Extension of imprinted silencing in the Igf2r cluster in extra-embryonic tissues

Geprägte Gene kommen in Gruppen vor, welche meist von einer makro-nichtprotein- kodierende (mnk) RNA reguliert werden. Die inneren Gene der Gruppe können von der mnk RNA überlappt werden und zeigen im gesamten Embryo und in extra-embryonalen Geweben geprägte Expression. Die äußeren Gene der Gruppe werden nicht von der mnk RNA überlappt und zeigen nur in extra-embryonalen Geweben eine geprägte Expression. Extraembryonales Gewebe besteht aus unterschiedlichen Zelltypen und kann von mütterlichem Gewebe kontaminiert werden. Dadurch kann die genomische Prägung des untersuchten Gens durch biallelische Expression im kontaminierenden Gewebe maskiert werden. Um Kontaminationen zu vermeiden, haben wir eine effiziente Methode entwickelt, um eine pure Population von viszeralem Endoderm (VE) aus dem Dottersack zu isolieren. VE ist somit eine gutes extra-embryonales Model- Gewebe um neue geprägte Gene zu finden. Die monoallelische “T-hairpin” Deletion, welche die gut charakterisierte geprägte Igf2r Gengruppe einschließt, wird genutzt um im VE nach neuen geprägten Genen zu suchen. In der Igf2r Gengruppe reguliert die mnk RNA Airn die Expression der geprägten Gene. Um zu testen, ob ein geprägtes Gen zu der Igf2r Gengruppe gehört, habe ich untersucht ob dieses von Airn reguliert wird. Dabei habe ich Wildtyp Embryonen mit Embryonen verglichen, welche eine gekürzte, nicht funktionelle Variante von Airn exprimieren. Mit diesem Ansatz habe ich 15 Gene getestet, wovon drei vorranging vom mütterlichen Allele exprimiert werden. Zusätzlich zeigen meine vorläufigen Daten, dass das in der Plazenta mütterliche geprägte Gen Pde10a von Airn reguliert wird. Zusammenfassend zeigen meine Ergebnisse, dass die Igf2r Gruppe nicht wie bisher angenommen 440Kb groß ist, sondern sich in extra-embryonalen Gewebe auf bis zu 4Mb ausbreitet.Imprinted genes occur in clusters, several of which are regulated by a macro long non-protein-coding (lnc) RNA. Inner genes within the cluster can be overlapped by the macro lncRNA and show multi-lineage (ML) imprinted expression in the embryo and extra-embryonic tissues, while outer genes are not overlapped by the macro lncRNA and show extra-embryonic-lineage (EXEL) specific imprinted expression. Extra-embryonic tissues are a mixture of ML and EXEL cell types, so imprinted expression may be masked by biallelic expression in ML tissues if a whole organ is examined. We have developed an efficient method to isolate a pure population of visceral endoderm (VE), an EXEL cell type, providing a system to discover new imprinted genes as EXEL tissues show imprinted expression of both, ML and EXEL genes, and masking is avoided. Using this system I have taken advantage of the T-hairpin uniparental deletion to systematically check for the limits of the well-characterised Igf2r cluster. Imprinted genes can be definitively shown to be part of these Igf2r cluster by determining if they are regulated by the Airn macro lncRNA. This is done by comparing wildtype embryos with those containing a truncated and non-functional Airn. Using this system I have tested 15 genes and identified three genes showing biased imprinted expression in VE and shown that they are part of the Igf2r cluster. In addition, I have preliminary data confirming a recent report that Pde10a shows imprinted expression in placenta, and have shown that imprinted silencing is regulated by Airn. Taken together these results extend the known region showing imprinted expression in the Igf2r cluster from 440Kb up to 4Mb in extra-embryonic tissues

