Symptoms in advanced pancreatic cancer are of importance for energy intake

Purpose Cancer cachexia and low energy intake (EI) probably contribute to weight loss in advanced pancreatic cancer (PC). However, little is known about the actual EI in this disease. Aims were to assess EI, weight loss and symptoms during the disease course and investigate associations between symptoms and EI. Methods Thirty-nine patients (21 males) with advanced PC were consecutively included and followed every 4 weeks until the end of life. A 24-h dietary recall was used to assess EI. The Edmonton Symptom Assessment System (ESAS) and the PC-specific health-related quality of life questionnaire (QLQ-PAN26) were used for symptom assessment. Results Median age was 62 years (48–88), WHO performance status 1 (0–2) and survival 5 months (1–25). Seventeen (44 %) patients had unresectable cancer, 16 (41 %) metastatic and six (15 %) recurrent disease. Upon inclusion, 37 (95 %) reported weight loss (median 4.0 kg per month). During follow-up, median weight loss per month was <1.0 kg. Forty to 65 % had EI <29 kcal/kg/day (cut-off value for weight maintenance) during the observation period but they did not lose more weight than patients with EI ≥ 29 kcal. Strong negative correlations (r range) were found between EI and pain (0.51–0.61), fatigue (0.54–0.67), oral dryness (0.61–0.64) and loss of appetite (0.53–0.71). Conclusion In this study, several symptoms influenced EI negatively. Low EI did not completely explain weight loss in this patient group, but careful monitoring and early follow-up of symptoms may be important interventions to reduce weight loss in advanced PC

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Studier av interaksjonen mellom oncoproteinet c-Myb og kromatin remodelleringsfaktoren Mi2.

Sammendrag: I en celle foregår det tusenvis av forskjellige prosesser og interaksjoner til enhver tid, og regulering av genaktivitet har stor betydning i dette samspillet. Mange faktorer er med på å regulere transkripsjon, og blant dem finnes også transkripsjonsfaktoren c-Myb. c-Myb er involvert regulering av gener som har betydning for proliferering (cellevekst), differensiering (modning) og apoptose (programmert celledød). Faktorer som er involvert i regulering av gener virker sammen i et komplisert nettverk av proteiner. Protein-protein interaksjoner spiller derfor en avgjørende rolle for de fleste biologiske prosesser. For å kunne forstå noe av dette samspillet må man først finne ut hvilke faktorer som faktisk samarbeider eller direkte regulerer hverandre. For å identifisere nye interaksjonspartnere til c-Myb utførte Øyvind Dahle en tohybrid screening i gjær. Resultatet var fem nye potensielle interaksjonspartnere til c-Myb. En av disse var Mi2, eller nærmere bestemt det C-terminale domenet av dette proteinet, og det er c-Mybs interaksjon med dette proteinet som er studert i denne oppgaven. Det c-Myb-interagerende domenet av Mi2 har vi kalt MID, for Myb interacting domain . Mi2 er en kromatin remodelleringsfaktor assosiert med repressorkomplekset NuRD. Målet med denne oppgaven var å verifisere og karakterisere Mi2 som interaksjonspartner til c-Myb. Dette ble gjort ved in vitro forsøk. Først ble interaksjonen mellom c-Myb og MID bekreftet og lokalisert til FAETL-domenet av c-Myb. Interaksjonen med MID var like sterk for en onkogen variant av c-Myb (AMV v-Myb) som for c-Myb. Mutasjoner som blokkerer for sumoylering av c-Myb gir økt transaktivering. Disse mutasjonene i c-Myb påvirket ikke interaksjonen mellom MID og c-Myb. Videre ble interaksjonen med FAETL-domenet til c-Myb også bekreftet for Mi2 1096. Mi2 1096 er en forkortet versjon av Mi2, men inneholder alle predikerte funksjonelle domener. Mi2 1096 interagerte også med DNA-bindingsdomenet av c-Myb. En funksjonell konsekvens av interaksjonen ble også vist. c-Mybs DNA-bindingsaktivitet ble hemmet av MID in vitro. MID hadde imidlertid ingen påvirkning på c-Mybs binding til DNA når denne bindingen først var blitt dannet, og MID påvirket heller ikke bindingen av et isolert DBD av c-Myb til DNA. c-Myb er tradisjonelt kjent som en transkripsjonsaktivator, selv om enkelte tilfeller av repressjon er observert. En interaksjon med et repressorkompleks som NuRD vil kunne forklare hvordan c-Myb kan fungere som repressor. Fremtidige in vivo studier vil vise i hvilke biologiske prosesser denne interaksjonen har betydning

