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Purpose By-products from farmed fish contain large amounts of proteins and may be used for human consumption. The purpose of this study was to investigate cardiometabolic effects and metabolic tolerance in mice consuming fishmeal from salmon by-products, salmon filet or beef. Methods Female C57BL/6J mice were fed chow, as a healthy reference group, or a high-fat diet for 10 weeks to induce obesity and glucose intolerance. Obese mice were subsequently given isocaloric diets containing 50% of the dietary protein from salmon fishmeal, salmon filet or beef for 10 weeks. Mice were subjected to metabolic phenotyping, which included measurements of body composition, energy metabolism in metabolic cages and glucose tolerance. Lipid content and markers of hepatic toxicity were determined in plasma and liver. Hepatic gene and protein expression was determined with RNA sequencing and immunoblotting. Results Mice fed fishmeal, salmon filet or beef had similar food intake, energy consumption, body weight gain, adiposity, glucose tolerance and circulating levels of lipids and hepatic toxicity markers, such as p-ALT and p-AST. Fishmeal increased hepatic cholesterol levels by 35–36% as compared to salmon filet (p = 0.0001) and beef (p = 0.005). This was accompanied by repressed expression of genes involved in steroid and cholesterol metabolism and reduced levels of circulating Pcsk9. Conclusion Salmon fishmeal was well tolerated, but increased hepatic cholesterol content. The high cholesterol content in fishmeal may be responsible for the effects on hepatic cholesterol metabolism. Before introducing fishmeal from salmon by-products as a dietary component, it may be advantageous to reduce the cholesterol content in fishmeal.

The association between fish consumption and decreased risk of CVD is well documented. However, studies on health effects of fish consumption suggest that other components than n-3 PUFA have beneficial cardiometabolic effects, including effects on glucose metabolism. The aim of the present study was to investigate effects of salmon fish protein on cardiometabolic risk markers in a double-blind, randomised controlled parallel trial. We hypothesised that daily intake of a salmon fish protein supplement for 8 weeks would improve glucose tolerance in persons with increased risk of type 2 diabetes mellitus (T2DM). Our primary outcome measure was serum glucose (s-glucose) 2 h after a standardised oral glucose tolerance test. In total, eighty-eight adults with elevated s-glucose levels were randomised to 7·5 g of salmon fish protein/d or placebo, and seventy-four participants were included in the analysis. We found no significant effect of salmon fish protein supplementation on our primary outcome or other markers related to glucose tolerance, serum lipids, weight or blood pressure compared with placebo. The present study does not support the hypothesis that daily intake of a salmon fish protein supplement for 8 weeks improves glucose tolerance in persons with increased risk of T2DM.

Purpose The main aim of the present study was to examine the effect of a fish protein supplement made from by-products from production of Atlantic salmon, on blood concentration of micronutrients. Methods We conducted an 8-week double-blind parallel-group randomised controlled trial. In total, 88 adults were randomised to a salmon fish protein supplement or placebo, and 74 participants were included in the analysis of vitamin D, omega-3, vitamin B12, selenium, folate, zinc, homocysteine and mercury. Results During the intervention period, geometric mean (GSD) of serum vitamin B12 concentrations increased from 304 (1.40) to 359 (1.42) pmol/L in the fish protein group (P vs. controls = 0.004) and mean (SD) serum selenium increased from 1.18 (0.22) to 1.30 (0.20) μmol/L (P vs. controls = 0.002). The prevalence of low vitamin B12 status (B12 pmol/L) decreased from 15.4 to 2.6% in the fish protein group, while increasing from 5.9 to 17.6% in the placebo group (P = 0.045). There was no difference between the groups in serum levels of the other micronutrients measured. Conclusion Including a salmon fish protein supplement in the daily diet for 8 weeks, increases serum vitamin B12 and selenium concentrations. From a sustainability perspective, by-products with high contents of micronutrients and low contents of contaminants, could be a valuable dietary supplement or food ingredient in populations with suboptimal intake. Trail Registration The study was registered at ClinicalTrials.gov (ID: NCT03764423) on June 29th 2018.

