Gene Regulatory Networks: Modeling, Intervention and Context

abstract: Biological systems are complex in many dimensions as endless transportation and communication networks all function simultaneously. Our ability to intervene within both healthy and diseased systems is tied directly to our ability to understand and model core functionality. The progress in increasingly accurate and thorough high-throughput measurement technologies has provided a deluge of data from which we may attempt to infer a representation of the true genetic regulatory system. A gene regulatory network model, if accurate enough, may allow us to perform hypothesis testing in the form of computational experiments. Of great importance to modeling accuracy is the acknowledgment of biological contexts within the models -- i.e. recognizing the heterogeneous nature of the true biological system and the data it generates. This marriage of engineering, mathematics and computer science with systems biology creates a cycle of progress between computer simulation and lab experimentation, rapidly translating interventions and treatments for patients from the bench to the bedside. This dissertation will first discuss the landscape for modeling the biological system, explore the identification of targets for intervention in Boolean network models of biological interactions, and explore context specificity both in new graphical depictions of models embodying context-specific genomic regulation and in novel analysis approaches designed to reveal embedded contextual information. Overall, the dissertation will explore a spectrum of biological modeling with a goal towards therapeutic intervention, with both formal and informal notions of biological context, in such a way that will enable future work to have an even greater impact in terms of direct patient benefit on an individualized level.Dissertation/ThesisPh.D. Computer Science 201

Context-specific gene regulatory networks subdivide intrinsic subtypes of breast cancer

<p>Abstract</p> <p>Background</p> <p>Breast cancer is a highly heterogeneous disease with respect to molecular alterations and cellular composition making therapeutic and clinical outcome unpredictable. This diversity creates a significant challenge in developing tumor classifications that are clinically reliable with respect to prognosis prediction.</p> <p>Results</p> <p>This paper describes an unsupervised context analysis to infer context-specific gene regulatory networks from 1,614 samples obtained from publicly available gene expression data, an extension of a previously published methodology. We use the context-specific gene regulatory networks to classify the tumors into clinically relevant subgroups, and provide candidates for a finer sub-grouping of the previously known intrinsic tumors with a focus on Basal-like tumors. Our analysis of pathway enrichment in the key contexts provides an insight into the biological mechanism underlying the identified subtypes of breast cancer.</p> <p>Conclusions</p> <p>The use of context-specific gene regulatory networks to identify biological contexts from heterogenous breast cancer data set was able to identify genomic drivers for subgroups within the previously reported intrinsic subtypes. These subgroups (contexts) uphold the clinical relevant features for the intrinsic subtypes and were associated with increased survival differences compared to the intrinsic subtypes. We believe our computational approach led to the generation of novel rationalized hypotheses to explain mechanisms of disease progression within sub-contexts of breast cancer that could be therapeutically exploited once validated.</p

DNA methylation and transcriptional control in memory formation, persistence and suppression

Memory formation is a complex process regulated by various molecular mechanisms, including unique transcriptional signatures and epigenetic factors. In addition, the brain is equipped with mechanisms that not only promote, but actively constrict memory formation. While the role of epigenetic modifications, such as DNA methylation, in cognition has been established, there are still significant gaps in our understanding of the specific functions of individual DNA methyltransferases (Dnmts) and how their downstream effectors orchestrate memory. Moreover, the molecular mechanisms underlying memory persistence and memory suppression remain largely unexplored. I investigated the role of specific Dnmts in long-term memory formation, highlighting their unique functions and downstream effects. Additionally, I explored how DNA methylation contributes to the transfer of information from the hippocampus to the cortex for long-term storage and the stabilisation of cortical engrams to drive memory persistence. First, I examined the involvement of Dnmt3a1, the predominant Dnmt3a isoform in the adult brain, in hippocampus-dependent long-term memory formation. I identified an activity-regulated Dnmt3a1-dependent gene expression program and found a downstream effector gene (Neuropilin-1) with a previously undescribed function in memory formation. Intriguingly, I found that despite a common requirement for memory formation, Dnmt3a1 and Dnmt3a2 regulate this process via distinct mechanisms - Nrp1 overexpression rescued Dnmt3a1, but not Dnmt3a2, knockdown-driven impairments in memory formation. Next, I investigated the molecular mechanisms underlying memory persistence and systems consolidation, the gradual transfer of information from the hippocampus to the cortex. By modulating DNA methylation processes in the dorsal hippocampus, a short-lasting memory could be converted into a long-lasting one. The applied manipulation resulted in improved reactivation of cortical engrams and increased fear generalisation, mimicking the characteristics of remote memory. These findings provide compelling evidence for the facilitatory role of DNA methylation in memory information transfer to the cortex for long-term storage. Furthermore, I examined the temporal expression patterns of immediate early genes (IEGs), specifically neuronal PAS domain protein 4 (Npas4), and its potential role in memory suppression. My investigation revealed that highly salient stimuli induced a biphasic expression of Npas4 in the hippocampus, with the later phase dependent on NMDA receptor activity. Notably, this later phase of Npas4 expression restricted memory consolidation, suggesting a role in balancing the formation of highly salient memories and preventing the development of maladaptive behaviours. These findings highlighted the intricate regulatory network by which experience salience modulates IEG expression and thereby fine-tunes memory consolidation. Overall, this study uncovered the unique functions of distinct Dnmts in memory formation and persistence and shed light on the associated mechanisms that are responsible to facilitate the transfer of information required for long-term storage. This comprehensive understanding of the molecular processes underlying memory formation contributes to our broader knowledge of memory consolidation and may have implications for therapeutic interventions targeting memory-related disorders

