Inhibiting Smad2/3 signaling in pluripotent mouse embryonic stem cells enhances endoderm formation by increasing transcriptional priming of lineage-specifying target genes.

BACKGROUND: Pluripotent embryonic stem cells (ESCs) offer great potential for regenerative medicine. However, efficient in vitro generation of specific desired cell types is still a challenge. We previously established that Smad2/3 signaling, essential for endoderm formation, regulates target gene expression by counteracting epigenetic repression mediated by Polycomb Repressive Complex 2 (PRC2). Although this mechanism has been demonstrated during differentiation and reprogramming, little is known of its role in pluripotent cells. RESULTS: Chromatin immunoprecipitation-deep sequencing of undifferentiated mouse ESCs inhibited for Smad2/3 signaling identified Prdm14, important for protecting pluripotency, as a target gene. Although Prdm14 accumulates the normally repressive PRC2 deposited histone modification H3K27me3 under these conditions, surprisingly, expression increases. Analysis indicates that increased H3K27me3 leads to increased binding of PRC2 accessory component Jarid2 and recruitment of RNA polymerase II. Similar increases were found at the Nodal endoderm target gene Eomes but it remained unexpressed in pluripotent cells as normal. Upon differentiation, however, Eomes expression was significantly higher than in cells that had not been inhibited for signaling before differentiation. In addition, endoderm formation was markedly increased. CONCLUSIONS: Blocking Smad2/3 signaling in pluripotent stem cells results in epigenetic changes that enhance the capacity for endoderm differentiation. Developmental Dynamics 245:807-815, 2016. © 2016 Wiley Periodicals, Inc.

Polycomb determines responses to smad2/3 signaling in embryonic stem cell differentiation and in reprogramming.

Integration of extrinsic signals, epigenetic regulators, and intrinsic transcription factors establishes pluripotent stem cell identity. Interplay between these components also underlies the capacity of stem cells to undergo differentiation, and of differentiated cells to re-establish the pluripotent state in direct reprogramming. Polycomb repressive complexes are epigenetic regulators that play key roles in stem cell identity and in differentiated cell fates. Smad2 and Smad3 (Smad2/3), the intracellular mediators of the Nodal/Activin/transforming growth factor (TGF) ß cell-cell signaling pathway also are implicated in stem cell pluripotency and in differentiation. Here, we show that Polycomb imposes responses to Smad2/3-mediated signaling to selectively regulate expression of the master pluripotency factor Oct 4 during initiation of differentiation, but not in the self-renewing pluripotent ground state. During reprogramming back to the ground state, we find that the enhancement of reprogramming efficiency stemming from blocking Nodal/Activin/TGFß signaling also depends on Polycomb. These context-dependent responses to Smad2/3 imposed by Polycomb action provide a mechanism for selective gene regulation that can reconcile the apparently conflicting roles of this signaling pathway in pluripotency, differentiation, and reprogramming.

Nodal signaling recruits the histone demethylase Jmjd3 to counteract polycomb-mediated repression at target genes.

Both intercellular signaling and epigenetic mechanisms regulate embryonic development, but it is unclear how they are integrated to establish and maintain lineage-specific gene expression programs. Here, we show that a key function of the developmentally essential Nodal-Smads2/3 (Smad2 and Smad3) signaling pathway is to recruit the histone demethylase Jmjd3 to target genes, thereby counteracting repression by Polycomb. Smads2/3 bound to Jmjd3 and recruited it to chromatin in a manner that was dependent on active Nodal signaling. Knockdown of Jmjd3 alone substantially reduced Nodal target gene expression, whereas in the absence of Polycomb, target loci were expressed independently of Nodal signaling. These data establish a role for Polycomb in imposing a dependency on Nodal signaling for the expression of target genes and reveal how developmental signaling integrates with epigenetic processes to control gene expression.

The chromatin remodeling factor Mi-2alpha acts as a novel co-activator for human c-Myb.

The c-Myb protein belongs to a group of early hematopoietic transcription factors that are important for progenitor generation and proliferation. These factors have been hypothesized to participate in establishing chromatin patterns specific for hematopoietic genes. In a two-hybrid screening we identified the chromatin remodeling factor Mi-2alpha as an interaction partner for human c-Myb. The main interacting domains were mapped to the N-terminal region of Mi-2alpha and the DNA-binding domain of c-Myb. Surprisingly, functional analysis revealed that Mi-2alpha, previously studied as a subunit in the NuRD co-repressor complex, enhanced c-Myb-dependent reporter activation. Consistently, knock-down of endogenous Mi-2alpha in c-Myb-expressing K562 cells, led to down-regulation of the c-Myb target genes NMU and ADA. When wild-type and helicase-dead Mi-2alpha were compared, the Myb-Mi-2alpha co-activation appeared to be independent of the ATPase/DNA helicase activity of Mi-2alpha. The rationale for the unexpected co-activator function seems to lie in a dual function of Mi-2alpha, by which this factor is able to repress transcription in a helicase-dependent and activate in a helicase-independent fashion, as revealed by Gal4-tethering experiments. Interestingly, desumoylation of c-Myb potentiated the Myb-Mi-2alpha transactivational co-operation, as did co-transfection with p300.

