Biesbrock, K. V.; Haws, S. A.; Cormaty, H.; Sridharan, R.; Denu, J. M.

Central metabolites function as essential co substrates for chromatin modifying enzymes, directly linking cellular metabolism to chromatin regulation. Accordingly, whole cell fluctuations in co substrate availabilities have been shown to promote diverse phenotypes through chromatin dependent mechanisms. There is emerging evidence that metabolic enzymes producing co substrates for chromatin modifying enzymes can exist in the nucleus, suggesting that nucleus-specific metabolite availability regulates chromatin state. Here, we developed CRISPRm (CRISPR metabolite) to assess how nucleus specific metabolic perturbations influence chromatin function. Five dCas9 metabolic enzyme fusions (i.e., dCas9 ACSS2, NMNAT1, MAT2A, GDH, and AHCY) were used to modulate nuclear levels of essential co substrates involved in histone (de)acetylation and (de)methylation reactions. Transient expression of all dCas9 fusions in HEK293T cells induced distinct global changes in gene expression patterns, with dCas9 ACSS2 (acetyl-CoA producing) and NMNAT1 (NAD+ producing) eliciting large opposing changes in gene expression, suggesting transcriptional responses to nuclear acetyl CoA and NAD+ production may be directly facilitated by acetylation or deacetylation reactions, respectively. Targeting dCas9 ACSS2 and NMNAT1 to promoters of select candidate genes revealed enhanced transcriptional modulation. dCas9 ACSS2 upregulated, and dCas9 NMNAT1 downregulated genes showed basal enrichment of H3K9ac, H3K18ac, H3K27ac, H3K4me3, and p300, suggesting these genomic loci reside within epigenetic environments susceptible to fluctuations in acetyl-CoA and NAD+ availability. Of significant genes altered, dCas9-MAT2A (SAM producing) increased expression of 72% whereas dCAS9-GDH (alpha-ketoglutarate producing) decreased expression of 79%. Surprisingly, dCAS9-AHCY (SAH hydrolysis) led to down-regulation of shared genes up-regulated by dCas9-MAT2A. The observations amongst the methylation-specific enzymes revealed unexpected and unique gene-regulatory sensitivities to SAM, SAH and alpha-ketoglutarate. Together, these results demonstrate the utility of CRISPRm in studying nuclear metabolic regulation of transcription and provide strong evidence that perturbations in nuclear co-substrates do not lead to a large mass- action changes in chromatin acetylation/methylation but rather to modulation of select chromatin-modifying enzymes with targeted transcription responses.