Modulation of heat shock transcription factor 1 as a therapeutic target for small molecule intervention in neurodegenerative disease.

Neurodegenerative diseases such as Huntington disease are devastating disorders with no therapeutic approaches to ameliorate the underlying protein misfolding defect inherent to poly-glutamine (polyQ) proteins. Given the mounting evidence that elevated levels of protein chaperones suppress polyQ protein misfolding, the master regulator of protein chaperone gene transcription, HSF1, is an attractive target for small molecule intervention. We describe a humanized yeast-based high-throughput screen to identify small molecule activators of human HSF1. This screen is insensitive to previously characterized activators of the heat shock response that have undesirable proteotoxic activity or that inhibit Hsp90, the central chaperone for cellular signaling and proliferation. A molecule identified in this screen, HSF1A, is structurally distinct from other characterized small molecule human HSF1 activators, activates HSF1 in mammalian and fly cells, elevates protein chaperone expression, ameliorates protein misfolding and cell death in polyQ-expressing neuronal precursor cells and protects against cytotoxicity in a fly model of polyQ-mediated neurodegeneration. In addition, we show that HSF1A interacts with components of the TRiC/CCT complex, suggesting a potentially novel regulatory role for this complex in modulating HSF1 activity. These studies describe a novel approach for the identification of new classes of pharmacological interventions for protein misfolding that underlies devastating neurodegenerative disease.

Recovery from an acute infection in C. elegans requires the GATA transcription factor ELT-2.

The mechanisms involved in the recognition of microbial pathogens and activation of the immune system have been extensively studied. However, the mechanisms involved in the recovery phase of an infection are incompletely characterized at both the cellular and physiological levels. Here, we establish a Caenorhabditis elegans-Salmonella enterica model of acute infection and antibiotic treatment for studying biological changes during the resolution phase of an infection. Using whole genome expression profiles of acutely infected animals, we found that genes that are markers of innate immunity are down-regulated upon recovery, while genes involved in xenobiotic detoxification, redox regulation, and cellular homeostasis are up-regulated. In silico analyses demonstrated that genes altered during recovery from infection were transcriptionally regulated by conserved transcription factors, including GATA/ELT-2, FOXO/DAF-16, and Nrf/SKN-1. Finally, we found that recovery from an acute bacterial infection is dependent on ELT-2 activity.

Explicit DNase sequence bias modeling enables high-resolution transcription factor footprint detection.

DNaseI footprinting is an established assay for identifying transcription factor (TF)-DNA interactions with single base pair resolution. High-throughput DNase-seq assays have recently been used to detect in vivo DNase footprints across the genome. Multiple computational approaches have been developed to identify DNase-seq footprints as predictors of TF binding. However, recent studies have pointed to a substantial cleavage bias of DNase and its negative impact on predictive performance of footprinting. To assess the potential for using DNase-seq to identify individual binding sites, we performed DNase-seq on deproteinized genomic DNA and determined sequence cleavage bias. This allowed us to build bias corrected and TF-specific footprint models. The predictive performance of these models demonstrated that predicted footprints corresponded to high-confidence TF-DNA interactions. DNase-seq footprints were absent under a fraction of ChIP-seq peaks, which we show to be indicative of weaker binding, indirect TF-DNA interactions or possible ChIP artifacts. The modeling approach was also able to detect variation in the consensus motifs that TFs bind to. Finally, cell type specific footprints were detected within DNase hypersensitive sites that are present in multiple cell types, further supporting that footprints can identify changes in TF binding that are not detectable using other strategies.

Quantitative genetics of CTCF binding reveal local sequence effects and different modes of X-chromosome association.

Associating genetic variation with quantitative measures of gene regulation offers a way to bridge the gap between genotype and complex phenotypes. In order to identify quantitative trait loci (QTLs) that influence the binding of a transcription factor in humans, we measured binding of the multifunctional transcription and chromatin factor CTCF in 51 HapMap cell lines. We identified thousands of QTLs in which genotype differences were associated with differences in CTCF binding strength, hundreds of them confirmed by directly observable allele-specific binding bias. The majority of QTLs were either within 1 kb of the CTCF binding motif, or in linkage disequilibrium with a variant within 1 kb of the motif. On the X chromosome we observed three classes of binding sites: a minority class bound only to the active copy of the X chromosome, the majority class bound to both the active and inactive X, and a small set of female-specific CTCF sites associated with two non-coding RNA genes. In sum, our data reveal extensive genetic effects on CTCF binding, both direct and indirect, and identify a diversity of patterns of CTCF binding on the X chromosome.

