University, Diss., 2006--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2005.

Techn. Universiẗat, Diss., 2001--Braunschweig.

Epigenetics refers to heritable changes in phenotype without alterations to the genotype. Epigenetic changes involve two main mechanisms: DNA methylation and histone modification. Methylation of DNA at cytosine bases is the best-studied epigenetic process to date. CpG methylation states are thought to be maintained throughout cell divisions. However, loss of DNA methylation or DNA demethylation has been observed in specific stages of mammalian development. Such prominent examples of developmental DNA demethylation processes occur in developing primordial germ cells and in preimplantation embryos. However, little is known about DNA methylation changes of other tissues in mammalian development. Therefore, the first aim of this PhD study was to investigate changing nuclear distributions and levels of DNA methylation during development in order to discover dynamic variations amongst developing mouse tissues. In addition, a transgenic MBD-GFP mouse was employed to visualise DNA methylation in tissues. Several hypothetical mechanisms for the enzymatic removal of 5mC have been proposed. One of the proposed candidates is Tet-mediated successive oxidation of 5mC to generate 5hmC, 5fC and 5caC. 5hmC has therefore been considered as a transient intermediate in an active cytosine demethylation pathway. Nevertheless, some studies suggest that 5hmC may also function as an epigenetic modification in its own right. Thus, the second aim of this study was to address the research question of how and where 5hmC originates during development. In order to be able to identify tissues undergoing dynamic nuclear changes in DNA methylation and hydroxymethylation states during early mouse development, new working protocols for immunodetection of 5mC and 5hmC on tissue cryosections were required. The protocol optimisation for 5mC immunodetection is discussed in greater detail in Chapter 3. It was found that DNA methylation immunostaining of cryosections required heat-mediated DNA denaturation, which was partly compatible with protein immunostaining. Next, Chapter 4 focuses on identifying tissues undergoing dynamic changes in 5mC and 5hmC patterns during development from E9.5 to E14.5 mouse embryonic stages, using optimised immunohistochemistry protocols. These protocols revealed interesting dynamic observations of 5mC and 5hmC in the developing cerebral neocortex, surface ectoderm, liver, red blood cells, diaphragm and heart. These findings suggested that dynamic changes of 5mC and 5hmC during neocortical and compact myocardial development were in good agreement with a model where the formation of 5hmC may correlate with the loss of old 5mC, but the observations were also consistent with an involvement of de novo methylation in the generation of 5hmC. In other developing tissues, including surface ectoderm, liver, red blood cells, diaphragm and cardiac trabeculae, dynamic changes in 5mC and 5hmC levels were in line with a model where the 5hmC may act as a new epigenetic mark that functions independently. The optimised protocol also confirmed DNA demethylation of the germ cells at E12.5. The presence of three Tet family enzymes (Tet1, Tet2, Tet3) and de novo methyltransferase DNMT3A in mouse E12.5 tissues is reported in the second part of Chapter 4. It was found that Tet1, Tet2, Tet3 and Dnmt3a were present at detectable levels in neocortex, liver, diaphragm and heart. Contrastingly, no apparent signals for Tet1, Tet2, Tet3 and Dnmt3a were observed in red blood cells. This result was expected due to the very low levels of 5hmC staining in E12.5 red blood cells. The third aim of the present study was to investigate the existence of crosstalk between various epigenetic mechanisms. Thus, Chapter 5 focuses on exploring the relationship between 5mC and repressive histone marks, H3K9me3 and H3K27me3. Histone methylation dynamics at H3K9 and H3K27 were observed during mouse fetal development in neocortex and heart. The overall distribution patterns of H3K9me3 and H3K27me3 demonstrated strong association with developmental changes in 5mC, suggesting that these three repressive epigenetic marks work in concert to establish a silenced state of heterochromatin. Chapter 6, on the other hand, focuses on visualising DNA methylation in tissues using mouse transgenic tools. It was found that brain, liver, heart and neural tube expressed high levels of GFP. But no apparent developmental dynamics of GFP was observed. In conclusion, this study will contribute scientific understanding of dynamic DNA methylation and nuclear heterochromatin organisation during mammalian development, and its role in the specification and maintenance of cell lineages forming tissues and organs. This knowledge will provide insight into current barriers to cell fate reprogramming, which will be of benefit to cell regenerative biomedical technologies.