RNA, the Epicenter of Genetic Information

The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers

Cancer epigenetics

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.A Epigenética traz um largo percurso histórico, desde a sua descoberta até á sua aplicação em práticas laboratoriais da vida real. Fundamentalmente, Epigenética traduz-se nas modificações químicas que o DNA sofre, mas que não alteram diretamente o código genético, alterando a regulação dos genes através de uma grande variedade de mecanismos capazes de modificar o estado da cromatina, bem como o das proteínas responsáveis pela sua transcrição. A metilação no DNA, as modificações nas histonas e os non-coding RNAs são os principais mecanismos através dos quais os traços epigenéticos são passados entre as gerações. Estas marcas epigenéticas são transmitidas de acordo com a sua “impressão genómica”, o processo pelo qual os genes “impressos” mantêm a sua informação genética e são capazes de a transmitir às novas linhagens de células, apesar de estas sofrerem um reset epigenético durante o seu desenvolvimento embrionário, de maneira a adquirir o seu estado de pluripotência. Como o Cancro mantém a sua posição no pódio, em segundo lugar entre as doenças que causam mais mortes anualmente, as variações epigenéticas nos cancros têm sido um campo de estudo muito abordado recentemente nesta área, de maneira a potenciar as terapêuticas e os procedimentos de diagnóstico. Através de cuidadosas investigações, foi descoberto que os tecidos tumorais apresentavam frequentemente marcas epigenéticas nas suas células, e os recentes avanços tecnológicos permitiram o estudo destes traços epigenéticos mutados em diferentes tipos de cancros. Foi ainda provado que as células tumorais sofrem um fenómeno idêntico ao reset epigenético, mas que é controlado pelas próprias células tumorais, o que lhes permite a continua renovação do seu estado de pluripotência, concedendo-lhes liberdade para se de-diferenciarem e trans-diferenciarem. Todos os traços epigenéticos previamente mencionados já foram encontrados alterados em diversas maneiras em diferentes tecidos cancerosos, tendo sido levadas a cabo diversas investigações no sentido de desenvolver procedimentos de diagnóstico e terapias com o âmbito de tratar estes cancros. Apesar de se considerar um campo relativamente recente, o estudo de variações epigenéticas no cancro já conta com bastantes procedimentos de diagnóstico e terapias que são aplicadas por todo o mundo.Epigenetics have come a long way since its discovery, through its application in real world practices. Epigenetics in its fundamental meaning, translates in chemical modifications in the DNA sequence that does not directly alter the DNA code, but instead alters its gene regulation through a variety of mechanisms that change the chromatins state, as well as the proteins responsible for its transcription. DNA methylation, histone modifications and non-coding RNAs are the main mechanisms through epigenetic traits are passed down from generation to generation. These epigenetic marks are passed down through generations according to its genomic imprinting, the process through which the imprinted genes maintain their epigenetic information and are able to transmit them to the new lineages of cells, although they go through an epigenetic reset process during embryonic development, in order to gain its pluripotent cell state. As Cancer still maintains its podium position as number two amongst the diseases which cause the most deaths annually, cancer epigenetics has been a recent field of study amongst cancer subjects, in order to enhance cancer therapies and diagnostic procedures. Through thoroughtfully cared investigations, it was found that tumorous tissues often present epigenetic marks in their cells, and recent advances in technical methods have allowed the study of these mutated expressed epigenetic traits along the different cancer types. It was also proven that tumorous cells undergo a similar process as epigenetic reset, but this one is controlled by the tumorous cell and allows it to continually regain its pluripotent state, granting it full liberty to de-differentiate and transdifferentiate. Each of the epigenetic traits have already been found to be present in numerous altered ways in various cancerous tissues, and therefore further investigations have already developed diagnostic and therapeutical procedures in order to treat this epigenetically modified cancers. Although a considerably recent field of study, cancer epigenetics already counts with multiple diagnostic and therapeutical methods being applied all over the world, and it only hopes to grow even further

Tracing the evolution of long non-coding RNAs: Principles of comparative transcriptomics for splice site conservation and biological applications

