Publications by authors named "Ayush Raman"

18 Publications

  • Page 1 of 1

Reprogramming of H3K9bhb at regulatory elements is a key feature of fasting in the small intestine.

Cell Rep 2021 Nov;37(8):110044

Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address:

β-hydroxybutyrate (β-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine β-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5 stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of β-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting.
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http://dx.doi.org/10.1016/j.celrep.2021.110044DOI Listing
November 2021

Reprogramming of bivalent chromatin states in NRAS mutant melanoma suggests PRC2 inhibition as a therapeutic strategy.

Cell Rep 2021 Jul;36(3):109410

Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA. Electronic address:

The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients.
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http://dx.doi.org/10.1016/j.celrep.2021.109410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8369408PMC
July 2021

Enhancer reprogramming in PRC2-deficient malignant peripheral nerve sheath tumors induces a targetable de-differentiated state.

Acta Neuropathol 2021 09 20;142(3):565-590. Epub 2021 Jul 20.

Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that frequently harbor genetic alterations in polycomb repressor complex 2 (PRC2) components-SUZ12 and EED. Here, we show that PRC2 loss confers a dedifferentiated early neural-crest phenotype which is exclusive to PRC2-mutant MPNSTs and not a feature of neurofibromas. Neural crest phenotype in PRC2 mutant MPNSTs was validated via cross-species comparative analysis using spontaneous and transgenic MPNST models. Systematic chromatin state profiling of the MPNST cells showed extensive epigenomic reprogramming or chromatin states associated with PRC2 loss and identified gains of active enhancer states/super-enhancers on early neural crest regulators in PRC2-mutant conditions around genomic loci that harbored repressed/poised states in PRC2-WT MPNST cells. Consistently, inverse correlation between H3K27me3 loss and H3K27Ac gain was noted in MPNSTs. Epigenetic editing experiments established functional roles for enhancer gains on DLX5-a key regulator of neural crest phenotype. Consistently, blockade of enhancer activity by bromodomain inhibitors specifically suppressed this neural crest phenotype and tumor burden in PRC2-mutant PDXs. Together, these findings reveal accumulation of dedifferentiated neural crest like state in PRC2-mutant MPNSTs that can be targeted by enhancer blockade.
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http://dx.doi.org/10.1007/s00401-021-02341-zDOI Listing
September 2021

Smart-RRBS for single-cell methylome and transcriptome analysis.

Nat Protoc 2021 08 9;16(8):4004-4030. Epub 2021 Jul 9.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system.
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http://dx.doi.org/10.1038/s41596-021-00571-9DOI Listing
August 2021

Chromatin state dynamics confers specific therapeutic strategies in enhancer subtypes of colorectal cancer.

Gut 2021 May 31. Epub 2021 May 31.

Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

Objective: Enhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described.

Design: We performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin.

Results: We demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such as and was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFβi, mTORi and SRCi) for EpiC groups.

Conclusion: Our data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.
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http://dx.doi.org/10.1136/gutjnl-2020-322835DOI Listing
May 2021

Extended-representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells.

Nat Biotechnol 2021 09 6;39(9):1086-1094. Epub 2021 May 6.

Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

The biological roles of DNA methylation have been elucidated by profiling methods based on whole-genome or reduced-representation bisulfite sequencing, but these approaches do not efficiently survey the vast numbers of non-coding regulatory elements in mammalian genomes. Here we present an extended-representation bisulfite sequencing (XRBS) method for targeted profiling of DNA methylation. Our design strikes a balance between expanding coverage of regulatory elements and reproducibly enriching informative CpG dinucleotides in promoters, enhancers and CTCF binding sites. Barcoded DNA fragments are pooled before bisulfite conversion, allowing multiplex processing and technical consistency in low-input samples. Application of XRBS to single leukemia cells enabled us to evaluate genetic copy number variations and methylation variability across individual cells. Our analysis highlights heterochromatic H3K9me3 regions as having the highest cell-to-cell variability in their methylation, likely reflecting inherent epigenetic instability of these late-replicating regions, compounded by differences in cell cycle stages among sampled cells.
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http://dx.doi.org/10.1038/s41587-021-00910-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8434949PMC
September 2021

A research parasite's perspective on establishing a baseline to avoid errors in secondary analyses.

Authors:
Ayush T Raman

Gigascience 2021 03;10(3)

Broad Institute of MIT and Harvard, 75 Ames St, Cambridge, MA 02142, USA.

