Differential Complex Formation via Paralogs in the Human Sin3 Protein Interaction Network.

Despite the continued analysis of HDAC inhibitors in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The presence of two Sin3 paralogs in humans, SIN3A and SIN3B, may result in a heterogeneous population of Sin3 complexes and contributes to our poor understanding of the functional attributes of these complexes. Here, we profile the interaction networks of SIN3A and SIN3B to gain insight into complex composition and organization. In accordance with existing data, we show that Sin3 paralog identity influences complex composition. Additionally, chemical cross-linking MS identifies domains that mediate interactions between Sin3 proteins and binding partners. The characterization of rare SIN3B proteoforms provides additional evidence for the existence of conserved and divergent elements within human Sin3 proteins. Together, these findings shed light on both the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize.

TNIP2 is a Hub Protein in the NF-κB Network with Both Protein and RNA Mediated Interactions.

The NF-κB family of transcription factors is pivotal in controlling cellular responses to environmental stresses; abnormal nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling features in many autoimmune diseases and cancers. Several components of the NF-κB signaling pathway have been reported to interact with the protein TNIP2 (also known as ABIN2), and TNIP2 can both positively and negatively regulate NF-κB- dependent transcription of target genes. However, the function of TNIP2 remains elusive and the cellular machinery associating with TNIP2 has not been systematically defined. Here we first used a broad MudPIT/Halo Affinity Purification Mass Spectrometry (AP-MS) approach to map the network of proteins associated with the NF-κB transcription factors, and establish TNIP2 as an NF-κB network hub protein. We then combined AP-MS with biochemical approaches in a more focused study of truncated and mutated forms of TNIP2 to map protein associations with distinct regions of TNIP2. NF-κB interacted with the N-terminal region of TNIP2. A central region of TNIP2 interacted with the endosomal sorting complex ESCRT-I via its TSG101 subunit, a protein essential for HIV-1 budding, and a single point mutant in TNIP2 disrupted this interaction. The major gene ontology category for TNIP2 associated proteins was mRNA metabolism, and several of these associations, like KHDRBS1, were lost upon depletion of RNA. Given the major association of TNIP2 with mRNA metabolism proteins, we analyzed the RNA content of affinity purified TNIP2 using RNA-Seq. Surprisingly, a specific limited number of mRNAs was associated with TNIP2. These RNAs were enriched for transcription factor binding, transcription factor cofactor activity, and transcription regulator activity. They included mRNAs of genes in the Sin3A complex, the Mediator complex, JUN, HOXC6, and GATA2. Taken together, our findings suggest an expanded role for TNIP2, establishing a link between TNIP2, cellular transport machinery, and RNA transcript processing.

Proteomic and Genomic Analyses of the Rvb1 and Rvb2 Interaction Network upon Deletion of R2TP Complex Components.

The highly conserved yeast R2TP complex, consisting of Rvb1, Rvb2, Pih1, and Tah1, participates in diverse cellular processes ranging from assembly of protein complexes to apoptosis. Rvb1 and Rvb2 are closely related proteins belonging to the AAA+ superfamily and are essential for cell survival. Although Rvbs have been shown to be associated with various protein complexes including the Ino80 and Swr1chromatin remodeling complexes, we performed a systematic quantitative proteomic analysis of their associated proteins and identified two additional complexes that associate with Rvb1 and Rvb2: the chaperonin-containing T-complex and the 19S regulatory particle of the proteasome complex. We also analyzed Rvb1 and Rvb2 purified from yeast strains devoid of PIH1 and TAH1. These analyses revealed that both Rvb1 and Rvb2 still associated with Hsp90 and were highly enriched with RNA polymerase II complex components. Our analyses also revealed that both Rvb1 and Rvb2 were recruited to the Ino80 and Swr1 chromatin remodeling complexes even in the absence of Pih1 and Tah1 proteins. Using further biochemical analysis, we showed that Rvb1 and Rvb2 directly interacted with Hsp90 as well as with the RNA polymerase II complex. RNA-Seq analysis of the deletion strains compared with the wild-type strains revealed an up-regulation of ribosome biogenesis and ribonucleoprotein complex biogenesis genes, down-regulation of response to abiotic stimulus genes, and down-regulation of response to temperature stimulus genes. A Gene Ontology analysis of the 80 proteins whose protein associations were altered in the PIH1 or TAH1 deletion strains found ribonucleoprotein complex proteins to be the most enriched category. This suggests an important function of the R2TP complex in ribonucleoprotein complex biogenesis at both the proteomic and genomic levels. Finally, these results demonstrate that deletion network analyses can provide novel insights into cellular systems.

Controlling for gene expression changes in transcription factor protein networks.

The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBß, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions.

The cohesin acetyltransferase Eco1 coordinates rDNA replication and transcription.

Eco1 is the acetyltransferase that establishes sister-chromatid cohesion during DNA replication. A budding yeast strain with an eco1 mutation that genocopies Roberts syndrome has reduced ribosomal DNA (rDNA) transcription and a transcriptional signature of starvation. We show that deleting FOB1--a gene that encodes a replication fork-blocking protein specific for the rDNA region--rescues rRNA production and partially rescues transcription genome-wide. Further studies show that deletion of FOB1 corrects the genome-wide replication defects, nucleolar structure, and rDNA segregation that occur in the eco1 mutant. Our study highlights that the presence of cohesin at the rDNA locus has a central role in controlling global DNA replication and gene expression.

