Biology subjects

Peterson, K. R.

Publications and source records attributed to Peterson, K. R..

3 recordsLinked to original sources

bioRxiv2022

Repurposing the CRISPR-Cas9 System for Targeted Chromatin O-linked β-N- acetylglucosamine Editing

Eukaryotic gene transcription is controlled by many proteins, including the basal transcription machinery, epigenetic chromatin remodeling complexes, and transcription cofactors. Chromatin and genome-mapping consortia identified O-linked {beta}-N-acetylglucosamine (O-GlcNAc) as an abundant chromatin post-translational modification involved in numerous transcriptional processes, including RNA polymerase function, epigenetic dynamics, and transcription factor activity. Thus, O-GlcNAc regulation of cis-regulatory elements is essential for proper gene expression. O-GlcNAc is a single N-acetylglucosamine sugar attached to serine or threonine residues in nuclear, cytoplasmic, or mitochondrial proteins. Two enzymes cycle O-GlcNAc on or off protein; O-GlcNAc transferase (OGT) adds the modification, and O-GlcNAcase (OGA) removes it. O-GlcNAcylation responds to inputs from multiple metabolic and stress pathways including glucose, amino acid, fatty acid, and nucleotide metabolism. Therefore, O-GlcNAc acts as a sensor of cellular homeostasis able to link environmental conditions with gene transcription; however, decoding the precise function of millions of O-GlcNAc regulated elements remains challenging. Technologies to readily manipulate O-GlcNAcylation at specific cis-regulatory elements for functional analysis without pleiotropic consequences are lacking. We have employed novel CRISPR-based gene targeting tools to probe the function of O-GlcNAc regulated cis-elements. First, we developed a programmable CRISPR-Cas9-based targeting system. This was accomplished by fusing a catalytically dead Cas9 (dCas9) to O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA), which allows for highly specific O-GlcNAc manipulation at chromatin cis-regulatory elements. Previously, we demonstrated that O-GlcNAc plays a role in regulating human A{gamma}-globin gene expression by regulating CHD4 function and the formation of the NuRD (Nucleosome Remodeling and Deacetylase) complex at the -566 GATA repressor-binding site. Thus, as a proof of principle and to further explore the function of O-GlcNAc in{gamma} -globin gene transcription, we targeted both dCas9-OGT and -OGA fusion proteins to the A{gamma}-globin gene promoter. When dCas9-OGT or dCas9-OGA was targeted to the -566 GATA silencer site of the A{gamma}-globin promoter, gene expression decreased or increased, respectively. This data strongly correlates with our previous findings and implicates O-GlcNAc cycling in{gamma} -globin gene regulation. Importantly, this method can be employed to investigate O-GlcNAc events known to exist within the eukaryotic genome in a highly specific manner. Together, this tool will be fundamental in elucidating the function of O-GlcNAc in gene transcription.

biochemistry↗

bioRxiv2019

An intergenic non-coding RNA promoter required for histone modifications in the human β-globin chromatin domain

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human {beta}-globin genes. Mutational analyses in {beta}-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult {delta}- and {beta}-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring {varepsilon}- and {gamma}-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of {beta}-like globin gene transcription during development.

genetics↗

bioRxiv2019

Mutational mimics of allosteric effectors: a genome editing design to validate allosteric drug targets

Development of drugs that allosterically regulate enzyme functions to treat disease is a costly venture. Screening mutations that mimic allosteric effectors in vitro will identify therapeutic regulatory targets enhancing the likelihood of developing a disease treatment at a reasonable cost. We demonstrate the potential of this approach utilizing human liver pyruvate kinase (hLPYK) as a model. Inhibition of hLPYK was the first desired outcome of our screen. We identified individual point mutations that: 1) mimicked allosteric inhibition by alanine, 2) mimicked inhibition by protein phosphorylation, and 3) prevented binding of fructose-1,6-bisphosphate (Fru-1,6-BP). Our second desired screening outcome was activation of hLPYK. We identified individual point mutations that: 1) prevented hLPYK from binding alanine, the allosteric inhibitor, 2) prevented inhibitory protein phosphorylation, or 3) mimicked allosteric activation by Fru-1,6-BP. Combining the three activating point mutations produced a constitutively activated enzyme that was unresponsive to regulators. Expression of a mutant hLPYK transgene containing these three mutations in a mouse model was not lethal. Thus, mutational mimics of allosteric effectors will be useful to confirm whether allosteric activation of hLPYK will control glycolytic flux in the diabetic liver to reduce hepatic glucose production and, in turn, reduce or prevent hyperglycemia.

biochemistry↗