Mutations in IKBKAP, encoding a subunit of Elongator, cause familial dysautonomia (FD), a severe neurodevelopmental disease with complex clinical characteristics. Elongator was previously linked not only with transcriptional elongation and histone acetylation but also with other cellular processes. Here, we used RNA interference (RNAi) and fibroblasts from FD patients to identify Elongator target genes and study the role of Elongator in transcription. Strikingly, whereas Elongator is recruited to both target and nontarget genes, only target genes display histone H3 hypoacetylation and progressively lower RNAPII density through the coding region in FD cells. Interestingly, several target genes encode proteins implicated in cell motility. Indeed, characterization of IKAP/hELP1 RNAi cells, FD fibroblasts, and neuronal cell-derived cells uncovered defects in this cellular function upon Elongator depletion. These results indicate that defects in Elongator function affect transcriptional elongation of several genes and that the ensuing cell motility deficiencies may underlie the neuropathology of FD patients.

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

Human Elongator complex was purified to virtual homogeneity from HeLa cell extracts. The purified factor can exist in two forms: a six-subunit complex, holo-Elongator, which has histone acetyltransferase activity directed against histone H3 and H4, and a three-subunit core form, which does not have histone acetyltransferase activity despite containing the catalytic Elp3 subunit. Elongator is a component of early elongation complexes formed in HeLa nuclear extracts and can interact directly with RNA polymerase II in solution. Several human homologues of the yeast Elongator subunits were identified as subunits of the human Elongator complex, including StIP1 (STAT-interacting protein 1) and IKAP (IKK complex-associated protein). Mutations in IKAP can result in the severe human disorder familial dysautonomia, raising the possibility that this disease might be due to compromised Elongator function and therefore could be a transcription disorder.

The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5 and 3 parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitopespecific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.TFIIB plays a crucial role in preinitiation complex (PIC) assembly, providing a bridge between promoter-bound TFIID and RNA polymerase II (pol II)-TFIIF (reviewed in references 13 and 26). TFIIB is composed of two domains, a core domain with two alpha-helical direct repeats and an N-terminal region that has been shown by nuclear magnetic resonance spectrometry to contain a zinc ribbon motif (42). The structure of the core domain of TFIIB (TFIIBc), both as a free entity and in a complex with TATA-binding protein (TBP) and a TATA element, has been elucidated (2, 24). TFIIB makes non-sequencespecific contacts with DNA both upstream and downstream of the TATA box in this structure. In addition, TFIIB can make sequence-specific DNA contact with an element immediately upstream of the TATA box (18,33,39). This TFIIB recognition element (BRE) has been reported to be present in a subset of eukaryotic and archaeal promoters. At least one function of this element is to modulate the strength of the core promoter (7,18,33). Another function is in the determination of the orientation of the TFIIB-TBP-TATA complex that would project the zinc ribbon of TFIIB towards the transcription initiation site (4,22,39).Present data concerning the structure of TFIIB in a complex with TBP at a promoter have been limited to TFIIBc, which lacks both the zinc ribbon and the highly conserved spacer region. Several studies have reported that the N-and C-terminal regions of TFIIB are engaged in an intramolecular interaction (15,16,36,41). Indeed, conformation plays a critical role in the response of TFIIB to transcriptional activators (15,37,41). Thus, present structural models do not help us to understand the role of TFIIB conformation in the assembly of the PIC.TFIIB plays a central role in transcription start site selection (3, 14, 30). In fact, yeast TFIIB was cloned as the result of a genetic screen that generated a yeast mutant with an altered transcription start site phenotype (30). In addition, the unusual start site selection mechanism (scanner) specifi...