Em eucariotos, o DNA é organizado juntamente com histonas em um complexo nucleoproteico conhecido como cromatina, cuja unidade fundamental corresponde aos nucleossomos. A cromatina apresenta-se de duas maneiras: eucromatina, região estruturalmente menos condensada e, portanto, mais facilmente transcrita, e heterocromatina, região muito condensada e transcricionalmente silenciosa. Na forma de eucromatina, o acesso dos fatores de transcrição a regiões de DNA livres de nucleossomos é facilitado, enquanto que na forma de heterocromatina os fatores de transcrição não conseguem acessar o DNA para ativar ou reprimir a expressão gênica inferindo, assim, que o grau de compactação da cromatina interfere na regulação da expressão gênica e que o nucleossomo atua como silenciador gênico. Neste contexto, os objetivos foram identificar, mapear e caracterizar regiões em eucromatina na musculatura esquelética de bovinos da raça Nelore. As análises foram realizadas em relação ao músculo Longissimus dorsi pela técnica Assay for Transposase-Accessible Chromatin (ATAC-Seq), capaz de isolar regiões livres de nucleossomos a partir do mecanismo enzimático de transposição. A fim de otimizar o protocolo dessa metodologia para tecido muscular, foram testadas concentrações de 50 mil, 75 mil e 100 mil núcleos tratados com transposase. Destes, foram encontrados 6.811, 11.121 e 11.473 picos de eucromatina, respectivamente, e 6.212 regiões de cromatina aberta foram coincidentes nas três amostras. A associação entre regiões eucromáticas, expressão gênica e gordura intrasmuscular foi confirmada a partir da análise de sobreposição com transcriptional start sites (TSS), genes expressos em músculo esquelético, genes diferencialmente expressos (GDE) para gordura intramuscular e regiões de expression quantitative trait loci (eQTL) de tecido muscular reforçando, assim, o potencial regulatório das regiões de eucromatina. / In eukaryotes, DNA is organized along with histones in nucleoproteins complexes known as chromatin, which has nucleosomes as their fundamental unit. Chromatin exists in two forms: euchromatin, corresponding to a lightly condensed structure and an easily transcribed region, and heterochromatin, a highly condensed and transcriptionally silent region. In euchromatin form, transcription factors have free access to nucleosome-depleted DNA regions, while in heterochromatin the transcription factors can not access the DNA for activate or repress genic expression, which suggests that the chromatin compaction degree interferes with regulation of gene expression and that nucleosomes act as gene silencer. In this context, the aims of the present project were to identify, map and characterize euchromatin regions in the skeletal musculature of Nellore cattle. Analyzes were performed considering the muscle Longissimus dorsi using Assay for Transposase-Accessible Chromatin technique (ATAC-Seq), which isolates nucleosome-depleted regions throught transposition enzymatic mechanism. Differente transposase-treated nuclei concentrations were tested: 50 thousand, 75 thousand and 100 thousand. From these, 6.811, 11.121, and 11.473 euchromatin peaks were found, respectively, and 6.212 open chromatin regions were coincident among them. The association between euchromatic regions, gene expression and intrasmuscular fat was confirmed from the overlap analysis with transcriptional start sites (TSS), genes expressed in skeletal muscle, differentially expressed genes (GDE) for intramuscular fat and regions of expression quantitative trait loci (eQTL) of muscle tissue, reinforcing the regulatory potential of the euchromatin regions.

