Project description:Timely and selective recruitment of transcription factors to their appropriate DNA-binding sites represents a critical step in regulating gene activation; however the regulatory strategies underlying each factor’s effective recruitment to specific promoter and/or enhancer regions are not fully understood. Here, we identify an unexpected regulatory mechanism by which promoter-specific binding, and therefore function, of PPARG in adipocytes requires G protein Suppressor 2 (GPS2) to prime the local chromatin environment via inhibition of the ubiquitin ligase RNF8 and stabilization of the H3K9 histone demethylase KDM4A/JMJD2. Integration of genome-wide profiling data indicates that the pioneering activity of GPS2/KDM4A is required for PPARG mediated regulation of a specific transcriptional program, including the lipolytic enzymes ATGL and HSL. Hence, our findings reveal that GPS2 exerts a biologically important function in adipose tissue lipid mobilization by directly regulating ubiquitin signaling and indirectly modulating chromatin remodeling to prime selected genes for activation.
Project description:Timely and selective recruitment of transcription factors to their appropriate DNA-binding sites represents a critical step in regulating gene activation; however the regulatory strategies underlying each factor’s effective recruitment to specific promoter and/or enhancer regions are not fully understood. Here, we identify an unexpected regulatory mechanism by which promoter-specific binding, and therefore function, of PPARG in adipocytes requires G protein Suppressor 2 (GPS2) to prime the local chromatin environment via inhibition of the ubiquitin ligase RNF8 and stabilization of the H3K9 histone demethylase KDM4A/JMJD2. Integration of genome-wide profiling data indicates that the pioneering activity of GPS2/KDM4A is required for PPARG mediated regulation of a specific transcriptional program, including the lipolytic enzymes ATGL and HSL. Hence, our findings reveal that GPS2 exerts a biologically important function in adipose tissue lipid mobilization by directly regulating ubiquitin signaling and indirectly modulating chromatin remodeling to prime selected genes for activation.
Project description:Timely and selective recruitment of transcription factors to their appropriate DNA-binding sites represents a critical step in regulating gene activation; however, the regulatory strategies underlying each factor's effective recruitment to specific promoter and/or enhancer regions are not fully understood. Here, we identify an unexpected regulatory mechanism by which promoter-specific binding, and therefore function, of peroxisome proliferator-activator receptor ? (PPAR?) in adipocytes requires G protein suppressor 2 (GPS2) to prime the local chromatin environment via inhibition of the ubiquitin ligase RNF8 and stabilization of the H3K9 histone demethylase KDM4A/JMJD2. Integration of genome-wide profiling data indicates that the pioneering activity of GPS2/KDM4A is required for PPAR?-mediated regulation of a specific transcriptional program, including the lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). Hence, our findings reveal that GPS2 exerts a biologically important function in adipose tissue lipid mobilization by directly regulating ubiquitin signaling and indirectly modulating chromatin remodeling to prime selected genes for activation.
Project description:The yeast Gis1 protein is a transcriptional regulator belonging to the JMJD2/KDM4 subfamily of demethylases that contain a JmjC domain, which are highly conserved from yeast to humans. They have important functions in histone methylation, cellular signaling and tumorigenesis. Besides serving as a cofactor in many proteins, heme is known to directly regulate the activities of proteins ranging from transcriptional regulators to potassium channels. Here, we report a novel mechanism governing heme regulation of Gis1 transcriptional and histone demethylase activities. We found that two Gis1 modules, the JmjN + JmjC domain and the zinc finger (ZnF), can bind to heme specifically in vitro. In vivo functional analysis showed that the ZnF, not the JmjN + JmjC domain, promotes heme activation of transcriptional activity. Likewise, measurements of the demethylase activity of purified Gis1 proteins showed that full-length Gis1 and the JmjN + JmjC domain both possess demethylase activity. However, heme potentiates the demethylase activity of full-length Gis1, but not that of the JmjN + JmjC domain, which can confer heme activation of transcriptional activity in an unrelated protein. These results demonstrate that Gis1 represents a novel class of multi-functional heme sensing and signaling proteins, and that heme binding to the ZnF stimulates Gis1 demethylase and transcriptional activities.
Project description:Chromatin-modifying enzymes are predominantly nuclear; however, these factors are also localized to the cytoplasm, and very little is known about their role in this compartment. In this report, we reveal a non-chromatin-linked role for the lysine-specific demethylase KDM4A. We demonstrate that KDM4A interacts with the translation initiation complex and affects the distribution of translation initiation factors within polysome fractions. Furthermore, KDM4A depletion reduced protein synthesis and enhanced the protein synthesis suppression observed with mTOR inhibitors, which paralleled an increased sensitivity to these drugs. Finally, we demonstrate that JIB-04, a JmjC demethylase inhibitor, suppresses translation initiation and enhances mTOR inhibitor sensitivity. These data highlight an unexpected cytoplasmic role for KDM4A in regulating protein synthesis and suggest novel potential therapeutic applications for this class of enzyme.This report documents an unexpected cytoplasmic role for the lysine demethylase KDM4A. We demonstrate that KDM4A interacts with the translation initiation machinery, regulates protein synthesis and, upon coinhibition with mTOR inhibitors, enhances the translation suppression and cell sensitivity to these therapeutics.
