Fern E. Murdoch

The role of ligand in estrogen receptor regulation of gene expression

We believe that steroid binding is not required for receptor binding to DNA, but instead induces a conformational change in the receptor domains involved in the protein-protein interactions proposed above. Data from Hansen and Gorski (1986), and more recent studies (M. Fritsch and J. Gorski, unpublished results) strongly suggest that the steroid binding domain when bound to estrogens undergoes a dramatic change in conformation characterized by a loss of hydrophobic surface. This marked change in the steroid binding domain probably affects the so-called dimerization region located in this domain and thus the interaction of receptor with nuclear proteins in vivo. In our working model, ER is bound to specific DNA sequences or response elements of a variety of genes with or without estrogen. Ligand binding induces conformational changes in the steroid binding and perhaps other domains of the receptor that in turn change receptor interaction with the transcriptional machinery. The nature of this change is not at all clear at present, and the possibility of enzymatic modification of receptor or associated transcription factors should not be excluded. Whatever the mechanism of receptor action on transcription, we expect it kinetically will be closely related to the occupancy of the receptor with estrogen. Finally, any model of ER interactions with target genes also needs to account for the drastic ligand effect on the extractability of all ER from the nucleus.

High Histone Acetylation and Decreased Polycomb Repressive Complex 2 Member Levels Regulate Gene Specific Transcriptional Changes During Early Embryonic Stem Cell Differentiation Induced by Retinoic Acid

Histone modifications play a crucial role during embryonic stem (ES) cell differentiation. During differentiation, binding of polycomb repressive complex 2 (PRC2), which mediates trimethylation of lysine 27 on histone H3 (K27me3), is lost on developmental genes that are transcriptionally induced. We observed a global decrease in K27me3 in as little as 3 days after differentiation of mouse ES cells induced by retinoic acid (RA) treatment. The global levels of the histone K27 methyltransferase EZH2 also decreased with RA treatment. A loss of EZH2 binding and K27me3 was observed locally on PRC2 target genes induced after 3 days of RA, including Nestin. In contrast, direct RA‐responsive genes that are rapidly induced, such as Hoxa1, showed a loss of EZH2 binding and K27me3 after only a few hours of RA treatment. Following differentiation induced by leukemia inhibitor factor (LIF) withdrawal without RA, Hoxa1 was not transcriptionally activated. Small interfering RNA‐mediated knockdown of EZH2 resulted in loss of K27me3 during LIF withdrawal, but the Hoxa1 gene remained transcriptionally silent after loss of this repressive mark. Induction of histone hyperacetylation overrode the repressive K27me3 modification and resulted in Hoxa1 gene expression. Together, these data show that there are multiple temporal phases of derepression of PRC2 target genes during ES cell differentiation and that other epigenetic marks (specifically, increased acetylation of histones H3 and H4), in addition to derepression, are important for gene‐specific transcriptional activation. This report demonstrates the temporal interplay of various epigenetic changes in regulating gene expression during early ES cell differentiation.

The Role of Histone Acetylation in Regulating Early Gene Expression Patterns during Early Embryonic Stem Cell Differentiation

We have examined the role of histone acetylation in the very earliest steps of differentiation of mouse embryonic stem cells in response to withdrawal of leukemia inhibitory factor (LIF) as a differentiation signal. The cells undergo dramatic changes in morphology and an ordered program of gene expression changes representing differentiation to all three germ layers over the first 3-5 days of LIF withdrawal. We observed a global increase in acetylation on histone H4 and to a lesser extent on histone H3 over this time period. Treatment of the cells with trichostatin A (TSA), a histone deacetylase inhibitor, induced changes in morphology, gene expression, and histone acetylation that mimicked differentiation induced by withdrawal of LIF. We examined localized histone acetylation in the regulatory regions of genes that were transcriptionally either active in undifferentiated cells, induced during differentiation, or inactive under all treatments. There was striking concordance in the histone acetylation patterns of specific genes induced by both TSA and LIF withdrawal. Increased histone acetylation in local regions correlated best with induction of gene expression. Finally, TSA treatment did not support the maintenance or progression of differentiation. Upon removal of TSA, the cells reverted to the undifferentiated phenotype. We concluded that increased histone acetylation at specific genes played a role in their expression, but additional events are required for maintenance of differentiated gene expression and loss of the pluripotent state.

