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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

Characterization of HMGB4 and HMGB4L1.(a) Alignment of rat TP4/HMGB4L1 and HMGB4 amino acid sequences. HMGB-boxes A and B are underlined. Red letters indicate identical amino acids. An alternative allele in position 34 of TP4/HMGB4L1 is either a tyrosine or a isoleucine. Amino acids marked in italics have been identified by amino terminal amino acid sequencing7. Domain structures and number of amino acids of rat HMGB1, HMGB4 and TP4/HMGB4L1 are shown in the schematic picture. (b) Western –blot of recombinant mouse HMGB4. Recombinant HMGB4 was detected with Ponceau S –staining and with anti-HMGB4 and anti-HMGB4L1 antibodies. The antibodies did not detect recombinant HMGB1. (c) Northern Blot -analysis of the mouse HMGB4 transcript. Total RNA samples were isolated from adult mouse testes and analyzed via Northern Blot, using a probe derived from the entire coding sequence of mouse HMGB4. The probe detected a 1.1 kb band. Ethidum bromide stained ribosomal RNA is shown. (d) Northern Blot -analysis of rat HMGB4L1 transcript. Total RNA samples were isolated from adult rat testes and from developing rat testes, and analyzed via Northern Blot, using a probe derived from the entire coding sequence of rat HMGB4L1. The probe detected a 0.9 kb band in samples derived from testes of sexually mature rat. Ethidum bromide stained ribosomal RNA is shown. 3 w = 3 week old rat, 5 w = 5 week old rat. (e) Immunohistochemical staining of rat HMGB4L1 protein and in situ hybridization of mouse HMGB4 mRNA in adult testes. Anti-HMGB4L1 polyclonal antibody staining revealed intense HMGB4L1 expression in elongated spermatids (red). Haematoxylin was used as a counterstain. Control sections were stained without the primary antibody. HMGB4 mRNA localized to round and elongated spermatids. The control section shows adult mouse testes incubated with the sense probe. Bars represent 50 μm. (f) Expression of HMGB4 and HMGB4L1 mRNA in cultured rat neurons. Neuronal cells from the hippocampus and the cerebellum were cultured, and both HMGB4 and HMGB4L1 expression (arrows) was detected with RT-PCR. +RT =  reverse transcribed, -RT =  without reverse transcription.
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f1: Characterization of HMGB4 and HMGB4L1.(a) Alignment of rat TP4/HMGB4L1 and HMGB4 amino acid sequences. HMGB-boxes A and B are underlined. Red letters indicate identical amino acids. An alternative allele in position 34 of TP4/HMGB4L1 is either a tyrosine or a isoleucine. Amino acids marked in italics have been identified by amino terminal amino acid sequencing7. Domain structures and number of amino acids of rat HMGB1, HMGB4 and TP4/HMGB4L1 are shown in the schematic picture. (b) Western –blot of recombinant mouse HMGB4. Recombinant HMGB4 was detected with Ponceau S –staining and with anti-HMGB4 and anti-HMGB4L1 antibodies. The antibodies did not detect recombinant HMGB1. (c) Northern Blot -analysis of the mouse HMGB4 transcript. Total RNA samples were isolated from adult mouse testes and analyzed via Northern Blot, using a probe derived from the entire coding sequence of mouse HMGB4. The probe detected a 1.1 kb band. Ethidum bromide stained ribosomal RNA is shown. (d) Northern Blot -analysis of rat HMGB4L1 transcript. Total RNA samples were isolated from adult rat testes and from developing rat testes, and analyzed via Northern Blot, using a probe derived from the entire coding sequence of rat HMGB4L1. The probe detected a 0.9 kb band in samples derived from testes of sexually mature rat. Ethidum bromide stained ribosomal RNA is shown. 3 w = 3 week old rat, 5 w = 5 week old rat. (e) Immunohistochemical staining of rat HMGB4L1 protein and in situ hybridization of mouse HMGB4 mRNA in adult testes. Anti-HMGB4L1 polyclonal antibody staining revealed intense HMGB4L1 expression in elongated spermatids (red). Haematoxylin was used as a counterstain. Control sections were stained without the primary antibody. HMGB4 mRNA localized to round and elongated spermatids. The control section shows adult mouse testes incubated with the sense probe. Bars represent 50 μm. (f) Expression of HMGB4 and HMGB4L1 mRNA in cultured rat neurons. Neuronal cells from the hippocampus and the cerebellum were cultured, and both HMGB4 and HMGB4L1 expression (arrows) was detected with RT-PCR. +RT =  reverse transcribed, -RT =  without reverse transcription.

Mentions: Database searches revealed that both human and mouse have a single copy of the HMGB4-gene217, whereas the rat has two HMGB4-like genes, one on chromosome 5 and another one on the X-chromosome [coding for proteins HMGB4 (NP_001102933) and predicted high mobility group protein B4 –like (XP_006227590), respectively, in the NCBI database]. The predicted protein coded by the gene on the rat X-chromosome was identified as TP47 (accession number AAB24466). The amino acid sequences of the rat HMGB4 and TP4 are 66% identical and 82% similar (Fig. 1a refs 2 and 7). TP4 is renamed high mobility group box 4 protein –like 1 (HMGB4L1) in this study. Comparison of HMGB4 and HMGB4L1 to the amino acid sequence of the archetype of the HMGB-proteins, HMGB1, revealed that rat HMGB4 is 42% identical and 67% similar, and HMGB4L1 is 43% identical and 66% similar.


HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation
Characterization of HMGB4 and HMGB4L1.(a) Alignment of rat TP4/HMGB4L1 and HMGB4 amino acid sequences. HMGB-boxes A and B are underlined. Red letters indicate identical amino acids. An alternative allele in position 34 of TP4/HMGB4L1 is either a tyrosine or a isoleucine. Amino acids marked in italics have been identified by amino terminal amino acid sequencing7. Domain structures and number of amino acids of rat HMGB1, HMGB4 and TP4/HMGB4L1 are shown in the schematic picture. (b) Western –blot of recombinant mouse HMGB4. Recombinant HMGB4 was detected with Ponceau S –staining and with anti-HMGB4 and anti-HMGB4L1 antibodies. The antibodies did not detect recombinant HMGB1. (c) Northern Blot -analysis of the mouse HMGB4 transcript. Total RNA samples were isolated from adult mouse testes and analyzed via Northern Blot, using a probe derived from the entire coding sequence of mouse HMGB4. The probe detected a 1.1 kb band. Ethidum bromide stained ribosomal RNA is shown. (d) Northern Blot -analysis of rat HMGB4L1 transcript. Total RNA samples were isolated from adult rat testes and from developing rat testes, and analyzed via Northern Blot, using a probe derived from the entire coding sequence of rat HMGB4L1. The probe detected a 0.9 kb band in samples derived from testes of sexually mature rat. Ethidum bromide stained ribosomal RNA is shown. 3 w = 3 week old rat, 5 w = 5 week old rat. (e) Immunohistochemical staining of rat HMGB4L1 protein and in situ hybridization of mouse HMGB4 mRNA in adult testes. Anti-HMGB4L1 polyclonal antibody staining revealed intense HMGB4L1 expression in elongated spermatids (red). Haematoxylin was used as a counterstain. Control sections were stained without the primary antibody. HMGB4 mRNA localized to round and elongated spermatids. The control section shows adult mouse testes incubated with the sense probe. Bars represent 50 μm. (f) Expression of HMGB4 and HMGB4L1 mRNA in cultured rat neurons. Neuronal cells from the hippocampus and the cerebellum were cultured, and both HMGB4 and HMGB4L1 expression (arrows) was detected with RT-PCR. +RT =  reverse transcribed, -RT =  without reverse transcription.
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f1: Characterization of HMGB4 and HMGB4L1.(a) Alignment of rat TP4/HMGB4L1 and HMGB4 amino acid sequences. HMGB-boxes A and B are underlined. Red letters indicate identical amino acids. An alternative allele in position 34 of TP4/HMGB4L1 is either a tyrosine or a isoleucine. Amino acids marked in italics have been identified by amino terminal amino acid sequencing7. Domain structures and number of amino acids of rat HMGB1, HMGB4 and TP4/HMGB4L1 are shown in the schematic picture. (b) Western –blot of recombinant mouse HMGB4. Recombinant HMGB4 was detected with Ponceau S –staining and with anti-HMGB4 and anti-HMGB4L1 antibodies. The antibodies did not detect recombinant HMGB1. (c) Northern Blot -analysis of the mouse HMGB4 transcript. Total RNA samples were isolated from adult mouse testes and analyzed via Northern Blot, using a probe derived from the entire coding sequence of mouse HMGB4. The probe detected a 1.1 kb band. Ethidum bromide stained ribosomal RNA is shown. (d) Northern Blot -analysis of rat HMGB4L1 transcript. Total RNA samples were isolated from adult rat testes and from developing rat testes, and analyzed via Northern Blot, using a probe derived from the entire coding sequence of rat HMGB4L1. The probe detected a 0.9 kb band in samples derived from testes of sexually mature rat. Ethidum bromide stained ribosomal RNA is shown. 3 w = 3 week old rat, 5 w = 5 week old rat. (e) Immunohistochemical staining of rat HMGB4L1 protein and in situ hybridization of mouse HMGB4 mRNA in adult testes. Anti-HMGB4L1 polyclonal antibody staining revealed intense HMGB4L1 expression in elongated spermatids (red). Haematoxylin was used as a counterstain. Control sections were stained without the primary antibody. HMGB4 mRNA localized to round and elongated spermatids. The control section shows adult mouse testes incubated with the sense probe. Bars represent 50 μm. (f) Expression of HMGB4 and HMGB4L1 mRNA in cultured rat neurons. Neuronal cells from the hippocampus and the cerebellum were cultured, and both HMGB4 and HMGB4L1 expression (arrows) was detected with RT-PCR. +RT =  reverse transcribed, -RT =  without reverse transcription.
Mentions: Database searches revealed that both human and mouse have a single copy of the HMGB4-gene217, whereas the rat has two HMGB4-like genes, one on chromosome 5 and another one on the X-chromosome [coding for proteins HMGB4 (NP_001102933) and predicted high mobility group protein B4 –like (XP_006227590), respectively, in the NCBI database]. The predicted protein coded by the gene on the rat X-chromosome was identified as TP47 (accession number AAB24466). The amino acid sequences of the rat HMGB4 and TP4 are 66% identical and 82% similar (Fig. 1a refs 2 and 7). TP4 is renamed high mobility group box 4 protein –like 1 (HMGB4L1) in this study. Comparison of HMGB4 and HMGB4L1 to the amino acid sequence of the archetype of the HMGB-proteins, HMGB1, revealed that rat HMGB4 is 42% identical and 67% similar, and HMGB4L1 is 43% identical and 66% similar.

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