SAF-A Has a Role in Transcriptional Regulation ofOct4in ES Cells Through Promoter Binding

Abstract

Methodologies to reprogram somatic cells into patient-specific pluripotent cells, which could potentially be used in personalized drug discovery and cell replacement therapies, are currently under development. Oct4 activation is essential for successful reprogramming and pluripotency of embryonic stem (ES) cells, albeit molecular details of Oct4 activation are not completely understood. Here we report that endogenous SAF-A is involved in regulation of Oct4 expression, binds the Oct4 proximal promoter in ES cells, and dissociates from the promoter upon early differentiation induced by LIF withdrawal. Depletion of SAF-A decreases Oct4 expression even in the presence of LIF, and results in an increase of the mesodermal marker Brachyury. The overexpression of wild-type human SAF-A rescues the mouse knock-down phenotype and results in increased Oct4 level. We also demonstrate that endogenous SAF-A interacts with the C-terminal domain (CTD) of endogenous RNA polymerase II and that the interaction is independent of CTD phosphorylation and mRNA. Moreover, we show that SAF-A exist in complexes with transcription factors Sox2 and Oct4 as well as STAT3 in ES cells. The number of endogenous SAF-A:Oct4 and SAF-A:Sox2 complexes decreases upon LIF depletion. These discoveries allow us to propose a model for activation of Oct4 transcription.

Xmas ESC: A new female embryonic stem cell system that reveals the BAF complex as a key regulator of the establishment of X chromosome inactivation

“…To overcome the variable percentage of XO cells in the starting populations, each experiment was normalised to the matched Nons control ( Figure 5D). Unexpectedly, knockdown of Hnrnpu caused a rapid loss of double positivity prior to weaning into differentiation media, likely due to the requirement of Hnrnpu for pluripotency (Vizlin-Hodzic et al, 2011) and suggesting Xmas ESCs may be useful for the identification of novel pluripotency factors. To circumvent the role of Hnrnpu in pluripotency and instead test its function in XCI, we transduced cells with shRNA at day 2 of differentiation, so that knockdown takes effect following exit from pluripotency.…”

Functions of Heterogeneous Nuclear Ribonucleoproteins in Stem Cell Potency and Differentiation

“…hnRNP U like-1 protein (hnRNPUL1) is considered as a novel surface molecule marker on undifferentiated human ES cells [60]. In addition, hnRNP U maintains ES cell pluripotency as a modulator of pluripotency factor OCT4 through direct binding to OCT4 proximal promoter and activation of OCT4 gene expression [68]. …”

Generation of Patient Specific Stem Cells: A Human Model System

“…SAF-A was found on the Oct4 promoter only when the gene is actively transcribed in murine ESCs, depending on LIF, and gene silencing of SAF-A in ESCs resulted in down regulation of Oct4 (Vizlin-Hodzic et al, 2011). Other Oct4 modulators have been reported that in similarity with SAF-A are in complex with RNA polymerase II (Ding et al, 2009;Ponnusamy et al, 2009).…”