The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. While the active mark H3K4me3 is present around the transcription start site on the gene, the function of the H3K36me3 mark is unknown. A number of H3K36-specific histone methyltransferases (HMTs) have been identified, however the focus of this study is the HMT Hypb. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at a subset of genes. Upon differentiation, we observe that H3K36me3 becomes enriched at the 3’ end of several muscle-specific genes. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of the H3K36 HMT Hypb was performed in muscle myoblasts using shRNA. Upon Hypb knockdown, we were surprised to observe enhanced myogenesis. N-ChIP was also performed on differentiated Hypb knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. N-ChIP data show a drop in H3K36me3 enrichment levels on myogenin and Ckm genes. The possible occupancy of Hypb on the coding regions of muscle-specific genes was experimentally observed by cross-linked chromatin immunoprecipitation (X-ChIP) on differentiated C2C12 cells and subsequently confirmed by X-ChIP on knockdown lines where the occupancy was lost. A model is proposed that links the observed phenotype with H3K36me3.

Histone H3 lysine 4 (H3K4) methylation is an evolutionarily conserved mark commonly associated with transcription activation in eukaryotes. In mammals, this post-translational modification is deposited by the KMT2 family of H3K4 methyltransferases. Biochemical studies have shown that the enzymatic activity of the KMT2 enzymes is regulated by a core complex of four evolutionarily conserved proteins: WDR5, RbBP5, ASH2L and DPY30, collectively known as WRAD, which are all important for global H3K4 methylation. However, how these proteins interact and regulate the activity of the KMT2 enzymes is not well investigated. During my PhD, I have used structural and biochemical approaches to determine the interactions underlying formation of the core complex and regulation of KMT2 enzymatic activity. My research have shown that 1) WDR5 uses two peptide-binding clefts on opposite sides of its β-propeller domain to bridge the KMT2 enzymes to the regulatory subunit RbBP5, 2) the WDR5 peptidyl-arginine-binding cleft exhibits plasticity to accommodate the binding of all KMT2 enzymes and 3) RbBP5 S350 phosphorylation stimulates formation of the RbBP5-ASH2L complex and H3K4 methylation by the mammalian KMT2 enzymes. Collectively, these studies have provided the structural basis for understanding the important interactions governing KMT2 complex assembly and activity.

The nucleoplasmin (NPM) family of acidic histone chaperones and the FK506-binding (FKBP) peptidyl proline isomerases are both linked to chromatin regulation. In vertebrates, NPM and FKBP domains are found on separate proteins. In fungi, NPM-like and FKBP domains are expressed as a single polypeptide in nucleoplasmin-like FKBP (NPL-FKBP) histone chaperones. Saccharomyces cerevisiae has two NPL-FKBPs: Fpr3 and Fpr4. These paralogs are 72% similar and are clearly derived from a common ancestral gene. This suggests that they may have redundant functions. Their retention over millions of years of evolution also implies that each must contribute non-redundantly to organism fitness. The redundant and separate biological functions of these chromatin regulators have not been studied. In this dissertation I take a systems biology approach to fill this knowledge gap. First, I refine the powerful synthetic genetic array (SGA) method of annotating gene-gene interactions, making it amenable for the analyses of paralogous genes. Using these ‘paralog-SGA’ screens I define distinct genetic interactions unique to either Fpr3 or Fpr4, shared genetic interactions common to both paralogs, and masked genetic interactions which are direct evidence for processes where these enzymes are functionally redundant. I provide transcriptomic evidence that Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. I identify an important role for Fpr4 at the 5’ ends of protein coding genes and the non-transcribed spacers of ribosomal DNA. Finally, I show that yeast lacking Fpr4 exhibit a genome instability phenotype at rDNA, implying that this histone chaperone regulates chromatin structure and DNA access at this locus. Collectively, these data demonstrate that Fpr3 and Fpr4 operate separately, cooperatively and redundantly to regulate a variety of chromatin environments. This work is the first comprehensive and comparative study of NPL-FKBP chaperones and as such represents a significant contribution to our understanding of their biological functions. / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22746号 / 