The homeodomain of Oct4 is a dimeric binder of methylated CpG elements

Abstract

Oct4 is essential to maintain pluripotency and has a pivotal role in establishing the germline. Its DNA-binding POU domain was recently found to bind motifs with methylated CpG elements normally associated with epigenetic silencing. However, the mode of binding and the consequences of this capability has remained unclear. Here, we show that Oct4 binds to a compact palindromic DNA element with a methylated CpG core (CpGpal) in alternative states of pluripotency and during cellular reprogramming towards induced pluripotent stem cells (iPSCs). During cellular reprogramming, typical Oct4 bound enhancers are uniformly demethylated, with the prominent exception of the CpGpal sites where DNA methylation is often maintained. We demonstrate that Oct4 cooperatively binds the CpGpal element as a homodimer, which contrasts with the ectoderm-expressed POU factor Brn2. Indeed, binding to CpGpal is Oct4-specific as other POU factors expressed in somatic cells avoid this element. Binding assays combined with structural analyses and molecular dynamic simulations show that dimeric Oct4-binding to CpGpal is driven by the POU-homeodomain whilst the POU-specific domain is detached from DNA. Collectively, we report that Oct4 exerts parts of its regulatory function in the context of methylated DNA through a DNA recognition mechanism that solely relies on its homeodomain.

Epigenomic insights into common human disease pathology

“…Whilst some classes of TF were inhibited by DNAm, others, especially those with critical roles in embryonic and organismal development, showed a preference for binding to templates containing methylated CpGs. This latter grouping included the Yamanaka pluripotency factor Oct4, binding a mCpG TFBS motif [ 107 ] and, additionally it has been proposed to exhibit a preference for 5hmC [ 108 ]. Although Oct4 is not exclusive to this motif, as it recognises other TFBSs that do not contain a CpG [ 106 ].…”

Transcriptional Coactivator BOB1 (OBF1, OCA-B) Modulates the Specificity of DNA Recognition by the POU-Domain Factors OCT1 and OCT2 in a Monomeric Configuration

“…It has been demonstrated that the majority of TF pair sites involve a large overlap between the recognition motifs of individual TFs, resulting in the recognition of composite sites that markedly differ from the individual TF motifs [9]. This principle fully applies to the cooperation between OCT1/2 and BOB1, which was shown more than two decades ago [10][11][12][13][14] but has not been explored in detail since that finding. It has been demonstrated that while OCT1/2 binds the well-known canonical octamer ATGCAAAT and related sequences, only a subset of these sequences allows the formation of ternary complexes with BOB1.…”

“…In addition, a stringent requirement for adenine in the fifth position of the octamer motif has been demonstrated for the recruitment of BOB1 by OCT1 [11]. The other classes of POU domain-interacting DNA elements, such as the recently discovered CpGpal [12], have to be yet examined for their ability to recruit BOB1. A notable attempt to explore the specificity of the ternary complex formation was made 20 years ago using the derivatives of the palindromic Oct factor recognition element (PORE) sequence ATTTGAAATGCAAAT from an intronic enhancer of the osteopontin (Spp1) gene [14].…”