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Decoding the Epigenetic Landscape by the Histone Readers: Implications in Human Diseases

Epigenetic modifications in DNA and core histones of chromatin fine-tune the underlying gene expression programs. These modifications are highly dynamic and can be causally related to normal homeostasis of the organism and the pathobiological state. The epigenetic modifications are operated through a class of proteins termed as “chromatin reader/effector” which lead to differential recruitment of other regulatory factors. The research focus of Chromatin Dynamics Laboratory is to understand some of these epigenetic readers in the context of cellular functions and their possible connection to the disease, including metabolic as well as infectious diseas

Researchers from the Chromatin Dynamics Laboratory have recently shown that Plant Homeo Domain (PHD) finger containing protein TCF19 is a novel glucose and insulin responsive transcription factor that modulates histone post-translational modifications to regulate glucose homeostasis in hepatocytes. Microarray analysis on TCF19 depleted cells showed a global effect on metabolic pathways and interestingly the gluconeogenic genes were significantly upregulated. More in-depth analysis revealed how TCF19 exerts a repressive effect on the gluconeogenic genes by integrating the hormonal and metabolic cues via its PHD finger interactions with chromatin.
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Figure 1: (a) Transcriptional activity of three key gluconeogenic genes in HepG2 cells: Glucose-6 Phosphatase (G6PC), Fructose 1,6 bisphosphatase (FBP1) and Pyruvate-Carboxy Kinase 1 (PCK1) was quantified in the presence of known activators (Dexamethasone/cMAP), repressor (insulin), either singly or in combination, with overexpression of either  TCF19-full length ( TCF19 FL) or TCF19 ΔPHD FLAG tagged constructs . (b) Transcription levels of the three gluconeogenic genes were quantified by real time PCR with mRNA isolated from HepG2 treated with TCF19 siRNA, under influence of Dexamethasone/cMAP or repressor insulin either singly, or in combination. (c) Heat maps of expression values for differentially expressed genes on TCF19 knockdown under High glucose condition. (p value ≤ 0.05, fold change ≥ 1.5). Down-regulated genes are marked in green and up-regulated genes are marked in red. (d) Effect of knockdown of TCF19 in HepG2, Huh7 and HepaRG cells on core gluconeogenic genes. TCF19 siRNA was transfected in HepG2 cells under hyperglycemic condition and Normoglycemic condition. Non-targeting siRNA used as negative control. Fold change calculated over mRNA levels of HepG2 cells treated with scrambled siRNA and 18S rRNA was used for normalization. Experiments were repeated thrice, and error bar represents standard deviation. Unpaired Students t-test was used to determine p value (*p<0.05, **p<0.01).

Physical interaction of TCF19 with components of a deacetylase complex: NuRD (Nucleosome Remodelling and Deacetylase), and their co-recruitment onto promoters of gluconeogenic genes in high glucose conditions suggests that the observed repression is possibly mediated in concert with NuRD complex.. Thus, their investigation establishes that TCF19 is an important target in modulating the glucose homeostasis in cells.
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Figure 2: ChIP assays were done in HepG2 cells maintained under low glucose condition (5mM glucose) or high glucose condition (40mM glucose). As a template for recruitment, approx. 500bp of the region, upstream of the transcription start site of each of the three key gluconeogenic genes was selected and previously reported primers were used. pp: proximal promoter region, 3’ untranslated region (3’UTR) of individual genes were used as negative control. NAV1.2 promoter is used as a glucose non-responsive control for TCF19. (a) Recruitment of TCF19 was found to be enhanced in high glucose conditions. (b) Depletion of activation mark H3K9Me3 following high glucose induction at the  gluconeogenic promoter site. (c) Binding of CHD4 (NuRD component) to promoter region was reduced on siRNA mediated knockdown of TCF19 in HepG2 cells under high glucose condition. (d) Depletion of TCF19 in HepG2 cells maintained in either low glucose or high glucose conditions causes increase in transcription activation mark H3K9Me3.
The prevalence global health problems like type 1 diabetes, type 2 diabetes, obesity and other metabolic disorders has garnered a lot of public attention, and increasing studies indicate an epigenetic regulation can influence metabolic disease progression. In this laboratory, the researchers have adapted a multipronged strategy to understand the interdependence of epigenetic regulation and metabolic homeostasis.