The main aim of the present study was to examine the effect of a fish protein supplement made from by-products from production of Atlantic salmon, on blood concentration of micronutrients.We conducted an 8-week double-blind parallel-group randomised controlled trial. In total, 88 adults were randomised to a salmon fish protein supplement or placebo, and 74 participants were included in the analysis of vitamin D, omega-3, vitamin B12, selenium, folate, zinc, homocysteine and mercury.During the intervention period, geometric mean (GSD) of serum vitamin B12 concentrations increased from 304 (1.40) to 359 (1.42) pmol/L in the fish protein group (P vs. controls = 0.004) and mean (SD) serum selenium increased from 1.18 (0.22) to 1.30 (0.20) μmol/L (P vs. controls = 0.002). The prevalence of low vitamin B12 status (B12 148-221 pmol/L) decreased from 15.4 to 2.6% in the fish protein group, while increasing from 5.9 to 17.6% in the placebo group (P = 0.045). There was no difference between the groups in serum levels of the other micronutrients measured.Including a salmon fish protein supplement in the daily diet for 8 weeks, increases serum vitamin B12 and selenium concentrations. From a sustainability perspective, by-products with high contents of micronutrients and low contents of contaminants, could be a valuable dietary supplement or food ingredient in populations with suboptimal intake.The study was registered at ClinicalTrials.gov (ID: NCT03764423) on June 29th 2018.

High mobility group A2 (HMGA2) is a chromatin-associated protein involved in the regulation of stem cell function, embryogenesis and cancer development. Although the protein does not contain a consensus SUMOylation site, it is shown to be SUMOylated. In this study, we demonstrate that the first lysine residue in the reported K66KAE SUMOylation motif in HMGA2 can be methylated in vitro and in vivo by the Set7/9 methyltransferase. By editing the lysine, the increased hydrophobicity of the resulting 6-N-methyl-lysine transforms the sequence into a consensus SUMO motif. This post-translational editing dramatically increases the subsequent SUMOylation of this site. Furthermore, similar putative methylation-dependent SUMO motifs are found in a number of other chromatin factors, and we confirm methylation-dependent SUMOylation of a site in one such protein, the Polyhomeotic complex 1 homolog (PHC1). Together, these results suggest that crosstalk between methylation and SUMOylation is a general mode for regulation of chromatin function.

The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXR&alpha, and ChREBP&alpha, interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXR&alpha, co-activates ChREBP&alpha, and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP but not for LXR, indicating that ChREBP&alpha, recruits LXR&alpha, to chromatin in trans. The two factors interact via their key activation domains, the low glucose inhibitory domain (LID) of ChREBP&alpha, and the ligand-binding domain (LBD) of LXR&alpha, While unliganded LXR&alpha, ligand-bound LXR&alpha, surprisingly represses ChREBP&alpha, activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXR&alpha, ChREBP&alpha, interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXR&alpha, in responding to nutritional cues that was overlooked due to LXR lipogenesis-promoting function.

Bacheloroppgave i bygg, Institutt for byggfag, Høgskulen på Vestlandet, campus Bergen BYG150

The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in liver. We have previously shown that LXR regulates ChREBP transcription and activity; however, the underlying mechanisms are unclear. In the current study, we demonstrate that LXRα and ChREBPα interact physically, and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα, and regulates ChREBP-specific target genesin vitroandin vivo. This co-activation is dependent on functional recognition elements for ChREBP, but not for LXR, indicating that ChREBPα recruits LXRα to chromatin intrans. The two factors interact via their key activation domains; ChREBPα’s low glucose inhibitory domain (LID) and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXRα that was overlooked due to LXR lipogenesis-promoting function.

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20LWKPGAQDASSQAQGGSSCILRE42. However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826–832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11–13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT.

