Influence of sex differences on microRNA gene regulation in disease.

Sexual dimorphism is observed in most human diseases. The difference in the physiology and genetics between sexes can contribute tremendously to the disease prevalence, severity, and outcome. Both hormonal and genetic differences between males and females can lead to differences in gene expression patterns that can influence disease risk and course. MicroRNAs have emerged as potential regulatory molecules in all organisms. They can have a broad effect on every aspect of physiology, including embryogenesis, metabolism, and growth and development. Numerous microRNAs have been identified and elucidated to play a key role in cardiovascular diseases, as well as in neurological and autoimmune disorders. This is especially important as microRNA-based tools can be exploited as beneficial therapies for disease treatment and prevention. Sex steroid hormones as well as X-linked genes can have a considerable influence on the regulation of microRNAs. However, there are very few studies highlighting the role of microRNAs in sex biased diseases. This review attempts to summarize differentially regulated microRNAs in males versus females in different diseases and calls for more attention in this underexplored area that should set the basis for more effective therapeutic strategies for sexually dimorphic diseases

Super-Enhancer-Mediated RNA Processing Revealed by Integrative MicroRNA Network Analysis

Super-enhancers are an emerging subclass of regulatory regions controlling cell identity and disease genes. However, their biological function and impact on miRNA networks are unclear. Here, we report that super-enhancers drive the biogenesis of master miRNAs crucial for cell identity by enhancing both transcription and Drosha/DGCR8-mediated primary miRNA (pri-miRNA) processing. Super-enhancers, together with broad H3K4me3 domains, shape a tissue-specific and evolutionarily conserved atlas of miRNA expression and function. CRISPR/Cas9 genomics revealed that super-enhancer constituents act cooperatively and facilitate Drosha/DGCR8 recruitment and pri-miRNA processing to boost cell-specific miRNA production. The BET-bromodomain inhibitor JQ1 preferentially inhibits super-enhancer-directed cotranscriptional pri-miRNA processing. Furthermore, super-enhancers are characterized by pervasive interaction with DGCR8/Drosha and DGCR8/Drosha-regulated mRNA stability control, suggesting unique RNA regulation at super-enhancers. Finally, super-enhancers mark multiple miRNAs associated with cancer hallmarks. This study presents principles underlying miRNA biology in health and disease and an unrecognized higher-order property of super-enhancers in RNA processing beyond transcription. Keywords: microRNA; super-enhancer; broad H3K4me3 domain; Drosha; DGCR8; Brd4; cancerUnited States. Public Health Service (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant P30-CA14051

Heterogeneity and multi-omics features of alternative lengthening of telomeres

Telomere maintenance mechanisms (TMM) are crucial for cancer cells as they are required for their unlimited proliferation capacity. While most cancers reactivate the reverse transcriptase telomerase, a significant fraction of tumors maintains telomere length without it. These cancers employ the alternative lengthening of telomeres (ALT) pathway, which relies on DNA repair and recombination to extend telomere repeats. ALT presence is primarily confirmed with the goldstandard C-circle assay, which quantifies extrachromosomal telomere repeats that are only found in ALT-positive cells. Mutations within the repeat repressor ATRX/DAXX/H3.3 are overrepresented in ALT cancers, which are believed to further telomere dysfunction and gene expression programs in ALT. ALT presence has an impact on long-term survival in cancer, and detection of ALT in omics data is currently lacking. Although much progress has been made in the past few decades in elucidating the ALT mechanism, the details concerning ALT are still unknown. This thesis addresses this question from three angles: (i) Describing ALT activity heterogeneity in primary tumor samples; (ii) Using sequencing readouts to define ALT and extract a characteristic signature; (iii) Inhibiting epigenetic modifiers with drugs and observing their effect on viability in ALT cells. Firstly, I quantified C-circle levels in 687 primary tumor biopsies from sarcomas. The heterogeneous distribution indicates that ALT-activity can vary about tenfold within the same tumor entity. Next, I conducted ATAC-seq and RNA-seq of long and short RNAs in ALT positive and negative cell lines from pediatric glioblastoma and osteosarcoma to find shared ALT features. Information on open chromatin regions, transcriptome, miRNA, transposable elements, and piRNA was extracted from these data. From the ATAC-seq data, it was found that ALT+ cell lines had predominantly increased chromatin accessibility in non-coding regions. This change may be driven by AP-1 and RUNX transcription factors (TF), whilst downregulated accessible regions result from reduced SOX TFs. From transcriptomic data, it was revealed that immune TFs were enriched in upregulated ALT genes. This led to the identification of NFATC2 as a potential ALT biomarker, as it was found in differential expression and transcriptome TF motif analysis. The immune TFs may be induced by genetic instability, yet the multi-omics ALT signature indicated that the cell lines have a reduced response to oxidative stress as well. These factors may cooperate in inducing a heightened inflammatory state that drives chromatin accessibility and gene expression. Differential miRNAs were extracted and could explain both TERT and SOX downregulation and RUNX upregulation, indicating another gene regulatory mechanism employed by ALT cell lines. Furthermore, an integrative multi-omics analysis was performed to extract an ALT signature. ALT-positive cells displayed a characteristic signature that was influenced by transcriptome, miRNA, and chromatin accessibility. As more upregulated open regions in ATAC data were observed, epigenetic drugs for repressive marks were applied to assess the relationship between ALT activity and cell viability. I found that inhibition of repressive H3K27me3 and DNA methylation was correlated with an ALT-specific lethality and survival, respectively. Another aberrant epigenetic feature found in ALT, an H3.3S31p chromosome-wide signal during mitosis, was studied with different inhibitors to elucidate which kinase is responsible for its establishment. The HASPIN kinase was identified to reduce H3.3S31 phosphorylation upon treatment with a corresponding inhibitor. This kinase is involved in chromosomal segregation and links ALT genetic instability to DNA damage signaling during mitosis

