Characterization And Function Of CTCF Mediated Chromatin Interactions During MyoD Induced Transdifferentiation

Cellular transdifferentiation is a type of reprogramming,a process in which one type of terminally differentiated cell is directly transformed into another,and a phenomenon that existed originally in tissue regeneration.Current researches obtained target cells in one step through over-expression of a factor or the addition of small molecule compounds in vitro,which provides a theoretical basis for understanding cell fate decisions and seed cells for artificially grown meat production.Therefore,how to accomplish cellular transdifferentiation efficiently in vitro is an urgent issue to be addressed.Cellular transdifferentiation occurs when changes in gene transcription levels.In fact,the regulation of gene expression is closely related to epigenetic modification and chromatin conformation,and the interaction of these two is the driving force in determining cell fate.Here we employ MyoD-induced transdifferentiation of fibroblasts as a model to illustrate the epigenetic obstacles and from which we can find strategies to enhance the generation of target muscle cells.In this study,we collected HiChIP,ChIP-Seq and RNA-Seq data from different cells and analyzed the characteristics of histone modification,MyoD and CTCF mediated chromatin structure.The cells were divided into six cell populations according to their status and treatment conditions:fibroblast,fibroblast＿control,fibroblast＿MyoD,myoblast,fibroblast＿MyoD shCon and fibroblast＿MyoD shCTCF.We explored the epigenetic changes during transdifferentiation by integrated analysis of the three kinds of data and finally found a method to facilitate the transdifferentiation efficiency through rearrangement of chromatin conformation and affects multiple gene expression.This approach is more convenient and widely applicable than the previous approach that tested one gene alone and provide a new strategy to improve the efficiency of cellular reprogramming.The results are as follows:1.MyoD induces incomplete reprogramming and myogenic differentiationTo induce reprogramming in fibroblasts,we established a MyoD-inducible in vitro transdifferentiation system.But in comparison to C2C12 murine myoblasts,the myogenic differentiation efficiency is much lower and numerous genes which are refractory to reprogramming remain unchanged in transcription in MyoD transformed fibroblasts indicating incomplete reprogramming.2.MyoD-induced transition of chromatin states during transdifferentiationChIP-seq of three histone markers(H3K27ac,H3K4me3 and H3K27me3)were performed in fibroblast and fibroblast＿MyoD cells and the chromatin states were identified as active,repressive and not determined(ND).Overall,the global chromatin appears to be activated during transdifferentiation as shown by a modest shift towards the active chromatin distributions while the repressive and ND regions were decreased.Interestingly,there is an activation of substantial portion of repressive and ND regions after MyoD binding and following myogenic genes were activated.Among the active state,enhancers identified by H3K27ac ChIP-Seq uncovered that enhancer activation is associated with the upregulation of adjacent genes during transdifferentiation.In addition,we found the changes in histone modifications had an effect on CTCF binding that with H3K4me3 and H3K27ac modifications were more likely to be lost.3.MyoD directly established chromatin interactions to drive the myogenic transdifferentiationWe carried out BL-HiChIP experiments in fibroblast＿MyoD cells to specifically identify MyoD-mediated chromatin loops using the flag antibody.The majority of MyoD-mediated loops were enhancer-enhancer interactions(52%).We found that the alterations of expression level in MyoD＿enhancer genes were significantly higher than that of MyoD＿promoter genes,indicating MyoD-mediated chromatin interactions as a major driver for myogenic gene activation rather than the direct promoter binding induced transcription.We also found examples showing the MyoD-mediated chromatin loops directly tether distal enhancer to the gene promoter and thus contributing to myogenic gene activation during transdifferentiation.They are Has2,Ca3 and their associated enhancers.In addition,we also demonstrated that MyoD and CTCF can be involved in the formation of chromatin interactions as co-binding factors during transdifferentiation.4.CTCF anchored loops control the cell-specific genes during transdifferentiationWe found that about 50% CTCF-mediated chromatin loops were silenced in fibroblasts,whereas in fibroblast＿MyoD cells,40% CTCF-mediated chromatin loops were newly established.Further analysis of the relationship between dynamic loops and gene expression revealed that CTCF anchored loops control the cell-specific genes.We selected the most upregulated genes and performed loss-of-function experiment by knockout the CTCF binding sites on the corresponding loop anchors verifying that the gained CTCF-mediated chromatin interactions favor the reprogramming,assessed by RT-qPCR,3C-qPCR and immunofluorescence.In addition,we presented two genes associated with the loss of CTCF loops,FBN2 and P3h2,and it is the loss of CTCF loops that leads to decreased gene expression.5.Temporal inhibition of CTCF reassembles the chromatin interactions and facilitates cell transdifferentiationWe adopted the temporal adenovirus shRNA knockdown tool to reduce the CTCF levels which presumably disrupt the CTCF-mediated chromatin interactions during transdifferentiation.We found significantly more MHC positive myotubes under CTCF knockdown conditions compared to control as shown by the differentiation index,indicating that myogenic capability of the fibroblast＿MyoD was substantially improved after transient CTCF inhibition.Accordingly,the comparative transcriptomics of shCTCF and shCon fibroblast MyoD cells revealed that CTCF inhibition could further increase the myogenic gene expression and reduce the fibrogenic gene expression.Furthermore,we found majority of the persistent CTCF loops were missed after CTCF inhibition through CTCF BL-HiChIP,4C-Seq and 3C-PCR,containing three genes: Serpinb1 b,Pcdhb15 and Pcdhb16,which highly expressed in fibroblast cells but low in muscle cells and was repressed after shCTCF.We further demonstrated that expression changes of CTCF were consistent with fibrogenic gene expression on the early inhibition stage using Smart-Seq data which demonstrate that inhibition of CTCF diminishes the identity of fibroblasts and thus promotes cell identity transformation.To further evidence this conclusion,we also tested in pig and chicken cells and the results showed consistent.6.Application of transdifferentiation cellsIn order to provide a wider source of cells for application,small molecule compounds were used for in vitro transdifferentiation,and the results showed that the small molecule compounds could induce transdifferentiation of fibroblasts to myogenic cells in porcine,chicken and mouse cells.We first seeded the chicken transdifferentiation cells in methacrylamide gelatin for three-dimensional culture and the results showed that the cells grow well and form three-dimensional myotubes.Accordingly,we analyzed the multi-omics data and investigated the characteristics and changes during transdifferentiation.Finally,we found a transformed method to facilitate the efficiency which provided experimental data and seed cell materials for the synthesis of artificially grown meat.