Identifying the molecular systems that influence cognitive resilience to Alzheimer\u27s disease in genetically diverse mice.

Individual differences in cognitive decline during normal aging and Alzheimer\u27s disease (AD) are common, but the molecular mechanisms underlying these distinct outcomes are not fully understood. We utilized a combination of genetic, molecular, and behavioral data from a mouse population designed to model human variation in cognitive outcomes to search for the molecular mechanisms behind this population-wide variation. Specifically, we used a systems genetics approach to relate gene expression to cognitive outcomes during AD and normal aging. Statistical causal-inference Bayesian modeling was used to model systematic genetic perturbations matched with cognitive data that identified astrocyte and microglia molecular networks as drivers of cognitive resilience to AD. Using genetic mapping, we identifie

Comprehensive Behavioral Analysis of Calcium/Calmodulin-Dependent Protein Kinase IV Knockout Mice

Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO) mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain sensitivity, prepulse inhibition, attention, or depression-like behavior. Consistent with previous reports, CaMKIV KO mice exhibited impaired retention in a fear conditioning test 28 days after training. In contrast, however, CaMKIV KO mice did not show any testing performance deficits in passive avoidance, one of the most commonly used fear memory paradigms, 28 days after training, suggesting that remote fear memory is intact. CaMKIV KO mice exhibited intact spatial reference memory learning in the Barnes circular maze, and normal spatial working memory in an eight-arm radial maze. CaMKIV KO mice also showed mildly decreased anxiety-like behavior, suggesting that CaMKIV is involved in regulating emotional behavior. These findings indicate that CaMKIV might not be essential for fear memory or spatial memory, although it is possible that the activities of other neural mechanisms or signaling pathways compensate for the CaMKIV deficiency

Phenotypic Characterization of Transgenic Mice Overexpressing Neuregulin-1

BACKGROUND: Neuregulin-1 (NRG1) is one of the susceptibility genes for schizophrenia and implicated in the neurotrophic regulation of GABAergic and dopaminergic neurons, myelination, and NMDA receptor function. Postmortem studies often indicate a pathologic association of increased NRG1 expression or signaling with this illness. However, the psychobehavioral implication of NRG1 signaling has mainly been investigated using hypomorphic mutant mice for individual NRG1 splice variants. METHODOLOGY/PRINCIPAL FINDINGS: To assess the behavioral impact of hyper NRG1 signaling, we generated and analyzed two independent mouse transgenic (Tg) lines carrying the transgene of green fluorescent protein (GFP)-tagged type-1 NRG1 cDNA. The promoter of elongation-factor 1α gene drove ubiquitous expression of GFP-tagged NRG1 in the whole brain. As compared to control littermates, both heterozygous NRG1-Tg lines showed increased locomotor activity, a nonsignificant trend toward decreasing prepulse inhibition, and decreased context-dependent fear learning but exhibited normal levels of tone-dependent learning. In addition, social interaction scores in both Tg lines were reduced in an isolation-induced resident-intruder test. There were also phenotypic increases in a GABAergic marker (parvalbumin) as well as in myelination markers (myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase) in their frontal cortex, indicating the authenticity of NRG1 hyper-signaling, although there were marked decreases in tyrosine hydroxylase levels and dopamine content in the hippocampus. CONCLUSIONS: These findings suggest that aberrant hyper-signals of NRG1 also disrupt various cognitive and behavioral processes. Thus, neuropathological implication of hyper NRG1 signaling in psychiatric diseases should be evaluated with further experimentation

