Int J Stem Cells.  2024 Nov;17(4):427-436. 10.15283/ijsc23202.

Usp7 Regulates Glial Lineage Cell-Specific Transcription Factors by Modulating Histone H2B Monoubiquitination

• Kim DH
• Kim SL
• Singh V
• Ramakrishna S

• KMID: 2562362
• DOI: http://doi.org/10.15283/ijsc23202

Abstract

Histone H2B monoubiquitination (H2Bub1) is a dynamic posttranslational modification which are linked to DNA damage and plays a key role in a wide variety of regulatory transcriptional programs. Cancer cells exhibit a variety of epigenetic changes, particularly any aberrant H2Bub1 has frequently been associated with the development of tumors. Nevertheless, our understanding of the mechanisms governing the histone H2B deubiquitination and their associated functions during stem cell differentiation remain only partially understood. In this study, we wished to investigate the role of deubiquitinating enzymes (DUBs) on H2Bub1 regulation during stem cell differentiation. In a search for potential DUBs for H2B monoubiquitination, we identified Usp7, a ubiquitin-specific protease that acts as a negative regulator of H2B ubiquitination during the neuronal differentiation of mouse embryonic carcinoma cells. Loss of function of the Usp7 gene by a CRISPR/Cas9 system during retinoic acid-mediated cell differentiation contributes to the increase in H2Bub1. Furthermore, knockout of the Usp7 gene particularly elevated the expression of neuronal differentiation related genes including astryocyte-specific markers and oligodendrocyte-specific markers. In particular, glial lineage cell-specific transcription factors including oligodendrocyte transcription factor 2, glial fibrillary acidic protein, and SRY-box transcription factor 10 was significantly upregulated during neuronal differentiation. Thus, our findings suggest a novel role of Usp7 in gliogenesis in mouse embryonic carcinoma cells.

Keyword

Deubiquitinase; Epigenetics; Gliogenesis; Histone H2A; Histone H2B; Transcriptional regulation

Figure

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  Fig. 1 Potential ubiquitin-specific protease (USP) candidates regulating H2B monoubiquitination. (A) The effect of CRISPR/Cas9-mediated knockout (KO) of each USP candidate (Usp7, Usp12, Usp21, and Usp37) on the expression level of histone H2B monoubiquitination (H2Bub1) was estimated by Western blotting. (B) The expression of H2Bub1 was graphically represented. *p<0.05, ***p<0.001 by paired t-test. (C) Schematic representation of retinoic acid (RA)-induced P19 cell neuronal differentiation schedule. P19 cells formed aggregates for four days with 10 μM of RA. Aggregates were collected and seeded in cell culture plates on day 4 and harvested at the indicated time points. (D) RA-induced neuronal differentiation of P19 cells at different days. Samples were collected at the indicated time points and the expressions of Oct3/4 and βIII-tubulin protein were analyzed by Western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed as a loading control. (E) Fluorescence microscopy of P19 cell aggregates after transfection of Cas9 tagged with red fluorescent protein (RFP) along with single guide RNA (sgRNA) targeting Usp7. Scale bar=100 μm.

• 

  Fig. 2 Usp7 knockout (KO)-mediated negative regulation of H2B ubiquitination during retinoic acid (RA)-induced neuronal differentiation. (A) The histone H2B monoubiquitination (H2Bub1) protein expression levels in the control and Usp7 KO P19 cells during RA-induced neuronal differentiation analyzed by Western blot. Differentiated samples were collected on the indicated days. The protein expression of H2Bub1 and total H2B were analyzed by Western blot. (B) RA-induced neuronal differentiation in P19 cells. Differentiated samples were collected on the indicated days and the protein expression of Usp7 were analyzed by Western blot. (C) The messenger RNA (mRNA) expression of Usp7 in control P19 cells was estimated by real-time polymerase chain reaction during RA-induced differentiation. Error bars were derived from three independent experiments. N=3. ***p<0.001.

• 

  Fig. 3 The expression of transcription factors related to mouse neurogenesis upon Usp7 knockout (KO). (A-C) Relative expression levels of oligodendrocyte-specific transcription factors (A) Olig2, (B) Sox10, and (C) O4 messe-nger RNA (mRNA) were analyzed by real-time polymerase chain reaction (PCR). Samples were collected on day 9 after differentiation from control and Usp7 KO P19 cells. Results were normalized with the level of GAPDH. (D-F) Relative expression of astrocyte-specific transcription factor (D) GFAP, (E) ALDH1L1, and (F) S100β were analyzed by real-time PCR. Error bars were derived from three independent experiments. N=3. ***p<0.001.

• 

  Fig. 4 Knockout (KO) of Usp7 alters histone H2B monoubiquitination (H2Bub1) of genes involved in neuronal differentiation. (A-C) Relative expression levels of neuronal lineage cell-specific transcription factors (A) Ascl1, (B) Ngn1, (C) NeuroD1, and (D, E) marker genes (D) Map2 and (E) βIII-tubulin messenger RNA (mRNA) were analyzed with real-time polymerase chain reaction (PCR). Samples were collected on day 9 after differentiation from control and Usp7 KO P19 cells. Results were normalized with the level of GAPDH. (F-H) Samples were collected on day 9 after differentiation from control and Usp7 KO P19 cells and subjected to chromatin immunoprecipitation analysis with antibodies specific for H2Bub1 and H2B. Immunoprecipitated DNA was quantified by real-time PCR with primers specific for the promoter regions of (F) Olig2, (G) GFAP, and (H) Pax6. Error bars were derived from three independent experiments. N=3. RA: retinoic acid. *p<0.05, **p<0.01, ***p<0.001.

Reference

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