The membrane bound receptor tyrosine kinase Her2 is overexpressed in approximately 30% of human breast cancers which correlates with poor prognosis. Her2-induced signaling pathways include MAPK and PI3K/Akt, of which the latter has been shown to be critical for Her2+ breast cancer cell growth and survival. Additionally, the NF-κB pathway has been shown to be activated downstream of Her2 overexpression, however the mechanisms leading to this activation are not currently clear. Using Her2+/ER- breast cancer cells, we show that Her2 activates NF-κB through the canonical pathway which, surprisingly, involves IKKα. Knockdown of IKKα led to a significant decrease in transcription levels of multiple NF-κB-regulated cytokine and chemokine genes. siRNA-mediated knockdown of IKKα resulted in a decrease in cancer cell invasion, but had no effect on cell proliferation. Inhibition of the PI3K/Akt pathway had no effect on NF-κB activation, but significantly inhibited cell proliferation. Our study suggests different roles for the NF-κB and PI3K pathways downstream of Her2, leading to changes in invasion and proliferation of breast cancer cells. Additionally this work indicates the importance of IKKα as a mediator of Her2-induced tumor progression.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

Assembly of the spliceosome onto pre-mRNA is a dynamic process involving the ordered exchange of snRNPs to form the catalytically active spliceosome. These ordered rearrangements have recently been shown to occur cotranscriptionally, while the RNA polymerase is still actively engaged with the chromatin template. We previously demonstrated that the histone acetyltransferase Gcn5 is required for U2 snRNP association with the branchpoint. Here we provide evidence that histone acetylation and deacetylation facilitate proper cotranscriptional association of spliceosomal snRNPs. As with GCN5, mutation or deletion of Gcn5-targeted histone H3 residues leads to synthetic lethality when combined with deletion of the genes encoding the U2 snRNP components Lea1 or Msl1. Gcn5 associates throughout intron-containing genes and, in the absence of the histone deacetylases Hos3 and Hos2, enhanced histone H3 acetylation is observed throughout the body of genes. Deletion of histone deacetylaces also results in persistent association of the U2 snRNP and a severe defect in the association of downstream factors. These studies show that cotranscriptional spliceosome rearrangements are driven by dynamic changes in the acetylation state of histones and provide a model whereby yeast spliceosome assembly is tightly coupled to histone modification.pre-mRNA splicing | Saccharomyces cerevisiae R emoval of noncoding sequences from premessenger RNA is achieved by the activity of a dynamic ribonucleoprotein complex, the spliceosome. As the spliceosomal snRNPs sequentially recognize sequences in the pre-mRNA, the spliceosome undergoes ATP-dependent rearrangement of its RNA and protein components. Recently, it has been recognized that splicing can occur cotranscriptionally, while the RNA polymerase is still actively engaged with the chromatin template.The monomeric units of chromatin, nucleosomes, are comprised of DNA wrapped around the core histones H3, H4, H2A, and H2B. Histones undergo extensive posttranslational modification on their N-terminal tails that affect compaction of DNA and binding of regulatory factors. Although it is known that splicing occurs cotranscriptionally, it is far less well understood how changes in this chromatin template affect the reaction. Analysis of tiling array data has suggested that nucleosomes and, according to several of these studies, specific histone modifications are enriched in exon sequences, suggesting that there may be specific histone "marks" that are associated with splicing signals (1-7). Additionally, proteins that bind to methylated histones (H3K4me3 and H3K36me3) have been shown to facilitate the recruitment of snRNPs to the nascent transcript and influence efficiency of splicing and alternative splicing (8, 9). The chromatin-bound mammalian SWI/SNF complex associates with components of the spliceosome and affects alternative splicing (10, 11). Although these studies suggest a role for histone modifications in mammalian alternative splicing, there has been little analysis of their roles in constitu...

BackgroundActivation of the transcription factor NF-κB by cytokines is rapid, mediated through the activation of the IKK complex with subsequent phosphorylation and degradation of the inhibitory IκB proteins. The IKK complex is comprised of two catalytic subunits, IKKα and IKKβ, and a regulatory protein known as NEMO. Using cells from mice that are genetically deficient in IKKβ or IKKα, or using a kinase inactive mutant of IKKβ, it has been proposed that IKKβ is critical for TNF-induced IκB phosphorylation/degradation through the canonical pathway while IKKα has been shown to be involved in the non-canonical pathway for NF-κB activation. These conclusions have led to a focus on development of IKKβ inhibitors for potential use in inflammatory disorders and cancer.MethodologyAnalysis of NF-κB activation in response to TNF in MEFs reveals that IKKβ is essential for efficient phosphorylation and subsequent degradation of IκBα, yet IKKα contributes to the NF-κB activation response in these cells as measured via DNA binding assays. In HeLa cells, both IKKα and IKKβ contribute to IκBα phosphorylation and NF-κB activation. A kinase inactive mutant of IKKβ, which has been used as evidence for the critical importance of IKKβ in TNF-induced signaling, blocks activation of NF-κB induced by IKKα, even in cells that are deficient in IKKβ.ConclusionsThese results demonstrate the importance of IKKα in canonical NF-κB activation, downstream of cytokine treatment of cells. The experiments suggest that IKKα will be a therapeutic target in inflammatory disorders.

The discovery that many intron-containing genes can be cotranscriptionally spliced has led to an increased understanding of how splicing and transcription are intricately intertwined. Cotranscriptional splicing has been demonstrated in a number of different organisms and has been shown to play roles in coordinating both constitutive and alternative splicing. The nature of cotranscriptional splicing suggests that changes in transcription can dramatically affect splicing, and new evidence suggests that splicing can, in turn, influence transcription. In this chapter, we discuss the mechanisms and consequences of cotranscriptional splicing and introduce some of the tools used to measure this process.