Many mammalian genes exhibit circadian expression patterns concordant with periodic binding of transcription factors, chromatin modifications, and chromosomal interactions. Here we investigate whether chromatin periodically associates with nuclear lamins. Entrainment of the circadian clock is accompanied, in mouse liver, by a net gain of lamin B1–chromatin interactions genome-wide, after which the majority of lamina-associated domains (LADs) are conserved during the circadian cycle. By tailoring a bioinformatics pipeline designed to identify periodic gene expression patterns, we also observe hundreds of variable lamin B1–chromatin interactions among which oscillations occur at 64 LADs, affecting one or both LAD extremities or entire LADs. Only a small subset of these oscillations however exhibit highly significant 12, 18, 24, or 30 h periodicity. These periodic LADs display oscillation asynchrony between their 5′ and 3′ borders, and are uncoupled from periodic gene expression within or in the vicinity of these LADs. Periodic gene expression is also unrelated to variations in gene-to-nearest LAD distances detected during the circadian cycle. Accordingly, periodic genes, including central clock-control genes, are located megabases away from LADs throughout circadian time, suggesting stable residence in a transcriptionally permissive chromatin environment. We conclude that periodic LADs are not a dominant feature of variable lamin B1–chromatin interactions during the circadian cycle in mouse liver. Our results also suggest that periodic hepatic gene expression is not regulated by rhythmic chromatin associations with the nuclear lamina.

Source at https://doi.org/10.1016/j.bmc.2019.07.032. The peroxisome proliferator activated receptors (PPARs) are important drug targets in treatment of metabolic and inflammatory disorders. Fibrates, acting as PPARα agonists, have been widely used lipid-lowering agents for decades. However, the currently available PPARα targeting agents show low subtype-specificity and consequently a search for more potent agonists have emerged. In this study, previously isolated oxohexadecenoic acids from the marine algae Chaetoceros karianus were used to design a PPARα-specific analogue. Herein we report the design, synthesis, molecular modelling studies and biological evaluations of the novel 3,5-disubstituted isoxazole analogue 6-(5-heptyl-1,2-oxazol-3-yl)hexanoic acid (1), named ADAM. ADAM shows a clear receptor preference and significant dose-dependent activation of PPARα (EC50 = 47 µM) through its ligand-binding domain (LBD). Moreover, ADAM induces expression of important PPARα target genes, such as CPT1A, in the Huh7 cell line and primary mouse hepatocytes. In addition, ADAM exhibits a moderate ability to regulate PPARγ target genes and drive adipogenesis. Molecular modelling studies indicated that ADAM docks its carboxyl group into opposite ends of the PPARα and -γ LBD. ADAM interacts with the receptor-activating polar network of amino acids (Tyr501, His447 and Ser317) in PPARα, but not in PPARγ LBD. This may explain the lack of PPARγ agonism, and argues for a PPARα-dependent adipogenic function. Such compounds are of interest towards developing new lipid-lowering remedies.

Source at https://doi.org/10.1016/j.ejmech.2018.06.034. Obesity and associated disorders such as metabolic syndrome and type 2 diabetes (T2D) have reached epidemic proportions. Several natural products have been reported as Peroxisome Proliferator-Activated Receptor (PPAR) agonists, functioning as lead compounds towards developing new anti-diabetic drugs due to adverse side effects of existing PPAR drugs. We recently isolated and identified (7E)-9-oxohexadec-7-enoic acid (1) and (10E)-9-oxohexadec-10-enoic acid (2) from the marine algae Chaetoceros karianus. Herein we report the total synthesis, pharmacological characterization, and biological evaluations of these naturally occurring oxo-fatty acids (oFAs). The syntheses of 1 and 2 afforded sufficient material for extensive biological evaluations. Both oFAs show an appreciable dose-dependent activation of PPARα and -γ, with EC50 values in the micromolar range, and an ability to regulate important PPAR target genes in hepatocytes and adipocytes. Moreover, both 1 and 2 are able to drive adipogenesis when evaluated in the Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell model, but with lowered expression of adipocyte markers and reduced lipid accumulation compared to the drug rosiglitazone. This seems to be caused by a transient upregulation of PPARγ and C/EBPα expression. Importantly, whole transcriptome analysis shows that both compounds induce anti-diabetic gene programs in adipocytes by upregulating insulin-sensitizing adipokines and repressing pro-inflammatory cytokines.

