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 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 twohybrid 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.Keywords: c-Myb; transcription; SUMO-1; Ubc9; PIASy.The c-Myb transcription factor plays a central role in the regulation of cell growth and differentiation, in particular in hematopoietic progenitor cells (reviewed in [1]). Homozygous null c-Myb/Rag1 chimerical mice are blocked in early T-cell development, while mice with a c-myb null mutation display severe hematopoietic defects leading to in utero death at E15 [2,3]. The c-Myb protein consists of an N-terminal DNA-binding domain (DBD), a central transactivation domain (TAD) and a C-terminal negative regulatory domain (NRD). The DBD of c-Myb is comprised of the three imperfect repeats: R 1 , R 2 and R 3 , each related to the helix-turn-helix motif [4][5][6][7].Oncogenic alterations, as found in AMV v-Myb, include both N-and C-terminal deletions as well as point mutations [8]. AMV v-myb is a potent and cell-type specific oncogene that transforms target cells in the macrophage lineage and induces monocytic leukemia [8,9]. Several studies have attempted to define oncogenic determinants of v-myb. N-and C-terminal deletions remove several sites of protein modification, including an N-terminal CK2 phosphorylation site (S11 and S12) [10], and a putative MAPK-site (S528) [11][12][13] as well as acetylation sites [14,15] located in the deleted portion of the C-terminal NRD. In addition, specific point mutations in v-My...

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-2␣ as an interaction partner for human c-Myb. The main interacting domains were mapped to the N-terminal region of Mi-2␣ and the DNA-binding domain of c-Myb. Surprisingly, functional analysis revealed that Mi-2␣, previously studied as a subunit in the NuRD corepressor complex, enhanced c-Myb-dependent reporter activation. Consistently, knock-down of endogenous Mi-2␣ 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-2␣ were compared, the Myb-Mi-2␣ co-activation appeared to be independent of the ATPase/DNA helicase activity of Mi-2␣. The rationale for the unexpected co-activator function seems to lie in a dual function of Mi-2␣, 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-2␣ transactivational co-operation, as did co-transfection with p300.

The c-Myb oncoprotein is a DNA-binding transcription factor with a key role in early stages of hematopoiesis. To expand our knowledge of partners cooperating with c-Myb, we performed a yeast two-hybrid screening with full-length c-Myb as bait. Here, we report FLICEassociated huge protein (FLASH)/CASP8AP2 as a novel Myb-interacting protein. We show that FLASH interacts with the DNA-binding domain of c-Myb and enhances c-Myb-dependent reporter activity and expression of endogenous c-Myb target genes. Chromatin immunoprecipitation assays revealed that FLASH and c-Myb both associate with the MYC promoter region as well as with the intronic enhancer of the c-Myb target gene ADA. Furthermore, siRNA knock-down of FLASH or c-Myb both result in a reduction of MYC and ADA expression. The co-activator effect is mediated through the C-terminal part of FLASH, which binds c-Myb. The FLASH-induced enhancement is comparable with the increase seen with the c-Myb co-activator p300. We find FLASH localized in discrete nuclear speckles in several cell lines, co-localized with c-Myb in active RNA polymerase II foci. These results imply a novel molecular mechanism of regulation of c-Myb activity. We propose that c-Myb cooperates with FLASH in foci associated with active RNA polymerase II, leading to enhancement of Myb-dependent gene activation.

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 CD8α gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8α 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.

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.

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