MIR retrotransposon sequences provide insulators to the human genome

2015 Aug 11;112(32):E4428-37

Wang J, Vince-Garcia, Seruggia D, Molto E, Fernandez-Minan A, Neto A, Lee E, Gomez-Skarmeta JL, Montoliu L, Lunyak VV, Jordan IK.

Insulators are regulatory elements that help to organize eukaryotic chromatin via enhancer-blocking and chromatin barrier activity. Although there are several examples of transposable element (TE)-derived insulators, the contribution of TEs to human insulators has not been systematically explored. Mammalian-wide interspersed repeats (MIRs) are a conserved family of TEs that have substantial regulatory capacity and share sequence characteristics with tRNA-related insulators. We sought to evaluate whether MIRs can serve as insulators in the human genome. We applied a bioinformatic screen using genome sequence and functional genomic data from CD4(+) T cells to identify a set of 1,178 predicted MIR insulators genome-wide. These predicted MIR insulators were computationally tested to serve as chromatin barriers and regulators of gene expression in CD4(+) T cells. The activity of predicted MIR insulators was experimentally validated using in vitro and in vivo enhancer-blocking assays. MIR insulators are enriched around genes of the T-cell receptor pathways and reside at T-cell-specific boundaries of repressive and active chromatin. A total of 58% of the MIR insulators predicted here show evidence of T-cell specific chromatin barrier and gene regulatory activity. MIR insulators appear to be CCCTC-binding factor (CTCF) independent and show distinct local chromatin environment and marked peaks for RNA Pol III and a number of histone modifications, suggesting that MIR insulators recruit transcriptional complexes and chromatin modifying enzymes in situ to help establish chromatin and regulatory domains in the human genome. The provisioning of insulators by MIRs across the human genome suggests a specific mechanism by which TE sequences can be used to modulate gene regulatory networks.

The full study is accessible here. 

Transcriptional profiling of interleukin-2-primed human adipose derived mesenchymal stem cells revealed dramatic changes in stem cells response imposed by replicative senescence

Oncotarget. 2015 Jul 20; 6(20):17938-57.

Ping Niu, Aibek Smagul, Lu Wang, Aiman Sadvakas, Ying Sha, Laura M. Pérez, Aliya Nussupbekova, Aday Amirbekov, Akan A. Akanov, Beatriz G. Gálvez, I. King Jordan, and Victoria V. Lunyak.

Inflammation is a double-edged sword with both detrimental and beneficial consequences. Understanding of the mechanisms of crosstalk between the inflammatory milieu and human adult mesenchymal stem cells is an important basis for clinical efforts. Here, we investigate changes in the transcriptional response of human adipose-derived stem cells to physiologically relevant levels of IL-2 (IL-2 priming) upon replicative senescence. Our data suggest that replicative senescence might dramatically impede human mesenchymal stem cell (MSC) function via global transcriptional deregulation in response to IL-2. We uncovered a novel senescence- associated transcriptional signature in human adipose-derived MSCs hADSCs after exposure to pro-inflammatory environment: significant enhancement of the expression of the genes encoding potent growth factors and cytokines with anti-inflammatory and migration-promoting properties, as well as genes encoding angiogenic and anti- apoptotic promoting factors, all of which could participate in the establishment of a unique microenvironment. We observed transcriptional up-regulation of critical components of the nitric oxide synthase pathway (iNOS) in hADSCs upon replicative senescence suggesting, that senescent stem cells can acquire metastasis-promoting properties via stem cell-mediated immunosuppression. Our study highlights the importance of age as a factor when designing cell-based or pharmacological therapies for older patients and predicts measurable biomarkers characteristic of an environment that is conducive to cancer cells invasiveness and metastasis. 

The full study and its related press release is accessible here. 

Developmentally regulated activation of a SINE B2 repeat as a domain boundary in organogenesis.

Science. 2007 Jul 13;317(5835):248-51

Lunyak VV, Prefontaine GG, Núñez E, Cramer T, Ju BG, Ohgi KA, Hutt K, Roy R, García-Díaz A, Zhu X, Yung Y, Montoliu L, Glass CK, Rosenfeld MG.

