Cell proliferation and differentiation are regulated by growth regulatory factors such as transforming growth factor-beta (TGF-beta) and the liphophilic hormone vitamin D. TGF-beta causes activation of SMAD proteins acting as coactivators or transcription factors in the nucleus. Vitamin D controls transcription of target genes through the vitamin D receptor (VDR). Smad3, one of the SMAD proteins downstream in the TGF-beta signaling pathway, was found in mammalian cells to act as a coactivator specific for ligand-induced transactivation of VDR by forming a complex with a member of the steroid receptor coactivator-1 protein family in the nucleus. Thus, Smad3 may mediate cross-talk between vitamin D and TGF-beta signaling pathways.

One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identi®ed a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor a (hERa) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hERa A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogenbound hERa in MCF7 cells and in partially puri®ed complexes associated with hERa from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hERa and TIF2 in the nucleus. The presence of p72/ p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hERa. These ®nd-ings indicate that p72/p68 acts as an ER subtypeselective coactivator through ERa AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.

Cited2 is a cAMP-responsive element-binding protein (CBP)͞p300 interacting transcriptional modulator and a proposed negative regulator for hypoxia-inducible factor (HIF)-1␣ through its competitive binding with HIF-1␣ to CBP͞p300. Disruption of the gene encoding Cited2 is embryonic lethal because of defects in the development of heart and neural tube. Morphological and Doppler echocardiographic analyses of Cited2 ؊/؊ embryos reveal severe cardiovascular abnormalities, including pulmonic arterial stenosis and ventricular septal defects accompanied by high peak outflow velocities, features of the human congenital cardiac defect termed tetralogy of Fallot. The mRNA levels of several HIF-1␣-responsive genes, such as vascular endothelial growth factor (VEGF), Glut1, and phosphoglycerate kinase 1, increased in the Cited2 ؊/؊ hearts. The increase of VEGF levels is significant, because defects in the Cited2 ؊/؊ embryos closely resemble the major defects observed in the VEGF transgenic embryos. Finally, compared with wild-type, cultured fibroblasts from Cited2 ؊/؊ embryos demonstrate an enhanced expression of HIF-1␣-responsive genes under hypoxic conditions. These observations suggest that functional loss of Cited2 is responsible for defects in heart and neural tube development, in part because of the modulation of HIF-1 transcriptional activities in the absence of Cited2. These findings demonstrate that Cited2 is an indispensable regulatory gene during prenatal development.

Disrupted ERK1/2 (MAPK3/MAPK1) MAPK signaling has been associated with several developmental syndromes in humans; however, mutations in ERK1 or ERK2 have not been described. We demonstrate haplo-insufficient ERK2 expression in patients with a novel Ϸ1 Mb micro-deletion in distal 22q11.2, a region that includes ERK2. These patients exhibit conotruncal and craniofacial anomalies that arise from perturbation of neural crest development and exhibit defects comparable to the DiGeorge syndrome spectrum. Remarkably, these defects are replicated in mice by conditional inactivation of ERK2 in the developing neural crest. Inactivation of upstream elements of the ERK cascade (B-Raf and C-Raf, MEK1and MEK2) or a downstream effector, the transcription factor serum response factor resulted in analogous developmental defects. Our findings demonstrate that mammalian neural crest development is critically dependent on a RAF/MEK/ERK/ serum response factor signaling pathway and suggest that the craniofacial and cardiac outflow tract defects observed in patients with a distal 22q11.2 micro-deletion are explained by deficiencies in neural crest autonomous ERK2 signaling.22q11 microdeletion ͉ human syndromes ͉ MAP kinase