Em eucariotos, o DNA é organizado juntamente com histonas em um complexo nucleoproteico conhecido como cromatina, cuja unidade fundamental corresponde aos nucleossomos. A cromatina apresenta-se de duas maneiras: eucromatina, região estruturalmente menos condensada e, portanto, mais facilmente transcrita, e heterocromatina, região muito condensada e transcricionalmente silenciosa. Na forma de eucromatina, o acesso dos fatores de transcrição a regiões de DNA livres de nucleossomos é facilitado, enquanto que na forma de heterocromatina os fatores de transcrição não conseguem acessar o DNA para ativar ou reprimir a expressão gênica inferindo, assim, que o grau de compactação da cromatina interfere na regulação da expressão gênica e que o nucleossomo atua como silenciador gênico. Neste contexto, os objetivos foram identificar, mapear e caracterizar regiões em eucromatina na musculatura esquelética de bovinos da raça Nelore. As análises foram realizadas em relação ao músculo Longissimus dorsi pela técnica Assay for Transposase-Accessible Chromatin (ATAC-Seq), capaz de isolar regiões livres de nucleossomos a partir do mecanismo enzimático de transposição. A fim de otimizar o protocolo dessa metodologia para tecido muscular, foram testadas concentrações de 50 mil, 75 mil e 100 mil núcleos tratados com transposase. Destes, foram encontrados 6.811, 11.121 e 11.473 picos de eucromatina, respectivamente, e 6.212 regiões de cromatina aberta foram coincidentes nas três amostras. A associação entre regiões eucromáticas, expressão gênica e gordura intrasmuscular foi confirmada a partir da análise de sobreposição com transcriptional start sites (TSS), genes expressos em músculo esquelético, genes diferencialmente expressos (GDE) para gordura intramuscular e regiões de expression quantitative trait loci (eQTL) de tecido muscular reforçando, assim, o potencial regulatório das regiões de eucromatina. / In eukaryotes, DNA is organized along with histones in nucleoproteins complexes known as chromatin, which has nucleosomes as their fundamental unit. Chromatin exists in two forms: euchromatin, corresponding to a lightly condensed structure and an easily transcribed region, and heterochromatin, a highly condensed and transcriptionally silent region. In euchromatin form, transcription factors have free access to nucleosome-depleted DNA regions, while in heterochromatin the transcription factors can not access the DNA for activate or repress genic expression, which suggests that the chromatin compaction degree interferes with regulation of gene expression and that nucleosomes act as gene silencer. In this context, the aims of the present project were to identify, map and characterize euchromatin regions in the skeletal musculature of Nellore cattle. Analyzes were performed considering the muscle Longissimus dorsi using Assay for Transposase-Accessible Chromatin technique (ATAC-Seq), which isolates nucleosome-depleted regions throught transposition enzymatic mechanism. Differente transposase-treated nuclei concentrations were tested: 50 thousand, 75 thousand and 100 thousand. From these, 6.811, 11.121, and 11.473 euchromatin peaks were found, respectively, and 6.212 open chromatin regions were coincident among them. The association between euchromatic regions, gene expression and intrasmuscular fat was confirmed from the overlap analysis with transcriptional start sites (TSS), genes expressed in skeletal muscle, differentially expressed genes (GDE) for intramuscular fat and regions of expression quantitative trait loci (eQTL) of muscle tissue, reinforcing the regulatory potential of the euchromatin regions.

Cis-regulatorische Elemente wie Promotoren und Enhancer, die die Regulation der Transkription von Genen steuern, befinden sich in Regionen des dekondensierten Chromatins. DNase-seq und ATAC-seq sind weit verbreitete Verfahren, um solche offenen Chromatinregionen genomweit zu untersuchen. Die einzel-Nukleotid-Auflösung von DNase-seq wurde des Weiteren genutzt, um Transkriptionsfaktor-Bindungsstellen (TFBS) in regulatorischen Regionen durch TF-Footprinting zu bestimmen. Kürzlich durchgeführte Studien haben jedoch gezeigt, dass DNase I einen Sequenzbias aufweist, welcher nachteilige Auswirkungen auf die Footprinting-Effizienz hat. Auch wurden das Footprinting und die Auswirkungen des Sequenzbias auf ATAC-seq noch nicht umfassend untersucht. In dieser Arbeit nehme ich einen systematischen Vergleich der beiden Methoden vor und zeige, dass die beiden Methoden unterschiedliche Sequenzbiases haben und korrigiere diese protokollspezifischen Biases beim Footprinting. Der Einfluss von Bias-Korrekturen der Footprinting