Principal Investigator Harvey Lodish
Using the in-vitro erythroid culture system developed in the Lodish lab, Senthil Raja Jayapal, who is a joint graduate student in the Lodish lab and in the labs of Bing Lim at the Genome Institute of Singapore and Philipp Kaldis at Institute of Molecular and Cell Biology in Singapore, is investigating the role of cell cycle proteins and epigenetic modifications of histones in late stage erythroid maturation. In order to study the relationship between proliferation and differentiation programs during terminal erythroid maturation, he initially chose to focus on c-myc, which directs proliferation in many cell types and is down regulated during terminal differentiation when cells withdraw from cell cycle. The protein levels of c-myc are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G1-phase and enucleation, suggesting possible roles for c-myc in one or both of these processes. Surprisingly, ectopic c-myc expression had a dose dependent effect on terminal erythroid maturation. Ectopic expression of c-myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown, but specifically blocked erythroid nuclear condensation and enucleation. When over-expressed at levels much higher than physiological, c-myc blocked erythroid differentiation completely and the cells continued to proliferate in culture with an early erythroblast morphology. These studies revealed important roles for c-myc in erythroid cells independent of its cell cycle regulatory functions.
Since histone deacetylation has been associated with erythroid nuclear condensation and enucleation, he compared the changes in acetylation status of histones H3 and H4 in erythroid cells with physiological levels of ectopic myc expression that are specifically blocked in enucleation, relative to untreated wild type erythroblasts. c-myc ectopic expression prevented deacetylation at several lysine residues that are normally deacetylated during erythroid maturation. By transcriptional profiling, one specific histone acetyl transferase (HAT) was shown to be upregulated by ectopic myc expression. The level of this HAT, like that of c-myc, normally decreases dramatically during late stage erythroid maturation. Chromatin immunoprecipitation assays demonstrated binding of c-myc to the promoter region of this HAT, indicating that it is a direct myc target in erythroid cells. Over-expression of this HAT inhibits nuclear condensation and enucleation specifically without affecting other aspects of terminal erythroid differentiation, and prevents histone deacetylation similar to ectopic c-myc expression. These data support a model where histone deacetylation associated with down regulation of c-myc and this HAT is essential for chromatin condensation and enucleation in mammalian erythroid cells. Currently, he is investigating the roles of other HATs and HDACs in terminal erythroid differentiation.