Craniofacial anomalies are common birth defects in the U.S., with orofacial clefts alone affecting 6,800 infants annually. In mice, mutations in more than 600 genes, many encoding transcriptional regulators, are associated with craniofacial anomalies. CREB-binding protein (CBP) and the closely related p300 (CBP/p300 collectively) are transcriptional coactivators that interact physically or functionally with ~15% of the estimated 2,000 mammalian transcriptional regulatory proteins. Haploinsufficiency (one null allele) of CBP, and to a lesser degree p300, causes Rubinstein-Taybi Syndrome (RTS), characterized by craniofacial anomalies, broad thumbs and toes, and mental and growth retardation. Several unique domains in CBP/p300 bind to the activation domains of numerous transcription factors, but the insufficient functions of mutant CBP (and p300) that lead to RTS are unclear. Mice deficient for one of these domains in CBP, CH1, have craniofacial anomalies reminiscent of CBP haploinsufficient mice, the latter being a model for RTS. The CH1 domain is therefore one of the essential domains of CBP needed for craniofacial development. The broad, long-term goal is to address how the CH1 domain mediates normal craniofacial development. The objective of the current proposal is to determine the role of the CBP/p300 CH1 domain in mediating craniofacial-critical transcription. The central hypothesis is that the CH1 domain is a major """"""""hub"""""""" that connects transcription factors and target genes in a regulatory network that is essential for craniofacial development. Discovery of such networks will help identify candidate genes and pathways involved in nonsyndromic orofacial clefting, for example, where the underlying multigenic mechanisms are poorly understood.
Specific aim 1 is to identify the known craniofacial-determining transcription factors that require the CH1 domain for transactivation function and CBP/p300-interaction using mouse cells in transient transfection assays.
Specific aim 2 is to use CH1 domain-deficient primary cells to discover the craniofacial-determining genes that require the CH1 domain by measuring endogenous gene expression using microarrays and real-time RT-PCR, and CBP/p300 recruitment using chromatin immunoprecipitation. The significance of a CH1/craniofacial network will be to illuminate the signaling and transcriptional mechanisms that regulate craniofacial gene transcription, and to reveal genetic interactions that underlie craniofacial development. Moreover, demonstration of a highly connected craniofacial network would suggest that the CH1 domain, and more generally CBP and p300, are likely to act as genetic modifiers in multigenic nonsyndromic orofacial anomalies. In the long-term, the network will help identify genes and pathways involved in human craniofacial anomalies and lead to improved diagnosis and treatment.
Many instances of craniofacial anomalies, such as cleft lip and palate, are associated with multiple gene abnormalities, and this complexity hampers understanding the fundamental causes. The creation of a gene interaction network will help identify similarly regulated genes that are important for the formation of the skull and face. Such knowledge will lead to a new view of this complicated developmental process, and possibly to improved diagnosis and treatment of skull and facial anomalies.
