{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T12:18:51Z","timestamp":1778674731375,"version":"3.51.4"},"posted":{"date-parts":[[2026,5,10]]},"group-title":"Biochemistry","reference-count":65,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,10]],"date-time":"2026-05-10T00:00:00Z","timestamp":1778371200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH","award":["R35 GM149279"],"award-info":[{"award-number":["R35 GM149279"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  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 (\n                  <jats:italic>i.e<\/jats:italic>\n                  ., 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\n                  <jats:sup>+<\/jats:sup>\n                  producing) eliciting large opposing changes in gene expression, suggesting transcriptional responses to nuclear acetyl-CoA and NAD\n                  <jats:sup>+<\/jats:sup>\n                  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\n                  <jats:sup>+<\/jats:sup>\n                  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.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Highlights<\/jats:title>\n                  <jats:list list-type=\"bullet\">\n                    <jats:list-item>\n                      <jats:p>CRISPRm is a novel, modular dCas9-effector platform that enables interrogation of the metabolism-epigenome axis<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>dCas9-ACSS2, -NMNAT1, -MAT2A, -AHCY, and -GDH induce distinct transcriptional programs.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Targeting CRISPRm to promoters enhances transcriptional responses.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>dCas9-ACSS2 and -NMNAT1 sensitive gene promoters exhibit unique enrichment of chromatin features, including H3K9ac, H3K18ac, H3K27ac, H3K4me3 and p300.<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>","DOI":"10.64898\/2026.05.10.724078","type":"posted-content","created":{"date-parts":[[2026,5,11]],"date-time":"2026-05-11T06:10:13Z","timestamp":1778479813000},"source":"Crossref","is-referenced-by-count":0,"title":["dCas9-metabolic enzyme fusions modulate global and locus-specific gene expression"],"prefix":"10.64898","author":[{"given":"Kellen V.","family":"Biesbrock","sequence":"first","affiliation":[{"name":"Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, 53715, USA"},{"name":"Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Spencer A.","family":"Haws","sequence":"additional","affiliation":[{"name":"Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, 53715, USA"},{"name":"Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Harshini","family":"Cormaty","sequence":"additional","affiliation":[{"name":"Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, 53715, USA"},{"name":"Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, 53792, USA"},{"name":"Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, 53705, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rupa","family":"Sridharan","sequence":"additional","affiliation":[{"name":"Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, 53715, USA"},{"name":"Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, 53792, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9415-0365","authenticated-orcid":false,"given":"John M.","family":"Denu","sequence":"additional","affiliation":[{"name":"Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, 53715, USA"},{"name":"Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA"},{"name":"University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin, Madison, WI 53705, USA"},{"name":"University of Wisconsin Comprehensive Diabetes Center, Madison, WI, 53705, USA"},{"name":"Wisconsin Nathan Shock Center of Excellence in the Basic Biology of Aging, Madison, WI, 53705, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026051304454700000_2026.05.10.724078v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2015.08.024"},{"key":"2026051304454700000_2026.05.10.724078v1.2","doi-asserted-by":"crossref","first-page":"111837","DOI":"10.1016\/j.intimp.2024.111837","article-title":"S-Adenosylmethionine (SAM) diet promotes innate immunity via histone H3K4me3 complex","volume":"131","year":"2024","journal-title":"Int. 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