JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

<div><p>The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule GSK-J4. Our results propose a potential role of JMJD3 in lineage conversion of BM cells into hepatic lineage.</p></div

Binding of JMJD3 to HTFs in the presence of GSKJ4 inhibitor.

<p>Relative enrichment of H3K27me3 and JMJD3 at promoters of (A) HNF4α (B) HNF3α (C) HNF3β (D) HNF1α(E) CEBPα and (F) GATA4 determined by ChIP-qPCR. Number of experiment = 3. p value < 0.05 was considered as significant change. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173977#pone.0173977.s017" target="_blank">S5 Table</a> for IgG control values.</p

Genome-wide transcriptional remodeling in BM-derived hepatocytes relative to Lin^- BM cells.

<p>(A) Heat map of differentially regulated hepatocyte-specific functional genes and mesenchymal and hematopoietic lineage specific genes in BM derived hepatocytes– 1 month (B1, B2) and 5 month (G1, G3) with respect to control–Lin^- BMCs (L3, L4). (B) Scatter plot analysis generated by R program comparing the log2 transformed normalized expression values of the filtered genes in Lin^- BMCs and BM-derived hepatocytes (1 month). (C) Scatter plot analysis generated by ‘R’ program comparing the log2 transformed normalized expression values of the filtered genes in primary and BM-derived hepatocytes (5 month). Number of experiment = 2 (each experiment was based on pooled cells of 3 mice).</p

Chromatin modifying enzymes in reprogramming of Lin^- BMCs to hepatocytes.

<p>(A) Expression of some chromatin modifying enzymes in BM-derived hepatocytes. Expression levels normalized to that of primary hepatocytes were used to calculate relative fold change. (B) ChIP-qPCR analyses of EZH2 at the promoters of hepatic transcription factors and hematopoietic genes in BM-derived hepatocytes. Enrichment of the marks in the immuno-precipitated samples over input samples was calculated. (C) ChIP-qPCR analyses of JMJD3 at the promoters of hepatic transcription factors and hematopoietic genes in BM-derived hepatocytes. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173977#pone.0173977.s016" target="_blank">S4 Table</a> for IgG control values.</p

Generation of hepatocyte-like cells from Lin^- BM cells.

<p>(A) Immuno-histochemical analysis of recipient mice liver sections after transplantation. <i>Upper panel</i>: Images (600× magnification) showing donor derived hepatocytes (anti-GFP/donkey anti-mouse Alexa fluor 488 and anti-albumin /donkey anti-goat Alexa fluor 594). <i>Middle panel</i>: Images (630×2.8 zoom magnification) showing donor derived hepatocytes (anti-GFP/donkey anti-mouse Alexa fluor 488 and anti-HNF4α /donkey anti-goat Alexa fluor 594). <i>Lower panel</i>: IHC analysis of liver sections (630× magnification) from recipient showing donor derived hepatocytes which shows no CD45 expression (black arrow) and CD45 expressing non-parenchymal cells (white arrow). Number of experiment = 3. (B) Interphase FISH analysis of X and Y chromosome in BM-derived hepatocytes after 5 month of transplantation. The cells were analyzed for the presence of X (Red, platinum bright 550) and Y (Green, platinum bright 495) chromosomes in the nuclei (blue). (C) Cytogenetic analysis showing the percentage of donor cells that did not contain Y chromosome. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). (D) RT-qPCR analysis showing expressions of hepatic marker genes in BM-derived hepatocytes relative to expression in primary hepatocytes. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). (E) RT-qPCR analysis showing expression of various hepatic transcription factors in BM-derived hepatocytes relative to expression in primary hepatocytes. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). * No Ct.</p

Histone modifications at promoters of hepatic transcription factors in BM-derived hepatocytes.

<p>BM-derived hepatocytes were isolated after 5 months of transplantation for ChIP-qPCR analysis. Lin^-CD45⁺ and Lin^-CD45^- BMCs served as negative controls and primary hepatocytes as positive control. ChIP-qPCR analysis of (A) H3K4me3, (B) H3K9Ac, (C) H3K27me3 and (D) H3K9me3 at the promoters of hepatic transcription factors in BM-derived hepatocytes. Enrichment of the marks in the immuno-precipitated samples over input samples was calculated. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173977#pone.0173977.s015" target="_blank">S3 Table</a> for IgG control values.</p