PLoS One. 2016 Feb 18;11(2):e0149502. doi: 10.1371/journal.pone.0149502.

SETD7 Regulates the Differentiation of Human Embryonic Stem Cells

Julio Castaño1, Cristina Morera1, Borja Sesé1, Stephanie Boue1,2, Carles Bonet-Costa3, Merce Martí1, Alicia Roque3,4, Albert Jordan3, Maria J. Barrero1,5

1 Center for Regenerative Medicine in Barcelona, (CMRB), Barcelona, 08003, Spain,

2 Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, 28029, Spain,

3 Institut de Biologia Molecular de Barcelona (IBMB-CSIC), Barcelona, 08028, Spain,

4 Departamento de Bioquímica y Biología Molecular, Facultad de Biociencias, Universidad Autónoma de Barcelona, Cerdanyola, 08193, Barcelona, Spain,

5 Spanish National Cancer Research Center (CNIO), Madrid, 28029, Spain

 

Abstract

The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation.

PMID: 26890252

 

Supplement:

The generation of specialized cell types from human pluripotent cells in the laboratory can provide an unlimited source of cells and tissues useful for transplantation and therefore holds a great promise for regenerative medicine. Successful therapies depend on the generation of functional cell types that have enough plasticity to survive and repopulate the damaged tissues with a low risk of forming tumors. However, obtaining these cell types remains challenging partially due to the limited knowledge exiting on the mechanisms involved in differentiation. Identifying relevant players of differentiation, especially those that can be pharmacologically targeted, appears critical to develop and improve strategies for obtaining differentiated cell types with therapeutic potential.

 

 

Figure1

Figure 1. SETD7 is needed for the proper differentiation of human embryonic stem cells. (A) Human embryonic stem cells transduced with a shRNA targeting SETD7 (shSETD7) or a control shRNA (shSCR) grown under self-renewal conditions (d0) show no morphological differences. At day 4 of embryoid body (EB) differentiation in suspension control EBs show cystic structures characteristic of differentiation while EBs knock down for SETD7 do not, suggesting that differentiation is affected by the SETD7 knock down. (B) Microarray data shows the expression of pluripotency and differentiation-related genes in control and SETD7 knock down embryonic stem cells in self-renewal conditions (d0) and at a days 8 (d8) and 15 (d15) of in vitro differentiation. Knocking down the expression of SETD7 causes a delay in both the silencing of pluripotency genes and the induction of differentiation genes.

 

We identified SETD7 as a critical player in human embryonic stem cell differentiation. SETD7 is expressed at very low levels in human embryonic stem cells (hESC) grown under self-renewal conditions. Interestingly, its expression is strongly induced when these cells go into differentiation programs suggesting a potential role in differentiation. Blocking the induction or the activity of SETD7 affects the in vitro differentiation of hESC, which is characterized by both morphological defects and delays in the silencing of pluripotency genes and in the induction of differentiation makers (Figure 1). The relevance of SETD7 for differentiation is not only limited to hESC, but also blocking the expression of SETD7 during zebrafish development has dramatic consequences such as increased mortality of the embryos and other important developmental defects (Figure 2).

 

 

Figure2

Figure 2. Setd7 is needed for the proper development of zebrafish. The expression of Setd7 in zebrafish embryos was blocked using morpholinos that specifically target Setd7 (Setd7-MO). Setd7-MO injected embryos suffered decreased viability, developmental delays, tail defects, pericardium edema and yolk sac edema compared to control conditions (Control-MO). Experiments were performed by C. Fabregat at the Center for Regenerative Medicine in Barcelona.

 

SETD7 is a lysine methyltransferase that was initially described as a histone methyltransferase involved in gene activation [1]. However, many non-histone methylation targets have been described during recent years making hard to understand which is the major molecular target of SETD7 in vivo [2]. We set up to identify additional methylation partners that might help to decipher the role of SETD7 during differentiation. We found that SETD7 methylates linker histone H1 in vitro and that knock down of SETD7 causes a decrease in the recruitment of H1 to pluripotency genes, which might eventually lead to defects on their silencing during differentiation (Figure 3). Although conformational analysis shows that methylation of H1 by SETD7 causes structural changes in vitro further studies will be needed to determine effects in vivo. Additionally, methylation of additional targets might also play a role in differentiation and therefore a more precise dissection of the differentiation process is granted.

Inhibitors of the catalytic activity of SETD7 have been recently developed [3]. Chemical activation or inhibition of enzymes involved in differentiation offers the opportunity to modulate the process of differentiation and might be used in combination with other chemicals known to favor differentiation towards specific cell types. In this regard, our work shows that SETD7 is important for the pan-differentiation of hESC into derivatives of the three germ layers, but it is still unknown if the modulation of SETD7 activity could facilitate the differentiation of human embryonic stem cells towards very specific cell types relevant for therapy.

 

 

Figure3

Figure 3. Proposed mechanism for the role of SETD7 in differentiation. SETD7 is induced during differentiation, methylates histone H1 and facilitates its recruitment to pluripotency genes to mediate their silencing. Since SETD7 can methylated multiple targets other mechanisms could also account for its effects in differentiation.

 

References

  1.          Wang H, Cao R, Xia L, Erdjument-Bromage H, Borchers C, Tempst P, et al. Purification and Functional Characterization of a Histone H3-Lysine 4-Specific Methyltransferase. Mol Cell. 2001;8: 1207–1217. doi:http://dx.doi.org/10.1016/S1097-2765(01)00405-1
  2.           Pradhan S, Chin HG, Estève P-O, Jacobsen SE. SET7/9 mediated methylation of non-histone proteins in mammalian cells. Epigenetics. NIH Public Access; 2009;4: 383–7. Available: http://www.ncbi.nlm.nih.gov/pubmed/19684477
  3.           Barsyte-Lovejoy D, Li F, Oudhoff MJ, Tatlock JH, Dong A, Zeng H, et al. (R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells. Proc Natl Acad Sci U S A. 2014;111: 12853–8. doi:10.1073/pnas.1407358111

 

Acknowledgements

Authors would like to thank C. Fabregat for the experiments performed in zebrafish.

This work was funded through different funding agencies supported by the Spanish Government and Fundación CELLEX

 

 

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