Project description:Human zygotes are difficult to obtain for research because of limited resources and ethical debates. Corrected human tripronuclear (ch3PN) zygotes obtained by removal of the extra pronucleus from abnormally fertilized tripronuclear (3PN) zygotes are considered an alternative resource for basic scientific research. In the present study, eight-cell and blastocyst formation efficiency were significantly lower in both 3PN and ch3PN embryos than in normal fertilized (2PN) embryos, while histone H3 lysine 9 trimethylation (H3K9me3) levels were much higher. It was speculated that the aberrant H3K9me3 level detected in ch3PN embryos may be related to low developmental competence. Microinjection of 1000?ng/µl lysine-specific demethylase 4A (KDM4A) mRNA effectively reduced the H3K9me3 level and significantly increased the developmental competence of ch3PN embryos. The quality of ch3PN zygotes improved as the grading criteria, cell number and pluripotent expression significantly increased in response to KDM4A mRNA injection. Developmental genes related to zygotic genome activation (ZGA) were also upregulated. These results indicate that KDM4A activates the transcription of the ZGA program by enhancing the expression of related genes, promoting epigenetic modifications and regulating the developmental potential of ch3PN embryos. The present study will facilitate future studies of ch3PN embryos and could provide additional options for infertile couples.
Project description:Recent discoveries of histone demethylases demonstrate that histone methylation is reversible. However, mechanisms governing the targeting and regulation of histone demethylation remain elusive. Here we report that a Drosophila melanogaster JmjC domain-containing protein, dKDM4A, is a histone H3K36 demethylase. dKDM4A specifically demethylates H3K36me2 and H3K36me3 both in vitro and in vivo. Affinity purification and mass spectrometry analysis revealed that heterochromatin protein 1a (HP1a) associates with dKDMA4A. We found that the chromo shadow domain of HP1a and a HP1-interacting motif of dKDM4A are responsible for this interaction. HP1a stimulates the histone H3K36 demethylation activity of dKDM4A, and this stimulation depends on the H3K9me-binding motif of HP1a. Finally, we provide in vivo evidence suggesting that HP1a and dKDM4A interact with each other and that loss of HP1a leads to an increased level of histone H3K36me3. Collectively, these results suggest a function of HP1a in transcription facilitating H3K36 demethylation at transcribed and/or heterochromatin regions.
Project description:The JmjC histone lysine demethylases (KDMs) are epigenetic regulators involved in the removal of methyl groups from post-translationally modified lysyl residues within histone tails, modulating gene transcription. These enzymes require molecular oxygen for catalytic activity and, as 2-oxoglutarate (2OG)-dependent oxygenases, are related to the cellular oxygen sensing HIF hydroxylases PHD2 and FIH. Recent studies have indicated that the activity of some KDMs, including the pseudogene-encoded KDM4E, may be sensitive to changing oxygen concentrations. Here, we report detailed analysis of the effect of oxygen availability on the activity of the KDM4 subfamily member KDM4A, importantly demonstrating a high level of O2 sensitivity both with isolated protein and in cells. Kinetic analysis of the recombinant enzyme revealed a high KMapp(O2) of 173 ± 23 μM, indicating that the activity of the enzyme is able to respond sensitively to a reduction in oxygen concentration. Furthermore, immunofluorescence experiments in U2OS cells conditionally overexpressing KDM4A showed that the cellular activity of KDM4A against its primary substrate, H3K9me3, displayed a graded response to depleting oxygen concentrations in line with the data obtained using isolated protein. These results suggest that KDM4A possesses the potential to act as an oxygen sensor in the context of chromatin modifications, with possible implications for epigenetic regulation in hypoxic disease states. Importantly, this correlation between the oxygen sensitivity of the catalytic activity of KDM4A in biochemical and cellular assays demonstrates the utility of biochemical studies in understanding the factors contributing to the diverse biological functions and varied activity of the 2OG oxygenases.
Project description:Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. We demonstrate that dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. We conclude that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions.
Project description:Histone demethylases are epigenetic modulators that play key roles in regulating gene expression related to many critical cellular functions and are emerging as promising therapeutic targets in a number of tumor types. We previously identified histone demethylase family members as overexpressed in the pediatric sarcoma, rhabdomyosarcoma. Here we show high sensitivity of rhabdomyosarcoma cells to a pan-histone demethylase inhibitor, JIB-04 and identify a key role for the histone demethylase KDM4B in rhabdomyosarcoma cell growth through an RNAi-screening approach. Decreasing KDM4B levels affected cell cycle progression and transcription of G1/S and G2/M checkpoint genes including CDK6 and CCNA2, which are bound by KDM4B in their promoter regions. However, after sustained knockdown of KDM4B, rhabdomyosarcoma cell growth recovered. We show that this can be attributed to acquired molecular compensation via recruitment of KDM4A to the promoter regions of CDK6 and CCNA2 that are otherwise bound by KDM4B. Furthermore, upfront silencing of both KDM4B and KDM4A led to RMS cell apoptosis, not seen by reducing either alone. To circumvent compensation and elicit stronger therapeutic responses, our study supports targeting histone demethylase sub-family proteins through selective poly-pharmacology as a therapeutic approach.