Ergebnisse ist für DNase-seq größer als für ATAC-seq, und Footprinting mit DNase-seq führt zu besseren Ergebnissen in unserer Datensätze. Trotz dieser Unterschiede zeige ich, dass die Integration replizierter Experimente die Ableitung von qualitativ hochwertigen Footprints ermöglicht, wobei die beiden Techniken weitgehend übereinstimmen. Diese Techniken werden ferner eingesetzt, um die cis-regulatorischen Elemente zu charakterisieren, die die Embryogenese der Fruchtfliege Drosophila melanogaster bestimmen. Durch die Verwendung von Embryonen die sich im richtigen Entwicklungsstadium befinden, sowie gewebespezifischer Kernsortierung mit offenem Chromatin-Profiling können zeitlich und gewebespezifisch aufgelöste vermeintliche cis-regulatorische Elemente definiert werden. Zusammengenommen demonstrieren diese Analysen die Fähigkeit der offenen Chromatin-Profilierung und der Computeranalyse zur Aufklärung der Mechanismen der Genregulation. / Cis-regulatory elements such as promoters and enhancers, that govern transcriptional gene regulation, reside in regions of open chromatin. DNase-seq and ATAC-seq are broadly used methods to assay open chromatin regions genome-wide. The single nucleotide resolution of DNase-seq has been further exploited to infer transcription factor binding sites (TFBS) in regulatory regions through TF footprinting. However, recent studies have demonstrated the sequence bias of DNase I and its adverse effects on footprinting efficiency. Furthermore, footprinting and the impact of sequence bias have not been extensively studied for ATAC-seq. In this thesis, I undertake a systematic comparison of the two methods and demonstrate that the two methods have distinct sequence biases and correct for these protocol-specific biases when performing footprinting. The impact of bias correction on footprinting performance is greater for DNase-seq than for ATAC-seq, and footprinting with DNase-seq leads to better performance in our datasets. Despite these differences, I show that integrating replicate experiments allows the inference of high-quality footprints, with substantial agreement between the two techniques. These techniques are further employed to characterize the cis-regulatory elements governing the embryogenesis of a complex organism, the fruit fly Drosophila melanogaster. Combining tight staging of embryos and tissue-specific nuclear sorting with open chromatin profiling, enables the definition of temporally and tissue-specifically resolved putative cis-regulatory elements. Taken together, these analyses demonstrate the power of open chromatin profiling and computational analysis in elucidating the mechanisms of transcriptional gene regulation.

Haematopoietic stem cells (HSCs) maintain the vertebrate blood system throughout life. Exploiting their clinical potential requires a thorough understanding of the natural origins of the HSCs. They first arise from the haemogenic endothelium (HE), located in the main embryonic artery, the dorsal aorta. Our understanding of the genetic mechanisms underlying HE specification remains incomplete, but one of the crucial transcription factors is Gata2. We found that a conserved enhancer of zebrafish gata2a gene (i4 enhancer) is active in vivo specifically in endothelial cells, including the HE. To unravel the function of gata2a in specifying the HSCs, we have targeted the i4 enhancer with CRISPR/Cas9, generating the first reported genomic deletion of an endogenous cis-regulatory region in zebrafish. Deletion of the i4 enhancer leads to a decrease in endothelial gata2a expression and a concomitant transient decrease in the number of HSCs. This is marked by an early decrease in the expression of gata2b, a gata2a paralogue previously shown to be required for the initiation of the haematopoietic programme. Our results suggest non-redundant roles of both zebrafish gata2 paralogues in programming of HSCs, providing insights into different roles of GATA2 throughout the programming of HSCs. We also confirmed the previously reported loss of HSCs upon MO-mediated knockdown of lmo4a, associated with increased gata2a expression in HE. We validated the increase in gata2a levels in TALEN-generated lmo4a mutants. To identify the links between lmo4a, gata2a and the HE programming, we have profiled the transcriptome of lmo4a-deficient endothelial cells, including the HE. Our results suggest that Lmo4a may be a global regulator of the transcriptional programming of the HE. Moreover, Wnt signalling pathway may regulate gata2a downstream of lmo4a. This provides novel insights into the gene regulatory network orchestrating the generation of HSCs in the embryo.

