be230cLecture5

Nkx25 promoter contains an enhancer 9kb that is

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Unformatted text preview: H3K9 H3K14 activation[22] activation[23] H3K27 H3K79 H4K20 H2BK5 activation[23] activation[23][24] activation[23] activation[23] repression[25] repression[25] activation[24] activation[26] repression[23] repression[23] activation,[24] repression[23] repression[25] activation[26] activation[26] acetylation EpigeneJc AcJvaJon of Cardiomyocyte Gene Program JK Takeuchi & BG Bruneau Nature 459, 708 ­11 (2009) •  •  •  •  Components of the SWI/SNF chromaJn remodeling complex important in acJvaJng cardiogenesis within early embryonic mesoderm in situ Baf60b and c (but not a) idenJfied as cardiogenic Requires the transcripJon factor Gata4, augmented by Tbx5 SWI/SNF opens chromaJn, making accessible to transcripJonal machinery What is Cardiac Commitment? During Embryo and ESC Cardiogenesis, InhibiJng Nodal/TGFβ and BMP Triggers an EpigeneJc Switch for Cardiomyocyte DifferenJaJon Endoderm Cerberus-1 Uncommitted Flk1+, MesP1+ Multipotent Progenitors Cer1 siRNA ( Cardiogenesis) Nkx2.5+, Mef2c+ Committed Cardiac Precursors Cardiomyocytes Cerberus-1 (and other Nodal & BMP inhibitors) Nodal/TGFβ Baf60c BMP4 Tbx5 GATA4 SWI/SNF Complex GATA4 Nkx2.5 Closed Tbx5 Baf60c Nkx2.5 Open 11 4/15/13 Does Cerberus-1 Remodel Chromatin via Baf60c? •  Nkx2.5 promoter contains an enhancer (-9kb) that is sufficient to direct expression throughout the early heart field at E7 An enhancer at -2.8kb directs stomach, thyroid and pharynx expression •  Chromatin Accessibility Assay ESCs ± siRNAs Nuclei MboII digestion MboII resistant (closed) •  Upstream promoter elements are responsible for regionalized transcription in the heart MboII sensitive (open) Genomic DNA extraction and Q-PCR to detect presence or absence of DNA fragments Cerberus ­1, via Baf60c, Opens ChromaJn of the Nkx2.5 Cardiac Enhancer and AcJvates TranscripJon ESCs ± siRNAs Nuclei MboII digestion closed MboII resistant (closed) MboII sensitive (open) Genomic DNA extraction and Q-PCR to detect presence or absence of DNA fragments open Baf60c Can Rescue shCer1 Effect on ChromaJn 160 undifferentiated 140 siCer1+pcDNA siNeg+pcDNA siCer1+Baf60c 120 closed siCer1+Baf60c+Gata4 siCer1+Baf60C+Tbx5 acc% 100 siCer1+Gata4+Tbx5 80 60 open 40 20 0 Nkx2.5 promoter -9kb Nkx2.5 promoter -2.8kb 12 4/15/13 InhibiJng Nodal/TGFβ and BMP Triggers an EpigeneJc Switch for Cardiomyocyte DifferenJaJon Example of Using a hESC  ­>...
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This document was uploaded on 04/09/2014.

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