Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Summary

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Abstract

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

Introduction

The heart is the first organ to be formed and to become functional in the embryo. The heart is built from many cell lineages that arise from the first and second embryonic heart fields¹. From the post-fertilization blastocyst stage up to the shaped heart, embryonic cells have thus to make many cell fate decisions. Gene transcription is regulated in a time- and space-dependent manner and is a key biological process that underlies cell fate decision during embryonic development. Such a process is mediated by specific transcription factors which bind regulatory regions within the genome including enhancers and promoters of cardiac constitutive genes. DNA is wr....

Protocol

1. DNA-protein Cross-linking

1. Fix in 15 ml tubes harvested-ES cells (2 x 10⁶ cells for regular ChIP, 2 x 10⁵ cells for microChIP), embryoid bodies (EBs) generated from ES cells and embryonic heart tissues dissected from E9.5 mouse embryos (atrioventricular canal, outflow tract and ventricle) using 1% formaldehyde in PBS for cells or in permeabilization PB2 buffer for embryonic tissues. Place the tubes on orbital shaker at a speed of 60 rpm at room temperature for exactly 10 min.
2. Stop the cross-linking reaction by adding glycine to a final concentration of 125 mM and incubate for 5 min at room temperature on the orbi....

Results

Figure 1A illustrates first the preparation of DNA-binding beads and quality control using DNA of different sizes (1 kb ladder). 0ne, 2 and 2.5 volumes (1 to 3) of beads was added to one volume of the sample to purify high and low molecular size DNA fragments.

Figures 1 B,C,D are typical examples of DNA gels from whole sonicated DNA extracted from mouse ES cells, embryoid bodies or a cardiac emb.......

Discussion

Epigenetics has become an important field of research in developmental biology. How a genetic program is activated in embryonic cells to allow the cells to acquiring a specific identity within an embryonic lineage has been for long time a key question for developmental biologists.

ChIP has been broadly used within the last years and combined to DNA sequencing following improvement in resolution of sequencing. This has become a powerful technique in order to investigate in a genome wide depende.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Formaldehyde	Sigma	F8775	Cell Fixation
Glycine	Sigma	G8898	Cross-link stop
Aprotinin	Fluka	10820	Proteases inhibitor
Leupeptin hemisulfate	Sigma	L2882	Proteases inhibitor
PMSF	Sigma	P7626	Proteases inhibitor
Protein A magnetic beads	Life technologies	10001D	Immunoprecipitation
SPRI magnetic beads	Thermo Scientific	15002-01	DNA purification
Proteinase K	Life technologies	25530-015	Protein digestion
DNA BR standard	Life technologies	Q32850	Calibration range
Syber green	Molecular Probes	S-11484	DNA quantification
TE buffer	Invitrogen	P7589	DNA quantification
PBX 1x	Life technologies	14190-094	Washing
DNase RNase free water	Life technologies	10977-035	Dilution
Axygen tube	Axygen	MCT-175-C	ChIP purifiction
Antibody	Company	Reference	ChIP concentration
H3K27ac	Abcam	ab4729	3 µg for ESC and EBs, 1 µg for tissues
H3K4me1	Diagenode	C15410194 (pAb-194-050)	3 µg for ESC and EBs, 1 µg for tissues
H3K36me3	Diagenode	C15410058 (pAb-058-050)	3 µg for ESC and EBs, 1 µg for tissues
H3K9me2	Diagenode	C15410060 (pAb-060-050)	3 µg for ESC and EBs, 1 µg for tissues
H3K4me3	Diagenode	C15410030 (pAb-030-050)	3 µg for ESC and EBs, 1 µg for tissues
H3K27me3	Diagenode	C15410069 (pAb-069-050)	3 µg for ESC and EBs, 1 µg for tissues