Characterization of PPAR-gamma 1 and PPAR-gamma 2 in Knockin and Knockout Mouse Models

The global epidemic of obesity and type II diabetes has led to a growing interest in the underlying mechanisms of metabolic diseases. The peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear receptor superfamily, and is vital for the transcriptional regulation of adipogenesis, insulin sensitivity and lipid metabolism. In the mouse model, it has been demonstrated that global knockout of PPARγ leads to severe metabolic disturbance, resulting in embryonic lethality. However, the specific regulatory roles of its two protein isoforms, PPARγ1 and PPARγ2, remain uncertain, due to limitations of reagents and appropriate mouse models. To investigate the hypothesis that PPARγ1 and PPARγ2 are functionally distinct, we generated PPARγ1 and PPARγ2 tagged mice using CRISPR-Cas9 technology. PPARγ1 and PPARγ2 specific knockout mice were also generated incidentally during this process, via aberrant recombination. By reverse-transcription quantitative PCR (RT-qPCR), and western blot, we confirmed the presence of the appropriate tags in our PPARγ1 and PPARγ2 tagged mice, with no significant disruption to mRNA or protein expression. Furthermore, we found that PPARγ1 mRNA and protein expression levels were reduced in our PPARγ1 knockout model, compared to the wild type. Interestingly, we found that there was a complete loss of PPARγ2 protein expression, despite an increase in PPARγ2 mRNA expression in our PPARγ2 knockout model. These data suggest that we have successfully generated PPARγ1 and PPARγ2 knockin and knockout mice. Our mouse models provide a valuable tool to study the individual roles of PPARγ1 and PPARγ2 in adipogenesis, insulin sensitivity and metabolic disease

Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver

BACKGROUND: Metabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated. RESULTS: We performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the ‘single sample independence’ (SSI) test that greatly reduced the number of false-positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state. Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the two metabolic states are associated with co-localization of additional transcription factors at CREB sites. CONCLUSIONS: Our results support a model in which CREB is constitutively bound to thousands of target genes, and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, and suggests a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx

Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage–induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage–induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint

HDAC3-Dependent Epigenetic Pathway Controls Lung Alveolar Epithelial Cell Remodeling and Spreading via miR-17-92 and TGF-β Signaling Regulation

SummaryThe terminal stages of pulmonary development, called sacculation and alveologenesis, involve both differentiation of distal lung endoderm progenitors and extensive cellular remodeling of the resultant epithelial lineages. These processes are coupled with dramatic expansion of distal airspace and surface area. Despite the importance of these late developmental processes and their relation to neonatal respiratory diseases, little is understood about the molecular and cellular pathways critical for their successful completion. We show that a histone deacetylase 3 (Hdac3)-mediated epigenetic pathway is critical for the proper remodeling and expansion of the distal lung saccules into primitive alveoli. Loss of Hdac3 in the developing lung epithelium leads to a reduction of alveolar type 1 cell spreading and a disruption of lung sacculation. Hdac3 represses miR-17-92 expression, a microRNA cluster that regulates transforming growth factor β (TGF-β) signaling. De-repression of miR-17-92 in Hdac3-deficient lung epithelium results in decreased TGF-β signaling activity. Importantly, inhibition of TGF-β signaling and overexpression of miR-17-92 can phenocopy the defects observed in Hdac3 null lungs. Conversely, loss of miR-17-92 expression rescues many of the defects caused by loss of Hdac3 in the lung. These studies reveal an intricate epigenetic pathway where Hdac3 is required to repress miR-17-92 expression to allow for proper TGF-β signaling during lung sacculation

The Coiled-coil Domain Is the Structural Determinant for Mammalian Homologues of Drosophila Sina-mediated Degradation of Promyelocytic Leukemia Protein and Other Tripartite Motif Proteins by the Proteasome

Mammalian homologues of Drosophila Seven in Absentia (SIAHs) target for proteasome-mediated degradation several factors involved in cell growth and tumorigenesis. Here we show that SIAH-1/2 binds and targets for proteasome-mediated degradation the putative tumor suppressor and tripartite motif (TRIM) family member PML, leading to the loss of its transcriptional co-activating properties and a reduction in the number of endogenous PML nuclear bodies. Association with PML requires the substrate-binding domain (SBD) of SIAH-1/2 through an interacting surface apparently distinct from those predicted by the structural studies, or shown experimentally to mediate binding to SIAH-associated factors. Within PML, the coiled-coil domain is required for Siah- and proteasome-mediated degradation, and deletions of regions critical for the integrity of this region impair the ability of Siah to trigger PML-RAR degradation. Fusion of the coiled-coil domain to heterologous proteins resulted in the capacity of mSiah-2 to target their degradation. All of the TRIM proteins tested were degraded upon mSiah-2 overexpression. Finally, we show that the fusion protein PML-RAR (that retains the coiled-coil domain), which causes acute promyelocytic leukemias, is also a potential substrate of mSiah-2. As a result of mSiah-2 overexpression and subsequent degradation of the fusion protein, the arrest in hematopoietic differentiation because of expression of PML-RAR is partially rescued. These results identify PML and other TRIMs as new factors post-translationally regulated by SIAH and involve the coiled-coil region of PML and of other SIAH substrates as a novel structural determinant for targeted degradation

Fast TeV variability from misaligned minijets in the jet of M87

The jet of the radio galaxy M87 is misaligned, resulting in a Doppler factor delta~1 for emission of plasma moving parallel to the jet. This makes the observed fast TeV flares on timescales of t_v~5R_g/c harder to understand as emission from the jet. In previous work, we have proposed a jets-in-a-jet model for the ultra-fast TeV flares with t_v<<R_g/c seen in Mrk 501 and PKS 2155-304. Here, we show that about half of the minijets beam their emission outside the jet cone. Minijets emitting off the jet axis result in rapidly evolving TeV (and maybe lower energy) flares that can be observed in nearby radio galaxies. The TeV flaring from M87 fits well into this picture, if M87 is a misaligned blazar.Comment: 9 pages, 5 figures, minor changes, MNRAS, accepte