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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.


Related in: MedlinePlus

Interactions of HMGB4 and HMGB4L1 with histones.(a,b) Binding of C6 -cell nuclear proteins to HMGB-protein affinity columns. Proteins in elution fractions were analyzed with silver stained SDS-PAGE. The bands, representing core histones, are indicted with marks on the left. a = HMGB1 –affinity column, b = HMGB4 –affinity column. (c) Core histones eluted from HMGB-protein affinity columns and histones were quantified with ELISA. Affinity chromatography was done as described above (n ≥ 3, *p < 0.05, ±SD; Ac = acetylated, 2Me = di-methylated, 3Me = tri-methylated (lysine)). (d) HMGB1 and HMGB4 differ in their histone H1 binding capacity. Affinity chromatography was done as described above and eluted proteins were analyzed with histone H1 ELISA. Elution peak areas were quantified (n = 3, ± SD, *p < 0.002). (e) HDACs bind to HMGB1 and HMGB4. Nuclear proteins were analyzed with affinity chromatography as described above except that eluted fractions from each column were pooled to form a single fraction. Relative HDAC and sirtuin activities in elution fractions were determined (n ≥ 3 in each experiment, ±SD). (f ) Immunofluorescence intensities of modified histone antibody stained HMGB4 or HMGB4L1 overexpressing cells. Rat glioblastoma C6- cells transiently expressing HMGB4-V5 or HMGB4L1-V5 were double-immunostained with anti-acetylated (K9/K14), anti-dimethylated K27 or anti-trimethylated K27 histone H3 antibodies and with anti-V5 antibodies. The correlation blots are shown. (g) Regulation of protein levels in neuronal precursor cells by HMGB4 shRNA. Human neuronal precursor NTERA-2 cl. D1 cells stably overexpressing HMGB4 shRNA or control nonspecific shRNA were analyzed in cell ELISA. Absorbance values of HMGB4 shRNA expressing cells were normalized to values of control shRNA expressing cells (n ≥ 3, ± SD, *p < 0.03). H2A K5 Ac = histone H2A acetylated lysine 5, H4 K8 Ac = histone H4 acetylated lysine 8.
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f3: Interactions of HMGB4 and HMGB4L1 with histones.(a,b) Binding of C6 -cell nuclear proteins to HMGB-protein affinity columns. Proteins in elution fractions were analyzed with silver stained SDS-PAGE. The bands, representing core histones, are indicted with marks on the left. a = HMGB1 –affinity column, b = HMGB4 –affinity column. (c) Core histones eluted from HMGB-protein affinity columns and histones were quantified with ELISA. Affinity chromatography was done as described above (n ≥ 3, *p < 0.05, ±SD; Ac = acetylated, 2Me = di-methylated, 3Me = tri-methylated (lysine)). (d) HMGB1 and HMGB4 differ in their histone H1 binding capacity. Affinity chromatography was done as described above and eluted proteins were analyzed with histone H1 ELISA. Elution peak areas were quantified (n = 3, ± SD, *p < 0.002). (e) HDACs bind to HMGB1 and HMGB4. Nuclear proteins were analyzed with affinity chromatography as described above except that eluted fractions from each column were pooled to form a single fraction. Relative HDAC and sirtuin activities in elution fractions were determined (n ≥ 3 in each experiment, ±SD). (f ) Immunofluorescence intensities of modified histone antibody stained HMGB4 or HMGB4L1 overexpressing cells. Rat glioblastoma C6- cells transiently expressing HMGB4-V5 or HMGB4L1-V5 were double-immunostained with anti-acetylated (K9/K14), anti-dimethylated K27 or anti-trimethylated K27 histone H3 antibodies and with anti-V5 antibodies. The correlation blots are shown. (g) Regulation of protein levels in neuronal precursor cells by HMGB4 shRNA. Human neuronal precursor NTERA-2 cl. D1 cells stably overexpressing HMGB4 shRNA or control nonspecific shRNA were analyzed in cell ELISA. Absorbance values of HMGB4 shRNA expressing cells were normalized to values of control shRNA expressing cells (n ≥ 3, ± SD, *p < 0.03). H2A K5 Ac = histone H2A acetylated lysine 5, H4 K8 Ac = histone H4 acetylated lysine 8.