To enhance reproducibility in scientific research, more and more datasets are becoming publicly available so that researchers can perform secondary analyses to investigate questions the original scientists had not posited. This increases the return on investment for the NIH and other funding bodies. These datasets, however, are not perfect, and a better understanding of the assumptions that shaped them is required. The 2020 Junior Research Parasite Award recognized our work that showed that the signal-to-noise ratio in a particular dataset had not been investigated, leading to an erroneous conclusion in the original research. In this commentary, I share the process that led to the identification of the problem and hopefully provide useful lessons for other research parasites.
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http://dx.doi.org/10.1093/gigascience/giab015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953484PMC
March 2021

Enhancer Reprogramming Confers Dependence on Glycolysis and IGF Signaling in KMT2D Mutant Melanoma.

Cell Rep 2020 10;33(3):108293

Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors.
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http://dx.doi.org/10.1016/j.celrep.2020.108293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649750PMC
October 2020

Accumulation of long-chain fatty acids in the tumor microenvironment drives dysfunction in intrapancreatic CD8+ T cells.

J Exp Med 2020 08;217(8)

Department of Experimental Oncology, IRCCS European Institute of Oncology, Milano, Italy.

CD8+ T cells are master effectors of antitumor immunity, and their presence at tumor sites correlates with favorable outcomes. However, metabolic constraints imposed by the tumor microenvironment (TME) can dampen their ability to control tumor progression. We describe lipid accumulation in the TME areas of pancreatic ductal adenocarcinoma (PDA) populated by CD8+ T cells infiltrating both murine and human tumors. In this lipid-rich but otherwise nutrient-poor TME, access to using lipid metabolism becomes particularly valuable for sustaining cell functions. Here, we found that intrapancreatic CD8+ T cells progressively accumulate specific long-chain fatty acids (LCFAs), which, rather than provide a fuel source, impair their mitochondrial function and trigger major transcriptional reprogramming of pathways involved in lipid metabolism, with the subsequent reduction of fatty acid catabolism. In particular, intrapancreatic CD8+ T cells specifically exhibit down-regulation of the very-long-chain acyl-CoA dehydrogenase (VLCAD) enzyme, which exacerbates accumulation of LCFAs and very-long-chain fatty acids (VLCFAs) that mediate lipotoxicity. Metabolic reprogramming of tumor-specific T cells through enforced expression of ACADVL enabled enhanced intratumoral T cell survival and persistence in an engineered mouse model of PDA, overcoming one of the major hurdles to immunotherapy for PDA.
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http://dx.doi.org/10.1084/jem.20191920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398173PMC
August 2020

Nr2f1 heterozygous knockout mice recapitulate neurological phenotypes of Bosch-Boonstra-Schaaf optic atrophy syndrome and show impaired hippocampal synaptic plasticity.

Hum Mol Genet 2020 03;29(5):705-715

Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX, USA.

Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) has been identified as an autosomal-dominant disorder characterized by a complex neurological phenotype, with high prevalence of intellectual disability and optic nerve atrophy/hypoplasia. The syndrome is caused by loss-of-function mutations in NR2F1, which encodes a highly conserved nuclear receptor that serves as a transcriptional regulator. Previous investigations to understand the protein's role in neurodevelopment have mostly used mouse models with constitutive and tissue-specific homozygous knockout of Nr2f1. In order to represent the human disease more accurately, which is caused by heterozygous NR2F1 mutations, we investigated a heterozygous knockout mouse model and found that this model recapitulates some of the neurological phenotypes of BBSOAS, including altered learning/memory, hearing defects, neonatal hypotonia and decreased hippocampal volume. The mice showed altered fear memory, and further electrophysiological investigation in hippocampal slices revealed significantly reduced long-term potentiation and long-term depression. These results suggest that a deficit or alteration in hippocampal synaptic plasticity may contribute to the intellectual disability frequently seen in BBSOAS. RNA-sequencing (RNA-Seq) analysis revealed significant differential gene expression in the adult Nr2f1+/- hippocampus, including the up-regulation of multiple matrix metalloproteases, which are known to be critical for the development and the plasticity of the nervous system. Taken together, our studies highlight the important role of Nr2f1 in neurodevelopment. The discovery of impaired hippocampal synaptic plasticity in the heterozygous mouse model sheds light on the pathophysiology of altered memory and cognitive function in BBSOAS.
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http://dx.doi.org/10.1093/hmg/ddz233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104670PMC
March 2020

Atypical plant homeodomain of UBR7 functions as an H2BK120Ub ligase and breast tumor suppressor.

Nat Commun 2019 03 28;10(1):1398. Epub 2019 Mar 28.

Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata, 700064, India.

