Reduced Representation Bisulfite Sequencing to identify targets of Dnmt4 during zebrafish hematopoietic stem cell development

Aniket V Gore: Isolation of DNA and RNA from FACS sorted HSCs

Valya Russanova: Bisulfite library preparation

MGL team : Bisulfite sequencing and analysis

Definitive Hematopoiesis in Zebrafish

32-36 hours post fertilization

• HSCs develop from the dorsal aorta

• True HSCs capable of generating all blood lineages

• The original pool of HSCs

• Conserved across all the vertebrates

AGM-Aorta, Gonad, Mesonephros region

Schematic of HSC Egress From the Dorsal Aorta

Notochord

32-34hpf

Dorsal Aorta

Cardinal Vein

Boisset J et al., Bertrand J et al and Kissa and Herbomel, Nature 464, 2010

Notochord

Dorsal Aorta

Cardinal Vein

Dnmt4 is Expressed by Hemogenic Endothelium

NH2

N

N

O

NH2

N

N

O

DNA methyltransferase

Addition of -CH3 at position 5of cytosine

cytosine 5-methylcytosine

DNA methyltransferase Dnmt

CH3

Cm G Cm G C C A A G G Cm G Cm G T G Cm G C G C G C C A A G G C G C G T G C G

Methylated DNA Unmethylated DNA

Bisulfite Treatmentand

Sequencing

C G C G C C A A G G C G C G T G C G T G T G T T A A G G T G T G T G T G

Bisulfite sequencing: One of the ways to check DNA methylation

Exon1CGI

62%

8%

277bp, 23 CpG

Con MO

Dnmt4 MO

Cmyb gene body methylation and expression is regulated by Dnmt4

qRT-PCR

Bisulfite Seq

1: gene expression changes,RNA seq from total RNA isolated from control and dnmt4 deficient HSCs

To study genome wide changes in DNA methylation and gene expression

2:DNA methylation changes,Reduced representation by bisulfite sequencing from DNA isolated from control and dnmt4 deficient HSCs

Isolation of GFP/RFP double positive HSC populationFrom the transgenic line

RFP ONLYEndothelial cells

RFP/GFP Double positive

HSCs

Marker 1

Marker 2

Marker 3

Con 1

Con 2

RT-PCR on FACS sorted cells from WT flk>mApple; cmyb>GFP 48 hpf embryos

Marker gene expression showed enrichment ofHSCs in double positive cell population

Two sets of DNA and RNA for GFP, RFP, dark andDouble positive cells

HSC markers

1: Cas9 in vitro synthesized RNA (Control)

2: Frozen cells, Trizol, phenol chloroform

3: Frozen cells, Trizol, phenol chloroform, DNAseI, Phenol chloroform, Phase lock

4: Frozen cells, Qiagen microRNA isolation kit

5: Live cells, Zymo DNA/RNA kit

6: Live cell, Zymo DNA/RNA kit, DNAseI treatment

Different techniques to isolate good quality total RNA

1: Con MO Red endothelial cells2: Con MO double positive HSCs (195ng) (86,383 cells)3: dnmt4 MO Red endothelial cells4: dnmt4 MO double positive HSCs (150ng) (42,712 cells)

Cells isolated from flk>mApple; cmyb>GFP embryos @48 hpf and DNA and RNA isolated fromThe same cells using Zymo Duet Kit

Sample 3: Con MO double positive HSCs(Total 62.5ng)Sample 4: dnmt4 MO double positive HSCs(Total 50ng)

From Valya

Total genomic DNA isolated from the same sorted cellsAnalyzed by picogreen for concentrationAnd by gel for quality

Con MO RFP

Dnmt4 MO RFP

Con MO double

Dnmt4 MO double