Faculty Bibliography

Cilia are evolutionarily conserved, membrane-bound, microtubular projections emanating from the cell surface. They are assembled on virtually all cell types in the human body, with very few exceptions, and several recent reviews have covered the topic in great detail [1-3]. The cilium is assembled from mature (mother) centrioles or basal bodies, which serve to nucleate growth of axonemes that give rise to two structurally distinct variants, motile and nonmotile cilia. Whereas motile cilia are typically found in large bundles and beat synchronously to generate fluid flow, primary cilia (with the exception of those found at the embryonic node) are generally immotile and are found as solitary organelles [3,4]. Remarkably, until recently, the primary cilium was considered a vestigial organelle without apparent biological function. However, research over the past decade has established that the primary cilium is capable of transducing essential signaling information from the extracellular milieu [1,5]. Defects in the cilium, and the structure from which it arises, the basal body, have been shown to cause a spectrum of diseases, ranging from developmental defects to obesity, diabetes, and cancer [6]. Many of these diseases, or ciliopathies, are manifested as genetic syndromes, such as Joubert syndrome, Bardet-Biedel (BBS), Meckel-Gruber (MKS), and Nephronophthisis (NPHP) [6], illustrating the importance of understanding cilium structure and function and the mechanisms required for its assembly. This review focuses primarily on recent advances in our understanding of the regulatory controls governing the assembly and maintenance of the primary cilium.

Cilia are hair-like protrusions found at the surface of most eukaryotic cells. They can be divided into two types, motile and non-motile. Motile cilia are found in a restricted number of cell types, are generally present in large numbers, and beat in a coordinated fashion to generate fluid flow or locomotion. Non-motile or primary cilia, on the other hand, are detected in many different cell types, appear once per cell, and primarily function to transmit signals from the extracellular milieu to the cell nucleus. Defects in cilia formation, function, or maintenance are known to cause a bewildering set of human diseases, or ciliopathies, typified by retinal degeneration, renal failure and cystic kidneys, obesity, liver dysfunction, and neurological disorders. A common denominator between motile and primary cilia is their structural similarity, as both types of cilia are composed of an axoneme, the ciliary backbone that is made up of microtubules emanating from a mother centriole/basal body anchored to the cell membrane, surrounded by a ciliary membrane continuous with the plasma membrane. This structural similarity is indicative of a universal mechanism of cilia assembly involving a common set of molecular players and a sophisticated, highly regulated series of molecular events. In this review, we will mainly focus on recent advances in our understanding of the regulatory mechanisms underlying cilia assembly, with special attention paid to the centriolar protein, CP110, its interacting partner Cep290, and the various downstream molecular players and events leading to intraflagellar transport (IFT), a process that mediates the bidirectional movement of protein cargos along the axoneme and that is essential for cilia formation and maintenance.

Centrosome duplication is critical for cell division, and genome instability can result if duplication is not restricted to a single round per cell cycle. Centrosome duplication is controlled in part by CP110, a centriolar protein that positively regulates centriole duplication while restricting centriole elongation and ciliogenesis. Maintenance of normal CP110 levels is essential, as excessive CP110 drives centrosome over-duplication and suppresses ciliogenesis, whereas its depletion inhibits centriole amplification and leads to highly elongated centrioles and aberrant assembly of cilia in growing cells. CP110 levels are tightly controlled, partly through ubiquitination by the ubiquitin ligase complex SCF(cyclin F) during G2 and M phases of the cell cycle. Here, using human cells, we report a new mechanism for the regulation of centrosome duplication that requires USP33, a deubiquitinating enzyme that is able to regulate CP110 levels. USP33 interacts with CP110 and localizes to centrioles primarily in S and G2/M phases, the periods during which centrioles duplicate and elongate. USP33 potently and specifically deubiquitinates CP110, but not other cyclin-F substrates. USP33 activity antagonizes SCF(cyclin F)-mediated ubiquitination and promotes the generation of supernumerary centriolar foci, whereas ablation of USP33 destabilizes CP110 and thereby inhibits centrosome amplification and mitotic defects. To our knowledge, we have identified the first centriolar deubiquitinating enzyme whose expression regulates centrosome homeostasis by countering cyclin-F-mediated destruction of a key substrate. Our results point towards potential therapeutic strategies for inhibiting tumorigenesis associated with centrosome amplification.

Centrosome duplication is a pivotal process required for cell division. In order to avoid genome instability, the duplication of centrosomes must be restricted to once per cell cycle. Different mechanisms that control centrosome duplication impinge on the regulation of CP110, an essential component of the centriole duplication process. Excessive CP110 drives centrosome over-duplication while loss of CP110 inhibits centrosome amplification. CP110 levels are controlled through ubiquitin mediated proteolysis by the SCF(cyclin F) during G2 and M phase of the cell cycle. From published mass spectrometry data, we have identified a de-ubiquitylating enzyme (DUB) as a CP110-interacting protein. We report a new mechanism to regulate centrosome duplication that entails DUB-dependent regulation of CP110 levels. Ubiquitylation and deubiquitylation cycles control CP110 stability and centrosome duplication. We further observe that the levels of this DUB and CP110 are markedly elevated in pancreatic ductal adenocarcinoma (PDAC), suggesting a rationale for inhibiting tumors associated with centrosome amplification. These studies have identified one of the first centriolar deubiquitinating enzymes whose expression regulates centrosome homeostasis by countering cyclin F-mediated destruction of a key centrosomal substrate

