Transcription of the genes for the human.histone proteins H4, H3,.H2A, H2B, and Hi is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified.Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp9l-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.Cell proliferation is initiated by a sequential series of growth factor-dependent events that activate cyclin-dependent kinases (CDKs), which mediate the onset of the cell cycle and progression into S phase (1, 2). There are two functional components to the G1/S phase transition point during the cell cycle. First, initiation of DNA replication necessitates adjustments in the activities of enzymes involved in nucleotide metabolism and DNA synthesis. Second, progression into early S phase requires induction of histone gene expression, because de novo synthesis of histone nucleosomal proteins is essential for the ordered packaging of newly replicated DNA into chromatin (3).Many genes that are functionally linked to cell cycle progression appear to be regulated by the E2F class of transcription factors, including genes encoding enzymes and regulatory factors involved in DNA synthesis (e.g., refs. 4-13). E2F factors are heterodimers composed of different pairs of E2F/DP proteins that are capable of forming higher order complexes with multiple cell cycle regulators including retinoblastoma protein (pRB)-related proteins (pRB/plO5, pRB-