chromatin-interaction-analysis-with-paired-end-tag-sequencing-chia

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Activation of Stat proteins by cytokines is initiated by their Src homology 2 (SH2) domain-mediated association with the cytokine receptors. Previously, we identified an essential role of the coiled-coil domain of Stat3 in binding of the receptor peptides derived from the interleukin-6 receptor subunit, gp130. In this study, we further investigated the molecular basis of this regulation. We found that the C-terminal domain of Stat3 negatively regulates its receptor binding activity only in the absence of the first alpha-helix of the coiled-coil domain, which leads to a hypothesis of intramolecular interaction. Physical interactions between the coiled-coil domain and the C-terminal domain, as well as the SH2 domain, were indeed detected. Furthermore, a sub-region of the C-terminal domain (amino acids 720-740), which is also involved in the interaction with the coiled-coil domain, was demonstrated to be critical for the regulation of the receptor binding. Correspondingly, phosphorylation on Ser-727 within this region inhibits this interaction. In agreement with the peptide binding results, both the coiled-coil domain and the C-terminal sub-region are necessary for the functional recruitment of Stat3 to the cellular gp130 in response to interleukin-6, suggesting that the interdomain interaction is a prerequisite for the SH2-mediated receptor binding in interleukin-6 signaling.

Interdomain interaction of Stat3 regulates its Src homology 2 domain-mediated receptor binding activity.

Interdomain Interaktion von Stat3 reguliert seine Src-Homologie 2-Dom&#xE4;ne-vermittelten Rezeptorbindungsaktivit&#xE4;t.

Signal transducer and activator of transcription 3 (Stat3) is a latent cytoplasmic transcription factor that can be activated by cytokines and growth factors. Stat3 plays important roles in cell growth, anti-apoptosis and cell transformation, and is constitutively active in various cancers. We examined its potential regulators by yeast two-hybrid screening. GRIM-19, a gene product related to interferon-beta- and retinoic acid-induced cancer cell death, was identified and demonstrated to interact with Stat3 in various cell types. The interaction is specific for Stat3, but not for Stat1 and Stat5a. The interaction regions in both proteins were mapped, and the cellular localization of the interaction was examined. GRIM-19 itself co-localizes with mitochondrial markers, and forms aggregates at the perinulear region with co-expressed Stat3, which inhibits Stat3 nuclear translocation stimulated by epidermal growth factor (EGF). GRIM-19 represses Stat3 transcriptional activity and its target gene expression, and also suppresses cell growth in Src-transformed cells and a Stat3-expressing cell line. Our data suggest that GRIM-19 is a novel negative regulator of Stat3.

GRIM-19, a death-regulatory gene product, suppresses Stat3 activity via functional interaction.

GRIM-19, ein Tod-regulatorische Genprodukt unterdr&#xFC;ckt Stat3-Aktivit&#xE4;t &#xFC;ber funktionelle Interaktion.

DNA methylation is a critical epigenetic regulator in mammalian development. Here, we present a whole-genome comparative view of DNA methylation using bisulfite sequencing of three cultured cell types representing progressive stages of differentiation: human embryonic stem cells (hESCs), a fibroblastic differentiated derivative of the hESCs, and neonatal fibroblasts. As a reference, we compared our maps with a methylome map of a fully differentiated adult cell type, mature peripheral blood mononuclear cells (monocytes). We observed many notable common and cell-type-specific features among all cell types. Promoter hypomethylation (both CG and CA) and higher levels of gene body methylation were positively correlated with transcription in all cell types. Exons were more highly methylated than introns, and sharp transitions of methylation occurred at exon-intron boundaries, suggesting a role for differential methylation in transcript splicing. Developmental stage was reflected in both the level of global methylation and extent of non-CpG methylation, with hESC highest, fibroblasts intermediate, and monocytes lowest. Differentiation-associated differential methylation profiles were observed for developmentally regulated genes, including the HOX clusters, other homeobox transcription factors, and pluripotence-associated genes such as POU5F1, TCF3, and KLF4. Our results highlight the value of high-resolution methylation maps, in conjunction with other systems-level analyses, for investigation of previously undetectable developmental regulatory mechanisms.

Dynamic changes in the human methylome during differentiation.