In the same issue, researchers from the Chromatin Dynamics Laboratory have also shown a mechanism by which Hepatitis B virus (HBV) can evade host immune-response by hijacking host protein Sp110 and therefore proposed the latter to be a novel target for antiviral therapy. Hepatitis B virus (HBV), belonging to Hepadnaviridae family, remains undetected in early infection as it does not induce the innate immune response and is known to be the cause of several hepatic diseases leading to cirrhosis and hepatocellular carcinoma. HBx, a multifunctional protein encoded by HBV, plays a crucial role in pathogenesis and replication of the virus. The nucleus of a mammalian cell has many sub-compartments like the nucleolus, nuclear speckles, Cajal bodies, Promyelocytic leukemia nuclear bodies (PML-NB) etc. In the recent publication, they have identified PML-NB protein Sp110 as a novel interactor of the viral protein HBx.  Accompanied by a deSUMOylase, HBx was shown to drive Sp110 out of the PML body. Further, the viral protein was found to utilize the chromatin binding property of Sp110 to get recruited to its target site.
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Figure 3: HBx shows cooperative association with Sp110 and drives it out of the PML-NB. (A) Co-immunofluorescence staining showing that Sp110 associates with HBx to exit the PML-NBs Panel (I) shows HBx (Alexa 488) does not co-localize with PML-NBs, stained with its marker protein PML (Alexa 594). Panel (II) shows the usual PML-NB distribution of Sp110 (Alexa 594) altered upon 1.3mer HBV transfection and Sp110 co-localizes with HBx. Panel (III) shows Sp110 remains in its PML-NB distribution upon 1.3mer HBV X-null transfection (B)  Co-Immunoprecipitation with α-HA antibody in HA-HBx transfected HepG2 cells, followed by immunoblotting with a α-Sp110 antibody, confirms the interaction between HBx and Sp110. (C) ChIP assay showed significantly decreased recruitment of HBx from its target gene promoters upon Sp110 silencing in HepG2.2.15 cells. MYCN, TAF5, BCL2L12, and PERP are HBx regulated genes and ACTB is the negative control.
Intriguingly, Sp110 knock-down led to a significant viral elimination by activation of type-I interferon response pathway.
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Figure 4: Sp110 knockdown leads viral elimination through Type I IFN response pathway activation (A) A drastic decrease in the amount of HBV DNA released in the culture supernatant (Viral load) was observed upon Sp110 silencing but no significant change was observed upon Sp100 knockdown. (B) A network obtained from STRING network analysis of differentially regulated genes (absolute fold change>=2.0; p<=0.05; minimum required interaction score: 0.7, high confidence) from microarray data for HepG2.2.15 Control siRNA and Sp110 siRNA samples. It revealed a hub of highly connected nodes, belonging to Type-I interferon response pathway. The network is based on interactions reported experimentally, in databases or proteins that are co-expressed. (C) Validation of some of the upregulation of the genes obtained from Type-I interferon response pathway by qRT-PCR.
Present antiviral therapies available include drugs targeting various stages of the HBV life cycle, but are limited by their viral sub-type specificity. Another mode of treatment involves immuno-modulatory ligands, which have low effectiveness as the virus intervenes in the response pathway. The researchers in this laboratory have found Sp110 to be one of the prime host factors which is exploited by HBV and is crucial for the pathogenesis. Thus, Sp110 might be considered as a potential therapeutic target which can overcome the plausible drawbacks of the currently available methods.
Both works were accepted for publication in the December 15th, 2017 issue of J. Biol. Chem. and have been featured on the Cover Page of the same issue.
Chromatin Dynamics Laboratory (PI: Dr. Chandrima Das), Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata-700064, West Bengal, India.
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