医博第4664号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 小川 誠司, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In eukaryotes, the enormously long DNA molecules need to be packaged together with histone proteins into nucleosomes and further into compact chromatin structures to fit it into the nucleus. This nuclear organisation interferes with all phases of transcription that require the polymerase to bind to DNA. During transcription – the process in which the hereditary information stored in DNA is transferred to many transportable RNA molecules - nucleosomes form a physical obstacle for polymerase progression. Thus, transcription is usually accompanied by processes mediating nucleosome destabilisation, including post-translational histone modifications (PTMs) or exchange of canonical histones by their variant forms. To the best of our knowledge, acetylation of histones has the highest capability to induce chromatin opening. The lysine modification can destabilise histone-DNA interactions within a nucleosome and can serve as a binding site for various chromatin remodelers that can modify the nucleosome composition. For example, H4 acetylation can impede chromatin folding and can stimulate the exchange of canonical H2A histone by its variant form H2A.Z at transcription start sites (TSSs) in many eukaryotes, including humans. As histone H4, H2A.Z can be post-translationally acetylated and as acetylated H4, acetylated H2A.Z is enriched at TSSs suggested to be critical for transcription. However, thus far, it has been difficult to study the cause and consequence of H2A.Z acetylation. Even though, genome-wide chromatin profiling studies such as ChIP-seq have already revealed the genomic localisation of many histone PTMs and variant proteins, they can only be used to study individual chromatin marks and not to identify all factors important for establishing a distinct chromatin structure. This would require a comprehensive understanding of all marks associated to a specific genomic locus. However, thus far, such analyses of locus-specific chromatin have only been successful for repetitive regions, such as telomeres. In my doctoral thesis, I used the unicellular parasite Trypanosoma brucei as a model system for chromatin biology and took advantage of its chromatin landscape with TSSs comprising already 7% of the total T. brucei genome (humans: 0.00000156%). Atypical for a eukaryote, the protein-coding genes are arranged in long polycistronic transcription units (PTUs). Each PTU is controlled by its own ~10 kb-wide TSS, that lies upstream of the PTU. As observed in other eukaryotes, TSSs are enriched with nucleosomes containing acetylated histones and the histone variant H2A.Z. This is why I used T. brucei to particularly investigate the TSS-specific chromatin structures and to identify factors involved in H2A.Z deposition and transcription regulation in eukaryotes. To this end, I established an approach for locus-specific chromatin isolation that would allow me to identify the TSSs- and non-TSS-specific chromatin marks. Later, combining the approach with a method for quantifying lysine-specific histone acetylation levels, I found H2A.Z and H4 acetylation enriched in TSSs-nucleosomes and mediated by the histone acetyltransferases HAT1 and HAT2. Depletion of HAT2 reduced the levels of TSS-specific H4 acetylation, affected targeted H2A.Z deposition and shifted the sites of transcription initiation. Whereas HAT1 depletion had only a minor effect on H2A.Z deposition, it had a strong effect on H2A.Z acetylation and transcription levels. My findings demonstrate a clear link between histone acetylation, H2A.Z deposition and transcription initiation in the early diverged unicellular parasite T. brucei, which was thus far not possible to determine in other eukaryotes. Overall, my study highlights the usefulness of T. brucei as a model system for studying chromatin biology. My findings allow the conclusion that H2A.Z regardless of its modification state defines sites of transcription initiation, whereas H2A.Z acetylation is essential co-factor for transcription initiation. Altogether, my data suggest that TSS-specific chromatin establishment is one of the earliest developed mechanisms to control transcription initiation in eukaryotes. / In Eukaryoten muss die genomische DNA zusammen mit Histonproteinen zu Nukleosomen und weiter zu kompakten Chromatinstrukturen verpackt werden, damit sie in den Zellkern passt. Diese Organisation behindert die Transkription bei jedem Schritt, bei dem die Polymerase an der DNA bindet. Während der Transkription – dem Prozess bei dem die in der DNA gespeicherte Erbinformation in viele transportable RNA Molekülen umgewandelt wird – stellen