Synergy between transcription factors operating together on complex promoters is a key aspect of gene activation. The ability of specific factors to synergize is restricted by sumoylation (synergy control, SC). Focusing on the haematopoietic transcription factor c-Myb, we found evidence for a strong SC linked to SUMO-conjugation in its negative regulatory domain (NRD), while AMV v-Myb has escaped this control. Mechanistic studies revealed a SUMO-dependent switch in the function of NRD. When NRD is sumoylated, the activity of c-Myb is reduced. When sumoylation is abolished, NRD switches into being activating, providing the factor with a second activation function (AF). Thus, c-Myb harbours two AFs, one that is constitutively active and one in the NRD being SUMO-regulated (SRAF). This double AF augments c-Myb synergy at compound natural promoters. A similar SUMO-dependent switch was observed in the regulatory domains of Sp3 and p53. We show that the change in synergy behaviour correlates with a SUMO-dependent differential recruitment of p300 and a corresponding local change in histone H3 and H4 acetylation. We therefore propose a general model for SUMO-mediated SC, where SUMO controls synergy by determining the number and strength of AFs associated with a promoter leading to differential chromatin signatures.

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.

The c-Myb transcription factor is an important regulator of hematopoietic cell development. c-Myb is expressed in immature hematopoietic cells and plays a direct role in lineage fate selection, cell cycle progression, and differentiation of myeloid as well as B- and T-lymphoid progenitor cells. As a DNA-binding transcription factor, c-Myb regulates specific gene programs through activation of target genes. Still, our understanding of these programs is incomplete. Here, we report a set of novel c-Myb target genes, identified using a combined approach: specific c-Myb knockdown by 2 different siRNAs and subsequent global expression profiling, combined with the confirmation of direct binding of c-Myb to the target promoters by ChIP assays. The combination of these 2 approaches, as well as additional validation such as cloning and testing the promoters in reporter assays, confirmed that MYADM, LMO2, GATA2, STAT5A, and IKZF1 are target genes of c-Myb. Additional studies, using chromosome conformation capture, demonstrated that c-Myb target genes may directly interact with each other, indicating that these genes may be coordinately regulated. Of the 5 novel target genes identified, 3 are transcription factors, and one is a transcriptional co-regulator, supporting a role of c-Myb as a master regulator controlling the expression of other transcriptional regulators in the hematopoietic system.

Enhancer regions and transcription start sites of estrogen-target regulated genes are connected by means of Estrogen Receptor long-range chromatin interactions. Yet, the complete molecular mechanisms controlling the transcriptional output of engaged enhancers and subsequent activation of coding genes remain elusive. Here, we report that CTCF binding to enhancer RNAs is enriched when breast cancer cells are stimulated with estrogen. CTCF binding to enhancer regions results in modulation of estrogen-induced gene transcription by preventing Estrogen Receptor chromatin binding and by hindering the formation of additional enhancer-promoter ER looping. Furthermore, the depletion of CTCF facilitates the expression of target genes associated with cell division and increases the rate of breast cancer cell proliferation. We have also uncovered a genomic network connecting loci enriched in cell cycle regulator genes to nuclear lamina that mediates the CTCF function. The nuclear lamina and chromatin interactions are regulated by estrogen-ER. We have observed that the chromatin loops formed when cells are treated with estrogen establish contacts with the nuclear lamina. Once there, the portion of CTCF associated with the nuclear lamina interacts with enhancer regions, limiting the formation of ER loops and the induction of genes present in the loop. Collectively, our results reveal an important, unanticipated interplay between CTCF and nuclear lamina to control the transcription of ER target genes, which has great implications in the rate of growth of breast cancer cells.

Transcriptional activity of the TATA-binding protein (TBP) is controlled by a variety of proteins. The BTAF1 protein (formerly known as TAF II 170/TAF-172 and the human ortholog of Saccharomyces cerevisiae Mot1p) and the NC2 complex composed of NC2␣ (DRAP1) and NC2␤ (Dr1) are able to bind to TBP directly and regulate RNA polymerase II transcription both positively and negatively. Here, we present evidence that the NC2␣ subunit interacts with BTAF1. In contrast, the NC2␤ subunit is not able to associate with BTAF1 and seems to interfere with the BTAF1-TBP interaction. Addition of NC2␣ or the NC2 complex can stimulate the ability of BTAF1 to interact with TBP. This function is dependent on the presence of ATP in cell extracts but does not involve the ATPase activity of BTAF1 nor phosphorylation of NC2␣. Together, our results constitute the first evidence of the physical cooperation between BTAF1 and NC2␣ in TBP regulation and provide a framework to understand transcription functions of NC2␣ and NC2␤ in vivo.Initiation of gene transcription by eukaryotic RNA polymerase II (pol II) is tightly controlled by a multitude of regulatory factors. The concerted action of these factors results in the formation of the pol II preinitiation complex (32, 33). Recent studies have deepened our knowledge of the regulation of the steps leading to this. It is also becoming clear that the mode and sequence of the recruitment of the basal transcription factors vary among promoters (7). The preinitiation complex consists of several basal transcription factors, including TATAbinding protein (TBP), which plays a central role in the assembly process. This is underscored by the fact that transcription of the majority of cellular genes in vivo requires TBP (11). In human cells several factors were shown to bind directly to and regulate the activity of TBP in pol II transcription. The beststudied factors are TBP-associated factors (TAFs), which together with TBP form the TFIID complex (25,40). Others include the BTAF1 protein (TAF II 170/TAF-172) and the NC2 (Dr1-DRAP1) complex.BTAF1 and its Saccharomyces cerevisiae ortholog Mot1p form stable complexes with TBP in cell extracts (37,38,43,44; for a review, see reference 35). The observation that a large proportion of TBP is complexed with BTAF1 as the B-TFIID complex gives rise to the notion that BTAF1 is an important regulator of TBP function (44). Indeed, in vitro studies show that the B-TFIID complex is able to bind promoter DNA and support transcription (18,34,44). On the other hand, BTAF1 and Mot1p proteins contain dATPase activity, which is involved in the dissociation of TBP from DNA in an ATPdependent stroke. This activity can explain their negative effect on transcription (5,34,36). In accordance with a dual role of Mot1p in transcription, mRNA expression profiling and mutational analyses indicate that Mot1p affects transcription both positively and negatively (1,6,10,12,15,26,27,39). The positive role of Mot1p is strengthened by observations that it is present at the sites of certai...