Eukaryotic cells exhibit an extensive transcriptional diversity. Only about a quarter of the total RNA in the human cell can be accounted for by messenger RNA (mRNA), which convey genetic code for protein generation. The remaining part of the transcriptome consists of rather heterogenous molecules. While some classes are well defined and have been shown to carry out distinct functions, ranging from housekeeping to complex regulatory tasks, a big fraction of the transcriptional output is categorized solely based on the lack of protein-coding capacity and transcript length. Several studies have shown, that as a group, mRNA-like long non-coding RNAs (lncRNAs), are under stabilizing selection, however at much weaker levels than mRNAs. The conservation at the level of primary sequence is even lower, blurring the contrast between exonic and intronics parts, which impedes traditional methods of genome-wide homology search. As a consequence their evolutionary history is a fairly unexplored field and apart from a few experimentally studied cases, the vast majority of them is reported to be poorly conserved. However, the pervasive transcription and the highly spatio-temporal specific expression patterns of lncRNAs suggests their functional importance and makes their evolutionary age and conservation patterns a topic of interest. By employing diverse computational methods, recent studies shed light on the common conservation of lncRNA’s secondary and gene structures, highlighting the significance of structural features on functionality. Splice sites, in particular, are frequently retained over very large evolutionary time scales, as they maintain the intron-exon-structure of the transcript. Consequently, the conservation of splice sites can be utilized in a comparative genomics approach to establish homology and predict evolutionarily well-conserved transcripts, regardless of their coding capacity. Since splice site conservation cannot be directly inferred from experimental evidence, in the course of this thesis a computational pipeline was established to generate comparative maps of splice sites based on multiple sequence alignments together with transcriptomics data. Scoring schemes for splice site motifs are employed to assess the conservation of orthologs. This resource can then be used to systemically study the conservation patterns of RNAs and their gene structures. This thesis will demonstrate the versatility of this method by showcasing biological applications of three distinct studies. First, a comprehensive annotation of the human transcriptome, from RefSeq, ESTs and GENCODE, was used to trace the evolution of human lncRNAs. A large majority of human lncRNAs is found to be conserved across Eutheria, and many hundreds originated before the divergence of marsupials and placental mammals. However, they exhibit a rapid turnover of their transcript structures, indicating that they are actual ancient components of the vertebrate genome with outstanding evolutionary plasticity. Additionally, a public web server was setup, which allows the user to retrieve sets of orthologous splice sites from pre-computed comparative splice site maps and inspect visualizations of their conservation in the respective species. Second, a more specific data set of non-colinearly spliced latimerian RNAs is studied to fathom the origins of atypical transcripts. RNA-seq data from two coelacanth species are analyzed, yielding thousands of circular and trans-spliced products, with a surprising exclusivity of the majority of their splice junctions to atypically spliced forms, that is they are not used in linear isoforms. The conservation analysis with comparative splice site maps yielded high conservation levels for both cir- cularizing and trans-connecting splice sites. This fact in combination with their abundance strongly suggests that atypical RNAs are evolutionarily old and of functional importance. Lastly, comparative splice site maps are used to investigate the role of lncRNAs in the evolution of the Alzheimer’s disease (AD). The human specificity of AD clearly points out a phylogenetic aspect of the disease, which makes the evolutionary analysis a very promising field of research. Protein- coding and non-protein-coding regions, that have been identified to be differentially expressed in AD patients, are analyzed for conservation of their splice site and evolution of their exon-intron-structure. Both non-coding and protein-coding AD-associated genes are shown to have evolved more rapidly in their gene structure than the genome at large. This supports the view of AD as a consequence of the recent rapid adaptive evolution of the human brain. This phylogenetic trait might have far reaching consequences with respect to the appropriateness of animal models and the development of disease-modifying strategies.Eukaryotische Zellen legen eine umfangreiche transkriptionelle Vielfalt an den Tag. Nur etwa ein Viertel der in der menschlichen Zelle enthaltenen RNA ist messenger RNA (mRNA), welche den genetischen Code für die Proteingenerierung übermittelt. Der verbleibende Anteil des Transkriptoms besteht aus eher heterogenen Molekülen. Während einigen