Modulation of a single amino acid in the reprogramming factor Kruppel-like factor 4 (KLF4) has been demonstrated to markedly improve natural transcription factor function and to result in faster and more effective reprogramming of somatic cells into induced pluripotent stem (iPS) cells.

This finding should offer significant cost-effective advantages in future regenerative medical applications, according to a multicenter Japanese study led by scientists at the RIKEN BioResource Research Center (BRC) in Tsukuba and reported in the December 14, 2021, edition of iScience.

"To the best of our knowledge, this is the first study to show that modulating KLF4 results in faster and more effective reprogramming of somatic cells into iPS cells," said study leader Yohei Hayashi, team leader of the iPS Cell Advanced Characterization and Development Team at RIKEN BRC.

Advances in reprogramming technology have provided unprecedented opportunities for regenerative medicine, enabling one somatic cell type to become other desired cell types by ectopic expression of factors, predominantly transcription factors.

These transcription factors cooperatively bind silenced parts of DNA and initiate active transcriptional events to regulate expression of downstream genes and epigenetic modification toward reprogrammed cell states.

Multiple studies have investigated additional factors, including epigenetic modifiers, culture conditions and reprogramming factor delivery methods, to improve reprogramming outcomes.

However, reprogramming technology remains associated with a poor and low yield of high-quality reprogrammed cells from primary somatic cells, resulting in delayed medical applications.

"This is primarily because the currently used processes of generating iPS cells using conventional methods are uncontrollably stochastic [randomly determined]," said Hayashi, who is also an associate professor in the Faculty of Medicine at Tsukuba University.

Modification or addition of transcriptional activity in reprogramming factors has been shown to enhance reprogramming results, with natural transcription factors generally still being used as reprogramming factors.

KLF4

KLF4 is a zinc-finger (ZnF) protein reprogramming factor with both transcriptional activation and repression domains that regulates downstream gene expression to control cell states. It is one of four critical transcription factors for iPS cell reprogramming identified in 2006 by Shinya Yamanaka, who won the Nobel Prize for his work in 2012.

Although KLF4 naturally regulates development and homeostasis of different tissues, including cancers, it also plays a central role in reprogramming somatic cells into iPS cells and other cell types.

Notably, in iPS cell reprogramming, KLF4 regulates genome-wide epigenetic status and reorganization of chromatin and its direct downstream transcription.

However, although the functionality of KLF4 in reprogramming is well understood, the structural basis of KLF4 enabling its unique reprogramming activity remains elusive.

Therefore, in their new study, the authors used a structure-function approach to investigate whether rational engineering of reprogramming factors might improve reprogramming.

"We analyzed the X-ray crystal structure of KLF4 protein bound to DNA, and identified target amino acids to make candidate variants, then tested these for their functional iPS cell reprogramming capability," explained Hayashi.

Focusing on the KLF4 ZnF DNA-binding domain to delineate its unique contribution to iPS cell generation, the researchers scanned alanine-substituted mutants of DNA-interacting amino acid residues in iPS cell generation.

Although many of these mutants lost or decreased their reprogramming activity, one mutant, KLF4 L507A, was shown to increase the speed and efficiency of iPS cell generation in both mouse and human somatic cells.

"The KLF4 L507A variant was shown to [accelerate] the generation of iPS cells, from a few days to a few weeks, and to increase the efficiency of iPS cell production several-fold, depending on delivery methods and culture conditions," noted Hayashi.

Moreover, by testing all of the variants at the L507 position, those with smaller amino acid residues in the KLF4 L507 position showed higher reprogramming efficiency.

This is a significant finding, as in a biophysical study such as this, "it is important to discover that the size of only one amino acid residue controls the efficiency of iPS cell generation," said Hayashi.

"Smaller amino acid residues, such as glycine and alanine, showed the highest reprogramming activity, while larger amino acid residues showed little or no reprogramming activity," he told BioWorld Science.

L507A was further demonstrated to bind more to promoters or enhancers of pluripotency genes, such as Klf5, and to drive gene expression of these genes during reprogramming.

"Using chromatin immunoprecipitation-sequencing, which detects actual genomic DNA bound by cell proteins, L507A was shown to bind more promoters of pluripotency genes, including Klf5, Dppa5a and Alp," said Hayashi.

"Also, using RNA sequencing, which detects global cellular transcriptional patterns, L507A was shown to increase RNA expression of these genes during reprogramming," he added.

"Together with molecular dynamics simulation data, these findings indicate that L507A should bind genomic DNA with a stabilized structure by additional hydrogen bonds to DNA molecules, which is important for revealing the molecular mechanism whereby L507A promotes iPS cell generation."

Collectively, these findings demonstrate how modifications in amino acid residues of DNA-binding domains enable next-generation reprogramming technology with engineered reprogramming factors.

These findings have important implications "for future regenerative medicine, which employs autologous transplantation derived from iPS cells generated from patients' somatic cells," said Hayashi.

In particular, "the KLF4 L507A variant will be useful for producing high-quality iPS cells more rapidly and efficiently by reducing the time and cost of their generation," he told BioWorld Science.

Looking forward, said Hayashi, "our team will be further developing somatic cell reprogramming technology toward the realization of improved regenerative medicine."