Distinct and Temporal Roles of Nucleosomal Remodeling and Histone Deacetylation in the Repression of the hTERT Gene

Abstract

Transcriptional silencing of the hTERT gene during HL60 cell differentiation was a biphasic process. The initial repression was accompanied by the loss of c-Myc binding and disappearance of a nucleosome-free region at the core promoter. The subsequent nucleosomal remodeling and histone modifications at the promoter stabilized this repression.

Abstract

hTERT, the human telomerase reverse transcriptase, is highly expressed in stem cells and embryonic tissues but undetectable in most adult somatic cells. To understand its repression mechanisms in somatic cells, we investigated the endogenous hTERT gene regulation during differentiation of human leukemic HL60 cells. Our study revealed that silencing of the hTERT promoter was a biphasic process. Within 24 h after initiation of differentiation, hTERT mRNA expression decreased dramatically, accompanied by increased expression of Mad1 gene and disappearance of a nucleosome-free region at the hTERT core promoter. Subsequent to this early repression, nucleosomal remodeling continued at the promoter and downstream region for several days, as demonstrated by micrococcal nuclease and restriction enzyme accessibility assays. This later nucleosomal remodeling correlated with stable silencing of the hTERT promoter. Progressive changes of core histone modifications occurred throughout the entire differentiation process. Surprisingly, inhibition of histone deacetylation at the hTERT promoter did not prevent hTERT repression or nucleosomal deposition, indicating that nucleosomal deposition at the core promoter, but not histone deacetylation, was the cause of transcriptional repression. Our data also suggested that succeeding nucleosomal remodeling and histone deacetylation worked in parallel to establish the stable repressive status of hTERT gene in human somatic cells.