CRITERIA FOR AN UPDATED CLASSIFICATION OF HUMAN TRANSCRIPTION FACTOR DNA-BINDING DOMAINS

<p>By binding to cis-regulatory elements in a sequence-specific manner, transcription factors regulate the activity of nearby genes. Here, we discuss the criteria for a comprehensive classification of human TFs based on their DNA-binding domains. In particular, classification of basic leucine zipper (bZIP) and zinc finger factors is exemplarily discussed. The resulting classification can be used as a template for TFs of other biological species.</p>

Epigenetická regulace genu PU.1 v rezistenci na léčbu 5-azacytidinem u akutní myeloidní leukémie

Hematopoéza je vysoce koordinovaný proces, ve kterém hematopoetická kmenová buňka dává vzniknout všem krevním buněčným elementům. Pro myeloidní a lymfoidní vývoj je nezbytná přísná regulace exprese transkripčního faktoru PU.1. Delece PU.1 u myši je letální a jeho deregulace během vývoje hematopoetických buněk je spojena s hematologickými malignitami jako je akutní myeloidní leukémie (AML) a myelodysplastický syndrom (MDS). MDS a AML jsou závažné poruchy krvetvorby charakterizované expanzí nezralých krevních buněk a nedostatkem diferencovaných funkčních buněk. V patofyziologii leukemogeneze hrají významnou roli nejenom genetické, ale také epigenetické aberace. Deregulace PU.1 související s epigenetickými změnami na regulačních oblastech PU.1 genu představuje intenzivně studovaný mechanismus. Moderní terapie MDS a určité skupiny AML pacientů je založena na léčbě DNA hypometylačními látkami jako je 5-azacytidine (AZA), který ovlivňuje, mimo jiné, i regulaci PU.1 genu. Léčba AZA však často selhává a mechanismy rezistence nejsou příliš známy. V této práci prezentujeme výsledky z klonů rezistentních na AZA připravených z MDS/AML buněčné linie OCI-M2. Analyzovali jsme DNA metylace a hydroxymetylace na klíčovém regulačním elementu genu PU.1 (URE). Zjistili jsme, že epigenetické modifikace na URE značně ovlivňují...Hematopoiesis is a highly orchestrated process, in which a single hematopoietic stem cell (HSC) gives a rise to all blood cellular components. For myeloid and lymphoid development precise controlled expression of the PU.1 transcription factor is needed. Deletion of PU.1 gene in mouse is lethal and its dysregulation during hematopoietic differentiation is associated with blood malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). MDS and AML are serious blood disorders characterized by expansion of immature blood cells and lack of differentiated functional cells. Not only genetic but also epigenetic aberrations represent a very important field for studying pathophysiology of leukemia genesis and dysregulation of the PU.1 gene represents intensively studied candidate mechanism. Modern therapy of selected MDS and subset of AML patients is based on treatment with DNA hypomethylating agent Azacytidine (AZA) interfering in PU.1 gene regulatory mechanism. However, poor response or resistance to this therapy often occurs. In this thesis we present data obtained from AZA-resistant clones of MDS/AML cell line OCI-M2. We analysed DNA methylation and DNA hydroxymethylation at the key regulatory element of the PU.1 gene (URE). We found that these epigenetic modifications at URE...Department of Cell BiologyKatedra buněčné biologiePřírodovědecká fakultaFaculty of Scienc

Analysis of transcription factor binding specificity using ChIP-seq data.

Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis

Development and Applications of Shape-Based DNA Motifs

Transcriptional regulation is imperative for proper development of multicellular organisms, and disregulation of this process can lead to genetic disease. Due to technical limitations, the full human regulome has not been assayed. Computational methods provide resources to fill the gaps in our understanding of these processes. Sequence based representations of transcription factor DNA motifs have long been used for this purpose. We developed a model based on estimates of DNA shape known as Structural Motifs, extending the position weight matrix to accommodate multiple continuous shape parameters at each position. Using expectation maximization, Structural Motifs are discovered de novo from transcription factor binding data, and these motifs are specific to their cognate factors. When considered jointly with sequence motifs, Structural Motifs improve classification of transcription factor binding sites. Joint models also provide insight into the readout mechanisms utilized by transcription factors. DNA shape is an important component of the protein-DNA interaction to consider and improves the computational predictions of transcription factor binding, elevating our understanding of the regulatory landscape