On October 1, EMBO Journal published the latest research results of Dr. Xingguo Liu’s and Dr Duanqing Pei’s groups from Guangzhou Institutes of Biomedicine and Health, CAS and Guangzhou Regenerative Medicine and Health Guangdong Laboratory, entitled “Heterochromatin loosening by the Oct4 linker region facilitates Klf4 binding and iPSC reprogramming”. They found that Oct4 acts as a pioneer factor that loosens heterochromatin and facilitates the binding of Klf4 and the expression of epithelial genes in early reprogramming, leading to enhanced mesenchymal-to-epithelial transition. Mechanistic studies revealed that Oct4 loosens heterochromatin by recruiting ATP-dependent chromatin remodeling proteins such as Brg1. These studies reveal a cooperation between Oct4 and Klf4 at the chromatin level that facilitates MET at the cellular level and shed light into the research of multiple factors in cell fate determination.
Chromatin can be divided into euchromatin and heterochromatin. The former is in the relaxed state and has transcription activity, while the latter is in the condensed state and has no transcription activity. The transition between them is the key in cell fate determination. Compared with somatic cells, pluripotent stem cells have an open chromatin state and less heterochromatin. Therefore, the success of Yamanaka factor reprogramming of somatic cells into iPSCs requires chromatin remodeling. The changes of chromatin during reprogramming process has been investigated extensively at different levels. However, how factor-dependent chromatin opening occurs remains unclear.
Dr. Liu focuses on the mechanistic studies of organelles regulating reprograming at the subcellular level. By using imaging and other methods, his team systematically elucidated the regulatory of pluripotency acquisition such as component remodeling of organelles (Xingguo Liu *, Autophagy, 2017), ion signal (Xingguo Liu *, Cell Metabolism, 2016, 2018), energy metabolism (Xingguo Liu *, Stem Cells, 2016), etc. For the nucleus, the research team developed a real-time and accurate imaging method, fluorescence recovery after photobleaching (FRAP), to quantify euchromatin and heterochromatin remodeling dynamics. They labeled HP1a with mCherry and histone H1 with GFP. HP1a-mCherry allowed people to distinguish heterochromatin and euchromatin. By selecting region of interest within HP1a foci, they performed FRAP of heterochromatin H1 and found that only heterochromatin, not euchromatin, undergoes a relaxation process at the early phase of reprogramming. Furthermore, they identified growth arrest and DNA damage-inducible protein a (Gadd45a) as a chromatin relaxer, which also enhances somatic cell reprogramming efficiency (Xingguo Liu* and Duanqing Pei*, EMBO Reports, 2016; Xingguo Liu*, Cell Death and Disease, 2017). On the basis of this research, Dr. Liu’s group has carried out continuous research and applied FRAP to answer the basic scientific question "which reprogramming factor(s) loosen heterochromatin of somatic cells".
Most of the target genes of Klf4 located in heterochromatin. Whether KLF4 can bind and activate the target genes determines whether the somatic cells can change from the mesenchymal state to the epithelial state to initiate reprogramming. Oct4, dependent on its L80 residue, can loosen heterochromatin to facilitate Klf4 binding and epithelial gene expression. A mutation in the Oct4 linker, L80A, which shows impaired interaction with Brg1, is inactive in heterochromatin loosening. Oct4-L80A also blocks the binding of Klf4 and retards MET. Finally, vitamin C or Gadd45a could rescue the reprogramming deficiency of Oct4-L80A by enhancing chromatin opening and Klf4 binding.
This study was carried out in collaboration with Professor Hans R Schöler from the Max Planck Institute of molecular biomedicine, Germany. This research was supported by the state key R & D projects, Chinese Academy of Sciences, National Natural Science Foundation of China, Guangdong Province and Guangzhou city.