Epigenetic Changes Correlate with Loss of Pluripotency Marker Expression During Retinoic Acid-induced Differentiation of NTERA-2 Cells

Antony W. Wood , Susan Kane, Christopher J. Fry, Raffi Manoukian, and Curtis D. Desilets

Identifying and investigating the molecular and biochemical events asso-ciated with changes in cell fate, such as those that occur during stem cell differentiation or somatic cell reprogramming, are fundamental goals in stem cell biology research. Achieving these objectives requires the devel-opment of highly specific and sensitive reagents that have been validated in a contextually appropriate manner. At Cell Signaling Technology, we are developing a portfolio of sensitive, rigorously validated antibody reagents designed to investigate the epigenetic, molecular, and biochemical events that underlie changes in cell fate. Here, we use retinoic acid (RA)-induced differentiation of NTERA-2 cells as a model to demonstrate the utility of our antibody reagents for interrogating the epigenetic and biochemical events associated with induced neuronal differentiation. Using chroma-tin immunoprecipitation, fluorescent immunocytochemistry and intracel-lular flow cytometry, we are able to demonstrate the predicted correla-tion between epigenetic changes at distinct genomic loci with changes in the expression of key pluripotency factors during directed differentia-tion. These data confirm the utility of these antibody reagents, employed across multiple technology platforms, for investigating the molecular mechanisms underlying cellular differentiation.

1. Lee, V.M. and Andrews, P.W. (1986) Differentiation of NTERA-2 clonal human embryonal carcinoma cells into neurons involves the induction of all three neuro-filament proteins. J. Neurosci. 6, 514–521.

Contact InformationAntony W. Wood, Ph.D. Cell Signaling Technology | 3 Trask Lane, Danvers, MA 01923email: antony.wood@cellsignal.comwww.cellsignal.com

Abstract

Immunocytochemical analysis shows reduction of pluripotency factor expression during differentiation

ChIP analyses show loss of epigenetic marks of active transcription at pluripotency loci during differentiation

Flow Cytometric analysis shows reduction of pluripotency factor expression during differentiation

Antibodies validated for target specificity

Reference

Chromatin Immunoprecipitation analysis of selected epigenetic marks and transcription factors in NTERA-2 cells during RA- induced neuronal differentiation. Data indicate relative binding to indicated loci in untreated cells (Day 0) and after treatment with RA for the indicated durations. Data are normalized to total Histone H3 signal. Data are means +/- SD of 3 experiments.

Confocal IF analysis of NTERA-2 cells, untreated (upper panels) or RA-treated (10 µM all-trans RA for 14 days, lower panel), us-ing antibodies against core pluripotency factors, or neuronal markers. Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Note the loss of pluripotency transcription factor expression (green) as cells undergo neuronal differentiation.

Flow cytometric analysis showing changes in pluripotency factor expres-sion during RA-induced neuronal dif-ferentiation of NTERA-2 cells. Upper panels: Bivariate scatter plots showing expression of Sox2 (y-axis) and Oct-4A (x-axis) after 0, 3, and 5 days exposure to RA. Lower panels: Bivariate scat-ter plots showing expression of SSEA4 (y-axis) and Nanog (x-axis) after 3 and 5 days treatment with RA. Results demon-strate a time-dependent decline in the expression of pluripotency factors and an embryonic marker protein (SSEA4), detectable as early as 3 days after exposure to RA .

©2014 Cell Signaling Technology. Inc. Cell Signaling Technology®, CST™, SimpleChIP® and XP® are trademarks of Cell Signaling Technology, Inc. Alexa Fluor® is a registered trademark of Life Technologies Corporation. DRAQ5® is a registered trademark of Biostatus Limited. Galios™ is a trademark of Beckman Coulter, Inc.

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Materials and MethodsCell Culture:NTERA-2 embryonal carcinoma cells were treated with all-trans retinoic acid (RA) for 0–14 days to induce neuronal differentiation as previously described (1). Cells were collected for analysis at multiple time points representing different stages of neuronal differentiation.

Fluorescent Immunocytochemistry:Cells were fixed for 15 min in 4% formaldehyde, permeabilized, blocked for 1 hr, then incubated overnight in primary antibody at 4°C. After washing, cells were incubated in secondary antibody for 1-2 hr at RT in the dark. Stained cells were imaged by laser scanning confocal microscopy.

Flow Cytometry:Cells were harvested, fixed, permeabilized, and processed according to the standard CST flow cytometry protocol. In brief, cell pellets were resuspended in 4% formal-dehyde (methanol-free) for 10 min at 37°C, followed by addition of ice cold 90% methanol for at least 30 min on ice. Cells were then immunostained for 1 hr at RT by suspension in an antibody cocktail containing Oct-4A Alexa Fluor® 488, Sox2 Alexa Fluor® 647, or Nanog Alexa Fluor® 594. After washing 2x with PBS/0.5% BSA, cells were analyzed on a 4 laser Galios™ flow cytometer (Beckman Coulter).

Chromatin Immunoprecipitation:Chromatin Immunoprecipitations were performed with cross-linked chromatin from NTERA-2 cells using the indicated antibodies, or 2 μl of Normal Rabbit IgG #2729, using the SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using gene-specific primers. The amount of immunoprecipitated DNA in each sample is represented as a percentage of the total input chromatin.

kDa

Sox2

NTERA2

NCCIT

200140100806050

40

30

20

kDa

Oct-4A

MESNTERA2

200

140

100

80

60

50

40

kDa

Nanog

NCCITNTE

RA-2

iPS

20014010080

60

50

40

30

20

100 101 102 103

Oct-4A Alexa Fluor® 488

100

101

102

103

Sox2

Ale

xa F

luor

® 6

47

Day 0

100 101 102 103

Oct-4A Alexa Fluor® 488

100

101

102

103

Sox

2 A

lexa

Flu

or®

647

Day 3

100 101 102 103

Oct-4A Alexa Fluor® 488

100

101

102

103

Sox

2 A

lexa

Flu

or® 6

47

Day 5

100 101 102 103

Nanog Alexa Fluor® 594

100

101

102

103

SS

EA

4 P

E

4.89 38.4

47.29.55

Day 3

100 101 102 103

Nanog Alexa Fluor® 594

100

101

102

103

SS

EA

4 P

E

20.2

2.5729.3

47.9

Day 5

Day 0

Sox2

SSEA4

Oct-4A

Nanog

Day 3

Day 3

Day 5

Day 5

Confocal IF analysis of NTERA-2 cells (upper), HeLa cells (middle) and human iPS cells (lower) using antibodies against core pluripotency factors. Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudo-color = DRAQ5® #4084.

NTERA-2

Sox2 Oct-4A Nanog

HeLa

iPS cells

Sox2 Oct-4A Nanog

Nanog Sox2 Oct-4A Neurofilament-L β3-Tubulin

00 1 1.5 2 4

1

2

3

4

5

6

Duration of exposure (days) to retinoic acid (RA, 10 ug/ml)

Per

cent

Inp

ut

OCT-4A

RNA Pol II

H3K4me3

H3K9me3

H3K27me3

Rabbit lgG

00 1 1.5 2 4

1

2

3

4

5

Per

cent

Inp

ut

00 1 1.5 2 4

2

4

6

8

10

12

Per

cent

Inp

ut