Nukleosomen ein physikalisches Hindernis für das Vorankommen der Polymerase dar. Aus diesem Grund wird die Transkription üblicherweise von Prozessen begleitet, die für die Destabilisierung der Nukleosomen sorgen, wie zum Beispiel post-translationale Modifizierung (PTM) der Histone oder der Austausch von kanonischen Histonproteinen durch eine ihrer Varianten. Soweit bisher bekannt ist Histonacetylierung am besten dafür geeignet, eine offene Chromatinstruktur bereit zu stellen. Die Lysinmodifizierung kann Interaktionen zwischen der DNA und den Histonen innerhalb eines Nukleosomes destabilisieren und als Andockstelle für einige Proteinkomplexe sogenannte Chromatin-Modellierer fungieren, die die Zusammensetzung eines Nukleosomes verändern können. Zum Beispiel, kann Acetylierung am Histon H4 das „Zusammenfalten“ des Chromatins erschweren und den Austausch von kanonischem H2A mit ihrer Variante H2A.Z an den Transkriptiosinitiationsstellen (TSSen) in vielen eukaryotischen Organismen, Menschen eingeschlossen, stimulieren. Wie Histon H4, kann auch H2A.Z post-translationell acetyliert werden und wie acetyliertes H4, findet man auch acetyliertes H2A.Z vor allem an TSSen. Deswegen geht man davon aus, dass es sehr wichtig für die Transkriptioninitiierung ist. Allerdings war es bisher nicht möglich, die Ursache und Wirkung von H2A.Z Acetylierung genauer zu untersuchen. Genom-weite Chromatinprofilstudien wie z.B. ChIP-Seq ermöglichen es die genomische Lokalisierung von vielen Histon-Modifizierungen und -Varianten zu bestimmen. Dennoch reichen sie nicht dafür aus alle Faktoren, die für die Bildung einer bestimmten Chromatinstruktur notwendig sind, gleichzeitig herauszufinden. Das würde voraussetzen, dass man alle Merkmale der genomischen Stelle kennt. Bisher waren Analysen von spezifischen Chromatinstellen nur erfolgreich, wenn das Chromatin von einer repetitiven Region, wie z.B. Telomeren, stammt. In meiner Doktorarbeit verwendete ich den einzelligen Parasiten Trypanosoma brucei als Modelsystem für Chromatinbiologie. Dabei machte ich mir dessen Chromatinorganisation zunutze, die eher untypisch für einen eukaryotische Organismus ist. TSSen machen hier ungefähr 7% des gesamten Genoms aus (Mensch: 0.00000156%). Protein-kodierende Gene sind in langen polycistronischen Transkriptionseinheiten (PTE) angeordnet. Jede dieser Einheiten besitzt eine eigene TSS, die vor der PTE liegt, und bis zu 10 kb lang sein kann. Jedoch, wie in anderen Eukaryoten, sind an den TSSen Nukleosomen angereichert, die sich durch acetylierte Histone und den Einbau der Histonvariante H2A.Z auszeichnen. Aus diesen Gründen verwendete ich T. brucei, um während meiner Doktorarbeit die Chromatinstrukturen, die TSSen auszeichnen, genauer zu untersuchen und die Faktoren, die bei der H2A.Z Positionierung und dadurch bei der Transkriptionsregulation in Eukaryoten eine Rolle spielen, herauszufinden. Dafür etablierte ich zuerst eine Methode, mit der man Chromatin von einer bestimmten genomischen Stelle isolieren kann und die es mir ermöglichen würde, die Merkmale von TSS-spezifischen und -unspezifischen Chromatin zu identifizieren. Später konnte ich das entwickelte Protokoll mit einer Methode zur Quantifizierung von Lysin-spezifischen Histonacetylierung kombinieren. Dadurch konnte ich herausfinden, dass Nukleosomen an trypanosomischen TSSen stark acetyliertes H2A.Z und H4 enthalten und dass für diese Modifizierungen die Histonacetyltransferasen HAT1 und HAT2 verantwortlich sind. Eine Reduzierung der HAT2-Levels führte zu einer Reduzierung von H4 Acetylierung, verschlechterte die gezielte H2A.Z Positionierung und führte dazu, dass die Transkriptioninitiierung sich verlagerte. Wohingegen eine Reduzierung von HAT1, die zwar nur einen kleinen Einfluss auf die H2A.Z Positionierung hatte, eine sehr starke Verringerung von acetyliertem H2A.Z und der Transkriptionsrate zur Folge hatte. Durch meine Ergebnisse konnte ich zeigen, dass in T. brucei, einem evolutionär divergenten eukaryotischem Organismus, die Prozesse der Histonacetylierung, H2A.Z Positionierung und Transkriptionsinitiierung sehr stark miteinander verbunden sind. Meine Arbeit ist des weiteren ein Beweis dafür, dass T. brucei ein sehr wichtiger Modellorganismus für die Forschung an Chromatin ist. Insgesamt erlauben meine Ergebnisse die Schlussfolgerung, dass H2A.Z, egal ob modifiziert oder nicht, ein Herausstellungsmerkmal für TSSen ist, während acetyliertes H2A.Z essentiell für die Transkriptionsinitiierung darstellt. Zusammengefasst, weisen die Daten meiner Doktorarbeit darauf hin, dass die Etablierung von bestimmten Chromatinstrukturen an TSSen eines der frühesten entwickelten Mechanismen zur Kontrolle der Transkriptionsinitiierung in Eukaryoten ist.