During development, genes are often transcribed in a temporally and spatially regulated manner. The temporal and spatial regulation of gene expression in mammalian development is linked to the establishment of functional chromatin domains.  The murine growth hormone gene is differentially expressed in the developing pituitary gland. Lunyak et al. now examine the region surrounding the growth hormone gene and show that a repeated DNA sequence (short interspersed nuclear element B2) in the growth hormone locus functions as an insulator to produce a boundary for chromatin domains and limit the action of regulatory factors such as enhancers and silencers.

A topoisomerase IIbeta-mediated dsDNA break required for regulated transcription.

Science. 2006 Jun 23;312(5781):1798-802.

Ju BG, Lunyak VV, Perissi V, Garcia-Bassets I, Rose DW, Glass CK, Rosenfeld MG.

The lengthy genomic DNA of a eukaryotic cell manages to fit within the relatively small confines of its nucleus by spooling. Twisting ribbons of DNA are tightly wrapped around core histone proteins, forming compact nucleosomes that constitute chromatin, the substance of chromosomes. Although this compacted structure is a means to handle space constraint, it presents a barrier to regulated genomic activity, including gene expression and maintenance of genomic integrity. However, cells are not adversely affected by such tight packaging because this highly structured assembly is compliant and dynamic, displaying varying degrees of compaction that allows regulated access to protein complexes in response to various stimuli. To operate within this overcrowded area, protein complexes locally remodel chromatin. But precisely how defined areas of chromatin adopt different conformations, allowing regulated access to specific DNA regions, is a captivating question. This manuscript provides molecular evidence that the enzyme DNA topoisomerase IIβ (TopoIIβ) activates transcription by generating a break in double-stranded DNA within a nucleosome. This enzyme, which is associated with a DNA-repair machinery, allows chromatin to relax, which is needed to drive gene expression.

No rest for REST: REST/NRSF regulation of neurogenesis.

Cell. 2005 May 20;121(4):499-501

Lunyak VV, Rosenfeld MG.

Epigenetic strategies control the orderly acquisition and maintenance of neuronal traits. A complex network of transcriptional repressors and co-repressors mediates gene specificity for these strategies. This review provides insight into the early lineage commitment events during neurogenesis. We discuss study that demonstrates that regulation of the REST/NRSF transcriptional repressor plays a fundamental role in the progression of pluripotent cells to lineage-restricted neural progenitors. But precisely how defined areas of chromatin adopt different conformations, allowing regulated access to specific DNA regions, is a captivating question. This manuscript provides molecular evidence that the enzyme DNA topoisomerase IIβ (TopoIIβ) activates transcription by generating a break in double-stranded DNA within a nucleosome. This enzyme, which is associated with a DNA-repair machinery, allows chromatin to relax, which is needed to drive gene expression.

Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response.

Genes Dev. 2006 Jun 1;20(11):1405-28.

Rosenfeld MG, Lunyak VV, Glass CK.

A decade of intensive investigation of coactivators and corepressors required for regulated actions of DNA-binding transcription factors has revealed a network of sequentially exchanged cofactor complexes that execute a series of enzymatic modifications required for regulated gene expression. These coregulator complexes possess "sensing" activities required for interpretation of multiple signaling pathways. In this review, we examine recent progress in understanding the functional consequences of "molecular sensor" and "molecular adaptor" actions of corepressor/ coactivator complexes in integrating signal-dependent programs of transcriptional responses at the molecular level. This strategy imposes a temporal order for modifying programs of transcriptional regulation in response to the cellular milieu, which is used to mediate developmental/homeostatic and pathological events.

Analysis of alternative splicing associated with aging and neurodegeneration in the human brain. 

Genome Res. 2011 Oct;21(10):1572-82. 

Tollervey JR, Wang Z, Hortobágyi T, Witten JT, Zarnack K, Kayikci M, Clark TA, Schweitzer AC, Rot G, Curk T, Zupan B, Rogelj B, Shaw CE, Ule J. 

