Faculty Bibliography
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110
Whole-genome screen identifies diverse pathways that negatively regulate ciliogenesis
We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a previously undisclosed, extensive network of genes linking integrin signaling and cellular adhesion to the extracellular matrix with inhibition of ciliation in both normal and cancer cells. Surprisingly, a cohort of genes encoding extracellular matrix (ECM) proteins was also identified. We characterized several ciliation inhibitory genes and showed that their silencing was accompanied by altered cytoskeletal organization and induction of ciliation, which restricts cell growth and migration in normal and breast cancer cells. Conversely, supplying an integrin ligand, vitronectin, to the ECM rescued the enhanced ciliation observed upon silencing this gene. Aberrant ciliation could also be suppressed through hyper-activation of the YAP/TAZ pathway, indicating a potential mechanistic basis for our findings. Our findings suggest an unanticipated reciprocal relationship between ciliation and cellular adhesion to the extracellular matrix and provide a resource that could vastly expand our understanding of controls involving "outside-in" and "inside-out" signaling that restrain cilium assembly.
PMID: 33206585
ISSN: 1939-4586
CID: 4672802
LncRNA RP11-19E11 is an E2F1 target required for proliferation and survival of basal breast cancer
Long non-coding RNAs (lncRNAs) play key roles in the regulation of breast cancer initiation and progression. LncRNAs are differentially expressed in breast cancer subtypes. Basal-like breast cancers are generally poorly differentiated tumors, are enriched in embryonic stem cell signatures, lack expression of estrogen receptor, progesterone receptor, and HER2 (triple-negative breast cancer), and show activation of proliferation-associated factors. We hypothesized that lncRNAs are key regulators of basal breast cancers. Using The Cancer Genome Atlas, we identified lncRNAs that are overexpressed in basal tumors compared to other breast cancer subtypes and expressed in at least 10% of patients. Remarkably, we identified lncRNAs whose expression correlated with patient prognosis. We then evaluated the function of a subset of lncRNA candidates in the oncogenic process in vitro. Here, we report the identification and characterization of the chromatin-associated lncRNA, RP11-19E11.1, which is upregulated in 40% of basal primary breast cancers. Gene set enrichment analysis in primary tumors and in cell lines uncovered a correlation between RP11-19E11.1 expression level and the E2F oncogenic pathway. We show that this lncRNA is chromatin-associated and an E2F1 target, and its expression is necessary for cancer cell proliferation and survival. Finally, we used lncRNA expression levels as a tool for drug discovery in vitro, identifying protein kinase C (PKC) as a potential therapeutic target for a subset of basal-like breast cancers. Our findings suggest that lncRNA overexpression is clinically relevant. Understanding deregulated lncRNA expression in basal-like breast cancer may lead to potential prognostic and therapeutic applications.
PMCID:6944689
PMID: 31934613
ISSN: 2374-4677
CID: 4264352
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2019:116(29):14583-14592.DOI: 10.1073/pnas.1904324116
Paf1C regulates RNA polymerase II progression by modulating elongation rate
Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions.
PMID: 31249142
ISSN: 1091-6490
CID: 3963942
Pax3 cooperates with Ldb1 to direct local chromosome architecture during myogenic lineage specification
Chromatin looping allows enhancer-bound regulatory factors to influence transcription. Large domains, referred to as topologically associated domains, participate in genome organization. However, the mechanisms underlining interactions within these domains, which control gene expression, are not fully understood. Here we report that activation of embryonic myogenesis is associated with establishment of long-range chromatin interactions centered on Pax3-bound loci. Using mass spectrometry and genomic studies, we identify the ubiquitously expressed LIM-domain binding protein 1 (Ldb1) as the mediator of looping interactions at a subset of Pax3 binding sites. Ldb1 is recruited to Pax3-bound elements independently of CTCF-Cohesin, and is necessary for efficient deposition of H3K4me1 at these sites and chromatin looping. When Ldb1 is deleted in Pax3-expressing cells in vivo, specification of migratory myogenic progenitors is severely impaired. These results highlight Ldb1 requirement for Pax3 myogenic activity and demonstrate how transcription factors can promote formation of sub-topologically associated domain interactions involved in lineage specification.
