Losing Heterochromatin to Determine Cell Fate
In the moments after fertilization, the tiny mass that is the embryo sets in motion developmental programs that will transform it into a human being. One of the earliest steps in human development is the formation of the germ layers. Humans and other animals in the Chordata phylum, have three germ layers: the endoderm, mesoderm, and ectoderm. Cells from these three layers give rise to all of the different cells in the body. But how exactly cells know how to differentiate into germ layer cells and then into hundreds of different cells, remains a mystery.
One clue may be in how the packing of our DNA changes during early development. Early in development, DNA is not packed very tightly, but upon differentiation, it compacts more, which is thought to restrict the expression of particular genes so that cells can differentiate into specific types of cells . Upon differentiation, an endoderm cell that will become a liver cell, for example, will only express genes that are important for liver cell function and not genes important for other cell functions. However, the dynamics of this compaction are not well understood.
In a ground-breaking study published this month in Science, Nicetto and colleagues at the University of Pennsylvania used two independent approaches to ask how chromatin compaction changes during development. To achieve this they assessed compacted chromatin during multiple stages of mouse development, from the embryo to the adult.
To unequivocally identify compacted chromatin – called heterochromatin – the authors used a technique called sonication-resistant heterochromatin sequencing (srHC-seq) . When they used srHC-seq on cells from the mouse endoderm and adult mouse liver and pancreatic cells, the authors saw a dramatic rearrangement of heterochromatin at protein-coding genes.
But what happens to chromatin compaction even earlier in development? To answer this question, the authors assessed the presence of the repressive chromatin mark H3K9me3 by chromatin immunoprecipitation with sequencing (ChIP-seq) in cells from the pregastrula, one of the earliest stages of development and occurring before the formation of the germ layers. They found that in pregastrula cells, protein-coding genes maintained low levels of H3K9me3. During germ layer development, however, the authors found that H3K9me3 levels rose, and in later developmental stages when cells had differentiated, H3K9me3 levels fell again. This suggests that DNA compaction is important in the germ layer before cells have committed to specific fates, but as cells differentiate, this compaction is relieved. This likely occurs to allow expression of specific genes important for the identity of that particular cell.
To see how DNA compaction affects expression of specific genes during development, the authors asked what happens to the expression of liver-specific genes as cells differentiate from endoderm into liver cells. Comparing readouts from srHC-seq, H3K9me3 ChIP-seq, and RNA-seq, they found that liver-specific genes in the endoderm are marked with srHC and H3K9me3. However, when these cells differentiate into liver cells, these liver-specific genes lose the srHC and H3K9me3 marks. This result supports the role of H3K9me3-marked heterochromatin as an important regulator of proper gene expression during differentiation.
In an effort to better understand this process, the authors addressed the question of DNA compaction during development using a second method. They generated mice with endodermal cells that had lost expression of Setdb1, one of the three histone methyltransferases that are responsible for adding H3K9me3 marks. Using single-cell RNA-seq (scRNA-seq) of normal or Setdb1 mutant liver cells, they found that the cell types clustered into groups with three different functions. Compared to normal cells, the Setdb1 mutant cells, however, had different numbers of cells in each of the three groups. Additionally, if the researchers took cells that expressed albumin, a normal feature of liver cells, they saw that none of the cells in the Setdb1 mutant group expressed liver cell markers, indicating that Setdb1 plays a role in regulating the differentiation of cells that will become liver cells.
Expanding on this experiment, the authors wanted to more fully address the role of H3K9me3 on development, so they generated a mouse with endodermal cells that had lost expression of all three methyltransferases that establish H3K9me3. By sc-RNA-seq, they found that the cells that had lost expression of all three methyltransferases clustered in a separate group from either the normal cells or the Setdb1 mutant cells. Additionally, the triple mutant mice grew to be much smaller than their siblings who did not have the mutations. The liver cells in these triple mutants had increased inflammation and retracted ducts, and there was a substantial loss of srHC and H3K9me3 across the genome in these cells. Finally, electron microscopy of liver cells from a one month old mouse showed a global loss of condensed chromatin in the triple mutant compared to a normal mouse. These results suggest that if proper H3K9me3 is not established during early development, liver cell development is impaired and there is an improper expression of liver-specific genes.
Through two independent approaches, this work shows that there are increased levels of heterochromatin at genes during early development and that during development H3K9me3-marked heterochromatin is lost in a cell-type specific manner to establish the fate of the cell. While they all begin with a blank slate, this study shows how epigenetic regulation of chromatin compaction is important in establishing the identity of our cells.
Original article: Nicetto D, Donahue G, Jain T, Peng T, Sidoli S, Sheng L, Montavon T, Becker JS, Grindheim JM, Blahnik K, Garcia BA, Tan K, Bonasio R, Jenuwein T, Zaret KS (2019). H3K9me3-heterochromatin loss at protein-coding genes enables developmental lineage specification. Science, 363(6424):294-297. doi: 10.1126/science.aau0583.
 Chen T, Dent SY (2014). Chromatin modifiers and remodellers: regulators of cellular differentiation. Nat Rev Genet, 15(2):93-106. doi: 10.1038/nrg3607.
 Becker JS, McCarthy RL, Sidoli S, Donahue G, Kaeding KE, He Z, Lin S, Garcia BA, Zaret KS (2017). Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes. Mol Cell, 68(6):1023-1037.e15. doi: 10.1016/j.molcel.2017.11.030.