Panobinostat Effectively Increases Histone Acetylation and Alters Chromatin Accessibility Landscape in Canine Embryonic Fibroblasts but Does Not Enhance Cellular Reprogramming

Abstract

Robust and reproducible protocols to efficiently reprogram adult canine cells to induced pluripotent stem cells are still elusive. Somatic cell reprogramming requires global chromatin remodeling that is finely orchestrated spatially and temporally. Histone acetylation and deacetylation are key regulators of chromatin condensation, mediated by histone acetyltransferases and histone deacetylases (HDACs), respectively. HDAC inhibitors have been used to increase histone acetylation, chromatin accessibility, and somatic cell reprogramming in human and mice cells. We hypothesized that inhibition of HDACs in canine fibroblasts would increase their reprogramming efficiency by altering the epigenomic landscape and enabling greater chromatin accessibility. We report that a combined treatment of panobinostat (LBH589) and vitamin C effectively inhibits HDAC function and increases histone acetylation in canine embryonic fibroblasts in vitro, with no significant cytotoxic effects. We further determined the effect of this treatment on global chromatin accessibility via Assay for Transposase-Accessible Chromatin using sequencing. Finally, the treatment did not induce any significant increase in cellular reprogramming efficiency. Although our data demonstrate that the unique epigenetic landscape of canine cells does not make them amenable to cellular reprogramming through the proposed treatment, it provides a rationale for a targeted, canine-specific, reprogramming approach by enhancing the expression of transcription factors such as CEBP.

Opening the “Black Box” Underlying Barriers to the Use of Canine Induced Pluripotent Stem Cells: A Narrative Review

“…Reprogramming vectors can be viral or nonviral, but viral vectors have been utilized in most canine reprogramming cases as summarized in Table 1 . To date, vectors used to generate ciPSCs have included unspecified retroviruses [ 2 , 15 , 21 , 33 , 35 ], lentiviruses [ 4 , 5 , 21 , 23 , 28 , 38 ], Sendai viruses [ 19 , 25 , 27 , 29 , 32 ], Venezuelan equine encephalitis (VEE) virus [ 24 ], and episomes [ 40 ].…”

“…3 ) [ 52 , 53 ]. Global changes to chromatin accessibility, resulting from epigenetic changes to these loci, are a key determinant of successful cellular reprogramming [ 2 , 28 , 32 , 53 , 54 ] ( Fig. 3 ).…”

“…In contrast to changes occurring during SCR in mice [ 54 ], alterations to the epigenome during ciPSC generation are yet to be well characterized. Recent studies have investigated chromatin dynamics during ciPSC generation [ 2 ] and the effects of manipulating various epigenetic regulators such as histone deacetylases (HDACs) [ 28 ] and ten-eleven translocation (TET) enzymes [ 32 ] that modulate chromatin accessibility by changing the degree of histone acetylation and DNA methylation, respectively. Other studies have investigated various combinations of small molecule compounds (SMCs) known to influence epigenetic regulators [ 24 , 25 , 29 , 32 , 48 , 58 ].…”

Induced pluripotent stem cells in companion animals: how can we move the field forward?

“…Optimization of iPSC generation has been further pursued in dogs. For instance, Moshref et al ( 99 ) hypothesized that HDAC inhibitors would increase chromatin accessibility and facilitate reprogramming of adult canine cells. These authors found that neither valproic acid nor sodium butyrate effectively inhibited canine HDAC.…”

“…These authors found that neither valproic acid nor sodium butyrate effectively inhibited canine HDAC. On the other hand, panobinostat, another HDAC inhibitor, significantly increased histone acetylation and improved chromatin accessibility but without evidence of increased efficiency of generating iPSCs ( 99 ). Furthermore, Kimura et al found that a cocktail of small molecules including some of the inhibitors mentioned (ROCK inhibitor, MEK inhibitor, GSK3b inhibitor, TGFβ antagonist, forskolin, and ascorbic acid) contributed to efficient generation of canine iPSCs ( 92 ).…”