RNA Exosome & Chromatin: The Yin & Yang of Transcription: A Dissertation

Eukaryotic genomes can produce two types of transcripts: protein-coding and non-coding RNAs (ncRNAs). Cryptic ncRNA transcripts are bona fide RNA Pol II products that originate from bidirectional promoters, yet they are degraded by the RNA exosome. Such pervasive transcription is prevalent across eukaryotes, yet its regulation and function is poorly understood. We hypothesized that chromatin architecture at cryptic promoters may regulate ncRNA transcription. Nucleosomes that flank promoters are highly enriched in two histone marks: H3-K56Ac and the variant H2A.Z, which make nucleosomes highly dynamic. These histone modifications are present at a majority of promoters and their stereotypic pattern is conserved from yeast to mammals, suggesting their evolutionary importance. Although required for inducing a handful of genes, their contribution to steady-state transcription has remained elusive. In this work, we set out to understand if dynamic nucleosomes regulate cryptic transcription and how this is coordinated with the RNA exosome. Remarkably, we find that H3-K56Ac promotes RNA polymerase II occupancy at a large number of protein coding and noncoding loci, yet neither histone mark has a significant impact on steady state mRNA levels in budding yeast. Instead, broad effects of H3-K56Ac or H2A.Z on levels of both coding and ncRNAs are only revealed in the absence of the nuclear RNA exosome. We show that H2A.Z functions with H3-K56Ac in chromosome folding, facilitating formation of Chromosomal Interaction Domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA levels, perhaps in part by regulating higher order chromatin structures. Together, these chromatin factors achieve a balance of RNA exosome activity (yin; negative) and Pol II (yang; positive) to maintain transcriptional homeostasis

Determining the Roles that DICER1 and Noncoding RNAs Play in Endometrial Tumorigenesis

Cancer is both a genetic and epigenetic disease. Changes in DNA methylation, histone modifications, and microRNA processing promote tumorigenesis, just as mutations in coding sequences of specific genes contribute to cancer development. In my thesis work I sought to determine the role that noncoding RNAs play in endometrial tumorigenesis. Aberrant methylation of the promoter region of the MLH1 DNA mismatch repair gene in endometrial cancer is associated with loss of MLH1 expression and a mutator phenotype in endometrial and other cancers. The molecular and cellular processes leading to aberrant methylation of the MLH1 promoter region are largely unknown. I tested the hypothesis that the EPM2AIP1 antisense transcript at the MLH1 locus could be involved in MLH1 transcriptional silencing. I characterized the MLH1/EPM2AIP1 bidirectional promoter region in endometrial cancer and normal cell lines and found an abundance of forward and reverse transcripts initiating from a large region of nucleosome-free DNA in expressing cells. The DICER1 protein, which is necessary for processing small RNAs involved in post-transcriptional silencing, is downregulated in many cancers, including endometrial cancer. I used genomic methods: RNA-Seq and MeDIP/MRE) to characterize the transcriptome and methylome of endometrial cancer cells depleted of DICER1. Using a combination of computational and wet lab methods I showed that reduced DICER1 triggers an interferon response in cancer cells because of accumulation of pre-microRNAs that activate immune sensors of viral dsRNA. The methylome of DICER1 knockdown cells revealed subtle changes in methylation, including decreased methylation at the Alu family of repetitive elements. Small RNAs processed by DICER1 may thus be involved in silencing repetitive regions. Non-coding RNA has effects on endometrial cancer cells that may contribute to tumorigenesis, such as influencing the active state of the MLH1 gene and modulating the immune response

Histone complement of a rapidly evolving chordate Oikopleura dioica: Developmental and sex-specific deployment of novel and universal histone variants and their posttranslational modifications

The packaging of DNA into nucleosomes is a fundamentally conserved property of the eukaryotic nucleus which is evident in the conservation of histone sequences. Nevertheless, it is now clear that histone sequence variants have diversified in many species to assume crucial roles in the regulation of gene expression, DNA repair, chromosome segregation and other processes. While considerable data exist on coding sequences of histones and some selected histone variants in a wide variety of organisms, the information available on total histone gene complements is much more limited. Oikopleura dioica (Od) is a dioecious marine urochordate that occupies a key phylogenetic position near the invertebrate-vertebrate transition with the smallest genome ever found in a chordate (70 Mb). Its short life cycle is characterized by a developmental switch between mitotic and endocycling cells, making O. dioica an attractive model to study the spatial and temporal use of histone variants and posttranslational histone modifications (PTMs) throughout development and in different cell cycle types. We have characterized the complete histone gene complement and the developmental expression of histone genes present in the first assembly of the O. dioica draft genome and identified the major Od PTMs by massspectrometric analysis. Furthermore, we analyzed the dynamics and distribution of phosphorylated H3 variants during mitosis and meiosis of O. dioica and the deposition of the centromeric variant OdCenH3 in mitotic and endocycling cells with respect to centromeric PTMs. The Od histone gene complement displays several features not known from other chordates, including male-specific variants in all of the core histone families, N-terminal H2A.Z splice variants, and a diverse array of H2A variants but absence of the near universal variant H2AX. The results here suggest significant plasticity in histone gene organization, the variation within histone families and the chromosomal distribution of mitotic PTMs within the chordate lineage. This further supports the view that histone gene complements may also evolve adaptively to the specific life history traits, cell cycle regulation and genome architecture of organisms