Coreceptor signal strength regulates positive selection but does not determine CD4/CD8 lineage choice in a physiologic in vivo model.

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.

PML mediates IFN-alpha-induced apoptosis in myeloma by regulating TRAIL induction.

Interferon (IFN) induces expression of proapoptotic genes and has been used in the clinical treatment of multiple myeloma. The promyelocytic leukemia (PML) gene is an IFN-induced target that encodes a tumor suppressor protein. PML protein is typically localized within discrete speckled nuclear structures termed PML nuclear bodies (NBs). Multiple myeloma cells demonstrate differential responses to IFN treatment, the mechanism of which is largely unknown. Herein, we show that growth inhibition effects of IFN-alpha in myeloma cells correlate with PML NBs and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induction, whereas known IFN targets including signal transducer and activator of transcription-1 (STAT1), STAT3, p38, and Daxx cannot account for these differential responses. RNAi silencing of PML blocks IFN-alpha-induced apoptosis in myeloma cells and correspondingly down-regulates TRAIL expression. Similarly, stable expression of a dominant negative TRAIL receptor DR5 partially blocks IFN-induced cell death. These results demonstrate that PML and TRAIL play important roles in IFN-induced apoptosis and identify TRAIL as a novel downstream transcriptional target of PML. Identification of PML and PML NBs as effectors of IFN responses provides insights into mechanisms by which tumor cells exhibit resistance to this class of agents and may prove useful in assessing treatment regimens.

c-Myb associates with PML in nuclear bodies in hematopoietic cells.

The c-Myb transcription factor plays a central role in the regulation of cell growth and differentiation of hematopoietic cells. Being the product of a proto-oncogene, one would expect c-Myb function to be modulated by signal transduction pathways, but our knowledge on such regulation of c-Myb is rather limited. Recently, we and others showed that c-Myb is subjected to sumoylation and that this posttranslational modification has considerable effect on c-Myb's activity. Interestingly, many proteins subjected to SUMO-1 conjugation associate with the promyelocytic leukemia (PML) protein and localize to PML nuclear bodies (PML NBs). Although the precise molecular function of PML NBs still remains to be defined, they seem to play a role in regulation of gene expression and are linked to specific cellular signaling. We show here that c-Myb localizes to PML NBs and that c-Myb interacts with PML as judged by immunofluorescence microcopy and co-immunoprecipitation experiments. Enforced expression of PML IV was shown to enhance c-Myb-dependent reporter activation. Our results imply a role for PML and possibly other components of PML NBs in regulating c-Myb's activity. This novel link between c-Myb and PML, two gene products being implicated in leukemic disorders, suggests that previously unknown mechanisms for regulating c-Myb's activity involving PML may exist.

Transactivation properties of c-Myb are critically dependent on two SUMO-1 acceptor sites that are conjugated in a PIASy enhanced manner.

The transcription factor v-Myb is a potent inducer of myeloid leukemias, and its cellular homologue c-Myb plays a crucial role in the regulation of hematopoiesis. Recently, Bies and coworkers (Bies, J., Markus, J. & Wolff, L. (2002) J. Biol. Chem, 277, 8999-9009) presented evidence that murine c-Myb can be sumoylated under overexpression conditions in COS7 cells when cotransfected with FLAG-tagged SUMO-1. Here we provide independent evidence that human c-Myb is also subject to SUMO-1 conjugation under more physiological conditions as revealed by coimmunoprecipitation analysis of Jurkat cells and transfected CV-1 cells. Analysis in an in vitro conjugation system showed that modification of the two sites K503 and K527 is interdependent. A two-hybrid screening revealed that the SUMO-1 conjugase Ubc9 is one of a few major Myb-interacting proteins. The moderate basal level of sumoylation was greatly enhanced by cotransfection of PIASy, an E3 ligase for SUMO-1. The functional consequence of abolishing sumoylation was enhanced activation both of a transiently transfected reporter gene and of a resident Myb-target gene. When single and double mutants were compared, we found a clear correlation between reduction in sumoylation and increase in transcriptional activation. Enhancing sumoylation by contransfection of PIASy had a negative effect on both Myb-induced and basal level reporter activation. Furthermore, PIASy caused a shift in nuclear distribution of c-Myb towards the insoluble matrix fraction. We propose that the negative influence on transactivation properties by the negative regulatory domain region of c-Myb depends on the sumoylation sites located here.