Involvement of ZFPIP/Zfp462 in chromatin integrity and survival of P19 pluripotent cells

Abstract

Toti- or pluripotent cells proliferation and/or differentiation have been shown to be strongly related to nuclear chromatin organization and structure over the last past years.

We have recently identified ZFPIP/Zfp462 as a zinc finger nuclear factor necessary for correct cell division during early embryonic developmental steps of vertebrates. We thus questioned whether this factor was playing a general role during cell division or if it was somehow involved in embryonic cell fate or differentiation.

To achieve this goal, we performed a knock-down experiment in the pluripotent P19 and differentiated 3T3 cell lines, both expressing endogenous ZFPIP/Zfp462. Using specific shRNA directed against ZFPIP/Zfp462 transcripts, we demonstrated that depletion of this protein induced cell death in P19 but had no effect in 3T3 cells. In addition, in the absence of the protein, the P19 cells exhibited a complete destructuration of pericentromeric domains associated with a redistribution of the HP1α proteins and an increase in DNA satellites transcribed RNAs level.

These data suggested an instrumental role of ZFPIP/Zfp462 in maintaining the chromatin structure of pluripotent cells.

Introduction

We have recently identified ZFPIP/Zfp462 as a vertebrate-specific gene encoding a nuclear zinc finger protein that strongly interacts with Pbx1 [1]. The 250 kDa ZFPIP/Zfp462 protein contains a strikingly important number (34) of C₂H₂ zinc fingers distributed throughout the sequence and a putative Nuclear Localization Signal (NLS). Previous data have shown that ZFPIP/Zfp462 mRNA is expressed during development in the mouse with a maximum yield between E10.5 to E12.5. This expression is particularly abundant in brain, somites and limbs buds [1], [2]. ZFPIP/Zfp462 appeared to be an essential developmental gene in Xenopus laevis. Indeed, knocked-down expression of ZFPIP/Zfp462 in X. laevis embryos leads to a rapid cell division arrest coupled with important nuclear chromatin disorders: mitotic cells undergo abnormal mitosis, with aberrant metaphase, anaphase, incomplete chromosome segregation or conjoined nuclei [3]. Furthermore, decrease of ZFPIP/Zfp462 levels seems to impair cell cycle, inducing cell death with a mechanism described as “mitotic catastrophe” [4].

Analysis of the chromatin/cell division disturbance in whole animal using morpholinos remains difficult because of the important cellular variability in developing embryos [5]. Therefore, analysis of such mechanism in a cell culture model is an alternative approach. To this aim, we sought for a model of ZFPIP/Zfp462 expressing cell line and selected out the murine embryonal carcinoma P19 cell line which has already been widely used to study early events of embryonic differentiation [6], [7]. Indeed, these cells resemble the inner cell mass of the early embryo and can differentiate into different cell types according to the culture conditions, such as fibroblastic, muscular or neural cells [8], [9].

With this work, we demonstrated that ZFPIP/Zfp462 expression was cell-specific. Indeed, the protein was detected in P19, ES or 3T3 whereas no expression of the gene was detected in several vertebrate cell lines. Using a shRNA approach, we demonstrated that depletion of the protein in the P19 embryonic cells induced abnormal nucleus phenotype that was not observed in differentiated depleted 3T3 cells. Moreover, ZFPIP/Zfp462-depleted P19 cells exhibited disruption of nuclear heterochromatin, correlated to a delocalization of HP1α and an increase of DNA satellites transcription. Following these abnormal nuclear cell processes, the P19 cells died within 24 h.

These results demonstrated that (i) the absence of ZFPIP/Zfp462 led to a disorganization of pericentromeric domains in P19 cells which were subsequently not able to divide correctly and that (ii) the gene appeared vital in P19 pluripotent cells but not in 3T3 cells. The overall data clearly showed that ZFPIP/Zfp462 was an essential gene involved in chromatin structure modelling mechanisms occurring during cell division of pluripotent cells.