The adaptor protein ARA55 and the nuclear kinase HIPK1 assist c-Myb in recruiting p300 to chromatin.

LIM-domain proteins, containing multiple cysteine-rich zinc finger-like motifs, have been shown to play diverse roles in several cellular processes. A common theme is that they mediate important protein-protein interactions that are key to their function. Androgen receptor-associated protein 55 (ARA55) belongs to this family of bridging proteins containing four C-terminal LIM domains. It has a dual role with functions both at focal adhesions and in the nucleus, apparently shuttling between the two compartments. In the present work, we have expanded our understanding of its nuclear functions by showing that it interacts with three nuclear regulators not previously linked to ARA55. We first identified ARA55 as a novel interaction partner of the nuclear kinase HIPK1 and found that ARA55, like HIPK1, also interacts with the transcription factor c-Myb. In search of a function for these associations, we observed that the coactivator p300 not only binds to c-Myb, but to ARA55 as well. When combined, c-Myb, p300, HIPK1 and ARA55 caused strong synergistic activation of a chromatinized reporter gene. In parallel, all partners, including p300, were efficiently recruited to chromatin at the c-Myb-bound promoter. Consistent with this cooperation, we found that c-Myb and ARA55 share a common set of target genes in an osteosarcoma cellular context. We propose that ARA55 and HIPK1 assist c-Myb in recruiting the coactivator and acetyltransferase p300 to chromatin.

The syndrome of central hypothyroidism and macroorchidism: IGSF1 controls TRHR and FSHB expression by differential modulation of pituitary TGFß and Activin pathways.

IGSF1 (Immunoglobulin Superfamily 1) gene defects cause central hypothyroidism and macroorchidism. However, the pathogenic mechanisms of the disease remain unclear. Based on a patient with a full deletion of IGSF1 clinically followed from neonate to adulthood, we investigated a common pituitary origin for hypothyroidism and macroorchidism, and the role of IGSF1 as regulator of pituitary hormone secretion. The patient showed congenital central hypothyroidism with reduced TSH biopotency, over-secretion of FSH at neonatal minipuberty and macroorchidism from 3 years of age. His markedly elevated inhibin B was unable to inhibit FSH secretion, indicating a status of pituitary inhibin B resistance. We show here that IGSF1 is expressed both in thyrotropes and gonadotropes of the pituitary and in Leydig and germ cells in the testes, but at very low levels in Sertoli cells. Furthermore, IGSF1 stimulates transcription of the thyrotropin-releasing hormone receptor (TRHR) by negative modulation of the TGFß1-Smad signaling pathway, and enhances the synthesis and biopotency of TSH, the hormone secreted by thyrotropes. By contrast, IGSF1 strongly down-regulates the activin-Smad pathway, leading to reduced expression of FSHB, the hormone secreted by gonadotropes. In conclusion, two relevant molecular mechanisms linked to central hypothyroidism and macroorchidism in IGSF1 deficiency are identified, revealing IGSF1 as an important regulator of TGFß/Activin pathways in the pituitary.

PIAS1 binds p300 and behaves as a coactivator or corepressor of the transcription factor c-Myb dependent on SUMO-status.

The PIAS proteins (Protein Inhibitor of Activated STATs) constitute a family of multifunctional nuclear proteins operating as SUMO E3 ligases and being involved in a multitude of interactions. They participate in a range of biological processes, also beyond their well-established role in the immune system and cytokine signalling. They act both as transcriptional corepressors and coactivators depending on the context. In the present work, we investigated mechanisms by which PIAS1 causes activation or repression of c-Myb dependent target genes. Analysis of global expression data shows that c-Myb and PIAS1 knockdowns affect a subset of common targets, but with a dual outcome consistent with a role of PIAS1 as either a corepressor or coactivator. Our mechanistic studies show that PIAS1 engages in a novel interaction with the acetyltransferase and coactivator p300. Interaction and ChIP analysis suggest a bridging function where PIAS1 enhances p300 recruitment to c-Myb-bound sites through interaction with both proteins. In addition, the E3 activity of PIAS1 enhances further its coactivation. Remarkably, the SUMO status of c-Myb had a decisive role, indicating a SUMO-dependent switch in the way PIAS1 affects c-Myb, either as a coactivator or corepressor. Removal of the two major SUMO-conjugation sites in c-Myb (2KR mutant), which enhances its activity significantly, turned PIAS1 into a corepressor. Also, p300 was less efficiently recruited to chromatin by c-Myb-2KR. We propose that PIAS1 acts as a "protein inhibitor of activated c-Myb" in the absence of SUMOylation while, in its presence, PIAS behaves as a "protein activator of repressed c-Myb".

PIAS1 interacts with FLASH and enhances its co-activation of c-Myb.