Examining epigenetic variation in the brain in mental illness

Mental health represents one of the most significant and increasing burdens to global public health. Depression and schizophrenia, among other mental illnesses, constitute strong risk factors for suicidality which results in over 800,000 deaths every year. The majority of suicides worldwide are indeed related to psychiatric diseases. A growing body of genetic, epigenetic and epidemiological evidence suggests that psychiatric disorders are highly complex phenotypes originating from the multilevel interplay between the strong genetic component and a range of environmental and psychosocial factors. Deeper understanding about the biology of the genome has led to increased interest for the role of non-sequence-based variation in the etiology of neuropsychiatric phenotypes, including suicidality. Epigenetic alterations and gene expression dysregulation have been repetitively reported in post-mortem brain of individuals who died by suicide. To date, however, studies characterizing disease-associated methylomic and transcriptomic variation in the brain have been limited by screening performed in bulk tissue and by the assessment of a single marker at a time. The main aim of this thesis was to investigate DNA methylation and miRNA expression differences in post-mortem brain associated with suicidality and unravel the complexity of epigenetic signals in a heterogeneous tissue like the human brain by developing a method to profile genomic variation at the resolution of individual neural cell types. The results here reported, provide further support for a suicide-specific epigenetic signature, independent from comorbidity with other psychiatric phenotypes, as well as confirming the strong bias perpetrated by bulk tissue studies hence the need to examine genomic variations in purified cell types. In summary, this thesis has identified a) a suicide-specific signal in two different epigenetic markers (DNA methylation and miRNA expression) and b) a protocol to simultaneously profile DNA methylation levels across three purified cell types in the healthy brain highlighting the utility of cell sorting for identifying cell type-driven epigenetic differences associated with etiological variation in complex psychiatric phenotypes.1) ARUK-PPG2018A-010 – “Developing approaches to address neural cell heterogeneity in genomic studies of Alzheimer's disease”. 2) SBF001\1011 - “Using functional epigenomics to dissect the molecular architecture of schizophrenia