Mentions: STRING database21 searches suggested that HMGB4 interacts with transcription regulatory factors, histone H2A and topoisomerase II (Supplementary Table S1). Therefore we next analyzed the role of nuclear proteins in HMGB4 functions. Binding of nuclear proteins, isolated from C6 glioblastoma cells, to HMGB1 and HMGB4 -affinity columns was studied, using a previously described method for recombinant HMGB –protein affinity column liquid chromatography22. Core histones bound to both HMGB1- and HMGB4-columns, whereas the linker histone H1 only bound to the HMGB1-column (Fig. 3a–d). The fractions were then analyzed for histone modifying enzymatic histone deacetylase (HDAC) and sirtuin activities. HDAC-activity was significantly increased in HMGB1 and HMGB4 elution fractions whereas sirtuin activity was not (Fig. 3e). Since HMGB4 associated with histones and HDACs we tested whether the transient ectopic expression of HMGB4 or HMGB4L1 in C6 cells or downregulation of endogenous HMGB4 in NTERA2 cl. D1 cells affects the histone post-translational modification. The intensities of anti-acetylated histone H3 K9/K14 and anti-methylated histone H3 K27 staining in HMGB4/HMGB4L1 overexpressing cells were quantified. Acetylated histone H3 K9/K14 and HMGB4/HMGB4L1 stainings were negatively correlated. In contrast, trimethylated histone H3 K27 and HMGB4/HMGB4L1 stainings were positively correlated. There was no correlation between dimethylated histone H3 K27 and HMGB4/HMGB4L1 stainings (Fig. 3f). To monitor the effect of HMGB4 loss on the regulation of protein levels we stably transfected NTERA-2 cl. D1 cells with HMGB4 shRNA. HMGB4 shRNA-expressing cells expressed lower levels of HMGB4 and elevated levels of acetylated histones H2A and H4 (Fig. 3g). Taken together, these results show that both HMGB1 and HMGB4 are associated with core histones and HDACs, and that HMGB4 and HMGB4L1 regulate post-translational modifications of histones in transformed cells derived from the central nervous system.


HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation
Interactions of HMGB4 and HMGB4L1 with histones.(a,b) Binding of C6 -cell nuclear proteins to HMGB-protein affinity columns. Proteins in elution fractions were analyzed with silver stained SDS-PAGE. The bands, representing core histones, are indicted with marks on the left. a = HMGB1 –affinity column, b = HMGB4 –affinity column. (c) Core histones eluted from HMGB-protein affinity columns and histones were quantified with ELISA. Affinity chromatography was done as described above (n ≥ 3, *p < 0.05, ±SD; Ac = acetylated, 2Me = di-methylated, 3Me = tri-methylated (lysine)). (d) HMGB1 and HMGB4 differ in their histone H1 binding capacity. Affinity chromatography was done as described above and eluted proteins were analyzed with histone H1 ELISA. Elution peak areas were quantified (n = 3, ± SD, *p < 0.002). (e) HDACs bind to HMGB1 and HMGB4. Nuclear proteins were analyzed with affinity chromatography as described above except that eluted fractions from each column were pooled to form a single fraction. Relative HDAC and sirtuin activities in elution fractions were determined (n ≥ 3 in each experiment, ±SD). (f ) Immunofluorescence intensities of modified histone antibody stained HMGB4 or HMGB4L1 overexpressing cells. Rat glioblastoma C6- cells transiently expressing HMGB4-V5 or HMGB4L1-V5 were double-immunostained with anti-acetylated (K9/K14), anti-dimethylated K27 or anti-trimethylated K27 histone H3 antibodies and with anti-V5 antibodies. The correlation blots are shown. (g) Regulation of protein levels in neuronal precursor cells by HMGB4 shRNA. Human neuronal precursor NTERA-2 cl. D1 cells stably overexpressing HMGB4 shRNA or control nonspecific shRNA were analyzed in cell ELISA. Absorbance values of HMGB4 shRNA expressing cells were normalized to values of control shRNA expressing cells (n ≥ 3, ± SD, *p < 0.03). H2A K5 Ac = histone H2A acetylated lysine 5, H4 K8 Ac = histone H4 acetylated lysine 8.
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f3: Interactions of HMGB4 and HMGB4L1 with histones.(a,b) Binding of C6 -cell nuclear proteins to HMGB-protein affinity columns. Proteins in elution fractions were analyzed with silver stained SDS-PAGE. The bands, representing core histones, are indicted with marks on the left. a = HMGB1 –affinity column, b = HMGB4 –affinity column. (c) Core histones eluted from HMGB-protein affinity columns and histones were quantified with ELISA. Affinity chromatography was done as described above (n ≥ 3, *p < 0.05, ±SD; Ac = acetylated, 2Me = di-methylated, 3Me = tri-methylated (lysine)). (d) HMGB1 and HMGB4 differ in their histone H1 binding capacity. Affinity chromatography was done as described above and eluted proteins were analyzed with histone H1 ELISA. Elution peak areas were quantified (n = 3, ± SD, *p < 0.002). (e) HDACs bind to HMGB1 and HMGB4. Nuclear proteins were analyzed with affinity chromatography as described above except that eluted fractions from each column were pooled to form a single fraction. Relative HDAC and sirtuin activities in elution fractions were determined (n ≥ 3 in each experiment, ±SD). (f ) Immunofluorescence intensities of modified histone antibody stained HMGB4 or HMGB4L1 overexpressing cells. Rat glioblastoma C6- cells transiently expressing HMGB4-V5 or HMGB4L1-V5 were double-immunostained with anti-acetylated (K9/K14), anti-dimethylated K27 or anti-trimethylated K27 histone H3 antibodies and with anti-V5 antibodies. The correlation blots are shown. (g) Regulation of protein levels in neuronal precursor cells by HMGB4 shRNA. Human neuronal precursor NTERA-2 cl. D1 cells stably overexpressing HMGB4 shRNA or control nonspecific shRNA were analyzed in cell ELISA. Absorbance values of HMGB4 shRNA expressing cells were normalized to values of control shRNA expressing cells (n ≥ 3, ± SD, *p < 0.03). H2A K5 Ac = histone H2A acetylated lysine 5, H4 K8 Ac = histone H4 acetylated lysine 8.
Mentions: STRING database21 searches suggested that HMGB4 interacts with transcription regulatory factors, histone H2A and topoisomerase II (Supplementary Table S1). Therefore we next analyzed the role of nuclear proteins in HMGB4 functions. Binding of nuclear proteins, isolated from C6 glioblastoma cells, to HMGB1 and HMGB4 -affinity columns was studied, using a previously described method for recombinant HMGB –protein affinity column liquid chromatography22. Core histones bound to both HMGB1- and HMGB4-columns, whereas the linker histone H1 only bound to the HMGB1-column (Fig. 3a–d). The fractions were then analyzed for histone modifying enzymatic histone deacetylase (HDAC) and sirtuin activities. HDAC-activity was significantly increased in HMGB1 and HMGB4 elution fractions whereas sirtuin activity was not (Fig. 3e). Since HMGB4 associated with histones and HDACs we tested whether the transient ectopic expression of HMGB4 or HMGB4L1 in C6 cells or downregulation of endogenous HMGB4 in NTERA2 cl. D1 cells affects the histone post-translational modification. The intensities of anti-acetylated histone H3 K9/K14 and anti-methylated histone H3 K27 staining in HMGB4/HMGB4L1 overexpressing cells were quantified. Acetylated histone H3 K9/K14 and HMGB4/HMGB4L1 stainings were negatively correlated. In contrast, trimethylated histone H3 K27 and HMGB4/HMGB4L1 stainings were positively correlated. There was no correlation between dimethylated histone H3 K27 and HMGB4/HMGB4L1 stainings (Fig. 3f). To monitor the effect of HMGB4 loss on the regulation of protein levels we stably transfected NTERA-2 cl. D1 cells with HMGB4 shRNA. HMGB4 shRNA-expressing cells expressed lower levels of HMGB4 and elevated levels of acetylated histones H2A and H4 (Fig. 3g). Taken together, these results show that both HMGB1 and HMGB4 are associated with core histones and HDACs, and that HMGB4 and HMGB4L1 regulate post-translational modifications of histones in transformed cells derived from the central nervous system.

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 &le;0.013 (n&thinsp;=&thinsp;3) in a microarray analysis and displayed strongest association with adhesion and histone H2A &ndash;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.


Related in: MedlinePlus