Droplet-based microfluidics is a powerful tool permitting massive-scale single-cell analysis in pico-/nano-liter water-in-oil droplets. It has been integrated into various library preparation techniques to accomplish high-throughput scRNA-seq, scDNA-seq, scATAC-seq, scChIP-seq, as well as scMulti-omics-seq. These advanced technologies have been providing unique and novel insights into both normal differentiation and disease development at single-cell level. In this thesis, we develop four new droplet-based tools for single-cell omics profiling. First, the developed Drop-BS is the first droplet-based platform to construct single-cell bisulfite sequencing libraries for DNA methylome profiling and allows production of BS library of 2,000-10,000 single cells within 2 d. We applied the technology to separately profile mixed cell lines, mouse brain tissues, and human brain tissues to reveal cell type heterogeneity. Second, the new Drop-ChIP platform only requires two steps of droplet generation to achieve multiple steps of reactions in droplets such as single-cell lysis, chromatin fragmentation, ChIP, and barcoding. Third, we aim to establish a droplet-based platform to accomplish high-throughput full-length RNA-seq (Drop-full-seq), which both current tube-based and droplet-based methods cannot realize. Last, we constructed an in-house droplet-based tool to assist single-cell ATAC-seq library preparation (Drop-ATAC), which provided a low-cost and facile protocol to conduct scATAC-seq in laboratories without the expensive instrument. / Doctor of Philosophy / Microfluidics is a collection of techniques to manipulate fluids in the micrometer scale. One of microfluidic techniques is called "droplet-based microfluidics". It can manipulate (i.e., generate, merge, sort, split, etc) pico-/nano-liter of water-in-oil droplets. First, since the water phase is separated by the continuous oil phase, these droplets are discrete and individual reactors. Second, droplet-based microfluidics can achieve highly parallel manipulation of thousands to millions of droplets. These two advantages make droplet-based microfluidics an ideal tool to perform single-cell assays. Over the past 10 years, various droplet-based platforms have been developed to study single-cell transcriptome, genome, epigenome, as well as multi-ome. To expand droplet-based tools for single-cell analysis, we aim to develop four novel platforms in this thesis. First, Drop-BS, by integrating droplet generation and droplet fusion techniques, can achieve high-throughput single-cell bisulfite sequencing library preparation. It can generate 10,000 single-cell BS libraries within 2 days which is difficult to achieve for conventional library preparation in tubes/microwells. Second, we developed a novel and facile Drop-ChIP platform to prepare single-cell ChIP-seq library. It is easy to operate since it only requires two steps of droplet generation. It also generates higher quality of data compared to previous work. In addition, we are working on the development and characterization of the other two droplet-based tools to achieve full-length single-cell RNA-seq and single-cell ATAC-seq.