The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

Background The concept of pioneer transcription factors is emerging as an essential part of the epigenetic regulation, taking place during cell development and differentiation. However, the precise molecular mechanism underlying pioneer factor activity remains poorly understood. We recently reported that the transcription factor c-Myb acts as a pioneer factor in haematopoiesis, and a point mutation in its DNA binding domain, D152V, is able to abrogate this function. Results Here, we show that specific histone modifications, including H3K27ac, prevent binding of c-Myb to histone tails, representing a novel effect of histone modifications, namely restricting binding of a pioneer factor to chromatin. Furthermore, we have taken advantage of the pioneer-defect D152V mutant to investigate mechanisms of c-Myb’s pioneer factor activity. We show that c-Myb-dependent transcriptional activation of a gene in inaccessible chromatin relies on c-Myb binding to histones, as well as on c-Myb interacting with the histone acetyltransferase p300. ChIP assays show that both wild type and the D152V mutant of c-Myb bind to a selected target gene at its promoter and enhancer, but only wild-type c-Myb causes opening and activation of the locus. Enhancement of histone acetylation restores activation of the same gene in the absence of c-Myb, suggesting that facilitating histone acetylation is a crucial part of the pioneer factor function of c-Myb. Conclusions We suggest a pioneer factor model in which c-Myb binds to regions of closed chromatin and then recruits histone acetyltransferases. By binding to histones, c-Myb facilitates histone acetylation, acting as a cofactor for p300 at c-Myb bound sites. The resulting H3K27ac leads to chromatin opening and detachment of c-Myb from the acetylated chromatin. We propose that the latter phenomenon, acetylation-induced chromatin dissociation, represents a mechanism for controlling the dynamics of pioneer factor binding to chromatin

Dissecting the transactivation domain (tAD) of the transcription factor c‐Myb to assess recent models of tAD function

Transcription factors use a DNA‐binding domain to localize their action and a transactivation domain (tAD) to stimulate activation of the associated gene. Recent work has renewed interest in how tADs activate genes, which remains poorly understood. Key features in the new models are exposure of short linear motifs (SLMs) and liquid–liquid phase separation (LLPS). Inspired by the new models for tAD function, we decided to revisit the tAD of the haematopoietic transcription factor c‐Myb by performing a mutational analysis to see how these new models fit and potentially explain the tAD behaviour of this master regulator. We know that c‐Myb has an acidic tAD, which contains a well‐characterized SLM in the form of a LxxLL motif. By testing 12 alanine‐scanning mutants and three mutants with major reorganization of its tAD in two mammalian reporter systems, we found a pattern of effects very close to what would be expected from the SLM‐exposure model, with strong effects exerted by both acidic replacements and SLM mutation. When the same mutants were tested in a yeast system, the pattern of effects was dramatically different, with the SLM mutation exerting no effect, and tAD behaviour was much less affected by small alterations, as would be expected from a LLPS model. These observations are discussed in the light of the two new tAD models, and a two‐step hypothesis for transactivation, combining both models, is proposed

MOESM3 of The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

Additional File 3: Figure S3. Isolated nuclei were extracted with 0.1% TX-100 and increasing NaCl concentrations, and soluble (S) and insoluble (P) nuclear fractions analysed by western blotting using anti-Ty antibodies to visualize c-Myb and c-Myb D152V. Densitometric analysis of salt extractions are shown in upper row for c-Myb (middle row) and c-Myb D152V (bottom row)

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

MOESM2 of The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

Additional File 2: Figure S2. The same experiment as shown in Fig. 3, but here RNA was analysed for expression of lysozyme (LYZ, Gallus gallus (Chicken)) measured by qRT-PCR. The values of RNA expression were normalized to the relative amount of the reference gene hprt

Chromatin occupancy and target genes of the haematopoietic master transcription factor MYB

The transcription factor MYB is a master regulator in haematopoietic progenitor cells and a pioneer factor affecting differentiation and proliferation of these cells. Leukaemic transformation may be promoted by high MYB levels. Despite much accumulated molecular knowledge of MYB, we still lack a comprehensive understanding of its target genes and its chromatin action. In the present work, we performed a ChIP-seq analysis of MYB in K562 cells accompanied by detailed bioinformatics analyses. We found that MYB occupies both promoters and enhancers. Five clusters (C1–C5) were found when we classified MYB peaks according to epigenetic profiles. C1 was enriched for promoters and C2 dominated by enhancers. C2-linked genes were connected to hematopoietic specific functions and had GATA factor motifs as second in frequency. C1 had in addition to MYB-motifs a significant frequency of ETS-related motifs. Combining ChIP-seq data with RNA-seq data allowed us to identify direct MYB target genes. We also compared ChIP-seq data with digital genomic footprinting. MYB is occupying nearly a third of the super-enhancers in K562. Finally, we concluded that MYB cooperates with a subset of the other highly expressed TFs in this cell line, as expected for a master regulato

MOESM1 of The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

Additional File 1: Figure S1. Peptide arrays containing 384 histone tail modification combinations incubated with GST-c-Myb-R3 (left) or GST-c-Myb-R3-D152V (right) and detected with anti-GST primary antibody