Evolutionary plasticity of segmentation clock networks.

The vertebral column is a conserved anatomical structure that defines the vertebrate phylum. The periodic or segmental pattern of the vertebral column is established early in development when the vertebral precursors, the somites, are rhythmically produced from presomitic mesoderm (PSM). This rhythmic activity is controlled by a segmentation clock that is associated with the periodic transcription of cyclic genes in the PSM. Comparison of the mouse, chicken and zebrafish PSM oscillatory transcriptomes revealed networks of 40 to 100 cyclic genes mostly involved in Notch, Wnt and FGF signaling pathways. However, despite this conserved signaling oscillation, the identity of individual cyclic genes mostly differed between the three species, indicating a surprising evolutionary plasticity of the segmentation networks.

Aneuploidy confers quantitative proteome changes and phenotypic variation in budding yeast.

Aneuploidy, referring here to genome contents characterized by abnormal numbers of chromosomes, has been associated with developmental defects, cancer and adaptive evolution in experimental organisms. However, it remains unresolved how aneuploidy impacts gene expression and whether aneuploidy could directly bring about phenotypic variation and improved fitness over that of euploid counterparts. Here we show, using quantitative mass spectrometry-based proteomics and phenotypic profiling, that levels of protein expression in aneuploid yeast strains largely scale with chromosome copy numbers, following the same trend as that observed for the transcriptome, and that aneuploidy confers diverse phenotypes. We designed a novel scheme to generate, through random meiotic segregation, 38 stable and fully isogenic aneuploid yeast strains with distinct karyotypes and genome contents between 1N and 3N without involving any genetic selection. Through quantitative growth assays under various conditions or in the presence of a panel of chemotherapeutic or antifungal drugs, we found that some aneuploid strains grew significantly better than euploid control strains under conditions suboptimal for the latter. These results provide strong evidence that aneuploidy directly affects gene expression at both the transcriptome and proteome levels and can generate significant phenotypic variation that could bring about fitness gains under diverse conditions. Our findings suggest that the fitness ranking between euploid and aneuploid cells is dependent on context and karyotype, providing the basis for the notion that aneuploidy can directly underlie phenotypic evolution and cellular adaptation.

Sex-dimorphic gene expression and ineffective dosage compensation of Z-linked genes in gastrulating chicken embryos.

BACKGROUND: Considerable progress has been made in our understanding of sex determination and dosage compensation mechanisms in model organisms such as C. elegans, Drosophila and M. musculus. Strikingly, the mechanism involved in sex determination and dosage compensation are very different among these three model organisms. Birds present yet another situation where the heterogametic sex is the female. Sex determination is still poorly understood in birds and few key determinants have so far been identified. In contrast to most other species, dosage compensation of bird sex chromosomal genes appears rather ineffective. RESULTS: By comparing microarrays from microdissected primitive streak from single chicken embryos, we identified a large number of genes differentially expressed between male and female embryos at a very early stage (Hamburger and Hamilton stage 4), long before any sexual differentiation occurs. Most of these genes are located on the Z chromosome, which indicates that dosage compensation is ineffective in early chicken embryos. Gene ontology analyses, using an enhanced annotation tool for Affymetrix probesets of the chicken genome developed in our laboratory (called Manteia), show that among these male-biased genes found on the Z chromosome, more than 20 genes play a role in sex differentiation. CONCLUSIONS: These results corroborate previous studies demonstrating the rather inefficient dosage compensation for Z chromosome in birds and show that this sexual dimorphism in gene regulation is observed long before the onset of sexual differentiation. These data also suggest a potential role of non-compensated Z-linked genes in somatic sex differentiation in birds.

Comparison of pattern detection methods in microarray time series of the segmentation clock.

While genome-wide gene expression data are generated at an increasing rate, the repertoire of approaches for pattern discovery in these data is still limited. Identifying subtle patterns of interest in large amounts of data (tens of thousands of profiles) associated with a certain level of noise remains a challenge. A microarray time series was recently generated to study the transcriptional program of the mouse segmentation clock, a biological oscillator associated with the periodic formation of the segments of the body axis. A method related to Fourier analysis, the Lomb-Scargle periodogram, was used to detect periodic profiles in the dataset, leading to the identification of a novel set of cyclic genes associated with the segmentation clock. Here, we applied to the same microarray time series dataset four distinct mathematical methods to identify significant patterns in gene expression profiles. These methods are called: Phase consistency, Address reduction, Cyclohedron test and Stable persistence, and are based on different conceptual frameworks that are either hypothesis- or data-driven. Some of the methods, unlike Fourier transforms, are not dependent on the assumption of periodicity of the pattern of interest. Remarkably, these methods identified blindly the expression profiles of known cyclic genes as the most significant patterns in the dataset. Many candidate genes predicted by more than one approach appeared to be true positive cyclic genes and will be of particular interest for future research. In addition, these methods predicted novel candidate cyclic genes that were consistent with previous biological knowledge and experimental validation in mouse embryos. Our results demonstrate the utility of these novel pattern detection strategies, notably for detection of periodic profiles, and suggest that combining several distinct mathematical approaches to analyze microarray datasets is a valuable strategy for identifying genes that exhibit novel, interesting transcriptional patterns.