The fundamental unit of chromatin is the nucleosome, which consists of a core of eight proteins wrapped by DNA. This core is composed of four pairs of histone proteins: H2A, H2B, H3, and H4. The DNA wraps around the protein core ~1.7 times, facilitating compaction of DNA length in the cell. Further, the location of nucleosomes makes genomic elements encoded in the DNA, such as promoters or enhancers, accessible or inaccessible to RNA polymerase and transcription factors. Thus, where nucleosomes are located (or positioned), can play a major role in transcription or other cellular processes. Additionally, histone proteins are frequently post-translationally modified, and these modifications further play a role in cellular processes, and in some cases are even required for specific protein function. What positions nucleosomes, and the downstream results of positioning or post-translational modifications (PTMs) is a topic of prolific study. Nucleosome formation is not random. In vivo it is believed that chromatin remodelers are the primary determinant of where nucleosomes form, while in vitro the DNA itself is the primary determinant. Formation of nucleosomes in vitro is a potent tool to elucidate fundamentals of chromatin. Considering that in vitro nucleosome formation is dependent on free energy, morphology and base composition of the DNA influence the free energy of formation. We found that the ends of linear DNA fragments were much more likely to have in vitro nucleosomes form on them. While this has the potential to bias results, based on our observations we could not find any significant alteration of the overall underlying DNA sequence composition due to the end preference observed. Histone proteins frequently receive the PTMs of methylation or acetylation. Histone methylation is typically indicative of repressed genes, while histone acetylation is typically indicative of active genes. In vivo the addition and removal of methylation and acetylation is highly dynamic. We hypothesized that the histone PTMs of methylation and acetylation also played a role in where nucleosomes formed. Comparing both in vivo and in vitro datasets, we observed strikingly similar patterns of nucleosomes for several histone methylations and acetylations, suggesting that these PTMs do indeed direct nucleosome formation. Upon further investigation, the underlying DNA sequence preferences change when compared to unmodified nucleosomes. This suggests that the genome is encoded to position these marks in locations where they are likely to be needed.