Source at http://dx.doi.org/10.3390/md15060148 The peroxisome proliferator-activated receptors (PPARs) function as ligand-activated transcription factors that convert signals in the form of lipids to physiological responses through the activation of metabolic target genes. Due to their key roles in lipid and carbohydrate metabolism, the PPARs are important drug targets. However, for several of the PPAR drugs currently in use, adverse side effects have been reported. In an effort to identify compounds from marine organisms that may serve as molecular scaffolds for the development of novel and safer PPAR-targeting drugs, we performed a bioassay-guided screening of organic extracts made from organisms supplied by the Norwegian Biobank of Arctic Marine Organisms (Marbank). Among several interesting hits, we identified two poorly described isomeric oxo-fatty acids from the microalgae Chaetoceros karianus for which we provide the first evidence that they might display dual specificity towards human PPARα and PPARγ. Principal component analysis showed that C. karianus stood out from other Chaetoceros species, both with respect to the metabolic profile and the PPAR activity. The isolation of these compounds holds the potential of uncovering a PPAR pharmacophore with tunable activity and specificity.

Arctic is contaminated with persistent organochlorine pollutants (POPs), and exposure to these compounds may differ between south and north in Norway. POPs may have negative impact on male reproductive characteristics. We compared serum levels of the CB-153 and p,p'-DDE in men who were born and had lived most of their lifetime south and north (close to or above the Arctic Circle) in Norway. We found no geographical differences in levels of CB-153 (south: 50 ng/g lipid (mean), north: 59 ng/g lipid; p = 0.27) or sperm parameters. However, the levels of AV-ODE were higher in south than in north (81 ng/g lipid (mean) vs. 66 ng/g lipid; p = 0.02), as were the levels of total and free testosterone. The FSH levels were lowest in south. A strong relationship between the CB-153 and the SHBG levels was observed. The regional differences observed for p,p'-DDE, testosterone and FSH were not reflected in the semen quality. (C) 2011 Elsevier Inc. All rights reserved. (Less)

c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Whereas sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb, no role for noncovalent binding of small ubiquitin-like modifier (SUMO) to c-Myb has been described. Based on the consensus SUMO-interacting motif (SIM), we identified and examined putative SIMs in human c-Myb. Interaction and reporter assays showed that the SIM in the in the transactivation domain of c-Myb (V 267 NIV) is functional. This motif is necessary for c-Myb to be able to interact noncovalently with SUMO, preferentially SUMO2/3. Destroying the SUMO-binding properties by mutation resulted in a large increase in the transactivation potential of c-Myb. Mutational analysis and overexpression of conjugation-defective SUMO argued against intramolecular repression caused by sumoylated CRD and in favor of SUMO-dependent repression in trans. Using both a myeloid cell line-based assay and a primary hematopoietic cell assay, we addressed the transforming abilities of SUMO binding and conjugation mutants. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhanced the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated conferred a higher proliferative ability than the wild-type and caused an effective differentiation block. This establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check. Refereed/Peer-reviewed

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.

It is generally thought that a single ejaculate is a bad predictor of semen quality of a subject, because of significant intra-individual variation. Therefore, we investigated the degree to which the results of a first semen analysis differ from that of a second analysis among men from a general population in Norway. In addition, we analysed how the two different semen results mirrored the overall semen quality assessment. A total of 199 volunteers participated in the study and delivered two semen samples with an interval of 6 months. The semen parameters were determined according to the World Health Organization (WHO) 1999 guidelines, which were also used to determine whether semen quality was normal or abnormal. In addition, the DNA fragmentation index (DFI) was determined using the Sperm Chromatin Structure Assay. The two samples from each individual were very similar with regard to standard semen parameters and DFI (r(s:) 0.67-0.72), and there were no significant systematic differences between the two samples. The result of the first sample (normal/abnormal) was highly predictive of the overall conclusion based on the two samples (sperm concentration: in 93% of the cases (95% confidence interval [CI]: 89%-96%); sperm motility: in 85% of the cases (95% CI: 79%-89%); overall semen quality: in 85% of the cases (95% CI: 80%-90%). In epidemiological studies, one ejaculate is a sufficient indicator of semen quality in a group of subjects. In a clinical situation, when the question is whether the semen quality is normal or not, the first ejaculate will, in at least 85% of cases, give a correct overall conclusion.