PMID: 31127120
ISSN: 2041-1723
CID: 3923022
Time-dependent Pax3-mediated chromatin remodeling and cooperation with Six4 and Tead2 specify the skeletal myogenic lineage in developing mesoderm
The transcriptional mechanisms driving lineage specification during development are still largely unknown, as the interplay of multiple transcription factors makes it difficult to dissect these molecular events. Using a cell-based differentiation platform to probe transcription function, we investigated the role of the key paraxial mesoderm and skeletal myogenic commitment factors-mesogenin 1 (Msgn1), T-box 6 (Tbx6), forkhead box C1 (Foxc1), paired box 3 (Pax3), Paraxis, mesenchyme homeobox 1 (Meox1), sine oculis-related homeobox 1 (Six1), and myogenic factor 5 (Myf5)-in paraxial mesoderm and skeletal myogenesis. From this study, we define a genetic hierarchy, with Pax3 emerging as the gatekeeper between the presomitic mesoderm and the myogenic lineage. By assaying chromatin accessibility, genomic binding and transcription profiling in mesodermal cells from mouse and human Pax3-induced embryonic stem cells and Pax3-null embryonic day (E)9.5 mouse embryos, we identified conserved Pax3 functions in the activation of the skeletal myogenic lineage through modulation of Hedgehog, Notch, and bone morphogenetic protein (BMP) signaling pathways. In addition, we demonstrate that Pax3 molecular function involves chromatin remodeling of its bound elements through an increase in chromatin accessibility and cooperation with sine oculis-related homeobox 4 (Six4) and TEA domain family member 2 (Tead2) factors. To our knowledge, these data provide the first integrated analysis of Pax3 function, demonstrating its ability to remodel chromatin in mesodermal cells from developing embryos and proving a mechanistic footing for the transcriptional hierarchy driving myogenesis.
PMID: 30807574
ISSN: 1545-7885
CID: 3698352
A distal centriolar protein network controls organelle maturation and asymmetry
A long-standing mystery in the centrosome field pertains to the origin of asymmetry within the organelle. The removal of daughter centriole-specific/enriched proteins (DCPs) and acquisition of distal appendages on the future mother centriole are two important steps in the generation of asymmetry. We find that DCPs are recruited sequentially, and their removal is abolished in cells lacking Talpid3 or C2CD3. We show that removal of certain DCPs constitutes another level of control for distal appendage (DA) assembly. Remarkably, we also find that Talpid3 forms a distal centriolar multi-functional hub that coordinates the removal of specific DCPs, DA assembly, and recruitment of ciliary vesicles through distinct regions mutated in ciliopathies. Finally, we show that Talpid3, C2CD3, and OFD1 differentially regulate the assembly of sub-distal appendages, the CEP350/FOP/CEP19 module, centriolar satellites, and actin networks. Our work extends the spatial and functional understanding of proteins that control organelle maturation and asymmetry, ciliogenesis, and human disease.
PMID: 30258116
ISSN: 2041-1723
CID: 3315762
The regulation of cilium assembly and disassembly in development and disease
The primary cilium is an antenna-like organelle assembled on most types of quiescent and differentiated mammalian cells. This immotile structure is essential for interpreting extracellular signals that regulate growth, development and homeostasis. As such, ciliary defects produce a spectrum of human diseases, termed ciliopathies, and deregulation of this important organelle also plays key roles during tumor formation and progression. Recent studies have begun to clarify the key mechanisms that regulate ciliary assembly and disassembly in both normal and tumor cells, highlighting new possibilities for therapeutic intervention. Here, we review these exciting new findings, discussing the molecular factors involved in cilium formation and removal, the intrinsic and extrinsic control of cilium assembly and disassembly, and the relevance of these processes to mammalian cell growth and disease.
PMID: 30224385
ISSN: 1477-9129
CID: 3301082
Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018
Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.
PMCID:5864239
PMID: 29362479
ISSN: 1476-5403
CID: 2929282
Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy
Autophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk.
PMCID:5526835
PMID: 28712572
ISSN: 1879-0445
CID: 2640362
Gating Ciliary Transport
Cilia lack the ability to synthesize proteins and thus require dynamic transport. Reporting in this issue of Developmental Cell, Kanie et al. (2017) shed light on the mechanism of transport by implicating CEP19, which is associated with an autosomal-recessive obesity syndrome when mutated, in the triggering of intraflagellar transport.
PMID: 28697332
ISSN: 1878-1551
CID: 2630422
