Comment[GEAAccession]	E-GEAD-570
MAGE-TAB Version	1.1
Investigation Title	Sall1/4 cooperatively interact with Myocd/Srf to promote cardiac cell proliferation through the regulation of Cdk/Cyclin genes
Experiment Description	Sall1 and Sall4, zinc finger transcription factors, are expressed in the progenitors of the second heart field (SHF) and in cardiomyocytes during the early stages of mouse development. To understand the function of Sall1/4 in heart development, we generated heart-specific Sall1/4-functional inhibition mice by forced expression of the truncated form of Sall4 (delta-Sall4) in the heart. The delta-Sall4-overexpression (OE) mice exhibited a hypoplastic right ventricle and outflow tract, both of which were derived from SHF, and a thinner ventricular wall. We found that the numbers of proliferative SHF progenitors and cardiomyocytes were dramatically reduced in delta-Sall4-OE mice. RNA-sequencing data showed that Sall1/4 act upstream of the Cdk/Cyclin genes and ventricular myocardial compaction-related genes, including Myocd and Srf. In addition, ChIP-sequencing and co-immunoprecipitation analyses revealed that Sall4 and Myocd form a transcriptional complex with Srf and directly bind to the upstream regulatory regions of the Cdk/Cyclin genes (Cdk1, Ccne2, and Ccnb1). These results suggest that Sall1/4 are critical for the proliferation of cardiac cells via regulation of Cdk/Cyclin genes interacting with Myocd/Srf.
Experimental Design	genotype design	binding site identification design	genotyping design
Experimental Factor Name	target sequence
Experimental Factor Type	target sequence
Person Last Name	Katano	Koshiba-Takeuchi	Takeuchi
Person First Name	Wataru	Kazuko	Jun
Person Affiliation	Koshiba Lab  Graduate School of Life Sciences, Toyo University		
Person Roles	submitter	submitter	submitter
Public Release Date	2023-11-30
Protocol Name	P-GEAD-1592	P-GEAD-1593	P-GEAD-1594	P-GEAD-1595	P-GEAD-1596
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	Tissue extracts from the embryonic heart (E9.5) were crosslinked with 1% formaldehyde for 10 min, and quenched with 0.125 M glycine for 5 min.	The chromatin solution was incubated with anti-H3K27ac-mouse antibody (MAB10309), anti-H3K27me3-mouse antibody (MAB10323), anti-Sall4-mouse antibody (PP-PPZ0601-00, Perseus Proteomics), anti-Myocardin-mouse antibody (MAB4028, R&D), and conjugated Magnet-Dynabeads M-280 (Life Technologies 11202D) at 4°C. The immunoprecipitated samples (Mahoro) were eluted from the beads, incubated to reverse cross-linking, and purified for DNA analysis.	DNA libraries were generated using NEBNext ChIP-Seq (Illumina) from 5 ng of ChIP-DNA	DNA libraries were sequenced on NovaSeq (Illumina).	Sequence data were trimmed using Trimmomatic (ver.0.38) and mapped using Bowtie2 (ver. 2.3.4.2). Enriched DNA data were obtained using MACS2 (ver. 2.2.6) and analyzed using igv (2.7.2).
SDRF File	E-GEAD-570.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB14787
Comment[Last Update Date]	2023-11-30