BACKGROUND: FLASH is a huge nuclear protein involved in various cellular functions such as apoptosis signalling, NF-κB activation, S-phase regulation, processing of histone pre-mRNAs, and co-regulation of transcription. Recently, we identified FLASH as a co-activator of the transcription factor c-Myb and found FLASH to be tightly associated with active transcription foci. As a huge multifunctional protein, FLASH is expected to have many interaction partners, some which may shed light on its function as a transcriptional regulator. RESULTS: To find additional FLASH-associated proteins, we performed a yeast two-hybrid (Y2H) screening with FLASH as bait and identified the SUMO E3 ligase PIAS1 as an interaction partner. The association appears to involve two distinct interaction surfaces in FLASH. We verified the interaction by Y2H-mating, GST pulldowns, co-IP and ChIP. FLASH and PIAS1 were found to co-localize in nuclear speckles. Functional assays revealed that PIAS1 enhances the intrinsic transcriptional activity of FLASH in a RING finger-dependent manner. Furthermore, PIAS1 also augments the specific activity of c-Myb, and cooperates with FLASH to further co-activate c-Myb. The three proteins, FLASH, PIAS1, and c-Myb, are all co-localized with active RNA polymerase II foci, resembling transcription factories. CONCLUSIONS: We conclude that PIAS1 is a common partner for two cancer-related nuclear factors, c-Myb and FLASH. Our results point to a functional cooperation between FLASH and PIAS1 in the enhancement of c-Myb activity in active nuclear foci.

A SUMO-regulated activation function controls synergy of c-Myb through a repressor-activator switch leading to differential p300 recruitment.

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.

HIPK1 interacts with c-Myb and modulates its activity through phosphorylation.

The transcription factor v-Myb is a potent inducer of myeloid leukaemias, and its cellular homologue c-Myb plays a crucial role in the regulation of haematopoiesis. In a yeast two-hybrid (Y2H) screening we identified the nuclear kinase HIPK1 as an interaction partner for human c-Myb. The interaction involves a C-terminal region of HIPK1, while a bipartite interaction surface was identified in c-Myb, including regions in its N-terminal DNA-binding domain as well as in its C-terminal region. HIPK1 and c-Myb co-localize in distinct nuclear foci upon co-transfection. c-Myb appears to be phosphorylated by HIPK1 in its negative regulatory domain as supported by both in vivo and in vitro data. A functional assay revealed that HIPK1 repressed the ability of c-Myb to activate a chromatin embedded target gene, mim-1, in haematopoetic cells. Our findings point to a novel link between an important kinase and a key regulator of haematopoiesis.

SUMO modification regulates the transcriptional activity of FLASH.

FLASH is a huge multifunctional nuclear protein that has been linked to apoptotic signalling, transcriptional control and Cajal body function. To gain further insight into the functions of the FLASH protein, we performed a yeast two-hybrid screening with FLASH as bait and identified the SUMO-conjugating enzyme Ubc9 as an interaction partner. The main interaction surface for Ubc9 was found in the C-terminal part of FLASH, which is also a target for sumoylation. We identified K1813 as the major sumoylation site in FLASH, being enhanced by the SUMO E3 ligases Pc2 and PIASy. Disruption of this SUMO-conjugation site did not change the speckled subnuclear localization of FLASH, but it caused a reduction in FLASH activity as measured in a Gal4-tethering assay. Interestingly, the SUMO-specific protease SENP1 activated FLASH in the same assay. Overall, our results point to a complex involvement of sumoylation in modulating the function of FLASH.

The transcription factor c-Myb affects pre-mRNA splicing.

c-Myb is a transcription factor which plays a key role in haematopoietic proliferation and lineage commitment. We raised the question of whether c-Myb may have abilities beyond the extensively studied transcriptional activation function. In this report we show that c-Myb influences alternative pre-mRNA splicing. This was seen by its marked effect on the 5'-splice site selection during E1A alternative splicing, while no effect of c-Myb was observed when reporters for the 3'-splice site selection or for the constitutive splicing process were tested. Moreover, co-immunoprecipitation experiments provided evidence for interactions between c-Myb and distinct components of the splicing apparatus, such as the general splicing factor U2AF(65) and hnRNPA1 involved in the 5'-splice site selection. The effect on 5'-splice site selection was abolished in the oncogenic variant v-Myb. Altogether, these data provide evidence that c-Myb may serve a previously unappreciated role in the coupling between transcription and splicing.

Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator.