Molecular Regulation of SATB1 in Regulatory T-cells

In this study we have identified SATB1, a nuclear protein that recruits chromatin-remodeling factors and regulates numerous genes, as a novel effector molecule in Treg cells. Our interest in SATB1 resulted from a genome wide expression profile of Treg cells and conventional T-cells (Tconv cells). SATB1 was a prominent candidate gene that constantly repressed in Treg cells and highly expressed in Tconv cells. The dominant repression of SATB1 expression in Treg cells could be confirmed at mRNA, protein, and single cell level under resting and different stimulation conditions in humans and mice. In contrast, SATB1 is expressed at high levels in Tconv cells and is further enhanced following physiological stimulation. The inverse expression pattern of FOXP3, the main transcription factor in shaping and maintaining Treg cell identity, in relation to SATB1 led us to hypothesize its active involvement in regulation of SATB1. On the one hand, induction of FOXP3 was associated with inhibition of SATB1. This could be demonstrated by induction of FOXP3 in naïve CD4+ T-cells converted to induced Treg cells (iTreg) or in CD4+ T-cells ectopically overexpressing FOXP3 after lentiviral transduction. On the other hand, using different genetic approaches loss of FOXP3 expression in Treg cells results in relieving the FOXP3-mediated repression and leads to an upregulation of SATB1. Furthermore, confocal microscopy on lymphocytes form scurfy and normal mice interestingly showed mutually excluding staining patterns. While the SATB1 signal is low in normal FOXP3-expressing thymocytes, it is high in thymocytes expressing a mutated non-functional FOXP3 from scurfy animals. FOXP3 as a transcription factor has been linked to direct binding to DNA, thereby regulating gene expression. To investigate whether FOXP3 can directly bind to the SATB1 genomic locus FOXP3-ChIP tiling arrays were performed. The analysis of tiling array data provided us with several putative FOXP3 binding sites in the promoter and intronic regions of the SATB1 locus which were confirmed by ChIP qRT-PCR. Furthermore, we were able to demonstrate high specificity of the binding and determine the binding coefficients of FOXP3 to several motifs in the SATB1 locus by filter retention assays. To assess whether this binding has functional relevance, we performed reporter assays and showed that FOXP3 reduces lucifierase activity for several binding regions clearly supporting that FOXP3 regulates SATB1 transcription by direct binding to the genomic locus. Interstingly, we showed that FOXP3 also controls SATB1 gene expression indirectly at post-transcriptional level via miRNAs. Indeed we identified several FOXP3 dependent miRNA that have been linked to post-transcriptional regulation of gene expression. FOXP3-ChIP tiling arrays showed FOXP3 peaks within these miRNAs loci. Furthermore, silencing of FOXP3 reversed this enrichment, whereas over-expression of FOXP3 induced their expression. Binding of FOXP3-dependent miRNAs to the 3´UTR of SATB1 in reporter assays confirms the suppressive effect of these miRNAs on SATB1 expression. An additional level of regulation of gene expression is exerted by epigenetic modifactions of the respective genomic locus. Epigenetic changes control the accessibility of a genomic locus by permissive or inhibitory histone modifications as well as methylation of CpG islands. Although, we did not observe differences in the methylation pattern of the CpG islands at the SATB1 locus between Treg cells and Tconv cells, we observed more permissive and less repressive histone marks at the SATB1 genomic locus in Tconv cells and the opposite in Treg cells which is in line with the expression data and aforementioned described regulatory mechanism of SATB1 expression in Treg cells. Besides the molecular mechanism regulating SATB1 expression in Treg cells, we further delineated the functional consequences of induction of SATB1 in Treg cells. Lentiviral over-expression of SATB1 in human and murine Treg cells resulted in the edition of gene expression and function of Treg cells. The striking observation was the abrogation of the capacity of Treg cells to suppress the proliferation of responder cells in vitro, in addition to the production of proinflammatory cytokines like IL-4 and IFN-γ. These findings suggested that Treg cells acquire an effector phenotype a finding which is further corroborated on a genome wide level. Gene expression profiles of SATB1 overexpressing Treg cells showed that many proinflammatory genes have been switched on upon induction of SATB1 expression in Treg cells which promotes skewing of regulatory towards effector programs. To further prove the antagonistic effect of SATB1 on the regulatory function of Treg cells in vivo, we adoptively transferred Treg cells overexpressing SATB1 with naïve CD4+ cells into RAG2-/- mice. In this experimental setting Treg cells failed to suppress inflammation in vivo and subsequently the mice developed colitis. In conclusion, SATB1 is an important effector molecule whose expression is tightly regulated in Treg cells. SATB1 upregulation in Treg cells results in aquisition of proinflammatory properties and attenuated suppressive function in vitro and in vivo. Therefore, FOXP3-mediated repression of SATB1 expression in Treg cells seems to be an important regulatory circuit crucial to maintain suppressive function of these cells

A Family of microRNAs Encoded by Myosin Genes Governs Myosin Expression and Muscle Performance

SummaryMyosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance

Molecular Mechanisms of piRNA Biogenesis and Function in Drosophila: A Dissertation

In the Drosophila germ line, PIWI-interacting RNAs (piRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. We examined the genetic requirements for the biogenesis and function of piRNAs in both female and male germ line. We found that piRNAs function through the PIWI, rather than the AGO, family Argonaute proteins, and the production of piRNAs requires neither microRNA (miRNA) nor small interfering RNA (siRNA) pathway machinery. These findings allowed the discovery of the third conserved small RNA silencing pathway, which is distinct from both the miRNA and RNAi pathways in its mechanisms of biogenesis and function. We also found piRNAs in flies are modified. We determined that the chemical structure of the 3´-terminal modification is a 2´-O-methyl group, and also demonstrated that the same modification occurs on the 3´ termini of siRNAs in flies. Furthermore, we identified the RNA methyltransferase Drosophila Hen1, which catalyzes 2´-O-methylation on both siRNAs and piRNAs. Our data suggest that 2´-O-methylation by Hen1 is the final step of biogenesis of both the siRNA pathway and piRNA pathway. Studies from the Hannon Lab and the Siomi Lab suggest a ping-pong amplification loop for piRNA biogenesis and function in the Drosophila germline. In this model, an antisense piRNA, bound to Aubergine or Piwi, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce a second antisense piRNA. We isolated the loss-of-function mutations in ago3, allowing a direct genetic test of this model. We found that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. Moreover, we also discovered a second Ago3-independent piRNA pathway in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line

Biogenesis and Stability of Germline Small RNAs in C. elegans.