wohldefinierten Klassen spezifische Funktionen zugeordnet werden können, welche von Zellhaushalt bis zu komplexen regulatorischen Aufgaben reichen, wird ein großer Teil der transkriptionellen Produktion ausschließlich auf Grundlage der fehlenden Kodierungskapazität und der Transkriptlänge kategorisiert. Einige Studien zeigten, dass mRNA-ähnliche lange nicht-kodierende RNA (lncRNA) als Gruppe unter stabilisierender Selektion stehen, wenn auch in einem weitaus geringeren Ausmaß als mRNAs. Die Konservierung auf Ebene der primären Sequenz ist sogar noch niedriger, wodurch der Kontrast zwischen exonischen und intronischen Elementen verschwimmt und Methoden der traditionellen Homologiesuche erschwert werden. Infolgedessen ist die evolutionäre Geschichte der lncRNAs ein recht unerforschtes Gebiet und abgesehen von ein paar vereinzelten Fallstudien wird die große Mehrheit als schwach konserviert vermeldet. Die tiefgreifende Transkription und die in Raum und Zeit hochspezifischen Expressionsmuster von lncRNA deuten jedoch auf deren funktionelle Bedeutung hin und machen ihr evolutionäres Alter und ihre Konservierungsmuster zu einem Thema von Interesse. Durch die Verwendung von computergestützten Methoden konnten jüngste Studien die verbreitete Konservierung von Sekundär- und Genstruktur von lncRNAs aufzeigen, was die Signifikanz von strukturellen Merkmalen in Bezug auf deren Funktionalität unterstreicht. Spleißstellen im besonderen werden oft über lange evolutionäre Zeitspannen erhalten, da sie die Intron-Exon-Struktur des Transkripts bewahren. Folglich, kann die Konservierung von Spleißstellen durch einen Ansatz der vergleichenden Genomik benutzt werden, um Homologie herzuleiten und evolutionär gut konservierte Transkripte unabhängig von deren Kodierungskapazität zu prognostizieren. Da es nicht möglich ist die Spleißstellenkonservierung direkt anhand von experimentellen Indikatoren abzulesen, wurde im Zuge dieser These eine computergestützte Methode entwickelt, welche, basierend auf multiplen Sequenzalignments und Transkriptomikdaten, “Vergleichskarten” von Spleißstellen erstellt. Ein Punktebewertungssystem für Spleißstellenmotive wird benutzt um die Konservierung der Orthologen zu beurteilen. Diese Resource kann anschließend verwendet werden um systematisch die Konservierungsmuster von RNAs und deren Genstrukturen zu untersuchen. Diese Arbeit wird die Vielseitigkeit dieser Methode demonstrieren, indem die biologische Anwendung in drei verschiedenen Studien präsentiert wird. Zuerst wird eine umfassende Annotation des menschlichen Transkriptoms, basierend auf RefSeq, EST und GENCODE, benutzt, um die Evolution von humanen lncRNAs nachzuvollziehen. Es konnte festgestellt werden, dass eine große Mehrheit der menschlichen lncRNAs innerhalb der Eutheria konserviert ist und mehrere hundert bereits vor der Auseinanderentwicklung von Beuteltieren und höheren Säugetieren entstanden. Dennoch zeigen sie eine rasante Veränderung in ihren Transkriptstrukturen, welche darauf hindeutet, dass sie tatsächlich alte Bestandteile von Vertebratengenomen mit bemerkenswerter evolutionärer Formbarkeit sind. Zusätzlich wurde ein öffentlicher Webserver aufgesetzt, der dem Nutzer ermöglicht Datensätze orthologer Spleißstellen aus vorgenerierten Vergleichskarten zu extrahieren und Visualisierungen der Konservierung in den jeweiligen Spezies zu betrachten. Als zweites wird ein spezifischerer Datensatz von nicht-linear gespleißten Latimeria-RNA untersucht um die Ursprünge untypischer Transkripte zu ergründen. Die Analyse der RNA-seq Daten zweier Exemplare des Quastenflossers ergab tausende zirkulärer und Transspleiß-Produkte, wobei die Mehrheit der Spleißverbindungen eine überraschende Exklusivität für untypisch gespleißte Formen aufzeigt, d.h. diese werden nicht für lineare Isoformen genutzt. Die Konservierungsanalyse mit Spleißstellen-Vergleichskarten ergibt hohe Konservierungsniveaus sowohl für zirkulärisierende als auch für trans-verbindende Spleißstellen. Diese Tatsache in Kombination mit ihrem häufigen Vorkommen, deutet stark darauf hin, dass untypische RNAs evolutionär alt und von funktioneller Bedeutung sind. Zuletzt werden Spleißstellen-Vergleichskarten benutzt um die Rolle von lncRNAs in der Evolution der Alzheimer-Krankheit (AK) zu untersuchen. Die Spezifität der AK auf den Menschen weist klar auf einen phylogenetischen Aspekt der Krankheit hin, was deren evolutionäre Analyse zu einem vielversprechenden Forschungsgebiet macht. Proteinkodierende und nicht-proteinkodierende Regionen, bei denen eine differentielle Expression in AK-Patienten erkannt wurde, werden auf die Konservierung ihrer Spleißstellen und Evolution ihrer Exon-Intron-Strukturen hin analysiert. Es kann nachgewiesen werden, dass sich die Genstruktur von sowohl nicht-kodierenden als auch von proteinkodierenden AK-assoziierten Genen schneller entwickelt als das Genom im Allgemeinen. Das unterstützt die Auffassung, dass AK die Folge einer kürzlichen rasanten adaptiven Evolution des menschlichen Gehirns ist. Diese phylogenetische Eigenschaft könnte weitreichende Konsequenzen in Bezug auf die Angemessenheit von Tiermodellen und die Entwicklung von krankheitsmodifizierenden Strategien haben

