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HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

View Article: PubMed Central - PubMed

ABSTRACT

HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

No MeSH data available.


Expression of HMGB4 and HMGB4L1 in cultured primary cells of rat brain.(a) Immunofluorescence staining of nestin and HMGB4L1 in 1d differentiated rat neuronal cells in vitro. Neurospheres were allowed to adhere and differentiate for 1 day and cells were immunofluorescently stained with anti-nestin and anti-HMGB4L1 antibodies. Cell nuclei were stained with DAPI. The staining controls for anti-nestin or anti-HMGB4L1 staining were done without primary antibody or with preimmune serum, respectively. Scale bar = 30 μm. (b) Immunofluorescence staining of HMGB4L1 and NeuN in 14d differentiated rat neuronal cells in vitro. Neurospheres were allowed to differentiate for 14 days and cells were immunofluorescently stained with anti-NeuN and anti-HMGB4L1 antibodies. Staining controls for anti-HMGB4L1 and anti-NeuN were done with pre-immune serum or without primary antibody, respectively. Scale bar = 20 μm. (c,d) Proximity ligation assay of neurospheres with anti-PAN-Histone and anti-HMGB4L1 antibodies. Cell nuclei of cultured neurospheres were stained with DAPI and a proximity ligation assay was performed with anti-PAN-Histone antibodies and preimmune control serum (c) or with anti-PAN-Histone antibodies and anti-HMGB4L1 antibodies (d). Scale bar = 20 μm.
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f6: Expression of HMGB4 and HMGB4L1 in cultured primary cells of rat brain.(a) Immunofluorescence staining of nestin and HMGB4L1 in 1d differentiated rat neuronal cells in vitro. Neurospheres were allowed to adhere and differentiate for 1 day and cells were immunofluorescently stained with anti-nestin and anti-HMGB4L1 antibodies. Cell nuclei were stained with DAPI. The staining controls for anti-nestin or anti-HMGB4L1 staining were done without primary antibody or with preimmune serum, respectively. Scale bar = 30 μm. (b) Immunofluorescence staining of HMGB4L1 and NeuN in 14d differentiated rat neuronal cells in vitro. Neurospheres were allowed to differentiate for 14 days and cells were immunofluorescently stained with anti-NeuN and anti-HMGB4L1 antibodies. Staining controls for anti-HMGB4L1 and anti-NeuN were done with pre-immune serum or without primary antibody, respectively. Scale bar = 20 μm. (c,d) Proximity ligation assay of neurospheres with anti-PAN-Histone and anti-HMGB4L1 antibodies. Cell nuclei of cultured neurospheres were stained with DAPI and a proximity ligation assay was performed with anti-PAN-Histone antibodies and preimmune control serum (c) or with anti-PAN-Histone antibodies and anti-HMGB4L1 antibodies (d). Scale bar = 20 μm.

Mentions: Since PPP1R14a is a marker of mature oligodendrocytes both in mammals and in zebrafish2829 we hypothesized that HMGB4 may have functions in differentiation of brain cells. This hypothesis is further supported by the fact that HMGB4 is expressed in the brain (Supplementary Figure S2) where its expression is associated to extracellular region processes (Table 1). In addition, polymorphisms in the HMGB4 gene locus have been associated with psychiatric diseases13141516. We found mRNA coding for HMGB4 and HMGB4L1 in cultured rat neurons (Fig. 1f). Immunofluorescence staining of rat brain cell cultures with an anti-HMGB4L1 antibody indicated that HMGB4L1 is co-expressed with nestin and neuronal nuclei antigen (NeuN) in neurospheres and in vitro differentiated neuronal cells, respectively (Fig. 6a,b). In neurosphere cultures HMGB4L1 co-localizes with histones in the cell nucleus (Fig. 6c,d). Although the expression of HMGB4 decreases during neurosphere differentiation into neurons5, our results indicate that differentiated neurons still express some HMGB4/HMGB4L1.


HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation
Expression of HMGB4 and HMGB4L1 in cultured primary cells of rat brain.(a) Immunofluorescence staining of nestin and HMGB4L1 in 1d differentiated rat neuronal cells in vitro. Neurospheres were allowed to adhere and differentiate for 1 day and cells were immunofluorescently stained with anti-nestin and anti-HMGB4L1 antibodies. Cell nuclei were stained with DAPI. The staining controls for anti-nestin or anti-HMGB4L1 staining were done without primary antibody or with preimmune serum, respectively. Scale bar = 30 μm. (b) Immunofluorescence staining of HMGB4L1 and NeuN in 14d differentiated rat neuronal cells in vitro. Neurospheres were allowed to differentiate for 14 days and cells were immunofluorescently stained with anti-NeuN and anti-HMGB4L1 antibodies. Staining controls for anti-HMGB4L1 and anti-NeuN were done with pre-immune serum or without primary antibody, respectively. Scale bar = 20 μm. (c,d) Proximity ligation assay of neurospheres with anti-PAN-Histone and anti-HMGB4L1 antibodies. Cell nuclei of cultured neurospheres were stained with DAPI and a proximity ligation assay was performed with anti-PAN-Histone antibodies and preimmune control serum (c) or with anti-PAN-Histone antibodies and anti-HMGB4L1 antibodies (d). Scale bar = 20 μm.
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f6: Expression of HMGB4 and HMGB4L1 in cultured primary cells of rat brain.(a) Immunofluorescence staining of nestin and HMGB4L1 in 1d differentiated rat neuronal cells in vitro. Neurospheres were allowed to adhere and differentiate for 1 day and cells were immunofluorescently stained with anti-nestin and anti-HMGB4L1 antibodies. Cell nuclei were stained with DAPI. The staining controls for anti-nestin or anti-HMGB4L1 staining were done without primary antibody or with preimmune serum, respectively. Scale bar = 30 μm. (b) Immunofluorescence staining of HMGB4L1 and NeuN in 14d differentiated rat neuronal cells in vitro. Neurospheres were allowed to differentiate for 14 days and cells were immunofluorescently stained with anti-NeuN and anti-HMGB4L1 antibodies. Staining controls for anti-HMGB4L1 and anti-NeuN were done with pre-immune serum or without primary antibody, respectively. Scale bar = 20 μm. (c,d) Proximity ligation assay of neurospheres with anti-PAN-Histone and anti-HMGB4L1 antibodies. Cell nuclei of cultured neurospheres were stained with DAPI and a proximity ligation assay was performed with anti-PAN-Histone antibodies and preimmune control serum (c) or with anti-PAN-Histone antibodies and anti-HMGB4L1 antibodies (d). Scale bar = 20 μm.
Mentions: Since PPP1R14a is a marker of mature oligodendrocytes both in mammals and in zebrafish2829 we hypothesized that HMGB4 may have functions in differentiation of brain cells. This hypothesis is further supported by the fact that HMGB4 is expressed in the brain (Supplementary Figure S2) where its expression is associated to extracellular region processes (Table 1). In addition, polymorphisms in the HMGB4 gene locus have been associated with psychiatric diseases13141516. We found mRNA coding for HMGB4 and HMGB4L1 in cultured rat neurons (Fig. 1f). Immunofluorescence staining of rat brain cell cultures with an anti-HMGB4L1 antibody indicated that HMGB4L1 is co-expressed with nestin and neuronal nuclei antigen (NeuN) in neurospheres and in vitro differentiated neuronal cells, respectively (Fig. 6a,b). In neurosphere cultures HMGB4L1 co-localizes with histones in the cell nucleus (Fig. 6c,d). Although the expression of HMGB4 decreases during neurosphere differentiation into neurons5, our results indicate that differentiated neurons still express some HMGB4/HMGB4L1.

View Article: PubMed Central - PubMed

ABSTRACT

HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

No MeSH data available.