Dev Cell. Song Yan group reveals a new mechanism by which transcription factors drive end-of-life differentiation of neurons through phase separation.

Image credit: Zhi ye the heterochromatin labeled by h3k9me3, an inhibitory histone, becomes highly condensed during cell differentiation, and its region expands significantly [1,2], forming an important barrier to prevent the fate reversal of differentiated cells.correspondingly, decompression of h3k9me3 + heterochromatin region can greatly improve the efficiency of cell reprogramming [3,4].previous studies have shown that the formation of h3k9me3 + heterochromatin depends on the synergistic action of h3k9me3 "reader" heterochromatin protein 1 (HP1) and H3K9 methyltransferase suv39h1, the "writer" of h3k9me3 [5,6]. However, the molecular mechanism of h3k9me3 + heterochromatin condensation and expansion is still unclear under physiological conditions.in terminal differentiated cells, how heterochromatin regulates the permanent silencing of specific genes is a mystery.on December 19, 2019, song Yan research group of School of life sciences of Peking University published a research paper entitled "mitotic implanation of the transcription factor propeller via phase separation drives terminal neural differentiation" online in the Journal of developmental cell.this study revealed a new phenomenon and mechanism that a transcription factor was "implanted" into mitotic chromosomes of neural precursor cells through liquid-liquid phase separation during the development of Drosophila melanogaster, and ensured neuronal terminal differentiation by promoting h3k9me3 + heterochromatin condensation.when a cell enters mitosis, the chromatin condenses to form a highly dense chromosome, and most of the key regulatory elements (including transcription factors) of gene transcription will be detached from the chromosome.in this work, the researchers found that the transcription factor Prospero (PROs) could form small aggregates (FOCI) and stay in mitotic chromosomes of Drosophila neural precursor cells (Fig. 1).pros, a highly conserved homeobox transcription factor, plays a key role in promoting cell terminal differentiation along with its mammalian homologous gene Prox1.Fig. 1. The transcription factor pros was implanted into the chromosomes of neural precursor cells by liquid-liquid phase separation. Then, why could pros remain on the chromosomes with dense structure? What are the physiological functions of this phenomenon? By means of fine whole brain fluorescence dynamic imaging, whole brain light bleaching recovery, optical drop and in vitro phase separation, the researchers have observed a surprising result, that is, the pros protein is implanted by liquid-liquid separation and retained in the metacentric heterochromatin region labeled by h3k9me3 of neural precursor cells (Fig. 1).when neural precursor cells enter the end of mitosis, Pros protein retained in heterochromatin domain recruits and concentrates H3K9me3 reader HP1a into phase separated aggregates (condensates), and promotes its transformation to low fluidity near gel state, thereby driving the condensation and expansion of H3K9me3+ heterochromatin in newly generated neurons (Fig. 2).Fig. 2. Transcription factor pros drives further DamID SEQ, DNA of neuron terminal differentiation researchers through liquid-liquid separation The results of fish and dynamic imaging showed that when neural precursor cells divided into two neurons, pros dissociated from h3k9me3 labeled heterochromatin region, and carried a part of hp1a to its key target genes (important genes for promoting stem cell self replication and promoting cell cycle), and then mediated the local condensation of chromatin region of these genes through hp1a, Turn off the expression of these genes.in addition, these pro target gene sites with chromatin condensation may be close to the heterochromatin regions rich in hp1a through hp1a mediated droplet fusion, thus further promoting the permanent silencing of these key genes and ensuring the terminal differentiation of neurons.it is worth mentioning that Prox1 may also adopt the strategy of "chromosome implantation driven heterochromatin condensation" to ensure the terminal differentiation of neurons.in addition, the researchers found that pros protein could form aggregates and remain on the mitotic chromosomes of Drosophila intestinal precursor cells.therefore, the new phenomenon and mechanism revealed by this study may represent the universal rule of heterochromatin condensation and terminal differentiation driven by transcription factors through chromosome implantation.as a self-organizing way in cells, liquid-liquid separation provides a new perspective for us to understand many biological phenomena. however, whether phase separation is really involved in the regulation of important biological processes under physiological conditions remains to be further confirmed [7,8]. through point mutation or deletion of the key sites of phase separation mediated by Pros protein, the researchers in this work have constructed a pros mutant with specific phase separation ability without affecting endogenous expression, nuclear entry ability and transcriptional activity. the specific loss of phase separation ability makes it impossible for pros to remain on the chromosome, which also loses its function of regulating heterochromatin condensation and promoting neuronal terminal differentiation. more importantly, the phase separation ability of pros can be restored by fusing with the internal disordered region (IDR) of known driving phase separation. The chromosome retention of pros and its ability to promote heterochromatin condensation and neuronal differentiation can be effectively restored. therefore, through specific mutation and replenishment experiments and rigorous quantitative analysis, this study established for the first time the causal relationship between the liquid-liquid phase transition of transcription factors and a series of important biological events under physiological conditions, providing strong evidence for the important physiological significance of phase separation in animal development. the researchers speculate that other transcription factors may use similar liquid-liquid phase separation strategy to achieve chromosome retention, and regulate the process of cell fate determination by remodeling the three-dimensional structure of chromatin. to sum up, the unexpected results of this study reveal that transcription factors cause the remodeling of heterochromatin structure through the changes of their biophysical properties, and then drive the terminal differentiation of cells. It provides a new perspective and new ideas for further exploring the dynamic changes and regulatory mechanisms of heterochromatin during cell differentiation. Liu Xiaodan (PTN grade 13) and Shen Jingwen (grade 16), doctoral students of School of life sciences, Peking University, are co first authors of the research results. researcher song Yan from the College of life sciences is the corresponding author of the paper. researcher Li pilong of School of life sciences of Tsinghua University and his doctoral students Xie leiming, doctoral students Huang Zuxian (PTN 16) and undergraduates Wei Zelin (grade 16), Li Yiyao (grade 17) and Zheng Xinhe (grade 17) also made important contributions to the research. Wen, B., Wu, H., Shinkai, Y., irizarry, R.A., and Feinberg, A.P. (2009), K.S. (2016). H3K9me3-Dependent Heterochromatin: Barrier to Cell Fate Changes. Trends Genet 32, 29-41.3. Matoba, S., Liu, Y., Lu, F., Iwabuchi, K.A., Shen, L., Inoue, A., and Zhang, Y. (2014). 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