| 1. |
- Caruso, Vanni, et al.
(författare)
-
mRNA GPR162 changes are associated with decreased food intake in rat, and its human genetic variants with impairments in glucose homeostasis in two Swedish cohorts
- 2016
-
Ingår i: Gene. - : Elsevier BV. - 0378-1119 .- 1879-0038. ; 581:2, s. 139-145
-
Tidskriftsartikel (refereegranskat)abstract
- G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating intercellular interactions of fundamental physiological importance for survival including regulation of food intake, blood pressure, and hormonal sensing signaling, among other roles. Homeostatic alterations in the physiological status of GPCRs are often associated with underlying causes of disease, and to date, several orphan GPCRs are still uncharacterized. Findings from our previous study demonstrate that the Rhodopsin family protein GPR162 is widely expressed in GABAergic as well as other neurons within the mouse hippocampus, whereas extensive expression is observed in hypothalamus, amygdala, and ventral tegmental area, regions strictly interconnected and involved in the regulation of energy homeostasis and hedonic feeding. In this study, we provide a further anatomical characterization of GPR162 in mouse brain via in situ hybridization as well as detailed mRNA expression in a panel of rat tissues complementing a specie-specific mapping of the receptor. We also provide an attempt to demonstrate a functional implication of GPR162 in food intake-related behavior via antisense knockdown studies. Furthermore, we performed human genetic studies in which for the first time, variants of the GPR162 gene were associated with impairments in glucose homeostasis.
|
|
| 2. |
- Crona, Filip, 1977-, et al.
(författare)
-
Brakeless can directly activate and repress trancription in early Drosophila embryos
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The Brakeless protein performs many important functions during development in Drosophila, but how it controls gene expression is not understood. We previously showed that Brakeless can function as a transcriptional co-repressor. In this work, we perform transcriptional profiling of brakeless mutant embryos. Unexpectedly, the majority of target genes are down-regulated in brakeless mutants. We demonstrate that genomic regions in close proximity to some of the affected genes are occupied by Brakeless, that over-expression of Brakeless causes a reciprocal effect on expression of these genes, and that the activator function of Brakeless is intact when an activation domain is fused to Brakeless. By contrast, Brakeless repressor function is neutralized by the activation domain. Together, this shows that Brakeless can both repress and activate gene expression. To identify protein interactions that result in gene repression or activation, a yeast two-hybrid screen was performed. We find that the Mediator complex subunit Med19 interacts with an evolutionarily conserved part of Brakeless. Interestingly, down-regulated but not up-regulated Brakeless target genes are also affected in Med19-depleted embryos. Our data provide support for a Brakeless activator function that regulates transcription by interacting with Med19.
|
|
| 3. |
- Crona, Filip, 1977-, et al.
(författare)
-
Gene regulation by the lysine demethylase KDM4A in Drosophila
- 2013
-
Ingår i: Developmental Biology. - : Elsevier BV. - 0012-1606 .- 1095-564X. ; 373:2, s. 453-463
-
Tidskriftsartikel (refereegranskat)abstract
- Lysine methylation of histones is associated with both transcriptionally active chromatin and with silent chromatin, depending on what residue is modified. Histone methyltransferases and demethylases ensure that histone methylations are dynamic and can vary depending on cell cycle- or developmental stage. KDM4A demethylates H3K36me3, a modification enriched in the 3' end of active genes. The genomic targets and the role of KDM4 proteins in development remain largely unknown. We therefore generated KDM4A mutant Drosophila, and identified 99 mis-regulated genes in first instar larvae. Around half of these genes were down-regulated and the other half up-regulated in dKDM4A mutants. Although heterochromatin protein 1a (HP1a) can stimulate dKDM4A demethylase activity in vitro, we find that they antagonize each other in control of dKDM4A-regulated genes. Appropriate expression levels for some dKDM4A-regulated genes rely on the demethylase activity of dKDM4A, whereas others do not. Surprisingly, although highly expressed, many demethylase-dependent and independent genes are devoid of H3K36me3 in wild-type as well as in dKDM4A mutant larvae, suggesting that some of the most strongly affected genes in dKDM4A mutant animals are not regulated by H3K36 methylation. By contrast, dKDM4A over-expression results in a global decrease in H3K36me3 levels and male lethality, which might be caused by impaired dosage compensation. Our results show that a modest increase in global H3K36me3 levels is compatible with viability, fertility, and the expression of most genes, whereas decreased H3K36me3 levels are detrimental in males.