Assisted Reproductive Technologies Disrupt Genomic Imprinting in Human and Mitochondria in Mouse Embryos

Infertile couples worldwide use assisted reproductive technologies (ARTs) to help conceive their own biological child. Due to the rising use of ARTs, there is continual emergence of new techniques implemented in human fertility clinics. When treatment is successful, there is an increased risk even within singletons for perinatal complications including preterm birth, intrauterine growth restriction, low and high birth weight and genomic imprinting disorders Beckwith Wiedemann Syndrome, Angelman Syndrome, and Silver-Russel Syndrome. Consequently, there is a need to investigate the effects of these treatments on the manipulated oocyte and preimplantation embryo. To address this, I first analyzed the combined effects of multiple ARTs on imprinted DNA methylation in human day 3 (6 to 8 cells) and blastocyst-stage embryos. As imprinted DNA methylation is acquired during gametogenesis and maintained throughout preimplantation development, I hypothesized that ARTs disrupt this regulation in donated, good quality, human preimplantation embryos. I observed that seventy-six percent of day 3 embryos and fifty percent of blastocysts exhibited perturbed imprinted methylation at the SNRPN, KCNQ1OT1 and/or H19 domains. This frequency was similar to that previously observed in the mouse, and importantly demonstrated that extended culture did not pose a greater risk for imprinting errors. Overall, human preimplantation embryos generated with ARTs possessed a high frequency of imprinted methylation errors. Next, I hypothesized that a single, indispensible ART treatment, ovarian stimulation, disrupts mitochondria in mouse oocytes and preimplantation embryos. Ovarian stimulation led to a decreased total and active mitochondrial pool in high hormone-treated oocytes, and an increase in the percentage of oocytes displaying mislocalization of active mitochondria. Although the total mitochondrial pool was unchanged in hormone-treated preimplantation embryos compared to controls, the active mitochondrial pool was decreased in hormone-treated 1-cell, 2-cell, morula and blastocysts. Ultimately, the lower active mitochondrial pool in treated embryos was associated with a decreased percentage of outer blastomeres containing high amounts of active mitochondria in morula and blastocysts. In blastocysts, this was associated with increased superoxide levels. Overall, my results provide novel insight onto ARTs-induced disruption of imprinted DNA methylation and mitochondria in human and mouse preimplantation embryos, respectively

Mutations in regulators of the epigenome and their effects on the DNA methylome

Genome-wide profiling for genetic alterations in cancer has identified mutations in genes that are associated with epigenetic programming of genomes for DNA methylation patterns, histone modifications patterns and the positioning of nucleosomes. Here a systematic evaluation of the available cancer genome profiling data established by large international consortia, in order to identify recurrently mutated genes or pathways was described. Using curated list of approximately 700 epigenetic regulators and currently available genome-wide datasets on genetic and epigenetic alterations in cancers, the distribution of alterations in epigenetic regulators was described. Epigenetic genes were classified as potential oncogenic or those with tumor-suppressor function based on the location of mutations relative to functional domains and their frequencies. A panel of 50 epigenetic genes, including: DNMTs, histones (H3F3A, HIST1H3B), histone editors (KDM5C, KDM6A) and writers (MLLs, SETD2, EZH2, ATM) that can promote epigenetic changes in cancer was identified. Using correlative analysis of publicly available methylation data with information on deregulated epigenetic driver genes, many identified subtype-specific methylation clusters were correlated with groups of up to 3 epigenetic regulators. This analysis provides a source for the identification and link between methylation groups and deregulated epigenetic genes. Major cancer specific methylation changes have been observed in promoters and gene bodies. Tissue-specific cancer methylation differences have been located in enhancers and regulatory regions of non-coding RNAs. Based on identified results, the major mechanism of non-coding RNA deregulation in cancer has been investigated on independent data cohort. Using integrative analysis of non-coding RNA in early-onset prostate cancer, non-coding RNAs were classified as tumor-suppressive and oncogenic. About 120 novel prostate cancer specific non-coding RNAs that have been epigenetically deregulated have been identified. Our study on the defects in regulators of the epigenome will help to understand mechanisms leading to distinct epigenetic patterns and will allow the molecular validation of defined correlations in experimental settings

