The processes culminating in commitment to the S phase in metazoan cells include the up-regulation of genes required for duplication of the genome. The histone genes that encode proteins required for packaging of the newly synthesized DNA in the two daughter cells are also up-regulated in this sequence of events. The metazoan histone gene family includes the nucleosomal histone genes H2a, H2b, H3, and H4, as well as the genes encoding linker histones (H1 genes). Histone genes are among the most highly expressed and are among the most highly conserved genes known; within histone classes, the protein sequence is remarkably conserved among all eukaryotes. The cellular resources required for histone synthesis in the duplicating cell parallel those required for making a copy of the genome.
In the mouse, the up-regulation of the histone gene family at the G1-S boundary requires the coordinated activation of the transcription of many genes. Although various histone promoter elements have been implicated in the G1-S-specific up-regulation of vertebrate histone gene expression, none of these is common to more than one or two histone classes. Because genes of all histone classes are coordinately up-regulated, it follows that a common pathway is responsible. Furthermore, a common point of interaction with this pathway must exist for histone genes of all classes.
We have identified a highly conserved transcription factor, YY1, on the basis of its interaction with an element found in the protein-encoding sequence of replication-dependent histone genes of all classes. This transcription factor, implicated as both an activator and a repressor of gene expression in different cellular and viral genetic contexts, is found in all animal cell types. YY1 has not been found in yeast, but cDNAs encoding the factor have been cloned in frogs, mice, humans and, very recently, fruit flies (5
). The DNA-binding domain of YY1 is at the carboxy-terminal end of the protein and is comprised of four potential zinc fingers. This region of the protein is 100% identical in frog, mouse, and human sequences, and the fruit fly YY1 DNA-binding domain is 97% identical to that of human YY1 (5
), a remarkable degree of identity.
We have proven that the in vitro histone α DNA-binding activity requires YY1 for α DNA-protein complex formation. The addition of YY1 antibodies to the binding reaction greatly diminished the level of the H3.2 α complex. Similarly, the removal of YY1 by immunodepletion of a mouse myeloma cell nuclear extract with YY1 antibodies also abolished the formation of the α complex.
We examined the ability of a well-characterized YY1-binding site, the adeno-associated virus P5-60 element, to compete with the histone α factor for binding of the replication-dependent H3.2 α element. This nonhistone element and unlabeled H3.2 α oligonucleotides both competed effectively, although the H3.2 α DNA was a better competitor than P5-60 for either the H3.2 α or the P5-60 probe. This result may indicate that a viral cofactor that facilitates the YY1 P5-60-binding activity is not present in normal cell extracts or that the histone α element is simply a better binding site for YY1 than the viral element.
In contrast, the replication-independent H3.3 oligonucleotides did not compete with the labeled H3.2 α probe for binding of the α factor, as we previously showed (3
), nor did the H3.3 oligonucleotides compete with the labeled P5-60 oligonucleotides. Interestingly, the P5-60 oligonucleotide sequence is 55% different from that of H3.2, whereas the H3.3 oligonucleotides used as competitors are 41% different from those of H3.2 (11 of 27 nt positions differ). The G+C content of the H3.2 oligonucleotides is 70%, whereas the G+C contents of the P5-60 and H3.3 oligonucleotides are lower but very similar (45 and 52%, respectively). The specificity of YY1 interactions with the H3.2 α and adeno-associated virus P5-60 sites confirms the results of the experiments with YY1 antibodies; the highly specific interaction of the histone α DNA-binding activity is due to the factor YY1.
We previously examined the effect of altering the histone α sequence on expression in vivo and DNA-protein interactions in vitro (3
). Because all deletions or mutations of the α element abolished the formation of the α-DNA complex in vitro and also caused a significant decrease in gene expression in vivo in stable transfectants, it is very likely that YY1 plays an in vivo role in the regulation of replication-dependent histone gene expression. Specifically, mutation of the replication-dependent H3.2 α element to yield the replication-independent H3.3 sequence caused a fourfold decrease in expression in vivo and a loss of formation of the α-DNA complex in vitro. Further in vivo evidence is that yeast reporter genes incorporating the H3.3 α sequence were not activated by the YY1-GAL4 fusion protein, whereas reporter genes containing the H3.2 α sequence in their promoters showed activated levels of expression in strains containing YY1-GAL4 fusion cDNAs.
