Embryonic stem cells (ESCs), derived from the inner cell mass (ICM) of the blastocyst-stage embryo, are pluripotent. They can differentiate into all cell types and self-renew indefinitely in vitro, which determines its great research value and broad prospect of clinical application. In recent years, direct reprogramming of somatic cells to induced pluripotent stem (iPS) cells further shortens the distance between pluripotent stem cell research and their clinical utilization for disease treatment. Intensive research over past decades has demonstrated that transcription factors Oct4 and Sox2 are key players in maintaining the pluripotent state of ESCs and establishing iPS cells. Small changes in their levels disrupt normal expression of their target genes and induce differentiation of ESCs. However, it remains elusive how protein levels of Oct4 and Sox2 as well as expression of their target genes are precisely controlled in ESCs. Recent studies indicate that additional factors are involved in regulation of target genes of Oct4 and Sox2 and that these factors function in a gene-specific manner. Here, we conduct affinity chromatography with the nuclear extract (NE) from F9 mouse embryonal carcinoma (EC) cells, using synthetic biotinylated oligonucleotides containing the binding sites of Oct4 and Sox2 on the 3’ UTR enhancer of fibroblast growth factor 4 (FGF4). We identify PARP1 as a novel cofactor of Oct4 and Sox2 to regulate expression of their target gene FGF4. We demonstrate for the first time that PARP1 binds the FGF4 enhancer in a sequence-specific manner and regulates FGF4 expression through poly(ADP-ribosyl)ation of Sox2 during ESCs differentiation. Our data show that PARP1 interacts with and poly(ADP-ribosyl)ates Sox2 directly, which may be a step required for dissociation of inhibitory Sox2 proteins from the FGF4 enhancer. In addition, PARP activity can promote ubiqutination and degradation of Sox2. When PARP1 activity is low or absent, the post-translational modifications of Sox2 are significantly reduced, and association of Sox2 with FGF4 enhancers increases, accompanied by an elevated level of Sox2 proteins and reduced expression of FGF4. Significantly, specific knockdown of Sox2 expression by RNA interference can considerably abrogate the inhibitory effect of the poly(ADP-ribose) polymerase inhibitor on FGF4 expression. Interestingly, PARP1 deficiency does not affect undifferentiated ESCs but compromises cell survival and/or growth when ESCs are induced into differentiation. Addition of FGF4 can partially rescue the phenotypes caused by PARP1 deficiency during ESCs differentiation. In addition, compared with PARP1+/+ differentiated cells, expression of Hand1，a marker gene of trophoblast giant cells, is higher in PARP1-/- differentiated cells. Moreover, expression of marker genes for the trophectoderm and primitive endoderm (FgfR2 and Hnf4) is lower in PARP1-/- differentiated cells. Notably, expression of these two marker genes also decreases in FGF4-/- blastocysts at E4.5. Importantly, addition of FGF4 can decrease expression of Hand1 and increase expression of FgfR2 and Hnf4. These results indicate that PARP1 and FGF4 are functionally correlated and the function of PARP1 is, at least, partially depends on FGF4.Taken together, the major findings from this study include: i) identifying a new cofactor associated with Oct4/Sox2 to regulate expression of their target genes; ii) providing the first example that PARP1-mediated modification of the transcription factor can lead to degradation through the proteasome; iii) demonstrating the importance of post-translational modifications in regulation of the Sox2 protein level as well as its function; iv) establishing the links among PARP1, Sox2 and FGF4. These findings will greatly facilitate our understanding how Oct4/Sox2 regulates expression of their target genes and maintains self-renewal in ESCs. Moreover, the results uncover new mechanisms through which PARP1 participitate transcription regulation, making us easier to understand the phenotypes observed in the absence of PARP1. Also, since Sox2 is one of key factors in somatic cell reprogramming, our study will provide new information for deriving iPS cells efficiently and elucidating the molecular network associated with reprogramming.
