HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells

Abstract

High‐mobility group box (HMGB)1 and HMGB2 proteins are the subject of intensive research because of their involvement in DNA replication, repair, transcription, differentiation, proliferation, cell signaling, inflammation, and tumor migration. Using inducible, stably transfected human embryonic stem cells (hESCs) capable of the short hairpin RNA‐mediated knockdown (KD) of HMGB1 and HMGB2, we provide evidence that deregulation of HMGB1 or HMGB2 expression in hESCs and their differentiated derivatives (neuroectodermal cells) results in distinct modulation of telomere homeostasis. Whereas HMGB1 enhances telomerase activity, HMGB2 acts as a negative regulator of telomerase activity in the cell. Stimulation of telomerase activity in the HMGB2‐deficient cells may be related to activation of the PI3K/protein kinase B/glycogen synthase kinase‐3β/ β‐catenin signaling pathways by HMGB1, augmented TERT/telomerase RNA subunit transcription, and possibly also because of changes in telomeric repeat‐containing RNA (TERRA) and TERRA‐polyA+ transcription. The impact of HMGB1/2 KD on telomerase transcriptional regulation observed in neuroectodermal cells is partially masked in hESCs by their pluripotent state. Our findings on differential roles of HMGB1 and HMGB2 proteins in regulation of telomerase activity may suggest another possible outcome of HMGB1 targeting in cells, which is currently a promising approach aiming at increasing the anticancer activity of cytotoxic agents.—Kučírek, M., Bagherpoor, A. J., Jaroš, J., Hampl, A., Štros, M. HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells. FASEB J. 33, 14307‐14324 (2019). https://www.fasebj.org

Nonhistone Proteins HMGB1 and HMGB2 Differentially Modulate the Response of Human Embryonic Stem Cells and the Progenitor Cells to the Anticancer Drug Etoposide

“…The prepared inducible HMGB-shRNA constructs were then tested to determine which had the most efficient HMGB silencing. The impact was investigated using at least two different shRNA HMGB constructs for a given HMGB gene along with “scrambled” shRNA controls previously tested in several human cell lines [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. The sense and antisense strands containing the shRNA-expressing sequence targeting human HMGB1 , HMGB2 , or both HMGB1 and HMGB2 were annealed and ligated into the BglII/HindIII-linearized pSuperior-puro vector ( ), as detailed in [ 10 ].…”

“…The establishment of neuroectodermal cells (hNECs) capable of rosette structure formation was detailed in our previous paper [ 10 ]. Briefly, hESCs were seeded at a density of 2‒5 × 10 3 cells/cm 2 on culture plates precoated with Matrigel™ in the presence of neural differentiation media (DMEM/F12/Neurobasal 1:1, N2, B27 (Invitrogen, Carlsbad, CA, USA), 2 mmol/L L-glutamine (all media components from Invitrogen), 1× MEM nonessential amino acids, 1% penicillin‒streptomycin and (composition described in [ 10 , 17 ]), and 5% CO 2 in a humidified atmosphere at 37 °C. After 5 days of culture, a specific neuroectodermal cell morphology emerged, and neuronal rosettes appeared after 8‒10 days.…”

“…Two distinct scenarios for the differentiation of hESCs into neuroectodermal cells (hNECs) were examined. HMGB silencing was performed before (Scenario A) or after (Scenario B) differentiation of hESCs into the hNECs, which was initiated as detailed in [ 10 , 17 ].…”

“…HMGB1 continues to be expressed in all embryonic and adult cells (reviewed in [ 4 , 11 , 12 ]). The critical roles of HMGB1/2 proteins in cell survival, embryonal neurogenesis, regulation of cell proliferation, differentiation, and telomere biology have been previously demonstrated [ 4 , 10 , 13 , 14 , 15 , 16 , 17 , 18 ].…”

“…therein). Our recent findings on the distinct modulation of cellular activity of telomerase by HMGB1 and HMGB2 [ 10 , 17 , 28 ] could be a starting point for developing new approaches aiming at the promotion of cell death in drug-treated cancer cells.…”