BACKGROUND: Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1. RESULTS: NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa. It is localized on chromosome 1 and consists of 6 exons. Analysis of the amino acid sequence revealed no known transcription activation domains or DNA binding regions, however, four nuclear receptor boxes (LXXLL), and four SH3-interaction motives in addition to numerous potential phosphorylation sites were found. Two nuclear export signals were identified, but no nuclear localization signal. In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney. Electrophoretic mobility shift analysis showed specific binding of NCU-G1 to an oligonucleotide representing the footprint 1 element of the human cellular retinol-binding protein 1 gene promoter. NCU-G1 was found to activate transcription from this promoter and required presence of the footprint 1 element. In transiently transfected Drosophila Schneider S2 cells, we demonstrated that NCU-G1 functions as a co-activator for ligand-activated PPAR-alpha, resulting in an increased expression of a CAT reporter gene under control of the peroxisome proliferator-activated receptor-alpha responsive acyl-CoA oxidase promoter. CONCLUSION: We propose that NCU-G1 is a dual-function protein capable of functioning as a transcription factor as well as a nuclear receptor co-activator.

Revisiting a selection of target genes for the hematopoietic transcription factor c-Myb using chromatin immunoprecipitation and c-Myb knockdown.

The transcription factor c-Myb is an important regulator of hematopoiesis required for proper development of most blood cell lineages in vertebrates. An increasing number of target genes for c-Myb are being published, although with little or no overlap between the lists of genes reported. This raises the question of which criteria a bona fide c-Myb-target gene should satisfy. In the present paper, we have analyzed a set of previously reported target genes using chromatin immunoprecipitation (ChIP) and siRNA-mediated knockdown. Among the seven well-studied c-Myb target genes that we analyzed by ChIP, only ADA, c-MYC and MAT2A seemed to be occupied by c-Myb under our experimental settings in the Myb-positive cell lines Jurkat and HL60. After siRNA-mediated knockdown of c-Myb expression, the expression levels of two out of three ChIP positive Myb target genes, ADA and c-MYC, were strongly affected. These results clearly demonstrate the importance of combining different methods for target gene validation and suggest that a combination of ChIP and c-Myb knockdown may represent a powerful approach to identify a core collection of c-Myb target genes.

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.

Thyrotrophin-releasing hormone receptor 1 and prothyrotrophin-releasing hormone mRNA expression in the central nervous system are regulated by suckling in lactating rats.

BACKGROUND: The accepted function of the hypothalamic peptide, thyrotrophin-releasing hormone (TRH), is to initiate release of thyrotrophin (TSH) from the pituitary. A physiological role for TRH in lactating rats has not yet been established. METHODS: Tissues were prepared from random-cycling and lactating rats and analysed using Northern blot, real time RT-PCR and quantitative in situ hybridisation. RESULTS: This study demonstrates that TRH receptor 1 (TRHR1) mRNA expression is up-regulated in the pituitary and in discrete nuclei of the hypothalamus in lactating rats, while proTRH mRNA expression levels are increased only in the hypothalamus. The results were corroborated by quantitative in situ analysis of proTRH and TRHR1. Bromocriptine, which reduced prolactin (PRL) concentrations in plasma of lactating and nursing rats, also counteracted the suckling-induced increase in TRHR1 mRNA expression in the hypothalamus, but had an opposite effect in the pituitary. These changes were confined to the hypothalamus and the amygdala in the brain. CONCLUSIONS: The present study shows that the mechanisms of suckling-induced lactation involve region-specific regulation of TRHR1 and proTRH mRNAs in the central nervous system notably at the hypothalamic level. The results demonstrate that continued suckling is critical to maintain plasma prolactin (PRL) levels as well as proTRH and TRHR1 mRNA expression in the hypothalamus. Increased plasma PRL levels may have a positive modulatory role on the proTRH/TRHR1 system during suckling.

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.

The human neuroendocrine thyrotropin-releasing hormone receptor promoter is activated by the haematopoietic transcription factor c-Myb.

Thyrotropin-releasing hormone (TRH) receptor (TRHR) is a G-protein-coupled receptor playing a crucial role in the anterior pituitary where it controls the synthesis and secretion of thyroid-stimulating hormone and prolactin. Its widespread presence not only in the central nervous system, but also in peripheral tissues, including thymus, indicates other important, but unknown, functions. One hypothesis is that the neuropeptide TRH could play a role in the immune system. We report here that the human TRHR promoter contains 11 putative response elements for the haematopoietic transcription factor c-Myb and is highly Myb-responsive in transfection assays. Analysis of Myb binding to putative response elements revealed one preferred binding site in intron 1 of the receptor gene. Transfection studies of promoter deletions confirmed that this high-affinity element is necessary for efficient Myb-dependent transactivation of reporter plasmids in CV-1 cells. The Myb-dependent activation of the TRHR promoter was strongly suppressed by expression of a dominant negative Myb-Engrailed fusion. In line with these observations, reverse transcriptase PCR analysis of rat tissues showed that the TRHR gene is expressed both in thymocytes and bone marrow. Furthermore, specific, high-affinity TRH agonist binding to cell-surface receptors was demonstrated in thymocytes and a haematopoietic cell line. Our findings imply a novel functional link between the neuroendocrine and the immune systems at the level of promoter regulation.