Across the animal kingdom, small, noncoding RNAs preserve and promote fertility by engaging Argonaute effector proteins to silence deleterious genetic elements. Generated in germline and inherited into progeny, endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) regulate vast suites of gametic and zygotic genes, yet remarkably little is known about how they are regulated. With an expanded repertoire of small RNA classes, Caenorhabditis elegans provides an ideal model for investigating how animals drive epigenetic inheritance of fertility-preserving germline small RNAs. The conserved methyltransferase HEN1 methylates small RNAs to prevent their degradation. Methylation of germline small RNAs enhances accumulation, promoting robust inheritance into progeny. All plant small RNAs are methylated, but animal HEN1 methylates only some small RNAs. The mechanisms of selective methylation were unknown. I identified the functional C. elegans ortholog of HEN1 and demonstrated that it methylates all piRNAs but only select subclasses of endo-siRNAs. I further found that particular endo-siRNAs are methylated in maternal, but not paternal, germlines. Through genetic and biochemical analyses, I showed that small RNA methylation status is likely dictated by the associated Argonaute. This established selective expression of divergent Argonautes as a novel mechanism for differentially stabilizing germline small RNAs, with significant implications for preferential inheritance of maternal epigenetic information. piRNAs are essential for animal fertility, but their expression mechanisms are poorly characterized. In collaboration with bioinformatician Mallory Freeberg, I showed that C. elegans male and female germlines express distinct piRNA subsets that evolve independently and differ in inheritance. A common sequence motif lies upstream of nematode piRNA loci. We discovered that this motif varies significantly between male and female piRNAs. Using a novel transgenic approach, I established that C. elegans piRNAs represent thousands of tiny, autonomous transcriptional units, rivaling coding genes in number. I further demonstrated that the upstream motif is required for piRNA expression and that variation at a single nucleotide position within this motif orchestrates selective male versus female germline enrichment and inheritance of piRNAs. These and additional included studies define novel factors and mechanisms involved in regulation of germline small RNAs and transgenerational transmission of their crucial epigenetic information.PHDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111471/1/acbilli_1.pd

RNA- and Chromatin-binding Proteins in Small RNA-mediated Gene Silencing

Gene expression is coordinately regulated at many levels to ensure the proper development and reproductive success of an organism. For example, RNA-binding proteins (RBPs) and microRNAs (miRNAs) mediate post-transcriptional gene silencing through recognition of target mRNA 3’ untranslated regions (UTRs). Pumilio/fem-3 binding factor (PUF) proteins are conserved from yeast to humans, where they bind PUF recognition elements (PREs) in mRNA 3’ UTRs to direct translational repression, mRNA storage, and transcript decay. Recent studies suggest that PUF proteins mediate more complex mechanisms of 3’UTR regulation. C. elegans puf-9 genetically interacts with the let-7 miRNA to regulate epidermal stem cell differentiation and vulval integrity at the larval-to-adult transition. However, the full extent of puf-9/miRNA interaction has not been explored. Here, we globally identify PUF-9 and miRNA binding sites in vivo, revealing that PUF-9 binding sites are enriched in 3’UTR regions with secondary structure, overlapping with miRNA binding sites. We show that the genetic interaction between puf-9 and let-7 is mediated by co-targeting of the lin-41 3’UTR. Altogether, our data are consistent with a model where PUF-9 and miRNAs bind to adjacent sites in structured 3’UTRs to co-regulate shared targets. In animals, germ cells are the only lineage that must be completely totipotent and indefinitely replenished at every generation. Together, small RNAs, histone modifying enzymes, and chromatin binding proteins control gene expression patterns required for germline function. The germline hereditary RNA interference (HRDE) pathway directs the establishment and maintenance of silenced heterochromatin. HRDE-1 Argonaute/small RNA complexes are passed down across generations to protect the immortal germ cell lineage. Here, we characterize the role of conserved chromatin binding protein MicrORChidia-1 (MORC-1) downstream of C. elegans siRNAs. We show that morc-1 is required for small RNA-guided nuclear silencing of operon pre-mRNAs. In addition, morc-1 is required for inheritance of RNAi across generations, but not for the biogenesis or inheritance of siRNAs themselves. Similar to hrde-1 Argonaute mutants, morc-1 mutants exhibit progressive sterility over multiple generations at elevated temperature and also mislocalize heterochromatin transgene reporters. Altogether, MORC-1 is likely an effector protein downstream of nuclear siRNA targeting.PHDHuman Genetics PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/137040/1/dxyang_1.pd