Roles of CTCF and YY1 in T Cell Receptor Gene Rearrangement And T Cell Development

Diversity of T cell receptors (TCR) and immunoglobulins (Ig) is generated by V(D)J recombination of antigen receptor (AgR) loci. The Tcra-Tcrd locus is of particular interest because it displays a nested organization of Tcrd and Tcra gene segments and V(D)J recombination follows an intricate developmental program to assemble both TCRδ and TCRα repertoires. However, the mechanisms that dictate the developmental regulation of V(D)J recombination of the Tcra-Tcrd locus remain unclear.

We have previously shown that CCCTC-binding factor (CTCF) regulates Tcra gene transcription and rearrangement through organizing chromatin looping between CTCF- binding elements (CBEs). This study is one of many showing that CTCF functions as a chromatin organizer and transcriptional regulator genome-wide. However, detailed understanding of the impact of specific CBEs is needed to fully comprehend the biological function of CTCF and how CTCF influences the generation of the TCR repertoire during thymocyte development. Thus, we generated several mouse models with genetically modified CBEs to gain insight into the CTCF-dependent regulation of the Tcra-Tcrd locus. We revealed a CTCF-dependent chromatin interaction network at the Tcra-Tcrd locus in double-negative thymocytes. Disruption of a discrete chromatin loop encompassing Dδ, Jδ and Cδ gene segments allowed a single Vδ segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. In addition, we showed that insertion of an ectopic CBE can modify chromatin interactions and disrupt the rearrangement of particular Vδ gene segments. Finally, we investigated the role of YY1 in early T cell development by conditionally deleting YY1 in developing thymocytes. We found that early ablation of YY1 caused severe developmental defects in the DN compartment due to a dramatic increase in DN thymocyte apoptosis. Furthermore, late ablation of YY1 resulted in increased apoptosis of DP thymocytes and a restricted TCRα repertoire. Mechanistically, we showed that p53 was upregulated in both DN and DP YY1-deficient thymocytes. Eliminating p53 in YY1-deficient thymocytes rescued the survival and developmental defects, indicating that these YY1-dependent defects were p53-mediated. We conclude that YY1 is required to maintain cell viability during thymocyte development by thwarting the accumulation of p53.

Overall, this thesis work has shown that CTCF-dependent looping provides a central framework for lineage- and developmental stage-specific regulation of Tcra-Tcrd gene expression and rearrangements. In addition, we identified YY1 as a novel regulator of thymocyte viability.

Promoting developmental transcription.

Animal growth and development depend on the precise control of gene expression at the level of transcription. A central role in the regulation of developmental transcription is attributed to transcription factors that bind DNA enhancer elements, which are often located far from gene transcription start sites. Here, we review recent studies that have uncovered significant regulatory functions in developmental transcription for the TFIID basal transcription factors and for the DNA core promoter elements that are located close to transcription start sites.

Co-regulation of nuclear respiratory factor-1 by NFkappaB and CREB links LPS-induced inflammation to mitochondrial biogenesis.

The nuclear respiratory factor-1 (NRF1) gene is activated by lipopolysaccharide (LPS), which might reflect TLR4-mediated mitigation of cellular inflammatory damage via initiation of mitochondrial biogenesis. To test this hypothesis, we examined NRF1 promoter regulation by NFκB, and identified interspecies-conserved κB-responsive promoter and intronic elements in the NRF1 locus. In mice, activation of Nrf1 and its downstream target, Tfam, by Escherichia coli was contingent on NFκB, and in LPS-treated hepatocytes, NFκB served as an NRF1 enhancer element in conjunction with NFκB promoter binding. Unexpectedly, optimal NRF1 promoter activity after LPS also required binding by the energy-state-dependent transcription factor CREB. EMSA and ChIP assays confirmed p65 and CREB binding to the NRF1 promoter and p65 binding to intron 1. Functionality for both transcription factors was validated by gene-knockdown studies. LPS regulation of NRF1 led to mtDNA-encoded gene expression and expansion of mtDNA copy number. In cells expressing plasmid constructs containing the NRF-1 promoter and GFP, LPS-dependent reporter activity was abolished by cis-acting κB-element mutations, and nuclear accumulation of NFκB and CREB demonstrated dependence on mitochondrial H(2)O(2). These findings indicate that TLR4-dependent NFκB and CREB activation co-regulate the NRF1 promoter with NFκB intronic enhancement and redox-regulated nuclear translocation, leading to downstream target-gene expression, and identify NRF-1 as an early-phase component of the host antibacterial defenses.

Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming.

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca(2+) transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies.

Chromatin accessibility mapping identifies mediators of basal transcription and retinoid-induced repression of OTX2 in medulloblastoma.

Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes.

beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.

One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.

Sequence and Structural Determinants of Specificity Differences between Paralogous Transcription Factors

Transcription factors (TFs) control the temporal and spatial expression of target genes by interacting with DNA in a sequence-specific manner. Recent advances in high throughput experiments that measure TF-DNA interactions in vitro and in vivo have facilitated the identification of DNA binding sites for thousands of TFs. However, it remains unclear how each individual TF achieves its specificity, especially in the case of paralogous TFs that recognize distinct target genomic sites despite sharing very similar DNA binding motifs. In my work, I used a combination of high throughput in vitro protein-DNA binding assays and machine-learning algorithms to characterize and model the binding specificity of 11 paralogous TFs from 4 distinct structural families. My work proves that even very closely related paralogous TFs, with indistinguishable DNA binding motifs, oftentimes exhibit differential binding specificity for their genomic target sites, especially for sites with moderate binding affinity. Importantly, the differences I identify in vitro and through computational modeling help explain, at least in part, the differential in vivo genomic targeting by paralogous TFs. Future work will focus on in vivo factors that might also be important for specificity differences between paralogous TFs, such as DNA methylation, interactions with protein cofactors, or the chromatin environment. In this larger context, my work emphasizes the importance of intrinsic DNA binding specificity in targeting of paralogous TFs to the genome.

Porcine endothelial cells cocultured with smooth muscle cells became procoagulant in vitro.

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Neuroprotection resulting from insufficiency of RANBP2 is associated with the modulation of protein and lipid homeostasis of functionally diverse but linked pathways in response to oxidative stress.

Oxidative stress is a deleterious stressor associated with a plethora of disease and aging manifestations, including neurodegenerative disorders, yet very few factors and mechanisms promoting the neuroprotection of photoreceptor and other neurons against oxidative stress are known. Insufficiency of RAN-binding protein-2 (RANBP2), a large, mosaic protein with pleiotropic functions, suppresses apoptosis of photoreceptor neurons upon aging and light-elicited oxidative stress, and promotes age-dependent tumorigenesis by mechanisms that are not well understood. Here we show that, by downregulating selective partners of RANBP2, such as RAN GTPase, UBC9 and ErbB-2 (HER2; Neu), and blunting the upregulation of a set of orphan nuclear receptors and the light-dependent accumulation of ubiquitylated substrates, light-elicited oxidative stress and Ranbp2 haploinsufficiency have a selective effect on protein homeostasis in the retina. Among the nuclear orphan receptors affected by insufficiency of RANBP2, we identified an isoform of COUP-TFI (Nr2f1) as the only receptor stably co-associating in vivo with RANBP2 and distinct isoforms of UBC9. Strikingly, most changes in proteostasis caused by insufficiency of RANBP2 in the retina are not observed in the supporting tissue, the retinal pigment epithelium (RPE). Instead, insufficiency of RANBP2 in the RPE prominently suppresses the light-dependent accumulation of lipophilic deposits, and it has divergent effects on the accumulation of free cholesterol and free fatty acids despite the genotype-independent increase of light-elicited oxidative stress in this tissue. Thus, the data indicate that insufficiency of RANBP2 results in the cell-type-dependent downregulation of protein and lipid homeostasis, acting on functionally interconnected pathways in response to oxidative stress. These results provide a rationale for the neuroprotection from light damage of photosensory neurons by RANBP2 insufficiency and for the identification of novel therapeutic targets and approaches promoting neuroprotection.

Oestrogen shuts the door on SOX9.

Oestrogen exerts a robust yet imperfectly understood effect on sexual development in vertebrate embryos. New work by Pask and colleagues in BMC Biology indicates that it may interfere with male development by preventing nuclear localization of SOX9, a master regulator of the testis differentiation pathway. See research article http://www.biomedcentral.com/1741-7007/8/113.