DNA-Methylierung ist ein kritischer epigenetische Regulator in der S&auml;ugetier-Entwicklung. Hier pr&auml;sentieren wir ganz-Genom komparativen Perspektive der DNA-Methylierung mit Bisulfit-Sequenzierung von drei kultivierten Zelltypen, die schrittweise Differenzierung: menschlicher embryonaler Stammzellen (Zellkulturen), eine fibroblastic differenzierte Ableitung der Zellkulturen und neonatale Fibroblasten. Als Referenz verglichen wir unsere Karten mit einer Methylome-Karte vollst&auml;ndig differenzierten Erwachsenen Zelle Art, Reifen peripheren mononukle&auml;ren Blutzellen (Monozyten). Wir beobachteten viele bemerkenswerte gemeinsame und Zelle-Typ-spezifische Eigenschaften unter allen Zelltypen. Projekttr&auml;ger Hypomethylierung (CG und CA) und h&ouml;heren Ebenen der Gen-K&ouml;rper-Methylierung wurden mit Transkription in alle Zelltypen positiv korreliert. Exon waren mehr hoch methylierte als Introns und scharfe &Uuml;berg&auml;nge der Methylierung ist aufgetreten bei Exon-Intron-Grenzen, schl&auml;gt eine Rolle f&uuml;r differenzielle Methylierung in Abschrift Splei&szlig;en. Entwicklungsstadium spiegelte sich sowohl auf der Ebene der globalen Methylierung und Umfang der nicht-CpG Methylierung mit hESC h&ouml;chste, Fibroblasten zwischen- und Monozyten niedrigsten. Differenzierung-assoziierte differenzielle Methylierung-Profile wurden f&uuml;r entwicklungspolitisch regulierter Gene, darunter die HOX-Cluster, andere Homeobox Transkriptionsfaktoren und Pluripotence-assoziierten Gene wie POU5F1 TCF3 und KLF4 beobachtet. Unsere Ergebnisse zeigen den Wert von hochaufl&ouml;senden Methylierung Landkarten, in Verbindung mit anderen Systemebene-Analysen zur Untersuchung von zuvor nicht nachweisbar Entwicklungsst&ouml;rungen Regelungsmechanismen.

Dynamische Ver&auml;nderungen der menschlichen Methylome w&auml;hrend der Differenzierung.

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.

Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation.

H&ouml;herer Ordnung chromosomale Organisation f&uuml;r Transkription Verordnung ist bei Eukaryoten oft missverstanden. Mit genomweiten Chromatin Interaktionsanalyse mit Paired-End-Tag-Sequenzierung (ChIA-PET), wir zugeordnet Langstrecken Chromatin-Interaktionen, die RNS-Polymerase II in menschlichen Zellen und ungedeckten weit verbreitete Projekttr&auml;ger-zentrierte intragenic, extragenic und intergenetischer Interaktionen zugeordnet. Diese Interaktionen aggregiert weiter in h&ouml;herer Ordnung Cluster aufgeteilt, wobei proximalen und distalen Gene durch Projekttr&auml;ger-Projekttr&auml;ger Interaktionen involviert waren. Die meisten Gene mit Projekttr&auml;ger-Projekttr&auml;ger Interaktionen waren aktiv und transkribierte kooperativ, und einige interagierende F&ouml;rderer konnte beeinflussen sich gegenseitig was bedeutet kombinatorischen Komplexit&auml;t transkriptionelle Steuerelemente. Vergleichende Analysen der verschiedenen Zelllinien zeigte, dass zellspezifische Chromatin Interaktionen k&ouml;nnte die strukturelle Rahmenbedingungen f&uuml;r zellspezifische Transkription bieten erhebliche Bereicherung der Vergr&ouml;&szlig;erer-Projekttr&auml;ger Interaktionen f&uuml;r zellspezifische Funktionen vorgeschlagen. Dar&uuml;ber hinaus wurden genetisch identifiziert Krankheit-verbundenen nichtkodierende Elemente gefunden, r&auml;umlich mit entsprechenden Gene durch weitr&auml;umige Interaktionen eingebunden werden. Insgesamt bietet unsere Studie Einblicke in Transkription Verordnung von dreidimensionalen Chromatin Interaktionen f&uuml;r Hauswirtschaft sowohl zellspezifische Gene in menschliche Zellen.

Article	Year
Interdomain interaction of Stat3 regulates its Src homology 2 domain-mediated receptor binding activity. The Journal of biological chemistry\| PubMed ID: 11872739	2002
GRIM-19, a death-regulatory gene product, suppresses Stat3 activity via functional interaction. The EMBO journal\| PubMed ID: 12628925	2003
Dynamic changes in the human methylome during differentiation. Genome research\| PubMed ID: 20133333	2010
Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cell\| PubMed ID: 22265404	2012

Chin Thing Ong

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