By using a combination of approaches that complement location analysis, we found several lines of evidence that RBP2, like pRB, plays a role in differentiation. We identified two functionally distinct classes of genes that are occupied by RBP2 in differentiation- dependent and -independent manners, which offer insights into the regulation of cell-cycle genes and mitochondria biogenesis in differentiating cells. The differentiation-dependent RBP2 binding correlates with high gene activity and is aimed at the cell-cycle genes that are destined for repression. RBP2 also regulates components of the mitochondrion and is necessary for normal mitochondrial function. Repression by RBP2 involves the direct effects on H3K4 methylation levels. Analysis of RBP2 target promoters and the distribution of nucleosomes with H3K4me3 modification suggest that RBP2 recognizes modules in the promoter regions in addition to H3K4me3. Our promoter sequence analysis indicates that these modules may be other transcription factors, including E2F and pRB family members. Consistent with the idea that RBP2 controls pRB-dependent genes, the correlation in the expression changes after depletion of RBP2 and reintroduction of pRB is highly significant and reflects the restored ability of cells to produce extracellular proteins and proteins with a function in development.
We provide evidence for changes in the expression of genes that are differentially occupied by RBP2 during differentiation (). In growth-arrested cells that start to express markers of differentiation, such as U937 cells at 96 hr under TPA treatment, RBP2 is recruited to cell-cycle genes. These genes, however, are initially highly expressed until a later point in differentiation progression, when their two- to three-fold decrease in expression coincides with recruitment of RBP2. The shift to the actively transcribed genes at 96 hr is consistent with the idea that RBP2 appears in a fraction of active chromatin after the induction of differentiation (Benevolenskaya et al., 2005
). RBP2 repression of E2F targets might be generally assisted by E2F4, p107, and p130 complexes to achieve full repression. RBP2 interacts with p130 in a yeast two-hybrid system (E.V.B., unpublished data), suggesting that it may not only be recruited to the same promoters as RBP2, but also be a part of its protein complexes. While Pax-9 has been identified in a yeast two-hybrid screen with PLU1 (Tan et al., 2003
), we found overrepresentation of Pax-6 binding sites in RBP2 targets. Analogous to the PLU1 action, RBP2 might act as a Pax protein corepressor in differentiated cells. Consistent with this notion, it may be possible that RBP2 needs additional factors to be present at the promoter for repression to occur. In support of this, there is preferential recruitment of RBP2 in differentiated cells to highly active promoters that possess particular chromatin configuration and promoter motifs. Further, because at least half of its “constitutive” RBP2 target genes retain nucleosomes with H3K4me3 and their expression remains at average level, it is possible that RBP2 is present in an active form capable of demethylation when unbound to promoter, which then resolves H3K4 methylation upon binding, or that, in the promoter-bound form, RBP2 demethylation activity may be antagonized by histone H3K4 methyltransferases. In addition, DNA contact RBP2 mutants that retain histone demethylase activity show reduced activation of RBP2-dependent BRD2
promoter (Tu et al., 2008
), supporting the idea that DNA binding contributes to RBP2 recruitment and demethylation of specific genes.
Analysis of human cells and tissues showed that RBP2 is bound to genes where high expression is a feature of pluripotency, CD105+ and CD34+ cells. CD105 expression is a putative marker for mesenchymal stem cells (MSCs). CD34+ subset is enriched for hematopoietic stem cells (HSCs) and early progenitor cells. Many RBP2 targets encode proteins involved in DNA metabolism, including various transcriptional regulators. Their transcriptional repression versus activation in MSCs and HSCs may be important in programming differentiation. These conclusions can provide an explanation for the abnormalities in RBP2−/−
mice, which demonstrate increased entry into G1 phase and decreased apoptosis in HSC, and an increased number of myeloid progenitor cells in S/G2/M (Klose et al., 2007
). RBP2 target genes are also overrepresented among genes preferentially expressed in human leukemia and lymphoma. RBP2 has recently been shown to be involved in a translocation in a patient with acute myeloid leukemia (van Zutven et al., 2006
). When taken together, these data suggest that RBP2 has a distinctive role in hematopoiesis.
The overall structure of the mitochondria of many cell types can change rapidly in response to different biological stimuli (Cerveny et al., 2007
), suggesting that mitochondrial dynamics are highly regulated. Sustained mitochondrial elongation induces senescence-associated phenotypic changes (Lee et al., 2007b
). So too, during cardiomyocyte differentiation, mitochondria form long tubules (Chung et al., 2007
). Formation of long tubules is highly relevant for mitochondrial function, because it improves mitochondrial transport and improves efficiency of energy transmission and Ca2+
dynamics between mitochondria. We found that mitochondrial length and the rigor of mitochondrial staining by the redox-sensitive dye correlated with RBP2 levels. Consistent with the idea that mitochondria of filamentous structure may facilitate the delivery of membrane potential to specific subcellular regions (Skulachev, 2001
), cells with RBP2 knockdown showed widespread staining by this dye. Also, repression of MFN2 is known to lead to a reduced mitochondrial membrane potential as well (Pich et al., 2005
). Interestingly, the role of MFN2 in mitochondrial energization is independent of its role in mitochondrial fusion. Therefore, RBP2 may be directly involved in the negative control of mitochondrial fusion and energization, at least in part, by reducing mitofusin gene expression. Downregulation of RBP2 triggers both processes that may contribute to “flat” cell phenotype in SAOS-2 cells, consistent with data on rat MFN2 protein, which functions as a cell proliferation suppressor (Chen et al., 2004
). RBP2 proteins are conserved from fungi to human. We propose that regulation of enzymes and ribosomes present in mitochondria by RBP2 is an ancient event in evolution. For free-living, single-celled organisms the critical criterion in the decision to divide in a given condition is nutrient supply. In yeast in response to hundreds of various growth conditions, the group of genes that behave in a coregulated manner encode mitochondrial proteins (Hughes et al., 2000
). In complex organisms, extracellular signals, such as growth factors and antimitogens, determine tissue- and cell-specific gene expression. Gene expression data analysis of RB−/−
cells with reintroduced pRB function or with reduced RBP2 function showed an overrepresentation of genes encoding cell surface and cell adhesion molecules, receptors, and molecules with growth factor activity. Thus, RBP2 inactivation leads, still in an unknown manner, to the increased expression of genes involved in development and in extracellular regulation (). Our combined analysis sheds light on the mechanisms of the differentiation transcriptional switch mediated by RBP2 and provides a framework for studies on the contribution of the pRB/RBP2 pathway to disease pathogenesis such as in leukemia.