Mutations in treacle lead to Treacher Collins syndrome (TCS), an autosomal dominant disorder of cranio-facial development. Treacle associates with upstream binding factor (UBF) to regulate rRNA gene (rDNA) transcription, but the precise mechanisms mediated by treacle remain elusive. Here we show that the central repeated domain of treacle binds with RNA polymerase I (Pol I), while that the treacle C-terminus is involved in rDNA promoter recognition and UBF recruitment. Knockdown of treacle resulted in dispersion of Pol I and UBF away from nucleolus, whereas interactions of treacle with Pol I and rDNA promoter were not disrupted by UBF depletion. These flndings indicate that treacle, but not UBF, is essential for nucleolar recruitment of Pol I transcription complex. Furthermore, C-terminally truncated treacle, mimicking TCS-associated mutations, failed to target to the nucleolus, possibly causing loss-of-function in the mutant treacle. Our observations support that TCS results from haploinsufficiency of treacle. (C) 2009 Elsevier Inc. All rights reserved.