La leucémie est une forme de cancer qui affecte les cellules du système hématopoïétique. Selon la lignée cellulaire affectée et la vitesse de développement du cancer, la leucémie peut être myéloïde ou lymphoïde, aiguë ou chronique, respectivement. Chez les enfants, elles sont souvent caractérisées par la présence de translocations chromosomiques, impliquant notamment le gène KMT2A. L'impact biologique de ces fusions de gènes, connues pour être des perturbateurs épigénétiques, est encore mal compris. Afin d’étudier spécifiquement les conséquences de la présence de fusion impliquant le gène KMT2A, un modèle leucémique humain chez la souris a été mis en place. Le modèle utilisé consiste à induire de manière rétrovirale l’expression d’une fusion oncogénique dans des cellules souches hématopoïétiques et progénitrices d’un unique donneur sain. Ces cellules sont ensuite injectées dans des souris immunodéficientes pour produire une leucémie aiguë myéloïde ou lymphoïde après quelques semaines. L’utilisation de ce modèle leucémique vise à définir les gènes qui sont régulés de manière épigénétique et essentiels dans le processus de leucémogenèse médié par une translocation chromosomique faisant intervenir le gène KMT2A. La première partie des travaux cartographie les changements génétiques et épigénétiques à chacun des stades de la leucémogénèse causée par la fusion KMT2A-MLLT3. Nous avons cartographié les changements épigénétiques tels que la méthylation de l’ADN (Methyl-seq), les modifications des histones (ChIP-seq) et l’accessibilité de la chromatine (ATAC-seq), puis les avons corrélés avec les niveaux d’expression des gènes (RNA-seq). Nous avons observé que les leucémies myéloïdes aiguës présentent un phénotype global d'hypométhylation tandis que les changements d'expression après l'addition de la fusion ont mis en évidence l’inactivation de gènes associés aux cellules souches et des altérations dans d'autres gènes impliqués dans la leucémogenèse tels que S100A8/9. Nos données d’ATAC-seq ont montré qu'il y avait relativement peu de changements spécifiques à la leucémie myéloïde aiguë et que la grande majorité correspondait à des régions de chromatine ouvertes et à des régions contenant des motifs pour des facteurs de transcription précédemment observés dans d'autres types de cellules sanguines. L’analyse des marques d’histones associées à des promoteurs actifs suggère également un potentiel rôle du récepteur CCR1 et de son ligand spécifique CCL23. Finalement, nos résultats suggèrent que la transformation leucémique par la fusion KMT2A-MLLT3 implique des modifications épigénétiques minimes qui requièrent également la coopération des réseaux transcriptionnels utilisés dans les cellules sanguines normales. La deuxième partie de cette thèse s’intéresse à la fusion de gènes KMT2A-MLLT4, une translocation chromosomique peu étudiée mais pour laquelle le pronostic vital des patients est connu pour être défavorable et pire que celui des patients porteurs de la fusion KMT2A-MLLT3. L’extension de notre modèle à la fusion KMT2A-MLLT4 nous permet d’appliquer les mêmes approches que précédemment et de détailler les différences génétiques et épigénétiques entre ces deux fusions, jusqu’à maintenant jamais caractérisées. Nous avons pu observer une baisse globale d’expression dans un groupe de gènes intervenant dans les processus ribosomaux et traductionnels. Par ailleurs, PROM1 (CD133) fait office de potentiel candidat biomarqueur permettant la distinction entre ces deux translocations chromosomiques tandis que le gène LPL pourrait jouer un rôle dans la leucémogenèse médiée par la fusion de gènes KMT2A-MLLT4. En conclusion, l’étude des mécanismes à chacun des stades du développement leucémique nous a fourni une meilleure compréhension des changements épigénétiques intervenant dans le processus de leucémogenèse causé par des réarrangements de type KMT2A. Une meilleure caractérisation de la pathophysiologie de la leucémie pourrait permettre d’explorer des avenues thérapeutiques plus ciblées. / Leukemia is a form of cancer that affects blood cells. Depending on the affected cell lineage and the rate at which the cancer grows, leukemia can be myeloid or lymphoid, or acute or chronic, respectively. In children, they are often characterized by the presence of chromosomal translocations, in particular involving the KMT2A gene. The biological impact of these gene fusions, known to be epigenetic disruptors, is still poorly understood. To study the consequences of the presence of gene fusions involving KMT2A, we have developed a human leukemia model. The model consists of transducing hematopoietic stem and progenitor cells (CD34+) from a single healthy donor with a