Chromatin plays a key role in determining gene expression and cellular states. The basic unit of chromatin is a nucleosome composed of ~147bp of DNA wrapped around a histone core with exposed N-terminal tails that help regulate gene expression programs through their post-translational modifications (PTMs). Facultative heterochromatin inheritance, a type of chromatin containing the repressive trimethylation of histone H3 at lysine 27 (H3K27me3) PTM, is necessary for maintaining cell fate, identity, and plasticity. The parental facultative heterochromatin gene repression is maintained during the S-phase by recycling parental nucleosomes containing H3K27me3 onto the daughter strands of DNA and spreading their PTM onto newly synthesized nucleosomes by the polycomb repressive complex 2 (PRC2). Dr. Escobar found that Nucleophosmin 1 (NPM1), a histone chaperone, assists in facultative heterochromatin domain inheritance through interactions with PRC2 in mouse embryonic stem cells. NPM1 is found mutated in roughly 30% of cases of acute myeloid leukemia (AML). Preliminary data finds a significant presence of cleaved NPM1 product at 20 kDa (p20) within the nucleus of normal hematopoietic stem cells (HSCs), but a lack of this NPM1 modification in AML cell lines. Cathepsin B has been shown to cleave normal NPM1 to produce a fragment at p20. I hypothesize that the lack of cleaved NPM1 product factors into leukemogenesis. To test this hypothesis, I have three aims; Aim 1 identifies the Cathepsin B cleavage site of NPM1 using in vitro cleaving assays; Aim 2 assesses the phenotype of HSCs containing a non-cleavable NPM1 mutant; and Aim 3 monitors the phenotype of AML cell lines when inducibly expressing Cathepsin B. This project allows for future investigations into how NPM1 modifications impact facultative heterochromatin inheritance of HSCs and AML cancer cells.

Chromatin, the complex of DNA-wrapped histone octamers that make up our chromosomes, is decorated with post-translational histone modifications (PTMs) that either increase or decrease transcriptional accessibility. Most regions have either predominantly active or repressive modifications that shape chromatin into euchromatin or heterochromatin, respectively. In addition to euchromatin and heterochromatin, some cells have poised chromatin that is decorated with both permissive and repressive modifications. While much is still unknown about a poised chromatin state, it is thought to permit swift changes in gene expression, which is a feature common in stem cells and lymphoid memory cells. Ultimately, the Escobar lab aims to determine the epigenetic mechanisms involved in maintaining the poised chromatin state of memory CD8+ T cells, and in line with this aim, plans to use a CRISPR-Cas12a biotinylation system to tag and precipitate poised chromatin regions for protein analysis and mechanistic studies. This project details the development and proof of concept of this CRISPR-Cas12a biotinylation system. Using traditional cloning techniques and a one-pot strategy to assemble CRISPR arrays, we will express and purify dCas12a-BirA+gRNA ribonucleoproteins (RNPs), introduce these CRISPR RNPs to mouse embryonic stem cells (mESCs), and perform CUT&RUN to verify effective biotinylation at specific chromatin loci. Preliminary results have demonstrated the successful purification of 5 CRISPR RNPs and a dCas12a-BirA control, as well as verified the presence of these CRISPR RNPs and the biotinylation of H3 upon delivery of this system to mESCs. Upon CUT&RUN analysis, we expect to see biotinylation of H3 at our targeted loci of interest. The completion of this validation step will allow us to apply this technology to any cell of interest, particularly CD8+ T cells, which may support significant insights to the mechanistic determinants of poised chromatin.