Previously, CTIP1 and CTIP2 were demonstrated to repress transcription in a predominantly TSA-insensitive manner, possibly because of recruitment of SIRT1 to CTIP1/2-responsive promoters, at least in transiently transfected cells, and in the context of the tk
). However, in the context of the minimal promoter, we found herein that transcriptional repression mediated by CTIP2 was partially sensitive to inhibition by TSA (see ), suggesting that TSA-insensitive, SIRT1-mediated histone deacetylation may not necessarily generalize to all CTIP2-responsive promoters and/or cell types. Other transcriptional repressors have been similarly found to function in TSA-sensitive and -insensitive manners, as well as in a cell- and promoter-dependent contexts. For example, the retinoblastoma tumor suppressor protein (Rb) represses expression of Cdc2
, topoisomerase IIα
, and thymidylate synthase in a TSA-sensitive manner, but Rb-mediated repression of cyclin A is not reversed by TSA, demonstrating that the mechanism of Rb-mediated transcriptional repression is promoter-specific (34
). Similarly, RE-1 silencing transcription factor (REST)-mediated repression of connexin36 was found to be reversible by TSA, whereas that of the two other REST target genes, BDNF
, was not (35
). Moreover, REST-mediated repression of connexin36 was TSA-sensitive only in pancreatic α
cells, but not in neuronal cells, indicating that REST represses transcription in both promoter- and cell type-specific manners (35
). The differential responses of promoters and cell types to TSA may be an important scheme for transcriptional repressors, perhaps including CTIP2, to function in transcriptional regulation. In addition to TSA-sensitive and NAD+
-dependent HDACs, histone and DNA methylation may also be involved in transcriptional repression (36
). As we did not observe complete inhibition of CTIP2-mediated repression by TSA in our present studies, we cannot exclude the possibility that CTIP2 may use other mechanism(s), in addition to TSA-sensitive histone deacetylation, to repress transcription in the context of the minimal promoter.
The CTIP2 complex in from SK-N-MC cells appeared to migrate with a peak centered between 669 and 1000 kDa (see ). In contrast, the size of CTIP2 complex in Jurkat cells was found be up to 2000 kDa (15
). Although we found that the NuRD complex proteins co-fractionated with CTIP2 in Jurkat cells (data not shown), the difference in apparent masses of the CTIP2 complexes in these two cell types possibly suggests differing compositions of CTIP2 complexes, which may be of functional significance.
The components of the NuRD complex were recruited to a CTIP2-targeted promoter in a CTIP2-dependent manner (see ), and were found to co-occupy the promoter of an endogenous CTIP2-target gene in SK-N-MC neuroblastoma cells (see ). This recruitment is likely mediated by the direct interaction of CTIP2 with the histone binding proteins, RbAp46 and/or RbAp48 (see ). Additionally, many other transcription factors involved in transcriptional repression have been reported to interact with different subunits of the NuRD complex (20
), suggesting the possibility that the NuRD complex is involved in many pathways leading to transcriptional repression. However, it is presently unknown if the potentially differential recruitment of the NuRD complex to a particular, nucleating transcription factor may result in the formation of a gene-specific repressor complex(es).
Extensive studies of the biological function of the NuRD complex have shown that the components of this complex are required for morphogenesis of Drosophila
), embryonic patterning, vulva development and signaling in Caenorrhabditis elegans
), and mouse embryogenesis (40
). In combination with the data from RT-PCR analysis illustrating the expression of MBD3, Mi2, HDAC1, and HDAC2 from a very early stage of embryonic development (41
), transcriptional silencing by the NuRD complex may play a significant role in embryonic development in many species ranging from nematodes to mammals. In addition, recent reports revealed an important role of the NuRD complex in control cell fate determination during B and T cell development (20
The crucial function of CTIP2 in the context T cell development, as well as in the development of corticospinal motor neurons (CSMN), suggests that this protein may play a global role during mammalian development. As both CTIP2 and the NuRD complex appear to play significant, and possibly convergent, roles in cell fate determination and differentiation, association of CTIP2 with this complex raises the possibility that the histone deacetylase and chromosome remodeling activities of the NuRD complex may be implicated in regulation of both T cell and CSMN specification and development by CTIP2. This hypothesis may be tested in vivo by analysis of compound mutant mice.
The cdk inhibitor p57KIP2
was newly identified in this report as one of the putative CTIP2 target genes in SK-N-MC neuroblastoma cells. The cdk inhibitor p57KIP2 is a putative tumor suppressor, and has the ability to associate with and inhibit the catalytic activity of a number of cyclin-cdk complexes (33
). The human p57KIP2
gene is paternally imprinted in both humans and mice, and the human p57KIP2
locus is on chromosome 11p15, a region that has been implicated in various sporadic human malignancies, and also in Beckwith-Wiedemann syndrome (43
Several studies suggest that p57KIP2 plays a distinct role in neuronal differentiation, which may or may not be related to the function of this protein as a cdk inhibitor. During embryogenesis, p57KIP2
is expressed in mitotic progenitor cells that migrate away from retinal ventricular zone, and in this context p57KIP2 appears to be required for proper exit from cell cycle (44
). Postnatally, however, p57KIP2
is expressed in a restricted population of amacrine neurons, and it has been proposed that p57KIP2 can influence cell fate specification and differentiation long after terminal mitosis (44
). In addition, p57KIP2 is expressed in postmitotic differentiating midbrain dopaminergic neurons and is required for the maturation of these cells (45
). Interestingly, the mechanism by which p57KIP2 promotes maturation of dopaminergic neuronal cells does not require cdk inhibitor activity but rather is achieved through the direct protein-protein interaction of p57KIP2 with orphan nuclear receptor Nurr1 (45
The likely recruitment of the NuRD complex to the promoter of p57KIP2 via direct interaction with CTIP2 suggests that the NuRD complex plays a role in transcriptional repression mediated by CTIP2 in a neuron-like context. At present, we do not know if CTIP2 directly regulates expression of p57KIP2 within neuronal subpopulations in vivo, or the possible contribution of the NuRD complex to this and other CTIP2-mediated transcriptional repressive events. Further studies employing the power of Ctip2-/- mice are necessary to clarify role(s) of this protein and the corresponding transcriptional repression pathway(s) in vivo.