Limits...
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

Nuclear localization of HMGB4 and HMGB4L1.(a) Rat glioblastoma C6 –cells transiently transfected with V5-tagged HMGB4 and HMGB4L1 were double immunostained with anti-V5-tag and anti-acetylated K9/K14 histone H3 antibodies. Immunofluorescence staining intensity of anti-acetylated K9/K14 histone H3 is reduced in C6 –cells transiently expressing HMGB4 or HMGB4L1. Arrows indicate HMGB4- and HMGB4L1-positive nuclei in double immunostained cells. Scale bar 20 = μm. (b) Single nucleotide polymorphism of the human HMGB4 gene. Genomic DNA of different human cell lines was amplified in PCR with primers specific to HMGB4 gene and subjected to Ban 1 restriction enzyme analysis. SNP rs10379 destroys Ban 1 cleavage site in the coding sequence of HMGB4 gene. Gel figure indicates that HT1080 and Jurkat cells are heterozygotes for rs10379 SNP. (c) Nuclear mobility FRAP analyses of wild type HMGB4 and rs10379 polymorphic human HMGB4 protein forms. NIH-3T3 -cells were transfected with an expression vector coding for the prominent allele or the polymorphic allele of human HMGB4-EGFP fusion proteins. One brightly fluorescent area in the nucleus with high expression of HMGB4-EGFP was bleached with three laser pulses and recovery of fluorescence was measured. Values from pre-bleach areas were determined as 1 and normalized values for bleached areas were calculated. The failing of full fluorescent recovery indicates the existence of an immobile fraction of HMGB4-EGFP in the nucleus. Triangles = wild type HMGB4, squares = SNP form of HMGB4; n = 5; ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036535&req=5

f2: Nuclear localization of HMGB4 and HMGB4L1.(a) Rat glioblastoma C6 –cells transiently transfected with V5-tagged HMGB4 and HMGB4L1 were double immunostained with anti-V5-tag and anti-acetylated K9/K14 histone H3 antibodies. Immunofluorescence staining intensity of anti-acetylated K9/K14 histone H3 is reduced in C6 –cells transiently expressing HMGB4 or HMGB4L1. Arrows indicate HMGB4- and HMGB4L1-positive nuclei in double immunostained cells. Scale bar 20 = μm. (b) Single nucleotide polymorphism of the human HMGB4 gene. Genomic DNA of different human cell lines was amplified in PCR with primers specific to HMGB4 gene and subjected to Ban 1 restriction enzyme analysis. SNP rs10379 destroys Ban 1 cleavage site in the coding sequence of HMGB4 gene. Gel figure indicates that HT1080 and Jurkat cells are heterozygotes for rs10379 SNP. (c) Nuclear mobility FRAP analyses of wild type HMGB4 and rs10379 polymorphic human HMGB4 protein forms. NIH-3T3 -cells were transfected with an expression vector coding for the prominent allele or the polymorphic allele of human HMGB4-EGFP fusion proteins. One brightly fluorescent area in the nucleus with high expression of HMGB4-EGFP was bleached with three laser pulses and recovery of fluorescence was measured. Values from pre-bleach areas were determined as 1 and normalized values for bleached areas were calculated. The failing of full fluorescent recovery indicates the existence of an immobile fraction of HMGB4-EGFP in the nucleus. Triangles = wild type HMGB4, squares = SNP form of HMGB4; n = 5; ± SD.

Mentions: We studied the functions of HMGB4 and HMGB4L1 using different cultured cell models. Similarly to HMGB4, HMGB4L1 localizes to the nucleus of cultured C6 cells (Fig. 2a). The mobility of EGFP –tagged HMGB –proteins in NIH-3T3 –cell nuclei was studied by the Fluorescence Recovery After Photobleaching (FRAP) assay using method described previously for the same cell line, which ectopically expresses HMGB –fusion proteins19. Half of the nucleus was photobleached and fluorescence recovery time was monitored. HMGB4-EGFP and HMGB4L1-EGFP had a similar mobility within the nucleus [half-time (t1/2) 5.3 ± 2.8 s and 8.3 ± 1.6 s, respectively]. Compared to HMGB4-EGFP and HMGB4L1-EGFP, the HMGB1-EGFP displayed a clearly higher mobility, with a t1/2 of 1.8 ± 0.5 s that is similar to t1/2 of HMGB1 as described by others1920. A human HMGB4 nonsynonymous polymorph rs10379 was detected with restriction enzyme analysis and the mobility of proteins coded by the prominent allele and by the polymorphic allele was analyzed by another FRAP assay (Fig. 2b,c). In this particular assay bleaching and monitoring of fluorescence recovery was focused to one bright nuclear area, smaller than half of the nucleus, to obtain increased resolution. There was no difference in mobility between the forms coded by the prominent allele or by the polymorphic allele. However, fluorescence suggested existence of an immobile HMGB4-EGFP fraction that had not recovered during the time period observed. These results show that the two closely related proteins, HMGB4 and HMGB4L1, bind tightly to nuclei of living cells in contrast to HMGB1, which binds much more weakly.


HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation
Nuclear localization of HMGB4 and HMGB4L1.(a) Rat glioblastoma C6 –cells transiently transfected with V5-tagged HMGB4 and HMGB4L1 were double immunostained with anti-V5-tag and anti-acetylated K9/K14 histone H3 antibodies. Immunofluorescence staining intensity of anti-acetylated K9/K14 histone H3 is reduced in C6 –cells transiently expressing HMGB4 or HMGB4L1. Arrows indicate HMGB4- and HMGB4L1-positive nuclei in double immunostained cells. Scale bar 20 = μm. (b) Single nucleotide polymorphism of the human HMGB4 gene. Genomic DNA of different human cell lines was amplified in PCR with primers specific to HMGB4 gene and subjected to Ban 1 restriction enzyme analysis. SNP rs10379 destroys Ban 1 cleavage site in the coding sequence of HMGB4 gene. Gel figure indicates that HT1080 and Jurkat cells are heterozygotes for rs10379 SNP. (c) Nuclear mobility FRAP analyses of wild type HMGB4 and rs10379 polymorphic human HMGB4 protein forms. NIH-3T3 -cells were transfected with an expression vector coding for the prominent allele or the polymorphic allele of human HMGB4-EGFP fusion proteins. One brightly fluorescent area in the nucleus with high expression of HMGB4-EGFP was bleached with three laser pulses and recovery of fluorescence was measured. Values from pre-bleach areas were determined as 1 and normalized values for bleached areas were calculated. The failing of full fluorescent recovery indicates the existence of an immobile fraction of HMGB4-EGFP in the nucleus. Triangles = wild type HMGB4, squares = SNP form of HMGB4; n = 5; ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036535&req=5

f2: Nuclear localization of HMGB4 and HMGB4L1.(a) Rat glioblastoma C6 –cells transiently transfected with V5-tagged HMGB4 and HMGB4L1 were double immunostained with anti-V5-tag and anti-acetylated K9/K14 histone H3 antibodies. Immunofluorescence staining intensity of anti-acetylated K9/K14 histone H3 is reduced in C6 –cells transiently expressing HMGB4 or HMGB4L1. Arrows indicate HMGB4- and HMGB4L1-positive nuclei in double immunostained cells. Scale bar 20 = μm. (b) Single nucleotide polymorphism of the human HMGB4 gene. Genomic DNA of different human cell lines was amplified in PCR with primers specific to HMGB4 gene and subjected to Ban 1 restriction enzyme analysis. SNP rs10379 destroys Ban 1 cleavage site in the coding sequence of HMGB4 gene. Gel figure indicates that HT1080 and Jurkat cells are heterozygotes for rs10379 SNP. (c) Nuclear mobility FRAP analyses of wild type HMGB4 and rs10379 polymorphic human HMGB4 protein forms. NIH-3T3 -cells were transfected with an expression vector coding for the prominent allele or the polymorphic allele of human HMGB4-EGFP fusion proteins. One brightly fluorescent area in the nucleus with high expression of HMGB4-EGFP was bleached with three laser pulses and recovery of fluorescence was measured. Values from pre-bleach areas were determined as 1 and normalized values for bleached areas were calculated. The failing of full fluorescent recovery indicates the existence of an immobile fraction of HMGB4-EGFP in the nucleus. Triangles = wild type HMGB4, squares = SNP form of HMGB4; n = 5; ± SD.
Mentions: We studied the functions of HMGB4 and HMGB4L1 using different cultured cell models. Similarly to HMGB4, HMGB4L1 localizes to the nucleus of cultured C6 cells (Fig. 2a). The mobility of EGFP –tagged HMGB –proteins in NIH-3T3 –cell nuclei was studied by the Fluorescence Recovery After Photobleaching (FRAP) assay using method described previously for the same cell line, which ectopically expresses HMGB –fusion proteins19. Half of the nucleus was photobleached and fluorescence recovery time was monitored. HMGB4-EGFP and HMGB4L1-EGFP had a similar mobility within the nucleus [half-time (t1/2) 5.3 ± 2.8 s and 8.3 ± 1.6 s, respectively]. Compared to HMGB4-EGFP and HMGB4L1-EGFP, the HMGB1-EGFP displayed a clearly higher mobility, with a t1/2 of 1.8 ± 0.5 s that is similar to t1/2 of HMGB1 as described by others1920. A human HMGB4 nonsynonymous polymorph rs10379 was detected with restriction enzyme analysis and the mobility of proteins coded by the prominent allele and by the polymorphic allele was analyzed by another FRAP assay (Fig. 2b,c). In this particular assay bleaching and monitoring of fluorescence recovery was focused to one bright nuclear area, smaller than half of the nucleus, to obtain increased resolution. There was no difference in mobility between the forms coded by the prominent allele or by the polymorphic allele. However, fluorescence suggested existence of an immobile HMGB4-EGFP fraction that had not recovered during the time period observed. These results show that the two closely related proteins, HMGB4 and HMGB4L1, bind tightly to nuclei of living cells in contrast to HMGB1, which binds much more weakly.

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