The roles of Plant Homeodomain (PHD) fingers in catalysis of histone modifications are unknown. We demonstrated that the PHD finger of Ubiquitin Protein Ligase E3 Component N-Recognin7 (UBR7) harbors E3 ubiquitin ligase activity toward monoubiquitination of histone H2B at lysine120 (H2BK120Ub). Purified PHD finger or full-length UBR7 monoubiquitinated H2BK120 in vitro, and loss of UBR7 drastically reduced H2BK120Ub genome-wide binding sites in MCF10A cells. Low UBR7 expression was correlated with occurrence of triple-negative breast cancer and metastatic tumors. Consistently, UBR7 knockdown enhanced the invasiveness, induced epithelial-to-mesenchymal transition and promoted metastasis. Conversely, ectopic expression of UBR7 restored these cellular phenotypes and reduced tumor growth. Mechanistically, UBR7 loss reduced H2BK120Ub levels on cell adhesion genes, including CDH4, and upregulated the Wnt/β-Catenin signaling pathway. CDH4 overexpression could partially revert UBR7-dependent cellular phenotypes. Collectively, our results established UBR7 as a histone H2B monoubiquitin ligase that suppresses tumorigenesis and metastasis of triple-negative breast cancer.
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http://dx.doi.org/10.1038/s41467-019-08986-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438984PMC
March 2019

Loss of histone acetylation and H3K4 methylation promotes melanocytic malignant transformation.

Mol Cell Oncol 2018 26;5(3):e1359229. Epub 2018 Feb 26.

Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX, USA.

Epigenetic mechanisms play essential roles in biological processes such as cell maintenance, differentiation, proliferation and apoptosis. A recent report from our laboratory showed that the loss of histone acetylation and H3K4 (Histone H3 Lysine 4) methylation in the proximal regions of cancer regulatory genes promote tumorigenesis.
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http://dx.doi.org/10.1080/23723556.2017.1359229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149988PMC
February 2018

Apparent bias toward long gene misregulation in MeCP2 syndromes disappears after controlling for baseline variations.

Nat Commun 2018 08 13;9(1):3225. Epub 2018 Aug 13.

Graduate Program in Quantitative and Computational Biosciences, Baylor College of Medicine, Houston, TX, 77030, USA.

Recent studies have suggested that genes longer than 100 kb are more likely to be misregulated in neurological diseases associated with synaptic dysfunction, such as autism and Rett syndrome. These length-dependent transcriptional changes are modest in MeCP2-mutant samples, but, given the low sensitivity of high-throughput transcriptome profiling technology, here we re-evaluate the statistical significance of these results. We find that the apparent length-dependent trends previously observed in MeCP2 microarray and RNA-sequencing datasets disappear after estimating baseline variability from randomized control samples. This is particularly true for genes with low fold changes. We find no bias with NanoString technology, so this long gene bias seems to be particular to polymerase chain reaction amplification-based platforms. In contrast, authentic long gene effects, such as those caused by topoisomerase inhibition, can be detected even after adjustment for baseline variability. We conclude that accurate characterization of length-dependent (or other) trends requires establishing a baseline from randomized control samples.
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http://dx.doi.org/10.1038/s41467-018-05627-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6089998PMC
August 2018

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues.

J Vis Exp 2018 04 5(134). Epub 2018 Apr 5.

Department of Genomic Medicine, University of Texas MD Anderson Cancer Center;

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.
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http://dx.doi.org/10.3791/56972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933412PMC
April 2018

Detecting hidden batch factors through data-adaptive adjustment for biological effects.

Bioinformatics 2018 04;34(7):1141-1147

Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030, USA.

Motivation: Batch effects are one of the major source of technical variations that affect the measurements in high-throughput studies such as RNA sequencing. It has been well established that batch effects can be caused by different experimental platforms, laboratory conditions, different sources of samples and personnel differences. These differences can confound the outcomes of interest and lead to spurious results. A critical input for batch correction algorithms is the knowledge of batch factors, which in many cases are unknown or inaccurate. Hence, the primary motivation of our paper is to detect hidden batch factors that can be used in standard techniques to accurately capture the relationship between gene expression and other modeled variables of interest.

Results: We introduce a new algorithm based on data-adaptive shrinkage and semi-Non-negative Matrix Factorization for the detection of unknown batch effects. We test our algorithm on three different datasets: (i) Sequencing Quality Control, (ii) Topotecan RNA-Seq and (iii) Single-cell RNA sequencing (scRNA-Seq) on Glioblastoma Multiforme. We have demonstrated a superior performance in identifying hidden batch effects as compared to existing algorithms for batch detection in all three datasets. In the Topotecan study, we were able to identify a new batch factor that has been missed by the original study, leading to under-representation of differentially expressed genes. For scRNA-Seq, we demonstrated the power of our method in detecting subtle batch effects.

