Small Molecules Modulate Chromatin Accessibility to Promote NEUROG2-Mediated Fibroblast-to-Neuron Reprogramming

Abstract

SummaryPro-neural transcription factors and small molecules can induce the reprogramming of fibroblasts into functional neurons; however, the immediate-early molecular events that catalyze this conversion have not been well defined. We previously demonstrated that neurogenin 2 (NEUROG2), forskolin (F), and dorsomorphin (D) can reprogram fibroblasts into functional neurons with high efficiency. Here, we used this model to define the genetic and epigenetic events that initiate an acquisition of neuronal identity. We demonstrate that NEUROG2 is a pioneer factor, FD enhances chromatin accessibility and H3K27 acetylation, and synergistic transcription activated by these factors is essential to successful reprogramming. CREB1 promotes neuron survival and acts with NEUROG2 to upregulate SOX4, which co-activates NEUROD1 and NEUROD4. In addition, SOX4 targets SWI/SNF subunits and SOX4 knockdown results in extensive loss of open chromatin and abolishes reprogramming. Applying these insights, adult human glioblastoma cell and skin fibroblast reprogramming can be improved using SOX4 or chromatin-modifying chemicals.

Next‐generation disease modeling with direct conversion: a new path to old neurons

“…Interestingly, while TGF/ALK/SMAD and GSK3b inhibition is thought to destabilize non-neuronal identities and promote neuronal fate-stabilizing signaling [61], forskolin was shown to directly prime chromatin accessibility for more efficient Ngn2-only conversion, a pioneer-enhancing activity previously ascribed exclusively to TFs (Fig. 1D) [33]. Subsequently, it has been shown that additional pathway modulations, including SIRT1 activation and HDAC inhibition, can be applied to increase the efficacy and maturity of the neuronal population obtained [62].…”

“…Chromatin accessibility and transcriptome data have suggested that Zfp238, Sox8, and Dlx3 are among the most important endogenous secondary TF genes downstream of Ascl1 [32]. Some data indicate that Ngn2, when using an appropriate conversion medium, not only binds most of the Ascl1 binding sites in fibroblasts, but also possesses many additional binding sites [30,33]. However, other data suggest that Ascl1 and Ngn2 possess divergent binding patterns that result in distinct chromatin states and different neuronal fates [34].…”

“…Converting cells from one germ layer to another, for instance, from mesoderm (fibroblasts) to ectoderm (neurons), requires substantial chromatin remodeling to make target-cell-type-specific genes accessible to TFs. Such changes of cellular identity and the absence of a defined progenitor cell-type stage indicate that iNs do not follow the typical developmental path from stem cell to postmitotic neuron, but rather represent a jump start of a then self-propelled neuronal program [33,41,48,91,99]. In recent years, several studies performing transcriptomic and epigenomic analysis on converting neurons gave rise to new insights and concepts that characterize fibroblast-toneuron conversion.…”

Dual modulation of neuron‐specific microRNAs and the REST complex promotes functional maturation of human adult induced neurons

“…Ascl1 belongs to the family of the basic helix-loop-helix family, and it specifically binds DNA sequences containing an E-box motif [10]. Also, microRNAs [13][14][15][16] and small molecules [17][18][19][20] have been shown to play a critical role in the conversion process. Many other TFs can also contribute to the reprogramming, such as brain-specific homeobox/POU domain protein 2 (Brn2) that does not access fibroblast chromatin actively on their own but it is recruited by Ascl1 [11,12].…”

“…Many other TFs can also contribute to the reprogramming, such as brain-specific homeobox/POU domain protein 2 (Brn2) that does not access fibroblast chromatin actively on their own but it is recruited by Ascl1 [11,12]. Also, microRNAs [13][14][15][16] and small molecules [17][18][19][20] have been shown to play a critical role in the conversion process. Overexpression of neuron-specific microRNA miR124 triggers a neuronal gene expression profile in a number of different cell types: HeLa cells, human embryonic carcinoma stem cells (NT2), mouse neuronal progenitor cells (N2A), human retinal epithelial cells (ARPE19), and primary mouse embryonic fibroblasts [21].…”