Diverse Roles of RNA-protein Interactions: From Viral Antagonism to Mammalian Development

RNA is a widely utilized and integrated component of core cellular function because of its abilities to recognize and hybridize to nucleic acid templates, spatially localize to different compartments within the cell, bind combinatorially to effector molecules, and in some cases directly catalyze chemical reactions. In this thesis, I describe three cases, illustrating the biomolecule’s unique importance in several different aspects of cellular homeostasis. Chapter 1 provides historical context for studying RNA-protein interactions within RNA biology and Virology. Chapter 2 details experiments in which we explored RNA as a central target of host cell takeover by SARS-CoV-2. In the process, we highlight the importance of RNA in many integral complexes within the cell, including components of the spliceosome, the eukaryotic ribosome, and signal recognition particle. Chapter 3 presents data from our consideration of RNA within the context of cis gene regulation. We specifically focus on a model RNA-binding protein, SMRT/HDAC1 Associated Repressor Protein (SHARP), and the paternally imprinted long non-coding RNA, Kcnq1ot1, as case studies. Chapter 4 describes our dissection of a transcriptional circuit involving SHARP and discusses implications of RNA-binding to developmentally sensitive circuits and processes. Finally, Chapter 5 poses new questions raised by these studies. Together these data emphasize the diverse and unique role RNA plays in cellular homeostasis and suggest additional roles in nuclear compartment stabilization and crosstalk.</p

EVOLUTION OF THE ZHX TRANSCRIPTION FACTOR FAMILY AND ANALYSIS OF ZHX2 TARGET GENES \u3cem\u3eCYP2A4\u3c/em\u3e AND \u3cem\u3eCYP2A5\u3c/em\u3e IN MOUSE LIVER

The liver is the largest internal organ and performs a wide variety of functions to maintain organismal homeostasis. While some liver functions are carried out by all hepatocytes, other functions are restricted to certain populations of hepatocytes within the liver. This phenomenon, called zonal gene regulation or liver zonation, controls may metabolic processes within the liver including ammonia detoxification; glucose homeostasis; bile acid and glutamine synthesis; and metabolism of xenobiotics, lipids, and amino acids. The liver also expresses many genes in a developmental or sex-biased manner. Some genes are expressed at higher levels early or late in development, or alternatively, in male or female liver. Several years ago, our lab identified a transcription factor called Zinc finger and homeoboxes 2 (Zhx2) based on its ability to control the silencing of genes that are normally expressed in the fetal liver. Zhx2 belongs to a small gene family that also includes Zhx1 and Zhx3. These four exon genes have a rather unique structure in that their entire protein coding region is located on an unusually large third exon. Preliminary studies indicate that these proteins are found only in vertebrates. I have performed a comprehensive analysis of Zhx proteins across a number of chordate species to determine their relationship throughout chordate evolution. Using multiple sequence alignment and phylogenetic tree-building, my studies have found that the primordial Zhx gene is most related to Zhx3 and that this gene exists in lower chordates including lancelet, sea squirt, and sea lamprey. Additional studies from our lab showed that Zhx2 regulates numerous hepatic genes in the adult liver, including cytochrome p450 (Cyp) genes as well as other genes that exhibit sex-biased expression. Previous studies have demonstrated that female-biased expression of Cyp2a4, is controlled, in part, by Zhx2. I have extended these studies to perform a comprehensive analysis of Cyp2a4 and the highly related Cyp2a5 gene. Despite the high similarity of these two Cyp genes, my data indicate that these genes exhibit different zonal expression patterns and are differentially regulated in the regenerating liver. In the course of these studies, I discovered and characterized antisense transcripts for both Cyp2a4 and Cyp2a5. Both Cyp2a4as and Cyp2a5as have positively correlated expression patterns compared to their sense counterparts. In contrast to Cyp2a4 and Cyp2a5, Cyp2a4as and Cyp2a5as show sex-biased expression patterns earlier in development, suggesting that they might contribute to later sex-biased patterns established for Cyp2a4 and Cyp2a5