Age is the most important risk factor for neurodegeneration; however, the effects of aging and neurodegeneration on gene expression in the human brain have most often been studied separately. Here, we analyzed changes in transcript levels and alternative splicing in the temporal cortex of indiv iduals of different ages who were cognitively normal, affected by frontotemporal lobar degeneration (FTLD), or affected by Alzheimer's disease (AD). We identified age-related splicing changes in cognitively normal individuals and found that these were present also in 95% of individuals with FTLD or AD, independent of their age. These changes were consistent with increased polypyrimidine tract binding protein (PTB)-dependent splicing activity. We also identified disease-specific splicing changes that were present in individuals with FTLD or AD, but not in cognitively normal individuals. These changes were consistent with the decreased neuro-oncological ventral antigen (NOVA)-dependent splicing regulation, and the decreased nuclear abundance of NOVA proteins. As expected, a dramatic down-regulation of neuronal genes was associated with disease, whereas a modest down-regulation of glial and neuronal genes was associated with aging. Whereas our data indicated that the age-related splicing changes are regulated independently of transcript-level changes, these two regulatory mechanisms affected expression of genes with similar functions, including metabolism and DNA repair. In conclusion, the alternative splicing changes identified in this study provide a new link between aging and neurodegeneration.

Corepressor-Dependent Silencing of Chromosomal Regions Encoding Neuronal Genes

Science. 2002 Nov 29;298(5599):1747-52.

Lunyak VV, Burgess R, Prefontaine GG, Nelson C, Sze SH, Chenoweth J, Schwartz P, Pevzner PA, Glass C, Mandel G, Rosenfeld MG.

For normal organ and tissue development and function, certain genes must be expressed at the appropriate place and time. For example, neural genes must be expressed in neural tissue but shut down in nonneural tissues. Lunyak et al. examined mechanisms by which neural-specific gene expression can be restricted from nonneural tissues. The zinc-finger transcription factor REST/NRSF can mediate extraneural restriction through two different mechanisms, one of which uses active repression via a histone deacetylation complex and one that involves gene silencing via DNA methylation and the recruitment of the corepressor CoREST and silencing machinery. The latter mechanism can mediate gene silencing of specific chromosomal regions, including gene clusters encompassing neuron-specific genes, some  

Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. 

Nat Neurosci. 2011 Apr;14(4):452-8.

Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, König J, Hortobágyi T, Nishimura AL, Zupunski V, Patani R, Chandran S, Rot G, Zupan B, Shaw CE, Ule J. 

TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.

Chromatin signature discovery via histone modification profile alignments

Nucleic Acids Res. 2012 Nov;40(21):10642-56

Wang J, Lunyak VV, Jordan IK.

We report on the development of an unsupervised algorithm for the genome-wide discovery and analysis of chromatin signatures. Our Chromatin-profile Alignment followed by Tree-clustering algorithm (ChAT) employs dynamic programming of combinatorial histone modification profiles to identify locally similar chromatin sub-regions and provides complementary utility with respect to existing methods. We applied ChAT to genomic maps of 39 histone modifications in human CD4(+) T cells to identify both known and novel chromatin signatures. ChAT was able to detect chromatin signatures previously associated with transcription start sites and enhancers as well as novel signatures associated with a variety of regulatory elements. Promoter-associated signatures discovered with ChAT indicate that complex chromatin signatures, made up of numerous co-located histone modifications, facilitate cell-type specific gene expression. The discovery of novel L1 retrotransposon-associated bivalent chromatin signatures suggests that these elements influence the mono-allelic expression of human genes by shaping the chromatin environment of imprinted genomic regions. Analysis of long gene-associated chromatin signatures point to a role for the H4K20me1 and H3K79me3 histone modifications in transcriptional pause release. The novel chromatin signatures and functional associations uncovered by ChAT underscore the ability of the algorithm to yield novel insight on chromatin-based regulatory mechanisms.

Adult stem cells: simply a tool for regenerative medicine or an additional piece in the puzzle of human aging?

Cell Cycle. 2011 Dec 15;10(24):4173-6

Tollervey JR, Lunyak VV.

Adult stem cells have taken center stage in current research related to regenerative medicine and pharmacogenomic studies seeking new therapeutic interventions. As we learn more about these cells, it is becoming apparent that the next big leap in our understanding of adult stem cell biology and adult stem cell aging will depend on the integration of approaches from various disciplines. Major advances and technological breakthroughs at the crossroad of fields such as biomaterials, genomics, epigenomics, and proteomics will enable the design of better tools to model human diseases, and warrant safe usage of adult stem cells in the clinic.

Epigenetics components of aging in the central nervous system. 

Neurotherapeutics. 2013 Oct;10(4):647-63

Zhao YQ, Jordan IK, Lunyak VV.

