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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.


HEK 293T -cells overexpressing HMGB4-EGFP have increased sensitivity to topoisomerase inhibitors and altered histone variant composition.(a) Growth of HEK 293T -cell clones with doxycycline induced HMGB4-EGFP or EGFP -expression. The expression of HMGB4-EGFP or EGFP in cells was induced over one week with doxycycline and viability was measured with CellTiterGlo Luminescent assay reagent via ATP quantification. (b) Growth of stable HEK 293T -cell clones, constantly expressing HMGB4-EGFP or EGFP. Cells were cultured for 72 h and viability was measured as described above. (c) Overexpression of HMGB4 increases cell sensitivity to topoisomerase inhibitors. Cells overexpressing HMGB4-EGFP were more sensitive to topoisomerease inhibitors than control cells expressing EGFP. Figure shows representative curves from two different experiments. (d) Quantification of histone H3 variants of HMGB4-EGFP or EGFP-expressing HEK 293T -cell clones. Histones were isolated from the cells and analyzed with RP-HPLC. Histone peaks were identified according to their relative retention times. Histone H3.1 eluted in two peaks (H3.1 I and H3.1 II). Curves are derived from three EGFP -control cell clone analyses and from three HMGB4-EGFP –cell clone analyses. (e) Maximal core histone peak heights of RP-HPLC (see above) were determined with the UNICORN- software. Peak height sum of core histones was determined as 100% and relative peak heights were calculated. The relative amount of histone H3.2 was elevated in the HMGB4-EGFP -expressing cells when compared to the control cells (n = 3, ±SD, *p < 0.05).
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f4: HEK 293T -cells overexpressing HMGB4-EGFP have increased sensitivity to topoisomerase inhibitors and altered histone variant composition.(a) Growth of HEK 293T -cell clones with doxycycline induced HMGB4-EGFP or EGFP -expression. The expression of HMGB4-EGFP or EGFP in cells was induced over one week with doxycycline and viability was measured with CellTiterGlo Luminescent assay reagent via ATP quantification. (b) Growth of stable HEK 293T -cell clones, constantly expressing HMGB4-EGFP or EGFP. Cells were cultured for 72 h and viability was measured as described above. (c) Overexpression of HMGB4 increases cell sensitivity to topoisomerase inhibitors. Cells overexpressing HMGB4-EGFP were more sensitive to topoisomerease inhibitors than control cells expressing EGFP. Figure shows representative curves from two different experiments. (d) Quantification of histone H3 variants of HMGB4-EGFP or EGFP-expressing HEK 293T -cell clones. Histones were isolated from the cells and analyzed with RP-HPLC. Histone peaks were identified according to their relative retention times. Histone H3.1 eluted in two peaks (H3.1 I and H3.1 II). Curves are derived from three EGFP -control cell clone analyses and from three HMGB4-EGFP –cell clone analyses. (e) Maximal core histone peak heights of RP-HPLC (see above) were determined with the UNICORN- software. Peak height sum of core histones was determined as 100% and relative peak heights were calculated. The relative amount of histone H3.2 was elevated in the HMGB4-EGFP -expressing cells when compared to the control cells (n = 3, ±SD, *p < 0.05).

Mentions: To further characterize HMGB4 function in somatic cells, doxycycline induced HMGB4-EGFP –overexpressing and constantly HMGB4-EGFP overexpressing HEK 293T -cells were generated through lentivirus infection. Both clones proliferated similarly when compared to EGFP –expressing controls (Fig. 4a,b). Constantly overexpressing HMGB4-EGFP and EGFP expressing cell clones were further analyzed in a drug sensitivity and resistance testing screen with 279 drugs from an oncology collection library23 (Institute for Molecular Medicine, Finland). The most promising hits included topoisomerase inhibitors that were selected for further validation. Inhibitors of topoisomerases induced the death of HMGB4 expressing cell cultures at lower concentrations than in control cell cultures (Fig. 4c). These results are consistent with STRING –search results that suggested HMGB4 interactions with topoisomerase 2A and 2B (Supplementary Table S1). Since our STRING search also suggested that HMGB4 interacts with histones (Supplementary Table S1) and HMGB4 associates with core histones (Fig. 3b,c) we analyzed the expression of histones and histone variants in HMGB4-expressing cells. The relative amount of histone H3.2 was elevated in the HMGB4-EGFP -expressing cells when compared to the control cells (Fig. 4d,e). No changes in the composition of the other core histone or the linker histone H1 protein fractions were observed in the RP-HPLC analyses. These results indicate that HMGB4 can induce changes in the sensitivity to topoisomerase inhibitors and in histone composition.


HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation
HEK 293T -cells overexpressing HMGB4-EGFP have increased sensitivity to topoisomerase inhibitors and altered histone variant composition.(a) Growth of HEK 293T -cell clones with doxycycline induced HMGB4-EGFP or EGFP -expression. The expression of HMGB4-EGFP or EGFP in cells was induced over one week with doxycycline and viability was measured with CellTiterGlo Luminescent assay reagent via ATP quantification. (b) Growth of stable HEK 293T -cell clones, constantly expressing HMGB4-EGFP or EGFP. Cells were cultured for 72 h and viability was measured as described above. (c) Overexpression of HMGB4 increases cell sensitivity to topoisomerase inhibitors. Cells overexpressing HMGB4-EGFP were more sensitive to topoisomerease inhibitors than control cells expressing EGFP. Figure shows representative curves from two different experiments. (d) Quantification of histone H3 variants of HMGB4-EGFP or EGFP-expressing HEK 293T -cell clones. Histones were isolated from the cells and analyzed with RP-HPLC. Histone peaks were identified according to their relative retention times. Histone H3.1 eluted in two peaks (H3.1 I and H3.1 II). Curves are derived from three EGFP -control cell clone analyses and from three HMGB4-EGFP –cell clone analyses. (e) Maximal core histone peak heights of RP-HPLC (see above) were determined with the UNICORN- software. Peak height sum of core histones was determined as 100% and relative peak heights were calculated. The relative amount of histone H3.2 was elevated in the HMGB4-EGFP -expressing cells when compared to the control cells (n = 3, ±SD, *p < 0.05).
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f4: HEK 293T -cells overexpressing HMGB4-EGFP have increased sensitivity to topoisomerase inhibitors and altered histone variant composition.(a) Growth of HEK 293T -cell clones with doxycycline induced HMGB4-EGFP or EGFP -expression. The expression of HMGB4-EGFP or EGFP in cells was induced over one week with doxycycline and viability was measured with CellTiterGlo Luminescent assay reagent via ATP quantification. (b) Growth of stable HEK 293T -cell clones, constantly expressing HMGB4-EGFP or EGFP. Cells were cultured for 72 h and viability was measured as described above. (c) Overexpression of HMGB4 increases cell sensitivity to topoisomerase inhibitors. Cells overexpressing HMGB4-EGFP were more sensitive to topoisomerease inhibitors than control cells expressing EGFP. Figure shows representative curves from two different experiments. (d) Quantification of histone H3 variants of HMGB4-EGFP or EGFP-expressing HEK 293T -cell clones. Histones were isolated from the cells and analyzed with RP-HPLC. Histone peaks were identified according to their relative retention times. Histone H3.1 eluted in two peaks (H3.1 I and H3.1 II). Curves are derived from three EGFP -control cell clone analyses and from three HMGB4-EGFP –cell clone analyses. (e) Maximal core histone peak heights of RP-HPLC (see above) were determined with the UNICORN- software. Peak height sum of core histones was determined as 100% and relative peak heights were calculated. The relative amount of histone H3.2 was elevated in the HMGB4-EGFP -expressing cells when compared to the control cells (n = 3, ±SD, *p < 0.05).
Mentions: To further characterize HMGB4 function in somatic cells, doxycycline induced HMGB4-EGFP –overexpressing and constantly HMGB4-EGFP overexpressing HEK 293T -cells were generated through lentivirus infection. Both clones proliferated similarly when compared to EGFP –expressing controls (Fig. 4a,b). Constantly overexpressing HMGB4-EGFP and EGFP expressing cell clones were further analyzed in a drug sensitivity and resistance testing screen with 279 drugs from an oncology collection library23 (Institute for Molecular Medicine, Finland). The most promising hits included topoisomerase inhibitors that were selected for further validation. Inhibitors of topoisomerases induced the death of HMGB4 expressing cell cultures at lower concentrations than in control cell cultures (Fig. 4c). These results are consistent with STRING –search results that suggested HMGB4 interactions with topoisomerase 2A and 2B (Supplementary Table S1). Since our STRING search also suggested that HMGB4 interacts with histones (Supplementary Table S1) and HMGB4 associates with core histones (Fig. 3b,c) we analyzed the expression of histones and histone variants in HMGB4-expressing cells. The relative amount of histone H3.2 was elevated in the HMGB4-EGFP -expressing cells when compared to the control cells (Fig. 4d,e). No changes in the composition of the other core histone or the linker histone H1 protein fractions were observed in the RP-HPLC analyses. These results indicate that HMGB4 can induce changes in the sensitivity to topoisomerase inhibitors and in histone composition.

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.