In this report, we demonstrate that expression of MRG15, a chromatin regulator, is required for both proliferation and differentiation of neural precursor cells. We have found that the number of these primary stem/progenitor cells is lower in the initial isolation of cells from the embryonic null brain, most likely because of the increased apoptosis observed in vivo in the histological analyses. We observed that the overall neural tube is thinner in Mrg15 null embryos and this is most likely due to the presence of fewer neural precursor cells. As a result when the cells are cultured in vitro the number of large spheres is decreased in cell cultures derived from Mrg15 deficient embryonic brain. Cells derived from the largest neurospheres, when subcultured, continue to exhibit defects in number of spheres greater than 50 μm at each subsequent passage. BrdU incorporation in Mrg15 deficient cells is reduced when compared with wild-type, however, apoptosis is not affected indicating that in vitro defects in neural precursor proliferation is the result of reduced growth rate and long term growth potential, but not increased cell death. The difference in results between in vivo and in vitro studies is not surprising as the milieu of cells in culture is very different from that in the embryo. For example, overlying non-neuronal tissues may provide extrinsic or non-cell autonomous signals that are necessary for cell survival in vivo. However, the final results are similar in that cell cycle progression and completion are affected in both cases, with a mitotic defect contributing to a decreased number of precursor cells in vivo. The fact that infection with an adenovirus expressing MRG15 causes increased BrdU incorporation in null cells in vitro, demonstrates that it is the deficiency in MRG15 that causes the proliferation defect we observed.
Differentiation into neurons was also affected in Mrg15 deficient neural precursor cells in vitro. They did not attach to the tissue culture dishes as well as wild-type and many of the cells remained in aggregates in differentiation media. This suggests that the abnormalities observed in the developing brain of Mrg15 deficient embryos are a result of cell-autonomous defects in these neural precursor cells. Abnormalities observed in many other tissues of Mrg15 deficient embryo may be caused by a similar molecular mechanism(s) as that seen in brain tissue, and the data we have regarding proliferative defects in MEFs derived from null and wild-type embryos supports this possibility.
MRG15 associates in complexes with the HAT Tip60 (Cai et al. 2005
; Cai et al. 2003
; Doyon et al. 2004
; Hayakawa et al. 2007
; Sardiu et al. 2008
) and also mSin3/HDAC (Doyon et al. 2004
; Hayakawa et al. 2007
; Yochum and Ayer 2002
) and is thereby involved in the regulation of gene expression by changing the acetylation status of histones surrounding target genes. Recently, Fazzio et al. have reported that the Tip60-p400 complex is important for maintenance of embryonic stem cell (ESC) identity (Fazzio et al. 2008
). Knockdown of either of these components of the complex in ESCs resulted in reduced growth rate, flattened cell morphology, changes in gene expression, and loss of ESC markers eg: alkaline phosphatase activity was decreased and embryoid body formation less efficient. Gene expression analyses demonstrated that cell cycle regulators and cell division related genes were down-regulated and differentiation and embryonic development related genes were up-regulated following knockdown of these genes. However, interestingly, MRG15 knockdown in ESCs did not have a significant phenotype. One possibility is that MRGX, a mammalian homolog of MRG15, may compensate for MRG15 function in these cells, because MRGX can also associate in complexes with Tip60 (Cai et al. 2003
; Hayakawa et al. 2007
; Lerin et al. 2006
; Sardiu et al. 2008
). Since Tip60 HAT is a multi-subunit complex, another possibility is that MRG15 in this complex is not required for ESCs but is important for tissue precursor cells, such as the neural precursor cells we have studied in this report.
In our study we have found that Mrg15
deficient neural precursor cells exhibit differentiation defects in addition to growth defects. Mrg15
deficient neural precursor cells appear to be maintained as stem-like aggregates in differentiation medium and differentiate into neurons less-efficiently than wild-type cells. It is known that hematopoietic competence is a rare property of neural stem cells and epigenetic alterations can cause fate switching (Morshead et al. 2002
). Thus, treatment of neurospheres with trichostatin A (an HDAC inhibitor) and 5-aza-2′-deoxycytidine (a DNA methyltransferase inhibitor) can yield a transplantable hematopoietic population (Schmittwolf et al. 2005
). Additionally, Mbd3, which is a component of the nucleosome remodeling and histone deacetylation (NuRD) complex, is essential for commitment to developmental lineages in ESCs (Kaji et al. 2006
). The NuRD complex contains at least seven subunits and HDAC1 and HDAC2 are catalytic subunits of this complex (Wade et al. 1999
; Zhang et al. 1999
). Mbd3 deficient ESCs are viable but fail to silence genes which are important for maintenance of ESCs, such as Oct4, Nanog, and Rex1, under differentiation conditions (without LIF). Mbd3 deficient ESCs also cannot form neuroectoderm in culture. Normal ESCs lose Oct4 expression and express markers of neural progenitors (nestin-positive) and postmitotic neurons (Tuj1-positive) after 10 days in differentiation conditions. However, the majority of Mbd3 deficient cells continue to express Oct4 under these conditions and retain a stem cell-like growth (Kaji et al. 2006
). It is also known that inhibition of HDAC activities in neural progenitors induce neural differentiation but inhibit glial differentiation (Hsieh and Gage 2004
; Hsieh et al. 2004
). Taken together these data suggest that co-repressor complexes involving HDACs are also important for cell-fate determination and differentiation of stem/precursor cells.
MRG15 is also a component of HDAC1 and HDAC2 containing complexes. Although it is known that this complex acts to suppress spurious intragenic transcription in budding yeast, the function in mammalian cells is still unclear. MRG15 containing HDAC complex(es) may also work as a repressor of expression of genes required for stem cells to maintain their stem cells status in addition to inhibiting incorrect transcription initiation. The defects in self-renewal and differentiation, we have observed in Mrg15 deficient neural precursor cells may therefore be the result of inactivation of two or more independent MRG15 containing complexes.
We here present evidence for a role of MRG15 in neural cell proliferation. In an earlier study using an antibody against the chromodomain of MRG15 protein, we had analyzed hippocampal tissue samples from histopathologically confirmed Alzheimer's disease (AD) and non-AD age matched controls. We observed specific labeling of large pyramidal neurons only in AD cases, and that there was significant overlap of immunostaining of this protein and phosphorylated tau (Raina et al. 2001
). Age matched normal controls showed no immunoreactivity. The presence of a positive chromatin remodeling, transcriptionally controlling protein in association with intraneuronal neurofibrillary pathology is consistent with the multiple gene expression changes that have been observed in such regions. It also provides support for the idea that neurons in AD re-enter the cell cycle. Interestingly, the expression of microRNAs (miRNAs), that have been implicated in brain development and neuronal specification, have recently been demonstrated to be altered in AD brain suggesting functional deficits occur at various stages of the disease (Cogswell et al. 2008
). Thus, chromatin remodeling and the resulting gene expression modifications could well be a contributing factor to the initiation and progression of AD.