Systematic Analysis of Posterior HOXA/HOXD Function in Mesenchymal Cells
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Lebenswissenschaftliche Fakultät
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Abstract
HOX-Gene sind essentielle Transkriptionsfaktoren (TFs), die den Körperplan, die Struktur und die Organbildung während der Entwicklung bestimmen. Diese komplexen Prozesse werden präzise von in verschachtelter Weise exprimierten HOX-Genen reguliert. In vitro Experimente zeigten
jedoch, dass die HOX-DNA-Bindungsdomäne stark konserviert ist und oft ähnliche DNA-Sequenzen bindet. Die niedrige biochemische Bindungsspezifität und die hochspezifischen Funktionen stehen oft im Widerspruch und bilden das Schlussthema des so genannten Hox-Paradoxons. Das Paradox besteht aufgrund der folgenden Hindernisse: hohe Proteinhomologie, unspezifischen Antikörper sowie die verschachtelte HOX-Expressionsmuster. Das Ziel dieser Arbeit war, diese Probleme zu
überwinden, die HOX-DNA-Bindung in kontrollierten und physiologischen Umstände zu untersuchen und die Bindung von neun Gliedmaßen-spezifischen posterioren HOXA und -D-TFs zu vergleichen. Zu diesem Zweck wurden neun Hühner-HOX-Gene (HOXA- und HOXD9-13) mit dem FLAG markiert und mittels Viren in Gliedmaßen-mesenchymalen Zellen exprimiert. Somit wurde der Vergleich unter identischen und kontrollierten Bedingungen ermöglicht. Im Einklang mit in vivo Funktionsdaten zeigten die HOX-Bindungsprofile, dass zwei direkte Paraloge (z. B. HOXA10 und D10) häufiger dieselben Regionen binden als zwei Nicht-Paraloge (z. B. HOXA9 und A13). Außerdem, die hier beschriebene HOX-DNA-Bindung unterscheidet sich von in vitro Bindung, was darauf hinweist, dass Kofaktoren für deren biologische Funktion wichtig sind. Zusätzlich ergab sich aus dem Bindungsvergleich, dass es zuvor unbekannte Unterschiede zwischen Bindungsweise von HOX-TFs gibt, die zumindest teilweise auf der Häufigkeit von direkter Bindung und Ko-Bindung mit anderen TFs beruhen. Schließlich wurde mit der Kombination von Genetik, Genomik und Biochemie einen neuen HOX-Kofaktor entdeckt, CTCF, der auf ein mögliches Wechselspiel zwischen der HOX-Zielregulation und der Chromatinarchitektur hindeutet.
HOX genes are essential developmental transcription factors (TFs) that pattern the animal body plan, their structures and organs. To precisely control these very diverse processes HOX genes are expressed in a nested fashion and regulate their targets in a context specific way. However, in vitro experiments indicated that HOX DNA binding domain (Homeodomain) is remarkably rigid and often binds very similar DNA sequences. This discrepancy between high functional specificity and low in vitro biochemical specificity is at the core of a problem termed Hox paradox. This paradox persists due to several biological and technical obstacles; namely high HOX protein homology and lack of sufficiently specific antibodies as well as nested HOX expression pattern. The aim of this study was to address these problems, study HOX-DNA binding in a controlled, Hox-native environment and to compare HOX-DNA binding of nine posterior vertebrate HOXA and HOXD TFs. To do this, nine chicken HOX genes (HOXA9-13 and HOXD9-13) were FLAG-tagged and virally expressed in chicken mesenchymal limb-derived cells enabling comparison of their binding in an identical setup and controlled conditions. HOX binding profiles uncovered two direct paralogues (i.e. HOXA10 and D10) bind more often same regions than two non-paralogues (i.e. HOXA9 and A13) reminiscent of in vivo functional data. Moreover, the here described in vivo HOX-DNA binding differs from in vitro binding, indicating the importance of cofactors and biological context for HOX binding and functional outcome. Additionally, binding comparison uncovered previously unknown differences between binding modes of HOX-TFs that at least partially rely on the abundance of direct binding and co-binding with other TFs. Finally, with combination of genetics, genomics and biochemistry a novel HOX cofactor, CCCTC binding factor (CTCF), was discovered suggesting potential interplay between HOX target regulation and chromatin architecture.
HOX genes are essential developmental transcription factors (TFs) that pattern the animal body plan, their structures and organs. To precisely control these very diverse processes HOX genes are expressed in a nested fashion and regulate their targets in a context specific way. However, in vitro experiments indicated that HOX DNA binding domain (Homeodomain) is remarkably rigid and often binds very similar DNA sequences. This discrepancy between high functional specificity and low in vitro biochemical specificity is at the core of a problem termed Hox paradox. This paradox persists due to several biological and technical obstacles; namely high HOX protein homology and lack of sufficiently specific antibodies as well as nested HOX expression pattern. The aim of this study was to address these problems, study HOX-DNA binding in a controlled, Hox-native environment and to compare HOX-DNA binding of nine posterior vertebrate HOXA and HOXD TFs. To do this, nine chicken HOX genes (HOXA9-13 and HOXD9-13) were FLAG-tagged and virally expressed in chicken mesenchymal limb-derived cells enabling comparison of their binding in an identical setup and controlled conditions. HOX binding profiles uncovered two direct paralogues (i.e. HOXA10 and D10) bind more often same regions than two non-paralogues (i.e. HOXA9 and A13) reminiscent of in vivo functional data. Moreover, the here described in vivo HOX-DNA binding differs from in vitro binding, indicating the importance of cofactors and biological context for HOX binding and functional outcome. Additionally, binding comparison uncovered previously unknown differences between binding modes of HOX-TFs that at least partially rely on the abundance of direct binding and co-binding with other TFs. Finally, with combination of genetics, genomics and biochemistry a novel HOX cofactor, CCCTC binding factor (CTCF), was discovered suggesting potential interplay between HOX target regulation and chromatin architecture.
Description
Keywords
HOX, Homeobox, Transkriptionsfaktoren, DNA-Bindung, Genregulation, Entwicklung, Development, ChIP-seq, HOX, Homeobox, Transcription factors, DNA-binding, Gene regulation, ChIP-seq
Dewey Decimal Classification
570 Biologie
References
Has Part: 10.1371/journal.pgen.1006567.s012
Citation
Jerković, Ivana.(2018). Systematic Analysis of Posterior HOXA/HOXD Function in Mesenchymal Cells. 10.18452/19467