In the experiments shown in Fig. , we examined in vivo, in unperturbed cycling cells, the role of the histone α element in correct temporal regulation of the replication-dependent mouse H3.2 gene. We showed that the dramatic increase in the amounts of histone mRNA as cells move forward in the cell cycle to the G1
-S boundary that is normally observed for replication-dependent histone genes is altered by mutation of the histone α element. A similar result was obtained by Harris et al. (17
) using a histone gene construct in which the 5′-flanking sequences were replaced with the promoter of a constitutive U1 snRNA promoter. These experiments can be directly compared to our studies because mitotic shakeoff was used to obtain synchronous cell populations for RNA analyses. They found that the level of transcripts from the U1-histone chimeric gene increased a total of 10-fold between the plating of mitotic cells and entry into the S phase, very similar to the 14-fold increase that we observed for the mutant H3.2αXba gene here. Because the posttranscriptional regulatory events that play an important role in the regulation of histone mRNA in the cell cycle are unaffected by the α mutation, we postulate that the decrease observed in the up-regulation of the mutant histone gene is due to an alteration in events required for transcription initiation, as was clearly the case for the U1-histone chimeric gene described by Harris et al. (17
). Because a 100% correlation is observed between sequence requirements for α-DNA complex formation in vitro and effects on gene expression in vivo, a role for YY1 in histone gene expression in vivo is strongly implicated.
This highly conserved transcription factor, YY1, has been the subject of extensive study in recent years. Its abundance and ubiquity make it an excellent candidate for an important global role in gene regulation in the metazoan cell. The 100% correlation between our in vitro studies of the histone α DNA-protein interaction (3
; this study) and in vivo studies of the role of the α element in wild-type histone gene expression (3
) is strong evidence that YY1 plays just such a role in the coordinated up-regulation of histone genes at the G1
-S boundary of the cell cycle.
Because the yeast one-hybrid experimental system is designed to circumvent the possibility that an additional factor(s) is required in the histone α-YY1 interaction, our experiments thus far do not rule out this possibility. Here, we have demonstrated that YY1 is necessary for the α-DNA interaction, but it is our hypothesis that other factors may also participate in the histone α-YY1 interaction. In previous experiments, we demonstrated the dependence of the histone α DNA-binding activity on the state of phosphorylation (22
). Phosphorylation on serine/threonine or tyrosine residues results in inhibition of the α DNA-binding activity (22
). Kinases present in crude nuclear extracts are capable of inhibitory phosphorylation at 37°C, and the α DNA-binding activity can be recovered by treatment with phosphatases. We have also shown that cyclin D1-dependent complexes are capable of either direct or indirect inhibitory phosphorylation that results in the loss of the histone α DNA-protein complex in vitro (22
Although there is some evidence that YY1 is a phosphoprotein (35
), only one report implicates phosphorylation in the regulation of YY1 interactions with DNA (2
). In this case, phosphatase treatment of Jurkat T-cell nuclear extracts abolished YY1 interactions with a viral DNA sequence. This result is the converse of our previous results (22
), in which phosphatase treatment restored the histone α DNA-binding activity.
Roles suggested for YY1 in gene expression include activation, repression, and initiation. A role for YY1 in nuclear matrix interactions has also been postulated (13
). The results just described, obtained by phosphatase treatment (YY1 DNA-binding activity in nuclear extracts), are examples of the complexity of YY1 interactions in the metazoan cell. One explanation for the variety of reported effects of YY1 on gene expression could be a requirement for interactions with additional factors in a gene- or pathway-specific manner in different genetic contexts. We are currently examining directly the effect on histone gene expression of YY1 overexpression in vivo. The identification of YY1 as the DNA-binding component of the histone α factor will facilitate our ongoing studies of the role of elements in the coding region of replication-dependent histone genes in the coordinated up-regulation of histone gene expression in the proliferating cell.