Themes in our Research

We employ a combination of cellular and molecular tools, as well as human and non-human primate model systems, to assess epigenetic signatures (predominantly DNA methylation, DNA hydroxymethylation, and small RNAs) through high-throughput sequencing methods to decipher epigenetic contributions to disease risk.

Immunofluorescence image of induced pluripotent stem cells, stained for OCT4, SOX2 and NANOG.

Stem-cell and direct conversion-based in vitro model systems

We utilize human pluripotent stem cell-derived cells (skeletal muscle cells, neurons, astrocytes, and 3D brain organoids), as well as fibroblast-derived neural cell types, in combination with molecular tools (e.g., CRISPR-dCas9) to study complex disorders (obesity, Alzheimer's disease, epilepsy, and bipolar disorder).

Histological image of the intact Baboon brain.

Human and non-human primate model systems

We employ baboons as a model for epilepsy, and have projects investigating their small RNA profiles, whole genome profiles, snRNAseq profiles, and EEGs, and are developing stem-cell-based models. We also investigate epigenetic profiles in human population studies for lifestyle disorders, including diabetes and obesity.

UMAP plot of a representative single-cell RNA-sequencing experiment.

Methodologies in high-throughput sequencing

Our group utilizes commercially available sequencing methods for DNA methylation, hydroxymethylation, miRNA, and mRNA sequencing across all projects. We are also interested in developing novel combinatorial sequencing approaches to profile the epigenome at single-cell resolution.

Featured Projects

01.

In vitro epigenetic editing to evaluate the impact of DNA methylation changes in complex disorders

DNA methylation plays a vital role in the regulation of gene expression and other cellular processes. Numerous studies have found links between abnormal patterns of methylation and certain human diseases. We are interested in assessing the effect of altered methylation at key CpG sites associated with complex disorders, including obesity and Alzheimer’s disease. We have developed an in-house method that effectively integrates viral vectors into the genome to stably express a mutated form of CRISPR/Cas9 (dCas9-DNMT3A and guide RNA)., allowing us to methylate specific loci throughout the genome. We have integrated this dCas9 system into stem cells derived from healthy controls and patients with Alzheimer’s disease, as well as from individuals predisposed to obesity, and have shown altered methylation patterns throughout differentiation into neurons (in AD) and skeletal muscle cells (in obesity). We are currently expanding our work to include the CRISPRon/CRISPRoff system to allow for more tightly regulated inducible methylation changes.

Immunofluorescence image of human iPSC-derived skeletal muscle cells, stained in green for Titin and blue for DAPI.

Image Credit: Emily Shrimpton

02.

Development of "age-in-a-dish" models to study age- and age-related neurodegenerative disorders

Age-related neurodegenerative disorders like Alzheimer’s disease affect millions worldwide and pose a major burden to the healthcare system. Neuronal and non-neuronal cells play an important role in disease progression and epigenetic modifications, such as DNA methylation, are known contributors to both healthy aging and neurodegeneration. While iPSC-derived models of AD provide valuable insight into the molecular basis for the disease, they lack the inherent ability to recapitulate age-associated DNA methylation, transcription, and cellular phenotypes that are highly relevant in such late-stage, age-associated brain disorders. Direct conversion of fibroblasts to neurons retains such age-associated methylomic and transcriptomic patterns. Our lab is trying to extend such work by developing an efficient direct conversion strategy for astrocytes and other non-neuronal cells, including microglia and oligodendrocytes. We plan to use this model to further elucidate the association of age and disease-related changes in DNA methylation with astrocyte functionality.

Immunofluorescence image of fibroblast-derived astrocytes, stained for Vimentin and GFAP.

Image Credit: Uchit Bhaskar

03.

Epigenetics of neuropsychiatric diseases through the lens of neurodevelopment

There is substantive evidence that risk for neuropsychiatric diseases may begin during neural development, and epigenetic mechanisms likely drive this risk. We are interested in the effect of DNA hydroxymethylation on disease risk, due to its integral role in early neural development and high specificity within the developing and adult brain. We have generated preliminary data that suggest hydroxymethylation signatures in known neuropsychiatric-related genes are altered during neural differentiation, and that hydroxymethylation patterns may differ between iPSC-derived neuronal cells of adolescents with bipolar I disorder and their unaffected siblings. We are interested in exploring the relationship between DNA hydroxymethylation, methylation, and gene expression. We aim to do this using a joint-combinatorial indexing-based approach and are currently developing sequencing-based strategies to resolve this at a single-cell resolution.

Representative immunostaining image of fibroblast-derived induced neurons, stained for MAP2 and Synapsin.

Image Credit: Uchit Bhaskar

04.

DNA methylation changes associated with metabolic syndrome and its components

Our lab has identified epigenome-wide DNA methylation changes associated with metabolic syndrome and its components (i.e., diabetes, obesity, dyslipidemia and hypertension) in a large Mexican American cohort. We identified CpG sites whose methylation profiles were heritable, associated with age and sex, and associated with several metabolic phenotypes. We are also studying the contribution of DNA methylation to obesity and energy expenditure in a childhood cohort of Mexican Americans. We are using MethylSeq to investigate the role of DNA methylation signatures in obesity, energy expenditure, and substrate utilization. Using findings from this study, we are currently validating their physiological relevance by employing our epigenetic editing tools in iPSC-derived skeletal muscle cells to understand the functional role of such DNA methylation changes on mitochondrial function and cellular bioenergetics.

Image of Manhattan plots, showing association of DNA methylation with Type 2 Diabetes related phenotypes, obtained from our publication.

Read our paper

05.

The role of miRNAs in brain structure and function

Prior research has shown miRNA expression is altered in neurological disorders. miRNAs can be found in relatively accessible body fluids (e.g., blood, CSF), making them suited as potential biomarkers of diseases. In the lab, we are working with a non-human primate model (baboons) to assess neuroanatomical variation through EEG/MRI and examine their associations with peripheral miRNA biomarkers and brain-region-specific gene expression patterns. We are also interested in extending these findings to in vitro model systems, including baboon iPSC-derived brain organoids, and extending this area of research toward preclinical models of epilepsy. Overall, our goal is to better define peripheral miRNA biomarkers that may also have implications for brain pathology and disease states.

Representative immunostaining image of a section of baboon iPSC-derived cortical organoid, stained for SOX2, PAX6.

Image Credit: Emily Shrimpton

(in collaboration with Hsieh Lab, UTSA)

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Carless Lab

Epigenetics of Complex Disorders

Contact: melanie.carless@utsa.edu

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