Dot1L methyltransferase activity is a barrier to acquisition of pluripotency but not transdifferentiation

Abstract

Pluripotent stem cells (PSCs) exist in a unique state poised to differentiate into any of the somatic cell types in part by maintaining an open chromatin structure. How these extraordinary cells suppress spurious transcription in the relative absence of repressive epigenetic modifications is poorly understood. PSCs are depleted for transcriptional elongation associated epigenetic modifications, primarily H3K79me2. Although H3K79me2 is found on thousands of genes, inhibiting the enzymatic activity of its methyltransferase, Dot1L results in few transcriptional changes, the majority of which are upregulated. During reprogramming of somatic cells to pluripotency, Dot1L inhibition (Dot1Li) at the midpoint is particularly effective at yielding greater number of colonies. Further, Dot1Li enhances reprogramming beyond early phases such as the mesenchymal to epithelial transition and from already epithelial cell types such as keratinocytes. Modulation of single genes from the DotLi transcriptional response cannot replace its function. Significantly, Dot1L inhibition does not enhance lineage conversion to neurons. Taken together, our results indicate that H3K79me is not a universal barrier of cell fate transitions but specifically protects cells from reverting to the pluripotent state.

DOT1L interaction partner AF10 controls patterning of H3K79 methylation and RNA polymerase II to maintain cell identity

“…As we previously found (Wille and Sridharan, 2020), most of the Dot1Li DE genes do not resemble their expression in ESCs (Fig 3A). This aberrant expression is observed with much lower magnitude in ΔAF10, which suggests that improper activation or failure to downregulate many of these genes does not impair reprogramming as Dot1Li is more efficient than deletion of AF10 in NANOG+ colony formation (Fig 2D).…”

“…To identify if AF10 has a temporal effect during pluripotency acquisition, AF10 was deleted coincident with reprogramming factor induction (day 0), at the midpoint (day 2) and later (day 4) of a reprogramming timecourse and assessed for NANOG+ colony formation on day 6 ( Fig 2G). Similar to Dot1L inhibition, loss of AF10 most potently increased early and midreprogramming ( Fig 2H, S2B) (Onder et al, 2012;Wille and Sridharan, 2020). Taken together, AF10 and Dot1L collaboratively maintain cellular identity during somatic cell reprogramming.…”

“…Therefore, pluripotency acquisition is an ideal platform to interrogate the mechanism of Dot1L and its associated proteins. To explore which of the Dot1L associated factors contribute to maintenance of somatic identity (Fig 1A), we examined their expression (Wille and Sridharan, 2020) in somatic cells – MEFs, cells on day 2 of reprogramming, cells on day 4 of reprogramming, and ESCs (Fig 1B). All assessed Dot1L associated factors were expressed throughout reprogramming, yet with different patterns (Fig 1B).…”

“…B. Expression of Dot1L associated factors determined by bulk RNA-Seq (Wille and Sridharan, 2020) in MEFs, day 2 of reprogramming, day 4 of reprogramming, and ESCs.…”

“…Published datasets. Bulk expression of Dot1L and associated factors was assessed from data previously generated (Wille and Sridharan, 2020). Single cell expression and co-expression of Dot1L and associated factors were analyzed as described (Tran et al, 2019) and can be obtained at NCBI GEO GSE108222.…”