Carvalho Mendez, Erick

Rehbini, Ohoud

Sindi, Abdulmajeed

West, Katherine L.

English

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Erick Carvalho Mendez1, Abdulmajeed Sindi2, Ohoud Rehbini2 and Katherine L. West2 
1Derpartamento de Ciências Biológicas, Universidade Estadual do Sudoeste da Bahia 
2College of Medical, Veterinary and Life Sciences, Institute of Cancer Sciences, University of Glasgow 
 
 
HMGN (High Mobility Nucleosome Binding) are nucleosome-binding 
proteins that play important roles in epigenetic processes and gene 
expression. Previous studies have shown HMGN3a protein to be nuclear, 
but a recent study has shown HMGN3 to be mostly present in the cytoplasm 
of P19 cells, which are malignant cells derived form teratocarcinomas that 
originate in gonads, but still resemble embryonic stem cells (ESCs). 
Alongside,  studies revealed that the expression level of HMGN proteins is 
tightly related to the differentiation process of both Xenopus and mice and 
that expression of HMGN1 and HMGN2 decrease during erythropoiesis, 
chondrogenesis, and myogenesis. Herein we address the cellular localization 
of HMGN3a in mouse ESCs, undifferentiated P19 cells, and neurons 
derived from differentiated P19 cells. Additionally we found that the 
expression of HMGN1 and HMGN2 proteins decreased at the late stages of 
neuronal differentiation in vitro, applying an adherent culture system in 
embryonic carcinoma cells (ECCs, P19; Nakayama et al, 2014*). Thus, this 
adherent culture system provides a model to investigate the role of HMGN 
proteins and epigenetic processes during neuronal differentiation.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
•  Nakayama Y, Wada A, Inoue R, Terasawa K, Kimura I, Nakamura N, 
Kurosaka A (2014). A rapid and efficient method for neuronal induction of 
the P19 embryonic carcinoma cell line. Journal of Neuroscience Methods. 
227; 100–106. 
  
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*Merge with DAPI (blue) and MAP2 (red) 
Immunofluorescence signal of HMGN3a in P19, E14 and 
neurons 
•   Immunofluoresence data showed HMGN3a is present in the cytoplasm and 
nucleus of P19 cells and P19-derived neuronal cells. HMGN1 and HMGN2 were 
predominantly localized in the nucleus of all the cells studied, as expected from 
previous work. The same immunofluorescence results were observed for E14 
cells.  
Introduction 
GFP-tagged HMGN3a in P19 cells 
HMGN1 and HMGN2 expression is reduced during 
neuronal differentiation in vitro 
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Conclusions and future work 
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Western blot data from two different experiments indicate a reduction in the 
expression of HMGN1 and HMGN2 after neural induction. Both proteins are 
found at high levels in undifferentiated ECCs, and are nearly undetectable in 
terminal stages of differentiation. 
• These results are consistent with a role for HMGN proteins in neural stem 
cells, and this is the subject of ongoing investigations.  
• New immunofluorescence assays with monoclonal antibodies would 
extinguish the artefacts that were observed with polyclonal antibodies, and 
corroborate the results obtained with the fusion protein HMGN3a-GFP.  
Reference 
HMGN3a localization and HMGNs expression 
in P19 embryonal carcinoma cells  
Neurons* E14 
P19 
HMGN3a 
Merge 
 with DAPI 
Transfection of HMGN3a-GFP and GFP showed the protein clearly localised 
to the nucleus. This data suggests that HMGN3 is actually localised in the 
nucleus, and that artefacts due to antibody specificity or the 
immunofluorescence technique gave rise to the cytoplasmic signal.  
Immunofluorescence indicate that HMGN1 and HMGN2 protein levels decrease 
as cell differentiate into post-mitotic neurons. This data is consistent with mRNA 
expression patterns from the Allen Brain Atlas, which shows high levels of 
HMGN expression in regions of proliferating neural stem and progenitor cells, but 
reduced HMGN expression in post-mitotic neuronal cells.  
HMGN2 
MAP2 HMGN1 Merge with DAPI 
DAPI 
DAPI GFP Merge 
Merge HMGN3a-GFP  
 
 
 
Acknowledgement 
Poster code: GHM0004 


HMGN3a Localization and HMGNs Expressoin in P19 Embryonal Carcinoma Cells

http://eprints.gla.ac.uk/175031/1/175031.pdf

HMGN3a Localization and HMGNs Expressoin in P19 Embryonal Carcinoma Cells

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