Lavelle, D. | Search bioRxiv

bioRxiv2023

Effect of the LSD1 Inhibitor RN-1 on gamma;-globin and Global Gene Expression during Erythroid Differentiation in Baboons (Papio anubis)

Increased levels of Fetal Hemoglobin reduce the severity of symptoms of patients with sickle cell disease and decrease the risk of death. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the {gamma}-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on {gamma}-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for {gamma}-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.

molecular biology↗

bioRxiv2019

The alternative reality of plant mitochondrial DNA

Plant mitochondrial genomes are usually assembled and displayed as circular maps based on the widely-held assumption that circular genome molecules are the primary form of mitochondrial DNA, despite evidence to the contrary. Many plant mitochondrial genomes have one or more pairs of large repeats that can act as sites for inter- or intramolecular recombination, leading to multiple alternative genomic arrangements (isoforms). Most mitochondrial genomes have been assembled using methods that were unable to capture the complete spectrum of isoforms within a species, leading to an incomplete inference of their structure and recombinational activity. To document and investigate underlying reasons for structural diversity in plant mitochondrial DNA, we used long-read (PacBio) and short-read (Illumina) sequencing data to assemble and compare mitochondrial genomes of domesticated (Lactuca sativa) and wild (L. saligna and L. serriola) lettuce species. This allowed us to characterize a comprehensive, complex set of isoforms within each species and to compare genome structures between species. Physical analysis of L. sativa mtDNA molecules by fluorescence microscopy revealed a variety of linear, branched linear, and circular structures. The mitochondrial genomes for L. sativa and L. serriola were identical in sequence and arrangement, and differed substantially from L. saligna, indicating that the mitochondrial genome structure did not change during domestication. From the isoforms evident in our data, we inferred that recombination occurs at repeats of all sizes at variable frequencies. The differences in genome structure between L. saligna and the two other lettuce species can be largely explained by rare recombination events that rearrange the structure. Our data demonstrate that representations of plant mitochondrial DNA as simple, genome-sized circular molecules are not accurate descriptions of their true nature and that in reality plant mitochondrial DNA is a complex, dynamic mixture of forms. Data AvailabilityBioProject: Organellar genomes of cultivated and wild lettuce (Lactuca) varieties PRJNA508811 https://www.ncbi.nlm.nih.gov/bioproject/508811 and other accessions as indicated through the text and supplemental data. FundingNSF grant MCB-1413152 to ACC and support from UC Davis to RWM.

genomics↗