Dynamic changes in histone H3 phosphoacetylation during early embryonic stem cell differentiation are directly mediated by mitogen- and stress-activated protein kinase 1 via activation of MAPK pathways

Embryonic stem (ES) cells are pluripotent cells capable of unlimited self-renewal and differentiation into the three embryonic germ layers under appropriate conditions. Mechanisms for control of the early period of differentiation, involving exit from the pluripotent state and lineage commitment, are not well understood. An emerging concept is that epigenetic histone modifications may play a role during this early period. We have found that upon differentiation of mouse ES cells by removal of the cytokine leukemia inhibitory factor, there is a global increase in coupled histone H3 phosphorylation (Ser-10)-acetylation (Lys-14) (H3 phosphoacetylation). We show that this occurs through activation of both the extracellular signal-regulated kinase (ERK) and p38 MAPK signaling pathways. Early ES cell differentiation is delayed using pharmacological inhibitors of the ERK and p38 pathways. One common point of convergence of these pathways is the activation of the mitogen- and stress-activated protein kinase 1 (MSK1). We show here that MSK1 is the critical mediator of differentiation-induced H3 phosphoacetylation using both the chemical inhibitor H89 and RNA interference. Interestingly, inhibition of H3 phosphoacetylation also alters gene expression during early differentiation. These results point to an important role for both epigenetic histone modifications and kinase pathways in modulating early ES differentiation.

Activation of Wnt/β-catenin Signaling in Distinct Histologic Subtypes of Human Germ Cell Tumors

The molecular signaling pathways mediating human germ cell tumor (GCT) formation and progression are poorly understood despite a large number of studies detailing recurrent cytogenetic abnormalities. Germ cell tumors consist of multiple histologic subtypes and can also be divided into infantile/childhood or adolescent/adult tumors as well as gonadal or nongonadal sites of origin. All of these parameters are important in defining clinical outcome and in understanding the pathogenesis of these tumors. We utilized complementary DNA (cDNA) microarray analysis to identify differences in signal transduction pathways between 2 histologic subtypes of malignant ovarian GCTs (dysgerminomas versus ovarian endodermal sinus tumors). Hierarchical cluster analysis using only the genes involved in Wnt/β-catenin signaling was able to distinguish these 2 tumor subtypes from each other. Wnt13 and β-catenin showed significant differential expression patterns between the 2 tumor subtypes, and the results were confirmed by semiquantitative reverse transcriptase–polymerase chain reaction. Additional GCTs were studied for the expression of other members of Wnt/β-catenin signaling, including Wnt13, frizzled, disheveled, low-density lipoprotein receptor-related protein 6, and β-catenin. Differential expression levels were identified for several histologic subtypes of human GCTs. Finally, we prepared tissue microarrays containing GCTs from 83 different patients and demonstrated high levels of β-catenin protein expression in 100% and nuclear accumulation in approximately 50% to 70% of all endodermal sinus tumors and immature teratomas (ITs). This pattern was independent of the patients age. No nuclear accumulation of β-catenin was observed in germinomas, embryonal carcinomas, or choriocarcinomas. These results indicate that activation of Wnt/β-catenin signaling plays an important role in the pathogenesis of 2 histologic subtypes of human GCTs.

CHROMATIN CONTEXT DOMINATES ESTROGEN REGULATION OF pS2 GENE EXPRESSION

Chromatin structure and transcription factor activity collaborate to set the transcription level of a gene. Our understanding of the relative contributions of each of these factors at a specific gene is limited. We studied the effects of an altered chromatin environment on the activity of the estrogen-responsive pS2 promoter. We created stable cell lines with the pS2 promoter situated in an alternative chromatin site in addition to it being in its native site. Both promoters were estrogen-responsive for estrogen receptor alpha (ERalpha) recruitment, but transcription was inducible only at the native site. At the recombinant site, transcription was high and constitutive. Higher histone H3 and H4 acetylation (acH3 and acH4), as well as trimethylated lysine 4 on histone H3 levels, was observed at the recombinant site compared to the native site in vehicle treated cells. Inhibition of histone deacetylases (HDACs) resulted in increased acH4, but only modest increases in acH3, ERalpha binding and basal transcription at the native pS2 site. Inhibiting HDACs had no effect on transcription from the recombinant site. These data suggest that highly active chromatin is not only permissive for transcription, but can override the requirement for the transcription factor at an inducible promoter.