Availability And Implementation: DASC R package is available via Bioconductor or at https://github.com/zhanglabNKU/DASC.

Contact: [email protected] or [email protected]

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btx635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6454417PMC
April 2018

Forniceal deep brain stimulation induces gene expression and splicing changes that promote neurogenesis and plasticity.

Elife 2018 03 23;7. Epub 2018 Mar 23.

Department of Neuroscience, Baylor College of Medicine, Houston, United States.

Clinical trials are currently underway to assess the efficacy of forniceal deep brain stimulation (DBS) for improvement of memory in Alzheimer's patients, and forniceal DBS has been shown to improve learning and memory in a mouse model of Rett syndrome (RTT), an intellectual disability disorder caused by loss-of-function mutations in . The mechanism of DBS benefits has been elusive, however, so we assessed changes in gene expression, splice isoforms, DNA methylation, and proteome following acute forniceal DBS in wild-type mice and mice lacking . We found that DBS upregulates genes involved in synaptic function, cell survival, and neurogenesis and normalized expression of ~25% of the genes altered in -null mice. Moreover, DBS induced expression of 17-24% of the genes downregulated in other intellectual disability mouse models and in post-mortem human brain tissue from patients with Major Depressive Disorder, suggesting forniceal DBS could benefit individuals with a variety of neuropsychiatric disorders.
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http://dx.doi.org/10.7554/eLife.34031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906096PMC
March 2018

TRIM28 and Interacting KRAB-ZNFs Control Self-Renewal of Human Pluripotent Stem Cells through Epigenetic Repression of Pro-differentiation Genes.

Stem Cell Reports 2017 12 30;9(6):2065-2080. Epub 2017 Nov 30.

Laboratory for Gene Therapy, Department of Cancer Immunology, Poznan University of Medical Sciences, 60-806 Poznan, Poland; Laboratory for Gene Therapy, Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, 61-866 Poznan, Poland. Electronic address:

Reprogramming to induced pluripotent stem cells (iPSCs) and differentiation of pluripotent stem cells (PSCs) are regulated by epigenetic machinery. Tripartite motif protein 28 (TRIM28), a universal mediator of Krüppel-associated box domain zinc fingers (KRAB-ZNFs), is known to regulate both processes; however, the exact mechanism and identity of participating KRAB-ZNF genes remain unknown. Here, using a reporter system, we show that TRIM28/KRAB-ZNFs alter DNA methylation patterns in addition to H3K9me3 to cause stable gene repression during reprogramming. Using several expression datasets, we identified KRAB-ZNFs (ZNF114, ZNF483, ZNF589) in the human genome that maintain pluripotency. Moreover, we identified target genes repressed by these KRAB-ZNFs. Mechanistically, we demonstrated that these KRAB-ZNFs directly alter gene expression of important developmental genes by modulating H3K9me3 and DNA methylation of their promoters. In summary, TRIM28 employs KRAB-ZNFs to evoke epigenetic silencing of its target differentiation genes via H3K9me3 and DNA methylation.
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http://dx.doi.org/10.1016/j.stemcr.2017.10.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785758PMC
December 2017

A FOXO3-IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses.

Nature 2012 Oct 16;490(7420):421-5. Epub 2012 Sep 16.

Seattle Biomedical Research Institute, Seattle, Washington 98109, USA.

Antiviral responses must be tightly regulated to defend rapidly against infection while minimizing inflammatory damage. Type 1 interferons (IFN-I) are crucial mediators of antiviral responses and their transcription is regulated by a variety of transcription factors; principal among these is the family of interferon regulatory factors (IRFs). The IRF gene regulatory networks are complex and contain multiple feedback loops. The tools of systems biology are well suited to elucidate the complex interactions that give rise to precise coordination of the interferon response. Here we have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. Genome-wide location analysis combined with gene deletion studies identified the Irf7 gene as a critical target of FOXO3. FOXO3 was identified as a negative regulator of Irf7 transcription and we have further demonstrated that FOXO3, IRF7 and IFN-I form a coherent feed-forward regulatory circuit. Our data suggest that the FOXO3-IRF7 regulatory circuit represents a novel mechanism for establishing the requisite set points in the interferon pathway that balances the beneficial effects and deleterious sequelae of the antiviral response.
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http://dx.doi.org/10.1038/nature11428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556990PMC
October 2012
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