To identify the compendium of distal regulatory elements that govern myogenic differentiation, we generated chromatin state maps based on histone modifications and recruitment of factors that typify enhancers in myoblasts and myotubes. We found a striking concordance between the locations of these newly defined enhancers, MyoD1-binding events, and noncoding RNA transcripts. These enhancers recruit several sequence-specific transcription factors in a spatially constrained manner around MyoD1-binding sites. Remarkably, MyoD1-null myoblasts show a wholesale loss of recruitment of these factors as well as diminished monomethylation of H3K4 (H3K4me1) and acetylation of H3K27 (H3K27ac) and reduced recruitment of Set7, an H3K4 monomethylase. Surprisingly, we found that H3K4me1, but not H3K27ac, could be restored by re-expression of MyoD1 in MyoD1(-/-) myoblasts, although re-expression of this factor in MyoD1-null myotubes restored both histone modifications. Our studies identified a role for MyoD1 in condition-specific enhancer assembly through recruitment of transcription factors and histone-modifying enzymes that shape muscle differentiation.

Malignant transformation of human prostatic epithelium is associated with loss of androgen receptor (AR) in the surrounding stroma. However, the function and mechanisms of AR signaling in prostate cancer (PCa) stroma remain elusive. Here we report that androgen and its receptor inhibit proliferation of prostate stromal cells through transcriptional suppression of cyclin B1 by Proteomics Pathway Array Analysis (PPAA), confirmed at mRNA and protein levels using AR negative or positive primary prostate stromal cells. Furthermore, AR showed a negative correlation with cyclin B1 expression in stroma of human PCa samples in vivo. Mechanistically, we identify cyclin B1 as a bona fide AR target gene in prostate stromal cells. The negative regulation of cyclin B1 by AR is mediated through switching between E2F1 and E2F4 on the promoter of cyclin B1. E2F1 binds to cyclin B1 promoter and maintains its expression and subsequent cell cycle progression in AR negative stromal cells or AR positive stromal cells when androgens are depleted. Upon stimulation with androgen in AR positive stromal cells, E2F1 is displaced from the binding site by AR and replaced with E2F4, leading to recruitment of the SMRT/HDAC3 co-repressor complex and repression of cyclin B1 at chromatin level. The switch between E2F1 and E2F4 at the E2F binding site of the cyclin B1 promoter coincides with an androgen-dependent interaction between AR and E2F1 as well as cytoplasmic to nuclear translocation of E2F4. Thus, we identified a novel mechanism for E2F factors in the regulation of cell cycle gene expression and cell cycle progression under the control of AR signaling.

Here we identify Neuralized homologue 4 (Neurl4) as a protein that interacts with CP110, a centrosomal protein that regulates centrosome duplication. Neurl4 uses a Neuralized homology repeat to preferentially localize to procentrioles and daughter centrioles. Neurl4 depletion results in ectopic microtubular organizing centres (MTOCs), leading to accumulation of CP110 and recruitment of a cohort of centrosomal proteins. We show that these ectopic MTOCs persist through mitosis and assemble aberrant mitotic spindles. Interestingly, Neurl4 promotes ubiquitylation of CP110, thereby destabilizing this protein. Our results indicate that Neurl4 counteracts accumulation of CP110, thereby maintaining normal centriolar homeostasis and preventing formation of ectopic MTOCs.

Numerous algorithms have been developed to analyze ChIP-Seq data. However, the complexity of analyzing diverse patterns of ChIP-Seq signals, especially for epigenetic marks, still calls for the development of new algorithms and objective comparisons of existing methods. We developed Qeseq, an algorithm to detect regions of increased ChIP read density relative to background. Qeseq employs critical novel elements, such as iterative recalibration and neighbor joining of reads to identify enriched regions of any length. To objectively assess its performance relative to other 14 ChIP-Seq peak finders, we designed a novel protocol based on Validation Discriminant Analysis (VDA) to optimally select validation sites and generated two validation datasets, which are the most comprehensive to date for algorithmic benchmarking of key epigenetic marks. In addition, we systematically explored a total of 315 diverse parameter configurations from these algorithms and found that typically optimal parameters in one dataset do not generalize to other datasets. Nevertheless, default parameters show the most stable performance, suggesting that they should be used. This study also provides a reproducible and generalizable methodology for unbiased comparative analysis of high-throughput sequencing tools that can facilitate future algorithmic development.

Ubiquitylation of H2B on lysine 120 (H2Bub) is associated with active transcriptional elongation. H2Bub has been implicated in histone cross talk and is generally regarded to be a prerequisite for trimethylation of histone 3 lysine 4 (H3K4me3) and H3K79 in both yeast and mammalian cells. We performed a genome-wide analysis of epigenetic marks during muscle differentiation, and strikingly, we observed a near-complete loss of H2Bub in the differentiated state. We examined the basis for global loss of this mark and found that the H2B ubiquitin E3 ligase, RNF20, was depleted from chromatin in differentiated myotubes, indicating that recruitment of this protein to genes substantially decreases upon differentiation. Remarkably, during the course of myogenic differentiation, we observed retention and acquisition of H3K4 trimethylation on a large number of genes in the absence of detectable H2Bub. The Set1 H3K4 trimethylase complex was efficiently recruited to a subset of genes in myotubes in the absence of detectable H2Bub, accounting in part for H3K4 trimethylation in myotubes. Our studies suggest that H3K4me3 deposition in the absence of detectable H2Bub in myotubes is mediated via Set1 and, perhaps, MLL complexes, whose recruitment does not require H2Bub. Thus, muscle cells represent a novel setting in which to explore mechanisms that regulate histone cross talk.