Sunlight and Herpes Virus

The Herpesviridae are a family of viruses widely spread in nature that can infect a wide variety of species. After the primary infection, the human alphaherpesvirinae sub-family remains quiescent in the nerve ganglia from which it can periodically reactivate, causing clinical manifestations. Although spontaneous recurrences are possible, a wide variety of internal and external triggers may lead to transformation of the Herpes Simplex and Varicella-Zoster Viruses from a dormant to a proliferative state. Sunlight is a potent stimulus for the alphaherpesvirinae reactivation. The purpose of this paper is to analyze various features of this correlation and several steps you can take to lower your risk of triggering a herpes outbreak after sun exposure. Learning how to reduce the recurrence is extremely important and it is necessary: to perform a gradual and progressive sun exposure; to know what garments to wear; to know the environmental conditions of exposure; to know each skin phototype; to use a protective product against UVB and UVA with sun protection factor suitable for each phototype and environmental conditions

The role of RFX transcription factors in neurons and in the human brain

RFX transcription factors (TFs) are conserved in animals, fungi and some amoebae, but not in algae, plants and protozoan species. The conservation is based on the protein sequence of the DNA binding domain (DBD). The RFX DBD recognizes and binds to a DNA sequence motif called the X-box. In addition to the DBD, most RFX TFs have a Dimerization domain (DIM). The DIM enables RFX TFs to form homo- or heterodimers in detecting the X-box motif, rendering the X-box often described as an imperfect palindromic sequence of two 6-bp half-sites with variable spacers. So far, RFX TFs are known to regulate gene transcription in cell cycle, DNA repair, immune response, collagen transcription, insulin production, spermatogenesis and hearing. In animals, the most common feature of RFX TFs is their regulation of ciliogenesis and the maintenance of specialized functions of ciliated cells. Cilia are hair-like cell protrusions. They are present in all animals but absent in many species of fungi, amoebae and flowering plants. Based on the inner structure, cilia can be divided into two types, the primary cilia (one cilium per cell) and the motile cilia (either as mono-cilia or multiple-cilia per cell). The primary cilia are less understood despite being present on nearly every cell in the human body. Humans have eight RFX genes (RFX1-8) which are expressed in diverse tissues and cell types. This thesis serves to expand knowledge of the RFX TF family in humans and their role in primary cilia and neurons, with interest in human brain development and function. We used databases (Paper I), human cell lines (Papers I and II) and the worm C. elegans (Paper III) as our materials for experimentation. In Paper I, we performed an extensive survey of RFX1-8 expression by transcription start site (TSS) counts from the FANTOM5 database. RFX1-4 and RFX7 are prominently expressed in different brain tissues and spinal cord, making them the reference RFX TFs for neurons and the human brain. Furthermore, we predicted the regulation preference of RFX TFs based on co-clustering expression analysis with known RFX target genes. We also analyzed the positioning of the X-box motifs in the human genome and uncovered potential upstream regulators of RFX genes. In Paper II, we explored the role of RFX TFs in the context of developmental dyslexia, a developmental disorder of the human brain. The dyslexia candidate genes DYX1C1, DCDC2 and KIAA0319 have functional X-box motifs in their promoter regions, as shown by luciferase reporter assay of wild-type versus mutated X-boxes. By siRNA knockdowns of RFX1-3, we showed a complex regulatory mechanism among RFX1-3 in regulating DYX1C1 and DCDC2. Additionally, both DYX1C1 and DCDC2 localize to the primary cilia. In Paper III, we performed microarray analysis of target genes of DAF-19, the sole RFX TF of C. elegans, at three developmental stages (3-fold embryo, L1-larvae and adult). At all stages, DAF-19-regulated target genes were significantly enriched in neurons. Using transcriptional GFP reporter constructs, we observed that DAF-19-dependent target genes (both activated and repressed) affected only neurons, both ciliated and non-ciliated. Altogether, we provided insight into the role of RFX TFs for primary cilia and neurons. We speculate that RFX TFs and primary cilia continue to play a defined role for mature neuron function in the human brain

CRISPRing the Human Genome for Functional Regulatory Elements

The sequence of DNA is a code that contains all the information that is required for life (as we know it). DNA is stored inside the nucleus of cells and its sequence is replicated during cell division to ensure that the genetic information is transmitted to the daughter cells. The information contained in DNA is copied into RNA by a process called transcription. RNA acts as a messenger (mRNA) to carry the information between the nucleus and the cytoplasm, where it is used as a template to produce proteins through a process called translation. Proteins are the main effectors of all biological functions in the cell. However, the information required to make proteins (called “coding DNA sequence”) comprises only a small portion (~2%) of the entire human genome sequence. For several decades, it was generally accepted that the remaining 98% of the genome sequence had no biological function and, because of that, it was dubbed “junk DNA”. The discovery of non-coding DNA sequences that control the expression of genes challenged this idea, and revealed that there is biological function beyond protein-coding sequences. These non-coding sequences are called “regulatory elements” and they are classified into four classes according to their function: promoters, enhancers, insulators and silencers. Among them, enhancers play a critical role in activating the expression of genes in response to intra- and extra-cellular stimuli – which is essential for the development of complex organisms. Previous studies suggest that the human genome might contain more than one million enhancers – a much higher number compared to the