Skeletal muscle development and growth involve significant changes in gene expression. The overall objective of this dissertation project was to identify transcription factors, enhancers, and mechanisms that control gene expression during skeletal muscle development and growth on a genome-wide scale. Three independent studies were conducted in this project. The objective of the first study was to identify potentially novel mechanisms that mediate myoblast differentiation, a process whereby the mononuclear muscle precursor cells myoblasts express skeletal muscle-specific genes and fuse with each other to form multinucleated myotubes. Comparing gene expression profiles in C2C12 cells, a widely used model of myoblasts, before and 6 days after induced myogenic differentiation by RNA sequencing (RNA-seq) revealed 11,046 differentially expressed genes, of which 5,615 and 5,431 were upregulated and downregulated, respectively. Functional enrichment analyses revealed that the upregulated genes were associated with biological processes or cellular components such as skeletal muscle contraction, autophagy, and sarcomere. In contrast, the downregulated genes were associated with biological processes or cellular components such as ribonucleoprotein complex biogenesis, mRNA processing, and ribosome. Western blot analyses showed an increased conversion of LC3-I to LC3-II protein during myoblast differentiation, further demonstrating the upregulation of autophagy during myoblast differentiation. Blocking the autophagic flux in C2C12 cells with chloroquine inhibited the expression of skeletal muscle-specific genes and the formation of myotubes, confirming a positive role of autophagy in myoblast differentiation and fusion. The aim of the second study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells, which are the myogenic precursor cells in adult skeletal muscle, into myotubes. In this study chromatin immunoprecipitation followed by sequencing (ChIP-seq) was used to identify active enhancers, i.e., genomic regions marked with histone modification H3K27ac (acetylation of lysine 27 of H3 histone protein). 19,027 and 47,669 H3K27ac-marked enhancers were identified from undifferentiated and differentiating bovine satellite cells, respectively. Of these enhancers, 5,882 and 35,723were specific to undifferentiated and differentiating bovine satellite cells, respectively while 13,199 were shared by both undifferentiated and differentiating bovine satellite cells. Many of the H3K27ac-marked enhancers specific to differentiating bovine satellite cells were associated with muscle structure and development genes and were enriched with binding sites for MyoD, AP-1, AP-4, KLF, TEAD, and MEF2 transcription factors. Through siRNA-mediated knockdown, AP-4 was found to be essential for differentiation of bovine satellite cells into myotubes. The objective of the third study was to identify enhancers and transcription factors that control differential gene expression in skeletal muscle between neonatal and adult cattle. First, RNA-seq was performed to compare gene expression profiles in skeletal muscle between neonatal calves and adult steers. This analysis identified 924 genes downregulated and 1,021 upregulated from calf to steer muscle. Among genes downregulated in steer muscle were myosin heavy chain3 (MYH3) and MYH8, and among genes upregulated in steer muscle were MYH7 and myoglobin. Surprisingly, many so-called adult muscle genes, such as MYH1 and MYH2, were not differentially expressed between calf and steer muscle. Gene ontology analyses showed that many genes downregulated in steer muscle are involved in protein synthesis and glycolysis and that many genes upregulated in steer muscle function in blood vessel development and immune cell activation. Next, ChIP-seq was performed to identify genomic regions marked with H3K27ac, i.e., active enhancers, in the skeletal muscle of neonatal calves and adult steers. This experiment led to the finding of 20,163 enhancers specifically active in the calf muscle, 14,909 enhancers specifically active in the steer muscle, and 27,002 enhancers active in both the calf and steer muscle. Motif enrichment analyses revealed the enrichment of binding sites for the KLF family and TEAD family transcription factors in enhancers active specifically in the calf muscle, the enrichment of binding sites for the FOXO family and the SMAD family transcription factors in enhancers specifically active in the steer muscle, and the enrichment of binding sites for the MRF family and MEF2 family transcription factors in enhancers active in both the calf and steer muscle. . These results shed light on the differences in gene expression and biology between newborn calf and adult steer skeletal muscle. These results also shed light on the enhancers and transcription factors that control these differences. / Doctor of Philosophy / Muscle is the central part of meat. So, to improve meat yield, it is essential to know how muscle development is controlled. Muscle development, also called myogenesis, starts with muscle progenitor cells developing into myoblasts. Myoblasts then differentiate and fuse with each other to form myotubes. Myotubes undergo hypertrophy and form functional muscle fibers. During myogenesis, each step involves significant changes in gene expression. Gene expression is controlled mainly by proteins called transcription factors. The overall goal of this project was to identify transcription factors and DNA sequences bound by these factors that control gene expression during muscle development. This project consisted of three studies. In the first study, we used the RNA sequencing (RNA-seq) technique to find genes differentially expressed in myoblasts between before and after terminal differentiation. Analyzing the RNA-seq data led to the discovery that autophagy, a 'self-eating' biological process, is required for myoblast differentiation. In the second study, we used a technique called chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify genomic regions called active enhancers in differentiating bovine myoblasts. This work led to the identification of thousands of active enhancers and dozens of transcription factors binding to these genomic regions that control the differentiation of bovine myoblasts. In the third study, we combined RNA-seq and ChIP-seq to explore the genes and genomic regions controlling muscle transition from newborn calves to adult cattle. This part of the project led to the finding of thousands of genes differentially expressed and thousands of genomic regions differentially activated between newborn calf and adult steer muscle.