retrovirus bearing an oncogenic fusion. These cells are injected into immunodeficient mice to produce acute myeloid or lymphoid leukemia after a few weeks. By using this model, we aim to define genes that are epigenetically regulated and essential in the process of leukemogenesis mediated by KMT2A gene fusions. The first part of this thesis characterized the genetic and epigenetic changes at each step of leukemogenesis caused by KMT2A-MLLT3 gene fusion. We investigated epigenetic changes such as DNA methylation (Methyl-seq), histone marks (ChIP-seq), and chromatin accessibility (ATAC-seq) and correlated these with expression changes (RNA-seq). We observed that acute myeloid leukemias exhibit a profound hypomethylation phenotype while expression changes after addition of the fusion highlighted the loss of stem cell associated genes and alterations in other genes implicated in leukemogenesis such as S100A8/9 in the early stages of leukemic transformation. Our ATAC-seq data showed that there were relatively few changes specific to acute myeloid leukemia and that the vast majority corresponded to open chromatin regions and clusters of transcription factors previously seen in other types of blood cells. Examination of ChIP-seq data for active histone marks revealed that leukemia specific expression of the chemokine CCL23 can enable autocrine signalling through its cognate receptor, CCR1. Our results suggest that KMT2A-MLLT3 induces minimal changes in the epigenome while co-opting the normal transcriptional machinery to drive leukemogenesis. The second part of this thesis focuses on KMT2A-MLLT4 gene fusion, another chromosomal translocation for which the vital prognosis of patients is known to be worse than that of patients carrying the KMT2A-MLLT3 fusion. The extension of our model to the KMT2A-MLLT4 fusion allows us to apply the same approaches and to characterize the genetic and epigenetic differences between these two different leukemias. We were able to observe a dramatic decrease in the expression level of a group of genes involved in ribosomal and translational processes. Furthermore, PROM1 (CD133) acts as a potential biomarker candidate which might be used to make the distinction between these two leukemias. LPL gene might play a role in leukemogenesis mediated by KMT2A-MLLT4 gene fusion. In conclusion, studying the mechanisms at each stage of leukemic development has provided us with a better understanding of the epigenetic changes involved in the process of leukemogenesis mediated by KMT2A rearrangements. A better characterization of the pathophysiology of leukemia could make it possible to eventually develop more targeted therapeutic treatments.

Syftet med detta projekt var att undersöka hur vanligt använda cancerläkemedel påverkar mRNA-uttryck och kromatinorganisation i humana erytroleukemiceller. Som modell användes K562-celler från en patient i blastocystkris (2), för att utvärdera leukemicellernas svar på cancerläkemedel vinblastin och doxorubicin. Vinblastin och doxorubicin valdes på grund av deras distinkta mekanismer i cancercellen: medan doxorubicin interkaleras i DNA, hämmar topoisomeras II-aktivitet vilket orsakar celldöd, riktar vinblastin sig mot mikrotubuli för att stoppa mitotisk delning och proliferation. Uttryck av mRNA undersöktes i celler vid 0-timmar, 6-timmar och 24-timmar drogbehandling, samt efter en veckas återhämtning från 24-timmars drogbehandling. Kromatintillgänglighet med ATAC-seq undersöktes i K562-celler vid 0- timmar, 1-timmar, 6-timmar, 24-timmar och 24-timmar + en veckas återhämtning. Därefter utfördes DNA (ATAC-seq) och RNA (mRNA-seq) extraktion och biblioteksberedning på tre biologiska replikat, och öppna DNA-regioner samt mRNA expression undersöktes via sekvensering. Resultaten visade en stark korrelation mellan de biologiska replikaten, vilket indikerar att resultaten var upprepbara. Differentiellt uttryck av mRNA vid doxorubicin- och vinblastinbehandlingar utfördes genom att jämföra mRNA-nivåerna i läkemedelsbehandlade prover med obehandlade (0-timmar). Uppreglerade och nedreglerade gener identifierades och MA-grafer genererades för att visuellt analysera de differentiellt uttryckta generna vid olika tidpunkter efter läkemedelsbehandling och en veckas återhämtning. För att hitta anrikningar av funktionella genkategorier bland de läkemedelsinducerade eller -undertryckta generna, utfördes genontologianalyser. Slutligen användes verktyget Integrative Genomics Viewer (IGV) för att visuellt utforska mRNA-nivåerna och deras differentiella uttrycksmönster under läkemedelsbehandlingar. För ATAC-seq utfördes inte detaljerad dataanalys på grund av tidsbegränsning, men genomets öppenhet undersöktes visuellt