Amelia W. Hall

Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan

Large, long‐lived species experience more lifetime cell divisions and hence a greater risk of spontaneous tumor formation than smaller, short‐lived species. Large, long‐lived species are thus expected to evolve more elaborate tumor suppressor systems. In previous work, we showed that telomerase activity coevolves with body mass, but not lifespan, in rodents: telomerase activity is repressed in the somatic tissues of large rodent species but remains active in small ones. Without telomerase activity, the telomeres of replicating cells become progressively shorter until, at some critical length, cells stop dividing. Our findings therefore suggested that repression of telomerase activity mitigates the increased risk of cancer in larger‐bodied species but not necessarily longer‐lived ones. These findings imply that other tumor suppressor mechanisms must mitigate increased cancer risk in long‐lived species. Here, we examined the proliferation of fibroblasts from 15 rodent species with diverse body sizes and lifespans. We show that, consistent with repressed telomerase activity, fibroblasts from large rodents undergo replicative senescence accompanied by telomere shortening and overexpression of p16Ink4a and p21Cip1/Waf1 cycline‐dependent kinase inhibitors. Interestingly, small rodents with different lifespans show a striking difference: cells from small shorter‐lived species display continuous rapid proliferation, whereas cells from small long‐lived species display continuous slow proliferation. We hypothesize that cells of small long‐lived rodents, lacking replicative senescence, have evolved alternative tumor‐suppressor mechanisms that prevent inappropriate cell division in vivo and slow cell growth in vitro. Thus, large‐bodied species and small but long‐lived species have evolved distinct tumor suppressor mechanisms.

EF hands at the N-lobe of calmodulin are required for both SK channel gating and stable SK-calmodulin interaction

Small conductance calcium-activated potassium (SK) channels respond to intracellular Ca2+ via constitutively associated calmodulin (CaM). Previous studies have proposed a modular design for the interaction between CaM and SK channels. The C-lobe and the linker of CaM are thought to regulate the constitutive binding, whereas the N-lobe binds Ca2+ and gates SK channels. However, we found that coexpression of mutant CaM (E/Q) where the N-lobe has only one functional EF hand leads to rapid rundown of SK channel activity, which can be recovered with exogenously applied wild-type (WT), but not mutant, CaM. Our results suggest that the mutation at the N-lobe EF hand disrupts the stable interaction between CaM and SK channel subunits, such that mutant CaM dissociates from the channel complex when the inside of the membrane is exposed to CaM-free solution. The disruption of the stable interaction does not directly result from the loss of Ca2+-binding capacity because SK channels and WT CaM can stably interact in the absence of Ca2+. These findings question a previous conclusion that CaM where the N-lobe has only one functional EF hand can stably support the gating of SK channels. They cannot be explained by the current model of modular interaction between CaM and SK channels, and they imply a role for N-lobe EF hand residues in binding to the channel subunits. Additionally, we found that a potent enhancer for SK channels, 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309), enables the recovery of channel activity with CaM (E/Q), suggesting that NS309 stabilizes the interaction between CaM and SK channels. CaM (E/Q) can regulate Ca2+-dependent gating of SK channels in the presence of NS309, but with a lower apparent Ca2+ affinity than WT CaM.

Simultaneous SNP identification and assessment of allele-specific bias from ChIP-seq data.

BackgroundSingle nucleotide polymorphisms (SNPs) have been associated with many aspects of human development and disease, and many non-coding SNPs associated with disease risk are presumed to affect gene regulation. We have previously shown that SNPs within transcription factor binding sites can affect transcription factor binding in an allele-specific and heritable manner. However, such analysis has relied on prior whole-genome genotypes provided by large external projects such as HapMap and the 1000 Genomes Project. This requirement limits the study of allele-specific effects of SNPs in primary patient samples from diseases of interest, where complete genotypes are not readily available.ResultsIn this study, we show that we are able to identify SNPs de novo and accurately from ChIP-seq data generated in the ENCODE Project. Our de novo identified SNPs from ChIP-seq data are highly concordant with published genotypes. Independent experimental verification of more than 100 sites estimates our false discovery rate at less than 5%. Analysis of transcription factor binding at de novo identified SNPs revealed widespread heritable allele-specific binding, confirming previous observations. SNPs identified from ChIP-seq datasets were significantly enriched for disease-associated variants, and we identified dozens of allele-specific binding events in non-coding regions that could distinguish between disease and normal haplotypes.ConclusionsOur approach combines SNP discovery, genotyping and allele-specific analysis, but is selectively focused on functional regulatory elements occupied by transcription factors or epigenetic marks, and will therefore be valuable for identifying the functional regulatory consequences of non-coding SNPs in primary disease samples.