This review highlights recent discoveries that have shaped the emerging viewpoints in the field of epigenetic influences in the central nervous system (CNS), focusing on the following questions: (i) How is the CNS shaped during development when precursor cells transition into morphologically and molecularly distinct cell types, and is this event driven by epigenetic alterations?; ii) How do epigenetic pathways control CNS function?; (iii) What happens to "epigenetic memory" during aging processes, and do these alterations cause CNS dysfunction?; (iv) Can one restore normal CNS function by manipulating the epigenome using pharmacologic agents, and will this ameliorate aging-related neurodegeneration? These and other still unanswered questions remain critical to understanding the impact of multifaceted epigenetic machinery on the age-related dysfunction of CNS.

Epigenetic regulation of stem cell fate.

Hum Mol Genet. 2008 Apr 15;17.

Lunyak VV, Rosenfeld MG.

Stem cell-based regenerative medicine holds great promise for repair of diseased tissue. Modern directions in the field of epigenetic research aimed to decipher the epigenetic signals that give stem cells their unique ability to self-renew and differentiate into different cell types. However, this research is only the tip of the iceberg when it comes to writing an 'epigenetic instruction manual' for the ramification of molecular details of cell commitment and differentiation. In this review, we discuss the impact of the epigenetic research on our understanding of stem cell biology.

Depletion of nuclear histone H2A variants is associated with chronic DNA damage signaling upon drug-evoked senescence of human somatic cells. 

Aging. 2012 Nov;4(11):823-42.

Lopez MF , Tollervey J, Krastins B, Garces A, Sarracino D, Prakash A, Vogelsang M, Geesman G, Valderrama A, Jordan IK, Lunyak VV.

Cellular senescence is associated with global chromatin changes, altered gene expression, and activation of chronic DNA damage signaling. These events ultimately lead to morphological and physiological transformations in primary cells. In this study, we show that chronic DNA damage signals caused by genotoxic stress impact the expression of histones H2A family members and lead to their depletion in the nuclei of senescent human fibroblasts. Our data reinforce the hypothesis that progressive chromatin destabilization may lead to the loss of epigenetic information and impaired cellular function associated with chronic DNA damage upon drug-evoked senescence. We propose that changes in the histone biosynthesis and chromatin assembly may directly contribute to cellular aging. In addition, we also outline the method that allows for quantitative and unbiased measurement of these changes.

Epigenetics: judge, jury and executioner of stem cell fate.

Epigenetics. 2012 Aug;7(8):823-40

Tollervey JR, Lunyak VV.

Emerging evidence is shedding light on a large and complex network of epigenetic modifications at play in human stem cells. This "epigenetic landscape" governs the fine-tuning and precision of gene expression programs that define the molecular basis of stem cell pluripotency, differentiation and reprogramming. This review will focus on recent progress in our understanding of the processes that govern this landscape in stem cells, such as histone modification, DNA methylation, alterations of chromatin structure due to chromatin remodeling and non-coding RNA activity. Further investigation into stem cell epigenetics promises to provide novel advances in the diagnosis and treatment of a wide array of human diseases.

Regulation of vascular endothelial growth factor D by orphan receptors hepatocyte nuclear factor-4 alpha and chicken ovalbumin upstream promoter transcription factors 1 and 2.

Cancer Res. 2008 Jan 15;68(2):457-66.

Schäfer G, Wissmann C, Hertel J, Lunyak V, Höcker M.

