ABSTRACT
Establishment of the epithelial barrier is a key feature of healthy gut, and barrier disruption is a common
perpetuator of various gastrointestinal disorders, including inflammatory bowel diseases. Two major processes
regulate integrity, disruption, and repair of the gut barrier. One is assembly of epithelial tight junctions (TJ) and
adherens junctions (AJ); the other is intestinal epithelial cell (IEC) migration in normal and injured mucosa.
Remodeling of the actin cytoskeleton drives both TJ/AJ formation and IEC migration. Post-translational
modifications of actin molecules governs reversible assembly of actin monomers into the filaments and higher-
order cytoskeletal structures. Actin methylation on the His73 residue catalyzed by a specific SETD3
methyltransferase is an emerging regulator of cytoskeleton-dependent cellular processes. Roles of SETD3-
driven actin methylation in gut barrier integrity, disruption, and repair remain unexplored. Our preliminary data
demonstrates that SETD3 expression and actin His73 methylation are decreased in IEC isolated from
ulcerative colitis patients. SETD3 depletion in IEC monolayers leads to disruption of the epithelial barrier and
attenuation of epithelial wound healing. Furthermore, SETD3 knockout mice are more sensitive to dextran
sodium sulfate (DSS) induced colitis. This exciting preliminary data provides a strong scientific premise for the
following innovative hypothesis: SETD3-mediated actin methylation is a novel regulator of the intestinal
epithelial barrier assembly and function; decreased SETD3 expression in the inflamed mucosa
enhances leakiness and inhibits repair of the gut barrier.
This hypothesis will be tested in the following Aims: (1) to delineate the roles of SETD3 in establishment of the
intestinal epithelial barrier and assembly of apical junctions; (2) to determine the roles of SETD3 in regulating
intestinal epithelial cell migration; (3) to examine the roles of SETD3 in regulating disruption and restitution of
the intestinal epithelial barrier in vivo. The study will utilize in vitro IEC monolayers, ex vivo primary intestinal
organoids from SETD3-null mice cultured as 2-monolyers or in Gut-on-a-chip devise, as well as in vivo mouse
models of colitis and mucosal repair.
Roles of SETD3 will be examined by a combination of functional (IEC permeability measurements, wound
healing), biochemical (immunoblotting, immunoprecipitation), imaging (confocal microscopy, FRAP analysis,
FRET tension biosensors), and genetic (CRISPR/Cas9 gene editing, SETD3 mutagenesis and overexpression,
knockout mice) approaches. Significance: the proposed study will provide novel insights into the mechanisms
that regulate intestinal epithelial barrier integrity, injury and repair. It will also identify new therapeutic targets to
prevent breakdown and enhance reparation of the gut barrier in patients with digestive diseases