As a hetereodimer, hypoxia-inducible factor-1 (HIF-1) is composed of two bHLH/PAS-containing subunits, the constitutively presented α subunit and the key regulatory β protein whose stability as well as transcriptional activity is highly regulated by oxygen concentration. HIF-1 is the dominant executor during both physiological and pathophysiological hypoxia, exihibiting so many biological functions that it directly or indirectly regulates almost all hypoxia-related genes. As a result, HIF-1 becomes a famous factor and is reported updatedly almost every day, focusing on the identification and function of new target genes, the regulation of its own stability and transcriptional activity etc. In the past decades, there is increasing evicences showing that hypoxia and/or HIF-1 also modulate (induce or inhibit) differentiation in some kinds of tissue cells, such as hematopoietic stem cells, neural stem cells, myoblast, trophoblast, pulmonary artery adventitial fibroblasts and others. More recently, our group reported that moderate hypoxia and hypoxia-mimetic agents Cobalt Chloride (CoCl2) and Desferrioxamine (DFO) induce Acute Myeloid Leukemia (AML) cells to undergo differentiation in vitro and in vivo, possibly mediated by HIF-1α protein. Thus, this work attempts to further certify whether HIF-1α is the key factor during hypoxia-mediated leukemic cell differentiation, and whether HIF-1α protein is related to all-trans retinoic acid (ATRA)-induced leukemic differentiation. Furthermore, we investigate the molecular mechanisms by which HIF-1α triggers leukemic cell differentiation. To this end, the following original and interesting results were gotten: (1) HIF-1α is indeed the key factor in hypoxia-mediated leukemic cell differentiation. When the expression of HIF-1α was suppressed by specific short hairpin RNA (shRNA), hypoxia/hypoxia-mimetic agents-induced leukemic cell differentiation was remarkably antagonized. On the other hand, inducible expression of HIF-1α in U937 leukemic cells through tetracycline-off gene expression system (Tet-Off) directly triggered granulocytic differentiation in AML cells. (2) HIF-1α plays a role in ATRA-induced leukemic cell differentiation. ATRA rapidly increases endogenous and inducible expressed or CoCl2-stabilized HIF-1α protein of leukemic cells under normoxia. Importantly, we found that inhibited expression of HIF-1α by specific shRNAs partially but significantly blocked ATRA-induced AML cell differentiation. Reciprocally, differentiation-induced effect of ATRA is significantly enhanced by the conditional HIF-1α induction and HIF-1α-stabilizing CoCl2 treatment.(3) Hpoxia/HIF-1α-induced leukemic cell differentiation is independent of its own transcriptional activity but via enhancing the transcriptional activities of the hematopoietic-related transcription factors such as CCAAT/enhancer-binding protein α (C/EBPα) and Runx1. While inhibited expression of HIF-1α by RNAi did decrease the up-regulation of HIF-1 target genes in hypoxia, it failed to influence hypoxia/HIF-1α-mediated differentiation in the same AML cells. Furthermore, based on the expression of macrophage colony-stimulating factor receptor (M-CSFR) is significantly increased, which could be synergistically regulated by C/EBPα, Runx1 and PU.1, we demonstrated that HIF-1α enhanced their transcriptional activities by luciferase assay detection. Furthermore, knock-down of PU.1, Runx1 and C/EBPα by their specific shRNAs, greatly inhibits the differentiation effect of HIF-1α, and the differentiation cooperation of ATRA and HIF-1α induction. Taken together, based on the techniques such as Tet-Off and RNA interference (RNAi), this work provided the direct evidence for the first time that HIF-1α participates in hypoxia and hypoxia-mimetic agents-mediated leukemic cell differentiation. To the surprise, we found that hpoxia/HIF-1α-induced leukemic differentiation is independent of the transcriptional activity of HIF-1, but via enhancing the transcriptional activities of hematopoiesis-related regulators. Furthermore, we demonstrated that ATRA could rapidly stabilize HIF-1α protein, which in turn plays a role in ATRA-induced AML differentiaion. These findings not only extended the roles of HIF-1α, but also generated a particular pathway during hypoxia- and ATRA-mediated leukemic cell differentiation, which would shed new sights for understanding the leukemic cell differentiation and for recognizing new drug targets of differentiation therapy.