|
|
| 4. |
- Crona, Filip, 1977-
(författare)
-
Regulators of chromatin and transcription in Drosophila
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Development of multicellular organisms is achieved by organized temporal and spatial patterns of gene expression leading to cell differentiation. Chromatin regulators control how the DNA is utilized by altering access of proteins to DNA and thereby function as co-factors in transcription. Gene regulation also involves co-factors interacting with transcription factors at regulatory sequences of DNA. In this thesis, we have studied the in vivo role of three co-factors, CBP, dKDM4A and Brakeless, in regulating chromatin and transcription using Drosophila melanogaster. The CREB binding protein (CBP) belongs to histone acetyl transferases (HATs) and facilitates gene activation by many transcription factors. Our work has demonstrated that CBP occupies the genome preferentially together with Rel and Smad proteins controlling dorsal-ventral patterning in the Drosophila embryo. CBP occupancy generally correlates with gene expression but also occurs at silent genes without resulting in histone acetylation. KDM4A belongs to a family of JmjC domain proteins and demethylates H3K36me3, a histone modification enriched in the 3’end of active genes. We generated dKDM4A mutants with a global elevation of H3K36me3 levels and identify mis-regulated genes in first instar larvae. The data indicate that dKDM4A regulates some genes by mechanisms that do not involve H3K36 methylation. Further, over-expression of dKDM4A result in male lethality and globally reduced H3K36me3 levels, indicating impaired dosage compensation of the X-chromosome. Brakeless is a conserved co-factor participating in several important processes during development. We generated mutant brakeless embryos and identify direct genomic targets of Brakeless. To our surprise, Brakeless behaves as a direct activator for some genes but repressor in other cases. We also identify an interaction of Brakeless with the Mediator subunit Med19. In summary, these studies reveal unexpected roles for co-regulators in Drosophila development. The HAT CBP can bind silent genes without leading to histone acetylation. Brakeless has the ability to function both as a direct activator and repressor of transcription.
|
|
| 5. |
- Crona, Filip, et al.
(författare)
-
The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos
- 2015
-
Ingår i: Developmental Biology. - : Elsevier BV. - 0012-1606 .- 1095-564X. ; 407:1, s. 173-181
-
Tidskriftsartikel (refereegranskat)abstract
- The Brakeless protein performs many important functions during Drosophila development, but how it controls gene expression is poorly understood. We previously showed that Brakeless can function as a transcriptional co-repressor. In this work, we perform transcriptional profiling of brakeless mutant embryos. Unexpectedly, the majority of affected genes are down-regulated in brakeless mutants. We demonstrate that genomic regions in close proximity to some of these genes are occupied by Brakeless, that over-expression of Brakeless causes a reciprocal effect on expression of these genes, and that Brakeless remains an activator of the genes upon fusion to an activation domain. Together, our results show that Brakeless can both repress and activate gene expression. A yeast two-hybrid screen identified the Mediator complex subunit Med19 as interacting with an evolutionarily conserved part of Brakeless. Both down- and up-regulated Brakeless target genes are also affected in Med19-depleted embryos, but only down-regulated targets are influenced in embryos depleted of both Brakeless and Med19. Our data provide support for a Brakeless activator function that regulates transcription by interacting with Med19. We conclude that the transcriptional co-regulator Brakeless can either activate or repress transcription depending on context.
|
|
| 6. |
- Holmqvist, Per-Henrik, et al.
(författare)
-
Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos
- 2012
-
Ingår i: PLOS Genetics. - San Francisco : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 8:6
-
Tidskriftsartikel (refereegranskat)abstract
- CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire) ChIP-seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-beta/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorsoventral patterning and that CBP binds silent genes without causing histone hyperacetylation.
|
|
| 7. |
- Sreedharan, Smitha, et al.
(författare)
-
GPR162 is expressed in the hypothalamus and is involved in food intake related behaviour
- 2011
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The Rhodopsin family of G protein-coupled receptors (GPCRs) includes about 270 non-olfactory receptors and is the largest family of GPCRs. About sixty non-olfactory Rhodopsin GPCRs are still orphans without known ligands, and fairly little is known about their functions. In this study, we present molecular, neuroanatomical, genetic and behavioral data implicating a Rhodopsin family protein, GPR162, in the regulation of food intake-related behaviour and glucose homeostasis. The real-time PCR data show that GPR162 is predominantly expressed in the CNS. The in situ hybridization results confirmed significant expression of GPR162 in several hypothalamic sites, amygdala, substantia nigra and ventral tegmental area, among others regions. In line with the distribution of the GPR162 mRNA in the feeding circuitry, antisense oligo knockdown of GPR162 caused a significant reduction in food intake but no effect was observed towards reduction in body weight in rats. Our human genetics studies suggest that genetic variants of GPR162 affect glucose homeostasis. In conclusion, this study provides evidence linking the orphan GPR162 gene with the regulation of food intake-related behaviour.
|
|