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.

On the molecular level, essential fatty acid deficiency (EFAD) has been associated with induced fatty acid (FA) desaturase expression and activity in several tissues. However, there seem to be exceptions. In the present study, we examine the effects of EFAD in the male rat genital tract, combining FA analysis, gene expression studies, and morphological evaluation of epididymal spermatozoa. When feeding 21-day-old Wistar rats, a fat-free diet for 6 weeks, an increase in 18:1n-9 and 20:3n-9 and a concomitant decrease in the 18:2n-6 and 20:4n-6 species are seen in testis, as well as in liver. However, with regard to desaturase expression the rat testis seems to be unresponsive to EFAD conditions, in contrast to other organs studied. In the sexually mature testis none of the desaturases (SCD1, SCD2, D5D, or D6D) are induced in response to lowered contents of polyunsaturated FAs. This also applies tocaputepididymis, while EFAD sensitivity is regained incaudaepididymis, where the desaturases are upregulated. The FA profile of epididymal spermatozoa is increasingly affected by EFAD during the transport from testis tocaudaepididymis. Furthermore, a significant increase in the number of abnormal spermatozoa is observed incaudaepididymis.

Seasonal, daylight-dependent variation in human spermatozoa counts, with lowest values during summer, has been suggested. To test this hypothesis, we performed a longitudinal study of semen quality and reproductive hormone levels in Norwegian men living north and south of the Arctic Circle. An ejaculate and a serum specimen were obtained both in summer and in winter from 92 volunteers in Tromsoe (69° north latitude) and 112 in Oslo (60° north latitude). Semen analyses were performed, and serum was assayed for FSH and inhibin B. The median spermatozoa concentration in Tromsoe after adjustment for abstinence period length was 49 10 6 /ml in summer and 54 10 6 /ml in winter. Corresponding values for Oslo were 59 10 6 /ml and 54 10 6 /ml. The seasonal differences in spermatozoa concentration were not statistically significant, nor were significant differences observed in median total spermatozoa count, semen volume, percentage progressive motile spermatozoa, or FSH. In Tromsoe, but not Oslo, inhibin B concentration was slightly, but significantly (P 0.02) higher in winter than summer (229 ng/liter vs. 223 ng/liter). The length of the daylight period may have a slight impact on hormonal markers of spermatogenesis but does not cause substantial changes in spermatozoa numbers and motility. (J Clin Endocrinol Metab 89: 4397– 4402, 2004)

A growing number of estrogen receptor beta (ER beta) splice variants are reported. Several of these have been discovered in testis, but with few exceptions little is known about their cellular localization. The aim of this study was to identify and elucidate the mRNA expression pattern of the different ER beta splice variants in human testicular cells. Northern analysis was performed on whole testis and fractions enriched in germ cells from untreated men and from estrogen-treated men undergoing sex change surgery. Probes were constructed in order to systematically screen for and identify various ER beta splice variants. Several ER beta bands were observed in the human testis, in which splice variants constituted the major part of total ER beta transcripts. Interestingly, only two ER beta wild-type transcripts were detected. These seem to be virtually absent from the haploid germ cells and are probably mainly located in somatic cells and/or primary spermatocytes. Several novel ER beta deletion variants were found in high levels in the haploid germ cell fractions and were nearly absent in testicular cells from the estrogen-treated men. The cell-dependent distribution raises the question whether splice variants may have specific functions in spermatogenesis, and whether the differential splicing of ER beta is regulated in a cell-specific manner.

In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and Delta5- and Delta6-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of Delta5- and Delta6-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, Delta5- and Delta6-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. Delta5- and Delta6-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)2cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License., [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. © 2011 Alm-Kristiansen et al; licensee BioMed Central Ltd.

et al., 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. © The Author(s) 2011., This work was supported by the Norwegian Research Council [FUGE grant number 175364 and 158961]; the Norwegian Cancer Society [grant number 419436-107692-PR-2007-0148]; the Presidium of the Russian Academy of Sciences [grant MCB]; and the Ministry of Science and Education of the Russian Federation [contracts 16.740.11.0353 and 16.740.11.0483].

International audience; 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.