Vascular endothelial growth factor D has recently been linked to the control of lymphangiogenesis and lymphatic metastasis. The molecular determinants regulating vegf-D gene transcription, however, have not yet been identified. After isolation of 2 kb of 5'-flanking DNA of the human vegf-D gene, we identified a novel, atypical direct repeat (DR) element consisting of a consensus half-site (AGGTCA) at -125/-119 and a degenerated DR half-site (ATGTTA) at -99/-94 as sufficient and necessary for vegf-D transcription. The vegf-D DR element is bound and activated by the orphan receptors hepatocyte nuclear factor 4 alpha (HNF-4 alpha) and chicken ovalbumin upstream promoter transcription factor (COUP-TF)-1/COUP-TF2. Additionally, chromatin immunoprecipitation assays identified transcriptional coactivators cyclic AMP-responsive element binding protein-binding protein and glucocorticoid receptor interacting protein 1 at the vegf-D DR element and functional assays confirmed their stimulatory effect on the vegf-D promoter. Histone deacetylase inhibition by trichostatin A led to accumulation of acetylated histones H3/H4 at the vegf-D promoter, up-regulation of vegf-D mRNA levels, and transactivation of vegf-D promoter reporter gene constructs in cancer cell lines. This study for the first time describes the molecular determinants in cis and trans controlling vegf-D gene transcription and identifies interaction of HNF-4 alpha and COUP-TF1/COUP-TF2 with a proximal, atypical DR element as indispensable for vegf-D transcription. Moreover, our findings suggest that epigenetic control of histone acetylation represents an important determinant of vegf-D gene expression in cancer cells. These results provide novel insights into the molecular machinery controlling vegf-D gene expression and may add to a better understanding of the regulation of lymphangiogenesis in vascular development and cancer.

An induced Ets repressor complex regulates growth arrest during terminal macrophage differentiation.

Cell. 2002 Apr 19;109(2):169-80.

Klappacher GW, Lunyak VV, Sykes DB, Sawka-Verhelle D, Sage J, Brard G, Ngo SD, Gangadharan D, Jacks T, Kamps MP, Rose DW, Rosenfeld MG, Glass CK.

Defining the molecular mechanisms that coordinately regulate proliferation and differentiation is a central issue in development. Here, we describe a mechanism in which induction of the Ets repressor METS/PE1 links terminal differentiation to cell cycle arrest. Using macrophages as a model, we provide evidence that METS/PE1 blocks Ras-dependent proliferation without inhibiting Ras-dependent expression of cell type-specific genes by selectively replacing Ets activators on the promoters of cell cycle control genes. Antiproliferative effects of METS require its interaction with DP103, a DEAD box-containing protein that assembles a novel corepressor complex. Functional interactions between the METS/DP103 complex and E2F/ pRB family proteins are also necessary for inhibition of cellular proliferation, suggesting a combinatorial code that directs permanent cell cycle exit during terminal differentiation.


Inhibition of activated pericentromeric SINE/Alu repeat transcription in senescent human adult stem cells reinstates self-renewal.

Cell Cycle. 2011 Sep 1;10(17):3016-30

Wang J, Geesman GJ, Hostikka SL, Atallah M, Blackwell B, Lee E, Cook PJ, Pasaniuc B, Shariat G, Halperin E, Dobke M, Rosenfeld MG, Jordan IK, Lunyak VV.

Cellular aging is linked to deficiencies in efficient repair of DNA double strand breaks and authentic genome maintenance at the chromatin level. Aging poses a significant threat to adult stem cell function by triggering persistent DNA damage and ultimately cellular senescence. Senescence is often considered to be an irreversible process. Moreover, critical genomic regions engaged in persistent DNA damage accumulation are unknown. Here we report that 65% of naturally occurring repairable DNA damage in self-renewing adult stem cells occurs within transposable elements. Upregulation of Alu retrotransposon transcription upon ex vivo aging causes nuclear cytotoxicity associated with the formation of persistent DNA damage foci and loss of efficient DNA repair in pericentric chromatin. This occurs due to a failure to recruit of condensin I and cohesin complexes. Our results demonstrate that the cytotoxicity of induced Alu repeats is functionally relevant for the human adult stem cell aging. Stable suppression of Alu transcription can reverse the senescent phenotype, reinstating the cells' self-renewing properties and increasing their plasticity by altering so-called "master" pluripotency regulators.

Protein interactions with piALU RNA indicates putative participation of retroRNA in the cell cycle, DNA repair and chromatin assembly.

Mob Genet Elements. 2012 Jan 1;2(1):26-35.

Blackwell BJ, Lopez MF, Wang J, Krastins B, Sarracino D, Tollervey JR, Dobke M, Jordan IK, Lunyak VV.

Recent analyses suggest that transposable element-derived transcripts are processed to yield a variety of small RNA species that play critical functional roles in gene regulation and chromatin organization as well as genome stability and maintenance. Here we report a mass spectrometry analysis of an RNA-affinity complex isolation using a piRNA homologous sequence derived from Alu retrotransposal RNA. Our data point to potential roles for piALU RNAs in DNA repair, cell cycle and chromatin regulations.


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