Malith S Karunasiri’s research while affiliated with Howard Hughes Medical Institute and other places

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Publications (4)


Insights into Negative Regulation by the Glucocorticoid Receptor from Genome-Wide Profiling of Inflammatory Cistromes
  • Article

November 2012

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89 Reads

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243 Citations

Molecular Cell

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How the glucocorticoid receptor (GR) activates some genes while potently repressing others remains an open question. There are three current models for suppression: transrepression via GR tethering to AP-1/NF-κB sites, direct GR association with inhibitory elements (nGREs), and GR recruitment of the corepressor GRIP1. To gain insights into GR suppression, we used genomic analyses and genome-wide profiling of GR, p65, and c-Jun in LPS-stimulated macrophages. We show that GR mediates both activation and repression at tethered sites, GREs, and GRIP1-bound elements, indicating that motif classification is insufficient to predict regulatory polarity of GR binding. Interestingly, sites of GR repression utilize GRIP1's corepressor function and display reduced histone acetylation. Together, these findings suggest that while GR occupancy confers hormone responsiveness, the receptor itself may not participate in the regulatory effects. Furthermore, transcriptional outcome is not established by sequence but is influenced by epigenetic regulators, context, and other unrecognized regulatory determinants.


The Bcl6-SMRT/NCoR Cistrome Represses Inflammation to Attenuate Atherosclerosis

March 2012

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145 Reads

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112 Citations

Cell Metabolism

Chronic inflammation is a hallmark of atherosclerosis, but its transcriptional underpinnings are poorly understood. We show that the transcriptional repressor Bcl6 is an anti-inflammatory regulator whose loss in bone marrow of Ldlr(-/-) mice results in severe atherosclerosis and xanthomatous tendonitis, a virtually pathognomonic complication in patients with familial hypercholesterolemia. Disruption of the interaction between Bcl6 and SMRT or NCoR with a peptide inhibitor in vitro recapitulated atherogenic gene changes in mice transplanted with Bcl6-deficient bone marrow, pointing to these cofactors as key mediators of Bcl6 inflammatory suppression. Using ChIP-seq, we reveal the SMRT and NCoR corepressor cistromes, each consisting of over 30,000 binding sites with a nearly 50% overlap. While the complete cistromes identify a diversity of signaling pathways, the Bcl6-bound subcistromes for each corepressor are highly enriched for NF-κB-driven inflammatory and tissue remodeling genes. These results reveal that Bcl6-SMRT/NCoR complexes constrain immune responses and contribute to the prevention of atherosclerosis.


Exercise and PGC-1??-Independent Synchronization of Type I Muscle Metabolism and Vasculature by ERR??

March 2011

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129 Reads

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171 Citations

Cell Metabolism

How type I skeletal muscle inherently maintains high oxidative and vascular capacity in the absence of exercise is unclear. We show that nuclear receptor ERRγ is highly expressed in type I muscle and, when transgenically expressed in anaerobic type II muscles (ERRGO mice), dually induces metabolic and vascular transformation in the absence of exercise. ERRGO mice show increased expression of genes promoting fat metabolism, mitochondrial respiration, and type I fiber specification. Muscles in ERRGO mice also display an activated angiogenic program marked by myofibrillar induction and secretion of proangiogenic factors, neovascularization, and a 100% increase in running endurance. Surprisingly, the induction of type I muscle properties by ERRγ does not involve PGC-1α. Instead, ERRγ genetically activates the energy sensor AMPK in mediating the metabovascular changes in ERRGO mice. Therefore, ERRγ represents a previously unrecognized determinant that specifies intrinsic vascular and oxidative metabolic features that distinguish type I from type II muscle.


Figure 1. Bcl-6 coregulates the Tlr4-elicited gene expression program. (A) Representation of genes significantly induced or repressed in expression microarrays from wild-type (Bcl-6 +/+ ) and knockout (Bcl-6 À/À ) BMDMs versus genes significantly altered in wild-type BMDMs by exposure to LPS (100 ng/mL) for 6 h. (B,C) Functional categorization of Bcl-6-regulated genes (B) or Bcl-6-and LPS-coregulated genes (C) depicted as percentages of total. (D) qPCR of microarray-identified Bcl-6-and LPS-coregulated genes in wild-type and knockout BMDMs at 0, 2, or 6 h following exposure to LPS (100 ng/ mL). The mean relative expression 6 SD compared with wild-type BMDMs at baseline (0 h) is listed.
Figure 2. ChIP-seq reveals extensive colocalization of Bcl-6 with NF-kB. (A) Distribution of Bcl-6 and NF-kB p65-binding sites in BMDMs and their overlap with other cistromes as indicated. (B) Graphical depiction of the number of genes identified with significantly altered expression in Bcl-6 À/À versus Bcl-6 +/+ BMDMs (yellow), the number of genes annotated with Bcl-6-binding sites by ChIP-seq (blue), and the intersections of these gene sets in unstimulated BMDMs. (C) Functional classification of genes identified with binding sites for Bcl-6, NF-kB, or proximal Bcl-6 and NF-kB by ChIP-seq in unstimulated and LPS-stimulated BMDMs. (D) Schematic representing Tlr4-initiated transcriptional programs that are NF-kB p65-controlled (left) and NF-kB p65-independent (right) based on p65 ChIP-seq annotation. (Left) Bcl-6 regulation of Tlr4 responses is concentrated in genes annotated with proximal Bcl-6 and NF-kB sites. (Right) Bcl-6 binding is infrequent in Tlr4 target genes controlled by other transcription factors (TFs).
Figure 3. ChIP-seq and epigenetic analysis reveals Bcl-6 and NF- k B coregulation of inflammatory genes. ( A ) ChIP-seq tracks for acetylated H3, monomethylated H3K4 (H3K4me1), and RNA polymerase II (RNA pol II) in unstimulated BMDMs as well as p300, Pu.1, NF À k B p65, and Bcl-6 in unstimulated or LPS-stimulated (100 ng/mL for 3 h) BMDMs along the Il-1 gene cluster. For factors sequenced with and without exposure to LPS, track heights were normalized to the number of aligned reads. ( B , C ) ChIP qPCR of acetylated H3 normalized to H3 ( B ), and Hdac3 enrichment relative to input chromatin in wild-type and knockout BMDMs 
Figure 4. Model of macrophage Bcl-6 in inflammatory control. Bcl-6 restrains inflammation through transcriptional repression via histone deacetylation at inflammatory gene enhancers, in close proximity to sites inducibly bound by NF- k B and the histone acetyltransferase p300 following Tlr4 stimulation. In the setting of TLR signals, loss of Bcl-6 and its corepressor, Hdac3, results in unopposed NF- k B transcriptional activation and hyperinflammatory responses. 
Bcl-6 and NF- B cistromes mediate opposing regulation of the innate immune response
  • Article
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December 2010

