety-six i7 (read 1) and ninety-six i5 (read 2) barcodes allow unique labeling of up to 9,216 distinct samples. We created three independent Python scripts: SampleSheet.py automates construction of Illumina(R) Sample Sheets encoding up to 9,216 barcode:sample relationships; ImputedGenotypes.py defines alleles and imputes genotypes from demultiplexed fastq files; CollatedMotifs.py flags transcription factor recognition motif matches altered in alleles relative to a reference sequence.\n\nConclusionsCode-enabled definition of alleles and regulatory motifs in sequenced, demultiplexed amplicons facilitates evaluation of genetic diversity in up to 9,216 distinct samples. Here, we demonstrate the utility of three scripts in analysis of cell populations targeted by Cas9 for disruption of glucocorticoid receptor (GR) binding sites near FKBP5, a GR-regulated gene in the human adenocarcinoma cell line A549. SampleSheet.py, ImputedGenotypes.py, and CollatedMotifs.py operate independently and are broadly applicable beyond the case described here.

molecular biology↗

bioRxiv2019

Small molecule inhibitors of a human recombination-associated ATPase, RAD54

Homologous recombination (HR) is a principal support pathway for DNA replication and for recovery from DNA breaks and interstrand crosslinks, making it a rational target for inhibition in cancer therapy. The ATPase RAD54 functions in molecular events that promote DNA sequence-preservation during HR-mediated damage repair, including homology search, DNA strand exchange, and transition to DNA repair synthesis within a displacement loop intermediate. We developed a high-throughput biochemical screen to identify small-molecule inhibitors of human RAD54, using a phosphate detection assay to monitor RAD54 ATPase activity in the presence of double-stranded DNA (dsDNA). After filtering potential DNA intercalators and frequent hitters, we identified two chemotypes that reproducibly inhibited RAD54 ATPase in vitro. We evaluated these chemotypes for inhibition of RAD54-dsDNA binding and cancer cell survival. A halogenated carbazole/dihydroacridine scaffold inhibited a panel of SWI2/SNF2-related ATPases but not VCP/p97, an unrelated ATPase. Small molecules that interfere with key steps in HR-- such as inhibitors of RAD54--may expose DNA repair-dependent vulnerabilities in cancer cells.

biochemistry↗