genom IGV. Sammantaget inducerade doxorubicinbehandling en långsamt men långvarig förändring av genuttrycket, vilket involverade flera olika biologiska processer. Doxorubicinbehandlade K562-celler ändrade genuttryck att stöda kemoresistens snarare än att inducera apoptos eller celldöd. Behandlingen hade en långvarig inverkan på mRNA-nivåer som sträckte över återhämtningsveckan. Den totala uttrycksförändringen i återhämtningsproverna var förknippad med återhämtning av tumörigena egenskaper och återställning av mekanismener som stöder cellernas tillväxt. Vinblastine förorsakade snabb ökning av mRNA involverade i cytoskelettet. Vid 24-timmars vinblastinbehandling upplevde tumörcellerna stress på grund av grovt elongerad struktur, och de inducerade gener som stöder tumörbildning. En ökning av totala mRNA-nivåer detekterades i vinblastinbehandlade K562-leukemiceller, vilket var särskilt tydligt under återhämtningen. Resultaten visade att cellerna som överlevde vinblastinbehandling fokuserade på att återställa sin strukturella form. Sammantaget visade resultaten att monoterapi inte fungerar effektivt mot leukemiceller eftersom K562-leukemiceller inte bara överlevde läkemedelsbehandlingarna utan också inducerade mRNA som är involverade i resistens mot läkemedelsbehandlingar. / The primary objective of this project is to investigate how commonly used cancer drugs affect mRNA expression and chromatin organization in human erythroleukemia cells. As a model, K562 cells derived from a patient in blastocyst crisis (2) were utilized, evaluating the leukemia cells’ cellular responses to cancer medicines vinblastine and doxorubicin. Vinblastine and doxorubicin were chosen due to the distinct pathways they target in the cell: while doxorubicin intercalates into DNA and inhibits topoisomerase II activity, which eventually cause cell death, vinblastine targets microtubules to stops mitotic division and excessive proliferation. Expression of mRNA was investigated in cells harvested at 0h, 6h, 24h and 24h + one week recovery. Chromatin accessibility with ATAC-seq was investigated in K562 cells harvested at 0h, 1h, 6h, 24h and 24h + one week recovery. Then DNA (ATAC-seq) and RNA (mRNA-seq) extraction and library preparation were performed on three replicates, and the genome-wide results was investigated via sequencing. The results showed a strong correlation between the biological replicates, indicating that the experimental conditions were sustained in these biological variables. Differential Expression of mRNA upon doxorubicin and vinblastine treatments was performed by comparing the mRNA levels in drug-treated samples to non-treated (0h) upregulated and down regulated genes were identified and MA plots generated to visually analyze the differentially expressed genes at different time points after drug treatment and one week recovery. To find enrichments of functional gene categories among the drug-induced or -repressed genes, gene ontology analyses were performed. Finally, the Integrative genomics viewer (IGV) tool was used to visually explore the mRNA levels and their differential expression pattern during drug treatments. For ATAC-seq, detailed data analysis was not performed due to limitation of time, and data was only visually explored through IGV. Taken together, doxorubicin treatment showed slow initial response within 6h followed by an extensive change in gene expression in 24h, involving several different biological processes. The response was more inclined towards chemoresistance rather than inducing apoptosis or cell death. There was a sustained increase in mRNA levels of doxorubicin treated leukemia cells during recovery week. The overall expression change in the recovery samples was majorly linked with not only gaining back the tumourigenic properties and restoring the mechanism which were affected by doxorubicin action but, based on changes in mRNA expression, it looks like doxorubicin treatment made the tumour cells more aggressive. The initial, 6h, response to vinblastine increases mRNAs involved in cytoskeleton. Upon 24h vinblastine treatment the tumour cells experienced stress due to shear force and structural deformity, and they induced genes supporting tumourigenesis. An increase in total mRNA levels was detected in vinblastine-treated K562 leukemia cells, which was particularly evident during recovery. The results indicated that the cells that survived vinblastine treatment focused on recovering its structural form. Overall, the results indicated that monotherapy does not effectively work against leukemia cells as K562 leukemia cells not only survived the drug treatments but also induced mRNAs involved in resistance against drug treatment.