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448 Reads

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242 Citations

Genes & Development

In the macrophage, toll-like receptors (TLRs) are key sensors that trigger signaling cascades to activate inflammatory programs via the NF-κB gene network. However, the genomic network targeted by TLR/NF-κB activation and the molecular basis by which it is restrained to terminate activation and re-establish quiescence is poorly understood. Here, using chromatin immunoprecipitation sequencing (ChIP-seq), we define the NF-κB cistrome, which is comprised of 31,070 cis-acting binding sites responsive to lipopolysaccharide (LPS)-induced signaling. In addition, we demonstrate that the transcriptional repressor B-cell lymphoma 6 (Bcl-6) regulates nearly a third of the Tlr4-regulated transcriptome, and that 90% of the Bcl-6 cistrome is collapsed following Tlr4 activation. Bcl-6-deficient macrophages are acutely hypersensitive to LPS and, using comparative ChIP-seq analyses, we found that the Bcl-6 and NF-κB cistromes intersect, within nucleosomal distance, at nearly half of Bcl-6-binding sites in stimulated macrophages to promote opposing epigenetic modifications of the local chromatin. These results reveal a genomic strategy for controlling the innate immune response in which repressive and inductive cistromes establish a dynamic balance between macrophage quiescence and activation via epigenetically marked cis-regulatory elements.

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Citations (4)


... Recently, it has been suggested that for most GR-mediated repression of inflammation signaling, direct DNA binding of the receptor is required ( Figure 1C). In macrophages stimulated with inflammatory lipopolysaccharides, only one fifth of GR-mediated repression was determined to be related to tethering events, with the majority being regulated by direct DNA binding of GR (45). A study with zinc finger point-mutated GR unable to bind DNA, provided evidence that even though tethering is maintained, direct DNA binding is necessary to assemble coregulator complexes and convey transcriptional repression through GR (46). ...

Reference:

Glucocorticoid receptor action in prostate cancer: the role of transcription factor crosstalk
Insights into Negative Regulation by the Glucocorticoid Receptor from Genome-Wide Profiling of Inflammatory Cistromes
  • Citing Article
  • November 2012

Molecular Cell

... Chronic inflammation was a hallmark of atherosclerosis to the extent that acute transcriptional induction is a driver of inflammation. From early fatty streaks to mature lesions, MØ played a critical role in atherogenesis while the BCL6 played a pivotal role in MØ quiescence and the regulation of inflammation 38 . Previous research had found that BCL6 reduces oxidized LDL and TLR-induced inflammation and atherogenesis, indicating that BCL6 plays an important role in reducing early vascular lesion development 39 . ...

The Bcl6-SMRT/NCoR Cistrome Represses Inflammation to Attenuate Atherosclerosis
  • Citing Article
  • March 2012

Cell Metabolism

... Furthermore, ERRα coordinates muscle recovery and repair [75,76]. Other than that, estrogen-related receptor gamma (ERRγ) has a role in type I muscle fibers and promotes mitochondrial biogenesis and the oxidative assimilation framework [77,78]. ERRγ-regulated pathways allow ERRγ to coordinate the control of type I myosin expression and the high oxidative assimilation framework. ...

Exercise and PGC-1??-Independent Synchronization of Type I Muscle Metabolism and Vasculature by ERR??
  • Citing Article
  • March 2011

Cell Metabolism

... First, changes in transcriptional programs may occur in response to specific stimuli, and second, metabolic reprogramming causes gene-specific transcriptional changes and alterations. Signal-regulated transcription factors activate different macrophage gene expression programs by binding to numerous cis-regulatory regions [125][126][127]. The major transcription factors involved in modulating macrophage functions are signal transducer and activator of transcription (STATs) [128], NF-κβ [129,130], PPAR-γ [131,132], IRFs [133,134], AP-1(activator protein) [135], and cAMP-responsive element-binding protein (CREB) [136]. ...

Bcl-6 and NF- B cistromes mediate opposing regulation of the innate immune response

Genes & Development