Mammalian Brain As a Network of Networks

Acknowledgements AZ, SG and AL acknowledge support from the Russian Science Foundation (16-12-00077). Authors thank T. Kuznetsova for Fig. 6.Peer reviewedPublisher PD

Conditional Gene Editing in Presynaptic Extinction-ensemble Cells via the CRISPR-SaCas9 System

The CRISPR-Cas9 enables efficient gene editing in various cell types, including post-mitotic neurons. However, neuronal ensembles in the same brain region can still be functionally or anatomically different, and such heterogeneity requires gene editing in specific neuronal populations. We recently developed a CRISPR-SaCas9 system-based technique. Combined with activity-dependent cell-labeling methods and anterograde/retrograde adeno-associated virus (AAV) vectors, this technique achieves function- and projection-specific gene editing in the mammalian brain. We showed that perturbing cbp (CREB-binding protein) in extinction-ensemble neurons among amygdala-projecting infralimbic cortex (IL) cells impaired fear extinction learning, demonstrating the high efficiency in regulation of extinction learning with CRISPR-Cas9. Here, we describe a detailed protocol of gene perturbation in presynaptic extinction-ensemble neurons in adult rats, including gRNA design, gRNA evaluation in vitro, stereotaxic AAV injection, and contextual fear conditioning. The high specificity and efficiency of projection- and function-specific CRISPR-SaCas9 system can be widely applied in neural circuitry studies

Linking Acetyl-Coa Metabolism and Histone Acetylation to Dynamic Gene Regulation in Yeast and Mouse Hippocampus

A compelling body of evidence suggests an intimate relationship between metabolic state and chromatin regulation. This link is manifested in key metabolites that participate in biochemical pathways as intermediates, and function as cofactors to regulate chromatin modifying enzymes. Of particular interest is the metabolite acetyl-CoA, given its central role as an intermediate of cellular energy metabolism and key determinant of all histone acetylation. How nuclear acetyl-CoA levels are regulated to, in turn, control histone acetylation is under intense investigation, and holds promise for increased understanding of the molecular mechanisms adapting gene expression to internal and external stimuli. We studied the relationship between histone modification dynamics and the dramatic transcriptional changes that occur during nutrient‐induced cell cycle re-entry from quiescence in the yeast S. cerevisiae. ChIP‐seq and SILAC-based mass spec revealed genome‐wide shifts in histone acetylation at growth and stress genes as cells exit quiescence and transcription dramatically changes. Strikingly, however, the patterns of histone methylation remain intact. We conclude that histone acetylation, in contrast to methylation, rapidly responds to metabolic state, driving growth gene transcription in nutrient-induced cell cycle re-entry. Next, we set out to investigate how nuclear acetyl-CoA is regulated to control histone acetylation in mammalian cells. We reveal a previously unknown function of the central metabolic enzyme acetyl-CoA synthetase 2 (ACSS2) as a chromatin-bound transcriptional coactivator that stimulates histone acetylation and gene expression. We show that ACSS2 is a critical and direct regulator of histone acetylation in neurons and of long-term mammalian memory. Genome-wide, ACSS2 binding corresponds with increased histone acetylation and gene expression of key neuronal genes. Our data indicate that ACSS2 functions as a chromatin-bound co-activator to increase local concentrations of acetyl-CoA, to locally promote histone acetylation for transcription of neuron-specific genes. Remarkably, in vivo attenuation of hippocampal ACSS2 expression in adult mice impairs long-term spatial memory, a cognitive process reliant on histone acetylation. ACSS2 reduction in hippocampus also leads to a defect in upregulation of key neuronal genes involved in memory. These findings reveal a unique connection between cellular metabolism and neural plasticity, and establish a link between generation of acetyl-CoA and neuronal chromatin regulation