An eQTL analysis of the human glioblastoma multiforme genome

In this paper we use eQTL mapping to identify associations between gene dysregulation and single nucleotide polymorphism (SNP) genotypes in glioblastoma multiforme (GBM). A set of 532,954 SNPs was evaluated as predictors of the expression levels of 22,279 expression probes. We identified SNPs associated with fold change in expression level rather than raw expression levels in the tumor. Following adjustment for false discovery rate, the complete set of probes yielded 9257 significant associations (p<0.05). We found 18 eQTLs that were missense mutations. Many of the eQTLs in the non-coding regions of a gene, or linked to nearby genes, had large numbers of significant associations (e.g. 321 for RNASE3, 101 for BNC2). Functional enrichment analysis revealed that the expression probes in significant associations were involved in signal transduction, transcription regulation, membrane function, and cell cycle regulation. These results suggest several loci that may serve as hubs in gene regulatory pathways associated with GBM.

Conserved properties of individual Ca2+-binding sites in calmodulin.

Significance Calmodulin is essential for sensing intracellular Ca2+ in eukaryotic cells. Calmodulin modulates hundreds of effectors, and it has a highly conserved protein sequence. Humans have three identical copies, but a change in either the protein sequence or the protein expression level of any one of the three copies can cause life-threatening disease. We analyzed calmodulin sequences across eukaryotes and compared biophysical properties and structures to show that all of calmodulin’s four Ca2+-binding sites have conserved properties that distinguish them from one another. Calmodulin (CaM) is a Ca2+-sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca2+ concentration into cellular signals by binding to a wide range of target proteins in a Ca2+-dependent manner. We do not fully understand how CaM performs its role as a high-fidelity signal transducer for more than 300 target proteins, but diversity among its four Ca2+-binding sites, called EF-hands, may contribute to CaM’s functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca2+ signals using four specialized EF-hands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease.

Bivalent Chromatin Domains in Glioblastoma Reveal a Subtype-Specific Signature of Glioma Stem Cells

Glioblastoma multiforme (GBM) can be clustered by gene expression into four main subtypes associated with prognosis and survival, but enhancers and other gene-regulatory elements have not yet been identified in primary tumors. Here, we profiled six histone modifications and CTCF binding as well as gene expression in primary gliomas and identified chromatin states that define distinct regulatory elements across the tumor genome. Enhancers in mesenchymal and classical tumor subtypes drove gene expression associated with cell migration and invasion, whereas enhancers in proneural tumors controlled genes associated with a less aggressive phenotype in GBM. We identified bivalent domains marked by activating and repressive chromatin modifications. Interestingly, the gene interaction network from common (subtype-independent) bivalent domains was highly enriched for homeobox genes and transcription factors and dominated by SHH and Wnt signaling pathways. This subtype-independent signature of early neural development may be indicative of poised dedifferentiation capacity in glioblastoma and could provide potential targets for therapy.Significance: Enhancers and bivalent domains in glioblastoma are regulated in a subtype-specific manner that resembles gene regulation in glioma stem cells. Cancer Res; 78(10); 2463-74. ©2018 AACR.

EF hands at the N-lobe of calmodulin are required for both SK channel gating and stable SK–calmodulin interaction

1. 1. Li, 2. et al . 2009. J. Gen. Physiol. doi: 10.1085/jgp.200910295 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1085%252Fjgp.200910295%26rft_id%253Dinfo%253Apmid%252F19752189%26rft.genre%253Darticle%26rft_val_fmt%