Dynamic Switching of Active Promoter and Enhancer Domains Regulates Tet1 and Tet2 Expression during Cell State Transitions between Pluripotency and Differentiation

Abstract

The Tet 5-methylcytosine dioxygenases catalyze DNA demethylation by producing 5-hydroxymethylcytosine and further oxidized products. Tet1 and Tet2 are highly expressed in mouse pluripotent cells and downregulated to different extents in somatic cells, but the transcriptional mechanisms are unclear. Here we defined the promoter and enhancer domains in Tet1 and Tet2. Within a 15-kb "superenhancer" of Tet1, there are two transcription start sites (TSSs) with different activation patterns during development. A 6-kb promoter region upstream of the distal TSS is highly active in naive pluripotent cells, autonomously reports Tet1 expression in a transgenic system, and rapidly undergoes DNA methylation and silencing upon differentiation in cultured cells and native epiblast. A second TSS downstream, associated with a constitutively weak CpG-rich promoter, is activated by a neighboring enhancer in naive embryonic stem cells (ESCs) and primed epiblast-like cells (EpiLCs). Tet2 has a CpG island promoter with pluripotency-independent activity and an ESC-specific distal intragenic enhancer; the latter is rapidly downregulated in EpiLCs. Our study reveals distinct modes of transcriptional regulation at Tet1 and Tet2 during cell state transitions of early development. New transgenic reporters using Tet1 and Tet2 cis-regulatory domains may serve to distinguish nuanced changes in pluripotent states and the underlying epigenetic variations.T he study of transcription regulation is fundamental to understand how gene expression and phenotype are regulated during development. ENCODE (encyclopedia of DNA elements) studies recently revealed that the mammalian genome is more complex than previously annotated, in which different transcription start sites (TSSs) often mark core promoters, both intra-and intergenic, to drive the expression of alternative mRNA isoforms (1, 2). Distal elements known as enhancers are often recruitment platforms for cell-type-specific transcription factors and interact with promoter-bound factors to stabilize the association of RNA polymerase II (Pol II) at TSSs (3).Embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of mouse blastocysts represent a unique model of transcription regulation. The widespread presence of "open" chromatin achieved through feed-forward and autoregulatory loops involving transcription factors appears crucial for the transcriptome to adapt rapidly to inductive differentiation signals, allowing the cells to generate all early embryonic lineages (4, 5). This pluripotent state is dependent on the master transcription factors Oct4, Sox2, and Nanog (OSN), which often bind cooperatively at target sites on enhancers. In early mammalian development, the triad of OSN, by binding to different enhancers, orchestrates two states of pluripotency, the "naive" and "primed" conditions resembling the pre-and postimplantation epiblasts, respectively (6, 7). Furthermore, OSN together with Klf4, Esrrb, and Mediator coactivator, all highly expressed in naive ESCs, densely occupy extended d...

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